Ants as flower visitors and their effects on pollinator behavior
and plant reproduction
by
Adam Richard Cembrowski
A thesis submitted in conformity with the requirements
for the degree of Masters of Science
Ecology and Evolutionary Biology
University of Toronto
copy Copyright by Adam Cembrowski 2013
ii
Ants as flower visitors and their effects on pollinator behavior and plant
reproduction
Adam Cembrowski
Masters of Science
Ecology and Evolutionary Biology
University of Toronto
2013
Abstract
Ants regularly visit flowers but they may decrease plant reproductive success by competing with
pollinators or damaging reproductive structures However how ants may exert these costs needs
further clarification In Chapter 1 I review the literature finding that flower-visiting ants often
have neutral effects on plant fitness In Chapter 2 using artificial flowers with male and female
function I investigate how interference competition between flower-visiting Myrmica rubra ants
and Bombus impatiens bumblebees changes pollen analogue movement patterns Ant presence
and scent significantly reduced pollen analogue donation and reception because bees avoided
flowers with ant cues In Chapter 3 to assess the frequency of palynivory among ants I
conducted an acetolysis survey of 75 neo- and paleo-tropical ant species Ants consistently
contained low numbers of pollen grains suggesting opportunistic pollen consumption may be
widespread Altogether ant visitation may be costly but the mechanism depends on the plant
pollinator and ant identities
iii
Acknowledgments
Science is never an individual undertaking Numerous people made the completion of this thesis
possible First and foremost Megan Frederickson gave me the freedom to pursue research
outside of her realm of expertise was extremely patient my ever-changing interests and
supported the decisions I made in my degree My committee also deserves strong thanks James
Thomson was instrumental in my studies giving me lab space supplies and most importantly
time and guidance when I had questions about anything pollination bee or flower related Ben
Gilbert gave statistics help and was helpful with coding questions As well thank you to my
examination committee Spencer Barrett and Helen Rodd
The Frederickson and Thomson lab groups helped me immeasurably giving encouragement
guidance and reality checks when I went too far off track Thank you to Kyle Turner Lina
Arcila Hernandez Kirsten Prior Jane Ogilvie Alison Parker Eric Youngerman and Rebecca
Batstone Kyle especially helped me in all regards both inside and outside of school and was
without question the best field roommate and officemate I could have had Several volunteers
work study students and our lab tech fed ants washed artificial flowers and counted tiny objects
saving me countless hours particularly Harry Rusnock Shannon Meadley Dunphy Margaret
Thompson and Jackie Day I would also like to highlight Marcus Guorui Tan in particular for
his work collaborating with me and running trials when I was in out of town and being
unwaveringly positive about our research Our experiment would not have been nearly as
successful without him
Several people in the department and associated with the lab provided feedback jokes and
friendship without which grad school would have been a depressing place So thank you Natalie
Jones Rachel Germaine Kelly Carscadden Alex de Serrano Jordan Pleet Aaron Hall Susana
Wadgymar Emily Austen Eddie Ho Amanda Gorton Dorina Szuroczki Jenn Coughlan Jon
Sanders and Gabe Miller
People not in the department and both inside and outside of ecology made my time here possible
and more enjoyable than otherwise Thanks to the Tuner family for taking me in during holidays
Maureen Murray and Matt Mazowita for everything and my old supervisor Colleen Cassady St
Clair for helping me get where I am
iv
Lastly my family did an astounding amount for me in letting me cultivate my love of biology
buying me books letting me keep strange animals and getting me outdoors So thank you to my
brothers John and Mark my dad George and my mom Kay And to my partner Katherine for
her unbelievable patience both in regards to my academic life and in her willingness to adapt
hers for mine thank you This could not have happened without you
ldquoVor Allem sind aus der Reihe der fluumlgellosen Insecten die weitverbreiteten fluumlgellosen Ameisen
sehr unwillkommene Gaumlste der Bluumlthen Und dennoch sind gerade sie nach dem Nectar der
Bluumlthen in hohem Grade luumlsternhelliprdquo
ldquoOf all the wingless insects it is the widely dispersed ants that are the most unwelcome guests to
flowers And yet they are the very ones which have the greatest longing for nectarhelliprdquo
A Kerner 1878 Flowers and their Unbidden Guests
v
Table of Contents
Acknowledgments iii
List of Tables vii
List of Figures viii
Chapter 1 Ants as flower visitors and their effects on pollinator behavior and plant
reproduction 1
Abstract 1
Introduction 2
1 Ant interactions with flowers and floral visitors 3
2 Ants as pollinators 12
3 Floral defenses against ants 14
4 Future directions 20
References 24
Tables 39
Chapter 2 Ants and ant scent reduce bumblebee pollination of artificial flowers 54
Abstract 54
Introduction 55
Methods 57
Results 62
Discussion 63
Acknowledgements 66
References 68
Figures 73
Chapter 3 Not just for the bees pollen consumption is common among tropical ants 75
vi
Abstract 75
Introduction 75
Methods 77
Results 78
Discussion 79
Acknowledgements 82
References 83
Tables 86
Figures 91
Concluding Remarks 93
Copyright Acknowledgments 96
vii
List of Tables
Table 11 List of studies of flower-visiting ants that consumed floral rewards or interacted with
pollinators and included a measure of fitness39
Table 12 List of studies that have either demonstrated ant pollination through exclusion
experiments or whose results strongly suggest it48
Table 31 Numbers of adult worker and larval ants examined for each species collected in Peru
and numbers of pollen grains found87
Table 32 Numbers of adult worker and larval ants examined for each species collected in Papua
New Guinea and numbers of pollen grains found90
viii
List of Figures
Figure 21 Photograph of artificial flower lids with anther and stigma74
Figure 22 Dye transferred (mean plusmn 1SE) by B impatiens a) in the presence and absence of M
rubra ants and b) with or without M rubra scent75
Figure 31 Photographs of pollen grains found inside ants92
Figure 32 Relationship between trophic level and pollen presence for neotropical
ant species93
1
Chapter 1 Ants as flower visitors and their effects on pollinator behavior and plant
reproduction
Cembrowski AR and Frederickson ME
Planned submission to a journal such as Oikos or Insectes Sociaux
ARC wrote the manuscript with input from MEF
Abstract
Ants often provide a range of benefits to flowering plants particularly by reducing herbivory
However when ants visit flowers they can deter pollinators or damage floral structures
Currently it is unclear how often and in what direction flower-visiting ants affect plant
reproductive success A review of the literature shows that ants can help or hinder pollination
processes but overall flower-visiting ants most commonly have a non-significant net effect on
plant fitness Although ants may directly harm flowers or indirectly affect pollination by
consuming floral nectar or harassing pollinators they can also benefit plants by attacking
florivores or predators of pollinators Ants occasionally act as pollinators themselves though ant
secretions often kill pollen grains There is growing evidence that many plants have traits that
allow them to avoid or minimize the negative effects of flower-visiting ants More research is
needed to determine if these traits provide fitness benefits to plants by preventing ant visitation
or if they function as generalized repellents of floral antagonists
2
Introduction
Ants and angiosperms share a long evolutionary history Ant diversification closely followed that
of flowering plants as ants took advantage of the large prey numbers that angiosperms supported
and the new habitats they provided (Moreau 2006) Subsequently numerous angiosperm
lineages evolved intimate associations with ants that function as an indirect or biotic form of
plant defense Food rewards such as extrafloral nectar or pearl bodies or housing in the form of
domatia attract ants to plants where they reduce herbivory and increase plant fitness (reviewed
in Rosumek et al 2009) However the traits that make ants good plant defenders particularly
their abundance attraction to sugar sources and aggressiveness can also cause problems for
plant reproduction Ants attracted by floral nectar can compete with pollinators for resources
and defend flowers potentially reducing plant fitness (Ness 2006 Junker et al 2007) That ants
negatively affect plant reproduction is an old and pervasive hypothesis Kerner (1878) called ants
ldquomost unwelcome guestsrdquo on flowers and more recent authors have similarly expressed that ant
visitation to flowers reduces plant reproductive success (Ness 2006 Lach 2007 Willmer et al
2009)
Nonetheless studies assessing the impact of flower-visiting ants on plant reproductive
success have produced mixed results Ants can have negative impacts on pollination and flower
function (Galen 1983 Puterbaugh 1998 Ness 2006 Hansen and Muumlller 2009) However other
studies have found no effect or even strong benefits of ant visits to flowers (Rico-Gray and
Thien 1989 Altshuler 1999 Schuumlrch 2000 Ashman and King 2005) This discrepancy between
ants being ldquounwelcome guestsrdquo and their actual effects on plant reproduction requires
clarification Our goal here is to highlight the ways that flower-visiting ants may affect plant
reproduction both positively and negatively and to discern through a review of the literature
3
what impacts ants have on plant reproductive success First we discuss mechanisms by which
flower-visiting ants can directly and indirectly impact plant reproduction both through floral
damage and by mediating changes in pollinator or florivore behavior We end this section by
evaluating published research that collectively shows that flower-visiting ants have
predominantly neutral effects on plant fitness measures Next we discuss pollination by ants
focusing on the ldquoantibiotic hypothesisrdquo before describing how plants may prevent ants from
visiting flowers Lastly we discuss promising new avenues of research and highlight work that
still needs to be performed to give a more complete understanding of ants as flower visitors
1 Ant interactions with flowers and floral visitors
i) Damage to flowers
Ants can directly disrupt plant reproduction by damaging floral structures preventing either the
fertilization of ovules or the presentation of pollen Rarely ants forage on floral tissue itself for
example Puterbaugh (1998) found that Formica neorufibarbus ants chewed through the coronal
ring of Eritrichium aretiodes flowers to get lipids More often but still not commonly foraging
ants chew through reproductive tissue to access floral nectar This can cause sterilization of
whole flowers (Echium plantagineum Kirk 1984) or affect female function only if just styles are
damaged effectively creating male flowers (Polemonium viscosum Galen 1983) Antibiotic
secretions on ant integuments may also directly damage pollen grains but the fitness effects of
this are unclear (see 2 below)
Several species of Crematogaster and Allomerus ants sterilize their host plants by
attacking floral buds (Yu and Pierce 1998 Stanton et al 1999 Gaume et al 2005 Maleacute et al
2012 Table 11) These ants nest in ldquoant-plantsrdquo or ldquomyrmecophytesrdquomdashie plants that host ant
4
colonies in specialized structures (domatia) The sterilizing ants do not appear to consume floral
tissues (Yu and Pierce 1998) and two hypotheses may explain why ants destroy floral buds First
ant colony size is often limited by domatia availability on their host plant By destroying floral
buds ants can cause plants to divert resources from reproductive to vegetative growth increasing
domatia number and allowing for larger ant colonies (Yu amp Pierce 1998 Frederickson 2009)
Second when plant-ants must defend their colony from takeovers by other colonies they may
benefit from limiting access to their host plant By destroying buds that could develop into
flowers branches or leaves ants can prevent their host plant from contacting nearby plants
occupied by enemy ant colonies (Stanton et al 1999)
The impact of sterilization on lifetime plant fitness is unclear Ants may destroy all of
their host plantrsquos floral buds (Yu and Pierce 1998) completely preventing reproduction or only a
portion of them (Maleacute et al 2012) allowing some seed set However counter-intuitively this
sterilization behavior may provide long-term fitness benefits to plants (Frederickson 2009
Palmer et al 2010) Ant-plants often host several different ant species over their lifetimes some
of which are non-sterilizing If hosting a sterilizing species at a certain life stage increases
survival or growth relative to hosting a non-sterilizing species at that life stage plants may
increase lifetime fitness by largely forgoing current reproduction in favor of increasing future
reproduction (Palmer et al 2010) Why plants invest resources in producing flowers that are
quickly destroyed by ants is not known but the timing of flower destruction may be important If
ants attack flowers after anthers dehisce plants may still realize some male fitness via pollen
donation (Yu and Pierce 1998) Additionally if the cost of flower production is relatively minor
and ants sometimes ldquomissrdquo flowers (Edwards and Yu 2008) attempting limited reproduction
may be a better strategy than forgoing it entirely
5
ii) Competition with pollinators
Ant activity on flowers can dramatically alter pollinator behavior Ants can reduce the species
diversity frequency or duration of pollinator visits to flowers (Norment 1988 Tsuji et al 2004
Ness 2006 Junker et al 2007 Lach 2007 2008b Romero et al 2011 and see Chapter 2) These
changes are largely due to either exploitative competition (ie consumption of shared resources)
or interference competition (ie direct aggression) between ants and pollinators
a) Exploitative competition
Pollinators and ants frequently compete for floral nectar Flower-visiting ants are often classified
as nectar thieves (sensu Inouye 1980) because they typically enter flowers through the
ldquolegitimaterdquo opening and consume nectar but transfer no pollen (Wyatt 1980 Ness 2006
Chamberlain and Holland 2008) Ants can also act as primary nectar robbers chewing holes
through corollas to reach nectaries (Willmer and Corbet 1981 Koopowitz and Marchant 1998)
although they are more commonly secondary nectar robbers and use holes made by previous
robbers (Newman and Thomson 2005)
Like other flower visitors foraging ants will often leave nectar behind in flowers This
amount can be less (Bleil et al 2011) more (Fritz and Morse 1982) or similar to (Lach 2008a)
amounts left by other visitors Often ants are morphologically constrained from fully exploiting
nectar Unlike bees and butterflies ants do not have proboscises and must get close to nectar to
consume it Narrow corollas or nectar spurs can prevent ant access to some or all floral nectar
(Schemske 1980 Galen and Cuba 2001 and see below) Smaller ants may be better able to
6
access nectar (Newman and Thomson 2005 Agarwal and Rastogi 2007) but can be limited by
their crop size in the amount of nectar they can remove (Lach 2005) If foraging ants leave
enough nectar behind for a pollinator to recover the cost of visiting a flower ants may have
minimal effects on plant reproductive success (Maloof and Inouye 2000 Turner et al 2012)
Differences among ant species in behavior and diet also affect their consumption of floral
nectar Species vary in how many flowers they visit per inflorescence or per plant and in how
many ants will visit a flower simultaneously (Lach 2005 Junker et al 2007 Lach 2013)
Invasive antsmdashthought to be better at exploiting and defending resources than native ants
(Holway et al 2002)mdashoften recruit to flowers in large numbers sometimes several dozen or
more (Visser et al 1999 Hansen and Muumlller 2009) Overall many factors may influence how
heavily ants exploit floral nectar on plants including ant density plant height the spatial or
temporal distribution of floral nectar and the availability of alternative food resources for ants
(Cushman and Addicott 1991 Lach 2013)
Rarely ants and pollinators may compete for pollen We know of only a few studies that
have recorded ants harvesting pollen directly from anthers (Horskins and Turner 1999 Ness
2006 Byk and Del-Claro 2010) Other studies have found pollen grains in ants (eg Baroni
Urbani and de Andrade 1997 and see Chapter 3) but this may not indicate competition with
pollinators ants sometimes collect stray pollen grains from the environment (Creighton 1967)
One New World genus Cephalotes might be specially adapted for pollen consumption They
possess ldquoproventricular shieldsrdquo structures that may restrict solid food to the crop allowing
nestmates to exchange pollen grains via trophallaxis It is unclear if this structure evolved for this
purpose but Cephalotes are the most frequently documented palynivorous ants (eg Creighton
1967 Baroni Urbani and de Andrade 1997 Byk and Del-Claro 2010) How efficiently ants
7
digest pollen and whether ants like bees feed it to larvae as a protein source is unknown At
least two studies have found that ant larvae consume pollen providing some support for this idea
(Wheeler and Bailey 1920 and Chapter 3)
b) Interference competition
Ants are relatively unique among flower visitors in defending floral resources from competitors
(but see Willmer and Corbett 1981 Roubik 1982) This defense ranges from chance encounters
with ants attacking visitors when flower occupancy overlaps (Ashman and King 2005 Junker et
al 2007) to actively patrolling inflorescences or constructing protective galleries around
nectaries (Horvitz and Schemske 1984 Gaume et al 2005 Hansen and Muumlller 2009)
Interference competition even in the absence of exploitative competition is sufficient to alter
pollinator behavior (Chapter 2)
The strength and outcome of interference competition depends on the identities of the ant
and the pollinator Pollinators might not alter their visitation patterns in response to non-
aggressive ants (Junker et al 2007) but more antagonistic ants may elicit avoidance behavior
before or soon after the visitor lands (Willmer and Corbet 1982 Altshuler 1999 Schuumlrch et al
2000 Table 11) Pollinator size may help predict their reaction to ants because ants may deter
only small-bodied pollinators (Gonzaacutelvez et al 2012) It is not clear whether flower visitors view
ants as aggressive competitors or as potential predators rarely ants prey on pollinators (Schatz
and Wcislo 1999) but in most cases ants merely cause pollinators to relocate
How well pollinators recognize and avoid ant-visited flowers is unresolved Collectively
pollinators have lower visitation rates to flowers visited by ants (Romero et al 2011) This is
8
dependent on pollinator identity however dipterans do not appear to discriminate between ant-
visited and ant-free flowers while bees can identify and avoid ant-visited flowers Bees likely
use a combination of visual and olfactory cues to recognize ant-visited flowers They can
identify previous conspecific and some heterospecific insect visitors by the hydrocarbon
ldquofootprintsrdquo they leave during foraging (Stout and Goulson 2001) Bumblebees can detect ant
hydrocarbons although this has been shown only in the laboratory (Ballantyne and Willmer
2012 Chapter 2) No study has yet examined if bees can use sight alone to detect ants but
several suggest it (Junker et al 2007 Gonzaacutelvez et al 2012) Some bees may discriminate
between ant species when visiting flowers (eg Lach 2008b) which follows if not all ant-
pollinator interactions are equally costly The ability of bees to do so will affect how generalized
their avoidance behaviors will be If they can discriminate between species bees may avoid
specific flowers visited by more ldquocostlyrdquo ants However if they cannot bees might either avoid
all ant-visited flowers or none depending on how negative the average interaction with an ant is
iii) Interactions with flower and pollinator antagonists
Ants can defend the flowers they visit Numerous plant species have circumfloral or bracteal
nectaries (ie extrafloral nectaries around flowers) and ants attracted to these nectaries can
reduce bud ovule or flower predation increasing plant fitness (Inouye and Taylor 1979 Rico-
Gray and Thien 1989 Sugiura et al 2006) Although the presence of EFNs (extrafloral nectaries)
so close to floral nectaries (sometimes less than 1 cm Ness 2006) might increase discovery and
exploitation of floral nectar by ants only one study has investigated this hypothesis finding no
relationship between the proximity of EFNs to floral nectaries and antsrsquo use of floral nectar
(Galen 2005) Holes made by nectar robbers are functionally analogous to circumfloral nectaries
9
and can also attract ants that defend flowers (Newman and Thomson 2005) and nectar-thieving
ants can similarly prevent florivory in some systems (Lach 2007 Bleil et al 2011)
Ants can also reduce the abundance of flower-dwelling predators or parasites of
pollinators Ambush predators such as crab spiders often forage at flowers where they may
reduce pollinator visitation rates and plant fitness (Dukas 2001 Gonccedilalvez-Souza et al 2008)
Ants can decrease spider abundance and richness on plants (Rudgers 2004 Renault et al 2005
Nahas et al 2012) and some evidence suggests that they can do the same on flowers (Lach
2008) In select cases ants may also reduce the abundance of pollinator parasitoids (Holland et
al 2011) Whether ants increase plant reproductive success by removing pollinator antagonists
has not yet been examined but should be If ants create lsquoenemy-freersquo flowers pollinators might
prefer to land on flowers with ants
iv) Net effects
Table 11 compiles studies in which the authors recorded ants consuming floral rewards or
interacting with pollinators and measured some aspect of plant fitness (eg fruit or seed set
pollen deposition) We included only a single study per plant species unless subsequent studies
found conflicting results or measured the fitness of another plant sex Records of ant pollination
are included in a separate table (Table 12)
Table 11 reveals relatively few examples of flower-visiting ants negatively affecting
plant fitness given how costly ant visitation is assumed to be The net effect of ants on plant
reproductive success is actually quite variable with similar numbers of studies finding positive
negative or neutral effects of ant presence Surprisingly when ants do have negative effects it is
10
usually because they directly damage floral structures and not because they prevent pollinators
from visiting flowers and successfully transferring pollen Why are ants not more often negative
for plant reproduction when this is commonly assumed to be true
First ants may compensate for reducing pollinator visitation rates by improving
pollination quality As pollinators move between flowers on a plant they often deposit self-
pollen on stigmas (ie geitonogamy) In some plants with late-acting self-incompatibility ovules
fertilized by self-pollen are subsequently aborted reducing the number of seeds that can be
produced (Barrett 2002 and references therein) Geitonogamy can also reduce offspring viability
or vigor (de Jong et al 1993) and cause pollen discounting a reduction in the amount of pollen
available for outcrossing because of self-pollination (Barrett 2002) By competing with
pollinators ants might decrease rates of geitonogamous pollination if pollinators then visit fewer
sequential flowers within a plant Ants might also cause pollinators to fly further between plants
potentially increasing the genetic diversity of pollen arriving at stigmas Although such effects
have been reported for other floral larcenists (Maloof 2001 Irwin 2003) to our knowledge these
hypotheses have not been investigated in relation to ants
Second ants and pollinators do not always forage for the same resources possibly
allowing them to partition floral rewards If ants forage for nectar but not pollen and flowers can
be pollinated efficiently by visitors looking to gather mostly pollen and little or no nectar then
plant reproduction will not be affected by ants consuming floral nectar Junker et al (2010a)
found that honeybees foraged for both pollen and nectar in the absence of ants but reduced their
foraging on nectar but not pollen when ants could access flowers resulting in no difference in
fruit set between treatments Whether pollinators commonly adjust their foraging behaviors in
response to ants warrants further research
11
Third ants are somewhat unique among floral larcenists in that they often provide direct
benefits to the plants they visit In the studies listed in Table 11 ants sometimes decreased
herbivory on plants (Chamberlain and Holland 2008 Bleil et al 2011) or otherwise aided the
plant (eg ant presence repelling smaller less effective pollinators Gonzaacutelvez et al 2012) If the
positive effects of ants cancel or outweigh the negative effects of flower-visiting ants on
pollinators then ant visitation may be neutral or even positive for plant fitness
Fourth ants may impose fitness costs to plants not captured in the studies in Table 11
Nectar robbers have stronger negative effects when plants are pollen-limited (Burkle et al 2007)
but only one study in Table 11 assessed whether flowers would set more seed if they received
more pollen finding no evidence of pollen limitation (Lach 2008a Table 11) Even if ants
decrease pollinator visitation rates there is limited scope for ants to decrease plant reproductive
success when plants are not pollen-limited Furthermore nearly all the studies in Table 11
measured only female fitness Only three quantified aspects of male fitness (Wyatt 1980 Fritz
and Morse 1981 Galen and Geib 2007) and we know of only one other study in a system in
which ants occasionally pollinate that has also measured male fitness (Ashman 2000) All four
studies measured pollen or pollinia removal rates and none found any effect of flower-visiting
ants on male fitness However whether other measures of male fitness (eg seeds sired pollen
deposition patterns) would reveal similar or different effects needs investigation
Finally although floral defenses against ants (see 3 below) may prevent us from
observing the negative effects of ants on plant reproductive success we consider this unlikely
Several studies have examined interactions between ants and plants that share little or no
coevolutionary history usually in the context of ant invasions Although invasive ants often
recruit to flowers in larger numbers than native ants they do not appear to reduce plant
12
reproductive success (Blancafort and Goacutemez 2005 Lach 2007 Lach 2008a but see Hansen and
Muumlller 2009) And in Hawaii where angiosperms evolved for millions of years in the absence of
ants and are thought to have secondarily lost floral defenses against ants (Junker et al 2011a)
there is no evidence that the preponderance of recently introduced flower-visiting ants negatively
affects plant fitness (Junker et al 2010a Bleil et al 2011)
2 Ants as pollinators
Ants may directly benefit plant reproduction by acting as pollinators but this is a relatively rare
occurrence Ants have long been considered poor pollinators (Kerner 1878) Initially this was
based on morphological and behavioral differences between ants and recognized pollinators
Worker ants are wingless and often relatively hairless making them poor pollen dispersers As
well because many ants exhibit Ortstreue (literally ldquoplace fidelityrdquo) ants were thought to return
to the same branch or flower repeatedly effecting no cross pollination (Houmllldobler and Wilson
1990) Though largely true these traits do not necessarily preclude ants from acting as
pollinators (Table 12) Although they commonly have restricted foraging ranges (Houmllldobler and
Wilson 1990) some ants can effect outcrossing (eg Ashman and King 2005 Carvalheiro 2008)
and many ants are as hairy if not hairier than their pollinating counterparts
In the late 1970s the ldquoantibiotic hypothesisrdquo was put forward to explain the rarity of ant
pollination Ants use antibiotic secretions to protect themselves against the fungal and bacterial
pathogens common in their nests and environs Iwanami and Iwadare (1978) found that one of
these compounds myrmicacin also greatly reduces pollen germination rates Subsequently
Beattie et al (1984 1985) found that short periods of contact (le 30 minutes) with an antrsquos
integument caused pollen grains to fail to germinate Some of these pollen-killing compounds
13
were traced back to the ant metapleural gland which produces hygienic secretions (Houmllldobler
and Wilson 1990) although contact with ants lacking metapleural glands can also reduce pollen
viability (Beattie et al 1985 Dutton and Frederickson 2012) Ant mandibular or anal gland
secretions and potentially ant venom may likewise defend ants against pathogens and have
similar effects on pollen germination (Beattie et al 1985 Graystock and Hughes 2011)
Nonetheless ant pollination has been documented repeatedly There are at least 18 cases
in which ant pollination has been shown experimentally and five others in which observations
strongly suggest it (Table 12) The majority of these plants fit the ldquoant pollination syndromerdquo
originally proposed by Hickman (1974) dense stands of short plants with small flowers and
easily accessible nectaries Typically ant-pollinated plants are associated with hot and dry
climates (principally in the Mediterranean) but dry alpine environments may also favor the
evolution of ant pollination (eg Svensson 1985 Puterbaugh 1998 Schiestl and Glaser 2011) In
most cases ants appear to be the sole or major pollinator of the plant in question (Hickman 1974
Peakall et al 1990 de Vega et al 2009) although ants sometimes make only supplementary
contributions to pollination (Schuumlrch et al 2000 Ashman and King 2005) This suggests that co-
evolution between ants and plants may not be necessary for ants to be useful pollinatorsmdashwhich
is surprising given the potential negative effects of ants on plant reproduction
Ant-pollinated plants cope with ant secretions in a variety of ways Several plant species
may simply tolerate pollen damage (Gόmez and Zamora 1992 de Vega et al 2009) apparently
possessing no overt adaptations to prevent it These plants might receive enough pollen to
overcome reduced pollen viability Other plants may place pollen on ant body parts thought to
have fewer secretions thereby potentially escaping damage (eg on ant legs Ramsey 1995) or
attach pollen to ants with specialized structures avoiding direct contact with the ant integument
14
(Peakall et al 1990) It is not known whether pollen evolves resistance to ant secretions or
whether plants deposit pollen on ants which have less disruptive secretions although these
possibilities have been suggested elsewhere (eg Garciacutea et al 1995 Ramsey 1995)
One key question regarding the effects of ant secretions on pollen grains urgently needs
to be resolved Several studies have suggested that the pollen-killing secretions of ants may have
contributed to the evolution of floral defenses against ants (eg Wagner 2000 Galen and Geib
2007) For this to be true ants must decrease male fitness by damaging pollen grains before they
leave flowers and are deposited on receptive stigmas Only two studies have investigated the
effects of ant secretions on pollen still in anthers (Wagner 2000 Galen and Butchard 2003) and
both constrained ants unnaturally to flowers for significantly longer than ants would normally
stay on flowers Although these studies found strong reductions in pollen vigor it is unclear if
ant secretions decrease male fitness under natural ant visitation conditions
3 Floral defenses against ants
Plants are not passive recipients of flower visitors and have some ability to ldquochooserdquo which
organisms can access floral rewards Flowers resist ants using both structural and chemical traits
More thorough investigation is needed to determine whether these traits evolved to prevent or
minimize the negative effects of ants on plant reproductive success (ie whether these traits can
truly be considered defenses against ants see 4ia below) It is unlikely however that ants
experience reciprocal selection to circumvent floral resistance traits because the fitness benefits
to ants of accessing flowers are probably small Thus there may be little scope for
coevolutionary arms races between floral defenses against ants and ant counter-adaptations
15
i) Structural resistance traits
a) Restricting ant access
Numerous plant species have physical structures preventing ant access to flowers Thin pedicels
can limit antsrsquo gripping abilities and make ants easier to dislodge when flowers are moved by
wind (Gaume et al 2005) and waxy secretions can render stems too slippery for ants to climb
(Harley 1991) Structures surrounding flowers such as water moats or dense hairs may similarly
keep ants from accessing floral rewards (Willmer et al 2009) Many such traits function as broad
filters and prevent not only ants but most crawling insects from reaching flowers Thus it is
difficult to determine whether ants florivores or both acted as selective agents favoring the
evolution of physical barriers to flowers
Flower shape may also reduce ant-pollinator conflict Long narrow corollas or nectar
spurs can prevent all but the smallest ants from accessing floral nectaries (Bluumlthgen et al 2004
Agarwal and Rastogi 2007) while still allowing long-tongued pollinators access to rewards
Selection to exclude nectar-thieving ants in such a manner however may be counteracted by
pollinator preferences for wider corollas that allow easier flower handling (Galen and Cuba
2001) Additionally these types of flowers may be more prone to robbery (Irwin and Maloof
2002) limiting their usefulness in preventing nectar larceny
b) Separation in space or time
Plants may separate floral rewards and ants temporally or spatially to minimize ant impacts on
plant reproduction Several authors have proposed that myrmecophytes have adapted to the
16
sterilization or otherwise detrimental activities of ants by locating domatia or food rewards on
different branches than flowers (Izzo and Vasconcelos 2002 Raine et al 2002 Edwards and Yu
2008) Temporal separation of ant and pollinator activities could likewise reduce conflict though
evidence is limited Some acacia flowers dehisce during periods of low ant activity (Willmer and
Stone 1997 Nicklen and Wagner 2006) but it is unclear if this is in response to ants
temperature or pollinator activity Timing of nectar secretion may similarly reduce ant-pollinator
competition If ants are active prior to pollinators they may deplete floral nectar and reduce
pollinator visitation rates (Schaffer et al 1984) However if nectar is secreted when ants are
inactive pollination may occur unimpeded (Norment 1988) This temporal separation could
favor pollinators that are active when ants are not (eg Wyatt and Morse 1981) but this is
poorly documented Further studies particularly focusing on flowers with the potential for
nocturnal and diurnal pollination may provide more examples
c) Extrafloral nectaries (EFNs)
One hypothesis put forward to explain the evolution of EFNs proposes that EFNs ldquodistractrdquo ants
from consuming floral nectar (Kerner 1878 Wagner and Kay 2002) Wagner and Kay (2002)
tested this hypothesis by constructing model plants using artificial nectaries assigned at random
to represent either floral or extrafloral nectaries They found that the presence of EFNs could
reduce ant exploitation of floral nectar However as highlighted by Galen (2005) this design is
closer to a plant making a surplus of flowers some of which will avoid ant visitation if visitation
rates increase sublinearly with increasing flower number Producing extra flowers to escape ant
visitation appears to be a viable strategy in some systems (Ashman and King 2005 Galen 2005)
However Galen (2005) found that applying honey (simulating extrafloral nectar) to P viscosum
17
leaves had the opposite effect and increased ant visits to flowers Only two studies both in
Pachycereus schottii have found evidence for the distraction hypothesis under field conditions
(Chamberlain and Holland 2008 Holland et al 2011)
There is limited empirical support for the distraction hypothesis but abundant evidence
that EFNs attract ants that defend plants against herbivores (reviewed in Rosumek et al 2009)
To function as attractants the presence of EFNs must increase ant abundance on plants
However because of the increased numbers attracted EFNs would not be expected to reduce the
abundance of ants on nectar rewards or be able to satiate the ants recruited Only if ant
abundance stops increasing at a reward density much lower than that available on the plant
would EFNs serve to distract ants In this case the amount of rewards may overwhelm ants
leaving some flowers underexploited However even in P schottii ants still visited flowers
(Chamberlain and Holland 2008) In cases where ant abundance would continue to increase with
additional rewards it is unlikely that EFNs function as distractions
ii) Chemical resistance traits
a) Toxic nectar
One of the earliest proposed floral defenses against ants was toxic nectar (Janzen 1977)
However studies testing Janzenrsquos (1977) hypothesis uncovered only a very few isolated
examples (eg Catalpa speciosa Stephenson 1981 Crinum erubescens Guerrant and Fiedler
1981) in short most floral nectar is not toxic or repellent to ants (Baker and Baker 1978
Schubart and Anderson 1978) In fact ants sometimes prefer floral nectar over solutions with
equivalent sugar concentrations (Koptur and Truong 1998)
18
In contrast floral nectar can repel other flower visitors (eg birds Johnson et al 2006
lepidopterans Kessler and Baldwin 2007) Repellency is often caused by plant secondary
metabolites which may occur in nectar as a by-product of their production elsewhere in the
plant where they function as defenses against herbivory (Adler 2001) Alternatively toxic or
repellent nectar may be adaptive repellent nectar may promote effective pollination by reducing
the amount of reward any one pollinator takes spreading nectar over several pollinator visits
(Kessler and Baldwin 2007) or it may function as a filter selecting for specific pollinators
(Johnson et al 2006) The latter may explain why repellent nectar is not more common
legitimate pollinators in addition to floral larcenists may be deterred from visiting flowers with
repellent nectar This may be particularly true of nectars that would repel ants which are closely
related to bees nectar distasteful to ants may often also be distasteful to bees and those nectars
acting as a filter for bees may still be palatable to ants For example Prŷs-Jones and Willmer
(1992) found that the ammonia-containing nectar of Lathraea clandestina is readily consumed
by both bees and ants even though it deters birds that act as nectar thieves Although floral
nectar itself rarely repels ants Guerrant and Fielder (1981) found that the presence of macerated
petals within nectar can often keep ants away Alkaloids in petals may be responsible and might
explain why ants seldom act as primary nectar robbers these secondary compounds may prevent
ants from chewing through corollas
b) Floral volatiles
Repellent floral volatiles appear to be a more widespread form of chemical resistance against
ants These were reviewed recently by Willmer et al (2009) and will be discussed here only
briefly Numerous plant taxa produce floral scents repellent to ants including myrmecophilous
19
myrmecophytic and non-ant associated plants in both temperate and tropical ecosystems
(Willmer et al 2009 Junker et al 2011a Ballantyne and Willmer 2012) In three large studies
floral volatiles that repel ants were detected in about a third of all plants tested (Willmer et al
2009 Junker et al 2011a Ballantyne and Willmer 2012) Pollen is often the source of ant-
repellent volatile compounds (Willmer et al 2009 Ballantyne and Willmer 2012) but they can
be emitted from other floral parts including petals That pollen is often the source of repellence
could suggest that preventing ant visitation may be more important in protecting male than
female function Additionally because repellence would decrease as pollen is removed from
anthers ants may return and defend flowers more quickly than if repellent chemicals were in
longer-lasting structures (eg petals) In some cases floral volatiles are thought to mimic ant
alarm pheromones (Willmer et al 2009) However floral scents elicit inconsistent reactions
across ant species ranging from repellence to attraction (Agarwal and Rastogi 2007 Ballantyne
and Willmer 2012)
Producing structural or chemical resistance traits may be costly for plants Native
Hawaiian plants which have little recent coevolutionary history with ants appear to have
secondarily lost floral ant repellents (Junker et al 2011a) Both Willmer et al (2009) and Junker
et al (2011a) found that highly chemical repellent flowers did not have strong physical defenses
and vice versa suggesting trade-offs between the two (but see Ballantyne and Willmer 2012)
Like toxic nectar floral ant repellents might sometimes deter both pollinators and ants For
example Galen et al (2011) found that 2-phenylethanol in P viscosum floral scent repels both
flower-damaging F neorufibarbis ants and the plantrsquos main pollinator Bombus kirbyellus
Interestingly four recent studies have found that floral scents can also attract ants
(Agarwal and Rastogi 2007 Edwards and Yu 2008 Schiestl and Glaser 2011 Gonzaacutelvez et al
20
2012) One study (Schiestl and Glaser 2011) has shown that floral scents attract ants to a
potentially ant-pollinated plant and such systems may provide many more examples
4 Future directions
We have highlighted areas in need of further investigation throughout the review Here we focus
on several questions we consider high priorities for future research
i) Floral defenses against ants
a) Do floral resistance traits increase plant fitness
The existence ant-repellent floral traits is often taken as evidence that ants negatively affect plant
reproductive success However despite the abundance and variety of mechanisms excluding ants
from flowers few studies show that ants reduce plant fitness when they visit flowers (Table 11)
This absence is particularly apparent with repellent floral volatiles which may be the most
widespread floral resistance trait (Willmer et al 2009 Junker et al 2011a Ballantyne and
Willmer 2012) To our knowledge only one study has shown that floral ant repellents benefit
plants with repellent P viscosum flowers avoiding damaging ant visits (Galen et al 2011)
Research is urgently needed to investigate if these traits increase plant fitness by excluding ants
or if ant visitation is inconsequential More studies taking advantage of standing variation in
traits (Galen et al 2011) or creating it artificially (eg Junker et al 2010b) will allow us to
distinguish between the two
21
b) If ant visitation is often not costly what caused the evolution of repellent traits and
what maintains them Similarly are these repellent traits truly defenses against ant
visitation or are they generalized defenses against a range of floral antagonists
Ant-repellent floral traits are widespread and their production may be costly (Willmer et al
2009 Junker et al 2011a) The studies in Table 11 however indicate that flower-visiting ants
often do not disrupt plant reproduction particularly outside of myrmecophytes What then
selected for these traits One possibility is that these repellents are not directed solely at ants
Instead a community of antagonistic flower visitors may exert diffuse selection on plants
thereby selecting for and maintaining these traits Structural modifications may repel crawling
insects while chemical repellents such as floral volatiles may repel both flying and non-flying
insects (Junker et al 2011b) How much overlap there is in floral volatiles that repel both ants and
other antagonists needs investigation Until we examine how specific these defenses are to ants
we risk overstating the importance of ants in their evolution
ii) Do ants alter pollen movement patterns or reduce male fitness
We are aware of only four studies that have measured if ant visitation impacts male fitness
(Wyatt 1980 Fritz and Morse 1981 Ashman 2000 Galen and Geib 2007) While none of these
studies found an effect all measured only amounts of pollen or pollinia removed However this
measure does not adequately encompass male fitness alterations to pollen movement patterns
and eventual deposition location may be more important to male function than changes in
removal rates If as suggested by some studies (Altshuler 1999 Lach 2007) ants increase
pollinator relocation rates they may decrease pollen discounting increasing male fitness
However if this increased relocation reduces overall pollen export rates ant competition with
22
pollinators may depress male function Studies using pollen-tracking methods such as fluorescent
dyes will allow us to see how ants change pollen movement patterns and methods like paternity
analysis will reveal how competition between ants and pollinators alters male fitness
iii) Coevolution between ants and plants do myrmecophytes exhibit stronger more
specific defenses against ants
The floral traits of myrmecophytes are most likely to coevolve with ants because all plant
reproduction occurs in the presence of ants Their floral defenses would thus be expected to be
both stronger and more specific to their resident ants than those other plants visited by an array
of more opportunistic ant species Some evidence supports this Willmer et al (2009) found that
resident ants of acacias were repelled by flowers with more aggressive ant species showing
stronger repellence than less aggressive ants Additionally ants not normally associated with
acacias did not respond to acacia floral volatiles More studies are needed in other systems and
should take advantage of phylogenetic approaches examining floral traits in closely related
myrmecophytic and non-myrmecophytic plant species to determine whether ants commonly
select for floral defenses against ants in myrmecophytes
Acknowledgements
For comments and discussion we thank the Frederickson lab particularly Eric Youngerman
Lina Arcila Hernandez and Kyle Turner as well as Alison Parker and Jordan Pleet MEF
acknowledges financial support from an NSERC Discovery Grant a Connaught New Researcher
23
Award an Ontario Ministry of Economic Development and Innovation Early Researcher Award
and the University of Toronto ARC was supported by an Ontario Graduate Scholarship and
Sigma Xi
24
References
Adler L S 2001 The ecological significance of toxic nectar Oikos 91409ndash420
Agarwal V M and N Rastogi 2007 Role of floral repellents in the regulation of flower visits
of extrafloral nectary-visiting ants in an Indian crop plant Ecological Entomology 3359ndash
65
Altshuler D L 1999 Novel interactions of non-pollinating ants with pollinators and fruit
consumers in a tropical forest Oecologia 119600ndash606
Ashman TL 2000 Pollinator selectivity and its implications for the evolution of dioecy and
sexual dimorphism Ecology 812577-2591
Ashman TL and E A King 2005 Are flower-visiting ants mutualists or antagonists A study
in a gynodioecious wild strawberry American Journal of Botany 92891ndash5
Ballantyne G and P Willmer 2012 Floral visitors and ant scent marks noticed but not used
Ecological Entomology 37402-409
Ballantyne G and P Willmer 2012 Nectar theft and floral ant-repellence a link between
nectar volume and ant-repellent traits PloS One 7e43869
Baker H G and I Baker 1978 Ants and flowers Biotropica 1080
Barrett S C H 2002 Sexual interference of the floral kind Heredity 88154ndash159
Baroni Urbani C and M L de Andrade 1997 Pollen eating storing and spitting by ants
Naturwissenschaften 84256ndash258
25
Beattie A J C Turnbull R B Knox and E G Williams 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany
71421ndash426
Beattie A J C Turnbull T Hough S Jobson and R B Knox 1985 The vulnerability of
pollen and fungal spores to ant secretions evidence and some evolutionary implication
American Journal of Botany 72606ndash614
Blancafort X and C Goacutemez 2005 Consequences of the Argentine ant Linepithema humile
(Mayr) invasion on pollination of Euphorbia characias (L) (Euphorbiaceae) Acta
Oecologica 2849ndash55
Bleil R N Bluumlthgen and R R Junker 2011 Ant-plant mutualism in Hawailsquoi Invasive ants
reduce flower parasitism but also exploit floral nectar of the endemic shrub Vaccinium
reticulatum (Ericaceae) Pacific Science 65291ndash300
Bluumlthgen N G Gottsberger and K Fiedler 2004 Sugar and amino acid composition of ant-
attended nectar and honeydew sources from an Australian rainforest Austral Ecology
29418ndash429
Burkle L A R E Irwin and D A Newman 2007 Predicting the effects of nectar robbing on
plant reproduction implications of pollen limitation and plant mating system American
Journal of Botany 941935ndash1943
Byk J and K Del-Claro 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333ndash38
26
Caballero P C G Ossa W L Gonzaacuteles C Gonzaacutelez-Browne G Astorga M M Muruacutea and
R Medel 2013 Testing non-additive effects of nectar-robbing ants and hummingbird
pollination on the reproductive success of a parasitic plant Plant Ecology 214633ndash640
Carvalheiro L G E R M Barbosa and J Memmott 2008 Pollinator networks alien species
and the conservation of rare plants Trinia glauca as a case study Journal of Applied
Ecology 451419ndash1427
Chamberlain S A and J N Holland 2008 Density-mediated context-dependent consumer-
resource interactions between ants and extrafloral nectar plants Ecology 891364ndash1374
Creighton W S 1967 Studies on free colonies of Cryptocerus texanus Satschi (Hymenoptera
Formicidae) Psyche 7434ndash42
Cursach J and J Rita 2011 Implications of the reproductive biology of the narrow endemic
Naufraga balearica (Apiaceae) for its conservation status Plant Systematics and Evolution
298581ndash596
Cushman JH and Addicott JF In Huxley CR and DF Cutler (eds) 1st edition Ant-plant
interactions Oxford University Press Oxford
de Jong T J N M Waser and P G Klinkhamer 1993 Geitonogamy the neglected side of
selfing Trends in Ecology and Evolution 8321ndash325
de Vega C M Arista P L Oritz C M Herrera and S Talavera 2009 The ant-pollination
system of Cytinus hypocistis (Cytinaceae) a Mediterranean root holoparasite Annals of
Botany 4517ndash8
27
Dukas R 2001 Effects of perceived danger on flower choice by bees Ecology Letters 4327ndash
333
Dutton E M and M E Frederickson 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561ndash569
Edwards D P and D W Yu 2008 Tolerating castration by hiding flowers in plain sight
Behavioral Ecology and Sociobiology 6395ndash102
Frederickson M E 2009 Conflict over reproduction in an ant-plant symbiosis why Allomerus
octoarticulatus ants sterilize Cordia nodosa trees The American Naturalist 173675ndash81
Fritz R S and D H Morse 1981 Nectar parasitism of Asclepias syriaca by ants effect on
nectar levels pollinia insertion pollinaria removal and pod production Oecologia 50316ndash
319
Galen C 1983 The effects of nectar thieving ants on seedset in floral scent morphs of
Polemonium viscosum Oikos 41245ndash249
Galen C 2005 Catching ants with honey an experimental test of distraction and satiation as
alternative modes of escape from flower-damaging ants Oecologia 14480ndash87
Galen C and B Butchart 2003 Ants in your plants effects of nectar-thieves on pollen fertility
and seed-siring capacity in the alpine wildflower Polemonium viscosum Oikos 101521ndash
528
Galen C and J Cuba 2001 Down the tube pollinators predators and the evolution of flower
shape in the alpine skypilot Polemonium viscosum Evolution 551963-1971
28
Galen C and J C Geib 2007 Density-dependent effects of ants on selection for bumble bee
pollination in Polemonium viscosum Ecology 881202ndash1209
Galen C R Kaczorowski S L Todd J Geib and R A Raguso 2011 Dosage-dependent
impacts of a floral volatile compound on pollinators larcenists and the potential for floral
evolution in the alpine skypilot Polemonium viscosum The American Naturalist 177258ndash
272
Garciacutea M B R J Antor and X Espadaler 1995 Ant pollination of the palaeoendemic
dioecious Borderea pyrenaica (Dioscoreaceae) Plant Systematics and Evolution 19817ndash
27
Gaume L M Zacharias and R M Borges 2005 Ant ndash plant conflicts and a novel case of
castration parasitism in a myrmecophyte Evolutionary Ecology Research 7435ndash452
Graystock P and W O H Hughes 2011 Disease resistance in a weaver ant Polyrhachis
dives and the role of antibiotic-producing glands Behavioral Ecology and Sociobiology
652319ndash2327
Gόmez J M 2000 Effectiveness of ants as pollinators of Lobularia maritima effects on main
sequential fitness components of the host plant Oecologia 12290ndash97
Gόmez J M and R Zamora 1992 Pollination by ants consequences of the quantitative effects
on a mutualistic system Oecologia 91410ndash418
Gόmez J M R Zamora J A Hόdar and D Garciacutea 1996 Experimental study of pollination
by ants in Mediterranean high mountain and arid habitats Oecologia 105236ndash242
29
Gonccedilalves-Souza T P M Omena J C Souza and G Q Romero 2008 Trait-mediated effects
on flowers artificial spiders deceive pollinators and decrease plant fitness Ecology
892407ndash2413
Gonzaacutelvez F G L Santamariacutea R T Corlett and M A Rodriacuteguez-Gironeacutes 2012 Flowers
attract weaver ants that deter less effective pollinators Journal of Ecology 10178ndash85
Guerrant E O and P L Fiedler 1981 Flower defenses against nectar-pilferage by ants
Biotropica 1325ndash33
Harley R 1991 The greasy pole syndrome In Huxley CR and DF Cutler (eds) 1st edition
Ant-plant interactions Oxford University Press Oxford
Hansen D M and C B Muumlller 2009 Invasive ants disrupt gecko pollination and seed
dispersal of the endangered plant Roussea simplex in Mauritius Biotropica 41202ndash208
Hickman J C 1974 Pollination by ants a low-energy system Science 1841290ndash1292
Holland J N S a Chamberlain and T E X Miller 2011 Consequences of ants and
extrafloral nectar for a pollinating seed-consuming mutualism ant satiation floral
distraction or plant defense Oikos 120381ndash388
Houmllldobler B and Wilson E O 1990 The Ants 1st Edition Harvard University Press
Cambridge
Holway DA L Lach AV Suarez ND Tsutsui and TJ Case 2002 The causes and
consequences of ant invasions Annual Review of Ecology and Systematics 33181-233
30
Horskins K and Turner VB 1999 Resource use and foraging patterns of honeybees Apis
mellifera and native insects on flowers of Eucalyptus costata Australian Journal of
Ecology 24221-227
Horvitz C C and D W Schemske 1984 Effects of ants and an ant-tended herbivore on seed
production of a neotropical herb Ecology 651369ndash1378
Inouye DW 1980 The terminology of floral larceny Ecology 611251-1253
Inouye DW and O R J Taylor 1979 A temperate region plant-ant-seed predator system
consequences of extra floral nectar secretion by Helianthella quinquenervis Ecology 601ndash
7
Irwin RE 2003 Impact of nectar robbing on estimates of pollen flow conceptual predictions
and empirical outcomes Ecology 84485-495
Irwin RE and Maloof JE 2002 Variation in nectar robbing over time space and species
Oecologia 133525-533
Iwanami Y and T Iwadare 1978 Inhibiting effects of myrmicacin on pollen growth and pollen
tube mitosis Botanical Gazette 13942ndash45
Izzo TJ and Vasconcelos HL 2002 Cheating the cheater domatia loss minimizes the effects
of ant castration in an Amazonian ant plant Oecologia 133200-205
Janzen D H 1977 Why donrsquot ants visit flowers Biotropica 9252
Johnson SD Hargreaves AL and Brown M 2006 Dark bitter-tasting nectar functions as a
filter of flower visitors in a bird-pollinated plant Ecology 872709-2716
31
Junker R A Y C Chung and N Bluumlthgen 2007 Interaction between flowers ants and
pollinators additional evidence for floral repellence against ants Ecological Research
22665ndash670
Junker R R R Bleil C C Daehler and N Bluumlthgen 2010a Intra-floral resource partitioning
between endemic and invasive flower visitors consequences for pollinator effectiveness
Ecological Entomology 35760ndash767
Junker R R and N Bluumlthgen 2010b Floral scents repel facultative flower visitors but attract
obligate ones Annals of botany 105777ndash82
Junker R R C C Daehler S Doetteri A Keller and N Bluumlthgen 2011a Hawaiian ant ndash
flower networks nectar-thieving ants prefer undefended native over introduced plants with
floral defenses Ecological Monographs 81295ndash311
Junker R R J Gershenzon and S B Unsicker 2011b Floral odor bouquet loses its ant
repellent properties after inhibition of terpene biosynthesis Journal of Chemical Ecology
371323ndash31
Kawakita A and M Kato 2002 Floral biology and unique pollination system of root
holoparasites Balanophora kuroiwai and B tobiracola (Balanophraceae) American
Journal of Botany 891164ndash1170
Koopowitz H and TA Marchant 1998 Postpollination nectar reabsorption in the African
epiphyte Aerangis verdickii (Orchidaceae) American Journal of Botany 85508-512
Kirk W D J 1984 Ecological studies on Thrips imaginis Bagnall (Thysanoptera) in flowers of
Echium plantagineum L in Australia Australian Journal of Ecology 99ndash18
32
Kerner A 1878 Flowers and their unbidden guests C Kegan Paul London
Kessler D and I T Baldwin 2007 Making sense of nectar scents the effects of nectar
secondary metabolites on floral visitors of Nicotiana attenuata The Plant Journal 49840ndash
54
Koptur S and N Truong 1998 Facultative ant-plant interactions nectar sugar preferences of
introduced pest species in South Florida Biotropica 30179-189
Lach L 2005 Interference and exploitation competition of three nectar-thieving invasive ant
species Insectes Sociaux 52257ndash262
Lach L 2007 A mutualism with a native membracid facilitates pollinator displacement by
Argentine ants Ecology 881994ndash2004
Lach L 2008a Argentine ants displace floral arthropods in a biodiversity hotspot Diversity and
Distributions 14281ndash290
Lach L 2008b Floral visitation patterns of two invasive ant species and their effects on other
hymenopteran visitors Ecological Entomology 33155ndash160
Lach L 2013 A comparison of floral resource exploitation by native and invasive Argentine
ants Arthropod-Plant Interactions 7177-190
Luo C W K Li X M Chen Z Y Huang and K U N Li 2012 Ants contribute significantly
to the pollination of a biodiesel plant Jatropha curcas Environmental Entomology
411163ndash1168
33
Maleacute PJ G C Leroy A Dejean A Quilichini and J Orivel 2011 An ant symbiont directly
and indirectly limits its host plantrsquos reproductive success Evolutionary Ecology 2655ndash63
Maloof J E 2001 The effects of a bumble bee nectar robber on plant reproductive success and
pollinator behavior American Journal of Botany 881960ndash1965
Maloof J E and D W Inouye 2000 Are nectar robbers cheaters or mutualists Ecology
812651ndash2661
Moreau CS CD Bell R Vila SB Archibald and NE Pierce 2006 Phylogeny of the ants
diversification in the age of the angiosperms Science 312101-104
Nahas L M O Gonzaga and K Del-Claro 2012 Emergent impacts of ant and spider
interactions herbivory reduction in a tropical savanna tree Biotropica 44498ndash505
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506ndash514
Newman D A and J D Thomson 2005 Interactions among nectar robbing floral herbivory
and ant protection in Linaria vulgaris Oikos 110497ndash506
Nicklen E F and D Wagner 2006 Conflict resolution in an ant-plant interaction Acacia
constricta traits reduce ant costs to reproduction Oecologia 14881ndash7
Norment C J 1988 The effect of nectar-thieving ants on the reproductive success of Frasera
speciosa (Gentianaceae) American Midland Naturalist 120331ndash336
34
Orivel J L Lambs P-J G Maleacute C Leroy J Grangier T Otto A Quilichini et al 2011
Dynamics of the association between a long-lived understory myrmecophyte and its
specific associated ants Oecologia 165369ndash76
Palmer T M D F Doak M L Stanton J L Bronstein E T Kiers T P Young J R
Goheen et al 2010 Synergy of multiple partners including freeloaders increases host
fitness in a multispecies mutualism Proceedings of the National Academy of Sciences
10717234ndash9
Peakall R A J Beattie and S H James 1987 Pseudocopulation of an orchid by male ants a
test of two hypotheses accounting for the rarity of ant pollination Oecologia 73522ndash524
Peakall R and A J Beattie 1989 Pollination of the orchid Microtis parviflora R Br by
flightless worker ants Functional Ecology 3515ndash522
Peakall R C J Angus A J Beattie 1990 The significance of ant and plant traits for ant
pollination in Leporella fimbriata pollination Oecologia 84457ndash460
Puterbaugh M N 1998 The roles of ants as flower visitors experimental analysis in three
alpine plant species Oikos 8336ndash46
Prŷs-Jones OE and Willmer PG 1992 The biology of alkaline nectar in purple toothwort
(Lathraea clandenstina) ground level defenses Biological Journal of the Linnean Society
45373-388
Raine NE Willmer P and Stone GN 2002 Spatial structuring and floral avoidance
behaviour present in ant-pollinator conflict in a Mexican ant-acacia Ecology 833086-
3096
35
Ramsey M 1995 Ant pollination of the perennial herb Blandfordia grandiflora (Liliaceae)
Oikos 74265ndash272
Renault CK LM Buffa and Delfino MA 2005 An aphid-ant interaction effects on
different trophic levels Ecological Research 2071-74
Rico-Gray V and L B Thien 1989 Effect of different ant species on reproductive fitness of
Schomburgkia tibicinis (Orchidaceae) Oecologia 81487ndash489
Romero G Q P A P Antiqueira and J Koricheva 2011 A meta-analysis of predation risk
effects on pollinator behaviour PLoS One 6e20689
Rosumek FB FAO Silveira F de S Neves NP de U Barbosa L Diniz Y Oki F Pezzini
et al 2009 Ants on plants a meta-analysis of the role of ants as plant biotic defenses
Oecologia 160537ndash549
Roubik D W 1982 The ecological impact of nectar-robbing bees and pollinating
hummingbirds on a tropical shrub Ecology 63354ndash360
Rudgers J A 2004 Enemies of herbivores can shape plant traits selection in a facultative ant-
plant mutualism Ecology 85192ndash205
Schatz B and WT Wcislo 1999 Ambush predation by the ponerine ant Ectatomma ruidum
Roger (Formicidae) on a sweat bee Lasioglossum umbripenne (Halictidae) in Panama
Journal of Insect Behavior 12641-663
36
Schaffer W M D W Zeh S L Buchmann S Kleinhaus M V Schaffer and J Antrim
1983 Competition for nectar between introduced honey bees and native North American
bees and ants Ecology 64564ndash577
Schemske D W 1980 The evolutionary significance of extrafloral nectar production by Costus
woodsonii (Zingiberaceae) an experimental analysis of ant protection Journal of Ecology
68959ndash967
Schiestl F P and F Glaser 2011 Specific ant-pollination in an alpine orchid and the role of
floral scent in attracting pollinating ants Alpine Botany 1221ndash9
Schubart HOR AB Anderson 1978 Why donrsquot ants visit flowers A response to D H
Janzen Biotropica 10310-311
Schuumlrch S M Pfunder and B A Roy 2000 Effects of ants on the reproductive success of
Euphorbia cyparissias and associated pathogenic rust fungi Oikos 16ndash12
Stanton ML Palmer TM Young TP Evans A and Turner ML 1999 Sterilization and
canopy modification of swollen thorn acacia tree by a plant-ant Nature 401 578-581
Stephenson AG 1981 Toxic nectar deters nectar thieves of Catalpa speciosa American
Midland Naturalist 105381-383
Stout J C and D Goulson 2001 The use of conspecific and interspecific scent marks by
foraging bumblebees and honeybees Animal Behaviour 62183ndash189
Sugiura S T Abe and S Makino 2006 Loss of extrafloral nectary on an oceanic island plant
and its consequences for herbivory American Journal of Botany 93491ndash495
37
Svensson L 1985 An estimate of pollen carryover by ants in a natural population of
Scleranthus perennis L (Caryophyllaceae) Oecologia 66373ndash377
Tsuji K A Hasyim and K Nakamura 2004 Asian weaver ants Oecophylla smaragdina and
their repelling of pollinators Ecological Research 19669ndash673
Turner R C J J Midgley P Barnard R E Simmons and S D Johnson 2012 Experimental
evidence for bird pollination and corolla damage by ants in the short-tubed flowers of
Erica halicacaba (Ericaceae) South African Journal of Botany 7925ndash31
Visser D M G Wright and J H Giliomee 1996 The effect of the Argentine ant
Linepithema humile (Mayr) (Hymenoptera Formicidae) on flower-visiting insects of
Protea nitida Mill (Proteaceae) African Entomology 4285ndash287
Wagner D 2000 Pollen viability reduction as a potential cost of ant association for Acacia
constricta (Fabaceae) American Journal of Botany 87711ndash715
Wagner D and A Kay 2002 Do extrafloral nectaries distract ants from visiting flowers An
experimental test of an overlooked hypothesis Evolutionary Ecology Research 4293ndash305
Wang C Y Luo Y Tai D An and Y Kou 2008 Ants pollinate Neottia listeroides
(Orchidaceae) in Sichuan China 46836ndash846
Wyatt R 1980 The impact of nectar-robbing ants on the pollination system of Asclepias
curassavica Bulletin of the Torrey Botanical Club 10724ndash28
Wheeler WM and IW Bailey 1920 The feeding habits of Pseudomymine and other ants
Transactions of the American Philosophical Society 22235-279
38
Willmer P G and S A Corbet 1981 Temporal and microclimatic partitioning of the floral
resources of Justicia aurea amongst a concourse of pollen vectors and nectar robbers
Oecologia 5167ndash78
Willmer P G and G N Stone 1997 How aggressive ant-guards assist seed-set in Acacia
flowers Nature 388165ndash167
Willmer P G C V Nuttman N E Raine G N Stone J G Pattrick K Henson P Stillman
et al 2009 Floral volatiles controlling ant behaviour Functional Ecology 23888ndash900
Young T P C H Stubblefield and L a Isbell 1997 Ants on swollen-thorn acacias species
coexistence in a simple system Oecologia 10998ndash107
Yu D W and N E Pierce 1998 A castration parasite of an ant-plant mutualism Proceedings
of the Royal Society B Biological Sciences 265375ndash382
39
Tables
Table 11 Studies of ant-flower or ant-pollinator interactions that included a measure of plant fitness Each study system is listed only
once except when results varied between years or when different studies examined male and female fitness Significant effects of ants on
plant fitness are either lsquopositiversquo or lsquonegativersquo non-significant effects are lsquoneutralrsquo When ants significantly affected only one fitness
component but several were measured (eg fruit set and seed production) we still consider the result significant
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Acacia
drepanolobium
Crematogaster
nigriceps
Proportion
fruiting
Negative Destruction of floral buds Young et al 1997
Asclepias exaltata Several Pollinia inserted
removed
Negative for pollinia
insertion neutral for
pollinia removal
Exploitative competition Wyatt 1980 T
40
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Asclepias syriaca Several
Fruit set pollinia
removed
Negative for fruit
set neutral for
pollinia removal
Exploitative competition Fritz and Morse
1981 T
Cordia nodosa Allomerus cf
demerarae
Fruit set Negative Destruction of floral buds Yu and Pierce
1998
Costus woodsonii Wasmannia
auropunctata
Seed set Positive Unknown ant pollination
suggested (but unlikely)
Schemske 1980
Eritrichum aretioides F neorufibarbis
gelida
Seed set Negative Ants damaged flowers Puterbaugh 1998
41
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Euphorbia characias Several Seed set Neutral Plants in Argentine ant-
invaded area set less seed
than those in un-invaded
sites but an ant-exclusion
experiment did not produce
significant results
Blancafort and
Gόmez 2005
Ferocactus wislizeni Solenopsis xyloni
and others
Seed set Unknown negative Interference and exploitative
competition S xyloni
reduced seed set relative to
other ants but the study did
not include a no-ant control
Ness 2006
42
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Frasera speciosa F neorufibarbis
and Formica sp
Seed set Neutral Nocturnal pollination
occurring when ants were
less active may have
compensated for reduced
diurnal pollinator visitation
Norment 1988
Gypsophila struthium Several Seeds per
inflorescence
Neutral No effect despite large
numbers of ants on flowers
Gόmez et al
1996
Hirtella physophora Allomerus
decemarticulatus
Fruit set Negative Destruction of flowers Orivel et al
2011
Humboldtia brunonis Crematogaster
dohrni
Fruit set Negative Destruction of flowers Gaume et al
2005
43
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Lepidium subulatum Several Percentage of
ovules setting
seed
Positive Unknown Gόmez et al
1996
Leucospermum
conocarpodendron
Several Seed set Neutral Site not pollen limited Lach 2008
Linaria vulgaris Several ldquoFemale fitness
indexrdquo
(combination of
number of seeds
fruits flowers
and seed weight)
Positive Reduction in florivory and
seed predation nectar
robbing holes increased ant
visitation
Newman and
Thomson 2005
44
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Melastoma
malabathricum
Oecophylla
smaragdina
Seed set Positive Ants deterred less efficient
pollinators increasing
visitation by the more
efficient pollinator
Gonzaacutelvez et al
2012
Metrosideros
polymorpha
Several Fruit set Neutral Ants reduced nectar
collection rates by foraging
bees but had no effect on
pollen collection rates or
fruit set
Junker et al
2010a
Orexis alpine F neorufibarbis
gelida
Seed set Neutral Autogamy or not pollen
limited
Puterbaugh 1988
45
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Ouratea spectabilis Cephalotes
pusillus and others
Seeds per fruit Neutral positive C pusillus had no effect on
seed number but other ants
collectively increased seed
set
Byk and Del
Claro 2010
Pachycereus schottii Several Seed set Neutral positive In one year ants showed a
positive effect (seed
number 2008) but no effect
with increased sample sizes
Chamberlain and
Holland 2008
Polemonium
viscosum
F neorufibarbis
gelida
Seed set Negative Ants damaged styles Galen 1983
46
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Polemonium
viscosum
F neorufibarbis
gelida
Pollen removal Neutral Ants did not affect pollen
removal even at high ant
densities
Galen and Geib
2007 T
Protea nitida Linepithema
humile and others
Seed set Neutral positive L humile had no effect on
P nitida seed set but native
ants increased it in one year
ants may cause pollinators
to relocate
Lach 2007
Psychotria
limonensis
Ectatomma spp Fruit set Positive Suggested increased
pollinator relocation
Altshuler 1999
47
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Roussea simplex Technomyrmex
albipes
Seed set Negative Interference and exploitative
competition
Hansen and
Muumlller 2009
Tristerix aphyllus Several Fruit set Neutral Nectar robbing ants
minimally reduced nectar
levels and had no effect on
hummingbird visitation
Caballero et al
2013
Vaccinium
reticulatum
Several Fruit set Neutral Ants reduced florivory but
did not affect fruit set
Bleil et al 2011
T Study measured male fitness
Study of sterilizing ants showed short-term decrease in female fitness long-term effects are unclear (see text)
48
Table 12 Studies demonstrating either i) ant pollination through an exclusion experiment or ii) that ants transfer pollen (or pollen
analogue) between flowers strongly suggesting ant pollination
Plant species Ant species Ant pollination Evidence Reference
Alyssum purpureum Several Yes Exclusion experiment Gόmez et al 1996
Arenaria tetraquetra Several Yes Exclusion experiment Gόmez et al 1996
Balanophora kuroiwai Leptothorax sp Suggested Exclusion experiment and
observed pollen transfer
and pollen tube formation
but did not see if plants set
seed
Kawakita and Kato 2002
Blandfordia grandiflora Iridomyrmex sp Yes Exclusion experiment Ramsey 1995
Borderea pyrenaica Several Yes Exclusion experiment Garciacutea et al 1995
49
Plant species Ant species Ant pollination Evidence Reference
Cytinus hypocistis Several Yes Exclusion experiment de Vega et al 2009
Epipactis thunbergii Camponotus
japonicas
Suggested Observed transfer of
pollinia from ants to
orchids
Sugiura et al 2006
Euphorbia cyparissias Several Yes Exclusion experiment Schuumlrch et al 2000
Fragaria virginiana Several Very likely Ant-only treatments set
comparable seed to flying
pollinator-only treatment
seed set increased with
increasing ant visitation
Ashman and King 2005
Frasera speciosa Several Yes Exclusion experiment Norment 1988
Frankenia thymifolia Several Yes Exclusion experiment Gόmez et al 1996
50
Plant species Ant species Ant pollination Evidence Reference
Hormathophylla spinosa Proformica
longiseta
Yes Exclusion experiment Gόmez and Zamora 1992
Jatropha curcas
Several
Tapinoma
melanocephalum
most common
Yes Exclusion experiment Luo et al 2012
Leporella fimbriata Myrmecia urens Yes Observed pollinia transfer
and subsequently
confirmed as ant
pollination
Peakall et al 1987
51
Plant species Ant species Ant pollination Evidence Reference
Lobularia maritima Several
Camponotus
micans most
common
Yes Exclusion experiment Gόmez 2000
Microtis parviflora
Iridomyrmex
gracilis
Yes Observation used
laboratory colonies to
replicate observations
Peakall and Beattie 1989
Naufraga balearica
Several
especially
Plagiolepis
pygmaea and
Lasius grandis
Suggested Observation over three
years of study only ants
were observed visiting
ants carried pollen
Cursach and Rita 2011
52
Plant species Ant species Ant pollination Evidence Reference
Neottia listeroides Leptothoras sp
Paratrechina sp
Suggested Observed transfer of
pollinia from ants to
orchids
Wang et al 2008
Paronychia pulvinata Formica
neorufibarbis
gelida
Yes Exclusion experiment Puterbaugh 1998
Polygonum cascadense Formica argentea
Very likely Grew plants in greenhouse
open to flying visitors with
minimal seed set only
plants with access to ants
set significant seed
Hickman 1974
53
Plant species Ant species Ant pollination Evidence Reference
Retama sphaerocarpa Several Yes Exclusion experiment Gόmez et al 1996
Scleranthus perennis
Several
especially
Formica
rufibarbis
Suggested Ants transferred pollen
analogue to stigmas and
carried pollen on bodies
Svensson 1985
Sedum anglicum Proformica
longiseta
Yes Exclusion experiment Gόmez et al 1996
Trinia glauca Several Yes Exclusion experiment Carvalheiro et al 2008
54
Chapter 2 Ants and ant scent reduce bumblebee pollination of artificial flowers
Cembrowski AR Tan MG Thomson JD and Frederickson ME
In press The American Naturalist
All authors designed the experiment ARC and MGT performed the experiment ARC and MEF
performed statistical analyse
s and ARC wrote the manuscript with input from all authors
Abstract
Ants on flowers can disrupt pollination by consuming rewards or harassing pollinators but it is
difficult to disentangle the effects of these exploitative and interference forms of competition on
pollinator behavior Using highly rewarding and quickly replenishing artificial flowers that
simulate male or female function we allowed bumblebees (Bombus impatiens) to forage (1) on
flowers with or without ants (Myrmica rubra) and (2) on flowers with or without ant scent cues
Bumblebees transferred significantly more pollen analogue both to and from ant free flowers
demonstrating that interference competition with ants is sufficient to modify pollinator foraging
behavior Bees also removed significantly less pollen analogue from ant scented flowers than
from controls making this the first study to show that bees can use ant scent to avoid harassment
at flowers Ant effects on pollinator behavior possibly in addition to their effects on pollen
viability may contribute to the evolution of floral traits minimizing ant visitation
55
Introduction
Trait-mediated indirect interactions arise when a focal species causes phenotypic changes
including behavioral modifications in a second species and these effects cascade to still other
species (Werner and Peacor 2003) Although trait-mediated indirect interactions are often studied
in food webs (eg Werner and Peacor 2003 Preisser et al 2005) they are not limited to trophic
interactions For example the threat of predation can change the behavior of a mutualist thus
affecting its partners (Suttle 2003) In animal-pollinated plants predators can disrupt pollination
directly through density-mediated indirect interactions (Dukas 2005) or via trait-mediated
indirect interactions by changing pollinator behavior When predators are present pollinators
may switch to visiting less rewarding flowers or they may avoid flowers altogether potentially
reducing the amount of pollen donated or received by a flower and thus decreasing plant fitness
(Gonccedilalves-Souza 2008 Ings and Chittka 2009) Similarly competition for floral rewards with
other flower visitors may cause behavioral changes in pollinators (Maloof and Inouye 2000
Ohashi et al 2008) Previous research has shown that bees may spend less time visiting flowers
depleted of rewards (Thomson 1986) and may avoid flowers bearing scent marks that indicate
recent visits by other individuals of the same or different bee species as these flowers are likely
to be depleted (Stout and Goulson 2002)
Whereas competition between pollinators may not be particularly costly for the plant
because both competitors are likely to provide pollination services competition between
pollinators and organisms that do not usually provide a pollination service such as ants has a
greater potential to reduce plant fitness Ants are common floral visitors they are attracted to
flowers for nectar (Lach 2007) and in some cases pollen (Byk and Del Claro 2010) and will
often defend these resources against other flower visitors (Altshuler 1999) However ants rarely
56
contribute much useful pollination antibiotic secretions present on their cuticles kill pollen
grains (Beattie et al 1985 Dutton and Frederickson 2012) and when foraging in flowers ants
sometimes impede female function by damaging stigmas (Galen and Cuba 2001) We will
henceforth consider ants to be non-pollinators and will contrast them to animals like bees that
are pollinators Ants are capable of competing with pollinators in two ways (1) by consuming
floral rewards and reducing their availability to pollinators (ie exploitative competition) and (2)
by directly antagonizing and excluding pollinators from flowers (ie interference competition)
Many plants benefit from the presence of ants numerous plant species recruit ants using
extrafloral nectar food bodies or domatia because ants provide protection against folivores (Heil
and McKey 2003) Similarly some plants have extrafloral nectaries on or near reproductive
tissues that recruit ants that deter florivores (eg Inouye and Taylor 1979) However plants
may incur reproductive costs as a result of their association with ants Some ant species that nest
in myrmecophytes sterilize flowers to shunt plant resources from reproduction to vegetative
growth allowing for greater ant colony growth (Frederickson 2009) Also ants visiting
extrafloral nectaries may be attracted to floral nectaries where they can harass pollinators (Ness
2006) There is growing evidence that many angiosperms have evolved floral traits that prevent
ants from accessing flowers during anthesis (Willmer and Stone 1997 Ballantyne and Willmer
2012) The effects of flower-visiting ants can be substantial Ants can decrease the frequency
duration or species diversity of pollinator visits to flowers (Lach 2008 Hansen and Muumlller 2009
Junker et al 2010 Gonzaacutelvez et al 2012) all of which potentially impact pollen donation and
receipt In some studies changes to pollinator behavior induced by ants have also been linked to
seed set
It is difficult however to disentangle the intertwined effects of exploitative and
interference competition in nature If ants disrupt pollination plants may often evolve traits to
57
deter ants from flowers (Willmer et al 2009) From a practical standpoint this would then limit
our ability to study the effects of ants on pollinator visitation because ants would not visit
flowers Here we used artificial flowers to explore ant-bee interactions in the absence of floral
defenses against ants We used highly rewarding quickly replenishing artificial flowers to
examine how direct harassment (ie interference competition expressed through behavior)
changes bee foraging behavior and how this affects donation and receipt of a pollen analogue
(powdered food dye) while minimizing the effects of exploitative competition We predicted
that flowers visited by ants would both donate and receive less pollen analogue than flowers
without ants because bees would avoid flowers with ants or leave them sooner Because scent
plays a large role in bumblebee communication and flower choice (Stout and Goulson 2002) we
also tested whether the presence or absence of ant scent on artificial flowers would affect pollen
analogue donation because of changes in the foraging behavior of bumblebees
Methods
i) Subjects
Myrmica rubra is an invasive ant in eastern North America with a range and habitat preferences
overlapping that of Bombus impatiens a common bumblebee Because M rubra visits flowers
(A Cembrowski pers obs) the two species likely interact in nature We collected 12 M rubra
colonies in the fall of 2011 and 2012 from Toronto Ontario Canada and the surrounding area
and maintained them in environmental chambers on artificial diet (Dussutour and Simpson 2009)
and a 1410 LD schedule (light 600-2200) We used these colonies as sources of M rubra
workers for experiments
58
Workers from commercially supplied colonies of Bombus impatiens (Biobest Canada
Ltd) foraged on artificial flowers in flight cages (either 24 x 24 x 21 m or 79 x 34 x 20m) at
the University of Toronto We tested a total of five bee colonies four colonies were used in the
ant presence trials and two of these and one additional colony were used in the ant scent trials
Flight cages had overhead fluorescent lights attached to timers In contrast to most previous
studies in which bees have been trained and tested individually the entire worker force of a bee
colony was free to forage at will in our experiments Colonies were trained to forage on artificial
flowers for at least four days before being used in trials After being used for a trial the colony
was not used for at least two days to reduce dye carryover between trials Between trials
bumblebees were fed pollen and given sugar water
ii) Artificial flowers
The flowers (Fig 1 Thomson et al 2012 see also Makino 2008) consisted of glass jars filled
with 30 WV sucrose solution Sugar water travelled by capillary action up a sewing-thread
wick to a hole in a blue-painted lid accumulating in a knot that acts as a nectary Flowers
depleted by visitors were quickly replenished via capillary action and were non-rewarding only
very briefly after visits taking less than a minute to accumulate 05 microl note that bumblebee
visits to a single flower are often separated by several minutes or more (A Cembrowski
unpublished data)
Unlike most previous artificial flower designs these flowers allow for estimation of male
and female fitness because we can measure both the amount and type of dye received by female
flowers (see (d) Dye quantification below) In order to access nectar on a ldquomalerdquo flower a bee
must crawl through a circular ldquoantherrdquo of brush-like weather stripping material dusted in a
59
consistent manner with our pollen analogue powdered food dye (FDampC 5 or 6) which is
transferred to the bee in the process On a ldquofemalerdquo flower a bee crawls through a sticky plastic
reinforcement that functions as a ldquostigmardquo receiving dye from the beersquos body Male flowers
dispensed dye particles over multiple bee visits and could still dispense dye at the end of trials
They resemble many real flowers or flower heads in that pollen is dispensed gradually over time
but less dye is available for transfer with each subsequent bee visit (Harder and Thomson 1989)
iii) Flight cage trials
We conducted 6 ant scent trials between 22 March and 4 June 2012 and the remaining trials (16
ant presence trials and 9 ant scent trials) between 9 November 2012 and 6 January 2013 Ant
presence trials lasted for eight hours and ant scent trials lasted for four hours Artificial flowers
were prepared and placed individually in small plastic containers treated with Fluon (Insect-a-
Slip BioQuip Products Inc) to prevent ants from escaping In ant presence trials we used 32
flowers (16 male and 16 female flowers) arranged in an eight-by-four array with flowers spaced
45 cm apart between rows and 30 cm within rows Ant scent trials used 20 flowers (10 male and
10 female flowers) in a four-by-five array with flowers spaced 30 cm apart between and within
rows In all of the ant presence trials and 9 of the ant scent trials we counted the number of bees
actively foraging after one hour to get a measure of colony activity
a) Ant presence trials
We examined the effects of ants on the amount of dye donated by male flowers and received by
female flowers in sixteen trials testing four bee colonies four times each The containers of eight
randomly chosen male flowers and eight randomly chosen female flowers received 15 M rubra
60
workers each the other 16 flowers remained free of ants Isolated from their colonies M rubra
workers have nowhere to deposit sugar water they collect and therefore may become sated
nonetheless a force of 15 ant workers was enough to maintain visitation to the nectaries where
bees foraged while still rarely having more than one or two ants visit the nectary at any time As
ants attacked bees bees would occasionally carry or throw attacking ants off flowers but despite
this some ants maintained their presence at the nectary throughout the trials Because ants the
size of M rubra consume liquids slowly (~017 ndash 024 microlmin Davidson et al 2004) flowers
replenished nectar more quickly than ants consumed it We used two colors of dye (FDampC 5 and
FDampC 6) to differentiate male flowers with and without ants In two trials for each bee colony
anthers of male flowers with ants were coated with FDampC 5 dye while brushes of male flowers
lacking ants were coated with FDampC 6 dye In the other two trials we reversed dye colors to
control for effects of dye type and color Dye color was assigned in a random order Artificial
flowers were placed in randomly assigned positions in the array before we opened the colony
and allowed the bees to begin foraging
b) Scent trials
We explored the effect of ant scent on dye donation in fifteen trials testing three bee colonies in
six five and four trials each Bees were first exposed to ants by allowing the bees to forage for
eight hours on twenty flowers (ten male and ten female) of which five male flowers had 15 ants
and all others had none No dye was used during this ldquoexposurerdquo day We then collected all but
the five ant-free male flowers in the flight cage leaving these flowers to keep bees foraging
Next we individually stored five new male lids in Fluon-treated containers with ten M rubra
workers Five control male lids were put in identical containers lacking ants The following
61
morning we removed the remaining flowers and set out ten new male and female flowers in a
random spatial arrangement using the lids having or lacking ant scent Thus the ant-scented
flowers were not in the same positions as the ant-visited flowers on which the bees were trained
Only five male flowers had ant scent and the other 15 flowers (five male and ten female) did not
We used the same two dye colors as in i) to differentiate male flowers with and without ant
scent and randomized which color was used for ant-scented flowers between trials We collected
and replaced stigmas from female flowers after one hour and collected the stigmas again after
four hours In the first trial performed we also collected stigmas after two hours but due to the
small amount of dye transferred this was not repeated
iv) Dye quantification
After each trial we quantified the amount of dye transferred to female flowers using a
spectrophotometer We removed the stigmas from female flowers placed them in test tubes
added 51 mL of distilled water to each tube and vortexed each tube thoroughly to ensure the
dye was evenly diluted These were diluted further as needed if the absorbance exceeded the
sensitivity range of the spectrophotometer In the first six scent trials each stigma was analyzed
separately and the dye amounts were summed to obtain a total amount of each dye color
transferred to female flowers in each trial In all the ant presence trials and the other nine scent
trials we opted to treat the experiment as the unit of replication Therefore we combined stigmas
from each treatment type (ant-visited or ant-free) in a test tube and measured the total amount of
each dye color transferred in each treatment in each trial
Because we put different dye colors on male flowers with and without ants we could use
the amount of each dye color donated to all female flowers to measure the reproductive success
62
of ant-visited and ant-free male flowers The total amount of dye (of both colors) received by
female flowers with and without ants was our measure of female reproductive success We
calculated the amount of each dye color in the sample by measuring absorbance at 428 or 486
nm and converting absorbance to micrograms following computational methods for overlapping
spectra (Blanco et al 1989)
v) Statistical analyses
In two trials (one ant presence and one ant scent) most female flowers received no dye because
of low bee activity so we excluded these trials from analyses Because dye reception and
donation values were non-normally distributed we square-root transformed the data before
examining the effects of ant presence or ant scent on dye transfer in ANCOVAs For ant
presence trials we included ant presence on male and female flowers and their interaction as
main effects and the total amount of dye transferred in each trial as a covariate to account for
the large variation in overall dye transfer among trials For ant scent trials we included ant scent
as the main effect time (one or four hours) as a repeated measure the interaction between scent
and time and the total amount of dye transferred as a covariate Covariate by treatment (ant
presence or ant scent) interactions were never significant and so were excluded in final analyses
All analyses were conducted in JMP (v 1000)
Results
Bees usually started foraging within minutes of the beginning of the trial and continued until
flowers were collected from the flight cage or the lights were extinguished An average of 34 plusmn
012 and 40 plusmn 015 (mean plusmn SE) bees were foraging after an hour in the ant presence and ant
63
scent trials respectively Male flowers with ants donated significantly less dye than male flowers
lacking ants (Fig 22a F155 = 4019 p lt 00001) Similarly female flowers with ants received
significantly less dye than female flowers lacking ants (Fig 22a F155 = 461 p = 0036) There
was no significant interaction between ant presence on male flowers and ant presence on female
flowers (F155 = 118 p = 028) Flowers with ant scent donated significantly less dye than
flowers without ant scent (Fig 22b F125 = 11216 p lt 00001) There was no significant effect
of time (F125 = 121 p = 028) or interaction between time and scent (F125 = 111 p = 030) in
the model
Discussion
In this study ants altered bumblebee pollination behavior and exhibited trait-mediated indirect
interactions with (artificial) flowers Through interference competition M rubra workers
significantly affected the pattern of dye transfer by B impatiens causing a preferential flow of
dye from male flowers lacking ants to female flowers lacking ants If manifested in nature such
effects would reduce the reproductive success of plants visited by ants through both male and
female function
Ants could have changed the attractiveness of artificial flowers in at least two ways First
as ants interrupted or altogether prevented bumblebees from foraging at nectaries bumblebees
may have learned to avoid flowers with ants due to the relative inefficiency of foraging at these
flowers something bumblebees take into account (Heinrich 2004) Second bees attempt to
minimize their risk of being attacked during foraging bouts which ants did to visiting bees Ants
often attacked or harassed bees by biting grasping and appearing to sting visiting bees
preventing them from accessing the nectary or reducing their time on flowers (see online video
64
1) and bees sometimes avoided ant-tended flowers entirely This harassment was sometimes
physically traumatic (online video 1) and bees often appeared to have trouble flying after being
attacked by ants Previous research has demonstrated that bumblebees leave or avoid flowers
where they have been harassed (Jones and Dornhaus 2011) and avoid foraging where there is
visual or olfactory evidence of a predator or predation event (Abbott 2006 Goodale and Nieh
2012) Though it is unclear if bumblebees viewed ants as competitors or predators they
responded similarly to flowers having ants as they do to flowers housing predators (Gonccedilalves-
Souza et al 2008)
To avoid artificial flowers with ants bees likely used a combination of visual and
olfactory cues Previous research has shown that bees respond to conspicuous predators or
predator ldquodummiesrdquo on flowers (Suttle 2003 Gonccedilalves-Souza et al 2008) decreasing the
frequency and duration of their visits to these flowers Thus bees may have been able to avoid
flowers harboring ants by sight alone However even in the absence of ants bees still
preferentially visited flowers lacking ant scents (Fig 22b) suggesting that they had learned to
associate ant scent with harassment Bees are adept at associative learning (Wright and Schiestl
2009) and can learn to recognize unique scents left behind by both conspecific and heterospecific
flower visitors (Stout et al 1998) Bees can use these various scents often arising from tarsal
gland deposits (Stout et al 1998) to recognize recently visited flowers that are less likely to be
profitable (Stout and Goulson 2002) Recently Ballantyne and Willmer (2012) demonstrated that
bees learn to associate ant scents with unrewarding artificial flowers and decrease their visitation
to these flowers Our results complement their findings by showing that bees can associate ant
scent with harassment at otherwise rewarding flowers
In our study ant scent caused bees to adjust their foraging strategy and decreased the
amount of pollen analogue that was donated by ant-scented flowers Thus the effects of
65
interference competition with ants on flowers can extend beyond immediate interactions and may
have fitness consequences for plants even when ants are absent Like other olfactory cues these
effects are likely transitory (Stout et al 1998) Although more dye was still donated by male
flowers lacking ant scent than those with ant scent the ratio of dye donated was on average
closer to equality in hours 2-4 than in the first hour (Fig 22b) This effect may be partially
driven by dye depletion of male flowers without ant scent but the lack of a strong corresponding
decrease in flowers with ant scent suggests that visitation patterns became more similar We did
not test whether B impatiensrsquo avoidance of ant scent was an innate or a learned behavior but
previous work has shown that Bombus terrestris does not innately avoid flowers with ant scent
(Ballantyne and Willmer 2012)
Ants may be necessary for plant survival and growth but can be costly for plant
reproduction The evolution of ant attractants such as extrafloral nectaries in some plant lineages
suggests that the costs of ants can be outweighed by their protective abilities However in this
study flowers visited by ants received and donated significantly less dye although they did
retain some sexual function The net benefit of having ants depends on whether ants increase
plant fitness by reducing herbivory more than they decrease plant fitness by disrupting
pollination Alternatively plants may actually benefit from the costs of ants to plant
reproduction ants that castrate flowers may be better defenders increasing plant survival or
vegetative growth when plants are young allowing for increased reproduction later in life when
the plant is colonized by less aggressive non-castrating ants (Frederickson 2009 Palmer et al
2010) In some plant species pollinator harassment by ants may even be beneficial Altshuler
(1999) reported that Ectatomma ants greatly increased fruit set of Psychotria limonensis despite
reductions in pollinator visitation rates presumably due to increases in the rate of pollen
outcrossing Similarly Gonzaacutelvez et al (2012) found that the presence of Oecophylla
66
smaragdina ants on Melastoma malabathricum flowers reduced visitation by less effective
pollinators and increased visitation by more effective Xylocopa bees increasing plant fitness
Cases of ants being beneficial to flowers appear to be the minority and many plant
species have traits that limit ant access to flowers Several studies have detailed floral volatiles
that are thought to mimic ant alarm pheromones and thus repel ants (Junker and Bluumlthgen 2008
Willmer et al 2009) Other plants use structural modifications such as narrow corollas or
slippery stems to limit ant access to flowers (reviewed in Willmer et al 2009) Exploitative
competition between ants and pollinators (Lach 2005 Ballantyne and Willmer 2012) as well as
the lethal effects of ant antibiotic secretions on pollen grains (Beattie et al 1985 Dutton and
Frederickson 2012) may have resulted in selection on plants to reduce ant visits to flowers Our
results suggest that trait-mediated indirect interactions resulting from interference competition
between ants and bees may favor plants that defend their flowers and their pollinators against
ants
Acknowledgements
We thank H Rusnock and S Meadley-Dunphy for assistance J Dix for help with flight cage
construction and Biobest for bumblebee colonies We are grateful to the Thomson and
Frederickson labs for guidance particularly T Makino funded by JSPS for helping to refine
artificial flowers J Ogilvie for help with bees K Prior for M rubra colony collections and K
Turner for study design suggestions The comments of the editor and two anonymous reviewers
prompted improvements in the article especially with regards to study design MEF and JDT
acknowledge NSERC Discovery Grant funding The University of Torontorsquos Faculty of Arts and
Science supported MGRTrsquos participation
67
68
References
Abbott K R 2006 Bumblebees avoid flowers containing evidence of past predation events
Canadian Journal of Zoology 841240-1247
Altshuler D L 1999 Novel interactions of non-pollinating ants with pollinators and fruit
consumers in a tropical forest Oecologia 119600-606
Ballantyne G and Willmer P 2012 Floral visitors and ant scent marks noticed but not used
Ecological Entomology 37402-409
Beattie A J Turnbull C Knox R B and Williams E G 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany
71421-426
Blanco M Runiaga H Maspoch S and Tarin P 1989 A simple method for
spectrophotometric determination of two-components with overlapped spectra Journal of
Chemical Education 66178-180
Byk J amp Del Claro K 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333-38
Davidson D W S C Cook and R R Snelling 2004 Liquid-feeding performances of ants
(Formicidae) ecological and evolutionary implications Oecologia 139255ndash66
Dukas R 2005 Bumble bee predators reduce pollinator density and plant fitness Ecology
861401-1406
Dussutour A and Simpson S J 2009 Communal nutrition in ants Current Biology 19740-
744
69
Dutton E M and Frederickson M E 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561-569
Frederickson ME 2009 Conflict over reproduction in an ant-plant symbiosis why Allomerus
octoarticulatus ants sterilize Cordia nodosa trees American Naturalist 173675-681
Galen C and Cuba J 2001 Down the tube pollinators predators and the evolution of flower
shape in the alpine skypilot Polemonium viscosum Evolution 551963-1971
Gonccedilalves-Souza T Omena P M Souza J C and Romero G Q 2008 Trait-mediated
effects on flowers artificial spiders deceive pollinators and decrease plant fitness Ecology
892407-2413
Gonzaacutelvez F G Santamariacutea L Corlett R T and Rodriacuteguez-Gironeacutes M A 2012 Flowers
attract weaver ants that deter less effective pollinators Journal of Ecology 10178ndash85
Goodale E and Nieh JC 2012 Public use of olfactory information associated with predation
in two species of social bees Animal Behaviour 84919-924
Harder LD and Thomson JD 1989 Evolutionary options for maximizing pollen dispersal of
animal-pollinated plants American Naturalist 133323-344
Heil M and McKey D 2003 Protective ant-plant interactions as model systems in ecological
and evolutionary research Annual Review of Ecology Evolution and Systematics 34425-
453
Heinrich B 2004 Bumblebee economics 2nd
edn Harvard University Press Cambridge
Ings T C and Chittka L 2009 Predator crypsis enhances behaviourally mediated indirect
effects on plants by altering bumblebee foraging preferences Proceedings of the Royal
Society B 2762031-2036
70
Inouye D W and Taylor O R Jr 1979 A temperate region plant-ant-seed predator system
consequences of extra floral nectar secretion by Helianthella quinquenervis Ecology 601-
7
Junker R R and Bluumlthgen N 2008 Floral scents repel potentially nectar-thieving ants
Evolutionary Ecology Research 10295ndash308
Junker R R Bleil R Daehler C C and Bluumlthgen N 2010 Intra-floral resource partitioning
between endemic and invasive flower visitors consequences for pollinator effectiveness
Ecological Entomology 35760ndash767
Jones E I and Dornhaus A 2011 Predation risk makes bees reject rewarding flowers and
reduce foraging activity Behavioral Ecology and Sociobiology 651505-1511
Lach L 2005 Interference and exploitative competition of three nectar-thieving invasive ant
species Insect Sociaux 52257-262
Lach L 2007 A mutualism with a native membracid facilitates pollinator displacement by
Argentine ants Ecology 881994-2004
Lach L 2008 Argentine ants displace floral arthropods in a biodiversity hotspot Diversity and
Distributions 14281-290
Makino T T and Sakai S 2007Experience changes pollinator responses to floral display size
from size-based to reward-based foraging Functional Ecology 21854863
Maloof J E and Inouye D W 2000 Are nectar robbers cheaters or mutualists Ecology
812651ndash2661
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506-514
Ohashi K Leslie A and Thomson JD 2008 Trapline foraging by bumble bees V Effects of
experience and priority on competitive performance Behavioral Ecology 19936-948
71
Palmer T M Doak D F Stanton M L Bronstein J L Kiers E T Young T P Goheen
J R and Pringle R M 2010 Synergy of multiple partners including freeloaders
increases host fitness in a multispecies mutualism Proceedings of the National Academy
of Sciences 10717234-17239
Preisser E L Bolnick D I and Benard M E 2005 Scared to death The effects of
intimidation and consumption in predator-prey interactions Ecology 86501-509
Stout J C Goulson D and Allen J A 1998 Repellent scent-marking of flowers by a guild of
foraging bumblebees (Bombus spp) Behavioral Ecology and Sociobiology 43317-326
Stout J and Goulson D 2002 The influence of nectar secretion rates on the responses of
bumblebees (Bombus spp) to previously visited flowers Behavioral Ecology and
Sociobiology 52239-246
Suttle K B 2003 Pollinators as mediators of top-down effects on plants Ecology Letters
6688-694
Thomson JD 1986 Pollen transport and deposition by bumble bees in Erythronium influences
of floral nectar and bee grooming Journal of Ecology 74329-341
Thomson JD Ogilvie J E Makino T T Arisz A Raju S Rojas-Luengas V and Tan M
G R 2012 Estimating pollination success with novel artificial flowers effects of nectar
concentration Journal of Pollination Ecology 9108-114
Tsuji K Hasyim A and Nakamura K 2004 Asian weaver ants Oecophylla smaragdina and
their repelling of pollinators Ecological Research 19669-673
Ulrich S and Bertsch A 1990 Do foraging bumblebees scent-mark food sources and does it
matter Oecologia 82137-144
Werner E E and Peacor S D 2003 A review of trait-mediated indirect interactions in
ecological communities Ecology 841083-1100
72
Willmer P G Nuttman C V Raine N E Stone G N Pattrick J G Henson K Stillman
P McIlroy L Potts S G and Knudsen J T 2009 Floral volatiles controlling ant
behaviour Functional Ecology 23888-900
Wright G A and Schiestl F P 2009 The evolution of floral scent the influence of olfactory
learning by insect pollinators on the honest signaling of floral rewards Functional Ecology
23841-851
73
Figures
Figure 21 Photograph of artificial flower lids A = anther L = lid N = nectary R = region
where anther or stigma is placed S = stigma T = thread W = weight
74
Figure 22 Dye transferred (mean plusmn 1SE) per trial by B impatiens workers A) from artificial
male and to artificial female flowers in the presence (unfilled circles) or absence (filled circles)
of Myrmica rubra workers and B) from artificial male flowers with (unfilled circles) or without
(filled circles) M rubra scent to ant-free female flowers in the 1st or the 2
nd to 4
th hours of the
trials
75
Chapter 3 Not just for the bees pollen consumption is common among tropical
ants
Cembrowski AR Reurink G and Frederickson ME
Planned submission to Biotropica
ARC and MEF designed the study ARC and GR did laboratory work ARC performed statistical
analyses and wrote the manuscript with input from other authors
Abstract
Although palynivory or pollen consumption is widespread among other hymenopterans there
are few accounts of palynivory in ants To quantify how often ants consume pollen we subjected
adult workers and larvae from 75 species of neo- and paleo-tropical ants to acetolysis a process
that destroys most organic material but leaves behind pollen grains In over half of the species
we examined ants contained a few pollen grains (lt 5) and ants of several Camponotus species
contained abundant pollen grains (gt50) We tested for an association between trophic level and
palynivory using stable nitrogen isotope ratios (δ15
N) but we did not find a significant
correlation we found pollen grains in lsquoherbivorousrsquo omnivorous and carnivorous ants We
suggest that our results indicate sporadic opportunistic pollen consumption by ants in tropical
forests
Introduction
For those organisms that can digest it efficiently pollen can be an excellent food source
Depending on the plant species pollen can contain up to 60 protein as well as lipids
76
carbohydrates and numerous trace nutrients (Roulston and Cane 2000) Pollen feeding
(palynivory) occurs in many taxa including birds mammals and arthropods (Roulston and Cane
2000) but it is best known among the Hymenoptera particularly bees Although adult bees feed
mainly on plant exudates (ie nectar) nearly all bee species rely on pollen as the chief protein
source for developing larvae (Willmer 2011) Closely related to bees ants have similar
nutritional requirements adult workers need carbohydrates whereas larvae are fed a diet rich in
protein (Dussutour and Simpson 2009) Ants also exploit nectar heavily and regularly visit
flowers (eg Herrera et al 1984 Bluumlthgen et al 2004) but unlike bees are not believed to
widely consume pollen Instead ants are thought to receive most of the protein in their diet from
predation or scavenging (eg Floren et al 2002)
However ants may not be as reliant on animal protein sources as commonly assumed In
tropical forests arboreal ants can have similar δ15
N ratios a measure of trophic level as
herbivores suggesting limited carnivory and substantial feeding on plant-derived protein sources
(Davidson et al 2003) One potential protein source is pollen This idea is supported by scattered
accounts of pollen consumption by ants from other ecosystems including deserts (Ness 2006)
boreal forests (Czechowski et al 2009) and tropical savannas (Byk and Del Claro 2010) One
ant genus Cephalotes is widely recognized to consume pollen (Creighton 1967 Baroni Urbani
and de Andrade 1997 Byk and Del Claro 2010) However beyond this genus our knowledge of
pollen consumption by ants has largely been limited to direct observations (but see Czechowski
et al 2011) and it is unknown if palynivory is truly rare among ants or simply under-reported
We borrowed a technique commonly used in palynology acetolysis to assess the frequency of
pollen consumption by ants in 75 neo- and paleo-tropical ant species This technique allowed us
to examine many more ant species than we could have observed directly
77
Methods
Adult worker ants were collected by hand at baits or from leaf litter using Winkler extractors
while larvae along with adults were collected by hand from nests Collections were made by L
Arcila Hernandez and J Sanders in the primary tropical rainforest surrounding the Centro de
Investigaciόn y Capacitaciόn Rio Los Amigos (CICRA 12deg34rsquoS 70deg05rsquoW) in Peru in 2010-
2011 and by E Youngerman from lowland primary and secondary rainforests in Madang
Province Papua New Guinea (PNG) in 2011-2012 We sorted ant samples to morphospecies and
then identified them to genus and when possible to species using existing keys which were
unavailable for PNG ants Ant samples used for acetolysis were whole adult workers guts from
adult workers and whole larvae
We used acetolysis to look for pollen grains inside ants In acetolysis organic material is
dissolved in acetic anhydride and sulfuric acid leaving behind stained pollen exines When
possible we used adult and larval ants of the same species from different colonies or bait
locations in an attempt to sample more representatively We prepared samples by first washing
them in 70 ethanol to remove any pollen present on the samplersquos outer surface before placing
them individually in micro-centrifuge tubes with ~05mL of a 91 mixture of acetic anhydride
sulfuric acid We then heated each sample at 95degC for 20min allowing tissues to dissolve Next
we centrifuged samples at 1000 RPM for 5min and decanted the liquid supernatant before
adding ~05mL of glacial acetic acid to the precipitate centrifuging again as above We decanted
the samples and added a drop of distilled water
78
We examined a 150μL sub-sample of this solution for pollen grains under a light
microscope Larvae gave less than 150μL of solution following acetolysis so we sampled the
entire amount We identified pollen grains by their staining and their surface architecture For
most species we examined at least three adult workers and three larvae but for some species
fewer than three adult workers or larvae were available (Tables 31 and 32)
We investigated the relationship between trophic level as measured by δ15
N ratios and
pollen consumption by ants Stable isotope (SI) data was available for some (n =23) of the
Peruvian species from a separate project in which collections overlapped Samples were stored in
EtOH and dried at 50degC overnight We used heads legs and thoraxes (but not gasters) of ants
for SI analysis Often single individuals did not provide enough material for analysis and we
combined multiple individuals to achieve necessary weights up to 10 for small ant species
Samples were analyzed at the Boston University Stable Isotope Laboratory using a Finnigan
Delta-S analyzer For other Peruvian species for which we had samples but not corresponding
stable isotope data (n =25) we took the median δ15
N ratio value of that speciesrsquo genus and
assigned it to the species in question Using pollen presenceabsence data for these 48 species
we used logistic regression to correlate pollen presence and δ15
N ratio using R (v 2151)
Results
We found pollen grains in 37 of the 197 adult workers 20 of the 102 larvae and 38 of the 75 ant
species we examined (Tables 31 and 32) The proportion of individuals containing pollen did
not differ significantly between larvae and adults (χ2
1 = 002 p = 088) The number of pollen
grains found was often low (median = 3) but highly variable (range 1-468) Also pollen
79
presence was inconsistent among conspecific ant samples of species that contained pollen 31
had pollen in only one of the larvae or adults examined In only three species (two Cephalotes
and one Myrmelachista) did all examined adults contain pollen
Highly lsquoherbivorousrsquo carnivorous and omnivorous ants contained pollen grains (Fig
31) There was no correlation between δ15
N ratio and pollen presence (χ2 = 150 df = 1 p =
022) In fact several carnivorous species contained pollen grains (eg Odontomachus sp 1
Pachycondola sp1)
Discussion
Pollen consumption by ants appears to be more common than suspected The majority of species
(51) had at least one individual that had consumed pollen and in 20 multiple individuals
contained pollen Numbers of grains found tended to be low however with most individuals
containing only a few grains Ants may use pollen as only a small portion of their diet exploiting
a host of other protein-containing foods more heavily including other low- δ15
N foods (eg
honeydew epiphylls Houmllldobler and Wilson 1990 Davidson et al 2003)
There are a few reasons why pollen may not be heavily exploited by ants Pollen requires
much post-consumption processing to extract its nutrients These nutrients are located in the
pollenrsquos cytoplasm which is protected from direct digestion by the exines and intines of the
grains Organisms must either break through these layers or cause the pollen grains to germinate
or pseudo-germinate to access and digest the cytoplasm (Roulston and Cane 2000) This can be
achieved by consuming a sugar-containing solution (such as nectar) along with pollen However
animals that are not adapted to feed on pollen may not be very efficient at this process only
80
extracting nutrients from some of the grains (Herrera and Martiacutenez del Rio 1998) Thus while
ants can likely get some nutrients from pollen other forms of more easily processed protein may
be more attractive
Pollen may also pose handling difficulties for ants Despite the large number of studies
that have recorded ants visiting flowers (Chapter 1) only a few papers detail ants harvesting
pollen directly from anthers Byk and Del Claro (2010) reported that Cephalotes pusillus ants
removed almost all of the available pollen from Ouratea spectabilis flowers and at least two
other studies describe ants consuming pollen along with nectar from flowers (Horskins and
Turner 1999 Ness 2006) Unlike bees which possess both morphological (eg specialized hairs
for holding pollen) and behavioral adaptations (eg stereotyped pollen-packing motions) ants
have no apparent mechanisms to increase the amount of pollen harvested and are likely limited
by the number of grains they can ingest or carry Rather than collect pollen directly from flowers
it appears more likely that ants collect it haphazardly in the environment leading to a smaller
number of pollen grains consumed at any one time It is known that at least Cephalotes ants
engage in such behavior Creighton (1967) observed Cephalotes texanus collecting pollen grains
trapped by Citrus x paradisi leaf hairs
There is a chance that the small numbers of pollen grains we observed came from
secondary pollen consumption (ie consuming a palynivorous organism) or consumption of
liquid containing pollen While we cannot rule this out we believe our results represent actual
targeted pollen consumption by ants Cephalotes ants known palynivores (Creighton 1967
Baroni Urbani and de Andrade 1997) contained similar numbers of pollen grains as many of the
pollen-containing ant species that we examined As well in at least once instance ants that we
found containing pollen grains were in the process of preying on non-pollen consuming
81
organisms (eg a Pachycondola species that was carrying a termite in its mandibles) or were
attracted by sugar baits
Contrary to our expectations there was no relationship between an ant speciesrsquo δ15
N ratio
and whether it had consumed pollen (Fig 32) We expected palynivory to be more common in
herbivorous ants because pollen presents a widespread source of protein a potentially limiting
resource The lack of pattern might reflect how available pollen grains are in the environment
While other forms of food may be preferable ants tend to be opportunistic foragers (Carroll and
Janzen 1973) and there is likely little cost associated with the consumption of scattered pollen
grains during foraging trips beyond the volume they occupy in the gut Some genera were found
to consistently consume pollen In support of previous observations (Baroni Urbani and de
Andrade 1997 Byk and Del Claro 2010) Cephalotes species were among the most common
pollen consumers Camponotus species also consistently contained pollen grains of the eight
species examined five contained pollen and pollen was found in species from both Peru and
PNG Camponotus species also contained the two largest numbers of pollen grains found (468
and 271) Taken together these results suggest that they may be another as yet unrecognized
commonly palynivorous genus
In general larvae did not contain large numbers of pollen grains (ranging from 1-38
grains) This is counter to what is seen in bees where larvae are the main recipients of pollen
(Willmer 2011) This difference may be because ant larvae are continually fed via trophallaxis
by adults throughout their development whereas bee larval cells are provisioned with pollen and
then sealed while larvae complete their development If both adults and larvae are able to extract
nutrients from pollen we would expect to see a preferential flow of nutrients from pollen to
larvae but not necessarily the grains themselves
82
Unlike palynivory by organisms that also provide pollination services pollen
consumption by ants is unlikely to benefit plants Ants rarely act as pollinators (Beattie et al
2004) They have pollen-killing antibiotic secretions on their cuticles (Dutton and Frederickson
2012) and they can compete with other flower visitors reducing flower visitation rates (Ness
2006) The negative impacts of flower-visiting ants are thought to have led to the evolution of
ant-repellent floral volatiles (reviewed in Willmer 2009) These volatiles can pre-empt
competition between ants and pollinators and may often be located within the pollen itself
(Willmer 2009) If floral volatiles truly are a defense against ant visitation our results suggest an
additional evolutionary pressure to not only prevent ant-pollinator interactions but to also
protect plant gametes from occasional consumption by a non-pollinating visitor
Acknowledgements
We would like to thank Jon Sanders Lina Arcila Hernandez and Eric Youngerman for providing
samples the Thomson lab for use of supplies the Pierce lab for funding support during JSrsquo and
EYrsquos collections and the staff of CICRA and the New Guinea Binatang Research Center for
logistical support The Peruvian Ministry of Agriculture provided permits (Nos 394-2009-AG-
DGFFS-DGEFFS and 299-2011-AG-DGFFS-DGEFFS) MEF acknowledges financial support
from an NSERC Discovery Grant a Connaught New Researcher Award an Ontario Ministry of
Economic Development and Innovation Early Researcher Award and the University of Toronto
ARC was supported by an Ontario Graduate Scholarship and Sigma Xi
83
References
Baroni Urbani C and M L de Andrade 1997 Pollen eating storing and spitting by ants
Naturwissenschaften 84256ndash258
Beattie A J C Turnbull R B Knox and E G Williams 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany 71421ndash
426
Bluumlthgen N G Gottsberger and K Fiedler 2004 Sugar and amino acid composition of ant-
attended nectar and honeydew sources from an Australian rainforest Austral Ecology
29418ndash429
Byk J and K Del-Claro 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333ndash38
Carroll C R and D H Janzen 1973 Ecology of foraging ants Annual Review of Ecology and
Systematics 4231ndash257
Creighton W S 1967 Studies on free colonies of Cryptocerus texanus Satschi (Hymenoptera
Formicidae) Psyche 7434ndash42
Czechowski W B Markό and A Radchenko 2009 Rubbish dumps reveal the diet of ant
colonies Myrmica schencki Em and Myrmica rubra (L) (Hymenoptera Formicidae) as
facultative pollen-eaters Polish Journal of Ecology 56737ndash741
Czechowski WB Markoacute K Erős and E Csata 2011 Pollenivory in ants (Hymenoptera
Formicidae) seems to be much more common than it was thought Annales Zoologici
61519ndash525
84
Dussutour A and S J Simpson 2009 Communal nutrition in ants Current Biology 19740ndash4
Dutton E M and M E Frederickson 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561ndash569
Floren A A Biun and K E Linsenmair 2002 Arboreal ants as key predators in tropical
lowland rainforest trees Oecologia 131137ndash144
Grant B R 1996 Pollen digestion by Darwinrsquos finches and its importance for early breeding
Ecology 77489-499
Herrera L G and C Martiacutenez del Rio 1998 Pollen digestion by new world bats effects of
processing time and feeding habits Ecology 792828ndash2838
Houmllldobler B and Wilson E O 1990 The Ants 1st Edition Harvard University Press
Cambridge
Horskins K and Turner V B 1999 Resource use and foraging patterns of honeybees Apis
mellifera and native insects on flowers of Eucalyptus costata Australian Journal of
Ecology 24221-227
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506ndash514
Roulston T H I and J H Cane 2000 Pollen nutritional content and digestibility for animals
Plant Systematics 222187ndash209
Van Tets I G 1997 Extraction of nutrients from Protea pollen by African rodents Belgian
Journal of Zoology 12759-65
Willmer P G 2011 Pollination and Floral Ecology 1st Edition Princeton University Press
New Jersey
85
Willmer P G C V Nuttman N E Raine G N Stone J G Pattrick K Henson P Stillman
et al 2009 Floral volatiles controlling ant behaviour Functional Ecology 23888ndash900
86
Tables
Table 31 Numbers of adult worker and larval ants examined for each species collected in Peru
Numbers of pollen grains found in individual samples are given in parentheses after the sample
size when there were no grains no numbers are given
Genus Species Number of adults analyzed
(numbers of grains found)
Number of larvae analyzed
(numbers of grains found)
Dolichoderinae
Azteca sp 1 3 0
sp 2 2 0
sp 3 0 2
sp 4 3 (1) 0
sp 5 3 (2) 0
Dolichoderus decollatus 3 (66) 0
sp 1 3 0
sp 2 3 1 (2)
sp 3 3 3
sp 4 0 2
Ecitoninae
Labidus sp 1 3 0
Formicinae
Camponotus sp 1 3 3
sp 2 3 (82) 1 (2)
sp 3 3 (3) 0
sp 4 0 2
87
sp 5 8 (11) 0
sp 6 4 (7 16 468) 0
sp 7 5 (12) 0
sp 8 2 (1) 0
Paratrechina sp 1 4 (11) 0
Myrmelachista sp 1 3 (5 11 18) 0
sp 2 0 3
Trachymyrmex sp 1 3 (21) 0
sp 2 3 0
Myrmicinae
Atta sp 1 3 0
Cephalotes atratus 3 (2 2 21) 0
placidus 3 (1 4 21) 0
sp 1 3 0
sp 2 1 0
sp 3 2 0
Crematogaster sp 1 3 1
sp 2 3 (35) 0
sp 3 9 (7) 0
sp 4 2 0
Megalomyrmex sp 1 3 0
sp 2 0 2
sp 3 4 (7) 0
sp 4 3 0
Monomorium sp 1 0 3 (2)
88
Pheidole sp 1 0 3
sp 2 3 0
sp 3 0 3
Procryptocerus sp 1 3 0
Solenopsis sp 1 0 3
sp 2 3 (3 5) 0
Wasmannia sp 1 3 0
Ponerinae
Pachycondola sp 1 3 (3) 0
Odontomachus sp 1 3 (34) 0
Pseudomyrmicinae
Pseudomyrmex sp 1 3 0
sp 2 3 0
sp 3 3 (9) 0
sp 4 0 3
sp 5 0 3
89
Table 32 Numbers of adult worker and larval ants examined for each species collected in Papua
New Guinea Numbers of pollen grains found in individual samples are given in parentheses
after the sample size when there were no grains no numbers are given
Genus Species Number adults analyzed
(grains found)
Number larvae analyzed
(grains found)
Aenictinae
Aenictus sp 1 3 3 (1 1)
Dolichoderinae
Philidris sp 1 3 1 (4)
Tapinoma sp 1 2 3 (38)
Technomyrmex sp 1 0 3 (12 12)
Ectatomminae
Rhytidoponera sp 1 1 3 (2)
Formicinae
Acropyga sp 1 3 3 (2)
sp 2 3 3
sp 3 3 3
Anoplolepsis sp 1 3 (1) 3 (1)
Calomyrmex sp 1 3 3 (1)
Camponotus sp 1 3 (1 271) 3
sp 2 1 3
sp 3 2 0
Nylanderia sp 1 3 3
Opisthopsis sp 1 3 3
Polyrachis sp 1 1 3 (4)
90
Myrmicinae
Crematogaster sp 1 3 3
sp 2 3 (32) 3
Monomorium sp 1 3 (1) 3 (1)
sp 2 3 3 (1 1 1)
Pheidole sp 1 3 3
Pristomyrmex sp 1 3 3 (11)
Pyramica sp 1 3 0
Ponerinae
Odontomachus sp 1 3 3 (4)
91
Figures
Figure 31 Photographs of pollen grains found in two ant species a) Dolichoderus sp 2 and b)
Camponotus sp 6 Scale bars = 50 microm
92
Figure 32 Relationship between trophic level as measured by δ15
N ratio and pollen presence
for 48 neotropical ant species 1 Atta sp 5 Azteca spp 8 Camponotus spp 5 Cephalotes spp 4
Crematogaster spp 5 Dolichoderus spp 1 Labidus sp 3 Megalomyrmex spp 1 Monomorium
sp 2 Myrmelachista spp 1 Odontomachus sp 1 Pachycondola sp 1 Paratrechina sp 3
Pheidole sp 4 Pseudomyrmex spp 2 Solenopsis spp and 1 Wasmannia sp
93
Concluding Remarks
Ants have traditionally been considered to be unwelcome flower visitors they consume floral
rewards ostensibly meant for pollinators while rarely providing pollination services themselves
In this thesis I investigate the validity of this assumption and use an experimental study and an
observational study to examine two ways that ants may impact plant reproduction
In the first chapter I review the literature on ants as flower visitors I show that the common
assumption of ants always being categorically negative for plant reproduction is incorrect while
ants may reduce floral attractiveness to pollinators the net effect of ants on plant fitness
measures is variable ranging from positive to negative to neutral I also discuss ants as
pollinators and ways that plants may minimize ant visitation to flowers before finishing with a
section highlighting questions that still need investigation
In the second chapter I explore how interference competition with ants can structure bumblebee
foraging behavior and how this may alter pollen movement patterns Using artificial flowers with
male or female function I allowed Bombus impatiens bumblebees to forage on flowers that
either did or did not have Myrmica rubra ants on them Ant presence significantly reduced the
amount of pollen analogue donated and received by flowers Building on these results I found
that the presence of ant scent alone elicited similar changes to bumblebee behavior reducing the
amount of pollen analogue donated by male flowers My results show that interference
competition is sufficient to alter bumblebee foraging behavior and that the effects of ants on
pollinators may extend after ant visitation to flowers Further study is needed to determine if bees
are able to differentiate between ant species by visual or olfactory cues Their ability to do so
94
could affect how generalized their response to ant presence is and may help to better predict how
ants will affect plant reproduction
In the final chapter I investigate how common palynivory is amongst tropical ant species I used
acetolysis and light microscopy to examine whole ants larvae and ant guts from Papua New
Guinea and Peru I found that tropical ants consistently contain pollen grains albeit often in
small numbers (lt5) However some genera notably Camponotus and Cephalotes consistently
contained higher numbers providing the first evidence that Camponotus may be a palynivorous
genus The small numbers of pollen grains present may indicate that ants preferentially exploit
other more easily digestible high-protein foods over pollen Future studies should investigate
exactly what role pollen plays in ant diets by supplementing the diet of captive ant colonies with
pollen This would allow us to examine how pollen consumption affects colony demography as
well as how efficiently ants digest pollen and whether ants exhibit pollen preferences
I bring up several pressing and unanswered questions within this thesis and highlight three here
First numerous plant species possess chemical or physical traits assumed to increase plant
fitness by reducing ant visitation However this has rarely been tested and instead research has
focused predominately on documenting ant repellence We should move away from this and
begin examining plant fitness in the presence or absence of these repellent traits to see how
beneficial these traits truly are Second how flower visiting ants impact male fitness has been
almost completely ignored in favor of female fitness Future studies should investigate how ant
visitation affects both pollen donation and pollen movement patterns Lastly research involving
antibiotics present on ant cuticles and their effect on pollen viability has been too small in scope
often limited to demonstrating the pollen killing effects of ant secretions Investigation into
95
whether plants have evolved any resistance to the secretions of the local ant community or why
some plants appear unaffected by their ant partners may yield exciting results
In conclusion ants have the capacity to strongly affect plant reproduction both positively and
negatively The fact that these diametrically opposing forces exist means we cannot approach
flower-visiting ants as either positive or negative a priori Instead we should strive to work
towards a better understanding of what is occurring in the system of interest and what role ants
play within it
96
Copyright Acknowledgments
Chapter 2 ldquoAnts and ant scent reduce bumblebee pollination of artificial flowersrdquo is current in
press in The American Naturalist Request for its inclusion here was sought and approved
ii
Ants as flower visitors and their effects on pollinator behavior and plant
reproduction
Adam Cembrowski
Masters of Science
Ecology and Evolutionary Biology
University of Toronto
2013
Abstract
Ants regularly visit flowers but they may decrease plant reproductive success by competing with
pollinators or damaging reproductive structures However how ants may exert these costs needs
further clarification In Chapter 1 I review the literature finding that flower-visiting ants often
have neutral effects on plant fitness In Chapter 2 using artificial flowers with male and female
function I investigate how interference competition between flower-visiting Myrmica rubra ants
and Bombus impatiens bumblebees changes pollen analogue movement patterns Ant presence
and scent significantly reduced pollen analogue donation and reception because bees avoided
flowers with ant cues In Chapter 3 to assess the frequency of palynivory among ants I
conducted an acetolysis survey of 75 neo- and paleo-tropical ant species Ants consistently
contained low numbers of pollen grains suggesting opportunistic pollen consumption may be
widespread Altogether ant visitation may be costly but the mechanism depends on the plant
pollinator and ant identities
iii
Acknowledgments
Science is never an individual undertaking Numerous people made the completion of this thesis
possible First and foremost Megan Frederickson gave me the freedom to pursue research
outside of her realm of expertise was extremely patient my ever-changing interests and
supported the decisions I made in my degree My committee also deserves strong thanks James
Thomson was instrumental in my studies giving me lab space supplies and most importantly
time and guidance when I had questions about anything pollination bee or flower related Ben
Gilbert gave statistics help and was helpful with coding questions As well thank you to my
examination committee Spencer Barrett and Helen Rodd
The Frederickson and Thomson lab groups helped me immeasurably giving encouragement
guidance and reality checks when I went too far off track Thank you to Kyle Turner Lina
Arcila Hernandez Kirsten Prior Jane Ogilvie Alison Parker Eric Youngerman and Rebecca
Batstone Kyle especially helped me in all regards both inside and outside of school and was
without question the best field roommate and officemate I could have had Several volunteers
work study students and our lab tech fed ants washed artificial flowers and counted tiny objects
saving me countless hours particularly Harry Rusnock Shannon Meadley Dunphy Margaret
Thompson and Jackie Day I would also like to highlight Marcus Guorui Tan in particular for
his work collaborating with me and running trials when I was in out of town and being
unwaveringly positive about our research Our experiment would not have been nearly as
successful without him
Several people in the department and associated with the lab provided feedback jokes and
friendship without which grad school would have been a depressing place So thank you Natalie
Jones Rachel Germaine Kelly Carscadden Alex de Serrano Jordan Pleet Aaron Hall Susana
Wadgymar Emily Austen Eddie Ho Amanda Gorton Dorina Szuroczki Jenn Coughlan Jon
Sanders and Gabe Miller
People not in the department and both inside and outside of ecology made my time here possible
and more enjoyable than otherwise Thanks to the Tuner family for taking me in during holidays
Maureen Murray and Matt Mazowita for everything and my old supervisor Colleen Cassady St
Clair for helping me get where I am
iv
Lastly my family did an astounding amount for me in letting me cultivate my love of biology
buying me books letting me keep strange animals and getting me outdoors So thank you to my
brothers John and Mark my dad George and my mom Kay And to my partner Katherine for
her unbelievable patience both in regards to my academic life and in her willingness to adapt
hers for mine thank you This could not have happened without you
ldquoVor Allem sind aus der Reihe der fluumlgellosen Insecten die weitverbreiteten fluumlgellosen Ameisen
sehr unwillkommene Gaumlste der Bluumlthen Und dennoch sind gerade sie nach dem Nectar der
Bluumlthen in hohem Grade luumlsternhelliprdquo
ldquoOf all the wingless insects it is the widely dispersed ants that are the most unwelcome guests to
flowers And yet they are the very ones which have the greatest longing for nectarhelliprdquo
A Kerner 1878 Flowers and their Unbidden Guests
v
Table of Contents
Acknowledgments iii
List of Tables vii
List of Figures viii
Chapter 1 Ants as flower visitors and their effects on pollinator behavior and plant
reproduction 1
Abstract 1
Introduction 2
1 Ant interactions with flowers and floral visitors 3
2 Ants as pollinators 12
3 Floral defenses against ants 14
4 Future directions 20
References 24
Tables 39
Chapter 2 Ants and ant scent reduce bumblebee pollination of artificial flowers 54
Abstract 54
Introduction 55
Methods 57
Results 62
Discussion 63
Acknowledgements 66
References 68
Figures 73
Chapter 3 Not just for the bees pollen consumption is common among tropical ants 75
vi
Abstract 75
Introduction 75
Methods 77
Results 78
Discussion 79
Acknowledgements 82
References 83
Tables 86
Figures 91
Concluding Remarks 93
Copyright Acknowledgments 96
vii
List of Tables
Table 11 List of studies of flower-visiting ants that consumed floral rewards or interacted with
pollinators and included a measure of fitness39
Table 12 List of studies that have either demonstrated ant pollination through exclusion
experiments or whose results strongly suggest it48
Table 31 Numbers of adult worker and larval ants examined for each species collected in Peru
and numbers of pollen grains found87
Table 32 Numbers of adult worker and larval ants examined for each species collected in Papua
New Guinea and numbers of pollen grains found90
viii
List of Figures
Figure 21 Photograph of artificial flower lids with anther and stigma74
Figure 22 Dye transferred (mean plusmn 1SE) by B impatiens a) in the presence and absence of M
rubra ants and b) with or without M rubra scent75
Figure 31 Photographs of pollen grains found inside ants92
Figure 32 Relationship between trophic level and pollen presence for neotropical
ant species93
1
Chapter 1 Ants as flower visitors and their effects on pollinator behavior and plant
reproduction
Cembrowski AR and Frederickson ME
Planned submission to a journal such as Oikos or Insectes Sociaux
ARC wrote the manuscript with input from MEF
Abstract
Ants often provide a range of benefits to flowering plants particularly by reducing herbivory
However when ants visit flowers they can deter pollinators or damage floral structures
Currently it is unclear how often and in what direction flower-visiting ants affect plant
reproductive success A review of the literature shows that ants can help or hinder pollination
processes but overall flower-visiting ants most commonly have a non-significant net effect on
plant fitness Although ants may directly harm flowers or indirectly affect pollination by
consuming floral nectar or harassing pollinators they can also benefit plants by attacking
florivores or predators of pollinators Ants occasionally act as pollinators themselves though ant
secretions often kill pollen grains There is growing evidence that many plants have traits that
allow them to avoid or minimize the negative effects of flower-visiting ants More research is
needed to determine if these traits provide fitness benefits to plants by preventing ant visitation
or if they function as generalized repellents of floral antagonists
2
Introduction
Ants and angiosperms share a long evolutionary history Ant diversification closely followed that
of flowering plants as ants took advantage of the large prey numbers that angiosperms supported
and the new habitats they provided (Moreau 2006) Subsequently numerous angiosperm
lineages evolved intimate associations with ants that function as an indirect or biotic form of
plant defense Food rewards such as extrafloral nectar or pearl bodies or housing in the form of
domatia attract ants to plants where they reduce herbivory and increase plant fitness (reviewed
in Rosumek et al 2009) However the traits that make ants good plant defenders particularly
their abundance attraction to sugar sources and aggressiveness can also cause problems for
plant reproduction Ants attracted by floral nectar can compete with pollinators for resources
and defend flowers potentially reducing plant fitness (Ness 2006 Junker et al 2007) That ants
negatively affect plant reproduction is an old and pervasive hypothesis Kerner (1878) called ants
ldquomost unwelcome guestsrdquo on flowers and more recent authors have similarly expressed that ant
visitation to flowers reduces plant reproductive success (Ness 2006 Lach 2007 Willmer et al
2009)
Nonetheless studies assessing the impact of flower-visiting ants on plant reproductive
success have produced mixed results Ants can have negative impacts on pollination and flower
function (Galen 1983 Puterbaugh 1998 Ness 2006 Hansen and Muumlller 2009) However other
studies have found no effect or even strong benefits of ant visits to flowers (Rico-Gray and
Thien 1989 Altshuler 1999 Schuumlrch 2000 Ashman and King 2005) This discrepancy between
ants being ldquounwelcome guestsrdquo and their actual effects on plant reproduction requires
clarification Our goal here is to highlight the ways that flower-visiting ants may affect plant
reproduction both positively and negatively and to discern through a review of the literature
3
what impacts ants have on plant reproductive success First we discuss mechanisms by which
flower-visiting ants can directly and indirectly impact plant reproduction both through floral
damage and by mediating changes in pollinator or florivore behavior We end this section by
evaluating published research that collectively shows that flower-visiting ants have
predominantly neutral effects on plant fitness measures Next we discuss pollination by ants
focusing on the ldquoantibiotic hypothesisrdquo before describing how plants may prevent ants from
visiting flowers Lastly we discuss promising new avenues of research and highlight work that
still needs to be performed to give a more complete understanding of ants as flower visitors
1 Ant interactions with flowers and floral visitors
i) Damage to flowers
Ants can directly disrupt plant reproduction by damaging floral structures preventing either the
fertilization of ovules or the presentation of pollen Rarely ants forage on floral tissue itself for
example Puterbaugh (1998) found that Formica neorufibarbus ants chewed through the coronal
ring of Eritrichium aretiodes flowers to get lipids More often but still not commonly foraging
ants chew through reproductive tissue to access floral nectar This can cause sterilization of
whole flowers (Echium plantagineum Kirk 1984) or affect female function only if just styles are
damaged effectively creating male flowers (Polemonium viscosum Galen 1983) Antibiotic
secretions on ant integuments may also directly damage pollen grains but the fitness effects of
this are unclear (see 2 below)
Several species of Crematogaster and Allomerus ants sterilize their host plants by
attacking floral buds (Yu and Pierce 1998 Stanton et al 1999 Gaume et al 2005 Maleacute et al
2012 Table 11) These ants nest in ldquoant-plantsrdquo or ldquomyrmecophytesrdquomdashie plants that host ant
4
colonies in specialized structures (domatia) The sterilizing ants do not appear to consume floral
tissues (Yu and Pierce 1998) and two hypotheses may explain why ants destroy floral buds First
ant colony size is often limited by domatia availability on their host plant By destroying floral
buds ants can cause plants to divert resources from reproductive to vegetative growth increasing
domatia number and allowing for larger ant colonies (Yu amp Pierce 1998 Frederickson 2009)
Second when plant-ants must defend their colony from takeovers by other colonies they may
benefit from limiting access to their host plant By destroying buds that could develop into
flowers branches or leaves ants can prevent their host plant from contacting nearby plants
occupied by enemy ant colonies (Stanton et al 1999)
The impact of sterilization on lifetime plant fitness is unclear Ants may destroy all of
their host plantrsquos floral buds (Yu and Pierce 1998) completely preventing reproduction or only a
portion of them (Maleacute et al 2012) allowing some seed set However counter-intuitively this
sterilization behavior may provide long-term fitness benefits to plants (Frederickson 2009
Palmer et al 2010) Ant-plants often host several different ant species over their lifetimes some
of which are non-sterilizing If hosting a sterilizing species at a certain life stage increases
survival or growth relative to hosting a non-sterilizing species at that life stage plants may
increase lifetime fitness by largely forgoing current reproduction in favor of increasing future
reproduction (Palmer et al 2010) Why plants invest resources in producing flowers that are
quickly destroyed by ants is not known but the timing of flower destruction may be important If
ants attack flowers after anthers dehisce plants may still realize some male fitness via pollen
donation (Yu and Pierce 1998) Additionally if the cost of flower production is relatively minor
and ants sometimes ldquomissrdquo flowers (Edwards and Yu 2008) attempting limited reproduction
may be a better strategy than forgoing it entirely
5
ii) Competition with pollinators
Ant activity on flowers can dramatically alter pollinator behavior Ants can reduce the species
diversity frequency or duration of pollinator visits to flowers (Norment 1988 Tsuji et al 2004
Ness 2006 Junker et al 2007 Lach 2007 2008b Romero et al 2011 and see Chapter 2) These
changes are largely due to either exploitative competition (ie consumption of shared resources)
or interference competition (ie direct aggression) between ants and pollinators
a) Exploitative competition
Pollinators and ants frequently compete for floral nectar Flower-visiting ants are often classified
as nectar thieves (sensu Inouye 1980) because they typically enter flowers through the
ldquolegitimaterdquo opening and consume nectar but transfer no pollen (Wyatt 1980 Ness 2006
Chamberlain and Holland 2008) Ants can also act as primary nectar robbers chewing holes
through corollas to reach nectaries (Willmer and Corbet 1981 Koopowitz and Marchant 1998)
although they are more commonly secondary nectar robbers and use holes made by previous
robbers (Newman and Thomson 2005)
Like other flower visitors foraging ants will often leave nectar behind in flowers This
amount can be less (Bleil et al 2011) more (Fritz and Morse 1982) or similar to (Lach 2008a)
amounts left by other visitors Often ants are morphologically constrained from fully exploiting
nectar Unlike bees and butterflies ants do not have proboscises and must get close to nectar to
consume it Narrow corollas or nectar spurs can prevent ant access to some or all floral nectar
(Schemske 1980 Galen and Cuba 2001 and see below) Smaller ants may be better able to
6
access nectar (Newman and Thomson 2005 Agarwal and Rastogi 2007) but can be limited by
their crop size in the amount of nectar they can remove (Lach 2005) If foraging ants leave
enough nectar behind for a pollinator to recover the cost of visiting a flower ants may have
minimal effects on plant reproductive success (Maloof and Inouye 2000 Turner et al 2012)
Differences among ant species in behavior and diet also affect their consumption of floral
nectar Species vary in how many flowers they visit per inflorescence or per plant and in how
many ants will visit a flower simultaneously (Lach 2005 Junker et al 2007 Lach 2013)
Invasive antsmdashthought to be better at exploiting and defending resources than native ants
(Holway et al 2002)mdashoften recruit to flowers in large numbers sometimes several dozen or
more (Visser et al 1999 Hansen and Muumlller 2009) Overall many factors may influence how
heavily ants exploit floral nectar on plants including ant density plant height the spatial or
temporal distribution of floral nectar and the availability of alternative food resources for ants
(Cushman and Addicott 1991 Lach 2013)
Rarely ants and pollinators may compete for pollen We know of only a few studies that
have recorded ants harvesting pollen directly from anthers (Horskins and Turner 1999 Ness
2006 Byk and Del-Claro 2010) Other studies have found pollen grains in ants (eg Baroni
Urbani and de Andrade 1997 and see Chapter 3) but this may not indicate competition with
pollinators ants sometimes collect stray pollen grains from the environment (Creighton 1967)
One New World genus Cephalotes might be specially adapted for pollen consumption They
possess ldquoproventricular shieldsrdquo structures that may restrict solid food to the crop allowing
nestmates to exchange pollen grains via trophallaxis It is unclear if this structure evolved for this
purpose but Cephalotes are the most frequently documented palynivorous ants (eg Creighton
1967 Baroni Urbani and de Andrade 1997 Byk and Del-Claro 2010) How efficiently ants
7
digest pollen and whether ants like bees feed it to larvae as a protein source is unknown At
least two studies have found that ant larvae consume pollen providing some support for this idea
(Wheeler and Bailey 1920 and Chapter 3)
b) Interference competition
Ants are relatively unique among flower visitors in defending floral resources from competitors
(but see Willmer and Corbett 1981 Roubik 1982) This defense ranges from chance encounters
with ants attacking visitors when flower occupancy overlaps (Ashman and King 2005 Junker et
al 2007) to actively patrolling inflorescences or constructing protective galleries around
nectaries (Horvitz and Schemske 1984 Gaume et al 2005 Hansen and Muumlller 2009)
Interference competition even in the absence of exploitative competition is sufficient to alter
pollinator behavior (Chapter 2)
The strength and outcome of interference competition depends on the identities of the ant
and the pollinator Pollinators might not alter their visitation patterns in response to non-
aggressive ants (Junker et al 2007) but more antagonistic ants may elicit avoidance behavior
before or soon after the visitor lands (Willmer and Corbet 1982 Altshuler 1999 Schuumlrch et al
2000 Table 11) Pollinator size may help predict their reaction to ants because ants may deter
only small-bodied pollinators (Gonzaacutelvez et al 2012) It is not clear whether flower visitors view
ants as aggressive competitors or as potential predators rarely ants prey on pollinators (Schatz
and Wcislo 1999) but in most cases ants merely cause pollinators to relocate
How well pollinators recognize and avoid ant-visited flowers is unresolved Collectively
pollinators have lower visitation rates to flowers visited by ants (Romero et al 2011) This is
8
dependent on pollinator identity however dipterans do not appear to discriminate between ant-
visited and ant-free flowers while bees can identify and avoid ant-visited flowers Bees likely
use a combination of visual and olfactory cues to recognize ant-visited flowers They can
identify previous conspecific and some heterospecific insect visitors by the hydrocarbon
ldquofootprintsrdquo they leave during foraging (Stout and Goulson 2001) Bumblebees can detect ant
hydrocarbons although this has been shown only in the laboratory (Ballantyne and Willmer
2012 Chapter 2) No study has yet examined if bees can use sight alone to detect ants but
several suggest it (Junker et al 2007 Gonzaacutelvez et al 2012) Some bees may discriminate
between ant species when visiting flowers (eg Lach 2008b) which follows if not all ant-
pollinator interactions are equally costly The ability of bees to do so will affect how generalized
their avoidance behaviors will be If they can discriminate between species bees may avoid
specific flowers visited by more ldquocostlyrdquo ants However if they cannot bees might either avoid
all ant-visited flowers or none depending on how negative the average interaction with an ant is
iii) Interactions with flower and pollinator antagonists
Ants can defend the flowers they visit Numerous plant species have circumfloral or bracteal
nectaries (ie extrafloral nectaries around flowers) and ants attracted to these nectaries can
reduce bud ovule or flower predation increasing plant fitness (Inouye and Taylor 1979 Rico-
Gray and Thien 1989 Sugiura et al 2006) Although the presence of EFNs (extrafloral nectaries)
so close to floral nectaries (sometimes less than 1 cm Ness 2006) might increase discovery and
exploitation of floral nectar by ants only one study has investigated this hypothesis finding no
relationship between the proximity of EFNs to floral nectaries and antsrsquo use of floral nectar
(Galen 2005) Holes made by nectar robbers are functionally analogous to circumfloral nectaries
9
and can also attract ants that defend flowers (Newman and Thomson 2005) and nectar-thieving
ants can similarly prevent florivory in some systems (Lach 2007 Bleil et al 2011)
Ants can also reduce the abundance of flower-dwelling predators or parasites of
pollinators Ambush predators such as crab spiders often forage at flowers where they may
reduce pollinator visitation rates and plant fitness (Dukas 2001 Gonccedilalvez-Souza et al 2008)
Ants can decrease spider abundance and richness on plants (Rudgers 2004 Renault et al 2005
Nahas et al 2012) and some evidence suggests that they can do the same on flowers (Lach
2008) In select cases ants may also reduce the abundance of pollinator parasitoids (Holland et
al 2011) Whether ants increase plant reproductive success by removing pollinator antagonists
has not yet been examined but should be If ants create lsquoenemy-freersquo flowers pollinators might
prefer to land on flowers with ants
iv) Net effects
Table 11 compiles studies in which the authors recorded ants consuming floral rewards or
interacting with pollinators and measured some aspect of plant fitness (eg fruit or seed set
pollen deposition) We included only a single study per plant species unless subsequent studies
found conflicting results or measured the fitness of another plant sex Records of ant pollination
are included in a separate table (Table 12)
Table 11 reveals relatively few examples of flower-visiting ants negatively affecting
plant fitness given how costly ant visitation is assumed to be The net effect of ants on plant
reproductive success is actually quite variable with similar numbers of studies finding positive
negative or neutral effects of ant presence Surprisingly when ants do have negative effects it is
10
usually because they directly damage floral structures and not because they prevent pollinators
from visiting flowers and successfully transferring pollen Why are ants not more often negative
for plant reproduction when this is commonly assumed to be true
First ants may compensate for reducing pollinator visitation rates by improving
pollination quality As pollinators move between flowers on a plant they often deposit self-
pollen on stigmas (ie geitonogamy) In some plants with late-acting self-incompatibility ovules
fertilized by self-pollen are subsequently aborted reducing the number of seeds that can be
produced (Barrett 2002 and references therein) Geitonogamy can also reduce offspring viability
or vigor (de Jong et al 1993) and cause pollen discounting a reduction in the amount of pollen
available for outcrossing because of self-pollination (Barrett 2002) By competing with
pollinators ants might decrease rates of geitonogamous pollination if pollinators then visit fewer
sequential flowers within a plant Ants might also cause pollinators to fly further between plants
potentially increasing the genetic diversity of pollen arriving at stigmas Although such effects
have been reported for other floral larcenists (Maloof 2001 Irwin 2003) to our knowledge these
hypotheses have not been investigated in relation to ants
Second ants and pollinators do not always forage for the same resources possibly
allowing them to partition floral rewards If ants forage for nectar but not pollen and flowers can
be pollinated efficiently by visitors looking to gather mostly pollen and little or no nectar then
plant reproduction will not be affected by ants consuming floral nectar Junker et al (2010a)
found that honeybees foraged for both pollen and nectar in the absence of ants but reduced their
foraging on nectar but not pollen when ants could access flowers resulting in no difference in
fruit set between treatments Whether pollinators commonly adjust their foraging behaviors in
response to ants warrants further research
11
Third ants are somewhat unique among floral larcenists in that they often provide direct
benefits to the plants they visit In the studies listed in Table 11 ants sometimes decreased
herbivory on plants (Chamberlain and Holland 2008 Bleil et al 2011) or otherwise aided the
plant (eg ant presence repelling smaller less effective pollinators Gonzaacutelvez et al 2012) If the
positive effects of ants cancel or outweigh the negative effects of flower-visiting ants on
pollinators then ant visitation may be neutral or even positive for plant fitness
Fourth ants may impose fitness costs to plants not captured in the studies in Table 11
Nectar robbers have stronger negative effects when plants are pollen-limited (Burkle et al 2007)
but only one study in Table 11 assessed whether flowers would set more seed if they received
more pollen finding no evidence of pollen limitation (Lach 2008a Table 11) Even if ants
decrease pollinator visitation rates there is limited scope for ants to decrease plant reproductive
success when plants are not pollen-limited Furthermore nearly all the studies in Table 11
measured only female fitness Only three quantified aspects of male fitness (Wyatt 1980 Fritz
and Morse 1981 Galen and Geib 2007) and we know of only one other study in a system in
which ants occasionally pollinate that has also measured male fitness (Ashman 2000) All four
studies measured pollen or pollinia removal rates and none found any effect of flower-visiting
ants on male fitness However whether other measures of male fitness (eg seeds sired pollen
deposition patterns) would reveal similar or different effects needs investigation
Finally although floral defenses against ants (see 3 below) may prevent us from
observing the negative effects of ants on plant reproductive success we consider this unlikely
Several studies have examined interactions between ants and plants that share little or no
coevolutionary history usually in the context of ant invasions Although invasive ants often
recruit to flowers in larger numbers than native ants they do not appear to reduce plant
12
reproductive success (Blancafort and Goacutemez 2005 Lach 2007 Lach 2008a but see Hansen and
Muumlller 2009) And in Hawaii where angiosperms evolved for millions of years in the absence of
ants and are thought to have secondarily lost floral defenses against ants (Junker et al 2011a)
there is no evidence that the preponderance of recently introduced flower-visiting ants negatively
affects plant fitness (Junker et al 2010a Bleil et al 2011)
2 Ants as pollinators
Ants may directly benefit plant reproduction by acting as pollinators but this is a relatively rare
occurrence Ants have long been considered poor pollinators (Kerner 1878) Initially this was
based on morphological and behavioral differences between ants and recognized pollinators
Worker ants are wingless and often relatively hairless making them poor pollen dispersers As
well because many ants exhibit Ortstreue (literally ldquoplace fidelityrdquo) ants were thought to return
to the same branch or flower repeatedly effecting no cross pollination (Houmllldobler and Wilson
1990) Though largely true these traits do not necessarily preclude ants from acting as
pollinators (Table 12) Although they commonly have restricted foraging ranges (Houmllldobler and
Wilson 1990) some ants can effect outcrossing (eg Ashman and King 2005 Carvalheiro 2008)
and many ants are as hairy if not hairier than their pollinating counterparts
In the late 1970s the ldquoantibiotic hypothesisrdquo was put forward to explain the rarity of ant
pollination Ants use antibiotic secretions to protect themselves against the fungal and bacterial
pathogens common in their nests and environs Iwanami and Iwadare (1978) found that one of
these compounds myrmicacin also greatly reduces pollen germination rates Subsequently
Beattie et al (1984 1985) found that short periods of contact (le 30 minutes) with an antrsquos
integument caused pollen grains to fail to germinate Some of these pollen-killing compounds
13
were traced back to the ant metapleural gland which produces hygienic secretions (Houmllldobler
and Wilson 1990) although contact with ants lacking metapleural glands can also reduce pollen
viability (Beattie et al 1985 Dutton and Frederickson 2012) Ant mandibular or anal gland
secretions and potentially ant venom may likewise defend ants against pathogens and have
similar effects on pollen germination (Beattie et al 1985 Graystock and Hughes 2011)
Nonetheless ant pollination has been documented repeatedly There are at least 18 cases
in which ant pollination has been shown experimentally and five others in which observations
strongly suggest it (Table 12) The majority of these plants fit the ldquoant pollination syndromerdquo
originally proposed by Hickman (1974) dense stands of short plants with small flowers and
easily accessible nectaries Typically ant-pollinated plants are associated with hot and dry
climates (principally in the Mediterranean) but dry alpine environments may also favor the
evolution of ant pollination (eg Svensson 1985 Puterbaugh 1998 Schiestl and Glaser 2011) In
most cases ants appear to be the sole or major pollinator of the plant in question (Hickman 1974
Peakall et al 1990 de Vega et al 2009) although ants sometimes make only supplementary
contributions to pollination (Schuumlrch et al 2000 Ashman and King 2005) This suggests that co-
evolution between ants and plants may not be necessary for ants to be useful pollinatorsmdashwhich
is surprising given the potential negative effects of ants on plant reproduction
Ant-pollinated plants cope with ant secretions in a variety of ways Several plant species
may simply tolerate pollen damage (Gόmez and Zamora 1992 de Vega et al 2009) apparently
possessing no overt adaptations to prevent it These plants might receive enough pollen to
overcome reduced pollen viability Other plants may place pollen on ant body parts thought to
have fewer secretions thereby potentially escaping damage (eg on ant legs Ramsey 1995) or
attach pollen to ants with specialized structures avoiding direct contact with the ant integument
14
(Peakall et al 1990) It is not known whether pollen evolves resistance to ant secretions or
whether plants deposit pollen on ants which have less disruptive secretions although these
possibilities have been suggested elsewhere (eg Garciacutea et al 1995 Ramsey 1995)
One key question regarding the effects of ant secretions on pollen grains urgently needs
to be resolved Several studies have suggested that the pollen-killing secretions of ants may have
contributed to the evolution of floral defenses against ants (eg Wagner 2000 Galen and Geib
2007) For this to be true ants must decrease male fitness by damaging pollen grains before they
leave flowers and are deposited on receptive stigmas Only two studies have investigated the
effects of ant secretions on pollen still in anthers (Wagner 2000 Galen and Butchard 2003) and
both constrained ants unnaturally to flowers for significantly longer than ants would normally
stay on flowers Although these studies found strong reductions in pollen vigor it is unclear if
ant secretions decrease male fitness under natural ant visitation conditions
3 Floral defenses against ants
Plants are not passive recipients of flower visitors and have some ability to ldquochooserdquo which
organisms can access floral rewards Flowers resist ants using both structural and chemical traits
More thorough investigation is needed to determine whether these traits evolved to prevent or
minimize the negative effects of ants on plant reproductive success (ie whether these traits can
truly be considered defenses against ants see 4ia below) It is unlikely however that ants
experience reciprocal selection to circumvent floral resistance traits because the fitness benefits
to ants of accessing flowers are probably small Thus there may be little scope for
coevolutionary arms races between floral defenses against ants and ant counter-adaptations
15
i) Structural resistance traits
a) Restricting ant access
Numerous plant species have physical structures preventing ant access to flowers Thin pedicels
can limit antsrsquo gripping abilities and make ants easier to dislodge when flowers are moved by
wind (Gaume et al 2005) and waxy secretions can render stems too slippery for ants to climb
(Harley 1991) Structures surrounding flowers such as water moats or dense hairs may similarly
keep ants from accessing floral rewards (Willmer et al 2009) Many such traits function as broad
filters and prevent not only ants but most crawling insects from reaching flowers Thus it is
difficult to determine whether ants florivores or both acted as selective agents favoring the
evolution of physical barriers to flowers
Flower shape may also reduce ant-pollinator conflict Long narrow corollas or nectar
spurs can prevent all but the smallest ants from accessing floral nectaries (Bluumlthgen et al 2004
Agarwal and Rastogi 2007) while still allowing long-tongued pollinators access to rewards
Selection to exclude nectar-thieving ants in such a manner however may be counteracted by
pollinator preferences for wider corollas that allow easier flower handling (Galen and Cuba
2001) Additionally these types of flowers may be more prone to robbery (Irwin and Maloof
2002) limiting their usefulness in preventing nectar larceny
b) Separation in space or time
Plants may separate floral rewards and ants temporally or spatially to minimize ant impacts on
plant reproduction Several authors have proposed that myrmecophytes have adapted to the
16
sterilization or otherwise detrimental activities of ants by locating domatia or food rewards on
different branches than flowers (Izzo and Vasconcelos 2002 Raine et al 2002 Edwards and Yu
2008) Temporal separation of ant and pollinator activities could likewise reduce conflict though
evidence is limited Some acacia flowers dehisce during periods of low ant activity (Willmer and
Stone 1997 Nicklen and Wagner 2006) but it is unclear if this is in response to ants
temperature or pollinator activity Timing of nectar secretion may similarly reduce ant-pollinator
competition If ants are active prior to pollinators they may deplete floral nectar and reduce
pollinator visitation rates (Schaffer et al 1984) However if nectar is secreted when ants are
inactive pollination may occur unimpeded (Norment 1988) This temporal separation could
favor pollinators that are active when ants are not (eg Wyatt and Morse 1981) but this is
poorly documented Further studies particularly focusing on flowers with the potential for
nocturnal and diurnal pollination may provide more examples
c) Extrafloral nectaries (EFNs)
One hypothesis put forward to explain the evolution of EFNs proposes that EFNs ldquodistractrdquo ants
from consuming floral nectar (Kerner 1878 Wagner and Kay 2002) Wagner and Kay (2002)
tested this hypothesis by constructing model plants using artificial nectaries assigned at random
to represent either floral or extrafloral nectaries They found that the presence of EFNs could
reduce ant exploitation of floral nectar However as highlighted by Galen (2005) this design is
closer to a plant making a surplus of flowers some of which will avoid ant visitation if visitation
rates increase sublinearly with increasing flower number Producing extra flowers to escape ant
visitation appears to be a viable strategy in some systems (Ashman and King 2005 Galen 2005)
However Galen (2005) found that applying honey (simulating extrafloral nectar) to P viscosum
17
leaves had the opposite effect and increased ant visits to flowers Only two studies both in
Pachycereus schottii have found evidence for the distraction hypothesis under field conditions
(Chamberlain and Holland 2008 Holland et al 2011)
There is limited empirical support for the distraction hypothesis but abundant evidence
that EFNs attract ants that defend plants against herbivores (reviewed in Rosumek et al 2009)
To function as attractants the presence of EFNs must increase ant abundance on plants
However because of the increased numbers attracted EFNs would not be expected to reduce the
abundance of ants on nectar rewards or be able to satiate the ants recruited Only if ant
abundance stops increasing at a reward density much lower than that available on the plant
would EFNs serve to distract ants In this case the amount of rewards may overwhelm ants
leaving some flowers underexploited However even in P schottii ants still visited flowers
(Chamberlain and Holland 2008) In cases where ant abundance would continue to increase with
additional rewards it is unlikely that EFNs function as distractions
ii) Chemical resistance traits
a) Toxic nectar
One of the earliest proposed floral defenses against ants was toxic nectar (Janzen 1977)
However studies testing Janzenrsquos (1977) hypothesis uncovered only a very few isolated
examples (eg Catalpa speciosa Stephenson 1981 Crinum erubescens Guerrant and Fiedler
1981) in short most floral nectar is not toxic or repellent to ants (Baker and Baker 1978
Schubart and Anderson 1978) In fact ants sometimes prefer floral nectar over solutions with
equivalent sugar concentrations (Koptur and Truong 1998)
18
In contrast floral nectar can repel other flower visitors (eg birds Johnson et al 2006
lepidopterans Kessler and Baldwin 2007) Repellency is often caused by plant secondary
metabolites which may occur in nectar as a by-product of their production elsewhere in the
plant where they function as defenses against herbivory (Adler 2001) Alternatively toxic or
repellent nectar may be adaptive repellent nectar may promote effective pollination by reducing
the amount of reward any one pollinator takes spreading nectar over several pollinator visits
(Kessler and Baldwin 2007) or it may function as a filter selecting for specific pollinators
(Johnson et al 2006) The latter may explain why repellent nectar is not more common
legitimate pollinators in addition to floral larcenists may be deterred from visiting flowers with
repellent nectar This may be particularly true of nectars that would repel ants which are closely
related to bees nectar distasteful to ants may often also be distasteful to bees and those nectars
acting as a filter for bees may still be palatable to ants For example Prŷs-Jones and Willmer
(1992) found that the ammonia-containing nectar of Lathraea clandestina is readily consumed
by both bees and ants even though it deters birds that act as nectar thieves Although floral
nectar itself rarely repels ants Guerrant and Fielder (1981) found that the presence of macerated
petals within nectar can often keep ants away Alkaloids in petals may be responsible and might
explain why ants seldom act as primary nectar robbers these secondary compounds may prevent
ants from chewing through corollas
b) Floral volatiles
Repellent floral volatiles appear to be a more widespread form of chemical resistance against
ants These were reviewed recently by Willmer et al (2009) and will be discussed here only
briefly Numerous plant taxa produce floral scents repellent to ants including myrmecophilous
19
myrmecophytic and non-ant associated plants in both temperate and tropical ecosystems
(Willmer et al 2009 Junker et al 2011a Ballantyne and Willmer 2012) In three large studies
floral volatiles that repel ants were detected in about a third of all plants tested (Willmer et al
2009 Junker et al 2011a Ballantyne and Willmer 2012) Pollen is often the source of ant-
repellent volatile compounds (Willmer et al 2009 Ballantyne and Willmer 2012) but they can
be emitted from other floral parts including petals That pollen is often the source of repellence
could suggest that preventing ant visitation may be more important in protecting male than
female function Additionally because repellence would decrease as pollen is removed from
anthers ants may return and defend flowers more quickly than if repellent chemicals were in
longer-lasting structures (eg petals) In some cases floral volatiles are thought to mimic ant
alarm pheromones (Willmer et al 2009) However floral scents elicit inconsistent reactions
across ant species ranging from repellence to attraction (Agarwal and Rastogi 2007 Ballantyne
and Willmer 2012)
Producing structural or chemical resistance traits may be costly for plants Native
Hawaiian plants which have little recent coevolutionary history with ants appear to have
secondarily lost floral ant repellents (Junker et al 2011a) Both Willmer et al (2009) and Junker
et al (2011a) found that highly chemical repellent flowers did not have strong physical defenses
and vice versa suggesting trade-offs between the two (but see Ballantyne and Willmer 2012)
Like toxic nectar floral ant repellents might sometimes deter both pollinators and ants For
example Galen et al (2011) found that 2-phenylethanol in P viscosum floral scent repels both
flower-damaging F neorufibarbis ants and the plantrsquos main pollinator Bombus kirbyellus
Interestingly four recent studies have found that floral scents can also attract ants
(Agarwal and Rastogi 2007 Edwards and Yu 2008 Schiestl and Glaser 2011 Gonzaacutelvez et al
20
2012) One study (Schiestl and Glaser 2011) has shown that floral scents attract ants to a
potentially ant-pollinated plant and such systems may provide many more examples
4 Future directions
We have highlighted areas in need of further investigation throughout the review Here we focus
on several questions we consider high priorities for future research
i) Floral defenses against ants
a) Do floral resistance traits increase plant fitness
The existence ant-repellent floral traits is often taken as evidence that ants negatively affect plant
reproductive success However despite the abundance and variety of mechanisms excluding ants
from flowers few studies show that ants reduce plant fitness when they visit flowers (Table 11)
This absence is particularly apparent with repellent floral volatiles which may be the most
widespread floral resistance trait (Willmer et al 2009 Junker et al 2011a Ballantyne and
Willmer 2012) To our knowledge only one study has shown that floral ant repellents benefit
plants with repellent P viscosum flowers avoiding damaging ant visits (Galen et al 2011)
Research is urgently needed to investigate if these traits increase plant fitness by excluding ants
or if ant visitation is inconsequential More studies taking advantage of standing variation in
traits (Galen et al 2011) or creating it artificially (eg Junker et al 2010b) will allow us to
distinguish between the two
21
b) If ant visitation is often not costly what caused the evolution of repellent traits and
what maintains them Similarly are these repellent traits truly defenses against ant
visitation or are they generalized defenses against a range of floral antagonists
Ant-repellent floral traits are widespread and their production may be costly (Willmer et al
2009 Junker et al 2011a) The studies in Table 11 however indicate that flower-visiting ants
often do not disrupt plant reproduction particularly outside of myrmecophytes What then
selected for these traits One possibility is that these repellents are not directed solely at ants
Instead a community of antagonistic flower visitors may exert diffuse selection on plants
thereby selecting for and maintaining these traits Structural modifications may repel crawling
insects while chemical repellents such as floral volatiles may repel both flying and non-flying
insects (Junker et al 2011b) How much overlap there is in floral volatiles that repel both ants and
other antagonists needs investigation Until we examine how specific these defenses are to ants
we risk overstating the importance of ants in their evolution
ii) Do ants alter pollen movement patterns or reduce male fitness
We are aware of only four studies that have measured if ant visitation impacts male fitness
(Wyatt 1980 Fritz and Morse 1981 Ashman 2000 Galen and Geib 2007) While none of these
studies found an effect all measured only amounts of pollen or pollinia removed However this
measure does not adequately encompass male fitness alterations to pollen movement patterns
and eventual deposition location may be more important to male function than changes in
removal rates If as suggested by some studies (Altshuler 1999 Lach 2007) ants increase
pollinator relocation rates they may decrease pollen discounting increasing male fitness
However if this increased relocation reduces overall pollen export rates ant competition with
22
pollinators may depress male function Studies using pollen-tracking methods such as fluorescent
dyes will allow us to see how ants change pollen movement patterns and methods like paternity
analysis will reveal how competition between ants and pollinators alters male fitness
iii) Coevolution between ants and plants do myrmecophytes exhibit stronger more
specific defenses against ants
The floral traits of myrmecophytes are most likely to coevolve with ants because all plant
reproduction occurs in the presence of ants Their floral defenses would thus be expected to be
both stronger and more specific to their resident ants than those other plants visited by an array
of more opportunistic ant species Some evidence supports this Willmer et al (2009) found that
resident ants of acacias were repelled by flowers with more aggressive ant species showing
stronger repellence than less aggressive ants Additionally ants not normally associated with
acacias did not respond to acacia floral volatiles More studies are needed in other systems and
should take advantage of phylogenetic approaches examining floral traits in closely related
myrmecophytic and non-myrmecophytic plant species to determine whether ants commonly
select for floral defenses against ants in myrmecophytes
Acknowledgements
For comments and discussion we thank the Frederickson lab particularly Eric Youngerman
Lina Arcila Hernandez and Kyle Turner as well as Alison Parker and Jordan Pleet MEF
acknowledges financial support from an NSERC Discovery Grant a Connaught New Researcher
23
Award an Ontario Ministry of Economic Development and Innovation Early Researcher Award
and the University of Toronto ARC was supported by an Ontario Graduate Scholarship and
Sigma Xi
24
References
Adler L S 2001 The ecological significance of toxic nectar Oikos 91409ndash420
Agarwal V M and N Rastogi 2007 Role of floral repellents in the regulation of flower visits
of extrafloral nectary-visiting ants in an Indian crop plant Ecological Entomology 3359ndash
65
Altshuler D L 1999 Novel interactions of non-pollinating ants with pollinators and fruit
consumers in a tropical forest Oecologia 119600ndash606
Ashman TL 2000 Pollinator selectivity and its implications for the evolution of dioecy and
sexual dimorphism Ecology 812577-2591
Ashman TL and E A King 2005 Are flower-visiting ants mutualists or antagonists A study
in a gynodioecious wild strawberry American Journal of Botany 92891ndash5
Ballantyne G and P Willmer 2012 Floral visitors and ant scent marks noticed but not used
Ecological Entomology 37402-409
Ballantyne G and P Willmer 2012 Nectar theft and floral ant-repellence a link between
nectar volume and ant-repellent traits PloS One 7e43869
Baker H G and I Baker 1978 Ants and flowers Biotropica 1080
Barrett S C H 2002 Sexual interference of the floral kind Heredity 88154ndash159
Baroni Urbani C and M L de Andrade 1997 Pollen eating storing and spitting by ants
Naturwissenschaften 84256ndash258
25
Beattie A J C Turnbull R B Knox and E G Williams 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany
71421ndash426
Beattie A J C Turnbull T Hough S Jobson and R B Knox 1985 The vulnerability of
pollen and fungal spores to ant secretions evidence and some evolutionary implication
American Journal of Botany 72606ndash614
Blancafort X and C Goacutemez 2005 Consequences of the Argentine ant Linepithema humile
(Mayr) invasion on pollination of Euphorbia characias (L) (Euphorbiaceae) Acta
Oecologica 2849ndash55
Bleil R N Bluumlthgen and R R Junker 2011 Ant-plant mutualism in Hawailsquoi Invasive ants
reduce flower parasitism but also exploit floral nectar of the endemic shrub Vaccinium
reticulatum (Ericaceae) Pacific Science 65291ndash300
Bluumlthgen N G Gottsberger and K Fiedler 2004 Sugar and amino acid composition of ant-
attended nectar and honeydew sources from an Australian rainforest Austral Ecology
29418ndash429
Burkle L A R E Irwin and D A Newman 2007 Predicting the effects of nectar robbing on
plant reproduction implications of pollen limitation and plant mating system American
Journal of Botany 941935ndash1943
Byk J and K Del-Claro 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333ndash38
26
Caballero P C G Ossa W L Gonzaacuteles C Gonzaacutelez-Browne G Astorga M M Muruacutea and
R Medel 2013 Testing non-additive effects of nectar-robbing ants and hummingbird
pollination on the reproductive success of a parasitic plant Plant Ecology 214633ndash640
Carvalheiro L G E R M Barbosa and J Memmott 2008 Pollinator networks alien species
and the conservation of rare plants Trinia glauca as a case study Journal of Applied
Ecology 451419ndash1427
Chamberlain S A and J N Holland 2008 Density-mediated context-dependent consumer-
resource interactions between ants and extrafloral nectar plants Ecology 891364ndash1374
Creighton W S 1967 Studies on free colonies of Cryptocerus texanus Satschi (Hymenoptera
Formicidae) Psyche 7434ndash42
Cursach J and J Rita 2011 Implications of the reproductive biology of the narrow endemic
Naufraga balearica (Apiaceae) for its conservation status Plant Systematics and Evolution
298581ndash596
Cushman JH and Addicott JF In Huxley CR and DF Cutler (eds) 1st edition Ant-plant
interactions Oxford University Press Oxford
de Jong T J N M Waser and P G Klinkhamer 1993 Geitonogamy the neglected side of
selfing Trends in Ecology and Evolution 8321ndash325
de Vega C M Arista P L Oritz C M Herrera and S Talavera 2009 The ant-pollination
system of Cytinus hypocistis (Cytinaceae) a Mediterranean root holoparasite Annals of
Botany 4517ndash8
27
Dukas R 2001 Effects of perceived danger on flower choice by bees Ecology Letters 4327ndash
333
Dutton E M and M E Frederickson 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561ndash569
Edwards D P and D W Yu 2008 Tolerating castration by hiding flowers in plain sight
Behavioral Ecology and Sociobiology 6395ndash102
Frederickson M E 2009 Conflict over reproduction in an ant-plant symbiosis why Allomerus
octoarticulatus ants sterilize Cordia nodosa trees The American Naturalist 173675ndash81
Fritz R S and D H Morse 1981 Nectar parasitism of Asclepias syriaca by ants effect on
nectar levels pollinia insertion pollinaria removal and pod production Oecologia 50316ndash
319
Galen C 1983 The effects of nectar thieving ants on seedset in floral scent morphs of
Polemonium viscosum Oikos 41245ndash249
Galen C 2005 Catching ants with honey an experimental test of distraction and satiation as
alternative modes of escape from flower-damaging ants Oecologia 14480ndash87
Galen C and B Butchart 2003 Ants in your plants effects of nectar-thieves on pollen fertility
and seed-siring capacity in the alpine wildflower Polemonium viscosum Oikos 101521ndash
528
Galen C and J Cuba 2001 Down the tube pollinators predators and the evolution of flower
shape in the alpine skypilot Polemonium viscosum Evolution 551963-1971
28
Galen C and J C Geib 2007 Density-dependent effects of ants on selection for bumble bee
pollination in Polemonium viscosum Ecology 881202ndash1209
Galen C R Kaczorowski S L Todd J Geib and R A Raguso 2011 Dosage-dependent
impacts of a floral volatile compound on pollinators larcenists and the potential for floral
evolution in the alpine skypilot Polemonium viscosum The American Naturalist 177258ndash
272
Garciacutea M B R J Antor and X Espadaler 1995 Ant pollination of the palaeoendemic
dioecious Borderea pyrenaica (Dioscoreaceae) Plant Systematics and Evolution 19817ndash
27
Gaume L M Zacharias and R M Borges 2005 Ant ndash plant conflicts and a novel case of
castration parasitism in a myrmecophyte Evolutionary Ecology Research 7435ndash452
Graystock P and W O H Hughes 2011 Disease resistance in a weaver ant Polyrhachis
dives and the role of antibiotic-producing glands Behavioral Ecology and Sociobiology
652319ndash2327
Gόmez J M 2000 Effectiveness of ants as pollinators of Lobularia maritima effects on main
sequential fitness components of the host plant Oecologia 12290ndash97
Gόmez J M and R Zamora 1992 Pollination by ants consequences of the quantitative effects
on a mutualistic system Oecologia 91410ndash418
Gόmez J M R Zamora J A Hόdar and D Garciacutea 1996 Experimental study of pollination
by ants in Mediterranean high mountain and arid habitats Oecologia 105236ndash242
29
Gonccedilalves-Souza T P M Omena J C Souza and G Q Romero 2008 Trait-mediated effects
on flowers artificial spiders deceive pollinators and decrease plant fitness Ecology
892407ndash2413
Gonzaacutelvez F G L Santamariacutea R T Corlett and M A Rodriacuteguez-Gironeacutes 2012 Flowers
attract weaver ants that deter less effective pollinators Journal of Ecology 10178ndash85
Guerrant E O and P L Fiedler 1981 Flower defenses against nectar-pilferage by ants
Biotropica 1325ndash33
Harley R 1991 The greasy pole syndrome In Huxley CR and DF Cutler (eds) 1st edition
Ant-plant interactions Oxford University Press Oxford
Hansen D M and C B Muumlller 2009 Invasive ants disrupt gecko pollination and seed
dispersal of the endangered plant Roussea simplex in Mauritius Biotropica 41202ndash208
Hickman J C 1974 Pollination by ants a low-energy system Science 1841290ndash1292
Holland J N S a Chamberlain and T E X Miller 2011 Consequences of ants and
extrafloral nectar for a pollinating seed-consuming mutualism ant satiation floral
distraction or plant defense Oikos 120381ndash388
Houmllldobler B and Wilson E O 1990 The Ants 1st Edition Harvard University Press
Cambridge
Holway DA L Lach AV Suarez ND Tsutsui and TJ Case 2002 The causes and
consequences of ant invasions Annual Review of Ecology and Systematics 33181-233
30
Horskins K and Turner VB 1999 Resource use and foraging patterns of honeybees Apis
mellifera and native insects on flowers of Eucalyptus costata Australian Journal of
Ecology 24221-227
Horvitz C C and D W Schemske 1984 Effects of ants and an ant-tended herbivore on seed
production of a neotropical herb Ecology 651369ndash1378
Inouye DW 1980 The terminology of floral larceny Ecology 611251-1253
Inouye DW and O R J Taylor 1979 A temperate region plant-ant-seed predator system
consequences of extra floral nectar secretion by Helianthella quinquenervis Ecology 601ndash
7
Irwin RE 2003 Impact of nectar robbing on estimates of pollen flow conceptual predictions
and empirical outcomes Ecology 84485-495
Irwin RE and Maloof JE 2002 Variation in nectar robbing over time space and species
Oecologia 133525-533
Iwanami Y and T Iwadare 1978 Inhibiting effects of myrmicacin on pollen growth and pollen
tube mitosis Botanical Gazette 13942ndash45
Izzo TJ and Vasconcelos HL 2002 Cheating the cheater domatia loss minimizes the effects
of ant castration in an Amazonian ant plant Oecologia 133200-205
Janzen D H 1977 Why donrsquot ants visit flowers Biotropica 9252
Johnson SD Hargreaves AL and Brown M 2006 Dark bitter-tasting nectar functions as a
filter of flower visitors in a bird-pollinated plant Ecology 872709-2716
31
Junker R A Y C Chung and N Bluumlthgen 2007 Interaction between flowers ants and
pollinators additional evidence for floral repellence against ants Ecological Research
22665ndash670
Junker R R R Bleil C C Daehler and N Bluumlthgen 2010a Intra-floral resource partitioning
between endemic and invasive flower visitors consequences for pollinator effectiveness
Ecological Entomology 35760ndash767
Junker R R and N Bluumlthgen 2010b Floral scents repel facultative flower visitors but attract
obligate ones Annals of botany 105777ndash82
Junker R R C C Daehler S Doetteri A Keller and N Bluumlthgen 2011a Hawaiian ant ndash
flower networks nectar-thieving ants prefer undefended native over introduced plants with
floral defenses Ecological Monographs 81295ndash311
Junker R R J Gershenzon and S B Unsicker 2011b Floral odor bouquet loses its ant
repellent properties after inhibition of terpene biosynthesis Journal of Chemical Ecology
371323ndash31
Kawakita A and M Kato 2002 Floral biology and unique pollination system of root
holoparasites Balanophora kuroiwai and B tobiracola (Balanophraceae) American
Journal of Botany 891164ndash1170
Koopowitz H and TA Marchant 1998 Postpollination nectar reabsorption in the African
epiphyte Aerangis verdickii (Orchidaceae) American Journal of Botany 85508-512
Kirk W D J 1984 Ecological studies on Thrips imaginis Bagnall (Thysanoptera) in flowers of
Echium plantagineum L in Australia Australian Journal of Ecology 99ndash18
32
Kerner A 1878 Flowers and their unbidden guests C Kegan Paul London
Kessler D and I T Baldwin 2007 Making sense of nectar scents the effects of nectar
secondary metabolites on floral visitors of Nicotiana attenuata The Plant Journal 49840ndash
54
Koptur S and N Truong 1998 Facultative ant-plant interactions nectar sugar preferences of
introduced pest species in South Florida Biotropica 30179-189
Lach L 2005 Interference and exploitation competition of three nectar-thieving invasive ant
species Insectes Sociaux 52257ndash262
Lach L 2007 A mutualism with a native membracid facilitates pollinator displacement by
Argentine ants Ecology 881994ndash2004
Lach L 2008a Argentine ants displace floral arthropods in a biodiversity hotspot Diversity and
Distributions 14281ndash290
Lach L 2008b Floral visitation patterns of two invasive ant species and their effects on other
hymenopteran visitors Ecological Entomology 33155ndash160
Lach L 2013 A comparison of floral resource exploitation by native and invasive Argentine
ants Arthropod-Plant Interactions 7177-190
Luo C W K Li X M Chen Z Y Huang and K U N Li 2012 Ants contribute significantly
to the pollination of a biodiesel plant Jatropha curcas Environmental Entomology
411163ndash1168
33
Maleacute PJ G C Leroy A Dejean A Quilichini and J Orivel 2011 An ant symbiont directly
and indirectly limits its host plantrsquos reproductive success Evolutionary Ecology 2655ndash63
Maloof J E 2001 The effects of a bumble bee nectar robber on plant reproductive success and
pollinator behavior American Journal of Botany 881960ndash1965
Maloof J E and D W Inouye 2000 Are nectar robbers cheaters or mutualists Ecology
812651ndash2661
Moreau CS CD Bell R Vila SB Archibald and NE Pierce 2006 Phylogeny of the ants
diversification in the age of the angiosperms Science 312101-104
Nahas L M O Gonzaga and K Del-Claro 2012 Emergent impacts of ant and spider
interactions herbivory reduction in a tropical savanna tree Biotropica 44498ndash505
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506ndash514
Newman D A and J D Thomson 2005 Interactions among nectar robbing floral herbivory
and ant protection in Linaria vulgaris Oikos 110497ndash506
Nicklen E F and D Wagner 2006 Conflict resolution in an ant-plant interaction Acacia
constricta traits reduce ant costs to reproduction Oecologia 14881ndash7
Norment C J 1988 The effect of nectar-thieving ants on the reproductive success of Frasera
speciosa (Gentianaceae) American Midland Naturalist 120331ndash336
34
Orivel J L Lambs P-J G Maleacute C Leroy J Grangier T Otto A Quilichini et al 2011
Dynamics of the association between a long-lived understory myrmecophyte and its
specific associated ants Oecologia 165369ndash76
Palmer T M D F Doak M L Stanton J L Bronstein E T Kiers T P Young J R
Goheen et al 2010 Synergy of multiple partners including freeloaders increases host
fitness in a multispecies mutualism Proceedings of the National Academy of Sciences
10717234ndash9
Peakall R A J Beattie and S H James 1987 Pseudocopulation of an orchid by male ants a
test of two hypotheses accounting for the rarity of ant pollination Oecologia 73522ndash524
Peakall R and A J Beattie 1989 Pollination of the orchid Microtis parviflora R Br by
flightless worker ants Functional Ecology 3515ndash522
Peakall R C J Angus A J Beattie 1990 The significance of ant and plant traits for ant
pollination in Leporella fimbriata pollination Oecologia 84457ndash460
Puterbaugh M N 1998 The roles of ants as flower visitors experimental analysis in three
alpine plant species Oikos 8336ndash46
Prŷs-Jones OE and Willmer PG 1992 The biology of alkaline nectar in purple toothwort
(Lathraea clandenstina) ground level defenses Biological Journal of the Linnean Society
45373-388
Raine NE Willmer P and Stone GN 2002 Spatial structuring and floral avoidance
behaviour present in ant-pollinator conflict in a Mexican ant-acacia Ecology 833086-
3096
35
Ramsey M 1995 Ant pollination of the perennial herb Blandfordia grandiflora (Liliaceae)
Oikos 74265ndash272
Renault CK LM Buffa and Delfino MA 2005 An aphid-ant interaction effects on
different trophic levels Ecological Research 2071-74
Rico-Gray V and L B Thien 1989 Effect of different ant species on reproductive fitness of
Schomburgkia tibicinis (Orchidaceae) Oecologia 81487ndash489
Romero G Q P A P Antiqueira and J Koricheva 2011 A meta-analysis of predation risk
effects on pollinator behaviour PLoS One 6e20689
Rosumek FB FAO Silveira F de S Neves NP de U Barbosa L Diniz Y Oki F Pezzini
et al 2009 Ants on plants a meta-analysis of the role of ants as plant biotic defenses
Oecologia 160537ndash549
Roubik D W 1982 The ecological impact of nectar-robbing bees and pollinating
hummingbirds on a tropical shrub Ecology 63354ndash360
Rudgers J A 2004 Enemies of herbivores can shape plant traits selection in a facultative ant-
plant mutualism Ecology 85192ndash205
Schatz B and WT Wcislo 1999 Ambush predation by the ponerine ant Ectatomma ruidum
Roger (Formicidae) on a sweat bee Lasioglossum umbripenne (Halictidae) in Panama
Journal of Insect Behavior 12641-663
36
Schaffer W M D W Zeh S L Buchmann S Kleinhaus M V Schaffer and J Antrim
1983 Competition for nectar between introduced honey bees and native North American
bees and ants Ecology 64564ndash577
Schemske D W 1980 The evolutionary significance of extrafloral nectar production by Costus
woodsonii (Zingiberaceae) an experimental analysis of ant protection Journal of Ecology
68959ndash967
Schiestl F P and F Glaser 2011 Specific ant-pollination in an alpine orchid and the role of
floral scent in attracting pollinating ants Alpine Botany 1221ndash9
Schubart HOR AB Anderson 1978 Why donrsquot ants visit flowers A response to D H
Janzen Biotropica 10310-311
Schuumlrch S M Pfunder and B A Roy 2000 Effects of ants on the reproductive success of
Euphorbia cyparissias and associated pathogenic rust fungi Oikos 16ndash12
Stanton ML Palmer TM Young TP Evans A and Turner ML 1999 Sterilization and
canopy modification of swollen thorn acacia tree by a plant-ant Nature 401 578-581
Stephenson AG 1981 Toxic nectar deters nectar thieves of Catalpa speciosa American
Midland Naturalist 105381-383
Stout J C and D Goulson 2001 The use of conspecific and interspecific scent marks by
foraging bumblebees and honeybees Animal Behaviour 62183ndash189
Sugiura S T Abe and S Makino 2006 Loss of extrafloral nectary on an oceanic island plant
and its consequences for herbivory American Journal of Botany 93491ndash495
37
Svensson L 1985 An estimate of pollen carryover by ants in a natural population of
Scleranthus perennis L (Caryophyllaceae) Oecologia 66373ndash377
Tsuji K A Hasyim and K Nakamura 2004 Asian weaver ants Oecophylla smaragdina and
their repelling of pollinators Ecological Research 19669ndash673
Turner R C J J Midgley P Barnard R E Simmons and S D Johnson 2012 Experimental
evidence for bird pollination and corolla damage by ants in the short-tubed flowers of
Erica halicacaba (Ericaceae) South African Journal of Botany 7925ndash31
Visser D M G Wright and J H Giliomee 1996 The effect of the Argentine ant
Linepithema humile (Mayr) (Hymenoptera Formicidae) on flower-visiting insects of
Protea nitida Mill (Proteaceae) African Entomology 4285ndash287
Wagner D 2000 Pollen viability reduction as a potential cost of ant association for Acacia
constricta (Fabaceae) American Journal of Botany 87711ndash715
Wagner D and A Kay 2002 Do extrafloral nectaries distract ants from visiting flowers An
experimental test of an overlooked hypothesis Evolutionary Ecology Research 4293ndash305
Wang C Y Luo Y Tai D An and Y Kou 2008 Ants pollinate Neottia listeroides
(Orchidaceae) in Sichuan China 46836ndash846
Wyatt R 1980 The impact of nectar-robbing ants on the pollination system of Asclepias
curassavica Bulletin of the Torrey Botanical Club 10724ndash28
Wheeler WM and IW Bailey 1920 The feeding habits of Pseudomymine and other ants
Transactions of the American Philosophical Society 22235-279
38
Willmer P G and S A Corbet 1981 Temporal and microclimatic partitioning of the floral
resources of Justicia aurea amongst a concourse of pollen vectors and nectar robbers
Oecologia 5167ndash78
Willmer P G and G N Stone 1997 How aggressive ant-guards assist seed-set in Acacia
flowers Nature 388165ndash167
Willmer P G C V Nuttman N E Raine G N Stone J G Pattrick K Henson P Stillman
et al 2009 Floral volatiles controlling ant behaviour Functional Ecology 23888ndash900
Young T P C H Stubblefield and L a Isbell 1997 Ants on swollen-thorn acacias species
coexistence in a simple system Oecologia 10998ndash107
Yu D W and N E Pierce 1998 A castration parasite of an ant-plant mutualism Proceedings
of the Royal Society B Biological Sciences 265375ndash382
39
Tables
Table 11 Studies of ant-flower or ant-pollinator interactions that included a measure of plant fitness Each study system is listed only
once except when results varied between years or when different studies examined male and female fitness Significant effects of ants on
plant fitness are either lsquopositiversquo or lsquonegativersquo non-significant effects are lsquoneutralrsquo When ants significantly affected only one fitness
component but several were measured (eg fruit set and seed production) we still consider the result significant
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Acacia
drepanolobium
Crematogaster
nigriceps
Proportion
fruiting
Negative Destruction of floral buds Young et al 1997
Asclepias exaltata Several Pollinia inserted
removed
Negative for pollinia
insertion neutral for
pollinia removal
Exploitative competition Wyatt 1980 T
40
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Asclepias syriaca Several
Fruit set pollinia
removed
Negative for fruit
set neutral for
pollinia removal
Exploitative competition Fritz and Morse
1981 T
Cordia nodosa Allomerus cf
demerarae
Fruit set Negative Destruction of floral buds Yu and Pierce
1998
Costus woodsonii Wasmannia
auropunctata
Seed set Positive Unknown ant pollination
suggested (but unlikely)
Schemske 1980
Eritrichum aretioides F neorufibarbis
gelida
Seed set Negative Ants damaged flowers Puterbaugh 1998
41
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Euphorbia characias Several Seed set Neutral Plants in Argentine ant-
invaded area set less seed
than those in un-invaded
sites but an ant-exclusion
experiment did not produce
significant results
Blancafort and
Gόmez 2005
Ferocactus wislizeni Solenopsis xyloni
and others
Seed set Unknown negative Interference and exploitative
competition S xyloni
reduced seed set relative to
other ants but the study did
not include a no-ant control
Ness 2006
42
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Frasera speciosa F neorufibarbis
and Formica sp
Seed set Neutral Nocturnal pollination
occurring when ants were
less active may have
compensated for reduced
diurnal pollinator visitation
Norment 1988
Gypsophila struthium Several Seeds per
inflorescence
Neutral No effect despite large
numbers of ants on flowers
Gόmez et al
1996
Hirtella physophora Allomerus
decemarticulatus
Fruit set Negative Destruction of flowers Orivel et al
2011
Humboldtia brunonis Crematogaster
dohrni
Fruit set Negative Destruction of flowers Gaume et al
2005
43
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Lepidium subulatum Several Percentage of
ovules setting
seed
Positive Unknown Gόmez et al
1996
Leucospermum
conocarpodendron
Several Seed set Neutral Site not pollen limited Lach 2008
Linaria vulgaris Several ldquoFemale fitness
indexrdquo
(combination of
number of seeds
fruits flowers
and seed weight)
Positive Reduction in florivory and
seed predation nectar
robbing holes increased ant
visitation
Newman and
Thomson 2005
44
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Melastoma
malabathricum
Oecophylla
smaragdina
Seed set Positive Ants deterred less efficient
pollinators increasing
visitation by the more
efficient pollinator
Gonzaacutelvez et al
2012
Metrosideros
polymorpha
Several Fruit set Neutral Ants reduced nectar
collection rates by foraging
bees but had no effect on
pollen collection rates or
fruit set
Junker et al
2010a
Orexis alpine F neorufibarbis
gelida
Seed set Neutral Autogamy or not pollen
limited
Puterbaugh 1988
45
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Ouratea spectabilis Cephalotes
pusillus and others
Seeds per fruit Neutral positive C pusillus had no effect on
seed number but other ants
collectively increased seed
set
Byk and Del
Claro 2010
Pachycereus schottii Several Seed set Neutral positive In one year ants showed a
positive effect (seed
number 2008) but no effect
with increased sample sizes
Chamberlain and
Holland 2008
Polemonium
viscosum
F neorufibarbis
gelida
Seed set Negative Ants damaged styles Galen 1983
46
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Polemonium
viscosum
F neorufibarbis
gelida
Pollen removal Neutral Ants did not affect pollen
removal even at high ant
densities
Galen and Geib
2007 T
Protea nitida Linepithema
humile and others
Seed set Neutral positive L humile had no effect on
P nitida seed set but native
ants increased it in one year
ants may cause pollinators
to relocate
Lach 2007
Psychotria
limonensis
Ectatomma spp Fruit set Positive Suggested increased
pollinator relocation
Altshuler 1999
47
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Roussea simplex Technomyrmex
albipes
Seed set Negative Interference and exploitative
competition
Hansen and
Muumlller 2009
Tristerix aphyllus Several Fruit set Neutral Nectar robbing ants
minimally reduced nectar
levels and had no effect on
hummingbird visitation
Caballero et al
2013
Vaccinium
reticulatum
Several Fruit set Neutral Ants reduced florivory but
did not affect fruit set
Bleil et al 2011
T Study measured male fitness
Study of sterilizing ants showed short-term decrease in female fitness long-term effects are unclear (see text)
48
Table 12 Studies demonstrating either i) ant pollination through an exclusion experiment or ii) that ants transfer pollen (or pollen
analogue) between flowers strongly suggesting ant pollination
Plant species Ant species Ant pollination Evidence Reference
Alyssum purpureum Several Yes Exclusion experiment Gόmez et al 1996
Arenaria tetraquetra Several Yes Exclusion experiment Gόmez et al 1996
Balanophora kuroiwai Leptothorax sp Suggested Exclusion experiment and
observed pollen transfer
and pollen tube formation
but did not see if plants set
seed
Kawakita and Kato 2002
Blandfordia grandiflora Iridomyrmex sp Yes Exclusion experiment Ramsey 1995
Borderea pyrenaica Several Yes Exclusion experiment Garciacutea et al 1995
49
Plant species Ant species Ant pollination Evidence Reference
Cytinus hypocistis Several Yes Exclusion experiment de Vega et al 2009
Epipactis thunbergii Camponotus
japonicas
Suggested Observed transfer of
pollinia from ants to
orchids
Sugiura et al 2006
Euphorbia cyparissias Several Yes Exclusion experiment Schuumlrch et al 2000
Fragaria virginiana Several Very likely Ant-only treatments set
comparable seed to flying
pollinator-only treatment
seed set increased with
increasing ant visitation
Ashman and King 2005
Frasera speciosa Several Yes Exclusion experiment Norment 1988
Frankenia thymifolia Several Yes Exclusion experiment Gόmez et al 1996
50
Plant species Ant species Ant pollination Evidence Reference
Hormathophylla spinosa Proformica
longiseta
Yes Exclusion experiment Gόmez and Zamora 1992
Jatropha curcas
Several
Tapinoma
melanocephalum
most common
Yes Exclusion experiment Luo et al 2012
Leporella fimbriata Myrmecia urens Yes Observed pollinia transfer
and subsequently
confirmed as ant
pollination
Peakall et al 1987
51
Plant species Ant species Ant pollination Evidence Reference
Lobularia maritima Several
Camponotus
micans most
common
Yes Exclusion experiment Gόmez 2000
Microtis parviflora
Iridomyrmex
gracilis
Yes Observation used
laboratory colonies to
replicate observations
Peakall and Beattie 1989
Naufraga balearica
Several
especially
Plagiolepis
pygmaea and
Lasius grandis
Suggested Observation over three
years of study only ants
were observed visiting
ants carried pollen
Cursach and Rita 2011
52
Plant species Ant species Ant pollination Evidence Reference
Neottia listeroides Leptothoras sp
Paratrechina sp
Suggested Observed transfer of
pollinia from ants to
orchids
Wang et al 2008
Paronychia pulvinata Formica
neorufibarbis
gelida
Yes Exclusion experiment Puterbaugh 1998
Polygonum cascadense Formica argentea
Very likely Grew plants in greenhouse
open to flying visitors with
minimal seed set only
plants with access to ants
set significant seed
Hickman 1974
53
Plant species Ant species Ant pollination Evidence Reference
Retama sphaerocarpa Several Yes Exclusion experiment Gόmez et al 1996
Scleranthus perennis
Several
especially
Formica
rufibarbis
Suggested Ants transferred pollen
analogue to stigmas and
carried pollen on bodies
Svensson 1985
Sedum anglicum Proformica
longiseta
Yes Exclusion experiment Gόmez et al 1996
Trinia glauca Several Yes Exclusion experiment Carvalheiro et al 2008
54
Chapter 2 Ants and ant scent reduce bumblebee pollination of artificial flowers
Cembrowski AR Tan MG Thomson JD and Frederickson ME
In press The American Naturalist
All authors designed the experiment ARC and MGT performed the experiment ARC and MEF
performed statistical analyse
s and ARC wrote the manuscript with input from all authors
Abstract
Ants on flowers can disrupt pollination by consuming rewards or harassing pollinators but it is
difficult to disentangle the effects of these exploitative and interference forms of competition on
pollinator behavior Using highly rewarding and quickly replenishing artificial flowers that
simulate male or female function we allowed bumblebees (Bombus impatiens) to forage (1) on
flowers with or without ants (Myrmica rubra) and (2) on flowers with or without ant scent cues
Bumblebees transferred significantly more pollen analogue both to and from ant free flowers
demonstrating that interference competition with ants is sufficient to modify pollinator foraging
behavior Bees also removed significantly less pollen analogue from ant scented flowers than
from controls making this the first study to show that bees can use ant scent to avoid harassment
at flowers Ant effects on pollinator behavior possibly in addition to their effects on pollen
viability may contribute to the evolution of floral traits minimizing ant visitation
55
Introduction
Trait-mediated indirect interactions arise when a focal species causes phenotypic changes
including behavioral modifications in a second species and these effects cascade to still other
species (Werner and Peacor 2003) Although trait-mediated indirect interactions are often studied
in food webs (eg Werner and Peacor 2003 Preisser et al 2005) they are not limited to trophic
interactions For example the threat of predation can change the behavior of a mutualist thus
affecting its partners (Suttle 2003) In animal-pollinated plants predators can disrupt pollination
directly through density-mediated indirect interactions (Dukas 2005) or via trait-mediated
indirect interactions by changing pollinator behavior When predators are present pollinators
may switch to visiting less rewarding flowers or they may avoid flowers altogether potentially
reducing the amount of pollen donated or received by a flower and thus decreasing plant fitness
(Gonccedilalves-Souza 2008 Ings and Chittka 2009) Similarly competition for floral rewards with
other flower visitors may cause behavioral changes in pollinators (Maloof and Inouye 2000
Ohashi et al 2008) Previous research has shown that bees may spend less time visiting flowers
depleted of rewards (Thomson 1986) and may avoid flowers bearing scent marks that indicate
recent visits by other individuals of the same or different bee species as these flowers are likely
to be depleted (Stout and Goulson 2002)
Whereas competition between pollinators may not be particularly costly for the plant
because both competitors are likely to provide pollination services competition between
pollinators and organisms that do not usually provide a pollination service such as ants has a
greater potential to reduce plant fitness Ants are common floral visitors they are attracted to
flowers for nectar (Lach 2007) and in some cases pollen (Byk and Del Claro 2010) and will
often defend these resources against other flower visitors (Altshuler 1999) However ants rarely
56
contribute much useful pollination antibiotic secretions present on their cuticles kill pollen
grains (Beattie et al 1985 Dutton and Frederickson 2012) and when foraging in flowers ants
sometimes impede female function by damaging stigmas (Galen and Cuba 2001) We will
henceforth consider ants to be non-pollinators and will contrast them to animals like bees that
are pollinators Ants are capable of competing with pollinators in two ways (1) by consuming
floral rewards and reducing their availability to pollinators (ie exploitative competition) and (2)
by directly antagonizing and excluding pollinators from flowers (ie interference competition)
Many plants benefit from the presence of ants numerous plant species recruit ants using
extrafloral nectar food bodies or domatia because ants provide protection against folivores (Heil
and McKey 2003) Similarly some plants have extrafloral nectaries on or near reproductive
tissues that recruit ants that deter florivores (eg Inouye and Taylor 1979) However plants
may incur reproductive costs as a result of their association with ants Some ant species that nest
in myrmecophytes sterilize flowers to shunt plant resources from reproduction to vegetative
growth allowing for greater ant colony growth (Frederickson 2009) Also ants visiting
extrafloral nectaries may be attracted to floral nectaries where they can harass pollinators (Ness
2006) There is growing evidence that many angiosperms have evolved floral traits that prevent
ants from accessing flowers during anthesis (Willmer and Stone 1997 Ballantyne and Willmer
2012) The effects of flower-visiting ants can be substantial Ants can decrease the frequency
duration or species diversity of pollinator visits to flowers (Lach 2008 Hansen and Muumlller 2009
Junker et al 2010 Gonzaacutelvez et al 2012) all of which potentially impact pollen donation and
receipt In some studies changes to pollinator behavior induced by ants have also been linked to
seed set
It is difficult however to disentangle the intertwined effects of exploitative and
interference competition in nature If ants disrupt pollination plants may often evolve traits to
57
deter ants from flowers (Willmer et al 2009) From a practical standpoint this would then limit
our ability to study the effects of ants on pollinator visitation because ants would not visit
flowers Here we used artificial flowers to explore ant-bee interactions in the absence of floral
defenses against ants We used highly rewarding quickly replenishing artificial flowers to
examine how direct harassment (ie interference competition expressed through behavior)
changes bee foraging behavior and how this affects donation and receipt of a pollen analogue
(powdered food dye) while minimizing the effects of exploitative competition We predicted
that flowers visited by ants would both donate and receive less pollen analogue than flowers
without ants because bees would avoid flowers with ants or leave them sooner Because scent
plays a large role in bumblebee communication and flower choice (Stout and Goulson 2002) we
also tested whether the presence or absence of ant scent on artificial flowers would affect pollen
analogue donation because of changes in the foraging behavior of bumblebees
Methods
i) Subjects
Myrmica rubra is an invasive ant in eastern North America with a range and habitat preferences
overlapping that of Bombus impatiens a common bumblebee Because M rubra visits flowers
(A Cembrowski pers obs) the two species likely interact in nature We collected 12 M rubra
colonies in the fall of 2011 and 2012 from Toronto Ontario Canada and the surrounding area
and maintained them in environmental chambers on artificial diet (Dussutour and Simpson 2009)
and a 1410 LD schedule (light 600-2200) We used these colonies as sources of M rubra
workers for experiments
58
Workers from commercially supplied colonies of Bombus impatiens (Biobest Canada
Ltd) foraged on artificial flowers in flight cages (either 24 x 24 x 21 m or 79 x 34 x 20m) at
the University of Toronto We tested a total of five bee colonies four colonies were used in the
ant presence trials and two of these and one additional colony were used in the ant scent trials
Flight cages had overhead fluorescent lights attached to timers In contrast to most previous
studies in which bees have been trained and tested individually the entire worker force of a bee
colony was free to forage at will in our experiments Colonies were trained to forage on artificial
flowers for at least four days before being used in trials After being used for a trial the colony
was not used for at least two days to reduce dye carryover between trials Between trials
bumblebees were fed pollen and given sugar water
ii) Artificial flowers
The flowers (Fig 1 Thomson et al 2012 see also Makino 2008) consisted of glass jars filled
with 30 WV sucrose solution Sugar water travelled by capillary action up a sewing-thread
wick to a hole in a blue-painted lid accumulating in a knot that acts as a nectary Flowers
depleted by visitors were quickly replenished via capillary action and were non-rewarding only
very briefly after visits taking less than a minute to accumulate 05 microl note that bumblebee
visits to a single flower are often separated by several minutes or more (A Cembrowski
unpublished data)
Unlike most previous artificial flower designs these flowers allow for estimation of male
and female fitness because we can measure both the amount and type of dye received by female
flowers (see (d) Dye quantification below) In order to access nectar on a ldquomalerdquo flower a bee
must crawl through a circular ldquoantherrdquo of brush-like weather stripping material dusted in a
59
consistent manner with our pollen analogue powdered food dye (FDampC 5 or 6) which is
transferred to the bee in the process On a ldquofemalerdquo flower a bee crawls through a sticky plastic
reinforcement that functions as a ldquostigmardquo receiving dye from the beersquos body Male flowers
dispensed dye particles over multiple bee visits and could still dispense dye at the end of trials
They resemble many real flowers or flower heads in that pollen is dispensed gradually over time
but less dye is available for transfer with each subsequent bee visit (Harder and Thomson 1989)
iii) Flight cage trials
We conducted 6 ant scent trials between 22 March and 4 June 2012 and the remaining trials (16
ant presence trials and 9 ant scent trials) between 9 November 2012 and 6 January 2013 Ant
presence trials lasted for eight hours and ant scent trials lasted for four hours Artificial flowers
were prepared and placed individually in small plastic containers treated with Fluon (Insect-a-
Slip BioQuip Products Inc) to prevent ants from escaping In ant presence trials we used 32
flowers (16 male and 16 female flowers) arranged in an eight-by-four array with flowers spaced
45 cm apart between rows and 30 cm within rows Ant scent trials used 20 flowers (10 male and
10 female flowers) in a four-by-five array with flowers spaced 30 cm apart between and within
rows In all of the ant presence trials and 9 of the ant scent trials we counted the number of bees
actively foraging after one hour to get a measure of colony activity
a) Ant presence trials
We examined the effects of ants on the amount of dye donated by male flowers and received by
female flowers in sixteen trials testing four bee colonies four times each The containers of eight
randomly chosen male flowers and eight randomly chosen female flowers received 15 M rubra
60
workers each the other 16 flowers remained free of ants Isolated from their colonies M rubra
workers have nowhere to deposit sugar water they collect and therefore may become sated
nonetheless a force of 15 ant workers was enough to maintain visitation to the nectaries where
bees foraged while still rarely having more than one or two ants visit the nectary at any time As
ants attacked bees bees would occasionally carry or throw attacking ants off flowers but despite
this some ants maintained their presence at the nectary throughout the trials Because ants the
size of M rubra consume liquids slowly (~017 ndash 024 microlmin Davidson et al 2004) flowers
replenished nectar more quickly than ants consumed it We used two colors of dye (FDampC 5 and
FDampC 6) to differentiate male flowers with and without ants In two trials for each bee colony
anthers of male flowers with ants were coated with FDampC 5 dye while brushes of male flowers
lacking ants were coated with FDampC 6 dye In the other two trials we reversed dye colors to
control for effects of dye type and color Dye color was assigned in a random order Artificial
flowers were placed in randomly assigned positions in the array before we opened the colony
and allowed the bees to begin foraging
b) Scent trials
We explored the effect of ant scent on dye donation in fifteen trials testing three bee colonies in
six five and four trials each Bees were first exposed to ants by allowing the bees to forage for
eight hours on twenty flowers (ten male and ten female) of which five male flowers had 15 ants
and all others had none No dye was used during this ldquoexposurerdquo day We then collected all but
the five ant-free male flowers in the flight cage leaving these flowers to keep bees foraging
Next we individually stored five new male lids in Fluon-treated containers with ten M rubra
workers Five control male lids were put in identical containers lacking ants The following
61
morning we removed the remaining flowers and set out ten new male and female flowers in a
random spatial arrangement using the lids having or lacking ant scent Thus the ant-scented
flowers were not in the same positions as the ant-visited flowers on which the bees were trained
Only five male flowers had ant scent and the other 15 flowers (five male and ten female) did not
We used the same two dye colors as in i) to differentiate male flowers with and without ant
scent and randomized which color was used for ant-scented flowers between trials We collected
and replaced stigmas from female flowers after one hour and collected the stigmas again after
four hours In the first trial performed we also collected stigmas after two hours but due to the
small amount of dye transferred this was not repeated
iv) Dye quantification
After each trial we quantified the amount of dye transferred to female flowers using a
spectrophotometer We removed the stigmas from female flowers placed them in test tubes
added 51 mL of distilled water to each tube and vortexed each tube thoroughly to ensure the
dye was evenly diluted These were diluted further as needed if the absorbance exceeded the
sensitivity range of the spectrophotometer In the first six scent trials each stigma was analyzed
separately and the dye amounts were summed to obtain a total amount of each dye color
transferred to female flowers in each trial In all the ant presence trials and the other nine scent
trials we opted to treat the experiment as the unit of replication Therefore we combined stigmas
from each treatment type (ant-visited or ant-free) in a test tube and measured the total amount of
each dye color transferred in each treatment in each trial
Because we put different dye colors on male flowers with and without ants we could use
the amount of each dye color donated to all female flowers to measure the reproductive success
62
of ant-visited and ant-free male flowers The total amount of dye (of both colors) received by
female flowers with and without ants was our measure of female reproductive success We
calculated the amount of each dye color in the sample by measuring absorbance at 428 or 486
nm and converting absorbance to micrograms following computational methods for overlapping
spectra (Blanco et al 1989)
v) Statistical analyses
In two trials (one ant presence and one ant scent) most female flowers received no dye because
of low bee activity so we excluded these trials from analyses Because dye reception and
donation values were non-normally distributed we square-root transformed the data before
examining the effects of ant presence or ant scent on dye transfer in ANCOVAs For ant
presence trials we included ant presence on male and female flowers and their interaction as
main effects and the total amount of dye transferred in each trial as a covariate to account for
the large variation in overall dye transfer among trials For ant scent trials we included ant scent
as the main effect time (one or four hours) as a repeated measure the interaction between scent
and time and the total amount of dye transferred as a covariate Covariate by treatment (ant
presence or ant scent) interactions were never significant and so were excluded in final analyses
All analyses were conducted in JMP (v 1000)
Results
Bees usually started foraging within minutes of the beginning of the trial and continued until
flowers were collected from the flight cage or the lights were extinguished An average of 34 plusmn
012 and 40 plusmn 015 (mean plusmn SE) bees were foraging after an hour in the ant presence and ant
63
scent trials respectively Male flowers with ants donated significantly less dye than male flowers
lacking ants (Fig 22a F155 = 4019 p lt 00001) Similarly female flowers with ants received
significantly less dye than female flowers lacking ants (Fig 22a F155 = 461 p = 0036) There
was no significant interaction between ant presence on male flowers and ant presence on female
flowers (F155 = 118 p = 028) Flowers with ant scent donated significantly less dye than
flowers without ant scent (Fig 22b F125 = 11216 p lt 00001) There was no significant effect
of time (F125 = 121 p = 028) or interaction between time and scent (F125 = 111 p = 030) in
the model
Discussion
In this study ants altered bumblebee pollination behavior and exhibited trait-mediated indirect
interactions with (artificial) flowers Through interference competition M rubra workers
significantly affected the pattern of dye transfer by B impatiens causing a preferential flow of
dye from male flowers lacking ants to female flowers lacking ants If manifested in nature such
effects would reduce the reproductive success of plants visited by ants through both male and
female function
Ants could have changed the attractiveness of artificial flowers in at least two ways First
as ants interrupted or altogether prevented bumblebees from foraging at nectaries bumblebees
may have learned to avoid flowers with ants due to the relative inefficiency of foraging at these
flowers something bumblebees take into account (Heinrich 2004) Second bees attempt to
minimize their risk of being attacked during foraging bouts which ants did to visiting bees Ants
often attacked or harassed bees by biting grasping and appearing to sting visiting bees
preventing them from accessing the nectary or reducing their time on flowers (see online video
64
1) and bees sometimes avoided ant-tended flowers entirely This harassment was sometimes
physically traumatic (online video 1) and bees often appeared to have trouble flying after being
attacked by ants Previous research has demonstrated that bumblebees leave or avoid flowers
where they have been harassed (Jones and Dornhaus 2011) and avoid foraging where there is
visual or olfactory evidence of a predator or predation event (Abbott 2006 Goodale and Nieh
2012) Though it is unclear if bumblebees viewed ants as competitors or predators they
responded similarly to flowers having ants as they do to flowers housing predators (Gonccedilalves-
Souza et al 2008)
To avoid artificial flowers with ants bees likely used a combination of visual and
olfactory cues Previous research has shown that bees respond to conspicuous predators or
predator ldquodummiesrdquo on flowers (Suttle 2003 Gonccedilalves-Souza et al 2008) decreasing the
frequency and duration of their visits to these flowers Thus bees may have been able to avoid
flowers harboring ants by sight alone However even in the absence of ants bees still
preferentially visited flowers lacking ant scents (Fig 22b) suggesting that they had learned to
associate ant scent with harassment Bees are adept at associative learning (Wright and Schiestl
2009) and can learn to recognize unique scents left behind by both conspecific and heterospecific
flower visitors (Stout et al 1998) Bees can use these various scents often arising from tarsal
gland deposits (Stout et al 1998) to recognize recently visited flowers that are less likely to be
profitable (Stout and Goulson 2002) Recently Ballantyne and Willmer (2012) demonstrated that
bees learn to associate ant scents with unrewarding artificial flowers and decrease their visitation
to these flowers Our results complement their findings by showing that bees can associate ant
scent with harassment at otherwise rewarding flowers
In our study ant scent caused bees to adjust their foraging strategy and decreased the
amount of pollen analogue that was donated by ant-scented flowers Thus the effects of
65
interference competition with ants on flowers can extend beyond immediate interactions and may
have fitness consequences for plants even when ants are absent Like other olfactory cues these
effects are likely transitory (Stout et al 1998) Although more dye was still donated by male
flowers lacking ant scent than those with ant scent the ratio of dye donated was on average
closer to equality in hours 2-4 than in the first hour (Fig 22b) This effect may be partially
driven by dye depletion of male flowers without ant scent but the lack of a strong corresponding
decrease in flowers with ant scent suggests that visitation patterns became more similar We did
not test whether B impatiensrsquo avoidance of ant scent was an innate or a learned behavior but
previous work has shown that Bombus terrestris does not innately avoid flowers with ant scent
(Ballantyne and Willmer 2012)
Ants may be necessary for plant survival and growth but can be costly for plant
reproduction The evolution of ant attractants such as extrafloral nectaries in some plant lineages
suggests that the costs of ants can be outweighed by their protective abilities However in this
study flowers visited by ants received and donated significantly less dye although they did
retain some sexual function The net benefit of having ants depends on whether ants increase
plant fitness by reducing herbivory more than they decrease plant fitness by disrupting
pollination Alternatively plants may actually benefit from the costs of ants to plant
reproduction ants that castrate flowers may be better defenders increasing plant survival or
vegetative growth when plants are young allowing for increased reproduction later in life when
the plant is colonized by less aggressive non-castrating ants (Frederickson 2009 Palmer et al
2010) In some plant species pollinator harassment by ants may even be beneficial Altshuler
(1999) reported that Ectatomma ants greatly increased fruit set of Psychotria limonensis despite
reductions in pollinator visitation rates presumably due to increases in the rate of pollen
outcrossing Similarly Gonzaacutelvez et al (2012) found that the presence of Oecophylla
66
smaragdina ants on Melastoma malabathricum flowers reduced visitation by less effective
pollinators and increased visitation by more effective Xylocopa bees increasing plant fitness
Cases of ants being beneficial to flowers appear to be the minority and many plant
species have traits that limit ant access to flowers Several studies have detailed floral volatiles
that are thought to mimic ant alarm pheromones and thus repel ants (Junker and Bluumlthgen 2008
Willmer et al 2009) Other plants use structural modifications such as narrow corollas or
slippery stems to limit ant access to flowers (reviewed in Willmer et al 2009) Exploitative
competition between ants and pollinators (Lach 2005 Ballantyne and Willmer 2012) as well as
the lethal effects of ant antibiotic secretions on pollen grains (Beattie et al 1985 Dutton and
Frederickson 2012) may have resulted in selection on plants to reduce ant visits to flowers Our
results suggest that trait-mediated indirect interactions resulting from interference competition
between ants and bees may favor plants that defend their flowers and their pollinators against
ants
Acknowledgements
We thank H Rusnock and S Meadley-Dunphy for assistance J Dix for help with flight cage
construction and Biobest for bumblebee colonies We are grateful to the Thomson and
Frederickson labs for guidance particularly T Makino funded by JSPS for helping to refine
artificial flowers J Ogilvie for help with bees K Prior for M rubra colony collections and K
Turner for study design suggestions The comments of the editor and two anonymous reviewers
prompted improvements in the article especially with regards to study design MEF and JDT
acknowledge NSERC Discovery Grant funding The University of Torontorsquos Faculty of Arts and
Science supported MGRTrsquos participation
67
68
References
Abbott K R 2006 Bumblebees avoid flowers containing evidence of past predation events
Canadian Journal of Zoology 841240-1247
Altshuler D L 1999 Novel interactions of non-pollinating ants with pollinators and fruit
consumers in a tropical forest Oecologia 119600-606
Ballantyne G and Willmer P 2012 Floral visitors and ant scent marks noticed but not used
Ecological Entomology 37402-409
Beattie A J Turnbull C Knox R B and Williams E G 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany
71421-426
Blanco M Runiaga H Maspoch S and Tarin P 1989 A simple method for
spectrophotometric determination of two-components with overlapped spectra Journal of
Chemical Education 66178-180
Byk J amp Del Claro K 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333-38
Davidson D W S C Cook and R R Snelling 2004 Liquid-feeding performances of ants
(Formicidae) ecological and evolutionary implications Oecologia 139255ndash66
Dukas R 2005 Bumble bee predators reduce pollinator density and plant fitness Ecology
861401-1406
Dussutour A and Simpson S J 2009 Communal nutrition in ants Current Biology 19740-
744
69
Dutton E M and Frederickson M E 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561-569
Frederickson ME 2009 Conflict over reproduction in an ant-plant symbiosis why Allomerus
octoarticulatus ants sterilize Cordia nodosa trees American Naturalist 173675-681
Galen C and Cuba J 2001 Down the tube pollinators predators and the evolution of flower
shape in the alpine skypilot Polemonium viscosum Evolution 551963-1971
Gonccedilalves-Souza T Omena P M Souza J C and Romero G Q 2008 Trait-mediated
effects on flowers artificial spiders deceive pollinators and decrease plant fitness Ecology
892407-2413
Gonzaacutelvez F G Santamariacutea L Corlett R T and Rodriacuteguez-Gironeacutes M A 2012 Flowers
attract weaver ants that deter less effective pollinators Journal of Ecology 10178ndash85
Goodale E and Nieh JC 2012 Public use of olfactory information associated with predation
in two species of social bees Animal Behaviour 84919-924
Harder LD and Thomson JD 1989 Evolutionary options for maximizing pollen dispersal of
animal-pollinated plants American Naturalist 133323-344
Heil M and McKey D 2003 Protective ant-plant interactions as model systems in ecological
and evolutionary research Annual Review of Ecology Evolution and Systematics 34425-
453
Heinrich B 2004 Bumblebee economics 2nd
edn Harvard University Press Cambridge
Ings T C and Chittka L 2009 Predator crypsis enhances behaviourally mediated indirect
effects on plants by altering bumblebee foraging preferences Proceedings of the Royal
Society B 2762031-2036
70
Inouye D W and Taylor O R Jr 1979 A temperate region plant-ant-seed predator system
consequences of extra floral nectar secretion by Helianthella quinquenervis Ecology 601-
7
Junker R R and Bluumlthgen N 2008 Floral scents repel potentially nectar-thieving ants
Evolutionary Ecology Research 10295ndash308
Junker R R Bleil R Daehler C C and Bluumlthgen N 2010 Intra-floral resource partitioning
between endemic and invasive flower visitors consequences for pollinator effectiveness
Ecological Entomology 35760ndash767
Jones E I and Dornhaus A 2011 Predation risk makes bees reject rewarding flowers and
reduce foraging activity Behavioral Ecology and Sociobiology 651505-1511
Lach L 2005 Interference and exploitative competition of three nectar-thieving invasive ant
species Insect Sociaux 52257-262
Lach L 2007 A mutualism with a native membracid facilitates pollinator displacement by
Argentine ants Ecology 881994-2004
Lach L 2008 Argentine ants displace floral arthropods in a biodiversity hotspot Diversity and
Distributions 14281-290
Makino T T and Sakai S 2007Experience changes pollinator responses to floral display size
from size-based to reward-based foraging Functional Ecology 21854863
Maloof J E and Inouye D W 2000 Are nectar robbers cheaters or mutualists Ecology
812651ndash2661
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506-514
Ohashi K Leslie A and Thomson JD 2008 Trapline foraging by bumble bees V Effects of
experience and priority on competitive performance Behavioral Ecology 19936-948
71
Palmer T M Doak D F Stanton M L Bronstein J L Kiers E T Young T P Goheen
J R and Pringle R M 2010 Synergy of multiple partners including freeloaders
increases host fitness in a multispecies mutualism Proceedings of the National Academy
of Sciences 10717234-17239
Preisser E L Bolnick D I and Benard M E 2005 Scared to death The effects of
intimidation and consumption in predator-prey interactions Ecology 86501-509
Stout J C Goulson D and Allen J A 1998 Repellent scent-marking of flowers by a guild of
foraging bumblebees (Bombus spp) Behavioral Ecology and Sociobiology 43317-326
Stout J and Goulson D 2002 The influence of nectar secretion rates on the responses of
bumblebees (Bombus spp) to previously visited flowers Behavioral Ecology and
Sociobiology 52239-246
Suttle K B 2003 Pollinators as mediators of top-down effects on plants Ecology Letters
6688-694
Thomson JD 1986 Pollen transport and deposition by bumble bees in Erythronium influences
of floral nectar and bee grooming Journal of Ecology 74329-341
Thomson JD Ogilvie J E Makino T T Arisz A Raju S Rojas-Luengas V and Tan M
G R 2012 Estimating pollination success with novel artificial flowers effects of nectar
concentration Journal of Pollination Ecology 9108-114
Tsuji K Hasyim A and Nakamura K 2004 Asian weaver ants Oecophylla smaragdina and
their repelling of pollinators Ecological Research 19669-673
Ulrich S and Bertsch A 1990 Do foraging bumblebees scent-mark food sources and does it
matter Oecologia 82137-144
Werner E E and Peacor S D 2003 A review of trait-mediated indirect interactions in
ecological communities Ecology 841083-1100
72
Willmer P G Nuttman C V Raine N E Stone G N Pattrick J G Henson K Stillman
P McIlroy L Potts S G and Knudsen J T 2009 Floral volatiles controlling ant
behaviour Functional Ecology 23888-900
Wright G A and Schiestl F P 2009 The evolution of floral scent the influence of olfactory
learning by insect pollinators on the honest signaling of floral rewards Functional Ecology
23841-851
73
Figures
Figure 21 Photograph of artificial flower lids A = anther L = lid N = nectary R = region
where anther or stigma is placed S = stigma T = thread W = weight
74
Figure 22 Dye transferred (mean plusmn 1SE) per trial by B impatiens workers A) from artificial
male and to artificial female flowers in the presence (unfilled circles) or absence (filled circles)
of Myrmica rubra workers and B) from artificial male flowers with (unfilled circles) or without
(filled circles) M rubra scent to ant-free female flowers in the 1st or the 2
nd to 4
th hours of the
trials
75
Chapter 3 Not just for the bees pollen consumption is common among tropical
ants
Cembrowski AR Reurink G and Frederickson ME
Planned submission to Biotropica
ARC and MEF designed the study ARC and GR did laboratory work ARC performed statistical
analyses and wrote the manuscript with input from other authors
Abstract
Although palynivory or pollen consumption is widespread among other hymenopterans there
are few accounts of palynivory in ants To quantify how often ants consume pollen we subjected
adult workers and larvae from 75 species of neo- and paleo-tropical ants to acetolysis a process
that destroys most organic material but leaves behind pollen grains In over half of the species
we examined ants contained a few pollen grains (lt 5) and ants of several Camponotus species
contained abundant pollen grains (gt50) We tested for an association between trophic level and
palynivory using stable nitrogen isotope ratios (δ15
N) but we did not find a significant
correlation we found pollen grains in lsquoherbivorousrsquo omnivorous and carnivorous ants We
suggest that our results indicate sporadic opportunistic pollen consumption by ants in tropical
forests
Introduction
For those organisms that can digest it efficiently pollen can be an excellent food source
Depending on the plant species pollen can contain up to 60 protein as well as lipids
76
carbohydrates and numerous trace nutrients (Roulston and Cane 2000) Pollen feeding
(palynivory) occurs in many taxa including birds mammals and arthropods (Roulston and Cane
2000) but it is best known among the Hymenoptera particularly bees Although adult bees feed
mainly on plant exudates (ie nectar) nearly all bee species rely on pollen as the chief protein
source for developing larvae (Willmer 2011) Closely related to bees ants have similar
nutritional requirements adult workers need carbohydrates whereas larvae are fed a diet rich in
protein (Dussutour and Simpson 2009) Ants also exploit nectar heavily and regularly visit
flowers (eg Herrera et al 1984 Bluumlthgen et al 2004) but unlike bees are not believed to
widely consume pollen Instead ants are thought to receive most of the protein in their diet from
predation or scavenging (eg Floren et al 2002)
However ants may not be as reliant on animal protein sources as commonly assumed In
tropical forests arboreal ants can have similar δ15
N ratios a measure of trophic level as
herbivores suggesting limited carnivory and substantial feeding on plant-derived protein sources
(Davidson et al 2003) One potential protein source is pollen This idea is supported by scattered
accounts of pollen consumption by ants from other ecosystems including deserts (Ness 2006)
boreal forests (Czechowski et al 2009) and tropical savannas (Byk and Del Claro 2010) One
ant genus Cephalotes is widely recognized to consume pollen (Creighton 1967 Baroni Urbani
and de Andrade 1997 Byk and Del Claro 2010) However beyond this genus our knowledge of
pollen consumption by ants has largely been limited to direct observations (but see Czechowski
et al 2011) and it is unknown if palynivory is truly rare among ants or simply under-reported
We borrowed a technique commonly used in palynology acetolysis to assess the frequency of
pollen consumption by ants in 75 neo- and paleo-tropical ant species This technique allowed us
to examine many more ant species than we could have observed directly
77
Methods
Adult worker ants were collected by hand at baits or from leaf litter using Winkler extractors
while larvae along with adults were collected by hand from nests Collections were made by L
Arcila Hernandez and J Sanders in the primary tropical rainforest surrounding the Centro de
Investigaciόn y Capacitaciόn Rio Los Amigos (CICRA 12deg34rsquoS 70deg05rsquoW) in Peru in 2010-
2011 and by E Youngerman from lowland primary and secondary rainforests in Madang
Province Papua New Guinea (PNG) in 2011-2012 We sorted ant samples to morphospecies and
then identified them to genus and when possible to species using existing keys which were
unavailable for PNG ants Ant samples used for acetolysis were whole adult workers guts from
adult workers and whole larvae
We used acetolysis to look for pollen grains inside ants In acetolysis organic material is
dissolved in acetic anhydride and sulfuric acid leaving behind stained pollen exines When
possible we used adult and larval ants of the same species from different colonies or bait
locations in an attempt to sample more representatively We prepared samples by first washing
them in 70 ethanol to remove any pollen present on the samplersquos outer surface before placing
them individually in micro-centrifuge tubes with ~05mL of a 91 mixture of acetic anhydride
sulfuric acid We then heated each sample at 95degC for 20min allowing tissues to dissolve Next
we centrifuged samples at 1000 RPM for 5min and decanted the liquid supernatant before
adding ~05mL of glacial acetic acid to the precipitate centrifuging again as above We decanted
the samples and added a drop of distilled water
78
We examined a 150μL sub-sample of this solution for pollen grains under a light
microscope Larvae gave less than 150μL of solution following acetolysis so we sampled the
entire amount We identified pollen grains by their staining and their surface architecture For
most species we examined at least three adult workers and three larvae but for some species
fewer than three adult workers or larvae were available (Tables 31 and 32)
We investigated the relationship between trophic level as measured by δ15
N ratios and
pollen consumption by ants Stable isotope (SI) data was available for some (n =23) of the
Peruvian species from a separate project in which collections overlapped Samples were stored in
EtOH and dried at 50degC overnight We used heads legs and thoraxes (but not gasters) of ants
for SI analysis Often single individuals did not provide enough material for analysis and we
combined multiple individuals to achieve necessary weights up to 10 for small ant species
Samples were analyzed at the Boston University Stable Isotope Laboratory using a Finnigan
Delta-S analyzer For other Peruvian species for which we had samples but not corresponding
stable isotope data (n =25) we took the median δ15
N ratio value of that speciesrsquo genus and
assigned it to the species in question Using pollen presenceabsence data for these 48 species
we used logistic regression to correlate pollen presence and δ15
N ratio using R (v 2151)
Results
We found pollen grains in 37 of the 197 adult workers 20 of the 102 larvae and 38 of the 75 ant
species we examined (Tables 31 and 32) The proportion of individuals containing pollen did
not differ significantly between larvae and adults (χ2
1 = 002 p = 088) The number of pollen
grains found was often low (median = 3) but highly variable (range 1-468) Also pollen
79
presence was inconsistent among conspecific ant samples of species that contained pollen 31
had pollen in only one of the larvae or adults examined In only three species (two Cephalotes
and one Myrmelachista) did all examined adults contain pollen
Highly lsquoherbivorousrsquo carnivorous and omnivorous ants contained pollen grains (Fig
31) There was no correlation between δ15
N ratio and pollen presence (χ2 = 150 df = 1 p =
022) In fact several carnivorous species contained pollen grains (eg Odontomachus sp 1
Pachycondola sp1)
Discussion
Pollen consumption by ants appears to be more common than suspected The majority of species
(51) had at least one individual that had consumed pollen and in 20 multiple individuals
contained pollen Numbers of grains found tended to be low however with most individuals
containing only a few grains Ants may use pollen as only a small portion of their diet exploiting
a host of other protein-containing foods more heavily including other low- δ15
N foods (eg
honeydew epiphylls Houmllldobler and Wilson 1990 Davidson et al 2003)
There are a few reasons why pollen may not be heavily exploited by ants Pollen requires
much post-consumption processing to extract its nutrients These nutrients are located in the
pollenrsquos cytoplasm which is protected from direct digestion by the exines and intines of the
grains Organisms must either break through these layers or cause the pollen grains to germinate
or pseudo-germinate to access and digest the cytoplasm (Roulston and Cane 2000) This can be
achieved by consuming a sugar-containing solution (such as nectar) along with pollen However
animals that are not adapted to feed on pollen may not be very efficient at this process only
80
extracting nutrients from some of the grains (Herrera and Martiacutenez del Rio 1998) Thus while
ants can likely get some nutrients from pollen other forms of more easily processed protein may
be more attractive
Pollen may also pose handling difficulties for ants Despite the large number of studies
that have recorded ants visiting flowers (Chapter 1) only a few papers detail ants harvesting
pollen directly from anthers Byk and Del Claro (2010) reported that Cephalotes pusillus ants
removed almost all of the available pollen from Ouratea spectabilis flowers and at least two
other studies describe ants consuming pollen along with nectar from flowers (Horskins and
Turner 1999 Ness 2006) Unlike bees which possess both morphological (eg specialized hairs
for holding pollen) and behavioral adaptations (eg stereotyped pollen-packing motions) ants
have no apparent mechanisms to increase the amount of pollen harvested and are likely limited
by the number of grains they can ingest or carry Rather than collect pollen directly from flowers
it appears more likely that ants collect it haphazardly in the environment leading to a smaller
number of pollen grains consumed at any one time It is known that at least Cephalotes ants
engage in such behavior Creighton (1967) observed Cephalotes texanus collecting pollen grains
trapped by Citrus x paradisi leaf hairs
There is a chance that the small numbers of pollen grains we observed came from
secondary pollen consumption (ie consuming a palynivorous organism) or consumption of
liquid containing pollen While we cannot rule this out we believe our results represent actual
targeted pollen consumption by ants Cephalotes ants known palynivores (Creighton 1967
Baroni Urbani and de Andrade 1997) contained similar numbers of pollen grains as many of the
pollen-containing ant species that we examined As well in at least once instance ants that we
found containing pollen grains were in the process of preying on non-pollen consuming
81
organisms (eg a Pachycondola species that was carrying a termite in its mandibles) or were
attracted by sugar baits
Contrary to our expectations there was no relationship between an ant speciesrsquo δ15
N ratio
and whether it had consumed pollen (Fig 32) We expected palynivory to be more common in
herbivorous ants because pollen presents a widespread source of protein a potentially limiting
resource The lack of pattern might reflect how available pollen grains are in the environment
While other forms of food may be preferable ants tend to be opportunistic foragers (Carroll and
Janzen 1973) and there is likely little cost associated with the consumption of scattered pollen
grains during foraging trips beyond the volume they occupy in the gut Some genera were found
to consistently consume pollen In support of previous observations (Baroni Urbani and de
Andrade 1997 Byk and Del Claro 2010) Cephalotes species were among the most common
pollen consumers Camponotus species also consistently contained pollen grains of the eight
species examined five contained pollen and pollen was found in species from both Peru and
PNG Camponotus species also contained the two largest numbers of pollen grains found (468
and 271) Taken together these results suggest that they may be another as yet unrecognized
commonly palynivorous genus
In general larvae did not contain large numbers of pollen grains (ranging from 1-38
grains) This is counter to what is seen in bees where larvae are the main recipients of pollen
(Willmer 2011) This difference may be because ant larvae are continually fed via trophallaxis
by adults throughout their development whereas bee larval cells are provisioned with pollen and
then sealed while larvae complete their development If both adults and larvae are able to extract
nutrients from pollen we would expect to see a preferential flow of nutrients from pollen to
larvae but not necessarily the grains themselves
82
Unlike palynivory by organisms that also provide pollination services pollen
consumption by ants is unlikely to benefit plants Ants rarely act as pollinators (Beattie et al
2004) They have pollen-killing antibiotic secretions on their cuticles (Dutton and Frederickson
2012) and they can compete with other flower visitors reducing flower visitation rates (Ness
2006) The negative impacts of flower-visiting ants are thought to have led to the evolution of
ant-repellent floral volatiles (reviewed in Willmer 2009) These volatiles can pre-empt
competition between ants and pollinators and may often be located within the pollen itself
(Willmer 2009) If floral volatiles truly are a defense against ant visitation our results suggest an
additional evolutionary pressure to not only prevent ant-pollinator interactions but to also
protect plant gametes from occasional consumption by a non-pollinating visitor
Acknowledgements
We would like to thank Jon Sanders Lina Arcila Hernandez and Eric Youngerman for providing
samples the Thomson lab for use of supplies the Pierce lab for funding support during JSrsquo and
EYrsquos collections and the staff of CICRA and the New Guinea Binatang Research Center for
logistical support The Peruvian Ministry of Agriculture provided permits (Nos 394-2009-AG-
DGFFS-DGEFFS and 299-2011-AG-DGFFS-DGEFFS) MEF acknowledges financial support
from an NSERC Discovery Grant a Connaught New Researcher Award an Ontario Ministry of
Economic Development and Innovation Early Researcher Award and the University of Toronto
ARC was supported by an Ontario Graduate Scholarship and Sigma Xi
83
References
Baroni Urbani C and M L de Andrade 1997 Pollen eating storing and spitting by ants
Naturwissenschaften 84256ndash258
Beattie A J C Turnbull R B Knox and E G Williams 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany 71421ndash
426
Bluumlthgen N G Gottsberger and K Fiedler 2004 Sugar and amino acid composition of ant-
attended nectar and honeydew sources from an Australian rainforest Austral Ecology
29418ndash429
Byk J and K Del-Claro 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333ndash38
Carroll C R and D H Janzen 1973 Ecology of foraging ants Annual Review of Ecology and
Systematics 4231ndash257
Creighton W S 1967 Studies on free colonies of Cryptocerus texanus Satschi (Hymenoptera
Formicidae) Psyche 7434ndash42
Czechowski W B Markό and A Radchenko 2009 Rubbish dumps reveal the diet of ant
colonies Myrmica schencki Em and Myrmica rubra (L) (Hymenoptera Formicidae) as
facultative pollen-eaters Polish Journal of Ecology 56737ndash741
Czechowski WB Markoacute K Erős and E Csata 2011 Pollenivory in ants (Hymenoptera
Formicidae) seems to be much more common than it was thought Annales Zoologici
61519ndash525
84
Dussutour A and S J Simpson 2009 Communal nutrition in ants Current Biology 19740ndash4
Dutton E M and M E Frederickson 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561ndash569
Floren A A Biun and K E Linsenmair 2002 Arboreal ants as key predators in tropical
lowland rainforest trees Oecologia 131137ndash144
Grant B R 1996 Pollen digestion by Darwinrsquos finches and its importance for early breeding
Ecology 77489-499
Herrera L G and C Martiacutenez del Rio 1998 Pollen digestion by new world bats effects of
processing time and feeding habits Ecology 792828ndash2838
Houmllldobler B and Wilson E O 1990 The Ants 1st Edition Harvard University Press
Cambridge
Horskins K and Turner V B 1999 Resource use and foraging patterns of honeybees Apis
mellifera and native insects on flowers of Eucalyptus costata Australian Journal of
Ecology 24221-227
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506ndash514
Roulston T H I and J H Cane 2000 Pollen nutritional content and digestibility for animals
Plant Systematics 222187ndash209
Van Tets I G 1997 Extraction of nutrients from Protea pollen by African rodents Belgian
Journal of Zoology 12759-65
Willmer P G 2011 Pollination and Floral Ecology 1st Edition Princeton University Press
New Jersey
85
Willmer P G C V Nuttman N E Raine G N Stone J G Pattrick K Henson P Stillman
et al 2009 Floral volatiles controlling ant behaviour Functional Ecology 23888ndash900
86
Tables
Table 31 Numbers of adult worker and larval ants examined for each species collected in Peru
Numbers of pollen grains found in individual samples are given in parentheses after the sample
size when there were no grains no numbers are given
Genus Species Number of adults analyzed
(numbers of grains found)
Number of larvae analyzed
(numbers of grains found)
Dolichoderinae
Azteca sp 1 3 0
sp 2 2 0
sp 3 0 2
sp 4 3 (1) 0
sp 5 3 (2) 0
Dolichoderus decollatus 3 (66) 0
sp 1 3 0
sp 2 3 1 (2)
sp 3 3 3
sp 4 0 2
Ecitoninae
Labidus sp 1 3 0
Formicinae
Camponotus sp 1 3 3
sp 2 3 (82) 1 (2)
sp 3 3 (3) 0
sp 4 0 2
87
sp 5 8 (11) 0
sp 6 4 (7 16 468) 0
sp 7 5 (12) 0
sp 8 2 (1) 0
Paratrechina sp 1 4 (11) 0
Myrmelachista sp 1 3 (5 11 18) 0
sp 2 0 3
Trachymyrmex sp 1 3 (21) 0
sp 2 3 0
Myrmicinae
Atta sp 1 3 0
Cephalotes atratus 3 (2 2 21) 0
placidus 3 (1 4 21) 0
sp 1 3 0
sp 2 1 0
sp 3 2 0
Crematogaster sp 1 3 1
sp 2 3 (35) 0
sp 3 9 (7) 0
sp 4 2 0
Megalomyrmex sp 1 3 0
sp 2 0 2
sp 3 4 (7) 0
sp 4 3 0
Monomorium sp 1 0 3 (2)
88
Pheidole sp 1 0 3
sp 2 3 0
sp 3 0 3
Procryptocerus sp 1 3 0
Solenopsis sp 1 0 3
sp 2 3 (3 5) 0
Wasmannia sp 1 3 0
Ponerinae
Pachycondola sp 1 3 (3) 0
Odontomachus sp 1 3 (34) 0
Pseudomyrmicinae
Pseudomyrmex sp 1 3 0
sp 2 3 0
sp 3 3 (9) 0
sp 4 0 3
sp 5 0 3
89
Table 32 Numbers of adult worker and larval ants examined for each species collected in Papua
New Guinea Numbers of pollen grains found in individual samples are given in parentheses
after the sample size when there were no grains no numbers are given
Genus Species Number adults analyzed
(grains found)
Number larvae analyzed
(grains found)
Aenictinae
Aenictus sp 1 3 3 (1 1)
Dolichoderinae
Philidris sp 1 3 1 (4)
Tapinoma sp 1 2 3 (38)
Technomyrmex sp 1 0 3 (12 12)
Ectatomminae
Rhytidoponera sp 1 1 3 (2)
Formicinae
Acropyga sp 1 3 3 (2)
sp 2 3 3
sp 3 3 3
Anoplolepsis sp 1 3 (1) 3 (1)
Calomyrmex sp 1 3 3 (1)
Camponotus sp 1 3 (1 271) 3
sp 2 1 3
sp 3 2 0
Nylanderia sp 1 3 3
Opisthopsis sp 1 3 3
Polyrachis sp 1 1 3 (4)
90
Myrmicinae
Crematogaster sp 1 3 3
sp 2 3 (32) 3
Monomorium sp 1 3 (1) 3 (1)
sp 2 3 3 (1 1 1)
Pheidole sp 1 3 3
Pristomyrmex sp 1 3 3 (11)
Pyramica sp 1 3 0
Ponerinae
Odontomachus sp 1 3 3 (4)
91
Figures
Figure 31 Photographs of pollen grains found in two ant species a) Dolichoderus sp 2 and b)
Camponotus sp 6 Scale bars = 50 microm
92
Figure 32 Relationship between trophic level as measured by δ15
N ratio and pollen presence
for 48 neotropical ant species 1 Atta sp 5 Azteca spp 8 Camponotus spp 5 Cephalotes spp 4
Crematogaster spp 5 Dolichoderus spp 1 Labidus sp 3 Megalomyrmex spp 1 Monomorium
sp 2 Myrmelachista spp 1 Odontomachus sp 1 Pachycondola sp 1 Paratrechina sp 3
Pheidole sp 4 Pseudomyrmex spp 2 Solenopsis spp and 1 Wasmannia sp
93
Concluding Remarks
Ants have traditionally been considered to be unwelcome flower visitors they consume floral
rewards ostensibly meant for pollinators while rarely providing pollination services themselves
In this thesis I investigate the validity of this assumption and use an experimental study and an
observational study to examine two ways that ants may impact plant reproduction
In the first chapter I review the literature on ants as flower visitors I show that the common
assumption of ants always being categorically negative for plant reproduction is incorrect while
ants may reduce floral attractiveness to pollinators the net effect of ants on plant fitness
measures is variable ranging from positive to negative to neutral I also discuss ants as
pollinators and ways that plants may minimize ant visitation to flowers before finishing with a
section highlighting questions that still need investigation
In the second chapter I explore how interference competition with ants can structure bumblebee
foraging behavior and how this may alter pollen movement patterns Using artificial flowers with
male or female function I allowed Bombus impatiens bumblebees to forage on flowers that
either did or did not have Myrmica rubra ants on them Ant presence significantly reduced the
amount of pollen analogue donated and received by flowers Building on these results I found
that the presence of ant scent alone elicited similar changes to bumblebee behavior reducing the
amount of pollen analogue donated by male flowers My results show that interference
competition is sufficient to alter bumblebee foraging behavior and that the effects of ants on
pollinators may extend after ant visitation to flowers Further study is needed to determine if bees
are able to differentiate between ant species by visual or olfactory cues Their ability to do so
94
could affect how generalized their response to ant presence is and may help to better predict how
ants will affect plant reproduction
In the final chapter I investigate how common palynivory is amongst tropical ant species I used
acetolysis and light microscopy to examine whole ants larvae and ant guts from Papua New
Guinea and Peru I found that tropical ants consistently contain pollen grains albeit often in
small numbers (lt5) However some genera notably Camponotus and Cephalotes consistently
contained higher numbers providing the first evidence that Camponotus may be a palynivorous
genus The small numbers of pollen grains present may indicate that ants preferentially exploit
other more easily digestible high-protein foods over pollen Future studies should investigate
exactly what role pollen plays in ant diets by supplementing the diet of captive ant colonies with
pollen This would allow us to examine how pollen consumption affects colony demography as
well as how efficiently ants digest pollen and whether ants exhibit pollen preferences
I bring up several pressing and unanswered questions within this thesis and highlight three here
First numerous plant species possess chemical or physical traits assumed to increase plant
fitness by reducing ant visitation However this has rarely been tested and instead research has
focused predominately on documenting ant repellence We should move away from this and
begin examining plant fitness in the presence or absence of these repellent traits to see how
beneficial these traits truly are Second how flower visiting ants impact male fitness has been
almost completely ignored in favor of female fitness Future studies should investigate how ant
visitation affects both pollen donation and pollen movement patterns Lastly research involving
antibiotics present on ant cuticles and their effect on pollen viability has been too small in scope
often limited to demonstrating the pollen killing effects of ant secretions Investigation into
95
whether plants have evolved any resistance to the secretions of the local ant community or why
some plants appear unaffected by their ant partners may yield exciting results
In conclusion ants have the capacity to strongly affect plant reproduction both positively and
negatively The fact that these diametrically opposing forces exist means we cannot approach
flower-visiting ants as either positive or negative a priori Instead we should strive to work
towards a better understanding of what is occurring in the system of interest and what role ants
play within it
96
Copyright Acknowledgments
Chapter 2 ldquoAnts and ant scent reduce bumblebee pollination of artificial flowersrdquo is current in
press in The American Naturalist Request for its inclusion here was sought and approved
iii
Acknowledgments
Science is never an individual undertaking Numerous people made the completion of this thesis
possible First and foremost Megan Frederickson gave me the freedom to pursue research
outside of her realm of expertise was extremely patient my ever-changing interests and
supported the decisions I made in my degree My committee also deserves strong thanks James
Thomson was instrumental in my studies giving me lab space supplies and most importantly
time and guidance when I had questions about anything pollination bee or flower related Ben
Gilbert gave statistics help and was helpful with coding questions As well thank you to my
examination committee Spencer Barrett and Helen Rodd
The Frederickson and Thomson lab groups helped me immeasurably giving encouragement
guidance and reality checks when I went too far off track Thank you to Kyle Turner Lina
Arcila Hernandez Kirsten Prior Jane Ogilvie Alison Parker Eric Youngerman and Rebecca
Batstone Kyle especially helped me in all regards both inside and outside of school and was
without question the best field roommate and officemate I could have had Several volunteers
work study students and our lab tech fed ants washed artificial flowers and counted tiny objects
saving me countless hours particularly Harry Rusnock Shannon Meadley Dunphy Margaret
Thompson and Jackie Day I would also like to highlight Marcus Guorui Tan in particular for
his work collaborating with me and running trials when I was in out of town and being
unwaveringly positive about our research Our experiment would not have been nearly as
successful without him
Several people in the department and associated with the lab provided feedback jokes and
friendship without which grad school would have been a depressing place So thank you Natalie
Jones Rachel Germaine Kelly Carscadden Alex de Serrano Jordan Pleet Aaron Hall Susana
Wadgymar Emily Austen Eddie Ho Amanda Gorton Dorina Szuroczki Jenn Coughlan Jon
Sanders and Gabe Miller
People not in the department and both inside and outside of ecology made my time here possible
and more enjoyable than otherwise Thanks to the Tuner family for taking me in during holidays
Maureen Murray and Matt Mazowita for everything and my old supervisor Colleen Cassady St
Clair for helping me get where I am
iv
Lastly my family did an astounding amount for me in letting me cultivate my love of biology
buying me books letting me keep strange animals and getting me outdoors So thank you to my
brothers John and Mark my dad George and my mom Kay And to my partner Katherine for
her unbelievable patience both in regards to my academic life and in her willingness to adapt
hers for mine thank you This could not have happened without you
ldquoVor Allem sind aus der Reihe der fluumlgellosen Insecten die weitverbreiteten fluumlgellosen Ameisen
sehr unwillkommene Gaumlste der Bluumlthen Und dennoch sind gerade sie nach dem Nectar der
Bluumlthen in hohem Grade luumlsternhelliprdquo
ldquoOf all the wingless insects it is the widely dispersed ants that are the most unwelcome guests to
flowers And yet they are the very ones which have the greatest longing for nectarhelliprdquo
A Kerner 1878 Flowers and their Unbidden Guests
v
Table of Contents
Acknowledgments iii
List of Tables vii
List of Figures viii
Chapter 1 Ants as flower visitors and their effects on pollinator behavior and plant
reproduction 1
Abstract 1
Introduction 2
1 Ant interactions with flowers and floral visitors 3
2 Ants as pollinators 12
3 Floral defenses against ants 14
4 Future directions 20
References 24
Tables 39
Chapter 2 Ants and ant scent reduce bumblebee pollination of artificial flowers 54
Abstract 54
Introduction 55
Methods 57
Results 62
Discussion 63
Acknowledgements 66
References 68
Figures 73
Chapter 3 Not just for the bees pollen consumption is common among tropical ants 75
vi
Abstract 75
Introduction 75
Methods 77
Results 78
Discussion 79
Acknowledgements 82
References 83
Tables 86
Figures 91
Concluding Remarks 93
Copyright Acknowledgments 96
vii
List of Tables
Table 11 List of studies of flower-visiting ants that consumed floral rewards or interacted with
pollinators and included a measure of fitness39
Table 12 List of studies that have either demonstrated ant pollination through exclusion
experiments or whose results strongly suggest it48
Table 31 Numbers of adult worker and larval ants examined for each species collected in Peru
and numbers of pollen grains found87
Table 32 Numbers of adult worker and larval ants examined for each species collected in Papua
New Guinea and numbers of pollen grains found90
viii
List of Figures
Figure 21 Photograph of artificial flower lids with anther and stigma74
Figure 22 Dye transferred (mean plusmn 1SE) by B impatiens a) in the presence and absence of M
rubra ants and b) with or without M rubra scent75
Figure 31 Photographs of pollen grains found inside ants92
Figure 32 Relationship between trophic level and pollen presence for neotropical
ant species93
1
Chapter 1 Ants as flower visitors and their effects on pollinator behavior and plant
reproduction
Cembrowski AR and Frederickson ME
Planned submission to a journal such as Oikos or Insectes Sociaux
ARC wrote the manuscript with input from MEF
Abstract
Ants often provide a range of benefits to flowering plants particularly by reducing herbivory
However when ants visit flowers they can deter pollinators or damage floral structures
Currently it is unclear how often and in what direction flower-visiting ants affect plant
reproductive success A review of the literature shows that ants can help or hinder pollination
processes but overall flower-visiting ants most commonly have a non-significant net effect on
plant fitness Although ants may directly harm flowers or indirectly affect pollination by
consuming floral nectar or harassing pollinators they can also benefit plants by attacking
florivores or predators of pollinators Ants occasionally act as pollinators themselves though ant
secretions often kill pollen grains There is growing evidence that many plants have traits that
allow them to avoid or minimize the negative effects of flower-visiting ants More research is
needed to determine if these traits provide fitness benefits to plants by preventing ant visitation
or if they function as generalized repellents of floral antagonists
2
Introduction
Ants and angiosperms share a long evolutionary history Ant diversification closely followed that
of flowering plants as ants took advantage of the large prey numbers that angiosperms supported
and the new habitats they provided (Moreau 2006) Subsequently numerous angiosperm
lineages evolved intimate associations with ants that function as an indirect or biotic form of
plant defense Food rewards such as extrafloral nectar or pearl bodies or housing in the form of
domatia attract ants to plants where they reduce herbivory and increase plant fitness (reviewed
in Rosumek et al 2009) However the traits that make ants good plant defenders particularly
their abundance attraction to sugar sources and aggressiveness can also cause problems for
plant reproduction Ants attracted by floral nectar can compete with pollinators for resources
and defend flowers potentially reducing plant fitness (Ness 2006 Junker et al 2007) That ants
negatively affect plant reproduction is an old and pervasive hypothesis Kerner (1878) called ants
ldquomost unwelcome guestsrdquo on flowers and more recent authors have similarly expressed that ant
visitation to flowers reduces plant reproductive success (Ness 2006 Lach 2007 Willmer et al
2009)
Nonetheless studies assessing the impact of flower-visiting ants on plant reproductive
success have produced mixed results Ants can have negative impacts on pollination and flower
function (Galen 1983 Puterbaugh 1998 Ness 2006 Hansen and Muumlller 2009) However other
studies have found no effect or even strong benefits of ant visits to flowers (Rico-Gray and
Thien 1989 Altshuler 1999 Schuumlrch 2000 Ashman and King 2005) This discrepancy between
ants being ldquounwelcome guestsrdquo and their actual effects on plant reproduction requires
clarification Our goal here is to highlight the ways that flower-visiting ants may affect plant
reproduction both positively and negatively and to discern through a review of the literature
3
what impacts ants have on plant reproductive success First we discuss mechanisms by which
flower-visiting ants can directly and indirectly impact plant reproduction both through floral
damage and by mediating changes in pollinator or florivore behavior We end this section by
evaluating published research that collectively shows that flower-visiting ants have
predominantly neutral effects on plant fitness measures Next we discuss pollination by ants
focusing on the ldquoantibiotic hypothesisrdquo before describing how plants may prevent ants from
visiting flowers Lastly we discuss promising new avenues of research and highlight work that
still needs to be performed to give a more complete understanding of ants as flower visitors
1 Ant interactions with flowers and floral visitors
i) Damage to flowers
Ants can directly disrupt plant reproduction by damaging floral structures preventing either the
fertilization of ovules or the presentation of pollen Rarely ants forage on floral tissue itself for
example Puterbaugh (1998) found that Formica neorufibarbus ants chewed through the coronal
ring of Eritrichium aretiodes flowers to get lipids More often but still not commonly foraging
ants chew through reproductive tissue to access floral nectar This can cause sterilization of
whole flowers (Echium plantagineum Kirk 1984) or affect female function only if just styles are
damaged effectively creating male flowers (Polemonium viscosum Galen 1983) Antibiotic
secretions on ant integuments may also directly damage pollen grains but the fitness effects of
this are unclear (see 2 below)
Several species of Crematogaster and Allomerus ants sterilize their host plants by
attacking floral buds (Yu and Pierce 1998 Stanton et al 1999 Gaume et al 2005 Maleacute et al
2012 Table 11) These ants nest in ldquoant-plantsrdquo or ldquomyrmecophytesrdquomdashie plants that host ant
4
colonies in specialized structures (domatia) The sterilizing ants do not appear to consume floral
tissues (Yu and Pierce 1998) and two hypotheses may explain why ants destroy floral buds First
ant colony size is often limited by domatia availability on their host plant By destroying floral
buds ants can cause plants to divert resources from reproductive to vegetative growth increasing
domatia number and allowing for larger ant colonies (Yu amp Pierce 1998 Frederickson 2009)
Second when plant-ants must defend their colony from takeovers by other colonies they may
benefit from limiting access to their host plant By destroying buds that could develop into
flowers branches or leaves ants can prevent their host plant from contacting nearby plants
occupied by enemy ant colonies (Stanton et al 1999)
The impact of sterilization on lifetime plant fitness is unclear Ants may destroy all of
their host plantrsquos floral buds (Yu and Pierce 1998) completely preventing reproduction or only a
portion of them (Maleacute et al 2012) allowing some seed set However counter-intuitively this
sterilization behavior may provide long-term fitness benefits to plants (Frederickson 2009
Palmer et al 2010) Ant-plants often host several different ant species over their lifetimes some
of which are non-sterilizing If hosting a sterilizing species at a certain life stage increases
survival or growth relative to hosting a non-sterilizing species at that life stage plants may
increase lifetime fitness by largely forgoing current reproduction in favor of increasing future
reproduction (Palmer et al 2010) Why plants invest resources in producing flowers that are
quickly destroyed by ants is not known but the timing of flower destruction may be important If
ants attack flowers after anthers dehisce plants may still realize some male fitness via pollen
donation (Yu and Pierce 1998) Additionally if the cost of flower production is relatively minor
and ants sometimes ldquomissrdquo flowers (Edwards and Yu 2008) attempting limited reproduction
may be a better strategy than forgoing it entirely
5
ii) Competition with pollinators
Ant activity on flowers can dramatically alter pollinator behavior Ants can reduce the species
diversity frequency or duration of pollinator visits to flowers (Norment 1988 Tsuji et al 2004
Ness 2006 Junker et al 2007 Lach 2007 2008b Romero et al 2011 and see Chapter 2) These
changes are largely due to either exploitative competition (ie consumption of shared resources)
or interference competition (ie direct aggression) between ants and pollinators
a) Exploitative competition
Pollinators and ants frequently compete for floral nectar Flower-visiting ants are often classified
as nectar thieves (sensu Inouye 1980) because they typically enter flowers through the
ldquolegitimaterdquo opening and consume nectar but transfer no pollen (Wyatt 1980 Ness 2006
Chamberlain and Holland 2008) Ants can also act as primary nectar robbers chewing holes
through corollas to reach nectaries (Willmer and Corbet 1981 Koopowitz and Marchant 1998)
although they are more commonly secondary nectar robbers and use holes made by previous
robbers (Newman and Thomson 2005)
Like other flower visitors foraging ants will often leave nectar behind in flowers This
amount can be less (Bleil et al 2011) more (Fritz and Morse 1982) or similar to (Lach 2008a)
amounts left by other visitors Often ants are morphologically constrained from fully exploiting
nectar Unlike bees and butterflies ants do not have proboscises and must get close to nectar to
consume it Narrow corollas or nectar spurs can prevent ant access to some or all floral nectar
(Schemske 1980 Galen and Cuba 2001 and see below) Smaller ants may be better able to
6
access nectar (Newman and Thomson 2005 Agarwal and Rastogi 2007) but can be limited by
their crop size in the amount of nectar they can remove (Lach 2005) If foraging ants leave
enough nectar behind for a pollinator to recover the cost of visiting a flower ants may have
minimal effects on plant reproductive success (Maloof and Inouye 2000 Turner et al 2012)
Differences among ant species in behavior and diet also affect their consumption of floral
nectar Species vary in how many flowers they visit per inflorescence or per plant and in how
many ants will visit a flower simultaneously (Lach 2005 Junker et al 2007 Lach 2013)
Invasive antsmdashthought to be better at exploiting and defending resources than native ants
(Holway et al 2002)mdashoften recruit to flowers in large numbers sometimes several dozen or
more (Visser et al 1999 Hansen and Muumlller 2009) Overall many factors may influence how
heavily ants exploit floral nectar on plants including ant density plant height the spatial or
temporal distribution of floral nectar and the availability of alternative food resources for ants
(Cushman and Addicott 1991 Lach 2013)
Rarely ants and pollinators may compete for pollen We know of only a few studies that
have recorded ants harvesting pollen directly from anthers (Horskins and Turner 1999 Ness
2006 Byk and Del-Claro 2010) Other studies have found pollen grains in ants (eg Baroni
Urbani and de Andrade 1997 and see Chapter 3) but this may not indicate competition with
pollinators ants sometimes collect stray pollen grains from the environment (Creighton 1967)
One New World genus Cephalotes might be specially adapted for pollen consumption They
possess ldquoproventricular shieldsrdquo structures that may restrict solid food to the crop allowing
nestmates to exchange pollen grains via trophallaxis It is unclear if this structure evolved for this
purpose but Cephalotes are the most frequently documented palynivorous ants (eg Creighton
1967 Baroni Urbani and de Andrade 1997 Byk and Del-Claro 2010) How efficiently ants
7
digest pollen and whether ants like bees feed it to larvae as a protein source is unknown At
least two studies have found that ant larvae consume pollen providing some support for this idea
(Wheeler and Bailey 1920 and Chapter 3)
b) Interference competition
Ants are relatively unique among flower visitors in defending floral resources from competitors
(but see Willmer and Corbett 1981 Roubik 1982) This defense ranges from chance encounters
with ants attacking visitors when flower occupancy overlaps (Ashman and King 2005 Junker et
al 2007) to actively patrolling inflorescences or constructing protective galleries around
nectaries (Horvitz and Schemske 1984 Gaume et al 2005 Hansen and Muumlller 2009)
Interference competition even in the absence of exploitative competition is sufficient to alter
pollinator behavior (Chapter 2)
The strength and outcome of interference competition depends on the identities of the ant
and the pollinator Pollinators might not alter their visitation patterns in response to non-
aggressive ants (Junker et al 2007) but more antagonistic ants may elicit avoidance behavior
before or soon after the visitor lands (Willmer and Corbet 1982 Altshuler 1999 Schuumlrch et al
2000 Table 11) Pollinator size may help predict their reaction to ants because ants may deter
only small-bodied pollinators (Gonzaacutelvez et al 2012) It is not clear whether flower visitors view
ants as aggressive competitors or as potential predators rarely ants prey on pollinators (Schatz
and Wcislo 1999) but in most cases ants merely cause pollinators to relocate
How well pollinators recognize and avoid ant-visited flowers is unresolved Collectively
pollinators have lower visitation rates to flowers visited by ants (Romero et al 2011) This is
8
dependent on pollinator identity however dipterans do not appear to discriminate between ant-
visited and ant-free flowers while bees can identify and avoid ant-visited flowers Bees likely
use a combination of visual and olfactory cues to recognize ant-visited flowers They can
identify previous conspecific and some heterospecific insect visitors by the hydrocarbon
ldquofootprintsrdquo they leave during foraging (Stout and Goulson 2001) Bumblebees can detect ant
hydrocarbons although this has been shown only in the laboratory (Ballantyne and Willmer
2012 Chapter 2) No study has yet examined if bees can use sight alone to detect ants but
several suggest it (Junker et al 2007 Gonzaacutelvez et al 2012) Some bees may discriminate
between ant species when visiting flowers (eg Lach 2008b) which follows if not all ant-
pollinator interactions are equally costly The ability of bees to do so will affect how generalized
their avoidance behaviors will be If they can discriminate between species bees may avoid
specific flowers visited by more ldquocostlyrdquo ants However if they cannot bees might either avoid
all ant-visited flowers or none depending on how negative the average interaction with an ant is
iii) Interactions with flower and pollinator antagonists
Ants can defend the flowers they visit Numerous plant species have circumfloral or bracteal
nectaries (ie extrafloral nectaries around flowers) and ants attracted to these nectaries can
reduce bud ovule or flower predation increasing plant fitness (Inouye and Taylor 1979 Rico-
Gray and Thien 1989 Sugiura et al 2006) Although the presence of EFNs (extrafloral nectaries)
so close to floral nectaries (sometimes less than 1 cm Ness 2006) might increase discovery and
exploitation of floral nectar by ants only one study has investigated this hypothesis finding no
relationship between the proximity of EFNs to floral nectaries and antsrsquo use of floral nectar
(Galen 2005) Holes made by nectar robbers are functionally analogous to circumfloral nectaries
9
and can also attract ants that defend flowers (Newman and Thomson 2005) and nectar-thieving
ants can similarly prevent florivory in some systems (Lach 2007 Bleil et al 2011)
Ants can also reduce the abundance of flower-dwelling predators or parasites of
pollinators Ambush predators such as crab spiders often forage at flowers where they may
reduce pollinator visitation rates and plant fitness (Dukas 2001 Gonccedilalvez-Souza et al 2008)
Ants can decrease spider abundance and richness on plants (Rudgers 2004 Renault et al 2005
Nahas et al 2012) and some evidence suggests that they can do the same on flowers (Lach
2008) In select cases ants may also reduce the abundance of pollinator parasitoids (Holland et
al 2011) Whether ants increase plant reproductive success by removing pollinator antagonists
has not yet been examined but should be If ants create lsquoenemy-freersquo flowers pollinators might
prefer to land on flowers with ants
iv) Net effects
Table 11 compiles studies in which the authors recorded ants consuming floral rewards or
interacting with pollinators and measured some aspect of plant fitness (eg fruit or seed set
pollen deposition) We included only a single study per plant species unless subsequent studies
found conflicting results or measured the fitness of another plant sex Records of ant pollination
are included in a separate table (Table 12)
Table 11 reveals relatively few examples of flower-visiting ants negatively affecting
plant fitness given how costly ant visitation is assumed to be The net effect of ants on plant
reproductive success is actually quite variable with similar numbers of studies finding positive
negative or neutral effects of ant presence Surprisingly when ants do have negative effects it is
10
usually because they directly damage floral structures and not because they prevent pollinators
from visiting flowers and successfully transferring pollen Why are ants not more often negative
for plant reproduction when this is commonly assumed to be true
First ants may compensate for reducing pollinator visitation rates by improving
pollination quality As pollinators move between flowers on a plant they often deposit self-
pollen on stigmas (ie geitonogamy) In some plants with late-acting self-incompatibility ovules
fertilized by self-pollen are subsequently aborted reducing the number of seeds that can be
produced (Barrett 2002 and references therein) Geitonogamy can also reduce offspring viability
or vigor (de Jong et al 1993) and cause pollen discounting a reduction in the amount of pollen
available for outcrossing because of self-pollination (Barrett 2002) By competing with
pollinators ants might decrease rates of geitonogamous pollination if pollinators then visit fewer
sequential flowers within a plant Ants might also cause pollinators to fly further between plants
potentially increasing the genetic diversity of pollen arriving at stigmas Although such effects
have been reported for other floral larcenists (Maloof 2001 Irwin 2003) to our knowledge these
hypotheses have not been investigated in relation to ants
Second ants and pollinators do not always forage for the same resources possibly
allowing them to partition floral rewards If ants forage for nectar but not pollen and flowers can
be pollinated efficiently by visitors looking to gather mostly pollen and little or no nectar then
plant reproduction will not be affected by ants consuming floral nectar Junker et al (2010a)
found that honeybees foraged for both pollen and nectar in the absence of ants but reduced their
foraging on nectar but not pollen when ants could access flowers resulting in no difference in
fruit set between treatments Whether pollinators commonly adjust their foraging behaviors in
response to ants warrants further research
11
Third ants are somewhat unique among floral larcenists in that they often provide direct
benefits to the plants they visit In the studies listed in Table 11 ants sometimes decreased
herbivory on plants (Chamberlain and Holland 2008 Bleil et al 2011) or otherwise aided the
plant (eg ant presence repelling smaller less effective pollinators Gonzaacutelvez et al 2012) If the
positive effects of ants cancel or outweigh the negative effects of flower-visiting ants on
pollinators then ant visitation may be neutral or even positive for plant fitness
Fourth ants may impose fitness costs to plants not captured in the studies in Table 11
Nectar robbers have stronger negative effects when plants are pollen-limited (Burkle et al 2007)
but only one study in Table 11 assessed whether flowers would set more seed if they received
more pollen finding no evidence of pollen limitation (Lach 2008a Table 11) Even if ants
decrease pollinator visitation rates there is limited scope for ants to decrease plant reproductive
success when plants are not pollen-limited Furthermore nearly all the studies in Table 11
measured only female fitness Only three quantified aspects of male fitness (Wyatt 1980 Fritz
and Morse 1981 Galen and Geib 2007) and we know of only one other study in a system in
which ants occasionally pollinate that has also measured male fitness (Ashman 2000) All four
studies measured pollen or pollinia removal rates and none found any effect of flower-visiting
ants on male fitness However whether other measures of male fitness (eg seeds sired pollen
deposition patterns) would reveal similar or different effects needs investigation
Finally although floral defenses against ants (see 3 below) may prevent us from
observing the negative effects of ants on plant reproductive success we consider this unlikely
Several studies have examined interactions between ants and plants that share little or no
coevolutionary history usually in the context of ant invasions Although invasive ants often
recruit to flowers in larger numbers than native ants they do not appear to reduce plant
12
reproductive success (Blancafort and Goacutemez 2005 Lach 2007 Lach 2008a but see Hansen and
Muumlller 2009) And in Hawaii where angiosperms evolved for millions of years in the absence of
ants and are thought to have secondarily lost floral defenses against ants (Junker et al 2011a)
there is no evidence that the preponderance of recently introduced flower-visiting ants negatively
affects plant fitness (Junker et al 2010a Bleil et al 2011)
2 Ants as pollinators
Ants may directly benefit plant reproduction by acting as pollinators but this is a relatively rare
occurrence Ants have long been considered poor pollinators (Kerner 1878) Initially this was
based on morphological and behavioral differences between ants and recognized pollinators
Worker ants are wingless and often relatively hairless making them poor pollen dispersers As
well because many ants exhibit Ortstreue (literally ldquoplace fidelityrdquo) ants were thought to return
to the same branch or flower repeatedly effecting no cross pollination (Houmllldobler and Wilson
1990) Though largely true these traits do not necessarily preclude ants from acting as
pollinators (Table 12) Although they commonly have restricted foraging ranges (Houmllldobler and
Wilson 1990) some ants can effect outcrossing (eg Ashman and King 2005 Carvalheiro 2008)
and many ants are as hairy if not hairier than their pollinating counterparts
In the late 1970s the ldquoantibiotic hypothesisrdquo was put forward to explain the rarity of ant
pollination Ants use antibiotic secretions to protect themselves against the fungal and bacterial
pathogens common in their nests and environs Iwanami and Iwadare (1978) found that one of
these compounds myrmicacin also greatly reduces pollen germination rates Subsequently
Beattie et al (1984 1985) found that short periods of contact (le 30 minutes) with an antrsquos
integument caused pollen grains to fail to germinate Some of these pollen-killing compounds
13
were traced back to the ant metapleural gland which produces hygienic secretions (Houmllldobler
and Wilson 1990) although contact with ants lacking metapleural glands can also reduce pollen
viability (Beattie et al 1985 Dutton and Frederickson 2012) Ant mandibular or anal gland
secretions and potentially ant venom may likewise defend ants against pathogens and have
similar effects on pollen germination (Beattie et al 1985 Graystock and Hughes 2011)
Nonetheless ant pollination has been documented repeatedly There are at least 18 cases
in which ant pollination has been shown experimentally and five others in which observations
strongly suggest it (Table 12) The majority of these plants fit the ldquoant pollination syndromerdquo
originally proposed by Hickman (1974) dense stands of short plants with small flowers and
easily accessible nectaries Typically ant-pollinated plants are associated with hot and dry
climates (principally in the Mediterranean) but dry alpine environments may also favor the
evolution of ant pollination (eg Svensson 1985 Puterbaugh 1998 Schiestl and Glaser 2011) In
most cases ants appear to be the sole or major pollinator of the plant in question (Hickman 1974
Peakall et al 1990 de Vega et al 2009) although ants sometimes make only supplementary
contributions to pollination (Schuumlrch et al 2000 Ashman and King 2005) This suggests that co-
evolution between ants and plants may not be necessary for ants to be useful pollinatorsmdashwhich
is surprising given the potential negative effects of ants on plant reproduction
Ant-pollinated plants cope with ant secretions in a variety of ways Several plant species
may simply tolerate pollen damage (Gόmez and Zamora 1992 de Vega et al 2009) apparently
possessing no overt adaptations to prevent it These plants might receive enough pollen to
overcome reduced pollen viability Other plants may place pollen on ant body parts thought to
have fewer secretions thereby potentially escaping damage (eg on ant legs Ramsey 1995) or
attach pollen to ants with specialized structures avoiding direct contact with the ant integument
14
(Peakall et al 1990) It is not known whether pollen evolves resistance to ant secretions or
whether plants deposit pollen on ants which have less disruptive secretions although these
possibilities have been suggested elsewhere (eg Garciacutea et al 1995 Ramsey 1995)
One key question regarding the effects of ant secretions on pollen grains urgently needs
to be resolved Several studies have suggested that the pollen-killing secretions of ants may have
contributed to the evolution of floral defenses against ants (eg Wagner 2000 Galen and Geib
2007) For this to be true ants must decrease male fitness by damaging pollen grains before they
leave flowers and are deposited on receptive stigmas Only two studies have investigated the
effects of ant secretions on pollen still in anthers (Wagner 2000 Galen and Butchard 2003) and
both constrained ants unnaturally to flowers for significantly longer than ants would normally
stay on flowers Although these studies found strong reductions in pollen vigor it is unclear if
ant secretions decrease male fitness under natural ant visitation conditions
3 Floral defenses against ants
Plants are not passive recipients of flower visitors and have some ability to ldquochooserdquo which
organisms can access floral rewards Flowers resist ants using both structural and chemical traits
More thorough investigation is needed to determine whether these traits evolved to prevent or
minimize the negative effects of ants on plant reproductive success (ie whether these traits can
truly be considered defenses against ants see 4ia below) It is unlikely however that ants
experience reciprocal selection to circumvent floral resistance traits because the fitness benefits
to ants of accessing flowers are probably small Thus there may be little scope for
coevolutionary arms races between floral defenses against ants and ant counter-adaptations
15
i) Structural resistance traits
a) Restricting ant access
Numerous plant species have physical structures preventing ant access to flowers Thin pedicels
can limit antsrsquo gripping abilities and make ants easier to dislodge when flowers are moved by
wind (Gaume et al 2005) and waxy secretions can render stems too slippery for ants to climb
(Harley 1991) Structures surrounding flowers such as water moats or dense hairs may similarly
keep ants from accessing floral rewards (Willmer et al 2009) Many such traits function as broad
filters and prevent not only ants but most crawling insects from reaching flowers Thus it is
difficult to determine whether ants florivores or both acted as selective agents favoring the
evolution of physical barriers to flowers
Flower shape may also reduce ant-pollinator conflict Long narrow corollas or nectar
spurs can prevent all but the smallest ants from accessing floral nectaries (Bluumlthgen et al 2004
Agarwal and Rastogi 2007) while still allowing long-tongued pollinators access to rewards
Selection to exclude nectar-thieving ants in such a manner however may be counteracted by
pollinator preferences for wider corollas that allow easier flower handling (Galen and Cuba
2001) Additionally these types of flowers may be more prone to robbery (Irwin and Maloof
2002) limiting their usefulness in preventing nectar larceny
b) Separation in space or time
Plants may separate floral rewards and ants temporally or spatially to minimize ant impacts on
plant reproduction Several authors have proposed that myrmecophytes have adapted to the
16
sterilization or otherwise detrimental activities of ants by locating domatia or food rewards on
different branches than flowers (Izzo and Vasconcelos 2002 Raine et al 2002 Edwards and Yu
2008) Temporal separation of ant and pollinator activities could likewise reduce conflict though
evidence is limited Some acacia flowers dehisce during periods of low ant activity (Willmer and
Stone 1997 Nicklen and Wagner 2006) but it is unclear if this is in response to ants
temperature or pollinator activity Timing of nectar secretion may similarly reduce ant-pollinator
competition If ants are active prior to pollinators they may deplete floral nectar and reduce
pollinator visitation rates (Schaffer et al 1984) However if nectar is secreted when ants are
inactive pollination may occur unimpeded (Norment 1988) This temporal separation could
favor pollinators that are active when ants are not (eg Wyatt and Morse 1981) but this is
poorly documented Further studies particularly focusing on flowers with the potential for
nocturnal and diurnal pollination may provide more examples
c) Extrafloral nectaries (EFNs)
One hypothesis put forward to explain the evolution of EFNs proposes that EFNs ldquodistractrdquo ants
from consuming floral nectar (Kerner 1878 Wagner and Kay 2002) Wagner and Kay (2002)
tested this hypothesis by constructing model plants using artificial nectaries assigned at random
to represent either floral or extrafloral nectaries They found that the presence of EFNs could
reduce ant exploitation of floral nectar However as highlighted by Galen (2005) this design is
closer to a plant making a surplus of flowers some of which will avoid ant visitation if visitation
rates increase sublinearly with increasing flower number Producing extra flowers to escape ant
visitation appears to be a viable strategy in some systems (Ashman and King 2005 Galen 2005)
However Galen (2005) found that applying honey (simulating extrafloral nectar) to P viscosum
17
leaves had the opposite effect and increased ant visits to flowers Only two studies both in
Pachycereus schottii have found evidence for the distraction hypothesis under field conditions
(Chamberlain and Holland 2008 Holland et al 2011)
There is limited empirical support for the distraction hypothesis but abundant evidence
that EFNs attract ants that defend plants against herbivores (reviewed in Rosumek et al 2009)
To function as attractants the presence of EFNs must increase ant abundance on plants
However because of the increased numbers attracted EFNs would not be expected to reduce the
abundance of ants on nectar rewards or be able to satiate the ants recruited Only if ant
abundance stops increasing at a reward density much lower than that available on the plant
would EFNs serve to distract ants In this case the amount of rewards may overwhelm ants
leaving some flowers underexploited However even in P schottii ants still visited flowers
(Chamberlain and Holland 2008) In cases where ant abundance would continue to increase with
additional rewards it is unlikely that EFNs function as distractions
ii) Chemical resistance traits
a) Toxic nectar
One of the earliest proposed floral defenses against ants was toxic nectar (Janzen 1977)
However studies testing Janzenrsquos (1977) hypothesis uncovered only a very few isolated
examples (eg Catalpa speciosa Stephenson 1981 Crinum erubescens Guerrant and Fiedler
1981) in short most floral nectar is not toxic or repellent to ants (Baker and Baker 1978
Schubart and Anderson 1978) In fact ants sometimes prefer floral nectar over solutions with
equivalent sugar concentrations (Koptur and Truong 1998)
18
In contrast floral nectar can repel other flower visitors (eg birds Johnson et al 2006
lepidopterans Kessler and Baldwin 2007) Repellency is often caused by plant secondary
metabolites which may occur in nectar as a by-product of their production elsewhere in the
plant where they function as defenses against herbivory (Adler 2001) Alternatively toxic or
repellent nectar may be adaptive repellent nectar may promote effective pollination by reducing
the amount of reward any one pollinator takes spreading nectar over several pollinator visits
(Kessler and Baldwin 2007) or it may function as a filter selecting for specific pollinators
(Johnson et al 2006) The latter may explain why repellent nectar is not more common
legitimate pollinators in addition to floral larcenists may be deterred from visiting flowers with
repellent nectar This may be particularly true of nectars that would repel ants which are closely
related to bees nectar distasteful to ants may often also be distasteful to bees and those nectars
acting as a filter for bees may still be palatable to ants For example Prŷs-Jones and Willmer
(1992) found that the ammonia-containing nectar of Lathraea clandestina is readily consumed
by both bees and ants even though it deters birds that act as nectar thieves Although floral
nectar itself rarely repels ants Guerrant and Fielder (1981) found that the presence of macerated
petals within nectar can often keep ants away Alkaloids in petals may be responsible and might
explain why ants seldom act as primary nectar robbers these secondary compounds may prevent
ants from chewing through corollas
b) Floral volatiles
Repellent floral volatiles appear to be a more widespread form of chemical resistance against
ants These were reviewed recently by Willmer et al (2009) and will be discussed here only
briefly Numerous plant taxa produce floral scents repellent to ants including myrmecophilous
19
myrmecophytic and non-ant associated plants in both temperate and tropical ecosystems
(Willmer et al 2009 Junker et al 2011a Ballantyne and Willmer 2012) In three large studies
floral volatiles that repel ants were detected in about a third of all plants tested (Willmer et al
2009 Junker et al 2011a Ballantyne and Willmer 2012) Pollen is often the source of ant-
repellent volatile compounds (Willmer et al 2009 Ballantyne and Willmer 2012) but they can
be emitted from other floral parts including petals That pollen is often the source of repellence
could suggest that preventing ant visitation may be more important in protecting male than
female function Additionally because repellence would decrease as pollen is removed from
anthers ants may return and defend flowers more quickly than if repellent chemicals were in
longer-lasting structures (eg petals) In some cases floral volatiles are thought to mimic ant
alarm pheromones (Willmer et al 2009) However floral scents elicit inconsistent reactions
across ant species ranging from repellence to attraction (Agarwal and Rastogi 2007 Ballantyne
and Willmer 2012)
Producing structural or chemical resistance traits may be costly for plants Native
Hawaiian plants which have little recent coevolutionary history with ants appear to have
secondarily lost floral ant repellents (Junker et al 2011a) Both Willmer et al (2009) and Junker
et al (2011a) found that highly chemical repellent flowers did not have strong physical defenses
and vice versa suggesting trade-offs between the two (but see Ballantyne and Willmer 2012)
Like toxic nectar floral ant repellents might sometimes deter both pollinators and ants For
example Galen et al (2011) found that 2-phenylethanol in P viscosum floral scent repels both
flower-damaging F neorufibarbis ants and the plantrsquos main pollinator Bombus kirbyellus
Interestingly four recent studies have found that floral scents can also attract ants
(Agarwal and Rastogi 2007 Edwards and Yu 2008 Schiestl and Glaser 2011 Gonzaacutelvez et al
20
2012) One study (Schiestl and Glaser 2011) has shown that floral scents attract ants to a
potentially ant-pollinated plant and such systems may provide many more examples
4 Future directions
We have highlighted areas in need of further investigation throughout the review Here we focus
on several questions we consider high priorities for future research
i) Floral defenses against ants
a) Do floral resistance traits increase plant fitness
The existence ant-repellent floral traits is often taken as evidence that ants negatively affect plant
reproductive success However despite the abundance and variety of mechanisms excluding ants
from flowers few studies show that ants reduce plant fitness when they visit flowers (Table 11)
This absence is particularly apparent with repellent floral volatiles which may be the most
widespread floral resistance trait (Willmer et al 2009 Junker et al 2011a Ballantyne and
Willmer 2012) To our knowledge only one study has shown that floral ant repellents benefit
plants with repellent P viscosum flowers avoiding damaging ant visits (Galen et al 2011)
Research is urgently needed to investigate if these traits increase plant fitness by excluding ants
or if ant visitation is inconsequential More studies taking advantage of standing variation in
traits (Galen et al 2011) or creating it artificially (eg Junker et al 2010b) will allow us to
distinguish between the two
21
b) If ant visitation is often not costly what caused the evolution of repellent traits and
what maintains them Similarly are these repellent traits truly defenses against ant
visitation or are they generalized defenses against a range of floral antagonists
Ant-repellent floral traits are widespread and their production may be costly (Willmer et al
2009 Junker et al 2011a) The studies in Table 11 however indicate that flower-visiting ants
often do not disrupt plant reproduction particularly outside of myrmecophytes What then
selected for these traits One possibility is that these repellents are not directed solely at ants
Instead a community of antagonistic flower visitors may exert diffuse selection on plants
thereby selecting for and maintaining these traits Structural modifications may repel crawling
insects while chemical repellents such as floral volatiles may repel both flying and non-flying
insects (Junker et al 2011b) How much overlap there is in floral volatiles that repel both ants and
other antagonists needs investigation Until we examine how specific these defenses are to ants
we risk overstating the importance of ants in their evolution
ii) Do ants alter pollen movement patterns or reduce male fitness
We are aware of only four studies that have measured if ant visitation impacts male fitness
(Wyatt 1980 Fritz and Morse 1981 Ashman 2000 Galen and Geib 2007) While none of these
studies found an effect all measured only amounts of pollen or pollinia removed However this
measure does not adequately encompass male fitness alterations to pollen movement patterns
and eventual deposition location may be more important to male function than changes in
removal rates If as suggested by some studies (Altshuler 1999 Lach 2007) ants increase
pollinator relocation rates they may decrease pollen discounting increasing male fitness
However if this increased relocation reduces overall pollen export rates ant competition with
22
pollinators may depress male function Studies using pollen-tracking methods such as fluorescent
dyes will allow us to see how ants change pollen movement patterns and methods like paternity
analysis will reveal how competition between ants and pollinators alters male fitness
iii) Coevolution between ants and plants do myrmecophytes exhibit stronger more
specific defenses against ants
The floral traits of myrmecophytes are most likely to coevolve with ants because all plant
reproduction occurs in the presence of ants Their floral defenses would thus be expected to be
both stronger and more specific to their resident ants than those other plants visited by an array
of more opportunistic ant species Some evidence supports this Willmer et al (2009) found that
resident ants of acacias were repelled by flowers with more aggressive ant species showing
stronger repellence than less aggressive ants Additionally ants not normally associated with
acacias did not respond to acacia floral volatiles More studies are needed in other systems and
should take advantage of phylogenetic approaches examining floral traits in closely related
myrmecophytic and non-myrmecophytic plant species to determine whether ants commonly
select for floral defenses against ants in myrmecophytes
Acknowledgements
For comments and discussion we thank the Frederickson lab particularly Eric Youngerman
Lina Arcila Hernandez and Kyle Turner as well as Alison Parker and Jordan Pleet MEF
acknowledges financial support from an NSERC Discovery Grant a Connaught New Researcher
23
Award an Ontario Ministry of Economic Development and Innovation Early Researcher Award
and the University of Toronto ARC was supported by an Ontario Graduate Scholarship and
Sigma Xi
24
References
Adler L S 2001 The ecological significance of toxic nectar Oikos 91409ndash420
Agarwal V M and N Rastogi 2007 Role of floral repellents in the regulation of flower visits
of extrafloral nectary-visiting ants in an Indian crop plant Ecological Entomology 3359ndash
65
Altshuler D L 1999 Novel interactions of non-pollinating ants with pollinators and fruit
consumers in a tropical forest Oecologia 119600ndash606
Ashman TL 2000 Pollinator selectivity and its implications for the evolution of dioecy and
sexual dimorphism Ecology 812577-2591
Ashman TL and E A King 2005 Are flower-visiting ants mutualists or antagonists A study
in a gynodioecious wild strawberry American Journal of Botany 92891ndash5
Ballantyne G and P Willmer 2012 Floral visitors and ant scent marks noticed but not used
Ecological Entomology 37402-409
Ballantyne G and P Willmer 2012 Nectar theft and floral ant-repellence a link between
nectar volume and ant-repellent traits PloS One 7e43869
Baker H G and I Baker 1978 Ants and flowers Biotropica 1080
Barrett S C H 2002 Sexual interference of the floral kind Heredity 88154ndash159
Baroni Urbani C and M L de Andrade 1997 Pollen eating storing and spitting by ants
Naturwissenschaften 84256ndash258
25
Beattie A J C Turnbull R B Knox and E G Williams 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany
71421ndash426
Beattie A J C Turnbull T Hough S Jobson and R B Knox 1985 The vulnerability of
pollen and fungal spores to ant secretions evidence and some evolutionary implication
American Journal of Botany 72606ndash614
Blancafort X and C Goacutemez 2005 Consequences of the Argentine ant Linepithema humile
(Mayr) invasion on pollination of Euphorbia characias (L) (Euphorbiaceae) Acta
Oecologica 2849ndash55
Bleil R N Bluumlthgen and R R Junker 2011 Ant-plant mutualism in Hawailsquoi Invasive ants
reduce flower parasitism but also exploit floral nectar of the endemic shrub Vaccinium
reticulatum (Ericaceae) Pacific Science 65291ndash300
Bluumlthgen N G Gottsberger and K Fiedler 2004 Sugar and amino acid composition of ant-
attended nectar and honeydew sources from an Australian rainforest Austral Ecology
29418ndash429
Burkle L A R E Irwin and D A Newman 2007 Predicting the effects of nectar robbing on
plant reproduction implications of pollen limitation and plant mating system American
Journal of Botany 941935ndash1943
Byk J and K Del-Claro 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333ndash38
26
Caballero P C G Ossa W L Gonzaacuteles C Gonzaacutelez-Browne G Astorga M M Muruacutea and
R Medel 2013 Testing non-additive effects of nectar-robbing ants and hummingbird
pollination on the reproductive success of a parasitic plant Plant Ecology 214633ndash640
Carvalheiro L G E R M Barbosa and J Memmott 2008 Pollinator networks alien species
and the conservation of rare plants Trinia glauca as a case study Journal of Applied
Ecology 451419ndash1427
Chamberlain S A and J N Holland 2008 Density-mediated context-dependent consumer-
resource interactions between ants and extrafloral nectar plants Ecology 891364ndash1374
Creighton W S 1967 Studies on free colonies of Cryptocerus texanus Satschi (Hymenoptera
Formicidae) Psyche 7434ndash42
Cursach J and J Rita 2011 Implications of the reproductive biology of the narrow endemic
Naufraga balearica (Apiaceae) for its conservation status Plant Systematics and Evolution
298581ndash596
Cushman JH and Addicott JF In Huxley CR and DF Cutler (eds) 1st edition Ant-plant
interactions Oxford University Press Oxford
de Jong T J N M Waser and P G Klinkhamer 1993 Geitonogamy the neglected side of
selfing Trends in Ecology and Evolution 8321ndash325
de Vega C M Arista P L Oritz C M Herrera and S Talavera 2009 The ant-pollination
system of Cytinus hypocistis (Cytinaceae) a Mediterranean root holoparasite Annals of
Botany 4517ndash8
27
Dukas R 2001 Effects of perceived danger on flower choice by bees Ecology Letters 4327ndash
333
Dutton E M and M E Frederickson 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561ndash569
Edwards D P and D W Yu 2008 Tolerating castration by hiding flowers in plain sight
Behavioral Ecology and Sociobiology 6395ndash102
Frederickson M E 2009 Conflict over reproduction in an ant-plant symbiosis why Allomerus
octoarticulatus ants sterilize Cordia nodosa trees The American Naturalist 173675ndash81
Fritz R S and D H Morse 1981 Nectar parasitism of Asclepias syriaca by ants effect on
nectar levels pollinia insertion pollinaria removal and pod production Oecologia 50316ndash
319
Galen C 1983 The effects of nectar thieving ants on seedset in floral scent morphs of
Polemonium viscosum Oikos 41245ndash249
Galen C 2005 Catching ants with honey an experimental test of distraction and satiation as
alternative modes of escape from flower-damaging ants Oecologia 14480ndash87
Galen C and B Butchart 2003 Ants in your plants effects of nectar-thieves on pollen fertility
and seed-siring capacity in the alpine wildflower Polemonium viscosum Oikos 101521ndash
528
Galen C and J Cuba 2001 Down the tube pollinators predators and the evolution of flower
shape in the alpine skypilot Polemonium viscosum Evolution 551963-1971
28
Galen C and J C Geib 2007 Density-dependent effects of ants on selection for bumble bee
pollination in Polemonium viscosum Ecology 881202ndash1209
Galen C R Kaczorowski S L Todd J Geib and R A Raguso 2011 Dosage-dependent
impacts of a floral volatile compound on pollinators larcenists and the potential for floral
evolution in the alpine skypilot Polemonium viscosum The American Naturalist 177258ndash
272
Garciacutea M B R J Antor and X Espadaler 1995 Ant pollination of the palaeoendemic
dioecious Borderea pyrenaica (Dioscoreaceae) Plant Systematics and Evolution 19817ndash
27
Gaume L M Zacharias and R M Borges 2005 Ant ndash plant conflicts and a novel case of
castration parasitism in a myrmecophyte Evolutionary Ecology Research 7435ndash452
Graystock P and W O H Hughes 2011 Disease resistance in a weaver ant Polyrhachis
dives and the role of antibiotic-producing glands Behavioral Ecology and Sociobiology
652319ndash2327
Gόmez J M 2000 Effectiveness of ants as pollinators of Lobularia maritima effects on main
sequential fitness components of the host plant Oecologia 12290ndash97
Gόmez J M and R Zamora 1992 Pollination by ants consequences of the quantitative effects
on a mutualistic system Oecologia 91410ndash418
Gόmez J M R Zamora J A Hόdar and D Garciacutea 1996 Experimental study of pollination
by ants in Mediterranean high mountain and arid habitats Oecologia 105236ndash242
29
Gonccedilalves-Souza T P M Omena J C Souza and G Q Romero 2008 Trait-mediated effects
on flowers artificial spiders deceive pollinators and decrease plant fitness Ecology
892407ndash2413
Gonzaacutelvez F G L Santamariacutea R T Corlett and M A Rodriacuteguez-Gironeacutes 2012 Flowers
attract weaver ants that deter less effective pollinators Journal of Ecology 10178ndash85
Guerrant E O and P L Fiedler 1981 Flower defenses against nectar-pilferage by ants
Biotropica 1325ndash33
Harley R 1991 The greasy pole syndrome In Huxley CR and DF Cutler (eds) 1st edition
Ant-plant interactions Oxford University Press Oxford
Hansen D M and C B Muumlller 2009 Invasive ants disrupt gecko pollination and seed
dispersal of the endangered plant Roussea simplex in Mauritius Biotropica 41202ndash208
Hickman J C 1974 Pollination by ants a low-energy system Science 1841290ndash1292
Holland J N S a Chamberlain and T E X Miller 2011 Consequences of ants and
extrafloral nectar for a pollinating seed-consuming mutualism ant satiation floral
distraction or plant defense Oikos 120381ndash388
Houmllldobler B and Wilson E O 1990 The Ants 1st Edition Harvard University Press
Cambridge
Holway DA L Lach AV Suarez ND Tsutsui and TJ Case 2002 The causes and
consequences of ant invasions Annual Review of Ecology and Systematics 33181-233
30
Horskins K and Turner VB 1999 Resource use and foraging patterns of honeybees Apis
mellifera and native insects on flowers of Eucalyptus costata Australian Journal of
Ecology 24221-227
Horvitz C C and D W Schemske 1984 Effects of ants and an ant-tended herbivore on seed
production of a neotropical herb Ecology 651369ndash1378
Inouye DW 1980 The terminology of floral larceny Ecology 611251-1253
Inouye DW and O R J Taylor 1979 A temperate region plant-ant-seed predator system
consequences of extra floral nectar secretion by Helianthella quinquenervis Ecology 601ndash
7
Irwin RE 2003 Impact of nectar robbing on estimates of pollen flow conceptual predictions
and empirical outcomes Ecology 84485-495
Irwin RE and Maloof JE 2002 Variation in nectar robbing over time space and species
Oecologia 133525-533
Iwanami Y and T Iwadare 1978 Inhibiting effects of myrmicacin on pollen growth and pollen
tube mitosis Botanical Gazette 13942ndash45
Izzo TJ and Vasconcelos HL 2002 Cheating the cheater domatia loss minimizes the effects
of ant castration in an Amazonian ant plant Oecologia 133200-205
Janzen D H 1977 Why donrsquot ants visit flowers Biotropica 9252
Johnson SD Hargreaves AL and Brown M 2006 Dark bitter-tasting nectar functions as a
filter of flower visitors in a bird-pollinated plant Ecology 872709-2716
31
Junker R A Y C Chung and N Bluumlthgen 2007 Interaction between flowers ants and
pollinators additional evidence for floral repellence against ants Ecological Research
22665ndash670
Junker R R R Bleil C C Daehler and N Bluumlthgen 2010a Intra-floral resource partitioning
between endemic and invasive flower visitors consequences for pollinator effectiveness
Ecological Entomology 35760ndash767
Junker R R and N Bluumlthgen 2010b Floral scents repel facultative flower visitors but attract
obligate ones Annals of botany 105777ndash82
Junker R R C C Daehler S Doetteri A Keller and N Bluumlthgen 2011a Hawaiian ant ndash
flower networks nectar-thieving ants prefer undefended native over introduced plants with
floral defenses Ecological Monographs 81295ndash311
Junker R R J Gershenzon and S B Unsicker 2011b Floral odor bouquet loses its ant
repellent properties after inhibition of terpene biosynthesis Journal of Chemical Ecology
371323ndash31
Kawakita A and M Kato 2002 Floral biology and unique pollination system of root
holoparasites Balanophora kuroiwai and B tobiracola (Balanophraceae) American
Journal of Botany 891164ndash1170
Koopowitz H and TA Marchant 1998 Postpollination nectar reabsorption in the African
epiphyte Aerangis verdickii (Orchidaceae) American Journal of Botany 85508-512
Kirk W D J 1984 Ecological studies on Thrips imaginis Bagnall (Thysanoptera) in flowers of
Echium plantagineum L in Australia Australian Journal of Ecology 99ndash18
32
Kerner A 1878 Flowers and their unbidden guests C Kegan Paul London
Kessler D and I T Baldwin 2007 Making sense of nectar scents the effects of nectar
secondary metabolites on floral visitors of Nicotiana attenuata The Plant Journal 49840ndash
54
Koptur S and N Truong 1998 Facultative ant-plant interactions nectar sugar preferences of
introduced pest species in South Florida Biotropica 30179-189
Lach L 2005 Interference and exploitation competition of three nectar-thieving invasive ant
species Insectes Sociaux 52257ndash262
Lach L 2007 A mutualism with a native membracid facilitates pollinator displacement by
Argentine ants Ecology 881994ndash2004
Lach L 2008a Argentine ants displace floral arthropods in a biodiversity hotspot Diversity and
Distributions 14281ndash290
Lach L 2008b Floral visitation patterns of two invasive ant species and their effects on other
hymenopteran visitors Ecological Entomology 33155ndash160
Lach L 2013 A comparison of floral resource exploitation by native and invasive Argentine
ants Arthropod-Plant Interactions 7177-190
Luo C W K Li X M Chen Z Y Huang and K U N Li 2012 Ants contribute significantly
to the pollination of a biodiesel plant Jatropha curcas Environmental Entomology
411163ndash1168
33
Maleacute PJ G C Leroy A Dejean A Quilichini and J Orivel 2011 An ant symbiont directly
and indirectly limits its host plantrsquos reproductive success Evolutionary Ecology 2655ndash63
Maloof J E 2001 The effects of a bumble bee nectar robber on plant reproductive success and
pollinator behavior American Journal of Botany 881960ndash1965
Maloof J E and D W Inouye 2000 Are nectar robbers cheaters or mutualists Ecology
812651ndash2661
Moreau CS CD Bell R Vila SB Archibald and NE Pierce 2006 Phylogeny of the ants
diversification in the age of the angiosperms Science 312101-104
Nahas L M O Gonzaga and K Del-Claro 2012 Emergent impacts of ant and spider
interactions herbivory reduction in a tropical savanna tree Biotropica 44498ndash505
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506ndash514
Newman D A and J D Thomson 2005 Interactions among nectar robbing floral herbivory
and ant protection in Linaria vulgaris Oikos 110497ndash506
Nicklen E F and D Wagner 2006 Conflict resolution in an ant-plant interaction Acacia
constricta traits reduce ant costs to reproduction Oecologia 14881ndash7
Norment C J 1988 The effect of nectar-thieving ants on the reproductive success of Frasera
speciosa (Gentianaceae) American Midland Naturalist 120331ndash336
34
Orivel J L Lambs P-J G Maleacute C Leroy J Grangier T Otto A Quilichini et al 2011
Dynamics of the association between a long-lived understory myrmecophyte and its
specific associated ants Oecologia 165369ndash76
Palmer T M D F Doak M L Stanton J L Bronstein E T Kiers T P Young J R
Goheen et al 2010 Synergy of multiple partners including freeloaders increases host
fitness in a multispecies mutualism Proceedings of the National Academy of Sciences
10717234ndash9
Peakall R A J Beattie and S H James 1987 Pseudocopulation of an orchid by male ants a
test of two hypotheses accounting for the rarity of ant pollination Oecologia 73522ndash524
Peakall R and A J Beattie 1989 Pollination of the orchid Microtis parviflora R Br by
flightless worker ants Functional Ecology 3515ndash522
Peakall R C J Angus A J Beattie 1990 The significance of ant and plant traits for ant
pollination in Leporella fimbriata pollination Oecologia 84457ndash460
Puterbaugh M N 1998 The roles of ants as flower visitors experimental analysis in three
alpine plant species Oikos 8336ndash46
Prŷs-Jones OE and Willmer PG 1992 The biology of alkaline nectar in purple toothwort
(Lathraea clandenstina) ground level defenses Biological Journal of the Linnean Society
45373-388
Raine NE Willmer P and Stone GN 2002 Spatial structuring and floral avoidance
behaviour present in ant-pollinator conflict in a Mexican ant-acacia Ecology 833086-
3096
35
Ramsey M 1995 Ant pollination of the perennial herb Blandfordia grandiflora (Liliaceae)
Oikos 74265ndash272
Renault CK LM Buffa and Delfino MA 2005 An aphid-ant interaction effects on
different trophic levels Ecological Research 2071-74
Rico-Gray V and L B Thien 1989 Effect of different ant species on reproductive fitness of
Schomburgkia tibicinis (Orchidaceae) Oecologia 81487ndash489
Romero G Q P A P Antiqueira and J Koricheva 2011 A meta-analysis of predation risk
effects on pollinator behaviour PLoS One 6e20689
Rosumek FB FAO Silveira F de S Neves NP de U Barbosa L Diniz Y Oki F Pezzini
et al 2009 Ants on plants a meta-analysis of the role of ants as plant biotic defenses
Oecologia 160537ndash549
Roubik D W 1982 The ecological impact of nectar-robbing bees and pollinating
hummingbirds on a tropical shrub Ecology 63354ndash360
Rudgers J A 2004 Enemies of herbivores can shape plant traits selection in a facultative ant-
plant mutualism Ecology 85192ndash205
Schatz B and WT Wcislo 1999 Ambush predation by the ponerine ant Ectatomma ruidum
Roger (Formicidae) on a sweat bee Lasioglossum umbripenne (Halictidae) in Panama
Journal of Insect Behavior 12641-663
36
Schaffer W M D W Zeh S L Buchmann S Kleinhaus M V Schaffer and J Antrim
1983 Competition for nectar between introduced honey bees and native North American
bees and ants Ecology 64564ndash577
Schemske D W 1980 The evolutionary significance of extrafloral nectar production by Costus
woodsonii (Zingiberaceae) an experimental analysis of ant protection Journal of Ecology
68959ndash967
Schiestl F P and F Glaser 2011 Specific ant-pollination in an alpine orchid and the role of
floral scent in attracting pollinating ants Alpine Botany 1221ndash9
Schubart HOR AB Anderson 1978 Why donrsquot ants visit flowers A response to D H
Janzen Biotropica 10310-311
Schuumlrch S M Pfunder and B A Roy 2000 Effects of ants on the reproductive success of
Euphorbia cyparissias and associated pathogenic rust fungi Oikos 16ndash12
Stanton ML Palmer TM Young TP Evans A and Turner ML 1999 Sterilization and
canopy modification of swollen thorn acacia tree by a plant-ant Nature 401 578-581
Stephenson AG 1981 Toxic nectar deters nectar thieves of Catalpa speciosa American
Midland Naturalist 105381-383
Stout J C and D Goulson 2001 The use of conspecific and interspecific scent marks by
foraging bumblebees and honeybees Animal Behaviour 62183ndash189
Sugiura S T Abe and S Makino 2006 Loss of extrafloral nectary on an oceanic island plant
and its consequences for herbivory American Journal of Botany 93491ndash495
37
Svensson L 1985 An estimate of pollen carryover by ants in a natural population of
Scleranthus perennis L (Caryophyllaceae) Oecologia 66373ndash377
Tsuji K A Hasyim and K Nakamura 2004 Asian weaver ants Oecophylla smaragdina and
their repelling of pollinators Ecological Research 19669ndash673
Turner R C J J Midgley P Barnard R E Simmons and S D Johnson 2012 Experimental
evidence for bird pollination and corolla damage by ants in the short-tubed flowers of
Erica halicacaba (Ericaceae) South African Journal of Botany 7925ndash31
Visser D M G Wright and J H Giliomee 1996 The effect of the Argentine ant
Linepithema humile (Mayr) (Hymenoptera Formicidae) on flower-visiting insects of
Protea nitida Mill (Proteaceae) African Entomology 4285ndash287
Wagner D 2000 Pollen viability reduction as a potential cost of ant association for Acacia
constricta (Fabaceae) American Journal of Botany 87711ndash715
Wagner D and A Kay 2002 Do extrafloral nectaries distract ants from visiting flowers An
experimental test of an overlooked hypothesis Evolutionary Ecology Research 4293ndash305
Wang C Y Luo Y Tai D An and Y Kou 2008 Ants pollinate Neottia listeroides
(Orchidaceae) in Sichuan China 46836ndash846
Wyatt R 1980 The impact of nectar-robbing ants on the pollination system of Asclepias
curassavica Bulletin of the Torrey Botanical Club 10724ndash28
Wheeler WM and IW Bailey 1920 The feeding habits of Pseudomymine and other ants
Transactions of the American Philosophical Society 22235-279
38
Willmer P G and S A Corbet 1981 Temporal and microclimatic partitioning of the floral
resources of Justicia aurea amongst a concourse of pollen vectors and nectar robbers
Oecologia 5167ndash78
Willmer P G and G N Stone 1997 How aggressive ant-guards assist seed-set in Acacia
flowers Nature 388165ndash167
Willmer P G C V Nuttman N E Raine G N Stone J G Pattrick K Henson P Stillman
et al 2009 Floral volatiles controlling ant behaviour Functional Ecology 23888ndash900
Young T P C H Stubblefield and L a Isbell 1997 Ants on swollen-thorn acacias species
coexistence in a simple system Oecologia 10998ndash107
Yu D W and N E Pierce 1998 A castration parasite of an ant-plant mutualism Proceedings
of the Royal Society B Biological Sciences 265375ndash382
39
Tables
Table 11 Studies of ant-flower or ant-pollinator interactions that included a measure of plant fitness Each study system is listed only
once except when results varied between years or when different studies examined male and female fitness Significant effects of ants on
plant fitness are either lsquopositiversquo or lsquonegativersquo non-significant effects are lsquoneutralrsquo When ants significantly affected only one fitness
component but several were measured (eg fruit set and seed production) we still consider the result significant
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Acacia
drepanolobium
Crematogaster
nigriceps
Proportion
fruiting
Negative Destruction of floral buds Young et al 1997
Asclepias exaltata Several Pollinia inserted
removed
Negative for pollinia
insertion neutral for
pollinia removal
Exploitative competition Wyatt 1980 T
40
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Asclepias syriaca Several
Fruit set pollinia
removed
Negative for fruit
set neutral for
pollinia removal
Exploitative competition Fritz and Morse
1981 T
Cordia nodosa Allomerus cf
demerarae
Fruit set Negative Destruction of floral buds Yu and Pierce
1998
Costus woodsonii Wasmannia
auropunctata
Seed set Positive Unknown ant pollination
suggested (but unlikely)
Schemske 1980
Eritrichum aretioides F neorufibarbis
gelida
Seed set Negative Ants damaged flowers Puterbaugh 1998
41
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Euphorbia characias Several Seed set Neutral Plants in Argentine ant-
invaded area set less seed
than those in un-invaded
sites but an ant-exclusion
experiment did not produce
significant results
Blancafort and
Gόmez 2005
Ferocactus wislizeni Solenopsis xyloni
and others
Seed set Unknown negative Interference and exploitative
competition S xyloni
reduced seed set relative to
other ants but the study did
not include a no-ant control
Ness 2006
42
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Frasera speciosa F neorufibarbis
and Formica sp
Seed set Neutral Nocturnal pollination
occurring when ants were
less active may have
compensated for reduced
diurnal pollinator visitation
Norment 1988
Gypsophila struthium Several Seeds per
inflorescence
Neutral No effect despite large
numbers of ants on flowers
Gόmez et al
1996
Hirtella physophora Allomerus
decemarticulatus
Fruit set Negative Destruction of flowers Orivel et al
2011
Humboldtia brunonis Crematogaster
dohrni
Fruit set Negative Destruction of flowers Gaume et al
2005
43
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Lepidium subulatum Several Percentage of
ovules setting
seed
Positive Unknown Gόmez et al
1996
Leucospermum
conocarpodendron
Several Seed set Neutral Site not pollen limited Lach 2008
Linaria vulgaris Several ldquoFemale fitness
indexrdquo
(combination of
number of seeds
fruits flowers
and seed weight)
Positive Reduction in florivory and
seed predation nectar
robbing holes increased ant
visitation
Newman and
Thomson 2005
44
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Melastoma
malabathricum
Oecophylla
smaragdina
Seed set Positive Ants deterred less efficient
pollinators increasing
visitation by the more
efficient pollinator
Gonzaacutelvez et al
2012
Metrosideros
polymorpha
Several Fruit set Neutral Ants reduced nectar
collection rates by foraging
bees but had no effect on
pollen collection rates or
fruit set
Junker et al
2010a
Orexis alpine F neorufibarbis
gelida
Seed set Neutral Autogamy or not pollen
limited
Puterbaugh 1988
45
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Ouratea spectabilis Cephalotes
pusillus and others
Seeds per fruit Neutral positive C pusillus had no effect on
seed number but other ants
collectively increased seed
set
Byk and Del
Claro 2010
Pachycereus schottii Several Seed set Neutral positive In one year ants showed a
positive effect (seed
number 2008) but no effect
with increased sample sizes
Chamberlain and
Holland 2008
Polemonium
viscosum
F neorufibarbis
gelida
Seed set Negative Ants damaged styles Galen 1983
46
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Polemonium
viscosum
F neorufibarbis
gelida
Pollen removal Neutral Ants did not affect pollen
removal even at high ant
densities
Galen and Geib
2007 T
Protea nitida Linepithema
humile and others
Seed set Neutral positive L humile had no effect on
P nitida seed set but native
ants increased it in one year
ants may cause pollinators
to relocate
Lach 2007
Psychotria
limonensis
Ectatomma spp Fruit set Positive Suggested increased
pollinator relocation
Altshuler 1999
47
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Roussea simplex Technomyrmex
albipes
Seed set Negative Interference and exploitative
competition
Hansen and
Muumlller 2009
Tristerix aphyllus Several Fruit set Neutral Nectar robbing ants
minimally reduced nectar
levels and had no effect on
hummingbird visitation
Caballero et al
2013
Vaccinium
reticulatum
Several Fruit set Neutral Ants reduced florivory but
did not affect fruit set
Bleil et al 2011
T Study measured male fitness
Study of sterilizing ants showed short-term decrease in female fitness long-term effects are unclear (see text)
48
Table 12 Studies demonstrating either i) ant pollination through an exclusion experiment or ii) that ants transfer pollen (or pollen
analogue) between flowers strongly suggesting ant pollination
Plant species Ant species Ant pollination Evidence Reference
Alyssum purpureum Several Yes Exclusion experiment Gόmez et al 1996
Arenaria tetraquetra Several Yes Exclusion experiment Gόmez et al 1996
Balanophora kuroiwai Leptothorax sp Suggested Exclusion experiment and
observed pollen transfer
and pollen tube formation
but did not see if plants set
seed
Kawakita and Kato 2002
Blandfordia grandiflora Iridomyrmex sp Yes Exclusion experiment Ramsey 1995
Borderea pyrenaica Several Yes Exclusion experiment Garciacutea et al 1995
49
Plant species Ant species Ant pollination Evidence Reference
Cytinus hypocistis Several Yes Exclusion experiment de Vega et al 2009
Epipactis thunbergii Camponotus
japonicas
Suggested Observed transfer of
pollinia from ants to
orchids
Sugiura et al 2006
Euphorbia cyparissias Several Yes Exclusion experiment Schuumlrch et al 2000
Fragaria virginiana Several Very likely Ant-only treatments set
comparable seed to flying
pollinator-only treatment
seed set increased with
increasing ant visitation
Ashman and King 2005
Frasera speciosa Several Yes Exclusion experiment Norment 1988
Frankenia thymifolia Several Yes Exclusion experiment Gόmez et al 1996
50
Plant species Ant species Ant pollination Evidence Reference
Hormathophylla spinosa Proformica
longiseta
Yes Exclusion experiment Gόmez and Zamora 1992
Jatropha curcas
Several
Tapinoma
melanocephalum
most common
Yes Exclusion experiment Luo et al 2012
Leporella fimbriata Myrmecia urens Yes Observed pollinia transfer
and subsequently
confirmed as ant
pollination
Peakall et al 1987
51
Plant species Ant species Ant pollination Evidence Reference
Lobularia maritima Several
Camponotus
micans most
common
Yes Exclusion experiment Gόmez 2000
Microtis parviflora
Iridomyrmex
gracilis
Yes Observation used
laboratory colonies to
replicate observations
Peakall and Beattie 1989
Naufraga balearica
Several
especially
Plagiolepis
pygmaea and
Lasius grandis
Suggested Observation over three
years of study only ants
were observed visiting
ants carried pollen
Cursach and Rita 2011
52
Plant species Ant species Ant pollination Evidence Reference
Neottia listeroides Leptothoras sp
Paratrechina sp
Suggested Observed transfer of
pollinia from ants to
orchids
Wang et al 2008
Paronychia pulvinata Formica
neorufibarbis
gelida
Yes Exclusion experiment Puterbaugh 1998
Polygonum cascadense Formica argentea
Very likely Grew plants in greenhouse
open to flying visitors with
minimal seed set only
plants with access to ants
set significant seed
Hickman 1974
53
Plant species Ant species Ant pollination Evidence Reference
Retama sphaerocarpa Several Yes Exclusion experiment Gόmez et al 1996
Scleranthus perennis
Several
especially
Formica
rufibarbis
Suggested Ants transferred pollen
analogue to stigmas and
carried pollen on bodies
Svensson 1985
Sedum anglicum Proformica
longiseta
Yes Exclusion experiment Gόmez et al 1996
Trinia glauca Several Yes Exclusion experiment Carvalheiro et al 2008
54
Chapter 2 Ants and ant scent reduce bumblebee pollination of artificial flowers
Cembrowski AR Tan MG Thomson JD and Frederickson ME
In press The American Naturalist
All authors designed the experiment ARC and MGT performed the experiment ARC and MEF
performed statistical analyse
s and ARC wrote the manuscript with input from all authors
Abstract
Ants on flowers can disrupt pollination by consuming rewards or harassing pollinators but it is
difficult to disentangle the effects of these exploitative and interference forms of competition on
pollinator behavior Using highly rewarding and quickly replenishing artificial flowers that
simulate male or female function we allowed bumblebees (Bombus impatiens) to forage (1) on
flowers with or without ants (Myrmica rubra) and (2) on flowers with or without ant scent cues
Bumblebees transferred significantly more pollen analogue both to and from ant free flowers
demonstrating that interference competition with ants is sufficient to modify pollinator foraging
behavior Bees also removed significantly less pollen analogue from ant scented flowers than
from controls making this the first study to show that bees can use ant scent to avoid harassment
at flowers Ant effects on pollinator behavior possibly in addition to their effects on pollen
viability may contribute to the evolution of floral traits minimizing ant visitation
55
Introduction
Trait-mediated indirect interactions arise when a focal species causes phenotypic changes
including behavioral modifications in a second species and these effects cascade to still other
species (Werner and Peacor 2003) Although trait-mediated indirect interactions are often studied
in food webs (eg Werner and Peacor 2003 Preisser et al 2005) they are not limited to trophic
interactions For example the threat of predation can change the behavior of a mutualist thus
affecting its partners (Suttle 2003) In animal-pollinated plants predators can disrupt pollination
directly through density-mediated indirect interactions (Dukas 2005) or via trait-mediated
indirect interactions by changing pollinator behavior When predators are present pollinators
may switch to visiting less rewarding flowers or they may avoid flowers altogether potentially
reducing the amount of pollen donated or received by a flower and thus decreasing plant fitness
(Gonccedilalves-Souza 2008 Ings and Chittka 2009) Similarly competition for floral rewards with
other flower visitors may cause behavioral changes in pollinators (Maloof and Inouye 2000
Ohashi et al 2008) Previous research has shown that bees may spend less time visiting flowers
depleted of rewards (Thomson 1986) and may avoid flowers bearing scent marks that indicate
recent visits by other individuals of the same or different bee species as these flowers are likely
to be depleted (Stout and Goulson 2002)
Whereas competition between pollinators may not be particularly costly for the plant
because both competitors are likely to provide pollination services competition between
pollinators and organisms that do not usually provide a pollination service such as ants has a
greater potential to reduce plant fitness Ants are common floral visitors they are attracted to
flowers for nectar (Lach 2007) and in some cases pollen (Byk and Del Claro 2010) and will
often defend these resources against other flower visitors (Altshuler 1999) However ants rarely
56
contribute much useful pollination antibiotic secretions present on their cuticles kill pollen
grains (Beattie et al 1985 Dutton and Frederickson 2012) and when foraging in flowers ants
sometimes impede female function by damaging stigmas (Galen and Cuba 2001) We will
henceforth consider ants to be non-pollinators and will contrast them to animals like bees that
are pollinators Ants are capable of competing with pollinators in two ways (1) by consuming
floral rewards and reducing their availability to pollinators (ie exploitative competition) and (2)
by directly antagonizing and excluding pollinators from flowers (ie interference competition)
Many plants benefit from the presence of ants numerous plant species recruit ants using
extrafloral nectar food bodies or domatia because ants provide protection against folivores (Heil
and McKey 2003) Similarly some plants have extrafloral nectaries on or near reproductive
tissues that recruit ants that deter florivores (eg Inouye and Taylor 1979) However plants
may incur reproductive costs as a result of their association with ants Some ant species that nest
in myrmecophytes sterilize flowers to shunt plant resources from reproduction to vegetative
growth allowing for greater ant colony growth (Frederickson 2009) Also ants visiting
extrafloral nectaries may be attracted to floral nectaries where they can harass pollinators (Ness
2006) There is growing evidence that many angiosperms have evolved floral traits that prevent
ants from accessing flowers during anthesis (Willmer and Stone 1997 Ballantyne and Willmer
2012) The effects of flower-visiting ants can be substantial Ants can decrease the frequency
duration or species diversity of pollinator visits to flowers (Lach 2008 Hansen and Muumlller 2009
Junker et al 2010 Gonzaacutelvez et al 2012) all of which potentially impact pollen donation and
receipt In some studies changes to pollinator behavior induced by ants have also been linked to
seed set
It is difficult however to disentangle the intertwined effects of exploitative and
interference competition in nature If ants disrupt pollination plants may often evolve traits to
57
deter ants from flowers (Willmer et al 2009) From a practical standpoint this would then limit
our ability to study the effects of ants on pollinator visitation because ants would not visit
flowers Here we used artificial flowers to explore ant-bee interactions in the absence of floral
defenses against ants We used highly rewarding quickly replenishing artificial flowers to
examine how direct harassment (ie interference competition expressed through behavior)
changes bee foraging behavior and how this affects donation and receipt of a pollen analogue
(powdered food dye) while minimizing the effects of exploitative competition We predicted
that flowers visited by ants would both donate and receive less pollen analogue than flowers
without ants because bees would avoid flowers with ants or leave them sooner Because scent
plays a large role in bumblebee communication and flower choice (Stout and Goulson 2002) we
also tested whether the presence or absence of ant scent on artificial flowers would affect pollen
analogue donation because of changes in the foraging behavior of bumblebees
Methods
i) Subjects
Myrmica rubra is an invasive ant in eastern North America with a range and habitat preferences
overlapping that of Bombus impatiens a common bumblebee Because M rubra visits flowers
(A Cembrowski pers obs) the two species likely interact in nature We collected 12 M rubra
colonies in the fall of 2011 and 2012 from Toronto Ontario Canada and the surrounding area
and maintained them in environmental chambers on artificial diet (Dussutour and Simpson 2009)
and a 1410 LD schedule (light 600-2200) We used these colonies as sources of M rubra
workers for experiments
58
Workers from commercially supplied colonies of Bombus impatiens (Biobest Canada
Ltd) foraged on artificial flowers in flight cages (either 24 x 24 x 21 m or 79 x 34 x 20m) at
the University of Toronto We tested a total of five bee colonies four colonies were used in the
ant presence trials and two of these and one additional colony were used in the ant scent trials
Flight cages had overhead fluorescent lights attached to timers In contrast to most previous
studies in which bees have been trained and tested individually the entire worker force of a bee
colony was free to forage at will in our experiments Colonies were trained to forage on artificial
flowers for at least four days before being used in trials After being used for a trial the colony
was not used for at least two days to reduce dye carryover between trials Between trials
bumblebees were fed pollen and given sugar water
ii) Artificial flowers
The flowers (Fig 1 Thomson et al 2012 see also Makino 2008) consisted of glass jars filled
with 30 WV sucrose solution Sugar water travelled by capillary action up a sewing-thread
wick to a hole in a blue-painted lid accumulating in a knot that acts as a nectary Flowers
depleted by visitors were quickly replenished via capillary action and were non-rewarding only
very briefly after visits taking less than a minute to accumulate 05 microl note that bumblebee
visits to a single flower are often separated by several minutes or more (A Cembrowski
unpublished data)
Unlike most previous artificial flower designs these flowers allow for estimation of male
and female fitness because we can measure both the amount and type of dye received by female
flowers (see (d) Dye quantification below) In order to access nectar on a ldquomalerdquo flower a bee
must crawl through a circular ldquoantherrdquo of brush-like weather stripping material dusted in a
59
consistent manner with our pollen analogue powdered food dye (FDampC 5 or 6) which is
transferred to the bee in the process On a ldquofemalerdquo flower a bee crawls through a sticky plastic
reinforcement that functions as a ldquostigmardquo receiving dye from the beersquos body Male flowers
dispensed dye particles over multiple bee visits and could still dispense dye at the end of trials
They resemble many real flowers or flower heads in that pollen is dispensed gradually over time
but less dye is available for transfer with each subsequent bee visit (Harder and Thomson 1989)
iii) Flight cage trials
We conducted 6 ant scent trials between 22 March and 4 June 2012 and the remaining trials (16
ant presence trials and 9 ant scent trials) between 9 November 2012 and 6 January 2013 Ant
presence trials lasted for eight hours and ant scent trials lasted for four hours Artificial flowers
were prepared and placed individually in small plastic containers treated with Fluon (Insect-a-
Slip BioQuip Products Inc) to prevent ants from escaping In ant presence trials we used 32
flowers (16 male and 16 female flowers) arranged in an eight-by-four array with flowers spaced
45 cm apart between rows and 30 cm within rows Ant scent trials used 20 flowers (10 male and
10 female flowers) in a four-by-five array with flowers spaced 30 cm apart between and within
rows In all of the ant presence trials and 9 of the ant scent trials we counted the number of bees
actively foraging after one hour to get a measure of colony activity
a) Ant presence trials
We examined the effects of ants on the amount of dye donated by male flowers and received by
female flowers in sixteen trials testing four bee colonies four times each The containers of eight
randomly chosen male flowers and eight randomly chosen female flowers received 15 M rubra
60
workers each the other 16 flowers remained free of ants Isolated from their colonies M rubra
workers have nowhere to deposit sugar water they collect and therefore may become sated
nonetheless a force of 15 ant workers was enough to maintain visitation to the nectaries where
bees foraged while still rarely having more than one or two ants visit the nectary at any time As
ants attacked bees bees would occasionally carry or throw attacking ants off flowers but despite
this some ants maintained their presence at the nectary throughout the trials Because ants the
size of M rubra consume liquids slowly (~017 ndash 024 microlmin Davidson et al 2004) flowers
replenished nectar more quickly than ants consumed it We used two colors of dye (FDampC 5 and
FDampC 6) to differentiate male flowers with and without ants In two trials for each bee colony
anthers of male flowers with ants were coated with FDampC 5 dye while brushes of male flowers
lacking ants were coated with FDampC 6 dye In the other two trials we reversed dye colors to
control for effects of dye type and color Dye color was assigned in a random order Artificial
flowers were placed in randomly assigned positions in the array before we opened the colony
and allowed the bees to begin foraging
b) Scent trials
We explored the effect of ant scent on dye donation in fifteen trials testing three bee colonies in
six five and four trials each Bees were first exposed to ants by allowing the bees to forage for
eight hours on twenty flowers (ten male and ten female) of which five male flowers had 15 ants
and all others had none No dye was used during this ldquoexposurerdquo day We then collected all but
the five ant-free male flowers in the flight cage leaving these flowers to keep bees foraging
Next we individually stored five new male lids in Fluon-treated containers with ten M rubra
workers Five control male lids were put in identical containers lacking ants The following
61
morning we removed the remaining flowers and set out ten new male and female flowers in a
random spatial arrangement using the lids having or lacking ant scent Thus the ant-scented
flowers were not in the same positions as the ant-visited flowers on which the bees were trained
Only five male flowers had ant scent and the other 15 flowers (five male and ten female) did not
We used the same two dye colors as in i) to differentiate male flowers with and without ant
scent and randomized which color was used for ant-scented flowers between trials We collected
and replaced stigmas from female flowers after one hour and collected the stigmas again after
four hours In the first trial performed we also collected stigmas after two hours but due to the
small amount of dye transferred this was not repeated
iv) Dye quantification
After each trial we quantified the amount of dye transferred to female flowers using a
spectrophotometer We removed the stigmas from female flowers placed them in test tubes
added 51 mL of distilled water to each tube and vortexed each tube thoroughly to ensure the
dye was evenly diluted These were diluted further as needed if the absorbance exceeded the
sensitivity range of the spectrophotometer In the first six scent trials each stigma was analyzed
separately and the dye amounts were summed to obtain a total amount of each dye color
transferred to female flowers in each trial In all the ant presence trials and the other nine scent
trials we opted to treat the experiment as the unit of replication Therefore we combined stigmas
from each treatment type (ant-visited or ant-free) in a test tube and measured the total amount of
each dye color transferred in each treatment in each trial
Because we put different dye colors on male flowers with and without ants we could use
the amount of each dye color donated to all female flowers to measure the reproductive success
62
of ant-visited and ant-free male flowers The total amount of dye (of both colors) received by
female flowers with and without ants was our measure of female reproductive success We
calculated the amount of each dye color in the sample by measuring absorbance at 428 or 486
nm and converting absorbance to micrograms following computational methods for overlapping
spectra (Blanco et al 1989)
v) Statistical analyses
In two trials (one ant presence and one ant scent) most female flowers received no dye because
of low bee activity so we excluded these trials from analyses Because dye reception and
donation values were non-normally distributed we square-root transformed the data before
examining the effects of ant presence or ant scent on dye transfer in ANCOVAs For ant
presence trials we included ant presence on male and female flowers and their interaction as
main effects and the total amount of dye transferred in each trial as a covariate to account for
the large variation in overall dye transfer among trials For ant scent trials we included ant scent
as the main effect time (one or four hours) as a repeated measure the interaction between scent
and time and the total amount of dye transferred as a covariate Covariate by treatment (ant
presence or ant scent) interactions were never significant and so were excluded in final analyses
All analyses were conducted in JMP (v 1000)
Results
Bees usually started foraging within minutes of the beginning of the trial and continued until
flowers were collected from the flight cage or the lights were extinguished An average of 34 plusmn
012 and 40 plusmn 015 (mean plusmn SE) bees were foraging after an hour in the ant presence and ant
63
scent trials respectively Male flowers with ants donated significantly less dye than male flowers
lacking ants (Fig 22a F155 = 4019 p lt 00001) Similarly female flowers with ants received
significantly less dye than female flowers lacking ants (Fig 22a F155 = 461 p = 0036) There
was no significant interaction between ant presence on male flowers and ant presence on female
flowers (F155 = 118 p = 028) Flowers with ant scent donated significantly less dye than
flowers without ant scent (Fig 22b F125 = 11216 p lt 00001) There was no significant effect
of time (F125 = 121 p = 028) or interaction between time and scent (F125 = 111 p = 030) in
the model
Discussion
In this study ants altered bumblebee pollination behavior and exhibited trait-mediated indirect
interactions with (artificial) flowers Through interference competition M rubra workers
significantly affected the pattern of dye transfer by B impatiens causing a preferential flow of
dye from male flowers lacking ants to female flowers lacking ants If manifested in nature such
effects would reduce the reproductive success of plants visited by ants through both male and
female function
Ants could have changed the attractiveness of artificial flowers in at least two ways First
as ants interrupted or altogether prevented bumblebees from foraging at nectaries bumblebees
may have learned to avoid flowers with ants due to the relative inefficiency of foraging at these
flowers something bumblebees take into account (Heinrich 2004) Second bees attempt to
minimize their risk of being attacked during foraging bouts which ants did to visiting bees Ants
often attacked or harassed bees by biting grasping and appearing to sting visiting bees
preventing them from accessing the nectary or reducing their time on flowers (see online video
64
1) and bees sometimes avoided ant-tended flowers entirely This harassment was sometimes
physically traumatic (online video 1) and bees often appeared to have trouble flying after being
attacked by ants Previous research has demonstrated that bumblebees leave or avoid flowers
where they have been harassed (Jones and Dornhaus 2011) and avoid foraging where there is
visual or olfactory evidence of a predator or predation event (Abbott 2006 Goodale and Nieh
2012) Though it is unclear if bumblebees viewed ants as competitors or predators they
responded similarly to flowers having ants as they do to flowers housing predators (Gonccedilalves-
Souza et al 2008)
To avoid artificial flowers with ants bees likely used a combination of visual and
olfactory cues Previous research has shown that bees respond to conspicuous predators or
predator ldquodummiesrdquo on flowers (Suttle 2003 Gonccedilalves-Souza et al 2008) decreasing the
frequency and duration of their visits to these flowers Thus bees may have been able to avoid
flowers harboring ants by sight alone However even in the absence of ants bees still
preferentially visited flowers lacking ant scents (Fig 22b) suggesting that they had learned to
associate ant scent with harassment Bees are adept at associative learning (Wright and Schiestl
2009) and can learn to recognize unique scents left behind by both conspecific and heterospecific
flower visitors (Stout et al 1998) Bees can use these various scents often arising from tarsal
gland deposits (Stout et al 1998) to recognize recently visited flowers that are less likely to be
profitable (Stout and Goulson 2002) Recently Ballantyne and Willmer (2012) demonstrated that
bees learn to associate ant scents with unrewarding artificial flowers and decrease their visitation
to these flowers Our results complement their findings by showing that bees can associate ant
scent with harassment at otherwise rewarding flowers
In our study ant scent caused bees to adjust their foraging strategy and decreased the
amount of pollen analogue that was donated by ant-scented flowers Thus the effects of
65
interference competition with ants on flowers can extend beyond immediate interactions and may
have fitness consequences for plants even when ants are absent Like other olfactory cues these
effects are likely transitory (Stout et al 1998) Although more dye was still donated by male
flowers lacking ant scent than those with ant scent the ratio of dye donated was on average
closer to equality in hours 2-4 than in the first hour (Fig 22b) This effect may be partially
driven by dye depletion of male flowers without ant scent but the lack of a strong corresponding
decrease in flowers with ant scent suggests that visitation patterns became more similar We did
not test whether B impatiensrsquo avoidance of ant scent was an innate or a learned behavior but
previous work has shown that Bombus terrestris does not innately avoid flowers with ant scent
(Ballantyne and Willmer 2012)
Ants may be necessary for plant survival and growth but can be costly for plant
reproduction The evolution of ant attractants such as extrafloral nectaries in some plant lineages
suggests that the costs of ants can be outweighed by their protective abilities However in this
study flowers visited by ants received and donated significantly less dye although they did
retain some sexual function The net benefit of having ants depends on whether ants increase
plant fitness by reducing herbivory more than they decrease plant fitness by disrupting
pollination Alternatively plants may actually benefit from the costs of ants to plant
reproduction ants that castrate flowers may be better defenders increasing plant survival or
vegetative growth when plants are young allowing for increased reproduction later in life when
the plant is colonized by less aggressive non-castrating ants (Frederickson 2009 Palmer et al
2010) In some plant species pollinator harassment by ants may even be beneficial Altshuler
(1999) reported that Ectatomma ants greatly increased fruit set of Psychotria limonensis despite
reductions in pollinator visitation rates presumably due to increases in the rate of pollen
outcrossing Similarly Gonzaacutelvez et al (2012) found that the presence of Oecophylla
66
smaragdina ants on Melastoma malabathricum flowers reduced visitation by less effective
pollinators and increased visitation by more effective Xylocopa bees increasing plant fitness
Cases of ants being beneficial to flowers appear to be the minority and many plant
species have traits that limit ant access to flowers Several studies have detailed floral volatiles
that are thought to mimic ant alarm pheromones and thus repel ants (Junker and Bluumlthgen 2008
Willmer et al 2009) Other plants use structural modifications such as narrow corollas or
slippery stems to limit ant access to flowers (reviewed in Willmer et al 2009) Exploitative
competition between ants and pollinators (Lach 2005 Ballantyne and Willmer 2012) as well as
the lethal effects of ant antibiotic secretions on pollen grains (Beattie et al 1985 Dutton and
Frederickson 2012) may have resulted in selection on plants to reduce ant visits to flowers Our
results suggest that trait-mediated indirect interactions resulting from interference competition
between ants and bees may favor plants that defend their flowers and their pollinators against
ants
Acknowledgements
We thank H Rusnock and S Meadley-Dunphy for assistance J Dix for help with flight cage
construction and Biobest for bumblebee colonies We are grateful to the Thomson and
Frederickson labs for guidance particularly T Makino funded by JSPS for helping to refine
artificial flowers J Ogilvie for help with bees K Prior for M rubra colony collections and K
Turner for study design suggestions The comments of the editor and two anonymous reviewers
prompted improvements in the article especially with regards to study design MEF and JDT
acknowledge NSERC Discovery Grant funding The University of Torontorsquos Faculty of Arts and
Science supported MGRTrsquos participation
67
68
References
Abbott K R 2006 Bumblebees avoid flowers containing evidence of past predation events
Canadian Journal of Zoology 841240-1247
Altshuler D L 1999 Novel interactions of non-pollinating ants with pollinators and fruit
consumers in a tropical forest Oecologia 119600-606
Ballantyne G and Willmer P 2012 Floral visitors and ant scent marks noticed but not used
Ecological Entomology 37402-409
Beattie A J Turnbull C Knox R B and Williams E G 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany
71421-426
Blanco M Runiaga H Maspoch S and Tarin P 1989 A simple method for
spectrophotometric determination of two-components with overlapped spectra Journal of
Chemical Education 66178-180
Byk J amp Del Claro K 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333-38
Davidson D W S C Cook and R R Snelling 2004 Liquid-feeding performances of ants
(Formicidae) ecological and evolutionary implications Oecologia 139255ndash66
Dukas R 2005 Bumble bee predators reduce pollinator density and plant fitness Ecology
861401-1406
Dussutour A and Simpson S J 2009 Communal nutrition in ants Current Biology 19740-
744
69
Dutton E M and Frederickson M E 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561-569
Frederickson ME 2009 Conflict over reproduction in an ant-plant symbiosis why Allomerus
octoarticulatus ants sterilize Cordia nodosa trees American Naturalist 173675-681
Galen C and Cuba J 2001 Down the tube pollinators predators and the evolution of flower
shape in the alpine skypilot Polemonium viscosum Evolution 551963-1971
Gonccedilalves-Souza T Omena P M Souza J C and Romero G Q 2008 Trait-mediated
effects on flowers artificial spiders deceive pollinators and decrease plant fitness Ecology
892407-2413
Gonzaacutelvez F G Santamariacutea L Corlett R T and Rodriacuteguez-Gironeacutes M A 2012 Flowers
attract weaver ants that deter less effective pollinators Journal of Ecology 10178ndash85
Goodale E and Nieh JC 2012 Public use of olfactory information associated with predation
in two species of social bees Animal Behaviour 84919-924
Harder LD and Thomson JD 1989 Evolutionary options for maximizing pollen dispersal of
animal-pollinated plants American Naturalist 133323-344
Heil M and McKey D 2003 Protective ant-plant interactions as model systems in ecological
and evolutionary research Annual Review of Ecology Evolution and Systematics 34425-
453
Heinrich B 2004 Bumblebee economics 2nd
edn Harvard University Press Cambridge
Ings T C and Chittka L 2009 Predator crypsis enhances behaviourally mediated indirect
effects on plants by altering bumblebee foraging preferences Proceedings of the Royal
Society B 2762031-2036
70
Inouye D W and Taylor O R Jr 1979 A temperate region plant-ant-seed predator system
consequences of extra floral nectar secretion by Helianthella quinquenervis Ecology 601-
7
Junker R R and Bluumlthgen N 2008 Floral scents repel potentially nectar-thieving ants
Evolutionary Ecology Research 10295ndash308
Junker R R Bleil R Daehler C C and Bluumlthgen N 2010 Intra-floral resource partitioning
between endemic and invasive flower visitors consequences for pollinator effectiveness
Ecological Entomology 35760ndash767
Jones E I and Dornhaus A 2011 Predation risk makes bees reject rewarding flowers and
reduce foraging activity Behavioral Ecology and Sociobiology 651505-1511
Lach L 2005 Interference and exploitative competition of three nectar-thieving invasive ant
species Insect Sociaux 52257-262
Lach L 2007 A mutualism with a native membracid facilitates pollinator displacement by
Argentine ants Ecology 881994-2004
Lach L 2008 Argentine ants displace floral arthropods in a biodiversity hotspot Diversity and
Distributions 14281-290
Makino T T and Sakai S 2007Experience changes pollinator responses to floral display size
from size-based to reward-based foraging Functional Ecology 21854863
Maloof J E and Inouye D W 2000 Are nectar robbers cheaters or mutualists Ecology
812651ndash2661
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506-514
Ohashi K Leslie A and Thomson JD 2008 Trapline foraging by bumble bees V Effects of
experience and priority on competitive performance Behavioral Ecology 19936-948
71
Palmer T M Doak D F Stanton M L Bronstein J L Kiers E T Young T P Goheen
J R and Pringle R M 2010 Synergy of multiple partners including freeloaders
increases host fitness in a multispecies mutualism Proceedings of the National Academy
of Sciences 10717234-17239
Preisser E L Bolnick D I and Benard M E 2005 Scared to death The effects of
intimidation and consumption in predator-prey interactions Ecology 86501-509
Stout J C Goulson D and Allen J A 1998 Repellent scent-marking of flowers by a guild of
foraging bumblebees (Bombus spp) Behavioral Ecology and Sociobiology 43317-326
Stout J and Goulson D 2002 The influence of nectar secretion rates on the responses of
bumblebees (Bombus spp) to previously visited flowers Behavioral Ecology and
Sociobiology 52239-246
Suttle K B 2003 Pollinators as mediators of top-down effects on plants Ecology Letters
6688-694
Thomson JD 1986 Pollen transport and deposition by bumble bees in Erythronium influences
of floral nectar and bee grooming Journal of Ecology 74329-341
Thomson JD Ogilvie J E Makino T T Arisz A Raju S Rojas-Luengas V and Tan M
G R 2012 Estimating pollination success with novel artificial flowers effects of nectar
concentration Journal of Pollination Ecology 9108-114
Tsuji K Hasyim A and Nakamura K 2004 Asian weaver ants Oecophylla smaragdina and
their repelling of pollinators Ecological Research 19669-673
Ulrich S and Bertsch A 1990 Do foraging bumblebees scent-mark food sources and does it
matter Oecologia 82137-144
Werner E E and Peacor S D 2003 A review of trait-mediated indirect interactions in
ecological communities Ecology 841083-1100
72
Willmer P G Nuttman C V Raine N E Stone G N Pattrick J G Henson K Stillman
P McIlroy L Potts S G and Knudsen J T 2009 Floral volatiles controlling ant
behaviour Functional Ecology 23888-900
Wright G A and Schiestl F P 2009 The evolution of floral scent the influence of olfactory
learning by insect pollinators on the honest signaling of floral rewards Functional Ecology
23841-851
73
Figures
Figure 21 Photograph of artificial flower lids A = anther L = lid N = nectary R = region
where anther or stigma is placed S = stigma T = thread W = weight
74
Figure 22 Dye transferred (mean plusmn 1SE) per trial by B impatiens workers A) from artificial
male and to artificial female flowers in the presence (unfilled circles) or absence (filled circles)
of Myrmica rubra workers and B) from artificial male flowers with (unfilled circles) or without
(filled circles) M rubra scent to ant-free female flowers in the 1st or the 2
nd to 4
th hours of the
trials
75
Chapter 3 Not just for the bees pollen consumption is common among tropical
ants
Cembrowski AR Reurink G and Frederickson ME
Planned submission to Biotropica
ARC and MEF designed the study ARC and GR did laboratory work ARC performed statistical
analyses and wrote the manuscript with input from other authors
Abstract
Although palynivory or pollen consumption is widespread among other hymenopterans there
are few accounts of palynivory in ants To quantify how often ants consume pollen we subjected
adult workers and larvae from 75 species of neo- and paleo-tropical ants to acetolysis a process
that destroys most organic material but leaves behind pollen grains In over half of the species
we examined ants contained a few pollen grains (lt 5) and ants of several Camponotus species
contained abundant pollen grains (gt50) We tested for an association between trophic level and
palynivory using stable nitrogen isotope ratios (δ15
N) but we did not find a significant
correlation we found pollen grains in lsquoherbivorousrsquo omnivorous and carnivorous ants We
suggest that our results indicate sporadic opportunistic pollen consumption by ants in tropical
forests
Introduction
For those organisms that can digest it efficiently pollen can be an excellent food source
Depending on the plant species pollen can contain up to 60 protein as well as lipids
76
carbohydrates and numerous trace nutrients (Roulston and Cane 2000) Pollen feeding
(palynivory) occurs in many taxa including birds mammals and arthropods (Roulston and Cane
2000) but it is best known among the Hymenoptera particularly bees Although adult bees feed
mainly on plant exudates (ie nectar) nearly all bee species rely on pollen as the chief protein
source for developing larvae (Willmer 2011) Closely related to bees ants have similar
nutritional requirements adult workers need carbohydrates whereas larvae are fed a diet rich in
protein (Dussutour and Simpson 2009) Ants also exploit nectar heavily and regularly visit
flowers (eg Herrera et al 1984 Bluumlthgen et al 2004) but unlike bees are not believed to
widely consume pollen Instead ants are thought to receive most of the protein in their diet from
predation or scavenging (eg Floren et al 2002)
However ants may not be as reliant on animal protein sources as commonly assumed In
tropical forests arboreal ants can have similar δ15
N ratios a measure of trophic level as
herbivores suggesting limited carnivory and substantial feeding on plant-derived protein sources
(Davidson et al 2003) One potential protein source is pollen This idea is supported by scattered
accounts of pollen consumption by ants from other ecosystems including deserts (Ness 2006)
boreal forests (Czechowski et al 2009) and tropical savannas (Byk and Del Claro 2010) One
ant genus Cephalotes is widely recognized to consume pollen (Creighton 1967 Baroni Urbani
and de Andrade 1997 Byk and Del Claro 2010) However beyond this genus our knowledge of
pollen consumption by ants has largely been limited to direct observations (but see Czechowski
et al 2011) and it is unknown if palynivory is truly rare among ants or simply under-reported
We borrowed a technique commonly used in palynology acetolysis to assess the frequency of
pollen consumption by ants in 75 neo- and paleo-tropical ant species This technique allowed us
to examine many more ant species than we could have observed directly
77
Methods
Adult worker ants were collected by hand at baits or from leaf litter using Winkler extractors
while larvae along with adults were collected by hand from nests Collections were made by L
Arcila Hernandez and J Sanders in the primary tropical rainforest surrounding the Centro de
Investigaciόn y Capacitaciόn Rio Los Amigos (CICRA 12deg34rsquoS 70deg05rsquoW) in Peru in 2010-
2011 and by E Youngerman from lowland primary and secondary rainforests in Madang
Province Papua New Guinea (PNG) in 2011-2012 We sorted ant samples to morphospecies and
then identified them to genus and when possible to species using existing keys which were
unavailable for PNG ants Ant samples used for acetolysis were whole adult workers guts from
adult workers and whole larvae
We used acetolysis to look for pollen grains inside ants In acetolysis organic material is
dissolved in acetic anhydride and sulfuric acid leaving behind stained pollen exines When
possible we used adult and larval ants of the same species from different colonies or bait
locations in an attempt to sample more representatively We prepared samples by first washing
them in 70 ethanol to remove any pollen present on the samplersquos outer surface before placing
them individually in micro-centrifuge tubes with ~05mL of a 91 mixture of acetic anhydride
sulfuric acid We then heated each sample at 95degC for 20min allowing tissues to dissolve Next
we centrifuged samples at 1000 RPM for 5min and decanted the liquid supernatant before
adding ~05mL of glacial acetic acid to the precipitate centrifuging again as above We decanted
the samples and added a drop of distilled water
78
We examined a 150μL sub-sample of this solution for pollen grains under a light
microscope Larvae gave less than 150μL of solution following acetolysis so we sampled the
entire amount We identified pollen grains by their staining and their surface architecture For
most species we examined at least three adult workers and three larvae but for some species
fewer than three adult workers or larvae were available (Tables 31 and 32)
We investigated the relationship between trophic level as measured by δ15
N ratios and
pollen consumption by ants Stable isotope (SI) data was available for some (n =23) of the
Peruvian species from a separate project in which collections overlapped Samples were stored in
EtOH and dried at 50degC overnight We used heads legs and thoraxes (but not gasters) of ants
for SI analysis Often single individuals did not provide enough material for analysis and we
combined multiple individuals to achieve necessary weights up to 10 for small ant species
Samples were analyzed at the Boston University Stable Isotope Laboratory using a Finnigan
Delta-S analyzer For other Peruvian species for which we had samples but not corresponding
stable isotope data (n =25) we took the median δ15
N ratio value of that speciesrsquo genus and
assigned it to the species in question Using pollen presenceabsence data for these 48 species
we used logistic regression to correlate pollen presence and δ15
N ratio using R (v 2151)
Results
We found pollen grains in 37 of the 197 adult workers 20 of the 102 larvae and 38 of the 75 ant
species we examined (Tables 31 and 32) The proportion of individuals containing pollen did
not differ significantly between larvae and adults (χ2
1 = 002 p = 088) The number of pollen
grains found was often low (median = 3) but highly variable (range 1-468) Also pollen
79
presence was inconsistent among conspecific ant samples of species that contained pollen 31
had pollen in only one of the larvae or adults examined In only three species (two Cephalotes
and one Myrmelachista) did all examined adults contain pollen
Highly lsquoherbivorousrsquo carnivorous and omnivorous ants contained pollen grains (Fig
31) There was no correlation between δ15
N ratio and pollen presence (χ2 = 150 df = 1 p =
022) In fact several carnivorous species contained pollen grains (eg Odontomachus sp 1
Pachycondola sp1)
Discussion
Pollen consumption by ants appears to be more common than suspected The majority of species
(51) had at least one individual that had consumed pollen and in 20 multiple individuals
contained pollen Numbers of grains found tended to be low however with most individuals
containing only a few grains Ants may use pollen as only a small portion of their diet exploiting
a host of other protein-containing foods more heavily including other low- δ15
N foods (eg
honeydew epiphylls Houmllldobler and Wilson 1990 Davidson et al 2003)
There are a few reasons why pollen may not be heavily exploited by ants Pollen requires
much post-consumption processing to extract its nutrients These nutrients are located in the
pollenrsquos cytoplasm which is protected from direct digestion by the exines and intines of the
grains Organisms must either break through these layers or cause the pollen grains to germinate
or pseudo-germinate to access and digest the cytoplasm (Roulston and Cane 2000) This can be
achieved by consuming a sugar-containing solution (such as nectar) along with pollen However
animals that are not adapted to feed on pollen may not be very efficient at this process only
80
extracting nutrients from some of the grains (Herrera and Martiacutenez del Rio 1998) Thus while
ants can likely get some nutrients from pollen other forms of more easily processed protein may
be more attractive
Pollen may also pose handling difficulties for ants Despite the large number of studies
that have recorded ants visiting flowers (Chapter 1) only a few papers detail ants harvesting
pollen directly from anthers Byk and Del Claro (2010) reported that Cephalotes pusillus ants
removed almost all of the available pollen from Ouratea spectabilis flowers and at least two
other studies describe ants consuming pollen along with nectar from flowers (Horskins and
Turner 1999 Ness 2006) Unlike bees which possess both morphological (eg specialized hairs
for holding pollen) and behavioral adaptations (eg stereotyped pollen-packing motions) ants
have no apparent mechanisms to increase the amount of pollen harvested and are likely limited
by the number of grains they can ingest or carry Rather than collect pollen directly from flowers
it appears more likely that ants collect it haphazardly in the environment leading to a smaller
number of pollen grains consumed at any one time It is known that at least Cephalotes ants
engage in such behavior Creighton (1967) observed Cephalotes texanus collecting pollen grains
trapped by Citrus x paradisi leaf hairs
There is a chance that the small numbers of pollen grains we observed came from
secondary pollen consumption (ie consuming a palynivorous organism) or consumption of
liquid containing pollen While we cannot rule this out we believe our results represent actual
targeted pollen consumption by ants Cephalotes ants known palynivores (Creighton 1967
Baroni Urbani and de Andrade 1997) contained similar numbers of pollen grains as many of the
pollen-containing ant species that we examined As well in at least once instance ants that we
found containing pollen grains were in the process of preying on non-pollen consuming
81
organisms (eg a Pachycondola species that was carrying a termite in its mandibles) or were
attracted by sugar baits
Contrary to our expectations there was no relationship between an ant speciesrsquo δ15
N ratio
and whether it had consumed pollen (Fig 32) We expected palynivory to be more common in
herbivorous ants because pollen presents a widespread source of protein a potentially limiting
resource The lack of pattern might reflect how available pollen grains are in the environment
While other forms of food may be preferable ants tend to be opportunistic foragers (Carroll and
Janzen 1973) and there is likely little cost associated with the consumption of scattered pollen
grains during foraging trips beyond the volume they occupy in the gut Some genera were found
to consistently consume pollen In support of previous observations (Baroni Urbani and de
Andrade 1997 Byk and Del Claro 2010) Cephalotes species were among the most common
pollen consumers Camponotus species also consistently contained pollen grains of the eight
species examined five contained pollen and pollen was found in species from both Peru and
PNG Camponotus species also contained the two largest numbers of pollen grains found (468
and 271) Taken together these results suggest that they may be another as yet unrecognized
commonly palynivorous genus
In general larvae did not contain large numbers of pollen grains (ranging from 1-38
grains) This is counter to what is seen in bees where larvae are the main recipients of pollen
(Willmer 2011) This difference may be because ant larvae are continually fed via trophallaxis
by adults throughout their development whereas bee larval cells are provisioned with pollen and
then sealed while larvae complete their development If both adults and larvae are able to extract
nutrients from pollen we would expect to see a preferential flow of nutrients from pollen to
larvae but not necessarily the grains themselves
82
Unlike palynivory by organisms that also provide pollination services pollen
consumption by ants is unlikely to benefit plants Ants rarely act as pollinators (Beattie et al
2004) They have pollen-killing antibiotic secretions on their cuticles (Dutton and Frederickson
2012) and they can compete with other flower visitors reducing flower visitation rates (Ness
2006) The negative impacts of flower-visiting ants are thought to have led to the evolution of
ant-repellent floral volatiles (reviewed in Willmer 2009) These volatiles can pre-empt
competition between ants and pollinators and may often be located within the pollen itself
(Willmer 2009) If floral volatiles truly are a defense against ant visitation our results suggest an
additional evolutionary pressure to not only prevent ant-pollinator interactions but to also
protect plant gametes from occasional consumption by a non-pollinating visitor
Acknowledgements
We would like to thank Jon Sanders Lina Arcila Hernandez and Eric Youngerman for providing
samples the Thomson lab for use of supplies the Pierce lab for funding support during JSrsquo and
EYrsquos collections and the staff of CICRA and the New Guinea Binatang Research Center for
logistical support The Peruvian Ministry of Agriculture provided permits (Nos 394-2009-AG-
DGFFS-DGEFFS and 299-2011-AG-DGFFS-DGEFFS) MEF acknowledges financial support
from an NSERC Discovery Grant a Connaught New Researcher Award an Ontario Ministry of
Economic Development and Innovation Early Researcher Award and the University of Toronto
ARC was supported by an Ontario Graduate Scholarship and Sigma Xi
83
References
Baroni Urbani C and M L de Andrade 1997 Pollen eating storing and spitting by ants
Naturwissenschaften 84256ndash258
Beattie A J C Turnbull R B Knox and E G Williams 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany 71421ndash
426
Bluumlthgen N G Gottsberger and K Fiedler 2004 Sugar and amino acid composition of ant-
attended nectar and honeydew sources from an Australian rainforest Austral Ecology
29418ndash429
Byk J and K Del-Claro 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333ndash38
Carroll C R and D H Janzen 1973 Ecology of foraging ants Annual Review of Ecology and
Systematics 4231ndash257
Creighton W S 1967 Studies on free colonies of Cryptocerus texanus Satschi (Hymenoptera
Formicidae) Psyche 7434ndash42
Czechowski W B Markό and A Radchenko 2009 Rubbish dumps reveal the diet of ant
colonies Myrmica schencki Em and Myrmica rubra (L) (Hymenoptera Formicidae) as
facultative pollen-eaters Polish Journal of Ecology 56737ndash741
Czechowski WB Markoacute K Erős and E Csata 2011 Pollenivory in ants (Hymenoptera
Formicidae) seems to be much more common than it was thought Annales Zoologici
61519ndash525
84
Dussutour A and S J Simpson 2009 Communal nutrition in ants Current Biology 19740ndash4
Dutton E M and M E Frederickson 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561ndash569
Floren A A Biun and K E Linsenmair 2002 Arboreal ants as key predators in tropical
lowland rainforest trees Oecologia 131137ndash144
Grant B R 1996 Pollen digestion by Darwinrsquos finches and its importance for early breeding
Ecology 77489-499
Herrera L G and C Martiacutenez del Rio 1998 Pollen digestion by new world bats effects of
processing time and feeding habits Ecology 792828ndash2838
Houmllldobler B and Wilson E O 1990 The Ants 1st Edition Harvard University Press
Cambridge
Horskins K and Turner V B 1999 Resource use and foraging patterns of honeybees Apis
mellifera and native insects on flowers of Eucalyptus costata Australian Journal of
Ecology 24221-227
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506ndash514
Roulston T H I and J H Cane 2000 Pollen nutritional content and digestibility for animals
Plant Systematics 222187ndash209
Van Tets I G 1997 Extraction of nutrients from Protea pollen by African rodents Belgian
Journal of Zoology 12759-65
Willmer P G 2011 Pollination and Floral Ecology 1st Edition Princeton University Press
New Jersey
85
Willmer P G C V Nuttman N E Raine G N Stone J G Pattrick K Henson P Stillman
et al 2009 Floral volatiles controlling ant behaviour Functional Ecology 23888ndash900
86
Tables
Table 31 Numbers of adult worker and larval ants examined for each species collected in Peru
Numbers of pollen grains found in individual samples are given in parentheses after the sample
size when there were no grains no numbers are given
Genus Species Number of adults analyzed
(numbers of grains found)
Number of larvae analyzed
(numbers of grains found)
Dolichoderinae
Azteca sp 1 3 0
sp 2 2 0
sp 3 0 2
sp 4 3 (1) 0
sp 5 3 (2) 0
Dolichoderus decollatus 3 (66) 0
sp 1 3 0
sp 2 3 1 (2)
sp 3 3 3
sp 4 0 2
Ecitoninae
Labidus sp 1 3 0
Formicinae
Camponotus sp 1 3 3
sp 2 3 (82) 1 (2)
sp 3 3 (3) 0
sp 4 0 2
87
sp 5 8 (11) 0
sp 6 4 (7 16 468) 0
sp 7 5 (12) 0
sp 8 2 (1) 0
Paratrechina sp 1 4 (11) 0
Myrmelachista sp 1 3 (5 11 18) 0
sp 2 0 3
Trachymyrmex sp 1 3 (21) 0
sp 2 3 0
Myrmicinae
Atta sp 1 3 0
Cephalotes atratus 3 (2 2 21) 0
placidus 3 (1 4 21) 0
sp 1 3 0
sp 2 1 0
sp 3 2 0
Crematogaster sp 1 3 1
sp 2 3 (35) 0
sp 3 9 (7) 0
sp 4 2 0
Megalomyrmex sp 1 3 0
sp 2 0 2
sp 3 4 (7) 0
sp 4 3 0
Monomorium sp 1 0 3 (2)
88
Pheidole sp 1 0 3
sp 2 3 0
sp 3 0 3
Procryptocerus sp 1 3 0
Solenopsis sp 1 0 3
sp 2 3 (3 5) 0
Wasmannia sp 1 3 0
Ponerinae
Pachycondola sp 1 3 (3) 0
Odontomachus sp 1 3 (34) 0
Pseudomyrmicinae
Pseudomyrmex sp 1 3 0
sp 2 3 0
sp 3 3 (9) 0
sp 4 0 3
sp 5 0 3
89
Table 32 Numbers of adult worker and larval ants examined for each species collected in Papua
New Guinea Numbers of pollen grains found in individual samples are given in parentheses
after the sample size when there were no grains no numbers are given
Genus Species Number adults analyzed
(grains found)
Number larvae analyzed
(grains found)
Aenictinae
Aenictus sp 1 3 3 (1 1)
Dolichoderinae
Philidris sp 1 3 1 (4)
Tapinoma sp 1 2 3 (38)
Technomyrmex sp 1 0 3 (12 12)
Ectatomminae
Rhytidoponera sp 1 1 3 (2)
Formicinae
Acropyga sp 1 3 3 (2)
sp 2 3 3
sp 3 3 3
Anoplolepsis sp 1 3 (1) 3 (1)
Calomyrmex sp 1 3 3 (1)
Camponotus sp 1 3 (1 271) 3
sp 2 1 3
sp 3 2 0
Nylanderia sp 1 3 3
Opisthopsis sp 1 3 3
Polyrachis sp 1 1 3 (4)
90
Myrmicinae
Crematogaster sp 1 3 3
sp 2 3 (32) 3
Monomorium sp 1 3 (1) 3 (1)
sp 2 3 3 (1 1 1)
Pheidole sp 1 3 3
Pristomyrmex sp 1 3 3 (11)
Pyramica sp 1 3 0
Ponerinae
Odontomachus sp 1 3 3 (4)
91
Figures
Figure 31 Photographs of pollen grains found in two ant species a) Dolichoderus sp 2 and b)
Camponotus sp 6 Scale bars = 50 microm
92
Figure 32 Relationship between trophic level as measured by δ15
N ratio and pollen presence
for 48 neotropical ant species 1 Atta sp 5 Azteca spp 8 Camponotus spp 5 Cephalotes spp 4
Crematogaster spp 5 Dolichoderus spp 1 Labidus sp 3 Megalomyrmex spp 1 Monomorium
sp 2 Myrmelachista spp 1 Odontomachus sp 1 Pachycondola sp 1 Paratrechina sp 3
Pheidole sp 4 Pseudomyrmex spp 2 Solenopsis spp and 1 Wasmannia sp
93
Concluding Remarks
Ants have traditionally been considered to be unwelcome flower visitors they consume floral
rewards ostensibly meant for pollinators while rarely providing pollination services themselves
In this thesis I investigate the validity of this assumption and use an experimental study and an
observational study to examine two ways that ants may impact plant reproduction
In the first chapter I review the literature on ants as flower visitors I show that the common
assumption of ants always being categorically negative for plant reproduction is incorrect while
ants may reduce floral attractiveness to pollinators the net effect of ants on plant fitness
measures is variable ranging from positive to negative to neutral I also discuss ants as
pollinators and ways that plants may minimize ant visitation to flowers before finishing with a
section highlighting questions that still need investigation
In the second chapter I explore how interference competition with ants can structure bumblebee
foraging behavior and how this may alter pollen movement patterns Using artificial flowers with
male or female function I allowed Bombus impatiens bumblebees to forage on flowers that
either did or did not have Myrmica rubra ants on them Ant presence significantly reduced the
amount of pollen analogue donated and received by flowers Building on these results I found
that the presence of ant scent alone elicited similar changes to bumblebee behavior reducing the
amount of pollen analogue donated by male flowers My results show that interference
competition is sufficient to alter bumblebee foraging behavior and that the effects of ants on
pollinators may extend after ant visitation to flowers Further study is needed to determine if bees
are able to differentiate between ant species by visual or olfactory cues Their ability to do so
94
could affect how generalized their response to ant presence is and may help to better predict how
ants will affect plant reproduction
In the final chapter I investigate how common palynivory is amongst tropical ant species I used
acetolysis and light microscopy to examine whole ants larvae and ant guts from Papua New
Guinea and Peru I found that tropical ants consistently contain pollen grains albeit often in
small numbers (lt5) However some genera notably Camponotus and Cephalotes consistently
contained higher numbers providing the first evidence that Camponotus may be a palynivorous
genus The small numbers of pollen grains present may indicate that ants preferentially exploit
other more easily digestible high-protein foods over pollen Future studies should investigate
exactly what role pollen plays in ant diets by supplementing the diet of captive ant colonies with
pollen This would allow us to examine how pollen consumption affects colony demography as
well as how efficiently ants digest pollen and whether ants exhibit pollen preferences
I bring up several pressing and unanswered questions within this thesis and highlight three here
First numerous plant species possess chemical or physical traits assumed to increase plant
fitness by reducing ant visitation However this has rarely been tested and instead research has
focused predominately on documenting ant repellence We should move away from this and
begin examining plant fitness in the presence or absence of these repellent traits to see how
beneficial these traits truly are Second how flower visiting ants impact male fitness has been
almost completely ignored in favor of female fitness Future studies should investigate how ant
visitation affects both pollen donation and pollen movement patterns Lastly research involving
antibiotics present on ant cuticles and their effect on pollen viability has been too small in scope
often limited to demonstrating the pollen killing effects of ant secretions Investigation into
95
whether plants have evolved any resistance to the secretions of the local ant community or why
some plants appear unaffected by their ant partners may yield exciting results
In conclusion ants have the capacity to strongly affect plant reproduction both positively and
negatively The fact that these diametrically opposing forces exist means we cannot approach
flower-visiting ants as either positive or negative a priori Instead we should strive to work
towards a better understanding of what is occurring in the system of interest and what role ants
play within it
96
Copyright Acknowledgments
Chapter 2 ldquoAnts and ant scent reduce bumblebee pollination of artificial flowersrdquo is current in
press in The American Naturalist Request for its inclusion here was sought and approved
iv
Lastly my family did an astounding amount for me in letting me cultivate my love of biology
buying me books letting me keep strange animals and getting me outdoors So thank you to my
brothers John and Mark my dad George and my mom Kay And to my partner Katherine for
her unbelievable patience both in regards to my academic life and in her willingness to adapt
hers for mine thank you This could not have happened without you
ldquoVor Allem sind aus der Reihe der fluumlgellosen Insecten die weitverbreiteten fluumlgellosen Ameisen
sehr unwillkommene Gaumlste der Bluumlthen Und dennoch sind gerade sie nach dem Nectar der
Bluumlthen in hohem Grade luumlsternhelliprdquo
ldquoOf all the wingless insects it is the widely dispersed ants that are the most unwelcome guests to
flowers And yet they are the very ones which have the greatest longing for nectarhelliprdquo
A Kerner 1878 Flowers and their Unbidden Guests
v
Table of Contents
Acknowledgments iii
List of Tables vii
List of Figures viii
Chapter 1 Ants as flower visitors and their effects on pollinator behavior and plant
reproduction 1
Abstract 1
Introduction 2
1 Ant interactions with flowers and floral visitors 3
2 Ants as pollinators 12
3 Floral defenses against ants 14
4 Future directions 20
References 24
Tables 39
Chapter 2 Ants and ant scent reduce bumblebee pollination of artificial flowers 54
Abstract 54
Introduction 55
Methods 57
Results 62
Discussion 63
Acknowledgements 66
References 68
Figures 73
Chapter 3 Not just for the bees pollen consumption is common among tropical ants 75
vi
Abstract 75
Introduction 75
Methods 77
Results 78
Discussion 79
Acknowledgements 82
References 83
Tables 86
Figures 91
Concluding Remarks 93
Copyright Acknowledgments 96
vii
List of Tables
Table 11 List of studies of flower-visiting ants that consumed floral rewards or interacted with
pollinators and included a measure of fitness39
Table 12 List of studies that have either demonstrated ant pollination through exclusion
experiments or whose results strongly suggest it48
Table 31 Numbers of adult worker and larval ants examined for each species collected in Peru
and numbers of pollen grains found87
Table 32 Numbers of adult worker and larval ants examined for each species collected in Papua
New Guinea and numbers of pollen grains found90
viii
List of Figures
Figure 21 Photograph of artificial flower lids with anther and stigma74
Figure 22 Dye transferred (mean plusmn 1SE) by B impatiens a) in the presence and absence of M
rubra ants and b) with or without M rubra scent75
Figure 31 Photographs of pollen grains found inside ants92
Figure 32 Relationship between trophic level and pollen presence for neotropical
ant species93
1
Chapter 1 Ants as flower visitors and their effects on pollinator behavior and plant
reproduction
Cembrowski AR and Frederickson ME
Planned submission to a journal such as Oikos or Insectes Sociaux
ARC wrote the manuscript with input from MEF
Abstract
Ants often provide a range of benefits to flowering plants particularly by reducing herbivory
However when ants visit flowers they can deter pollinators or damage floral structures
Currently it is unclear how often and in what direction flower-visiting ants affect plant
reproductive success A review of the literature shows that ants can help or hinder pollination
processes but overall flower-visiting ants most commonly have a non-significant net effect on
plant fitness Although ants may directly harm flowers or indirectly affect pollination by
consuming floral nectar or harassing pollinators they can also benefit plants by attacking
florivores or predators of pollinators Ants occasionally act as pollinators themselves though ant
secretions often kill pollen grains There is growing evidence that many plants have traits that
allow them to avoid or minimize the negative effects of flower-visiting ants More research is
needed to determine if these traits provide fitness benefits to plants by preventing ant visitation
or if they function as generalized repellents of floral antagonists
2
Introduction
Ants and angiosperms share a long evolutionary history Ant diversification closely followed that
of flowering plants as ants took advantage of the large prey numbers that angiosperms supported
and the new habitats they provided (Moreau 2006) Subsequently numerous angiosperm
lineages evolved intimate associations with ants that function as an indirect or biotic form of
plant defense Food rewards such as extrafloral nectar or pearl bodies or housing in the form of
domatia attract ants to plants where they reduce herbivory and increase plant fitness (reviewed
in Rosumek et al 2009) However the traits that make ants good plant defenders particularly
their abundance attraction to sugar sources and aggressiveness can also cause problems for
plant reproduction Ants attracted by floral nectar can compete with pollinators for resources
and defend flowers potentially reducing plant fitness (Ness 2006 Junker et al 2007) That ants
negatively affect plant reproduction is an old and pervasive hypothesis Kerner (1878) called ants
ldquomost unwelcome guestsrdquo on flowers and more recent authors have similarly expressed that ant
visitation to flowers reduces plant reproductive success (Ness 2006 Lach 2007 Willmer et al
2009)
Nonetheless studies assessing the impact of flower-visiting ants on plant reproductive
success have produced mixed results Ants can have negative impacts on pollination and flower
function (Galen 1983 Puterbaugh 1998 Ness 2006 Hansen and Muumlller 2009) However other
studies have found no effect or even strong benefits of ant visits to flowers (Rico-Gray and
Thien 1989 Altshuler 1999 Schuumlrch 2000 Ashman and King 2005) This discrepancy between
ants being ldquounwelcome guestsrdquo and their actual effects on plant reproduction requires
clarification Our goal here is to highlight the ways that flower-visiting ants may affect plant
reproduction both positively and negatively and to discern through a review of the literature
3
what impacts ants have on plant reproductive success First we discuss mechanisms by which
flower-visiting ants can directly and indirectly impact plant reproduction both through floral
damage and by mediating changes in pollinator or florivore behavior We end this section by
evaluating published research that collectively shows that flower-visiting ants have
predominantly neutral effects on plant fitness measures Next we discuss pollination by ants
focusing on the ldquoantibiotic hypothesisrdquo before describing how plants may prevent ants from
visiting flowers Lastly we discuss promising new avenues of research and highlight work that
still needs to be performed to give a more complete understanding of ants as flower visitors
1 Ant interactions with flowers and floral visitors
i) Damage to flowers
Ants can directly disrupt plant reproduction by damaging floral structures preventing either the
fertilization of ovules or the presentation of pollen Rarely ants forage on floral tissue itself for
example Puterbaugh (1998) found that Formica neorufibarbus ants chewed through the coronal
ring of Eritrichium aretiodes flowers to get lipids More often but still not commonly foraging
ants chew through reproductive tissue to access floral nectar This can cause sterilization of
whole flowers (Echium plantagineum Kirk 1984) or affect female function only if just styles are
damaged effectively creating male flowers (Polemonium viscosum Galen 1983) Antibiotic
secretions on ant integuments may also directly damage pollen grains but the fitness effects of
this are unclear (see 2 below)
Several species of Crematogaster and Allomerus ants sterilize their host plants by
attacking floral buds (Yu and Pierce 1998 Stanton et al 1999 Gaume et al 2005 Maleacute et al
2012 Table 11) These ants nest in ldquoant-plantsrdquo or ldquomyrmecophytesrdquomdashie plants that host ant
4
colonies in specialized structures (domatia) The sterilizing ants do not appear to consume floral
tissues (Yu and Pierce 1998) and two hypotheses may explain why ants destroy floral buds First
ant colony size is often limited by domatia availability on their host plant By destroying floral
buds ants can cause plants to divert resources from reproductive to vegetative growth increasing
domatia number and allowing for larger ant colonies (Yu amp Pierce 1998 Frederickson 2009)
Second when plant-ants must defend their colony from takeovers by other colonies they may
benefit from limiting access to their host plant By destroying buds that could develop into
flowers branches or leaves ants can prevent their host plant from contacting nearby plants
occupied by enemy ant colonies (Stanton et al 1999)
The impact of sterilization on lifetime plant fitness is unclear Ants may destroy all of
their host plantrsquos floral buds (Yu and Pierce 1998) completely preventing reproduction or only a
portion of them (Maleacute et al 2012) allowing some seed set However counter-intuitively this
sterilization behavior may provide long-term fitness benefits to plants (Frederickson 2009
Palmer et al 2010) Ant-plants often host several different ant species over their lifetimes some
of which are non-sterilizing If hosting a sterilizing species at a certain life stage increases
survival or growth relative to hosting a non-sterilizing species at that life stage plants may
increase lifetime fitness by largely forgoing current reproduction in favor of increasing future
reproduction (Palmer et al 2010) Why plants invest resources in producing flowers that are
quickly destroyed by ants is not known but the timing of flower destruction may be important If
ants attack flowers after anthers dehisce plants may still realize some male fitness via pollen
donation (Yu and Pierce 1998) Additionally if the cost of flower production is relatively minor
and ants sometimes ldquomissrdquo flowers (Edwards and Yu 2008) attempting limited reproduction
may be a better strategy than forgoing it entirely
5
ii) Competition with pollinators
Ant activity on flowers can dramatically alter pollinator behavior Ants can reduce the species
diversity frequency or duration of pollinator visits to flowers (Norment 1988 Tsuji et al 2004
Ness 2006 Junker et al 2007 Lach 2007 2008b Romero et al 2011 and see Chapter 2) These
changes are largely due to either exploitative competition (ie consumption of shared resources)
or interference competition (ie direct aggression) between ants and pollinators
a) Exploitative competition
Pollinators and ants frequently compete for floral nectar Flower-visiting ants are often classified
as nectar thieves (sensu Inouye 1980) because they typically enter flowers through the
ldquolegitimaterdquo opening and consume nectar but transfer no pollen (Wyatt 1980 Ness 2006
Chamberlain and Holland 2008) Ants can also act as primary nectar robbers chewing holes
through corollas to reach nectaries (Willmer and Corbet 1981 Koopowitz and Marchant 1998)
although they are more commonly secondary nectar robbers and use holes made by previous
robbers (Newman and Thomson 2005)
Like other flower visitors foraging ants will often leave nectar behind in flowers This
amount can be less (Bleil et al 2011) more (Fritz and Morse 1982) or similar to (Lach 2008a)
amounts left by other visitors Often ants are morphologically constrained from fully exploiting
nectar Unlike bees and butterflies ants do not have proboscises and must get close to nectar to
consume it Narrow corollas or nectar spurs can prevent ant access to some or all floral nectar
(Schemske 1980 Galen and Cuba 2001 and see below) Smaller ants may be better able to
6
access nectar (Newman and Thomson 2005 Agarwal and Rastogi 2007) but can be limited by
their crop size in the amount of nectar they can remove (Lach 2005) If foraging ants leave
enough nectar behind for a pollinator to recover the cost of visiting a flower ants may have
minimal effects on plant reproductive success (Maloof and Inouye 2000 Turner et al 2012)
Differences among ant species in behavior and diet also affect their consumption of floral
nectar Species vary in how many flowers they visit per inflorescence or per plant and in how
many ants will visit a flower simultaneously (Lach 2005 Junker et al 2007 Lach 2013)
Invasive antsmdashthought to be better at exploiting and defending resources than native ants
(Holway et al 2002)mdashoften recruit to flowers in large numbers sometimes several dozen or
more (Visser et al 1999 Hansen and Muumlller 2009) Overall many factors may influence how
heavily ants exploit floral nectar on plants including ant density plant height the spatial or
temporal distribution of floral nectar and the availability of alternative food resources for ants
(Cushman and Addicott 1991 Lach 2013)
Rarely ants and pollinators may compete for pollen We know of only a few studies that
have recorded ants harvesting pollen directly from anthers (Horskins and Turner 1999 Ness
2006 Byk and Del-Claro 2010) Other studies have found pollen grains in ants (eg Baroni
Urbani and de Andrade 1997 and see Chapter 3) but this may not indicate competition with
pollinators ants sometimes collect stray pollen grains from the environment (Creighton 1967)
One New World genus Cephalotes might be specially adapted for pollen consumption They
possess ldquoproventricular shieldsrdquo structures that may restrict solid food to the crop allowing
nestmates to exchange pollen grains via trophallaxis It is unclear if this structure evolved for this
purpose but Cephalotes are the most frequently documented palynivorous ants (eg Creighton
1967 Baroni Urbani and de Andrade 1997 Byk and Del-Claro 2010) How efficiently ants
7
digest pollen and whether ants like bees feed it to larvae as a protein source is unknown At
least two studies have found that ant larvae consume pollen providing some support for this idea
(Wheeler and Bailey 1920 and Chapter 3)
b) Interference competition
Ants are relatively unique among flower visitors in defending floral resources from competitors
(but see Willmer and Corbett 1981 Roubik 1982) This defense ranges from chance encounters
with ants attacking visitors when flower occupancy overlaps (Ashman and King 2005 Junker et
al 2007) to actively patrolling inflorescences or constructing protective galleries around
nectaries (Horvitz and Schemske 1984 Gaume et al 2005 Hansen and Muumlller 2009)
Interference competition even in the absence of exploitative competition is sufficient to alter
pollinator behavior (Chapter 2)
The strength and outcome of interference competition depends on the identities of the ant
and the pollinator Pollinators might not alter their visitation patterns in response to non-
aggressive ants (Junker et al 2007) but more antagonistic ants may elicit avoidance behavior
before or soon after the visitor lands (Willmer and Corbet 1982 Altshuler 1999 Schuumlrch et al
2000 Table 11) Pollinator size may help predict their reaction to ants because ants may deter
only small-bodied pollinators (Gonzaacutelvez et al 2012) It is not clear whether flower visitors view
ants as aggressive competitors or as potential predators rarely ants prey on pollinators (Schatz
and Wcislo 1999) but in most cases ants merely cause pollinators to relocate
How well pollinators recognize and avoid ant-visited flowers is unresolved Collectively
pollinators have lower visitation rates to flowers visited by ants (Romero et al 2011) This is
8
dependent on pollinator identity however dipterans do not appear to discriminate between ant-
visited and ant-free flowers while bees can identify and avoid ant-visited flowers Bees likely
use a combination of visual and olfactory cues to recognize ant-visited flowers They can
identify previous conspecific and some heterospecific insect visitors by the hydrocarbon
ldquofootprintsrdquo they leave during foraging (Stout and Goulson 2001) Bumblebees can detect ant
hydrocarbons although this has been shown only in the laboratory (Ballantyne and Willmer
2012 Chapter 2) No study has yet examined if bees can use sight alone to detect ants but
several suggest it (Junker et al 2007 Gonzaacutelvez et al 2012) Some bees may discriminate
between ant species when visiting flowers (eg Lach 2008b) which follows if not all ant-
pollinator interactions are equally costly The ability of bees to do so will affect how generalized
their avoidance behaviors will be If they can discriminate between species bees may avoid
specific flowers visited by more ldquocostlyrdquo ants However if they cannot bees might either avoid
all ant-visited flowers or none depending on how negative the average interaction with an ant is
iii) Interactions with flower and pollinator antagonists
Ants can defend the flowers they visit Numerous plant species have circumfloral or bracteal
nectaries (ie extrafloral nectaries around flowers) and ants attracted to these nectaries can
reduce bud ovule or flower predation increasing plant fitness (Inouye and Taylor 1979 Rico-
Gray and Thien 1989 Sugiura et al 2006) Although the presence of EFNs (extrafloral nectaries)
so close to floral nectaries (sometimes less than 1 cm Ness 2006) might increase discovery and
exploitation of floral nectar by ants only one study has investigated this hypothesis finding no
relationship between the proximity of EFNs to floral nectaries and antsrsquo use of floral nectar
(Galen 2005) Holes made by nectar robbers are functionally analogous to circumfloral nectaries
9
and can also attract ants that defend flowers (Newman and Thomson 2005) and nectar-thieving
ants can similarly prevent florivory in some systems (Lach 2007 Bleil et al 2011)
Ants can also reduce the abundance of flower-dwelling predators or parasites of
pollinators Ambush predators such as crab spiders often forage at flowers where they may
reduce pollinator visitation rates and plant fitness (Dukas 2001 Gonccedilalvez-Souza et al 2008)
Ants can decrease spider abundance and richness on plants (Rudgers 2004 Renault et al 2005
Nahas et al 2012) and some evidence suggests that they can do the same on flowers (Lach
2008) In select cases ants may also reduce the abundance of pollinator parasitoids (Holland et
al 2011) Whether ants increase plant reproductive success by removing pollinator antagonists
has not yet been examined but should be If ants create lsquoenemy-freersquo flowers pollinators might
prefer to land on flowers with ants
iv) Net effects
Table 11 compiles studies in which the authors recorded ants consuming floral rewards or
interacting with pollinators and measured some aspect of plant fitness (eg fruit or seed set
pollen deposition) We included only a single study per plant species unless subsequent studies
found conflicting results or measured the fitness of another plant sex Records of ant pollination
are included in a separate table (Table 12)
Table 11 reveals relatively few examples of flower-visiting ants negatively affecting
plant fitness given how costly ant visitation is assumed to be The net effect of ants on plant
reproductive success is actually quite variable with similar numbers of studies finding positive
negative or neutral effects of ant presence Surprisingly when ants do have negative effects it is
10
usually because they directly damage floral structures and not because they prevent pollinators
from visiting flowers and successfully transferring pollen Why are ants not more often negative
for plant reproduction when this is commonly assumed to be true
First ants may compensate for reducing pollinator visitation rates by improving
pollination quality As pollinators move between flowers on a plant they often deposit self-
pollen on stigmas (ie geitonogamy) In some plants with late-acting self-incompatibility ovules
fertilized by self-pollen are subsequently aborted reducing the number of seeds that can be
produced (Barrett 2002 and references therein) Geitonogamy can also reduce offspring viability
or vigor (de Jong et al 1993) and cause pollen discounting a reduction in the amount of pollen
available for outcrossing because of self-pollination (Barrett 2002) By competing with
pollinators ants might decrease rates of geitonogamous pollination if pollinators then visit fewer
sequential flowers within a plant Ants might also cause pollinators to fly further between plants
potentially increasing the genetic diversity of pollen arriving at stigmas Although such effects
have been reported for other floral larcenists (Maloof 2001 Irwin 2003) to our knowledge these
hypotheses have not been investigated in relation to ants
Second ants and pollinators do not always forage for the same resources possibly
allowing them to partition floral rewards If ants forage for nectar but not pollen and flowers can
be pollinated efficiently by visitors looking to gather mostly pollen and little or no nectar then
plant reproduction will not be affected by ants consuming floral nectar Junker et al (2010a)
found that honeybees foraged for both pollen and nectar in the absence of ants but reduced their
foraging on nectar but not pollen when ants could access flowers resulting in no difference in
fruit set between treatments Whether pollinators commonly adjust their foraging behaviors in
response to ants warrants further research
11
Third ants are somewhat unique among floral larcenists in that they often provide direct
benefits to the plants they visit In the studies listed in Table 11 ants sometimes decreased
herbivory on plants (Chamberlain and Holland 2008 Bleil et al 2011) or otherwise aided the
plant (eg ant presence repelling smaller less effective pollinators Gonzaacutelvez et al 2012) If the
positive effects of ants cancel or outweigh the negative effects of flower-visiting ants on
pollinators then ant visitation may be neutral or even positive for plant fitness
Fourth ants may impose fitness costs to plants not captured in the studies in Table 11
Nectar robbers have stronger negative effects when plants are pollen-limited (Burkle et al 2007)
but only one study in Table 11 assessed whether flowers would set more seed if they received
more pollen finding no evidence of pollen limitation (Lach 2008a Table 11) Even if ants
decrease pollinator visitation rates there is limited scope for ants to decrease plant reproductive
success when plants are not pollen-limited Furthermore nearly all the studies in Table 11
measured only female fitness Only three quantified aspects of male fitness (Wyatt 1980 Fritz
and Morse 1981 Galen and Geib 2007) and we know of only one other study in a system in
which ants occasionally pollinate that has also measured male fitness (Ashman 2000) All four
studies measured pollen or pollinia removal rates and none found any effect of flower-visiting
ants on male fitness However whether other measures of male fitness (eg seeds sired pollen
deposition patterns) would reveal similar or different effects needs investigation
Finally although floral defenses against ants (see 3 below) may prevent us from
observing the negative effects of ants on plant reproductive success we consider this unlikely
Several studies have examined interactions between ants and plants that share little or no
coevolutionary history usually in the context of ant invasions Although invasive ants often
recruit to flowers in larger numbers than native ants they do not appear to reduce plant
12
reproductive success (Blancafort and Goacutemez 2005 Lach 2007 Lach 2008a but see Hansen and
Muumlller 2009) And in Hawaii where angiosperms evolved for millions of years in the absence of
ants and are thought to have secondarily lost floral defenses against ants (Junker et al 2011a)
there is no evidence that the preponderance of recently introduced flower-visiting ants negatively
affects plant fitness (Junker et al 2010a Bleil et al 2011)
2 Ants as pollinators
Ants may directly benefit plant reproduction by acting as pollinators but this is a relatively rare
occurrence Ants have long been considered poor pollinators (Kerner 1878) Initially this was
based on morphological and behavioral differences between ants and recognized pollinators
Worker ants are wingless and often relatively hairless making them poor pollen dispersers As
well because many ants exhibit Ortstreue (literally ldquoplace fidelityrdquo) ants were thought to return
to the same branch or flower repeatedly effecting no cross pollination (Houmllldobler and Wilson
1990) Though largely true these traits do not necessarily preclude ants from acting as
pollinators (Table 12) Although they commonly have restricted foraging ranges (Houmllldobler and
Wilson 1990) some ants can effect outcrossing (eg Ashman and King 2005 Carvalheiro 2008)
and many ants are as hairy if not hairier than their pollinating counterparts
In the late 1970s the ldquoantibiotic hypothesisrdquo was put forward to explain the rarity of ant
pollination Ants use antibiotic secretions to protect themselves against the fungal and bacterial
pathogens common in their nests and environs Iwanami and Iwadare (1978) found that one of
these compounds myrmicacin also greatly reduces pollen germination rates Subsequently
Beattie et al (1984 1985) found that short periods of contact (le 30 minutes) with an antrsquos
integument caused pollen grains to fail to germinate Some of these pollen-killing compounds
13
were traced back to the ant metapleural gland which produces hygienic secretions (Houmllldobler
and Wilson 1990) although contact with ants lacking metapleural glands can also reduce pollen
viability (Beattie et al 1985 Dutton and Frederickson 2012) Ant mandibular or anal gland
secretions and potentially ant venom may likewise defend ants against pathogens and have
similar effects on pollen germination (Beattie et al 1985 Graystock and Hughes 2011)
Nonetheless ant pollination has been documented repeatedly There are at least 18 cases
in which ant pollination has been shown experimentally and five others in which observations
strongly suggest it (Table 12) The majority of these plants fit the ldquoant pollination syndromerdquo
originally proposed by Hickman (1974) dense stands of short plants with small flowers and
easily accessible nectaries Typically ant-pollinated plants are associated with hot and dry
climates (principally in the Mediterranean) but dry alpine environments may also favor the
evolution of ant pollination (eg Svensson 1985 Puterbaugh 1998 Schiestl and Glaser 2011) In
most cases ants appear to be the sole or major pollinator of the plant in question (Hickman 1974
Peakall et al 1990 de Vega et al 2009) although ants sometimes make only supplementary
contributions to pollination (Schuumlrch et al 2000 Ashman and King 2005) This suggests that co-
evolution between ants and plants may not be necessary for ants to be useful pollinatorsmdashwhich
is surprising given the potential negative effects of ants on plant reproduction
Ant-pollinated plants cope with ant secretions in a variety of ways Several plant species
may simply tolerate pollen damage (Gόmez and Zamora 1992 de Vega et al 2009) apparently
possessing no overt adaptations to prevent it These plants might receive enough pollen to
overcome reduced pollen viability Other plants may place pollen on ant body parts thought to
have fewer secretions thereby potentially escaping damage (eg on ant legs Ramsey 1995) or
attach pollen to ants with specialized structures avoiding direct contact with the ant integument
14
(Peakall et al 1990) It is not known whether pollen evolves resistance to ant secretions or
whether plants deposit pollen on ants which have less disruptive secretions although these
possibilities have been suggested elsewhere (eg Garciacutea et al 1995 Ramsey 1995)
One key question regarding the effects of ant secretions on pollen grains urgently needs
to be resolved Several studies have suggested that the pollen-killing secretions of ants may have
contributed to the evolution of floral defenses against ants (eg Wagner 2000 Galen and Geib
2007) For this to be true ants must decrease male fitness by damaging pollen grains before they
leave flowers and are deposited on receptive stigmas Only two studies have investigated the
effects of ant secretions on pollen still in anthers (Wagner 2000 Galen and Butchard 2003) and
both constrained ants unnaturally to flowers for significantly longer than ants would normally
stay on flowers Although these studies found strong reductions in pollen vigor it is unclear if
ant secretions decrease male fitness under natural ant visitation conditions
3 Floral defenses against ants
Plants are not passive recipients of flower visitors and have some ability to ldquochooserdquo which
organisms can access floral rewards Flowers resist ants using both structural and chemical traits
More thorough investigation is needed to determine whether these traits evolved to prevent or
minimize the negative effects of ants on plant reproductive success (ie whether these traits can
truly be considered defenses against ants see 4ia below) It is unlikely however that ants
experience reciprocal selection to circumvent floral resistance traits because the fitness benefits
to ants of accessing flowers are probably small Thus there may be little scope for
coevolutionary arms races between floral defenses against ants and ant counter-adaptations
15
i) Structural resistance traits
a) Restricting ant access
Numerous plant species have physical structures preventing ant access to flowers Thin pedicels
can limit antsrsquo gripping abilities and make ants easier to dislodge when flowers are moved by
wind (Gaume et al 2005) and waxy secretions can render stems too slippery for ants to climb
(Harley 1991) Structures surrounding flowers such as water moats or dense hairs may similarly
keep ants from accessing floral rewards (Willmer et al 2009) Many such traits function as broad
filters and prevent not only ants but most crawling insects from reaching flowers Thus it is
difficult to determine whether ants florivores or both acted as selective agents favoring the
evolution of physical barriers to flowers
Flower shape may also reduce ant-pollinator conflict Long narrow corollas or nectar
spurs can prevent all but the smallest ants from accessing floral nectaries (Bluumlthgen et al 2004
Agarwal and Rastogi 2007) while still allowing long-tongued pollinators access to rewards
Selection to exclude nectar-thieving ants in such a manner however may be counteracted by
pollinator preferences for wider corollas that allow easier flower handling (Galen and Cuba
2001) Additionally these types of flowers may be more prone to robbery (Irwin and Maloof
2002) limiting their usefulness in preventing nectar larceny
b) Separation in space or time
Plants may separate floral rewards and ants temporally or spatially to minimize ant impacts on
plant reproduction Several authors have proposed that myrmecophytes have adapted to the
16
sterilization or otherwise detrimental activities of ants by locating domatia or food rewards on
different branches than flowers (Izzo and Vasconcelos 2002 Raine et al 2002 Edwards and Yu
2008) Temporal separation of ant and pollinator activities could likewise reduce conflict though
evidence is limited Some acacia flowers dehisce during periods of low ant activity (Willmer and
Stone 1997 Nicklen and Wagner 2006) but it is unclear if this is in response to ants
temperature or pollinator activity Timing of nectar secretion may similarly reduce ant-pollinator
competition If ants are active prior to pollinators they may deplete floral nectar and reduce
pollinator visitation rates (Schaffer et al 1984) However if nectar is secreted when ants are
inactive pollination may occur unimpeded (Norment 1988) This temporal separation could
favor pollinators that are active when ants are not (eg Wyatt and Morse 1981) but this is
poorly documented Further studies particularly focusing on flowers with the potential for
nocturnal and diurnal pollination may provide more examples
c) Extrafloral nectaries (EFNs)
One hypothesis put forward to explain the evolution of EFNs proposes that EFNs ldquodistractrdquo ants
from consuming floral nectar (Kerner 1878 Wagner and Kay 2002) Wagner and Kay (2002)
tested this hypothesis by constructing model plants using artificial nectaries assigned at random
to represent either floral or extrafloral nectaries They found that the presence of EFNs could
reduce ant exploitation of floral nectar However as highlighted by Galen (2005) this design is
closer to a plant making a surplus of flowers some of which will avoid ant visitation if visitation
rates increase sublinearly with increasing flower number Producing extra flowers to escape ant
visitation appears to be a viable strategy in some systems (Ashman and King 2005 Galen 2005)
However Galen (2005) found that applying honey (simulating extrafloral nectar) to P viscosum
17
leaves had the opposite effect and increased ant visits to flowers Only two studies both in
Pachycereus schottii have found evidence for the distraction hypothesis under field conditions
(Chamberlain and Holland 2008 Holland et al 2011)
There is limited empirical support for the distraction hypothesis but abundant evidence
that EFNs attract ants that defend plants against herbivores (reviewed in Rosumek et al 2009)
To function as attractants the presence of EFNs must increase ant abundance on plants
However because of the increased numbers attracted EFNs would not be expected to reduce the
abundance of ants on nectar rewards or be able to satiate the ants recruited Only if ant
abundance stops increasing at a reward density much lower than that available on the plant
would EFNs serve to distract ants In this case the amount of rewards may overwhelm ants
leaving some flowers underexploited However even in P schottii ants still visited flowers
(Chamberlain and Holland 2008) In cases where ant abundance would continue to increase with
additional rewards it is unlikely that EFNs function as distractions
ii) Chemical resistance traits
a) Toxic nectar
One of the earliest proposed floral defenses against ants was toxic nectar (Janzen 1977)
However studies testing Janzenrsquos (1977) hypothesis uncovered only a very few isolated
examples (eg Catalpa speciosa Stephenson 1981 Crinum erubescens Guerrant and Fiedler
1981) in short most floral nectar is not toxic or repellent to ants (Baker and Baker 1978
Schubart and Anderson 1978) In fact ants sometimes prefer floral nectar over solutions with
equivalent sugar concentrations (Koptur and Truong 1998)
18
In contrast floral nectar can repel other flower visitors (eg birds Johnson et al 2006
lepidopterans Kessler and Baldwin 2007) Repellency is often caused by plant secondary
metabolites which may occur in nectar as a by-product of their production elsewhere in the
plant where they function as defenses against herbivory (Adler 2001) Alternatively toxic or
repellent nectar may be adaptive repellent nectar may promote effective pollination by reducing
the amount of reward any one pollinator takes spreading nectar over several pollinator visits
(Kessler and Baldwin 2007) or it may function as a filter selecting for specific pollinators
(Johnson et al 2006) The latter may explain why repellent nectar is not more common
legitimate pollinators in addition to floral larcenists may be deterred from visiting flowers with
repellent nectar This may be particularly true of nectars that would repel ants which are closely
related to bees nectar distasteful to ants may often also be distasteful to bees and those nectars
acting as a filter for bees may still be palatable to ants For example Prŷs-Jones and Willmer
(1992) found that the ammonia-containing nectar of Lathraea clandestina is readily consumed
by both bees and ants even though it deters birds that act as nectar thieves Although floral
nectar itself rarely repels ants Guerrant and Fielder (1981) found that the presence of macerated
petals within nectar can often keep ants away Alkaloids in petals may be responsible and might
explain why ants seldom act as primary nectar robbers these secondary compounds may prevent
ants from chewing through corollas
b) Floral volatiles
Repellent floral volatiles appear to be a more widespread form of chemical resistance against
ants These were reviewed recently by Willmer et al (2009) and will be discussed here only
briefly Numerous plant taxa produce floral scents repellent to ants including myrmecophilous
19
myrmecophytic and non-ant associated plants in both temperate and tropical ecosystems
(Willmer et al 2009 Junker et al 2011a Ballantyne and Willmer 2012) In three large studies
floral volatiles that repel ants were detected in about a third of all plants tested (Willmer et al
2009 Junker et al 2011a Ballantyne and Willmer 2012) Pollen is often the source of ant-
repellent volatile compounds (Willmer et al 2009 Ballantyne and Willmer 2012) but they can
be emitted from other floral parts including petals That pollen is often the source of repellence
could suggest that preventing ant visitation may be more important in protecting male than
female function Additionally because repellence would decrease as pollen is removed from
anthers ants may return and defend flowers more quickly than if repellent chemicals were in
longer-lasting structures (eg petals) In some cases floral volatiles are thought to mimic ant
alarm pheromones (Willmer et al 2009) However floral scents elicit inconsistent reactions
across ant species ranging from repellence to attraction (Agarwal and Rastogi 2007 Ballantyne
and Willmer 2012)
Producing structural or chemical resistance traits may be costly for plants Native
Hawaiian plants which have little recent coevolutionary history with ants appear to have
secondarily lost floral ant repellents (Junker et al 2011a) Both Willmer et al (2009) and Junker
et al (2011a) found that highly chemical repellent flowers did not have strong physical defenses
and vice versa suggesting trade-offs between the two (but see Ballantyne and Willmer 2012)
Like toxic nectar floral ant repellents might sometimes deter both pollinators and ants For
example Galen et al (2011) found that 2-phenylethanol in P viscosum floral scent repels both
flower-damaging F neorufibarbis ants and the plantrsquos main pollinator Bombus kirbyellus
Interestingly four recent studies have found that floral scents can also attract ants
(Agarwal and Rastogi 2007 Edwards and Yu 2008 Schiestl and Glaser 2011 Gonzaacutelvez et al
20
2012) One study (Schiestl and Glaser 2011) has shown that floral scents attract ants to a
potentially ant-pollinated plant and such systems may provide many more examples
4 Future directions
We have highlighted areas in need of further investigation throughout the review Here we focus
on several questions we consider high priorities for future research
i) Floral defenses against ants
a) Do floral resistance traits increase plant fitness
The existence ant-repellent floral traits is often taken as evidence that ants negatively affect plant
reproductive success However despite the abundance and variety of mechanisms excluding ants
from flowers few studies show that ants reduce plant fitness when they visit flowers (Table 11)
This absence is particularly apparent with repellent floral volatiles which may be the most
widespread floral resistance trait (Willmer et al 2009 Junker et al 2011a Ballantyne and
Willmer 2012) To our knowledge only one study has shown that floral ant repellents benefit
plants with repellent P viscosum flowers avoiding damaging ant visits (Galen et al 2011)
Research is urgently needed to investigate if these traits increase plant fitness by excluding ants
or if ant visitation is inconsequential More studies taking advantage of standing variation in
traits (Galen et al 2011) or creating it artificially (eg Junker et al 2010b) will allow us to
distinguish between the two
21
b) If ant visitation is often not costly what caused the evolution of repellent traits and
what maintains them Similarly are these repellent traits truly defenses against ant
visitation or are they generalized defenses against a range of floral antagonists
Ant-repellent floral traits are widespread and their production may be costly (Willmer et al
2009 Junker et al 2011a) The studies in Table 11 however indicate that flower-visiting ants
often do not disrupt plant reproduction particularly outside of myrmecophytes What then
selected for these traits One possibility is that these repellents are not directed solely at ants
Instead a community of antagonistic flower visitors may exert diffuse selection on plants
thereby selecting for and maintaining these traits Structural modifications may repel crawling
insects while chemical repellents such as floral volatiles may repel both flying and non-flying
insects (Junker et al 2011b) How much overlap there is in floral volatiles that repel both ants and
other antagonists needs investigation Until we examine how specific these defenses are to ants
we risk overstating the importance of ants in their evolution
ii) Do ants alter pollen movement patterns or reduce male fitness
We are aware of only four studies that have measured if ant visitation impacts male fitness
(Wyatt 1980 Fritz and Morse 1981 Ashman 2000 Galen and Geib 2007) While none of these
studies found an effect all measured only amounts of pollen or pollinia removed However this
measure does not adequately encompass male fitness alterations to pollen movement patterns
and eventual deposition location may be more important to male function than changes in
removal rates If as suggested by some studies (Altshuler 1999 Lach 2007) ants increase
pollinator relocation rates they may decrease pollen discounting increasing male fitness
However if this increased relocation reduces overall pollen export rates ant competition with
22
pollinators may depress male function Studies using pollen-tracking methods such as fluorescent
dyes will allow us to see how ants change pollen movement patterns and methods like paternity
analysis will reveal how competition between ants and pollinators alters male fitness
iii) Coevolution between ants and plants do myrmecophytes exhibit stronger more
specific defenses against ants
The floral traits of myrmecophytes are most likely to coevolve with ants because all plant
reproduction occurs in the presence of ants Their floral defenses would thus be expected to be
both stronger and more specific to their resident ants than those other plants visited by an array
of more opportunistic ant species Some evidence supports this Willmer et al (2009) found that
resident ants of acacias were repelled by flowers with more aggressive ant species showing
stronger repellence than less aggressive ants Additionally ants not normally associated with
acacias did not respond to acacia floral volatiles More studies are needed in other systems and
should take advantage of phylogenetic approaches examining floral traits in closely related
myrmecophytic and non-myrmecophytic plant species to determine whether ants commonly
select for floral defenses against ants in myrmecophytes
Acknowledgements
For comments and discussion we thank the Frederickson lab particularly Eric Youngerman
Lina Arcila Hernandez and Kyle Turner as well as Alison Parker and Jordan Pleet MEF
acknowledges financial support from an NSERC Discovery Grant a Connaught New Researcher
23
Award an Ontario Ministry of Economic Development and Innovation Early Researcher Award
and the University of Toronto ARC was supported by an Ontario Graduate Scholarship and
Sigma Xi
24
References
Adler L S 2001 The ecological significance of toxic nectar Oikos 91409ndash420
Agarwal V M and N Rastogi 2007 Role of floral repellents in the regulation of flower visits
of extrafloral nectary-visiting ants in an Indian crop plant Ecological Entomology 3359ndash
65
Altshuler D L 1999 Novel interactions of non-pollinating ants with pollinators and fruit
consumers in a tropical forest Oecologia 119600ndash606
Ashman TL 2000 Pollinator selectivity and its implications for the evolution of dioecy and
sexual dimorphism Ecology 812577-2591
Ashman TL and E A King 2005 Are flower-visiting ants mutualists or antagonists A study
in a gynodioecious wild strawberry American Journal of Botany 92891ndash5
Ballantyne G and P Willmer 2012 Floral visitors and ant scent marks noticed but not used
Ecological Entomology 37402-409
Ballantyne G and P Willmer 2012 Nectar theft and floral ant-repellence a link between
nectar volume and ant-repellent traits PloS One 7e43869
Baker H G and I Baker 1978 Ants and flowers Biotropica 1080
Barrett S C H 2002 Sexual interference of the floral kind Heredity 88154ndash159
Baroni Urbani C and M L de Andrade 1997 Pollen eating storing and spitting by ants
Naturwissenschaften 84256ndash258
25
Beattie A J C Turnbull R B Knox and E G Williams 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany
71421ndash426
Beattie A J C Turnbull T Hough S Jobson and R B Knox 1985 The vulnerability of
pollen and fungal spores to ant secretions evidence and some evolutionary implication
American Journal of Botany 72606ndash614
Blancafort X and C Goacutemez 2005 Consequences of the Argentine ant Linepithema humile
(Mayr) invasion on pollination of Euphorbia characias (L) (Euphorbiaceae) Acta
Oecologica 2849ndash55
Bleil R N Bluumlthgen and R R Junker 2011 Ant-plant mutualism in Hawailsquoi Invasive ants
reduce flower parasitism but also exploit floral nectar of the endemic shrub Vaccinium
reticulatum (Ericaceae) Pacific Science 65291ndash300
Bluumlthgen N G Gottsberger and K Fiedler 2004 Sugar and amino acid composition of ant-
attended nectar and honeydew sources from an Australian rainforest Austral Ecology
29418ndash429
Burkle L A R E Irwin and D A Newman 2007 Predicting the effects of nectar robbing on
plant reproduction implications of pollen limitation and plant mating system American
Journal of Botany 941935ndash1943
Byk J and K Del-Claro 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333ndash38
26
Caballero P C G Ossa W L Gonzaacuteles C Gonzaacutelez-Browne G Astorga M M Muruacutea and
R Medel 2013 Testing non-additive effects of nectar-robbing ants and hummingbird
pollination on the reproductive success of a parasitic plant Plant Ecology 214633ndash640
Carvalheiro L G E R M Barbosa and J Memmott 2008 Pollinator networks alien species
and the conservation of rare plants Trinia glauca as a case study Journal of Applied
Ecology 451419ndash1427
Chamberlain S A and J N Holland 2008 Density-mediated context-dependent consumer-
resource interactions between ants and extrafloral nectar plants Ecology 891364ndash1374
Creighton W S 1967 Studies on free colonies of Cryptocerus texanus Satschi (Hymenoptera
Formicidae) Psyche 7434ndash42
Cursach J and J Rita 2011 Implications of the reproductive biology of the narrow endemic
Naufraga balearica (Apiaceae) for its conservation status Plant Systematics and Evolution
298581ndash596
Cushman JH and Addicott JF In Huxley CR and DF Cutler (eds) 1st edition Ant-plant
interactions Oxford University Press Oxford
de Jong T J N M Waser and P G Klinkhamer 1993 Geitonogamy the neglected side of
selfing Trends in Ecology and Evolution 8321ndash325
de Vega C M Arista P L Oritz C M Herrera and S Talavera 2009 The ant-pollination
system of Cytinus hypocistis (Cytinaceae) a Mediterranean root holoparasite Annals of
Botany 4517ndash8
27
Dukas R 2001 Effects of perceived danger on flower choice by bees Ecology Letters 4327ndash
333
Dutton E M and M E Frederickson 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561ndash569
Edwards D P and D W Yu 2008 Tolerating castration by hiding flowers in plain sight
Behavioral Ecology and Sociobiology 6395ndash102
Frederickson M E 2009 Conflict over reproduction in an ant-plant symbiosis why Allomerus
octoarticulatus ants sterilize Cordia nodosa trees The American Naturalist 173675ndash81
Fritz R S and D H Morse 1981 Nectar parasitism of Asclepias syriaca by ants effect on
nectar levels pollinia insertion pollinaria removal and pod production Oecologia 50316ndash
319
Galen C 1983 The effects of nectar thieving ants on seedset in floral scent morphs of
Polemonium viscosum Oikos 41245ndash249
Galen C 2005 Catching ants with honey an experimental test of distraction and satiation as
alternative modes of escape from flower-damaging ants Oecologia 14480ndash87
Galen C and B Butchart 2003 Ants in your plants effects of nectar-thieves on pollen fertility
and seed-siring capacity in the alpine wildflower Polemonium viscosum Oikos 101521ndash
528
Galen C and J Cuba 2001 Down the tube pollinators predators and the evolution of flower
shape in the alpine skypilot Polemonium viscosum Evolution 551963-1971
28
Galen C and J C Geib 2007 Density-dependent effects of ants on selection for bumble bee
pollination in Polemonium viscosum Ecology 881202ndash1209
Galen C R Kaczorowski S L Todd J Geib and R A Raguso 2011 Dosage-dependent
impacts of a floral volatile compound on pollinators larcenists and the potential for floral
evolution in the alpine skypilot Polemonium viscosum The American Naturalist 177258ndash
272
Garciacutea M B R J Antor and X Espadaler 1995 Ant pollination of the palaeoendemic
dioecious Borderea pyrenaica (Dioscoreaceae) Plant Systematics and Evolution 19817ndash
27
Gaume L M Zacharias and R M Borges 2005 Ant ndash plant conflicts and a novel case of
castration parasitism in a myrmecophyte Evolutionary Ecology Research 7435ndash452
Graystock P and W O H Hughes 2011 Disease resistance in a weaver ant Polyrhachis
dives and the role of antibiotic-producing glands Behavioral Ecology and Sociobiology
652319ndash2327
Gόmez J M 2000 Effectiveness of ants as pollinators of Lobularia maritima effects on main
sequential fitness components of the host plant Oecologia 12290ndash97
Gόmez J M and R Zamora 1992 Pollination by ants consequences of the quantitative effects
on a mutualistic system Oecologia 91410ndash418
Gόmez J M R Zamora J A Hόdar and D Garciacutea 1996 Experimental study of pollination
by ants in Mediterranean high mountain and arid habitats Oecologia 105236ndash242
29
Gonccedilalves-Souza T P M Omena J C Souza and G Q Romero 2008 Trait-mediated effects
on flowers artificial spiders deceive pollinators and decrease plant fitness Ecology
892407ndash2413
Gonzaacutelvez F G L Santamariacutea R T Corlett and M A Rodriacuteguez-Gironeacutes 2012 Flowers
attract weaver ants that deter less effective pollinators Journal of Ecology 10178ndash85
Guerrant E O and P L Fiedler 1981 Flower defenses against nectar-pilferage by ants
Biotropica 1325ndash33
Harley R 1991 The greasy pole syndrome In Huxley CR and DF Cutler (eds) 1st edition
Ant-plant interactions Oxford University Press Oxford
Hansen D M and C B Muumlller 2009 Invasive ants disrupt gecko pollination and seed
dispersal of the endangered plant Roussea simplex in Mauritius Biotropica 41202ndash208
Hickman J C 1974 Pollination by ants a low-energy system Science 1841290ndash1292
Holland J N S a Chamberlain and T E X Miller 2011 Consequences of ants and
extrafloral nectar for a pollinating seed-consuming mutualism ant satiation floral
distraction or plant defense Oikos 120381ndash388
Houmllldobler B and Wilson E O 1990 The Ants 1st Edition Harvard University Press
Cambridge
Holway DA L Lach AV Suarez ND Tsutsui and TJ Case 2002 The causes and
consequences of ant invasions Annual Review of Ecology and Systematics 33181-233
30
Horskins K and Turner VB 1999 Resource use and foraging patterns of honeybees Apis
mellifera and native insects on flowers of Eucalyptus costata Australian Journal of
Ecology 24221-227
Horvitz C C and D W Schemske 1984 Effects of ants and an ant-tended herbivore on seed
production of a neotropical herb Ecology 651369ndash1378
Inouye DW 1980 The terminology of floral larceny Ecology 611251-1253
Inouye DW and O R J Taylor 1979 A temperate region plant-ant-seed predator system
consequences of extra floral nectar secretion by Helianthella quinquenervis Ecology 601ndash
7
Irwin RE 2003 Impact of nectar robbing on estimates of pollen flow conceptual predictions
and empirical outcomes Ecology 84485-495
Irwin RE and Maloof JE 2002 Variation in nectar robbing over time space and species
Oecologia 133525-533
Iwanami Y and T Iwadare 1978 Inhibiting effects of myrmicacin on pollen growth and pollen
tube mitosis Botanical Gazette 13942ndash45
Izzo TJ and Vasconcelos HL 2002 Cheating the cheater domatia loss minimizes the effects
of ant castration in an Amazonian ant plant Oecologia 133200-205
Janzen D H 1977 Why donrsquot ants visit flowers Biotropica 9252
Johnson SD Hargreaves AL and Brown M 2006 Dark bitter-tasting nectar functions as a
filter of flower visitors in a bird-pollinated plant Ecology 872709-2716
31
Junker R A Y C Chung and N Bluumlthgen 2007 Interaction between flowers ants and
pollinators additional evidence for floral repellence against ants Ecological Research
22665ndash670
Junker R R R Bleil C C Daehler and N Bluumlthgen 2010a Intra-floral resource partitioning
between endemic and invasive flower visitors consequences for pollinator effectiveness
Ecological Entomology 35760ndash767
Junker R R and N Bluumlthgen 2010b Floral scents repel facultative flower visitors but attract
obligate ones Annals of botany 105777ndash82
Junker R R C C Daehler S Doetteri A Keller and N Bluumlthgen 2011a Hawaiian ant ndash
flower networks nectar-thieving ants prefer undefended native over introduced plants with
floral defenses Ecological Monographs 81295ndash311
Junker R R J Gershenzon and S B Unsicker 2011b Floral odor bouquet loses its ant
repellent properties after inhibition of terpene biosynthesis Journal of Chemical Ecology
371323ndash31
Kawakita A and M Kato 2002 Floral biology and unique pollination system of root
holoparasites Balanophora kuroiwai and B tobiracola (Balanophraceae) American
Journal of Botany 891164ndash1170
Koopowitz H and TA Marchant 1998 Postpollination nectar reabsorption in the African
epiphyte Aerangis verdickii (Orchidaceae) American Journal of Botany 85508-512
Kirk W D J 1984 Ecological studies on Thrips imaginis Bagnall (Thysanoptera) in flowers of
Echium plantagineum L in Australia Australian Journal of Ecology 99ndash18
32
Kerner A 1878 Flowers and their unbidden guests C Kegan Paul London
Kessler D and I T Baldwin 2007 Making sense of nectar scents the effects of nectar
secondary metabolites on floral visitors of Nicotiana attenuata The Plant Journal 49840ndash
54
Koptur S and N Truong 1998 Facultative ant-plant interactions nectar sugar preferences of
introduced pest species in South Florida Biotropica 30179-189
Lach L 2005 Interference and exploitation competition of three nectar-thieving invasive ant
species Insectes Sociaux 52257ndash262
Lach L 2007 A mutualism with a native membracid facilitates pollinator displacement by
Argentine ants Ecology 881994ndash2004
Lach L 2008a Argentine ants displace floral arthropods in a biodiversity hotspot Diversity and
Distributions 14281ndash290
Lach L 2008b Floral visitation patterns of two invasive ant species and their effects on other
hymenopteran visitors Ecological Entomology 33155ndash160
Lach L 2013 A comparison of floral resource exploitation by native and invasive Argentine
ants Arthropod-Plant Interactions 7177-190
Luo C W K Li X M Chen Z Y Huang and K U N Li 2012 Ants contribute significantly
to the pollination of a biodiesel plant Jatropha curcas Environmental Entomology
411163ndash1168
33
Maleacute PJ G C Leroy A Dejean A Quilichini and J Orivel 2011 An ant symbiont directly
and indirectly limits its host plantrsquos reproductive success Evolutionary Ecology 2655ndash63
Maloof J E 2001 The effects of a bumble bee nectar robber on plant reproductive success and
pollinator behavior American Journal of Botany 881960ndash1965
Maloof J E and D W Inouye 2000 Are nectar robbers cheaters or mutualists Ecology
812651ndash2661
Moreau CS CD Bell R Vila SB Archibald and NE Pierce 2006 Phylogeny of the ants
diversification in the age of the angiosperms Science 312101-104
Nahas L M O Gonzaga and K Del-Claro 2012 Emergent impacts of ant and spider
interactions herbivory reduction in a tropical savanna tree Biotropica 44498ndash505
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506ndash514
Newman D A and J D Thomson 2005 Interactions among nectar robbing floral herbivory
and ant protection in Linaria vulgaris Oikos 110497ndash506
Nicklen E F and D Wagner 2006 Conflict resolution in an ant-plant interaction Acacia
constricta traits reduce ant costs to reproduction Oecologia 14881ndash7
Norment C J 1988 The effect of nectar-thieving ants on the reproductive success of Frasera
speciosa (Gentianaceae) American Midland Naturalist 120331ndash336
34
Orivel J L Lambs P-J G Maleacute C Leroy J Grangier T Otto A Quilichini et al 2011
Dynamics of the association between a long-lived understory myrmecophyte and its
specific associated ants Oecologia 165369ndash76
Palmer T M D F Doak M L Stanton J L Bronstein E T Kiers T P Young J R
Goheen et al 2010 Synergy of multiple partners including freeloaders increases host
fitness in a multispecies mutualism Proceedings of the National Academy of Sciences
10717234ndash9
Peakall R A J Beattie and S H James 1987 Pseudocopulation of an orchid by male ants a
test of two hypotheses accounting for the rarity of ant pollination Oecologia 73522ndash524
Peakall R and A J Beattie 1989 Pollination of the orchid Microtis parviflora R Br by
flightless worker ants Functional Ecology 3515ndash522
Peakall R C J Angus A J Beattie 1990 The significance of ant and plant traits for ant
pollination in Leporella fimbriata pollination Oecologia 84457ndash460
Puterbaugh M N 1998 The roles of ants as flower visitors experimental analysis in three
alpine plant species Oikos 8336ndash46
Prŷs-Jones OE and Willmer PG 1992 The biology of alkaline nectar in purple toothwort
(Lathraea clandenstina) ground level defenses Biological Journal of the Linnean Society
45373-388
Raine NE Willmer P and Stone GN 2002 Spatial structuring and floral avoidance
behaviour present in ant-pollinator conflict in a Mexican ant-acacia Ecology 833086-
3096
35
Ramsey M 1995 Ant pollination of the perennial herb Blandfordia grandiflora (Liliaceae)
Oikos 74265ndash272
Renault CK LM Buffa and Delfino MA 2005 An aphid-ant interaction effects on
different trophic levels Ecological Research 2071-74
Rico-Gray V and L B Thien 1989 Effect of different ant species on reproductive fitness of
Schomburgkia tibicinis (Orchidaceae) Oecologia 81487ndash489
Romero G Q P A P Antiqueira and J Koricheva 2011 A meta-analysis of predation risk
effects on pollinator behaviour PLoS One 6e20689
Rosumek FB FAO Silveira F de S Neves NP de U Barbosa L Diniz Y Oki F Pezzini
et al 2009 Ants on plants a meta-analysis of the role of ants as plant biotic defenses
Oecologia 160537ndash549
Roubik D W 1982 The ecological impact of nectar-robbing bees and pollinating
hummingbirds on a tropical shrub Ecology 63354ndash360
Rudgers J A 2004 Enemies of herbivores can shape plant traits selection in a facultative ant-
plant mutualism Ecology 85192ndash205
Schatz B and WT Wcislo 1999 Ambush predation by the ponerine ant Ectatomma ruidum
Roger (Formicidae) on a sweat bee Lasioglossum umbripenne (Halictidae) in Panama
Journal of Insect Behavior 12641-663
36
Schaffer W M D W Zeh S L Buchmann S Kleinhaus M V Schaffer and J Antrim
1983 Competition for nectar between introduced honey bees and native North American
bees and ants Ecology 64564ndash577
Schemske D W 1980 The evolutionary significance of extrafloral nectar production by Costus
woodsonii (Zingiberaceae) an experimental analysis of ant protection Journal of Ecology
68959ndash967
Schiestl F P and F Glaser 2011 Specific ant-pollination in an alpine orchid and the role of
floral scent in attracting pollinating ants Alpine Botany 1221ndash9
Schubart HOR AB Anderson 1978 Why donrsquot ants visit flowers A response to D H
Janzen Biotropica 10310-311
Schuumlrch S M Pfunder and B A Roy 2000 Effects of ants on the reproductive success of
Euphorbia cyparissias and associated pathogenic rust fungi Oikos 16ndash12
Stanton ML Palmer TM Young TP Evans A and Turner ML 1999 Sterilization and
canopy modification of swollen thorn acacia tree by a plant-ant Nature 401 578-581
Stephenson AG 1981 Toxic nectar deters nectar thieves of Catalpa speciosa American
Midland Naturalist 105381-383
Stout J C and D Goulson 2001 The use of conspecific and interspecific scent marks by
foraging bumblebees and honeybees Animal Behaviour 62183ndash189
Sugiura S T Abe and S Makino 2006 Loss of extrafloral nectary on an oceanic island plant
and its consequences for herbivory American Journal of Botany 93491ndash495
37
Svensson L 1985 An estimate of pollen carryover by ants in a natural population of
Scleranthus perennis L (Caryophyllaceae) Oecologia 66373ndash377
Tsuji K A Hasyim and K Nakamura 2004 Asian weaver ants Oecophylla smaragdina and
their repelling of pollinators Ecological Research 19669ndash673
Turner R C J J Midgley P Barnard R E Simmons and S D Johnson 2012 Experimental
evidence for bird pollination and corolla damage by ants in the short-tubed flowers of
Erica halicacaba (Ericaceae) South African Journal of Botany 7925ndash31
Visser D M G Wright and J H Giliomee 1996 The effect of the Argentine ant
Linepithema humile (Mayr) (Hymenoptera Formicidae) on flower-visiting insects of
Protea nitida Mill (Proteaceae) African Entomology 4285ndash287
Wagner D 2000 Pollen viability reduction as a potential cost of ant association for Acacia
constricta (Fabaceae) American Journal of Botany 87711ndash715
Wagner D and A Kay 2002 Do extrafloral nectaries distract ants from visiting flowers An
experimental test of an overlooked hypothesis Evolutionary Ecology Research 4293ndash305
Wang C Y Luo Y Tai D An and Y Kou 2008 Ants pollinate Neottia listeroides
(Orchidaceae) in Sichuan China 46836ndash846
Wyatt R 1980 The impact of nectar-robbing ants on the pollination system of Asclepias
curassavica Bulletin of the Torrey Botanical Club 10724ndash28
Wheeler WM and IW Bailey 1920 The feeding habits of Pseudomymine and other ants
Transactions of the American Philosophical Society 22235-279
38
Willmer P G and S A Corbet 1981 Temporal and microclimatic partitioning of the floral
resources of Justicia aurea amongst a concourse of pollen vectors and nectar robbers
Oecologia 5167ndash78
Willmer P G and G N Stone 1997 How aggressive ant-guards assist seed-set in Acacia
flowers Nature 388165ndash167
Willmer P G C V Nuttman N E Raine G N Stone J G Pattrick K Henson P Stillman
et al 2009 Floral volatiles controlling ant behaviour Functional Ecology 23888ndash900
Young T P C H Stubblefield and L a Isbell 1997 Ants on swollen-thorn acacias species
coexistence in a simple system Oecologia 10998ndash107
Yu D W and N E Pierce 1998 A castration parasite of an ant-plant mutualism Proceedings
of the Royal Society B Biological Sciences 265375ndash382
39
Tables
Table 11 Studies of ant-flower or ant-pollinator interactions that included a measure of plant fitness Each study system is listed only
once except when results varied between years or when different studies examined male and female fitness Significant effects of ants on
plant fitness are either lsquopositiversquo or lsquonegativersquo non-significant effects are lsquoneutralrsquo When ants significantly affected only one fitness
component but several were measured (eg fruit set and seed production) we still consider the result significant
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Acacia
drepanolobium
Crematogaster
nigriceps
Proportion
fruiting
Negative Destruction of floral buds Young et al 1997
Asclepias exaltata Several Pollinia inserted
removed
Negative for pollinia
insertion neutral for
pollinia removal
Exploitative competition Wyatt 1980 T
40
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Asclepias syriaca Several
Fruit set pollinia
removed
Negative for fruit
set neutral for
pollinia removal
Exploitative competition Fritz and Morse
1981 T
Cordia nodosa Allomerus cf
demerarae
Fruit set Negative Destruction of floral buds Yu and Pierce
1998
Costus woodsonii Wasmannia
auropunctata
Seed set Positive Unknown ant pollination
suggested (but unlikely)
Schemske 1980
Eritrichum aretioides F neorufibarbis
gelida
Seed set Negative Ants damaged flowers Puterbaugh 1998
41
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Euphorbia characias Several Seed set Neutral Plants in Argentine ant-
invaded area set less seed
than those in un-invaded
sites but an ant-exclusion
experiment did not produce
significant results
Blancafort and
Gόmez 2005
Ferocactus wislizeni Solenopsis xyloni
and others
Seed set Unknown negative Interference and exploitative
competition S xyloni
reduced seed set relative to
other ants but the study did
not include a no-ant control
Ness 2006
42
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Frasera speciosa F neorufibarbis
and Formica sp
Seed set Neutral Nocturnal pollination
occurring when ants were
less active may have
compensated for reduced
diurnal pollinator visitation
Norment 1988
Gypsophila struthium Several Seeds per
inflorescence
Neutral No effect despite large
numbers of ants on flowers
Gόmez et al
1996
Hirtella physophora Allomerus
decemarticulatus
Fruit set Negative Destruction of flowers Orivel et al
2011
Humboldtia brunonis Crematogaster
dohrni
Fruit set Negative Destruction of flowers Gaume et al
2005
43
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Lepidium subulatum Several Percentage of
ovules setting
seed
Positive Unknown Gόmez et al
1996
Leucospermum
conocarpodendron
Several Seed set Neutral Site not pollen limited Lach 2008
Linaria vulgaris Several ldquoFemale fitness
indexrdquo
(combination of
number of seeds
fruits flowers
and seed weight)
Positive Reduction in florivory and
seed predation nectar
robbing holes increased ant
visitation
Newman and
Thomson 2005
44
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Melastoma
malabathricum
Oecophylla
smaragdina
Seed set Positive Ants deterred less efficient
pollinators increasing
visitation by the more
efficient pollinator
Gonzaacutelvez et al
2012
Metrosideros
polymorpha
Several Fruit set Neutral Ants reduced nectar
collection rates by foraging
bees but had no effect on
pollen collection rates or
fruit set
Junker et al
2010a
Orexis alpine F neorufibarbis
gelida
Seed set Neutral Autogamy or not pollen
limited
Puterbaugh 1988
45
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Ouratea spectabilis Cephalotes
pusillus and others
Seeds per fruit Neutral positive C pusillus had no effect on
seed number but other ants
collectively increased seed
set
Byk and Del
Claro 2010
Pachycereus schottii Several Seed set Neutral positive In one year ants showed a
positive effect (seed
number 2008) but no effect
with increased sample sizes
Chamberlain and
Holland 2008
Polemonium
viscosum
F neorufibarbis
gelida
Seed set Negative Ants damaged styles Galen 1983
46
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Polemonium
viscosum
F neorufibarbis
gelida
Pollen removal Neutral Ants did not affect pollen
removal even at high ant
densities
Galen and Geib
2007 T
Protea nitida Linepithema
humile and others
Seed set Neutral positive L humile had no effect on
P nitida seed set but native
ants increased it in one year
ants may cause pollinators
to relocate
Lach 2007
Psychotria
limonensis
Ectatomma spp Fruit set Positive Suggested increased
pollinator relocation
Altshuler 1999
47
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Roussea simplex Technomyrmex
albipes
Seed set Negative Interference and exploitative
competition
Hansen and
Muumlller 2009
Tristerix aphyllus Several Fruit set Neutral Nectar robbing ants
minimally reduced nectar
levels and had no effect on
hummingbird visitation
Caballero et al
2013
Vaccinium
reticulatum
Several Fruit set Neutral Ants reduced florivory but
did not affect fruit set
Bleil et al 2011
T Study measured male fitness
Study of sterilizing ants showed short-term decrease in female fitness long-term effects are unclear (see text)
48
Table 12 Studies demonstrating either i) ant pollination through an exclusion experiment or ii) that ants transfer pollen (or pollen
analogue) between flowers strongly suggesting ant pollination
Plant species Ant species Ant pollination Evidence Reference
Alyssum purpureum Several Yes Exclusion experiment Gόmez et al 1996
Arenaria tetraquetra Several Yes Exclusion experiment Gόmez et al 1996
Balanophora kuroiwai Leptothorax sp Suggested Exclusion experiment and
observed pollen transfer
and pollen tube formation
but did not see if plants set
seed
Kawakita and Kato 2002
Blandfordia grandiflora Iridomyrmex sp Yes Exclusion experiment Ramsey 1995
Borderea pyrenaica Several Yes Exclusion experiment Garciacutea et al 1995
49
Plant species Ant species Ant pollination Evidence Reference
Cytinus hypocistis Several Yes Exclusion experiment de Vega et al 2009
Epipactis thunbergii Camponotus
japonicas
Suggested Observed transfer of
pollinia from ants to
orchids
Sugiura et al 2006
Euphorbia cyparissias Several Yes Exclusion experiment Schuumlrch et al 2000
Fragaria virginiana Several Very likely Ant-only treatments set
comparable seed to flying
pollinator-only treatment
seed set increased with
increasing ant visitation
Ashman and King 2005
Frasera speciosa Several Yes Exclusion experiment Norment 1988
Frankenia thymifolia Several Yes Exclusion experiment Gόmez et al 1996
50
Plant species Ant species Ant pollination Evidence Reference
Hormathophylla spinosa Proformica
longiseta
Yes Exclusion experiment Gόmez and Zamora 1992
Jatropha curcas
Several
Tapinoma
melanocephalum
most common
Yes Exclusion experiment Luo et al 2012
Leporella fimbriata Myrmecia urens Yes Observed pollinia transfer
and subsequently
confirmed as ant
pollination
Peakall et al 1987
51
Plant species Ant species Ant pollination Evidence Reference
Lobularia maritima Several
Camponotus
micans most
common
Yes Exclusion experiment Gόmez 2000
Microtis parviflora
Iridomyrmex
gracilis
Yes Observation used
laboratory colonies to
replicate observations
Peakall and Beattie 1989
Naufraga balearica
Several
especially
Plagiolepis
pygmaea and
Lasius grandis
Suggested Observation over three
years of study only ants
were observed visiting
ants carried pollen
Cursach and Rita 2011
52
Plant species Ant species Ant pollination Evidence Reference
Neottia listeroides Leptothoras sp
Paratrechina sp
Suggested Observed transfer of
pollinia from ants to
orchids
Wang et al 2008
Paronychia pulvinata Formica
neorufibarbis
gelida
Yes Exclusion experiment Puterbaugh 1998
Polygonum cascadense Formica argentea
Very likely Grew plants in greenhouse
open to flying visitors with
minimal seed set only
plants with access to ants
set significant seed
Hickman 1974
53
Plant species Ant species Ant pollination Evidence Reference
Retama sphaerocarpa Several Yes Exclusion experiment Gόmez et al 1996
Scleranthus perennis
Several
especially
Formica
rufibarbis
Suggested Ants transferred pollen
analogue to stigmas and
carried pollen on bodies
Svensson 1985
Sedum anglicum Proformica
longiseta
Yes Exclusion experiment Gόmez et al 1996
Trinia glauca Several Yes Exclusion experiment Carvalheiro et al 2008
54
Chapter 2 Ants and ant scent reduce bumblebee pollination of artificial flowers
Cembrowski AR Tan MG Thomson JD and Frederickson ME
In press The American Naturalist
All authors designed the experiment ARC and MGT performed the experiment ARC and MEF
performed statistical analyse
s and ARC wrote the manuscript with input from all authors
Abstract
Ants on flowers can disrupt pollination by consuming rewards or harassing pollinators but it is
difficult to disentangle the effects of these exploitative and interference forms of competition on
pollinator behavior Using highly rewarding and quickly replenishing artificial flowers that
simulate male or female function we allowed bumblebees (Bombus impatiens) to forage (1) on
flowers with or without ants (Myrmica rubra) and (2) on flowers with or without ant scent cues
Bumblebees transferred significantly more pollen analogue both to and from ant free flowers
demonstrating that interference competition with ants is sufficient to modify pollinator foraging
behavior Bees also removed significantly less pollen analogue from ant scented flowers than
from controls making this the first study to show that bees can use ant scent to avoid harassment
at flowers Ant effects on pollinator behavior possibly in addition to their effects on pollen
viability may contribute to the evolution of floral traits minimizing ant visitation
55
Introduction
Trait-mediated indirect interactions arise when a focal species causes phenotypic changes
including behavioral modifications in a second species and these effects cascade to still other
species (Werner and Peacor 2003) Although trait-mediated indirect interactions are often studied
in food webs (eg Werner and Peacor 2003 Preisser et al 2005) they are not limited to trophic
interactions For example the threat of predation can change the behavior of a mutualist thus
affecting its partners (Suttle 2003) In animal-pollinated plants predators can disrupt pollination
directly through density-mediated indirect interactions (Dukas 2005) or via trait-mediated
indirect interactions by changing pollinator behavior When predators are present pollinators
may switch to visiting less rewarding flowers or they may avoid flowers altogether potentially
reducing the amount of pollen donated or received by a flower and thus decreasing plant fitness
(Gonccedilalves-Souza 2008 Ings and Chittka 2009) Similarly competition for floral rewards with
other flower visitors may cause behavioral changes in pollinators (Maloof and Inouye 2000
Ohashi et al 2008) Previous research has shown that bees may spend less time visiting flowers
depleted of rewards (Thomson 1986) and may avoid flowers bearing scent marks that indicate
recent visits by other individuals of the same or different bee species as these flowers are likely
to be depleted (Stout and Goulson 2002)
Whereas competition between pollinators may not be particularly costly for the plant
because both competitors are likely to provide pollination services competition between
pollinators and organisms that do not usually provide a pollination service such as ants has a
greater potential to reduce plant fitness Ants are common floral visitors they are attracted to
flowers for nectar (Lach 2007) and in some cases pollen (Byk and Del Claro 2010) and will
often defend these resources against other flower visitors (Altshuler 1999) However ants rarely
56
contribute much useful pollination antibiotic secretions present on their cuticles kill pollen
grains (Beattie et al 1985 Dutton and Frederickson 2012) and when foraging in flowers ants
sometimes impede female function by damaging stigmas (Galen and Cuba 2001) We will
henceforth consider ants to be non-pollinators and will contrast them to animals like bees that
are pollinators Ants are capable of competing with pollinators in two ways (1) by consuming
floral rewards and reducing their availability to pollinators (ie exploitative competition) and (2)
by directly antagonizing and excluding pollinators from flowers (ie interference competition)
Many plants benefit from the presence of ants numerous plant species recruit ants using
extrafloral nectar food bodies or domatia because ants provide protection against folivores (Heil
and McKey 2003) Similarly some plants have extrafloral nectaries on or near reproductive
tissues that recruit ants that deter florivores (eg Inouye and Taylor 1979) However plants
may incur reproductive costs as a result of their association with ants Some ant species that nest
in myrmecophytes sterilize flowers to shunt plant resources from reproduction to vegetative
growth allowing for greater ant colony growth (Frederickson 2009) Also ants visiting
extrafloral nectaries may be attracted to floral nectaries where they can harass pollinators (Ness
2006) There is growing evidence that many angiosperms have evolved floral traits that prevent
ants from accessing flowers during anthesis (Willmer and Stone 1997 Ballantyne and Willmer
2012) The effects of flower-visiting ants can be substantial Ants can decrease the frequency
duration or species diversity of pollinator visits to flowers (Lach 2008 Hansen and Muumlller 2009
Junker et al 2010 Gonzaacutelvez et al 2012) all of which potentially impact pollen donation and
receipt In some studies changes to pollinator behavior induced by ants have also been linked to
seed set
It is difficult however to disentangle the intertwined effects of exploitative and
interference competition in nature If ants disrupt pollination plants may often evolve traits to
57
deter ants from flowers (Willmer et al 2009) From a practical standpoint this would then limit
our ability to study the effects of ants on pollinator visitation because ants would not visit
flowers Here we used artificial flowers to explore ant-bee interactions in the absence of floral
defenses against ants We used highly rewarding quickly replenishing artificial flowers to
examine how direct harassment (ie interference competition expressed through behavior)
changes bee foraging behavior and how this affects donation and receipt of a pollen analogue
(powdered food dye) while minimizing the effects of exploitative competition We predicted
that flowers visited by ants would both donate and receive less pollen analogue than flowers
without ants because bees would avoid flowers with ants or leave them sooner Because scent
plays a large role in bumblebee communication and flower choice (Stout and Goulson 2002) we
also tested whether the presence or absence of ant scent on artificial flowers would affect pollen
analogue donation because of changes in the foraging behavior of bumblebees
Methods
i) Subjects
Myrmica rubra is an invasive ant in eastern North America with a range and habitat preferences
overlapping that of Bombus impatiens a common bumblebee Because M rubra visits flowers
(A Cembrowski pers obs) the two species likely interact in nature We collected 12 M rubra
colonies in the fall of 2011 and 2012 from Toronto Ontario Canada and the surrounding area
and maintained them in environmental chambers on artificial diet (Dussutour and Simpson 2009)
and a 1410 LD schedule (light 600-2200) We used these colonies as sources of M rubra
workers for experiments
58
Workers from commercially supplied colonies of Bombus impatiens (Biobest Canada
Ltd) foraged on artificial flowers in flight cages (either 24 x 24 x 21 m or 79 x 34 x 20m) at
the University of Toronto We tested a total of five bee colonies four colonies were used in the
ant presence trials and two of these and one additional colony were used in the ant scent trials
Flight cages had overhead fluorescent lights attached to timers In contrast to most previous
studies in which bees have been trained and tested individually the entire worker force of a bee
colony was free to forage at will in our experiments Colonies were trained to forage on artificial
flowers for at least four days before being used in trials After being used for a trial the colony
was not used for at least two days to reduce dye carryover between trials Between trials
bumblebees were fed pollen and given sugar water
ii) Artificial flowers
The flowers (Fig 1 Thomson et al 2012 see also Makino 2008) consisted of glass jars filled
with 30 WV sucrose solution Sugar water travelled by capillary action up a sewing-thread
wick to a hole in a blue-painted lid accumulating in a knot that acts as a nectary Flowers
depleted by visitors were quickly replenished via capillary action and were non-rewarding only
very briefly after visits taking less than a minute to accumulate 05 microl note that bumblebee
visits to a single flower are often separated by several minutes or more (A Cembrowski
unpublished data)
Unlike most previous artificial flower designs these flowers allow for estimation of male
and female fitness because we can measure both the amount and type of dye received by female
flowers (see (d) Dye quantification below) In order to access nectar on a ldquomalerdquo flower a bee
must crawl through a circular ldquoantherrdquo of brush-like weather stripping material dusted in a
59
consistent manner with our pollen analogue powdered food dye (FDampC 5 or 6) which is
transferred to the bee in the process On a ldquofemalerdquo flower a bee crawls through a sticky plastic
reinforcement that functions as a ldquostigmardquo receiving dye from the beersquos body Male flowers
dispensed dye particles over multiple bee visits and could still dispense dye at the end of trials
They resemble many real flowers or flower heads in that pollen is dispensed gradually over time
but less dye is available for transfer with each subsequent bee visit (Harder and Thomson 1989)
iii) Flight cage trials
We conducted 6 ant scent trials between 22 March and 4 June 2012 and the remaining trials (16
ant presence trials and 9 ant scent trials) between 9 November 2012 and 6 January 2013 Ant
presence trials lasted for eight hours and ant scent trials lasted for four hours Artificial flowers
were prepared and placed individually in small plastic containers treated with Fluon (Insect-a-
Slip BioQuip Products Inc) to prevent ants from escaping In ant presence trials we used 32
flowers (16 male and 16 female flowers) arranged in an eight-by-four array with flowers spaced
45 cm apart between rows and 30 cm within rows Ant scent trials used 20 flowers (10 male and
10 female flowers) in a four-by-five array with flowers spaced 30 cm apart between and within
rows In all of the ant presence trials and 9 of the ant scent trials we counted the number of bees
actively foraging after one hour to get a measure of colony activity
a) Ant presence trials
We examined the effects of ants on the amount of dye donated by male flowers and received by
female flowers in sixteen trials testing four bee colonies four times each The containers of eight
randomly chosen male flowers and eight randomly chosen female flowers received 15 M rubra
60
workers each the other 16 flowers remained free of ants Isolated from their colonies M rubra
workers have nowhere to deposit sugar water they collect and therefore may become sated
nonetheless a force of 15 ant workers was enough to maintain visitation to the nectaries where
bees foraged while still rarely having more than one or two ants visit the nectary at any time As
ants attacked bees bees would occasionally carry or throw attacking ants off flowers but despite
this some ants maintained their presence at the nectary throughout the trials Because ants the
size of M rubra consume liquids slowly (~017 ndash 024 microlmin Davidson et al 2004) flowers
replenished nectar more quickly than ants consumed it We used two colors of dye (FDampC 5 and
FDampC 6) to differentiate male flowers with and without ants In two trials for each bee colony
anthers of male flowers with ants were coated with FDampC 5 dye while brushes of male flowers
lacking ants were coated with FDampC 6 dye In the other two trials we reversed dye colors to
control for effects of dye type and color Dye color was assigned in a random order Artificial
flowers were placed in randomly assigned positions in the array before we opened the colony
and allowed the bees to begin foraging
b) Scent trials
We explored the effect of ant scent on dye donation in fifteen trials testing three bee colonies in
six five and four trials each Bees were first exposed to ants by allowing the bees to forage for
eight hours on twenty flowers (ten male and ten female) of which five male flowers had 15 ants
and all others had none No dye was used during this ldquoexposurerdquo day We then collected all but
the five ant-free male flowers in the flight cage leaving these flowers to keep bees foraging
Next we individually stored five new male lids in Fluon-treated containers with ten M rubra
workers Five control male lids were put in identical containers lacking ants The following
61
morning we removed the remaining flowers and set out ten new male and female flowers in a
random spatial arrangement using the lids having or lacking ant scent Thus the ant-scented
flowers were not in the same positions as the ant-visited flowers on which the bees were trained
Only five male flowers had ant scent and the other 15 flowers (five male and ten female) did not
We used the same two dye colors as in i) to differentiate male flowers with and without ant
scent and randomized which color was used for ant-scented flowers between trials We collected
and replaced stigmas from female flowers after one hour and collected the stigmas again after
four hours In the first trial performed we also collected stigmas after two hours but due to the
small amount of dye transferred this was not repeated
iv) Dye quantification
After each trial we quantified the amount of dye transferred to female flowers using a
spectrophotometer We removed the stigmas from female flowers placed them in test tubes
added 51 mL of distilled water to each tube and vortexed each tube thoroughly to ensure the
dye was evenly diluted These were diluted further as needed if the absorbance exceeded the
sensitivity range of the spectrophotometer In the first six scent trials each stigma was analyzed
separately and the dye amounts were summed to obtain a total amount of each dye color
transferred to female flowers in each trial In all the ant presence trials and the other nine scent
trials we opted to treat the experiment as the unit of replication Therefore we combined stigmas
from each treatment type (ant-visited or ant-free) in a test tube and measured the total amount of
each dye color transferred in each treatment in each trial
Because we put different dye colors on male flowers with and without ants we could use
the amount of each dye color donated to all female flowers to measure the reproductive success
62
of ant-visited and ant-free male flowers The total amount of dye (of both colors) received by
female flowers with and without ants was our measure of female reproductive success We
calculated the amount of each dye color in the sample by measuring absorbance at 428 or 486
nm and converting absorbance to micrograms following computational methods for overlapping
spectra (Blanco et al 1989)
v) Statistical analyses
In two trials (one ant presence and one ant scent) most female flowers received no dye because
of low bee activity so we excluded these trials from analyses Because dye reception and
donation values were non-normally distributed we square-root transformed the data before
examining the effects of ant presence or ant scent on dye transfer in ANCOVAs For ant
presence trials we included ant presence on male and female flowers and their interaction as
main effects and the total amount of dye transferred in each trial as a covariate to account for
the large variation in overall dye transfer among trials For ant scent trials we included ant scent
as the main effect time (one or four hours) as a repeated measure the interaction between scent
and time and the total amount of dye transferred as a covariate Covariate by treatment (ant
presence or ant scent) interactions were never significant and so were excluded in final analyses
All analyses were conducted in JMP (v 1000)
Results
Bees usually started foraging within minutes of the beginning of the trial and continued until
flowers were collected from the flight cage or the lights were extinguished An average of 34 plusmn
012 and 40 plusmn 015 (mean plusmn SE) bees were foraging after an hour in the ant presence and ant
63
scent trials respectively Male flowers with ants donated significantly less dye than male flowers
lacking ants (Fig 22a F155 = 4019 p lt 00001) Similarly female flowers with ants received
significantly less dye than female flowers lacking ants (Fig 22a F155 = 461 p = 0036) There
was no significant interaction between ant presence on male flowers and ant presence on female
flowers (F155 = 118 p = 028) Flowers with ant scent donated significantly less dye than
flowers without ant scent (Fig 22b F125 = 11216 p lt 00001) There was no significant effect
of time (F125 = 121 p = 028) or interaction between time and scent (F125 = 111 p = 030) in
the model
Discussion
In this study ants altered bumblebee pollination behavior and exhibited trait-mediated indirect
interactions with (artificial) flowers Through interference competition M rubra workers
significantly affected the pattern of dye transfer by B impatiens causing a preferential flow of
dye from male flowers lacking ants to female flowers lacking ants If manifested in nature such
effects would reduce the reproductive success of plants visited by ants through both male and
female function
Ants could have changed the attractiveness of artificial flowers in at least two ways First
as ants interrupted or altogether prevented bumblebees from foraging at nectaries bumblebees
may have learned to avoid flowers with ants due to the relative inefficiency of foraging at these
flowers something bumblebees take into account (Heinrich 2004) Second bees attempt to
minimize their risk of being attacked during foraging bouts which ants did to visiting bees Ants
often attacked or harassed bees by biting grasping and appearing to sting visiting bees
preventing them from accessing the nectary or reducing their time on flowers (see online video
64
1) and bees sometimes avoided ant-tended flowers entirely This harassment was sometimes
physically traumatic (online video 1) and bees often appeared to have trouble flying after being
attacked by ants Previous research has demonstrated that bumblebees leave or avoid flowers
where they have been harassed (Jones and Dornhaus 2011) and avoid foraging where there is
visual or olfactory evidence of a predator or predation event (Abbott 2006 Goodale and Nieh
2012) Though it is unclear if bumblebees viewed ants as competitors or predators they
responded similarly to flowers having ants as they do to flowers housing predators (Gonccedilalves-
Souza et al 2008)
To avoid artificial flowers with ants bees likely used a combination of visual and
olfactory cues Previous research has shown that bees respond to conspicuous predators or
predator ldquodummiesrdquo on flowers (Suttle 2003 Gonccedilalves-Souza et al 2008) decreasing the
frequency and duration of their visits to these flowers Thus bees may have been able to avoid
flowers harboring ants by sight alone However even in the absence of ants bees still
preferentially visited flowers lacking ant scents (Fig 22b) suggesting that they had learned to
associate ant scent with harassment Bees are adept at associative learning (Wright and Schiestl
2009) and can learn to recognize unique scents left behind by both conspecific and heterospecific
flower visitors (Stout et al 1998) Bees can use these various scents often arising from tarsal
gland deposits (Stout et al 1998) to recognize recently visited flowers that are less likely to be
profitable (Stout and Goulson 2002) Recently Ballantyne and Willmer (2012) demonstrated that
bees learn to associate ant scents with unrewarding artificial flowers and decrease their visitation
to these flowers Our results complement their findings by showing that bees can associate ant
scent with harassment at otherwise rewarding flowers
In our study ant scent caused bees to adjust their foraging strategy and decreased the
amount of pollen analogue that was donated by ant-scented flowers Thus the effects of
65
interference competition with ants on flowers can extend beyond immediate interactions and may
have fitness consequences for plants even when ants are absent Like other olfactory cues these
effects are likely transitory (Stout et al 1998) Although more dye was still donated by male
flowers lacking ant scent than those with ant scent the ratio of dye donated was on average
closer to equality in hours 2-4 than in the first hour (Fig 22b) This effect may be partially
driven by dye depletion of male flowers without ant scent but the lack of a strong corresponding
decrease in flowers with ant scent suggests that visitation patterns became more similar We did
not test whether B impatiensrsquo avoidance of ant scent was an innate or a learned behavior but
previous work has shown that Bombus terrestris does not innately avoid flowers with ant scent
(Ballantyne and Willmer 2012)
Ants may be necessary for plant survival and growth but can be costly for plant
reproduction The evolution of ant attractants such as extrafloral nectaries in some plant lineages
suggests that the costs of ants can be outweighed by their protective abilities However in this
study flowers visited by ants received and donated significantly less dye although they did
retain some sexual function The net benefit of having ants depends on whether ants increase
plant fitness by reducing herbivory more than they decrease plant fitness by disrupting
pollination Alternatively plants may actually benefit from the costs of ants to plant
reproduction ants that castrate flowers may be better defenders increasing plant survival or
vegetative growth when plants are young allowing for increased reproduction later in life when
the plant is colonized by less aggressive non-castrating ants (Frederickson 2009 Palmer et al
2010) In some plant species pollinator harassment by ants may even be beneficial Altshuler
(1999) reported that Ectatomma ants greatly increased fruit set of Psychotria limonensis despite
reductions in pollinator visitation rates presumably due to increases in the rate of pollen
outcrossing Similarly Gonzaacutelvez et al (2012) found that the presence of Oecophylla
66
smaragdina ants on Melastoma malabathricum flowers reduced visitation by less effective
pollinators and increased visitation by more effective Xylocopa bees increasing plant fitness
Cases of ants being beneficial to flowers appear to be the minority and many plant
species have traits that limit ant access to flowers Several studies have detailed floral volatiles
that are thought to mimic ant alarm pheromones and thus repel ants (Junker and Bluumlthgen 2008
Willmer et al 2009) Other plants use structural modifications such as narrow corollas or
slippery stems to limit ant access to flowers (reviewed in Willmer et al 2009) Exploitative
competition between ants and pollinators (Lach 2005 Ballantyne and Willmer 2012) as well as
the lethal effects of ant antibiotic secretions on pollen grains (Beattie et al 1985 Dutton and
Frederickson 2012) may have resulted in selection on plants to reduce ant visits to flowers Our
results suggest that trait-mediated indirect interactions resulting from interference competition
between ants and bees may favor plants that defend their flowers and their pollinators against
ants
Acknowledgements
We thank H Rusnock and S Meadley-Dunphy for assistance J Dix for help with flight cage
construction and Biobest for bumblebee colonies We are grateful to the Thomson and
Frederickson labs for guidance particularly T Makino funded by JSPS for helping to refine
artificial flowers J Ogilvie for help with bees K Prior for M rubra colony collections and K
Turner for study design suggestions The comments of the editor and two anonymous reviewers
prompted improvements in the article especially with regards to study design MEF and JDT
acknowledge NSERC Discovery Grant funding The University of Torontorsquos Faculty of Arts and
Science supported MGRTrsquos participation
67
68
References
Abbott K R 2006 Bumblebees avoid flowers containing evidence of past predation events
Canadian Journal of Zoology 841240-1247
Altshuler D L 1999 Novel interactions of non-pollinating ants with pollinators and fruit
consumers in a tropical forest Oecologia 119600-606
Ballantyne G and Willmer P 2012 Floral visitors and ant scent marks noticed but not used
Ecological Entomology 37402-409
Beattie A J Turnbull C Knox R B and Williams E G 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany
71421-426
Blanco M Runiaga H Maspoch S and Tarin P 1989 A simple method for
spectrophotometric determination of two-components with overlapped spectra Journal of
Chemical Education 66178-180
Byk J amp Del Claro K 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333-38
Davidson D W S C Cook and R R Snelling 2004 Liquid-feeding performances of ants
(Formicidae) ecological and evolutionary implications Oecologia 139255ndash66
Dukas R 2005 Bumble bee predators reduce pollinator density and plant fitness Ecology
861401-1406
Dussutour A and Simpson S J 2009 Communal nutrition in ants Current Biology 19740-
744
69
Dutton E M and Frederickson M E 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561-569
Frederickson ME 2009 Conflict over reproduction in an ant-plant symbiosis why Allomerus
octoarticulatus ants sterilize Cordia nodosa trees American Naturalist 173675-681
Galen C and Cuba J 2001 Down the tube pollinators predators and the evolution of flower
shape in the alpine skypilot Polemonium viscosum Evolution 551963-1971
Gonccedilalves-Souza T Omena P M Souza J C and Romero G Q 2008 Trait-mediated
effects on flowers artificial spiders deceive pollinators and decrease plant fitness Ecology
892407-2413
Gonzaacutelvez F G Santamariacutea L Corlett R T and Rodriacuteguez-Gironeacutes M A 2012 Flowers
attract weaver ants that deter less effective pollinators Journal of Ecology 10178ndash85
Goodale E and Nieh JC 2012 Public use of olfactory information associated with predation
in two species of social bees Animal Behaviour 84919-924
Harder LD and Thomson JD 1989 Evolutionary options for maximizing pollen dispersal of
animal-pollinated plants American Naturalist 133323-344
Heil M and McKey D 2003 Protective ant-plant interactions as model systems in ecological
and evolutionary research Annual Review of Ecology Evolution and Systematics 34425-
453
Heinrich B 2004 Bumblebee economics 2nd
edn Harvard University Press Cambridge
Ings T C and Chittka L 2009 Predator crypsis enhances behaviourally mediated indirect
effects on plants by altering bumblebee foraging preferences Proceedings of the Royal
Society B 2762031-2036
70
Inouye D W and Taylor O R Jr 1979 A temperate region plant-ant-seed predator system
consequences of extra floral nectar secretion by Helianthella quinquenervis Ecology 601-
7
Junker R R and Bluumlthgen N 2008 Floral scents repel potentially nectar-thieving ants
Evolutionary Ecology Research 10295ndash308
Junker R R Bleil R Daehler C C and Bluumlthgen N 2010 Intra-floral resource partitioning
between endemic and invasive flower visitors consequences for pollinator effectiveness
Ecological Entomology 35760ndash767
Jones E I and Dornhaus A 2011 Predation risk makes bees reject rewarding flowers and
reduce foraging activity Behavioral Ecology and Sociobiology 651505-1511
Lach L 2005 Interference and exploitative competition of three nectar-thieving invasive ant
species Insect Sociaux 52257-262
Lach L 2007 A mutualism with a native membracid facilitates pollinator displacement by
Argentine ants Ecology 881994-2004
Lach L 2008 Argentine ants displace floral arthropods in a biodiversity hotspot Diversity and
Distributions 14281-290
Makino T T and Sakai S 2007Experience changes pollinator responses to floral display size
from size-based to reward-based foraging Functional Ecology 21854863
Maloof J E and Inouye D W 2000 Are nectar robbers cheaters or mutualists Ecology
812651ndash2661
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506-514
Ohashi K Leslie A and Thomson JD 2008 Trapline foraging by bumble bees V Effects of
experience and priority on competitive performance Behavioral Ecology 19936-948
71
Palmer T M Doak D F Stanton M L Bronstein J L Kiers E T Young T P Goheen
J R and Pringle R M 2010 Synergy of multiple partners including freeloaders
increases host fitness in a multispecies mutualism Proceedings of the National Academy
of Sciences 10717234-17239
Preisser E L Bolnick D I and Benard M E 2005 Scared to death The effects of
intimidation and consumption in predator-prey interactions Ecology 86501-509
Stout J C Goulson D and Allen J A 1998 Repellent scent-marking of flowers by a guild of
foraging bumblebees (Bombus spp) Behavioral Ecology and Sociobiology 43317-326
Stout J and Goulson D 2002 The influence of nectar secretion rates on the responses of
bumblebees (Bombus spp) to previously visited flowers Behavioral Ecology and
Sociobiology 52239-246
Suttle K B 2003 Pollinators as mediators of top-down effects on plants Ecology Letters
6688-694
Thomson JD 1986 Pollen transport and deposition by bumble bees in Erythronium influences
of floral nectar and bee grooming Journal of Ecology 74329-341
Thomson JD Ogilvie J E Makino T T Arisz A Raju S Rojas-Luengas V and Tan M
G R 2012 Estimating pollination success with novel artificial flowers effects of nectar
concentration Journal of Pollination Ecology 9108-114
Tsuji K Hasyim A and Nakamura K 2004 Asian weaver ants Oecophylla smaragdina and
their repelling of pollinators Ecological Research 19669-673
Ulrich S and Bertsch A 1990 Do foraging bumblebees scent-mark food sources and does it
matter Oecologia 82137-144
Werner E E and Peacor S D 2003 A review of trait-mediated indirect interactions in
ecological communities Ecology 841083-1100
72
Willmer P G Nuttman C V Raine N E Stone G N Pattrick J G Henson K Stillman
P McIlroy L Potts S G and Knudsen J T 2009 Floral volatiles controlling ant
behaviour Functional Ecology 23888-900
Wright G A and Schiestl F P 2009 The evolution of floral scent the influence of olfactory
learning by insect pollinators on the honest signaling of floral rewards Functional Ecology
23841-851
73
Figures
Figure 21 Photograph of artificial flower lids A = anther L = lid N = nectary R = region
where anther or stigma is placed S = stigma T = thread W = weight
74
Figure 22 Dye transferred (mean plusmn 1SE) per trial by B impatiens workers A) from artificial
male and to artificial female flowers in the presence (unfilled circles) or absence (filled circles)
of Myrmica rubra workers and B) from artificial male flowers with (unfilled circles) or without
(filled circles) M rubra scent to ant-free female flowers in the 1st or the 2
nd to 4
th hours of the
trials
75
Chapter 3 Not just for the bees pollen consumption is common among tropical
ants
Cembrowski AR Reurink G and Frederickson ME
Planned submission to Biotropica
ARC and MEF designed the study ARC and GR did laboratory work ARC performed statistical
analyses and wrote the manuscript with input from other authors
Abstract
Although palynivory or pollen consumption is widespread among other hymenopterans there
are few accounts of palynivory in ants To quantify how often ants consume pollen we subjected
adult workers and larvae from 75 species of neo- and paleo-tropical ants to acetolysis a process
that destroys most organic material but leaves behind pollen grains In over half of the species
we examined ants contained a few pollen grains (lt 5) and ants of several Camponotus species
contained abundant pollen grains (gt50) We tested for an association between trophic level and
palynivory using stable nitrogen isotope ratios (δ15
N) but we did not find a significant
correlation we found pollen grains in lsquoherbivorousrsquo omnivorous and carnivorous ants We
suggest that our results indicate sporadic opportunistic pollen consumption by ants in tropical
forests
Introduction
For those organisms that can digest it efficiently pollen can be an excellent food source
Depending on the plant species pollen can contain up to 60 protein as well as lipids
76
carbohydrates and numerous trace nutrients (Roulston and Cane 2000) Pollen feeding
(palynivory) occurs in many taxa including birds mammals and arthropods (Roulston and Cane
2000) but it is best known among the Hymenoptera particularly bees Although adult bees feed
mainly on plant exudates (ie nectar) nearly all bee species rely on pollen as the chief protein
source for developing larvae (Willmer 2011) Closely related to bees ants have similar
nutritional requirements adult workers need carbohydrates whereas larvae are fed a diet rich in
protein (Dussutour and Simpson 2009) Ants also exploit nectar heavily and regularly visit
flowers (eg Herrera et al 1984 Bluumlthgen et al 2004) but unlike bees are not believed to
widely consume pollen Instead ants are thought to receive most of the protein in their diet from
predation or scavenging (eg Floren et al 2002)
However ants may not be as reliant on animal protein sources as commonly assumed In
tropical forests arboreal ants can have similar δ15
N ratios a measure of trophic level as
herbivores suggesting limited carnivory and substantial feeding on plant-derived protein sources
(Davidson et al 2003) One potential protein source is pollen This idea is supported by scattered
accounts of pollen consumption by ants from other ecosystems including deserts (Ness 2006)
boreal forests (Czechowski et al 2009) and tropical savannas (Byk and Del Claro 2010) One
ant genus Cephalotes is widely recognized to consume pollen (Creighton 1967 Baroni Urbani
and de Andrade 1997 Byk and Del Claro 2010) However beyond this genus our knowledge of
pollen consumption by ants has largely been limited to direct observations (but see Czechowski
et al 2011) and it is unknown if palynivory is truly rare among ants or simply under-reported
We borrowed a technique commonly used in palynology acetolysis to assess the frequency of
pollen consumption by ants in 75 neo- and paleo-tropical ant species This technique allowed us
to examine many more ant species than we could have observed directly
77
Methods
Adult worker ants were collected by hand at baits or from leaf litter using Winkler extractors
while larvae along with adults were collected by hand from nests Collections were made by L
Arcila Hernandez and J Sanders in the primary tropical rainforest surrounding the Centro de
Investigaciόn y Capacitaciόn Rio Los Amigos (CICRA 12deg34rsquoS 70deg05rsquoW) in Peru in 2010-
2011 and by E Youngerman from lowland primary and secondary rainforests in Madang
Province Papua New Guinea (PNG) in 2011-2012 We sorted ant samples to morphospecies and
then identified them to genus and when possible to species using existing keys which were
unavailable for PNG ants Ant samples used for acetolysis were whole adult workers guts from
adult workers and whole larvae
We used acetolysis to look for pollen grains inside ants In acetolysis organic material is
dissolved in acetic anhydride and sulfuric acid leaving behind stained pollen exines When
possible we used adult and larval ants of the same species from different colonies or bait
locations in an attempt to sample more representatively We prepared samples by first washing
them in 70 ethanol to remove any pollen present on the samplersquos outer surface before placing
them individually in micro-centrifuge tubes with ~05mL of a 91 mixture of acetic anhydride
sulfuric acid We then heated each sample at 95degC for 20min allowing tissues to dissolve Next
we centrifuged samples at 1000 RPM for 5min and decanted the liquid supernatant before
adding ~05mL of glacial acetic acid to the precipitate centrifuging again as above We decanted
the samples and added a drop of distilled water
78
We examined a 150μL sub-sample of this solution for pollen grains under a light
microscope Larvae gave less than 150μL of solution following acetolysis so we sampled the
entire amount We identified pollen grains by their staining and their surface architecture For
most species we examined at least three adult workers and three larvae but for some species
fewer than three adult workers or larvae were available (Tables 31 and 32)
We investigated the relationship between trophic level as measured by δ15
N ratios and
pollen consumption by ants Stable isotope (SI) data was available for some (n =23) of the
Peruvian species from a separate project in which collections overlapped Samples were stored in
EtOH and dried at 50degC overnight We used heads legs and thoraxes (but not gasters) of ants
for SI analysis Often single individuals did not provide enough material for analysis and we
combined multiple individuals to achieve necessary weights up to 10 for small ant species
Samples were analyzed at the Boston University Stable Isotope Laboratory using a Finnigan
Delta-S analyzer For other Peruvian species for which we had samples but not corresponding
stable isotope data (n =25) we took the median δ15
N ratio value of that speciesrsquo genus and
assigned it to the species in question Using pollen presenceabsence data for these 48 species
we used logistic regression to correlate pollen presence and δ15
N ratio using R (v 2151)
Results
We found pollen grains in 37 of the 197 adult workers 20 of the 102 larvae and 38 of the 75 ant
species we examined (Tables 31 and 32) The proportion of individuals containing pollen did
not differ significantly between larvae and adults (χ2
1 = 002 p = 088) The number of pollen
grains found was often low (median = 3) but highly variable (range 1-468) Also pollen
79
presence was inconsistent among conspecific ant samples of species that contained pollen 31
had pollen in only one of the larvae or adults examined In only three species (two Cephalotes
and one Myrmelachista) did all examined adults contain pollen
Highly lsquoherbivorousrsquo carnivorous and omnivorous ants contained pollen grains (Fig
31) There was no correlation between δ15
N ratio and pollen presence (χ2 = 150 df = 1 p =
022) In fact several carnivorous species contained pollen grains (eg Odontomachus sp 1
Pachycondola sp1)
Discussion
Pollen consumption by ants appears to be more common than suspected The majority of species
(51) had at least one individual that had consumed pollen and in 20 multiple individuals
contained pollen Numbers of grains found tended to be low however with most individuals
containing only a few grains Ants may use pollen as only a small portion of their diet exploiting
a host of other protein-containing foods more heavily including other low- δ15
N foods (eg
honeydew epiphylls Houmllldobler and Wilson 1990 Davidson et al 2003)
There are a few reasons why pollen may not be heavily exploited by ants Pollen requires
much post-consumption processing to extract its nutrients These nutrients are located in the
pollenrsquos cytoplasm which is protected from direct digestion by the exines and intines of the
grains Organisms must either break through these layers or cause the pollen grains to germinate
or pseudo-germinate to access and digest the cytoplasm (Roulston and Cane 2000) This can be
achieved by consuming a sugar-containing solution (such as nectar) along with pollen However
animals that are not adapted to feed on pollen may not be very efficient at this process only
80
extracting nutrients from some of the grains (Herrera and Martiacutenez del Rio 1998) Thus while
ants can likely get some nutrients from pollen other forms of more easily processed protein may
be more attractive
Pollen may also pose handling difficulties for ants Despite the large number of studies
that have recorded ants visiting flowers (Chapter 1) only a few papers detail ants harvesting
pollen directly from anthers Byk and Del Claro (2010) reported that Cephalotes pusillus ants
removed almost all of the available pollen from Ouratea spectabilis flowers and at least two
other studies describe ants consuming pollen along with nectar from flowers (Horskins and
Turner 1999 Ness 2006) Unlike bees which possess both morphological (eg specialized hairs
for holding pollen) and behavioral adaptations (eg stereotyped pollen-packing motions) ants
have no apparent mechanisms to increase the amount of pollen harvested and are likely limited
by the number of grains they can ingest or carry Rather than collect pollen directly from flowers
it appears more likely that ants collect it haphazardly in the environment leading to a smaller
number of pollen grains consumed at any one time It is known that at least Cephalotes ants
engage in such behavior Creighton (1967) observed Cephalotes texanus collecting pollen grains
trapped by Citrus x paradisi leaf hairs
There is a chance that the small numbers of pollen grains we observed came from
secondary pollen consumption (ie consuming a palynivorous organism) or consumption of
liquid containing pollen While we cannot rule this out we believe our results represent actual
targeted pollen consumption by ants Cephalotes ants known palynivores (Creighton 1967
Baroni Urbani and de Andrade 1997) contained similar numbers of pollen grains as many of the
pollen-containing ant species that we examined As well in at least once instance ants that we
found containing pollen grains were in the process of preying on non-pollen consuming
81
organisms (eg a Pachycondola species that was carrying a termite in its mandibles) or were
attracted by sugar baits
Contrary to our expectations there was no relationship between an ant speciesrsquo δ15
N ratio
and whether it had consumed pollen (Fig 32) We expected palynivory to be more common in
herbivorous ants because pollen presents a widespread source of protein a potentially limiting
resource The lack of pattern might reflect how available pollen grains are in the environment
While other forms of food may be preferable ants tend to be opportunistic foragers (Carroll and
Janzen 1973) and there is likely little cost associated with the consumption of scattered pollen
grains during foraging trips beyond the volume they occupy in the gut Some genera were found
to consistently consume pollen In support of previous observations (Baroni Urbani and de
Andrade 1997 Byk and Del Claro 2010) Cephalotes species were among the most common
pollen consumers Camponotus species also consistently contained pollen grains of the eight
species examined five contained pollen and pollen was found in species from both Peru and
PNG Camponotus species also contained the two largest numbers of pollen grains found (468
and 271) Taken together these results suggest that they may be another as yet unrecognized
commonly palynivorous genus
In general larvae did not contain large numbers of pollen grains (ranging from 1-38
grains) This is counter to what is seen in bees where larvae are the main recipients of pollen
(Willmer 2011) This difference may be because ant larvae are continually fed via trophallaxis
by adults throughout their development whereas bee larval cells are provisioned with pollen and
then sealed while larvae complete their development If both adults and larvae are able to extract
nutrients from pollen we would expect to see a preferential flow of nutrients from pollen to
larvae but not necessarily the grains themselves
82
Unlike palynivory by organisms that also provide pollination services pollen
consumption by ants is unlikely to benefit plants Ants rarely act as pollinators (Beattie et al
2004) They have pollen-killing antibiotic secretions on their cuticles (Dutton and Frederickson
2012) and they can compete with other flower visitors reducing flower visitation rates (Ness
2006) The negative impacts of flower-visiting ants are thought to have led to the evolution of
ant-repellent floral volatiles (reviewed in Willmer 2009) These volatiles can pre-empt
competition between ants and pollinators and may often be located within the pollen itself
(Willmer 2009) If floral volatiles truly are a defense against ant visitation our results suggest an
additional evolutionary pressure to not only prevent ant-pollinator interactions but to also
protect plant gametes from occasional consumption by a non-pollinating visitor
Acknowledgements
We would like to thank Jon Sanders Lina Arcila Hernandez and Eric Youngerman for providing
samples the Thomson lab for use of supplies the Pierce lab for funding support during JSrsquo and
EYrsquos collections and the staff of CICRA and the New Guinea Binatang Research Center for
logistical support The Peruvian Ministry of Agriculture provided permits (Nos 394-2009-AG-
DGFFS-DGEFFS and 299-2011-AG-DGFFS-DGEFFS) MEF acknowledges financial support
from an NSERC Discovery Grant a Connaught New Researcher Award an Ontario Ministry of
Economic Development and Innovation Early Researcher Award and the University of Toronto
ARC was supported by an Ontario Graduate Scholarship and Sigma Xi
83
References
Baroni Urbani C and M L de Andrade 1997 Pollen eating storing and spitting by ants
Naturwissenschaften 84256ndash258
Beattie A J C Turnbull R B Knox and E G Williams 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany 71421ndash
426
Bluumlthgen N G Gottsberger and K Fiedler 2004 Sugar and amino acid composition of ant-
attended nectar and honeydew sources from an Australian rainforest Austral Ecology
29418ndash429
Byk J and K Del-Claro 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333ndash38
Carroll C R and D H Janzen 1973 Ecology of foraging ants Annual Review of Ecology and
Systematics 4231ndash257
Creighton W S 1967 Studies on free colonies of Cryptocerus texanus Satschi (Hymenoptera
Formicidae) Psyche 7434ndash42
Czechowski W B Markό and A Radchenko 2009 Rubbish dumps reveal the diet of ant
colonies Myrmica schencki Em and Myrmica rubra (L) (Hymenoptera Formicidae) as
facultative pollen-eaters Polish Journal of Ecology 56737ndash741
Czechowski WB Markoacute K Erős and E Csata 2011 Pollenivory in ants (Hymenoptera
Formicidae) seems to be much more common than it was thought Annales Zoologici
61519ndash525
84
Dussutour A and S J Simpson 2009 Communal nutrition in ants Current Biology 19740ndash4
Dutton E M and M E Frederickson 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561ndash569
Floren A A Biun and K E Linsenmair 2002 Arboreal ants as key predators in tropical
lowland rainforest trees Oecologia 131137ndash144
Grant B R 1996 Pollen digestion by Darwinrsquos finches and its importance for early breeding
Ecology 77489-499
Herrera L G and C Martiacutenez del Rio 1998 Pollen digestion by new world bats effects of
processing time and feeding habits Ecology 792828ndash2838
Houmllldobler B and Wilson E O 1990 The Ants 1st Edition Harvard University Press
Cambridge
Horskins K and Turner V B 1999 Resource use and foraging patterns of honeybees Apis
mellifera and native insects on flowers of Eucalyptus costata Australian Journal of
Ecology 24221-227
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506ndash514
Roulston T H I and J H Cane 2000 Pollen nutritional content and digestibility for animals
Plant Systematics 222187ndash209
Van Tets I G 1997 Extraction of nutrients from Protea pollen by African rodents Belgian
Journal of Zoology 12759-65
Willmer P G 2011 Pollination and Floral Ecology 1st Edition Princeton University Press
New Jersey
85
Willmer P G C V Nuttman N E Raine G N Stone J G Pattrick K Henson P Stillman
et al 2009 Floral volatiles controlling ant behaviour Functional Ecology 23888ndash900
86
Tables
Table 31 Numbers of adult worker and larval ants examined for each species collected in Peru
Numbers of pollen grains found in individual samples are given in parentheses after the sample
size when there were no grains no numbers are given
Genus Species Number of adults analyzed
(numbers of grains found)
Number of larvae analyzed
(numbers of grains found)
Dolichoderinae
Azteca sp 1 3 0
sp 2 2 0
sp 3 0 2
sp 4 3 (1) 0
sp 5 3 (2) 0
Dolichoderus decollatus 3 (66) 0
sp 1 3 0
sp 2 3 1 (2)
sp 3 3 3
sp 4 0 2
Ecitoninae
Labidus sp 1 3 0
Formicinae
Camponotus sp 1 3 3
sp 2 3 (82) 1 (2)
sp 3 3 (3) 0
sp 4 0 2
87
sp 5 8 (11) 0
sp 6 4 (7 16 468) 0
sp 7 5 (12) 0
sp 8 2 (1) 0
Paratrechina sp 1 4 (11) 0
Myrmelachista sp 1 3 (5 11 18) 0
sp 2 0 3
Trachymyrmex sp 1 3 (21) 0
sp 2 3 0
Myrmicinae
Atta sp 1 3 0
Cephalotes atratus 3 (2 2 21) 0
placidus 3 (1 4 21) 0
sp 1 3 0
sp 2 1 0
sp 3 2 0
Crematogaster sp 1 3 1
sp 2 3 (35) 0
sp 3 9 (7) 0
sp 4 2 0
Megalomyrmex sp 1 3 0
sp 2 0 2
sp 3 4 (7) 0
sp 4 3 0
Monomorium sp 1 0 3 (2)
88
Pheidole sp 1 0 3
sp 2 3 0
sp 3 0 3
Procryptocerus sp 1 3 0
Solenopsis sp 1 0 3
sp 2 3 (3 5) 0
Wasmannia sp 1 3 0
Ponerinae
Pachycondola sp 1 3 (3) 0
Odontomachus sp 1 3 (34) 0
Pseudomyrmicinae
Pseudomyrmex sp 1 3 0
sp 2 3 0
sp 3 3 (9) 0
sp 4 0 3
sp 5 0 3
89
Table 32 Numbers of adult worker and larval ants examined for each species collected in Papua
New Guinea Numbers of pollen grains found in individual samples are given in parentheses
after the sample size when there were no grains no numbers are given
Genus Species Number adults analyzed
(grains found)
Number larvae analyzed
(grains found)
Aenictinae
Aenictus sp 1 3 3 (1 1)
Dolichoderinae
Philidris sp 1 3 1 (4)
Tapinoma sp 1 2 3 (38)
Technomyrmex sp 1 0 3 (12 12)
Ectatomminae
Rhytidoponera sp 1 1 3 (2)
Formicinae
Acropyga sp 1 3 3 (2)
sp 2 3 3
sp 3 3 3
Anoplolepsis sp 1 3 (1) 3 (1)
Calomyrmex sp 1 3 3 (1)
Camponotus sp 1 3 (1 271) 3
sp 2 1 3
sp 3 2 0
Nylanderia sp 1 3 3
Opisthopsis sp 1 3 3
Polyrachis sp 1 1 3 (4)
90
Myrmicinae
Crematogaster sp 1 3 3
sp 2 3 (32) 3
Monomorium sp 1 3 (1) 3 (1)
sp 2 3 3 (1 1 1)
Pheidole sp 1 3 3
Pristomyrmex sp 1 3 3 (11)
Pyramica sp 1 3 0
Ponerinae
Odontomachus sp 1 3 3 (4)
91
Figures
Figure 31 Photographs of pollen grains found in two ant species a) Dolichoderus sp 2 and b)
Camponotus sp 6 Scale bars = 50 microm
92
Figure 32 Relationship between trophic level as measured by δ15
N ratio and pollen presence
for 48 neotropical ant species 1 Atta sp 5 Azteca spp 8 Camponotus spp 5 Cephalotes spp 4
Crematogaster spp 5 Dolichoderus spp 1 Labidus sp 3 Megalomyrmex spp 1 Monomorium
sp 2 Myrmelachista spp 1 Odontomachus sp 1 Pachycondola sp 1 Paratrechina sp 3
Pheidole sp 4 Pseudomyrmex spp 2 Solenopsis spp and 1 Wasmannia sp
93
Concluding Remarks
Ants have traditionally been considered to be unwelcome flower visitors they consume floral
rewards ostensibly meant for pollinators while rarely providing pollination services themselves
In this thesis I investigate the validity of this assumption and use an experimental study and an
observational study to examine two ways that ants may impact plant reproduction
In the first chapter I review the literature on ants as flower visitors I show that the common
assumption of ants always being categorically negative for plant reproduction is incorrect while
ants may reduce floral attractiveness to pollinators the net effect of ants on plant fitness
measures is variable ranging from positive to negative to neutral I also discuss ants as
pollinators and ways that plants may minimize ant visitation to flowers before finishing with a
section highlighting questions that still need investigation
In the second chapter I explore how interference competition with ants can structure bumblebee
foraging behavior and how this may alter pollen movement patterns Using artificial flowers with
male or female function I allowed Bombus impatiens bumblebees to forage on flowers that
either did or did not have Myrmica rubra ants on them Ant presence significantly reduced the
amount of pollen analogue donated and received by flowers Building on these results I found
that the presence of ant scent alone elicited similar changes to bumblebee behavior reducing the
amount of pollen analogue donated by male flowers My results show that interference
competition is sufficient to alter bumblebee foraging behavior and that the effects of ants on
pollinators may extend after ant visitation to flowers Further study is needed to determine if bees
are able to differentiate between ant species by visual or olfactory cues Their ability to do so
94
could affect how generalized their response to ant presence is and may help to better predict how
ants will affect plant reproduction
In the final chapter I investigate how common palynivory is amongst tropical ant species I used
acetolysis and light microscopy to examine whole ants larvae and ant guts from Papua New
Guinea and Peru I found that tropical ants consistently contain pollen grains albeit often in
small numbers (lt5) However some genera notably Camponotus and Cephalotes consistently
contained higher numbers providing the first evidence that Camponotus may be a palynivorous
genus The small numbers of pollen grains present may indicate that ants preferentially exploit
other more easily digestible high-protein foods over pollen Future studies should investigate
exactly what role pollen plays in ant diets by supplementing the diet of captive ant colonies with
pollen This would allow us to examine how pollen consumption affects colony demography as
well as how efficiently ants digest pollen and whether ants exhibit pollen preferences
I bring up several pressing and unanswered questions within this thesis and highlight three here
First numerous plant species possess chemical or physical traits assumed to increase plant
fitness by reducing ant visitation However this has rarely been tested and instead research has
focused predominately on documenting ant repellence We should move away from this and
begin examining plant fitness in the presence or absence of these repellent traits to see how
beneficial these traits truly are Second how flower visiting ants impact male fitness has been
almost completely ignored in favor of female fitness Future studies should investigate how ant
visitation affects both pollen donation and pollen movement patterns Lastly research involving
antibiotics present on ant cuticles and their effect on pollen viability has been too small in scope
often limited to demonstrating the pollen killing effects of ant secretions Investigation into
95
whether plants have evolved any resistance to the secretions of the local ant community or why
some plants appear unaffected by their ant partners may yield exciting results
In conclusion ants have the capacity to strongly affect plant reproduction both positively and
negatively The fact that these diametrically opposing forces exist means we cannot approach
flower-visiting ants as either positive or negative a priori Instead we should strive to work
towards a better understanding of what is occurring in the system of interest and what role ants
play within it
96
Copyright Acknowledgments
Chapter 2 ldquoAnts and ant scent reduce bumblebee pollination of artificial flowersrdquo is current in
press in The American Naturalist Request for its inclusion here was sought and approved
v
Table of Contents
Acknowledgments iii
List of Tables vii
List of Figures viii
Chapter 1 Ants as flower visitors and their effects on pollinator behavior and plant
reproduction 1
Abstract 1
Introduction 2
1 Ant interactions with flowers and floral visitors 3
2 Ants as pollinators 12
3 Floral defenses against ants 14
4 Future directions 20
References 24
Tables 39
Chapter 2 Ants and ant scent reduce bumblebee pollination of artificial flowers 54
Abstract 54
Introduction 55
Methods 57
Results 62
Discussion 63
Acknowledgements 66
References 68
Figures 73
Chapter 3 Not just for the bees pollen consumption is common among tropical ants 75
vi
Abstract 75
Introduction 75
Methods 77
Results 78
Discussion 79
Acknowledgements 82
References 83
Tables 86
Figures 91
Concluding Remarks 93
Copyright Acknowledgments 96
vii
List of Tables
Table 11 List of studies of flower-visiting ants that consumed floral rewards or interacted with
pollinators and included a measure of fitness39
Table 12 List of studies that have either demonstrated ant pollination through exclusion
experiments or whose results strongly suggest it48
Table 31 Numbers of adult worker and larval ants examined for each species collected in Peru
and numbers of pollen grains found87
Table 32 Numbers of adult worker and larval ants examined for each species collected in Papua
New Guinea and numbers of pollen grains found90
viii
List of Figures
Figure 21 Photograph of artificial flower lids with anther and stigma74
Figure 22 Dye transferred (mean plusmn 1SE) by B impatiens a) in the presence and absence of M
rubra ants and b) with or without M rubra scent75
Figure 31 Photographs of pollen grains found inside ants92
Figure 32 Relationship between trophic level and pollen presence for neotropical
ant species93
1
Chapter 1 Ants as flower visitors and their effects on pollinator behavior and plant
reproduction
Cembrowski AR and Frederickson ME
Planned submission to a journal such as Oikos or Insectes Sociaux
ARC wrote the manuscript with input from MEF
Abstract
Ants often provide a range of benefits to flowering plants particularly by reducing herbivory
However when ants visit flowers they can deter pollinators or damage floral structures
Currently it is unclear how often and in what direction flower-visiting ants affect plant
reproductive success A review of the literature shows that ants can help or hinder pollination
processes but overall flower-visiting ants most commonly have a non-significant net effect on
plant fitness Although ants may directly harm flowers or indirectly affect pollination by
consuming floral nectar or harassing pollinators they can also benefit plants by attacking
florivores or predators of pollinators Ants occasionally act as pollinators themselves though ant
secretions often kill pollen grains There is growing evidence that many plants have traits that
allow them to avoid or minimize the negative effects of flower-visiting ants More research is
needed to determine if these traits provide fitness benefits to plants by preventing ant visitation
or if they function as generalized repellents of floral antagonists
2
Introduction
Ants and angiosperms share a long evolutionary history Ant diversification closely followed that
of flowering plants as ants took advantage of the large prey numbers that angiosperms supported
and the new habitats they provided (Moreau 2006) Subsequently numerous angiosperm
lineages evolved intimate associations with ants that function as an indirect or biotic form of
plant defense Food rewards such as extrafloral nectar or pearl bodies or housing in the form of
domatia attract ants to plants where they reduce herbivory and increase plant fitness (reviewed
in Rosumek et al 2009) However the traits that make ants good plant defenders particularly
their abundance attraction to sugar sources and aggressiveness can also cause problems for
plant reproduction Ants attracted by floral nectar can compete with pollinators for resources
and defend flowers potentially reducing plant fitness (Ness 2006 Junker et al 2007) That ants
negatively affect plant reproduction is an old and pervasive hypothesis Kerner (1878) called ants
ldquomost unwelcome guestsrdquo on flowers and more recent authors have similarly expressed that ant
visitation to flowers reduces plant reproductive success (Ness 2006 Lach 2007 Willmer et al
2009)
Nonetheless studies assessing the impact of flower-visiting ants on plant reproductive
success have produced mixed results Ants can have negative impacts on pollination and flower
function (Galen 1983 Puterbaugh 1998 Ness 2006 Hansen and Muumlller 2009) However other
studies have found no effect or even strong benefits of ant visits to flowers (Rico-Gray and
Thien 1989 Altshuler 1999 Schuumlrch 2000 Ashman and King 2005) This discrepancy between
ants being ldquounwelcome guestsrdquo and their actual effects on plant reproduction requires
clarification Our goal here is to highlight the ways that flower-visiting ants may affect plant
reproduction both positively and negatively and to discern through a review of the literature
3
what impacts ants have on plant reproductive success First we discuss mechanisms by which
flower-visiting ants can directly and indirectly impact plant reproduction both through floral
damage and by mediating changes in pollinator or florivore behavior We end this section by
evaluating published research that collectively shows that flower-visiting ants have
predominantly neutral effects on plant fitness measures Next we discuss pollination by ants
focusing on the ldquoantibiotic hypothesisrdquo before describing how plants may prevent ants from
visiting flowers Lastly we discuss promising new avenues of research and highlight work that
still needs to be performed to give a more complete understanding of ants as flower visitors
1 Ant interactions with flowers and floral visitors
i) Damage to flowers
Ants can directly disrupt plant reproduction by damaging floral structures preventing either the
fertilization of ovules or the presentation of pollen Rarely ants forage on floral tissue itself for
example Puterbaugh (1998) found that Formica neorufibarbus ants chewed through the coronal
ring of Eritrichium aretiodes flowers to get lipids More often but still not commonly foraging
ants chew through reproductive tissue to access floral nectar This can cause sterilization of
whole flowers (Echium plantagineum Kirk 1984) or affect female function only if just styles are
damaged effectively creating male flowers (Polemonium viscosum Galen 1983) Antibiotic
secretions on ant integuments may also directly damage pollen grains but the fitness effects of
this are unclear (see 2 below)
Several species of Crematogaster and Allomerus ants sterilize their host plants by
attacking floral buds (Yu and Pierce 1998 Stanton et al 1999 Gaume et al 2005 Maleacute et al
2012 Table 11) These ants nest in ldquoant-plantsrdquo or ldquomyrmecophytesrdquomdashie plants that host ant
4
colonies in specialized structures (domatia) The sterilizing ants do not appear to consume floral
tissues (Yu and Pierce 1998) and two hypotheses may explain why ants destroy floral buds First
ant colony size is often limited by domatia availability on their host plant By destroying floral
buds ants can cause plants to divert resources from reproductive to vegetative growth increasing
domatia number and allowing for larger ant colonies (Yu amp Pierce 1998 Frederickson 2009)
Second when plant-ants must defend their colony from takeovers by other colonies they may
benefit from limiting access to their host plant By destroying buds that could develop into
flowers branches or leaves ants can prevent their host plant from contacting nearby plants
occupied by enemy ant colonies (Stanton et al 1999)
The impact of sterilization on lifetime plant fitness is unclear Ants may destroy all of
their host plantrsquos floral buds (Yu and Pierce 1998) completely preventing reproduction or only a
portion of them (Maleacute et al 2012) allowing some seed set However counter-intuitively this
sterilization behavior may provide long-term fitness benefits to plants (Frederickson 2009
Palmer et al 2010) Ant-plants often host several different ant species over their lifetimes some
of which are non-sterilizing If hosting a sterilizing species at a certain life stage increases
survival or growth relative to hosting a non-sterilizing species at that life stage plants may
increase lifetime fitness by largely forgoing current reproduction in favor of increasing future
reproduction (Palmer et al 2010) Why plants invest resources in producing flowers that are
quickly destroyed by ants is not known but the timing of flower destruction may be important If
ants attack flowers after anthers dehisce plants may still realize some male fitness via pollen
donation (Yu and Pierce 1998) Additionally if the cost of flower production is relatively minor
and ants sometimes ldquomissrdquo flowers (Edwards and Yu 2008) attempting limited reproduction
may be a better strategy than forgoing it entirely
5
ii) Competition with pollinators
Ant activity on flowers can dramatically alter pollinator behavior Ants can reduce the species
diversity frequency or duration of pollinator visits to flowers (Norment 1988 Tsuji et al 2004
Ness 2006 Junker et al 2007 Lach 2007 2008b Romero et al 2011 and see Chapter 2) These
changes are largely due to either exploitative competition (ie consumption of shared resources)
or interference competition (ie direct aggression) between ants and pollinators
a) Exploitative competition
Pollinators and ants frequently compete for floral nectar Flower-visiting ants are often classified
as nectar thieves (sensu Inouye 1980) because they typically enter flowers through the
ldquolegitimaterdquo opening and consume nectar but transfer no pollen (Wyatt 1980 Ness 2006
Chamberlain and Holland 2008) Ants can also act as primary nectar robbers chewing holes
through corollas to reach nectaries (Willmer and Corbet 1981 Koopowitz and Marchant 1998)
although they are more commonly secondary nectar robbers and use holes made by previous
robbers (Newman and Thomson 2005)
Like other flower visitors foraging ants will often leave nectar behind in flowers This
amount can be less (Bleil et al 2011) more (Fritz and Morse 1982) or similar to (Lach 2008a)
amounts left by other visitors Often ants are morphologically constrained from fully exploiting
nectar Unlike bees and butterflies ants do not have proboscises and must get close to nectar to
consume it Narrow corollas or nectar spurs can prevent ant access to some or all floral nectar
(Schemske 1980 Galen and Cuba 2001 and see below) Smaller ants may be better able to
6
access nectar (Newman and Thomson 2005 Agarwal and Rastogi 2007) but can be limited by
their crop size in the amount of nectar they can remove (Lach 2005) If foraging ants leave
enough nectar behind for a pollinator to recover the cost of visiting a flower ants may have
minimal effects on plant reproductive success (Maloof and Inouye 2000 Turner et al 2012)
Differences among ant species in behavior and diet also affect their consumption of floral
nectar Species vary in how many flowers they visit per inflorescence or per plant and in how
many ants will visit a flower simultaneously (Lach 2005 Junker et al 2007 Lach 2013)
Invasive antsmdashthought to be better at exploiting and defending resources than native ants
(Holway et al 2002)mdashoften recruit to flowers in large numbers sometimes several dozen or
more (Visser et al 1999 Hansen and Muumlller 2009) Overall many factors may influence how
heavily ants exploit floral nectar on plants including ant density plant height the spatial or
temporal distribution of floral nectar and the availability of alternative food resources for ants
(Cushman and Addicott 1991 Lach 2013)
Rarely ants and pollinators may compete for pollen We know of only a few studies that
have recorded ants harvesting pollen directly from anthers (Horskins and Turner 1999 Ness
2006 Byk and Del-Claro 2010) Other studies have found pollen grains in ants (eg Baroni
Urbani and de Andrade 1997 and see Chapter 3) but this may not indicate competition with
pollinators ants sometimes collect stray pollen grains from the environment (Creighton 1967)
One New World genus Cephalotes might be specially adapted for pollen consumption They
possess ldquoproventricular shieldsrdquo structures that may restrict solid food to the crop allowing
nestmates to exchange pollen grains via trophallaxis It is unclear if this structure evolved for this
purpose but Cephalotes are the most frequently documented palynivorous ants (eg Creighton
1967 Baroni Urbani and de Andrade 1997 Byk and Del-Claro 2010) How efficiently ants
7
digest pollen and whether ants like bees feed it to larvae as a protein source is unknown At
least two studies have found that ant larvae consume pollen providing some support for this idea
(Wheeler and Bailey 1920 and Chapter 3)
b) Interference competition
Ants are relatively unique among flower visitors in defending floral resources from competitors
(but see Willmer and Corbett 1981 Roubik 1982) This defense ranges from chance encounters
with ants attacking visitors when flower occupancy overlaps (Ashman and King 2005 Junker et
al 2007) to actively patrolling inflorescences or constructing protective galleries around
nectaries (Horvitz and Schemske 1984 Gaume et al 2005 Hansen and Muumlller 2009)
Interference competition even in the absence of exploitative competition is sufficient to alter
pollinator behavior (Chapter 2)
The strength and outcome of interference competition depends on the identities of the ant
and the pollinator Pollinators might not alter their visitation patterns in response to non-
aggressive ants (Junker et al 2007) but more antagonistic ants may elicit avoidance behavior
before or soon after the visitor lands (Willmer and Corbet 1982 Altshuler 1999 Schuumlrch et al
2000 Table 11) Pollinator size may help predict their reaction to ants because ants may deter
only small-bodied pollinators (Gonzaacutelvez et al 2012) It is not clear whether flower visitors view
ants as aggressive competitors or as potential predators rarely ants prey on pollinators (Schatz
and Wcislo 1999) but in most cases ants merely cause pollinators to relocate
How well pollinators recognize and avoid ant-visited flowers is unresolved Collectively
pollinators have lower visitation rates to flowers visited by ants (Romero et al 2011) This is
8
dependent on pollinator identity however dipterans do not appear to discriminate between ant-
visited and ant-free flowers while bees can identify and avoid ant-visited flowers Bees likely
use a combination of visual and olfactory cues to recognize ant-visited flowers They can
identify previous conspecific and some heterospecific insect visitors by the hydrocarbon
ldquofootprintsrdquo they leave during foraging (Stout and Goulson 2001) Bumblebees can detect ant
hydrocarbons although this has been shown only in the laboratory (Ballantyne and Willmer
2012 Chapter 2) No study has yet examined if bees can use sight alone to detect ants but
several suggest it (Junker et al 2007 Gonzaacutelvez et al 2012) Some bees may discriminate
between ant species when visiting flowers (eg Lach 2008b) which follows if not all ant-
pollinator interactions are equally costly The ability of bees to do so will affect how generalized
their avoidance behaviors will be If they can discriminate between species bees may avoid
specific flowers visited by more ldquocostlyrdquo ants However if they cannot bees might either avoid
all ant-visited flowers or none depending on how negative the average interaction with an ant is
iii) Interactions with flower and pollinator antagonists
Ants can defend the flowers they visit Numerous plant species have circumfloral or bracteal
nectaries (ie extrafloral nectaries around flowers) and ants attracted to these nectaries can
reduce bud ovule or flower predation increasing plant fitness (Inouye and Taylor 1979 Rico-
Gray and Thien 1989 Sugiura et al 2006) Although the presence of EFNs (extrafloral nectaries)
so close to floral nectaries (sometimes less than 1 cm Ness 2006) might increase discovery and
exploitation of floral nectar by ants only one study has investigated this hypothesis finding no
relationship between the proximity of EFNs to floral nectaries and antsrsquo use of floral nectar
(Galen 2005) Holes made by nectar robbers are functionally analogous to circumfloral nectaries
9
and can also attract ants that defend flowers (Newman and Thomson 2005) and nectar-thieving
ants can similarly prevent florivory in some systems (Lach 2007 Bleil et al 2011)
Ants can also reduce the abundance of flower-dwelling predators or parasites of
pollinators Ambush predators such as crab spiders often forage at flowers where they may
reduce pollinator visitation rates and plant fitness (Dukas 2001 Gonccedilalvez-Souza et al 2008)
Ants can decrease spider abundance and richness on plants (Rudgers 2004 Renault et al 2005
Nahas et al 2012) and some evidence suggests that they can do the same on flowers (Lach
2008) In select cases ants may also reduce the abundance of pollinator parasitoids (Holland et
al 2011) Whether ants increase plant reproductive success by removing pollinator antagonists
has not yet been examined but should be If ants create lsquoenemy-freersquo flowers pollinators might
prefer to land on flowers with ants
iv) Net effects
Table 11 compiles studies in which the authors recorded ants consuming floral rewards or
interacting with pollinators and measured some aspect of plant fitness (eg fruit or seed set
pollen deposition) We included only a single study per plant species unless subsequent studies
found conflicting results or measured the fitness of another plant sex Records of ant pollination
are included in a separate table (Table 12)
Table 11 reveals relatively few examples of flower-visiting ants negatively affecting
plant fitness given how costly ant visitation is assumed to be The net effect of ants on plant
reproductive success is actually quite variable with similar numbers of studies finding positive
negative or neutral effects of ant presence Surprisingly when ants do have negative effects it is
10
usually because they directly damage floral structures and not because they prevent pollinators
from visiting flowers and successfully transferring pollen Why are ants not more often negative
for plant reproduction when this is commonly assumed to be true
First ants may compensate for reducing pollinator visitation rates by improving
pollination quality As pollinators move between flowers on a plant they often deposit self-
pollen on stigmas (ie geitonogamy) In some plants with late-acting self-incompatibility ovules
fertilized by self-pollen are subsequently aborted reducing the number of seeds that can be
produced (Barrett 2002 and references therein) Geitonogamy can also reduce offspring viability
or vigor (de Jong et al 1993) and cause pollen discounting a reduction in the amount of pollen
available for outcrossing because of self-pollination (Barrett 2002) By competing with
pollinators ants might decrease rates of geitonogamous pollination if pollinators then visit fewer
sequential flowers within a plant Ants might also cause pollinators to fly further between plants
potentially increasing the genetic diversity of pollen arriving at stigmas Although such effects
have been reported for other floral larcenists (Maloof 2001 Irwin 2003) to our knowledge these
hypotheses have not been investigated in relation to ants
Second ants and pollinators do not always forage for the same resources possibly
allowing them to partition floral rewards If ants forage for nectar but not pollen and flowers can
be pollinated efficiently by visitors looking to gather mostly pollen and little or no nectar then
plant reproduction will not be affected by ants consuming floral nectar Junker et al (2010a)
found that honeybees foraged for both pollen and nectar in the absence of ants but reduced their
foraging on nectar but not pollen when ants could access flowers resulting in no difference in
fruit set between treatments Whether pollinators commonly adjust their foraging behaviors in
response to ants warrants further research
11
Third ants are somewhat unique among floral larcenists in that they often provide direct
benefits to the plants they visit In the studies listed in Table 11 ants sometimes decreased
herbivory on plants (Chamberlain and Holland 2008 Bleil et al 2011) or otherwise aided the
plant (eg ant presence repelling smaller less effective pollinators Gonzaacutelvez et al 2012) If the
positive effects of ants cancel or outweigh the negative effects of flower-visiting ants on
pollinators then ant visitation may be neutral or even positive for plant fitness
Fourth ants may impose fitness costs to plants not captured in the studies in Table 11
Nectar robbers have stronger negative effects when plants are pollen-limited (Burkle et al 2007)
but only one study in Table 11 assessed whether flowers would set more seed if they received
more pollen finding no evidence of pollen limitation (Lach 2008a Table 11) Even if ants
decrease pollinator visitation rates there is limited scope for ants to decrease plant reproductive
success when plants are not pollen-limited Furthermore nearly all the studies in Table 11
measured only female fitness Only three quantified aspects of male fitness (Wyatt 1980 Fritz
and Morse 1981 Galen and Geib 2007) and we know of only one other study in a system in
which ants occasionally pollinate that has also measured male fitness (Ashman 2000) All four
studies measured pollen or pollinia removal rates and none found any effect of flower-visiting
ants on male fitness However whether other measures of male fitness (eg seeds sired pollen
deposition patterns) would reveal similar or different effects needs investigation
Finally although floral defenses against ants (see 3 below) may prevent us from
observing the negative effects of ants on plant reproductive success we consider this unlikely
Several studies have examined interactions between ants and plants that share little or no
coevolutionary history usually in the context of ant invasions Although invasive ants often
recruit to flowers in larger numbers than native ants they do not appear to reduce plant
12
reproductive success (Blancafort and Goacutemez 2005 Lach 2007 Lach 2008a but see Hansen and
Muumlller 2009) And in Hawaii where angiosperms evolved for millions of years in the absence of
ants and are thought to have secondarily lost floral defenses against ants (Junker et al 2011a)
there is no evidence that the preponderance of recently introduced flower-visiting ants negatively
affects plant fitness (Junker et al 2010a Bleil et al 2011)
2 Ants as pollinators
Ants may directly benefit plant reproduction by acting as pollinators but this is a relatively rare
occurrence Ants have long been considered poor pollinators (Kerner 1878) Initially this was
based on morphological and behavioral differences between ants and recognized pollinators
Worker ants are wingless and often relatively hairless making them poor pollen dispersers As
well because many ants exhibit Ortstreue (literally ldquoplace fidelityrdquo) ants were thought to return
to the same branch or flower repeatedly effecting no cross pollination (Houmllldobler and Wilson
1990) Though largely true these traits do not necessarily preclude ants from acting as
pollinators (Table 12) Although they commonly have restricted foraging ranges (Houmllldobler and
Wilson 1990) some ants can effect outcrossing (eg Ashman and King 2005 Carvalheiro 2008)
and many ants are as hairy if not hairier than their pollinating counterparts
In the late 1970s the ldquoantibiotic hypothesisrdquo was put forward to explain the rarity of ant
pollination Ants use antibiotic secretions to protect themselves against the fungal and bacterial
pathogens common in their nests and environs Iwanami and Iwadare (1978) found that one of
these compounds myrmicacin also greatly reduces pollen germination rates Subsequently
Beattie et al (1984 1985) found that short periods of contact (le 30 minutes) with an antrsquos
integument caused pollen grains to fail to germinate Some of these pollen-killing compounds
13
were traced back to the ant metapleural gland which produces hygienic secretions (Houmllldobler
and Wilson 1990) although contact with ants lacking metapleural glands can also reduce pollen
viability (Beattie et al 1985 Dutton and Frederickson 2012) Ant mandibular or anal gland
secretions and potentially ant venom may likewise defend ants against pathogens and have
similar effects on pollen germination (Beattie et al 1985 Graystock and Hughes 2011)
Nonetheless ant pollination has been documented repeatedly There are at least 18 cases
in which ant pollination has been shown experimentally and five others in which observations
strongly suggest it (Table 12) The majority of these plants fit the ldquoant pollination syndromerdquo
originally proposed by Hickman (1974) dense stands of short plants with small flowers and
easily accessible nectaries Typically ant-pollinated plants are associated with hot and dry
climates (principally in the Mediterranean) but dry alpine environments may also favor the
evolution of ant pollination (eg Svensson 1985 Puterbaugh 1998 Schiestl and Glaser 2011) In
most cases ants appear to be the sole or major pollinator of the plant in question (Hickman 1974
Peakall et al 1990 de Vega et al 2009) although ants sometimes make only supplementary
contributions to pollination (Schuumlrch et al 2000 Ashman and King 2005) This suggests that co-
evolution between ants and plants may not be necessary for ants to be useful pollinatorsmdashwhich
is surprising given the potential negative effects of ants on plant reproduction
Ant-pollinated plants cope with ant secretions in a variety of ways Several plant species
may simply tolerate pollen damage (Gόmez and Zamora 1992 de Vega et al 2009) apparently
possessing no overt adaptations to prevent it These plants might receive enough pollen to
overcome reduced pollen viability Other plants may place pollen on ant body parts thought to
have fewer secretions thereby potentially escaping damage (eg on ant legs Ramsey 1995) or
attach pollen to ants with specialized structures avoiding direct contact with the ant integument
14
(Peakall et al 1990) It is not known whether pollen evolves resistance to ant secretions or
whether plants deposit pollen on ants which have less disruptive secretions although these
possibilities have been suggested elsewhere (eg Garciacutea et al 1995 Ramsey 1995)
One key question regarding the effects of ant secretions on pollen grains urgently needs
to be resolved Several studies have suggested that the pollen-killing secretions of ants may have
contributed to the evolution of floral defenses against ants (eg Wagner 2000 Galen and Geib
2007) For this to be true ants must decrease male fitness by damaging pollen grains before they
leave flowers and are deposited on receptive stigmas Only two studies have investigated the
effects of ant secretions on pollen still in anthers (Wagner 2000 Galen and Butchard 2003) and
both constrained ants unnaturally to flowers for significantly longer than ants would normally
stay on flowers Although these studies found strong reductions in pollen vigor it is unclear if
ant secretions decrease male fitness under natural ant visitation conditions
3 Floral defenses against ants
Plants are not passive recipients of flower visitors and have some ability to ldquochooserdquo which
organisms can access floral rewards Flowers resist ants using both structural and chemical traits
More thorough investigation is needed to determine whether these traits evolved to prevent or
minimize the negative effects of ants on plant reproductive success (ie whether these traits can
truly be considered defenses against ants see 4ia below) It is unlikely however that ants
experience reciprocal selection to circumvent floral resistance traits because the fitness benefits
to ants of accessing flowers are probably small Thus there may be little scope for
coevolutionary arms races between floral defenses against ants and ant counter-adaptations
15
i) Structural resistance traits
a) Restricting ant access
Numerous plant species have physical structures preventing ant access to flowers Thin pedicels
can limit antsrsquo gripping abilities and make ants easier to dislodge when flowers are moved by
wind (Gaume et al 2005) and waxy secretions can render stems too slippery for ants to climb
(Harley 1991) Structures surrounding flowers such as water moats or dense hairs may similarly
keep ants from accessing floral rewards (Willmer et al 2009) Many such traits function as broad
filters and prevent not only ants but most crawling insects from reaching flowers Thus it is
difficult to determine whether ants florivores or both acted as selective agents favoring the
evolution of physical barriers to flowers
Flower shape may also reduce ant-pollinator conflict Long narrow corollas or nectar
spurs can prevent all but the smallest ants from accessing floral nectaries (Bluumlthgen et al 2004
Agarwal and Rastogi 2007) while still allowing long-tongued pollinators access to rewards
Selection to exclude nectar-thieving ants in such a manner however may be counteracted by
pollinator preferences for wider corollas that allow easier flower handling (Galen and Cuba
2001) Additionally these types of flowers may be more prone to robbery (Irwin and Maloof
2002) limiting their usefulness in preventing nectar larceny
b) Separation in space or time
Plants may separate floral rewards and ants temporally or spatially to minimize ant impacts on
plant reproduction Several authors have proposed that myrmecophytes have adapted to the
16
sterilization or otherwise detrimental activities of ants by locating domatia or food rewards on
different branches than flowers (Izzo and Vasconcelos 2002 Raine et al 2002 Edwards and Yu
2008) Temporal separation of ant and pollinator activities could likewise reduce conflict though
evidence is limited Some acacia flowers dehisce during periods of low ant activity (Willmer and
Stone 1997 Nicklen and Wagner 2006) but it is unclear if this is in response to ants
temperature or pollinator activity Timing of nectar secretion may similarly reduce ant-pollinator
competition If ants are active prior to pollinators they may deplete floral nectar and reduce
pollinator visitation rates (Schaffer et al 1984) However if nectar is secreted when ants are
inactive pollination may occur unimpeded (Norment 1988) This temporal separation could
favor pollinators that are active when ants are not (eg Wyatt and Morse 1981) but this is
poorly documented Further studies particularly focusing on flowers with the potential for
nocturnal and diurnal pollination may provide more examples
c) Extrafloral nectaries (EFNs)
One hypothesis put forward to explain the evolution of EFNs proposes that EFNs ldquodistractrdquo ants
from consuming floral nectar (Kerner 1878 Wagner and Kay 2002) Wagner and Kay (2002)
tested this hypothesis by constructing model plants using artificial nectaries assigned at random
to represent either floral or extrafloral nectaries They found that the presence of EFNs could
reduce ant exploitation of floral nectar However as highlighted by Galen (2005) this design is
closer to a plant making a surplus of flowers some of which will avoid ant visitation if visitation
rates increase sublinearly with increasing flower number Producing extra flowers to escape ant
visitation appears to be a viable strategy in some systems (Ashman and King 2005 Galen 2005)
However Galen (2005) found that applying honey (simulating extrafloral nectar) to P viscosum
17
leaves had the opposite effect and increased ant visits to flowers Only two studies both in
Pachycereus schottii have found evidence for the distraction hypothesis under field conditions
(Chamberlain and Holland 2008 Holland et al 2011)
There is limited empirical support for the distraction hypothesis but abundant evidence
that EFNs attract ants that defend plants against herbivores (reviewed in Rosumek et al 2009)
To function as attractants the presence of EFNs must increase ant abundance on plants
However because of the increased numbers attracted EFNs would not be expected to reduce the
abundance of ants on nectar rewards or be able to satiate the ants recruited Only if ant
abundance stops increasing at a reward density much lower than that available on the plant
would EFNs serve to distract ants In this case the amount of rewards may overwhelm ants
leaving some flowers underexploited However even in P schottii ants still visited flowers
(Chamberlain and Holland 2008) In cases where ant abundance would continue to increase with
additional rewards it is unlikely that EFNs function as distractions
ii) Chemical resistance traits
a) Toxic nectar
One of the earliest proposed floral defenses against ants was toxic nectar (Janzen 1977)
However studies testing Janzenrsquos (1977) hypothesis uncovered only a very few isolated
examples (eg Catalpa speciosa Stephenson 1981 Crinum erubescens Guerrant and Fiedler
1981) in short most floral nectar is not toxic or repellent to ants (Baker and Baker 1978
Schubart and Anderson 1978) In fact ants sometimes prefer floral nectar over solutions with
equivalent sugar concentrations (Koptur and Truong 1998)
18
In contrast floral nectar can repel other flower visitors (eg birds Johnson et al 2006
lepidopterans Kessler and Baldwin 2007) Repellency is often caused by plant secondary
metabolites which may occur in nectar as a by-product of their production elsewhere in the
plant where they function as defenses against herbivory (Adler 2001) Alternatively toxic or
repellent nectar may be adaptive repellent nectar may promote effective pollination by reducing
the amount of reward any one pollinator takes spreading nectar over several pollinator visits
(Kessler and Baldwin 2007) or it may function as a filter selecting for specific pollinators
(Johnson et al 2006) The latter may explain why repellent nectar is not more common
legitimate pollinators in addition to floral larcenists may be deterred from visiting flowers with
repellent nectar This may be particularly true of nectars that would repel ants which are closely
related to bees nectar distasteful to ants may often also be distasteful to bees and those nectars
acting as a filter for bees may still be palatable to ants For example Prŷs-Jones and Willmer
(1992) found that the ammonia-containing nectar of Lathraea clandestina is readily consumed
by both bees and ants even though it deters birds that act as nectar thieves Although floral
nectar itself rarely repels ants Guerrant and Fielder (1981) found that the presence of macerated
petals within nectar can often keep ants away Alkaloids in petals may be responsible and might
explain why ants seldom act as primary nectar robbers these secondary compounds may prevent
ants from chewing through corollas
b) Floral volatiles
Repellent floral volatiles appear to be a more widespread form of chemical resistance against
ants These were reviewed recently by Willmer et al (2009) and will be discussed here only
briefly Numerous plant taxa produce floral scents repellent to ants including myrmecophilous
19
myrmecophytic and non-ant associated plants in both temperate and tropical ecosystems
(Willmer et al 2009 Junker et al 2011a Ballantyne and Willmer 2012) In three large studies
floral volatiles that repel ants were detected in about a third of all plants tested (Willmer et al
2009 Junker et al 2011a Ballantyne and Willmer 2012) Pollen is often the source of ant-
repellent volatile compounds (Willmer et al 2009 Ballantyne and Willmer 2012) but they can
be emitted from other floral parts including petals That pollen is often the source of repellence
could suggest that preventing ant visitation may be more important in protecting male than
female function Additionally because repellence would decrease as pollen is removed from
anthers ants may return and defend flowers more quickly than if repellent chemicals were in
longer-lasting structures (eg petals) In some cases floral volatiles are thought to mimic ant
alarm pheromones (Willmer et al 2009) However floral scents elicit inconsistent reactions
across ant species ranging from repellence to attraction (Agarwal and Rastogi 2007 Ballantyne
and Willmer 2012)
Producing structural or chemical resistance traits may be costly for plants Native
Hawaiian plants which have little recent coevolutionary history with ants appear to have
secondarily lost floral ant repellents (Junker et al 2011a) Both Willmer et al (2009) and Junker
et al (2011a) found that highly chemical repellent flowers did not have strong physical defenses
and vice versa suggesting trade-offs between the two (but see Ballantyne and Willmer 2012)
Like toxic nectar floral ant repellents might sometimes deter both pollinators and ants For
example Galen et al (2011) found that 2-phenylethanol in P viscosum floral scent repels both
flower-damaging F neorufibarbis ants and the plantrsquos main pollinator Bombus kirbyellus
Interestingly four recent studies have found that floral scents can also attract ants
(Agarwal and Rastogi 2007 Edwards and Yu 2008 Schiestl and Glaser 2011 Gonzaacutelvez et al
20
2012) One study (Schiestl and Glaser 2011) has shown that floral scents attract ants to a
potentially ant-pollinated plant and such systems may provide many more examples
4 Future directions
We have highlighted areas in need of further investigation throughout the review Here we focus
on several questions we consider high priorities for future research
i) Floral defenses against ants
a) Do floral resistance traits increase plant fitness
The existence ant-repellent floral traits is often taken as evidence that ants negatively affect plant
reproductive success However despite the abundance and variety of mechanisms excluding ants
from flowers few studies show that ants reduce plant fitness when they visit flowers (Table 11)
This absence is particularly apparent with repellent floral volatiles which may be the most
widespread floral resistance trait (Willmer et al 2009 Junker et al 2011a Ballantyne and
Willmer 2012) To our knowledge only one study has shown that floral ant repellents benefit
plants with repellent P viscosum flowers avoiding damaging ant visits (Galen et al 2011)
Research is urgently needed to investigate if these traits increase plant fitness by excluding ants
or if ant visitation is inconsequential More studies taking advantage of standing variation in
traits (Galen et al 2011) or creating it artificially (eg Junker et al 2010b) will allow us to
distinguish between the two
21
b) If ant visitation is often not costly what caused the evolution of repellent traits and
what maintains them Similarly are these repellent traits truly defenses against ant
visitation or are they generalized defenses against a range of floral antagonists
Ant-repellent floral traits are widespread and their production may be costly (Willmer et al
2009 Junker et al 2011a) The studies in Table 11 however indicate that flower-visiting ants
often do not disrupt plant reproduction particularly outside of myrmecophytes What then
selected for these traits One possibility is that these repellents are not directed solely at ants
Instead a community of antagonistic flower visitors may exert diffuse selection on plants
thereby selecting for and maintaining these traits Structural modifications may repel crawling
insects while chemical repellents such as floral volatiles may repel both flying and non-flying
insects (Junker et al 2011b) How much overlap there is in floral volatiles that repel both ants and
other antagonists needs investigation Until we examine how specific these defenses are to ants
we risk overstating the importance of ants in their evolution
ii) Do ants alter pollen movement patterns or reduce male fitness
We are aware of only four studies that have measured if ant visitation impacts male fitness
(Wyatt 1980 Fritz and Morse 1981 Ashman 2000 Galen and Geib 2007) While none of these
studies found an effect all measured only amounts of pollen or pollinia removed However this
measure does not adequately encompass male fitness alterations to pollen movement patterns
and eventual deposition location may be more important to male function than changes in
removal rates If as suggested by some studies (Altshuler 1999 Lach 2007) ants increase
pollinator relocation rates they may decrease pollen discounting increasing male fitness
However if this increased relocation reduces overall pollen export rates ant competition with
22
pollinators may depress male function Studies using pollen-tracking methods such as fluorescent
dyes will allow us to see how ants change pollen movement patterns and methods like paternity
analysis will reveal how competition between ants and pollinators alters male fitness
iii) Coevolution between ants and plants do myrmecophytes exhibit stronger more
specific defenses against ants
The floral traits of myrmecophytes are most likely to coevolve with ants because all plant
reproduction occurs in the presence of ants Their floral defenses would thus be expected to be
both stronger and more specific to their resident ants than those other plants visited by an array
of more opportunistic ant species Some evidence supports this Willmer et al (2009) found that
resident ants of acacias were repelled by flowers with more aggressive ant species showing
stronger repellence than less aggressive ants Additionally ants not normally associated with
acacias did not respond to acacia floral volatiles More studies are needed in other systems and
should take advantage of phylogenetic approaches examining floral traits in closely related
myrmecophytic and non-myrmecophytic plant species to determine whether ants commonly
select for floral defenses against ants in myrmecophytes
Acknowledgements
For comments and discussion we thank the Frederickson lab particularly Eric Youngerman
Lina Arcila Hernandez and Kyle Turner as well as Alison Parker and Jordan Pleet MEF
acknowledges financial support from an NSERC Discovery Grant a Connaught New Researcher
23
Award an Ontario Ministry of Economic Development and Innovation Early Researcher Award
and the University of Toronto ARC was supported by an Ontario Graduate Scholarship and
Sigma Xi
24
References
Adler L S 2001 The ecological significance of toxic nectar Oikos 91409ndash420
Agarwal V M and N Rastogi 2007 Role of floral repellents in the regulation of flower visits
of extrafloral nectary-visiting ants in an Indian crop plant Ecological Entomology 3359ndash
65
Altshuler D L 1999 Novel interactions of non-pollinating ants with pollinators and fruit
consumers in a tropical forest Oecologia 119600ndash606
Ashman TL 2000 Pollinator selectivity and its implications for the evolution of dioecy and
sexual dimorphism Ecology 812577-2591
Ashman TL and E A King 2005 Are flower-visiting ants mutualists or antagonists A study
in a gynodioecious wild strawberry American Journal of Botany 92891ndash5
Ballantyne G and P Willmer 2012 Floral visitors and ant scent marks noticed but not used
Ecological Entomology 37402-409
Ballantyne G and P Willmer 2012 Nectar theft and floral ant-repellence a link between
nectar volume and ant-repellent traits PloS One 7e43869
Baker H G and I Baker 1978 Ants and flowers Biotropica 1080
Barrett S C H 2002 Sexual interference of the floral kind Heredity 88154ndash159
Baroni Urbani C and M L de Andrade 1997 Pollen eating storing and spitting by ants
Naturwissenschaften 84256ndash258
25
Beattie A J C Turnbull R B Knox and E G Williams 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany
71421ndash426
Beattie A J C Turnbull T Hough S Jobson and R B Knox 1985 The vulnerability of
pollen and fungal spores to ant secretions evidence and some evolutionary implication
American Journal of Botany 72606ndash614
Blancafort X and C Goacutemez 2005 Consequences of the Argentine ant Linepithema humile
(Mayr) invasion on pollination of Euphorbia characias (L) (Euphorbiaceae) Acta
Oecologica 2849ndash55
Bleil R N Bluumlthgen and R R Junker 2011 Ant-plant mutualism in Hawailsquoi Invasive ants
reduce flower parasitism but also exploit floral nectar of the endemic shrub Vaccinium
reticulatum (Ericaceae) Pacific Science 65291ndash300
Bluumlthgen N G Gottsberger and K Fiedler 2004 Sugar and amino acid composition of ant-
attended nectar and honeydew sources from an Australian rainforest Austral Ecology
29418ndash429
Burkle L A R E Irwin and D A Newman 2007 Predicting the effects of nectar robbing on
plant reproduction implications of pollen limitation and plant mating system American
Journal of Botany 941935ndash1943
Byk J and K Del-Claro 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333ndash38
26
Caballero P C G Ossa W L Gonzaacuteles C Gonzaacutelez-Browne G Astorga M M Muruacutea and
R Medel 2013 Testing non-additive effects of nectar-robbing ants and hummingbird
pollination on the reproductive success of a parasitic plant Plant Ecology 214633ndash640
Carvalheiro L G E R M Barbosa and J Memmott 2008 Pollinator networks alien species
and the conservation of rare plants Trinia glauca as a case study Journal of Applied
Ecology 451419ndash1427
Chamberlain S A and J N Holland 2008 Density-mediated context-dependent consumer-
resource interactions between ants and extrafloral nectar plants Ecology 891364ndash1374
Creighton W S 1967 Studies on free colonies of Cryptocerus texanus Satschi (Hymenoptera
Formicidae) Psyche 7434ndash42
Cursach J and J Rita 2011 Implications of the reproductive biology of the narrow endemic
Naufraga balearica (Apiaceae) for its conservation status Plant Systematics and Evolution
298581ndash596
Cushman JH and Addicott JF In Huxley CR and DF Cutler (eds) 1st edition Ant-plant
interactions Oxford University Press Oxford
de Jong T J N M Waser and P G Klinkhamer 1993 Geitonogamy the neglected side of
selfing Trends in Ecology and Evolution 8321ndash325
de Vega C M Arista P L Oritz C M Herrera and S Talavera 2009 The ant-pollination
system of Cytinus hypocistis (Cytinaceae) a Mediterranean root holoparasite Annals of
Botany 4517ndash8
27
Dukas R 2001 Effects of perceived danger on flower choice by bees Ecology Letters 4327ndash
333
Dutton E M and M E Frederickson 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561ndash569
Edwards D P and D W Yu 2008 Tolerating castration by hiding flowers in plain sight
Behavioral Ecology and Sociobiology 6395ndash102
Frederickson M E 2009 Conflict over reproduction in an ant-plant symbiosis why Allomerus
octoarticulatus ants sterilize Cordia nodosa trees The American Naturalist 173675ndash81
Fritz R S and D H Morse 1981 Nectar parasitism of Asclepias syriaca by ants effect on
nectar levels pollinia insertion pollinaria removal and pod production Oecologia 50316ndash
319
Galen C 1983 The effects of nectar thieving ants on seedset in floral scent morphs of
Polemonium viscosum Oikos 41245ndash249
Galen C 2005 Catching ants with honey an experimental test of distraction and satiation as
alternative modes of escape from flower-damaging ants Oecologia 14480ndash87
Galen C and B Butchart 2003 Ants in your plants effects of nectar-thieves on pollen fertility
and seed-siring capacity in the alpine wildflower Polemonium viscosum Oikos 101521ndash
528
Galen C and J Cuba 2001 Down the tube pollinators predators and the evolution of flower
shape in the alpine skypilot Polemonium viscosum Evolution 551963-1971
28
Galen C and J C Geib 2007 Density-dependent effects of ants on selection for bumble bee
pollination in Polemonium viscosum Ecology 881202ndash1209
Galen C R Kaczorowski S L Todd J Geib and R A Raguso 2011 Dosage-dependent
impacts of a floral volatile compound on pollinators larcenists and the potential for floral
evolution in the alpine skypilot Polemonium viscosum The American Naturalist 177258ndash
272
Garciacutea M B R J Antor and X Espadaler 1995 Ant pollination of the palaeoendemic
dioecious Borderea pyrenaica (Dioscoreaceae) Plant Systematics and Evolution 19817ndash
27
Gaume L M Zacharias and R M Borges 2005 Ant ndash plant conflicts and a novel case of
castration parasitism in a myrmecophyte Evolutionary Ecology Research 7435ndash452
Graystock P and W O H Hughes 2011 Disease resistance in a weaver ant Polyrhachis
dives and the role of antibiotic-producing glands Behavioral Ecology and Sociobiology
652319ndash2327
Gόmez J M 2000 Effectiveness of ants as pollinators of Lobularia maritima effects on main
sequential fitness components of the host plant Oecologia 12290ndash97
Gόmez J M and R Zamora 1992 Pollination by ants consequences of the quantitative effects
on a mutualistic system Oecologia 91410ndash418
Gόmez J M R Zamora J A Hόdar and D Garciacutea 1996 Experimental study of pollination
by ants in Mediterranean high mountain and arid habitats Oecologia 105236ndash242
29
Gonccedilalves-Souza T P M Omena J C Souza and G Q Romero 2008 Trait-mediated effects
on flowers artificial spiders deceive pollinators and decrease plant fitness Ecology
892407ndash2413
Gonzaacutelvez F G L Santamariacutea R T Corlett and M A Rodriacuteguez-Gironeacutes 2012 Flowers
attract weaver ants that deter less effective pollinators Journal of Ecology 10178ndash85
Guerrant E O and P L Fiedler 1981 Flower defenses against nectar-pilferage by ants
Biotropica 1325ndash33
Harley R 1991 The greasy pole syndrome In Huxley CR and DF Cutler (eds) 1st edition
Ant-plant interactions Oxford University Press Oxford
Hansen D M and C B Muumlller 2009 Invasive ants disrupt gecko pollination and seed
dispersal of the endangered plant Roussea simplex in Mauritius Biotropica 41202ndash208
Hickman J C 1974 Pollination by ants a low-energy system Science 1841290ndash1292
Holland J N S a Chamberlain and T E X Miller 2011 Consequences of ants and
extrafloral nectar for a pollinating seed-consuming mutualism ant satiation floral
distraction or plant defense Oikos 120381ndash388
Houmllldobler B and Wilson E O 1990 The Ants 1st Edition Harvard University Press
Cambridge
Holway DA L Lach AV Suarez ND Tsutsui and TJ Case 2002 The causes and
consequences of ant invasions Annual Review of Ecology and Systematics 33181-233
30
Horskins K and Turner VB 1999 Resource use and foraging patterns of honeybees Apis
mellifera and native insects on flowers of Eucalyptus costata Australian Journal of
Ecology 24221-227
Horvitz C C and D W Schemske 1984 Effects of ants and an ant-tended herbivore on seed
production of a neotropical herb Ecology 651369ndash1378
Inouye DW 1980 The terminology of floral larceny Ecology 611251-1253
Inouye DW and O R J Taylor 1979 A temperate region plant-ant-seed predator system
consequences of extra floral nectar secretion by Helianthella quinquenervis Ecology 601ndash
7
Irwin RE 2003 Impact of nectar robbing on estimates of pollen flow conceptual predictions
and empirical outcomes Ecology 84485-495
Irwin RE and Maloof JE 2002 Variation in nectar robbing over time space and species
Oecologia 133525-533
Iwanami Y and T Iwadare 1978 Inhibiting effects of myrmicacin on pollen growth and pollen
tube mitosis Botanical Gazette 13942ndash45
Izzo TJ and Vasconcelos HL 2002 Cheating the cheater domatia loss minimizes the effects
of ant castration in an Amazonian ant plant Oecologia 133200-205
Janzen D H 1977 Why donrsquot ants visit flowers Biotropica 9252
Johnson SD Hargreaves AL and Brown M 2006 Dark bitter-tasting nectar functions as a
filter of flower visitors in a bird-pollinated plant Ecology 872709-2716
31
Junker R A Y C Chung and N Bluumlthgen 2007 Interaction between flowers ants and
pollinators additional evidence for floral repellence against ants Ecological Research
22665ndash670
Junker R R R Bleil C C Daehler and N Bluumlthgen 2010a Intra-floral resource partitioning
between endemic and invasive flower visitors consequences for pollinator effectiveness
Ecological Entomology 35760ndash767
Junker R R and N Bluumlthgen 2010b Floral scents repel facultative flower visitors but attract
obligate ones Annals of botany 105777ndash82
Junker R R C C Daehler S Doetteri A Keller and N Bluumlthgen 2011a Hawaiian ant ndash
flower networks nectar-thieving ants prefer undefended native over introduced plants with
floral defenses Ecological Monographs 81295ndash311
Junker R R J Gershenzon and S B Unsicker 2011b Floral odor bouquet loses its ant
repellent properties after inhibition of terpene biosynthesis Journal of Chemical Ecology
371323ndash31
Kawakita A and M Kato 2002 Floral biology and unique pollination system of root
holoparasites Balanophora kuroiwai and B tobiracola (Balanophraceae) American
Journal of Botany 891164ndash1170
Koopowitz H and TA Marchant 1998 Postpollination nectar reabsorption in the African
epiphyte Aerangis verdickii (Orchidaceae) American Journal of Botany 85508-512
Kirk W D J 1984 Ecological studies on Thrips imaginis Bagnall (Thysanoptera) in flowers of
Echium plantagineum L in Australia Australian Journal of Ecology 99ndash18
32
Kerner A 1878 Flowers and their unbidden guests C Kegan Paul London
Kessler D and I T Baldwin 2007 Making sense of nectar scents the effects of nectar
secondary metabolites on floral visitors of Nicotiana attenuata The Plant Journal 49840ndash
54
Koptur S and N Truong 1998 Facultative ant-plant interactions nectar sugar preferences of
introduced pest species in South Florida Biotropica 30179-189
Lach L 2005 Interference and exploitation competition of three nectar-thieving invasive ant
species Insectes Sociaux 52257ndash262
Lach L 2007 A mutualism with a native membracid facilitates pollinator displacement by
Argentine ants Ecology 881994ndash2004
Lach L 2008a Argentine ants displace floral arthropods in a biodiversity hotspot Diversity and
Distributions 14281ndash290
Lach L 2008b Floral visitation patterns of two invasive ant species and their effects on other
hymenopteran visitors Ecological Entomology 33155ndash160
Lach L 2013 A comparison of floral resource exploitation by native and invasive Argentine
ants Arthropod-Plant Interactions 7177-190
Luo C W K Li X M Chen Z Y Huang and K U N Li 2012 Ants contribute significantly
to the pollination of a biodiesel plant Jatropha curcas Environmental Entomology
411163ndash1168
33
Maleacute PJ G C Leroy A Dejean A Quilichini and J Orivel 2011 An ant symbiont directly
and indirectly limits its host plantrsquos reproductive success Evolutionary Ecology 2655ndash63
Maloof J E 2001 The effects of a bumble bee nectar robber on plant reproductive success and
pollinator behavior American Journal of Botany 881960ndash1965
Maloof J E and D W Inouye 2000 Are nectar robbers cheaters or mutualists Ecology
812651ndash2661
Moreau CS CD Bell R Vila SB Archibald and NE Pierce 2006 Phylogeny of the ants
diversification in the age of the angiosperms Science 312101-104
Nahas L M O Gonzaga and K Del-Claro 2012 Emergent impacts of ant and spider
interactions herbivory reduction in a tropical savanna tree Biotropica 44498ndash505
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506ndash514
Newman D A and J D Thomson 2005 Interactions among nectar robbing floral herbivory
and ant protection in Linaria vulgaris Oikos 110497ndash506
Nicklen E F and D Wagner 2006 Conflict resolution in an ant-plant interaction Acacia
constricta traits reduce ant costs to reproduction Oecologia 14881ndash7
Norment C J 1988 The effect of nectar-thieving ants on the reproductive success of Frasera
speciosa (Gentianaceae) American Midland Naturalist 120331ndash336
34
Orivel J L Lambs P-J G Maleacute C Leroy J Grangier T Otto A Quilichini et al 2011
Dynamics of the association between a long-lived understory myrmecophyte and its
specific associated ants Oecologia 165369ndash76
Palmer T M D F Doak M L Stanton J L Bronstein E T Kiers T P Young J R
Goheen et al 2010 Synergy of multiple partners including freeloaders increases host
fitness in a multispecies mutualism Proceedings of the National Academy of Sciences
10717234ndash9
Peakall R A J Beattie and S H James 1987 Pseudocopulation of an orchid by male ants a
test of two hypotheses accounting for the rarity of ant pollination Oecologia 73522ndash524
Peakall R and A J Beattie 1989 Pollination of the orchid Microtis parviflora R Br by
flightless worker ants Functional Ecology 3515ndash522
Peakall R C J Angus A J Beattie 1990 The significance of ant and plant traits for ant
pollination in Leporella fimbriata pollination Oecologia 84457ndash460
Puterbaugh M N 1998 The roles of ants as flower visitors experimental analysis in three
alpine plant species Oikos 8336ndash46
Prŷs-Jones OE and Willmer PG 1992 The biology of alkaline nectar in purple toothwort
(Lathraea clandenstina) ground level defenses Biological Journal of the Linnean Society
45373-388
Raine NE Willmer P and Stone GN 2002 Spatial structuring and floral avoidance
behaviour present in ant-pollinator conflict in a Mexican ant-acacia Ecology 833086-
3096
35
Ramsey M 1995 Ant pollination of the perennial herb Blandfordia grandiflora (Liliaceae)
Oikos 74265ndash272
Renault CK LM Buffa and Delfino MA 2005 An aphid-ant interaction effects on
different trophic levels Ecological Research 2071-74
Rico-Gray V and L B Thien 1989 Effect of different ant species on reproductive fitness of
Schomburgkia tibicinis (Orchidaceae) Oecologia 81487ndash489
Romero G Q P A P Antiqueira and J Koricheva 2011 A meta-analysis of predation risk
effects on pollinator behaviour PLoS One 6e20689
Rosumek FB FAO Silveira F de S Neves NP de U Barbosa L Diniz Y Oki F Pezzini
et al 2009 Ants on plants a meta-analysis of the role of ants as plant biotic defenses
Oecologia 160537ndash549
Roubik D W 1982 The ecological impact of nectar-robbing bees and pollinating
hummingbirds on a tropical shrub Ecology 63354ndash360
Rudgers J A 2004 Enemies of herbivores can shape plant traits selection in a facultative ant-
plant mutualism Ecology 85192ndash205
Schatz B and WT Wcislo 1999 Ambush predation by the ponerine ant Ectatomma ruidum
Roger (Formicidae) on a sweat bee Lasioglossum umbripenne (Halictidae) in Panama
Journal of Insect Behavior 12641-663
36
Schaffer W M D W Zeh S L Buchmann S Kleinhaus M V Schaffer and J Antrim
1983 Competition for nectar between introduced honey bees and native North American
bees and ants Ecology 64564ndash577
Schemske D W 1980 The evolutionary significance of extrafloral nectar production by Costus
woodsonii (Zingiberaceae) an experimental analysis of ant protection Journal of Ecology
68959ndash967
Schiestl F P and F Glaser 2011 Specific ant-pollination in an alpine orchid and the role of
floral scent in attracting pollinating ants Alpine Botany 1221ndash9
Schubart HOR AB Anderson 1978 Why donrsquot ants visit flowers A response to D H
Janzen Biotropica 10310-311
Schuumlrch S M Pfunder and B A Roy 2000 Effects of ants on the reproductive success of
Euphorbia cyparissias and associated pathogenic rust fungi Oikos 16ndash12
Stanton ML Palmer TM Young TP Evans A and Turner ML 1999 Sterilization and
canopy modification of swollen thorn acacia tree by a plant-ant Nature 401 578-581
Stephenson AG 1981 Toxic nectar deters nectar thieves of Catalpa speciosa American
Midland Naturalist 105381-383
Stout J C and D Goulson 2001 The use of conspecific and interspecific scent marks by
foraging bumblebees and honeybees Animal Behaviour 62183ndash189
Sugiura S T Abe and S Makino 2006 Loss of extrafloral nectary on an oceanic island plant
and its consequences for herbivory American Journal of Botany 93491ndash495
37
Svensson L 1985 An estimate of pollen carryover by ants in a natural population of
Scleranthus perennis L (Caryophyllaceae) Oecologia 66373ndash377
Tsuji K A Hasyim and K Nakamura 2004 Asian weaver ants Oecophylla smaragdina and
their repelling of pollinators Ecological Research 19669ndash673
Turner R C J J Midgley P Barnard R E Simmons and S D Johnson 2012 Experimental
evidence for bird pollination and corolla damage by ants in the short-tubed flowers of
Erica halicacaba (Ericaceae) South African Journal of Botany 7925ndash31
Visser D M G Wright and J H Giliomee 1996 The effect of the Argentine ant
Linepithema humile (Mayr) (Hymenoptera Formicidae) on flower-visiting insects of
Protea nitida Mill (Proteaceae) African Entomology 4285ndash287
Wagner D 2000 Pollen viability reduction as a potential cost of ant association for Acacia
constricta (Fabaceae) American Journal of Botany 87711ndash715
Wagner D and A Kay 2002 Do extrafloral nectaries distract ants from visiting flowers An
experimental test of an overlooked hypothesis Evolutionary Ecology Research 4293ndash305
Wang C Y Luo Y Tai D An and Y Kou 2008 Ants pollinate Neottia listeroides
(Orchidaceae) in Sichuan China 46836ndash846
Wyatt R 1980 The impact of nectar-robbing ants on the pollination system of Asclepias
curassavica Bulletin of the Torrey Botanical Club 10724ndash28
Wheeler WM and IW Bailey 1920 The feeding habits of Pseudomymine and other ants
Transactions of the American Philosophical Society 22235-279
38
Willmer P G and S A Corbet 1981 Temporal and microclimatic partitioning of the floral
resources of Justicia aurea amongst a concourse of pollen vectors and nectar robbers
Oecologia 5167ndash78
Willmer P G and G N Stone 1997 How aggressive ant-guards assist seed-set in Acacia
flowers Nature 388165ndash167
Willmer P G C V Nuttman N E Raine G N Stone J G Pattrick K Henson P Stillman
et al 2009 Floral volatiles controlling ant behaviour Functional Ecology 23888ndash900
Young T P C H Stubblefield and L a Isbell 1997 Ants on swollen-thorn acacias species
coexistence in a simple system Oecologia 10998ndash107
Yu D W and N E Pierce 1998 A castration parasite of an ant-plant mutualism Proceedings
of the Royal Society B Biological Sciences 265375ndash382
39
Tables
Table 11 Studies of ant-flower or ant-pollinator interactions that included a measure of plant fitness Each study system is listed only
once except when results varied between years or when different studies examined male and female fitness Significant effects of ants on
plant fitness are either lsquopositiversquo or lsquonegativersquo non-significant effects are lsquoneutralrsquo When ants significantly affected only one fitness
component but several were measured (eg fruit set and seed production) we still consider the result significant
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Acacia
drepanolobium
Crematogaster
nigriceps
Proportion
fruiting
Negative Destruction of floral buds Young et al 1997
Asclepias exaltata Several Pollinia inserted
removed
Negative for pollinia
insertion neutral for
pollinia removal
Exploitative competition Wyatt 1980 T
40
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Asclepias syriaca Several
Fruit set pollinia
removed
Negative for fruit
set neutral for
pollinia removal
Exploitative competition Fritz and Morse
1981 T
Cordia nodosa Allomerus cf
demerarae
Fruit set Negative Destruction of floral buds Yu and Pierce
1998
Costus woodsonii Wasmannia
auropunctata
Seed set Positive Unknown ant pollination
suggested (but unlikely)
Schemske 1980
Eritrichum aretioides F neorufibarbis
gelida
Seed set Negative Ants damaged flowers Puterbaugh 1998
41
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Euphorbia characias Several Seed set Neutral Plants in Argentine ant-
invaded area set less seed
than those in un-invaded
sites but an ant-exclusion
experiment did not produce
significant results
Blancafort and
Gόmez 2005
Ferocactus wislizeni Solenopsis xyloni
and others
Seed set Unknown negative Interference and exploitative
competition S xyloni
reduced seed set relative to
other ants but the study did
not include a no-ant control
Ness 2006
42
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Frasera speciosa F neorufibarbis
and Formica sp
Seed set Neutral Nocturnal pollination
occurring when ants were
less active may have
compensated for reduced
diurnal pollinator visitation
Norment 1988
Gypsophila struthium Several Seeds per
inflorescence
Neutral No effect despite large
numbers of ants on flowers
Gόmez et al
1996
Hirtella physophora Allomerus
decemarticulatus
Fruit set Negative Destruction of flowers Orivel et al
2011
Humboldtia brunonis Crematogaster
dohrni
Fruit set Negative Destruction of flowers Gaume et al
2005
43
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Lepidium subulatum Several Percentage of
ovules setting
seed
Positive Unknown Gόmez et al
1996
Leucospermum
conocarpodendron
Several Seed set Neutral Site not pollen limited Lach 2008
Linaria vulgaris Several ldquoFemale fitness
indexrdquo
(combination of
number of seeds
fruits flowers
and seed weight)
Positive Reduction in florivory and
seed predation nectar
robbing holes increased ant
visitation
Newman and
Thomson 2005
44
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Melastoma
malabathricum
Oecophylla
smaragdina
Seed set Positive Ants deterred less efficient
pollinators increasing
visitation by the more
efficient pollinator
Gonzaacutelvez et al
2012
Metrosideros
polymorpha
Several Fruit set Neutral Ants reduced nectar
collection rates by foraging
bees but had no effect on
pollen collection rates or
fruit set
Junker et al
2010a
Orexis alpine F neorufibarbis
gelida
Seed set Neutral Autogamy or not pollen
limited
Puterbaugh 1988
45
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Ouratea spectabilis Cephalotes
pusillus and others
Seeds per fruit Neutral positive C pusillus had no effect on
seed number but other ants
collectively increased seed
set
Byk and Del
Claro 2010
Pachycereus schottii Several Seed set Neutral positive In one year ants showed a
positive effect (seed
number 2008) but no effect
with increased sample sizes
Chamberlain and
Holland 2008
Polemonium
viscosum
F neorufibarbis
gelida
Seed set Negative Ants damaged styles Galen 1983
46
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Polemonium
viscosum
F neorufibarbis
gelida
Pollen removal Neutral Ants did not affect pollen
removal even at high ant
densities
Galen and Geib
2007 T
Protea nitida Linepithema
humile and others
Seed set Neutral positive L humile had no effect on
P nitida seed set but native
ants increased it in one year
ants may cause pollinators
to relocate
Lach 2007
Psychotria
limonensis
Ectatomma spp Fruit set Positive Suggested increased
pollinator relocation
Altshuler 1999
47
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Roussea simplex Technomyrmex
albipes
Seed set Negative Interference and exploitative
competition
Hansen and
Muumlller 2009
Tristerix aphyllus Several Fruit set Neutral Nectar robbing ants
minimally reduced nectar
levels and had no effect on
hummingbird visitation
Caballero et al
2013
Vaccinium
reticulatum
Several Fruit set Neutral Ants reduced florivory but
did not affect fruit set
Bleil et al 2011
T Study measured male fitness
Study of sterilizing ants showed short-term decrease in female fitness long-term effects are unclear (see text)
48
Table 12 Studies demonstrating either i) ant pollination through an exclusion experiment or ii) that ants transfer pollen (or pollen
analogue) between flowers strongly suggesting ant pollination
Plant species Ant species Ant pollination Evidence Reference
Alyssum purpureum Several Yes Exclusion experiment Gόmez et al 1996
Arenaria tetraquetra Several Yes Exclusion experiment Gόmez et al 1996
Balanophora kuroiwai Leptothorax sp Suggested Exclusion experiment and
observed pollen transfer
and pollen tube formation
but did not see if plants set
seed
Kawakita and Kato 2002
Blandfordia grandiflora Iridomyrmex sp Yes Exclusion experiment Ramsey 1995
Borderea pyrenaica Several Yes Exclusion experiment Garciacutea et al 1995
49
Plant species Ant species Ant pollination Evidence Reference
Cytinus hypocistis Several Yes Exclusion experiment de Vega et al 2009
Epipactis thunbergii Camponotus
japonicas
Suggested Observed transfer of
pollinia from ants to
orchids
Sugiura et al 2006
Euphorbia cyparissias Several Yes Exclusion experiment Schuumlrch et al 2000
Fragaria virginiana Several Very likely Ant-only treatments set
comparable seed to flying
pollinator-only treatment
seed set increased with
increasing ant visitation
Ashman and King 2005
Frasera speciosa Several Yes Exclusion experiment Norment 1988
Frankenia thymifolia Several Yes Exclusion experiment Gόmez et al 1996
50
Plant species Ant species Ant pollination Evidence Reference
Hormathophylla spinosa Proformica
longiseta
Yes Exclusion experiment Gόmez and Zamora 1992
Jatropha curcas
Several
Tapinoma
melanocephalum
most common
Yes Exclusion experiment Luo et al 2012
Leporella fimbriata Myrmecia urens Yes Observed pollinia transfer
and subsequently
confirmed as ant
pollination
Peakall et al 1987
51
Plant species Ant species Ant pollination Evidence Reference
Lobularia maritima Several
Camponotus
micans most
common
Yes Exclusion experiment Gόmez 2000
Microtis parviflora
Iridomyrmex
gracilis
Yes Observation used
laboratory colonies to
replicate observations
Peakall and Beattie 1989
Naufraga balearica
Several
especially
Plagiolepis
pygmaea and
Lasius grandis
Suggested Observation over three
years of study only ants
were observed visiting
ants carried pollen
Cursach and Rita 2011
52
Plant species Ant species Ant pollination Evidence Reference
Neottia listeroides Leptothoras sp
Paratrechina sp
Suggested Observed transfer of
pollinia from ants to
orchids
Wang et al 2008
Paronychia pulvinata Formica
neorufibarbis
gelida
Yes Exclusion experiment Puterbaugh 1998
Polygonum cascadense Formica argentea
Very likely Grew plants in greenhouse
open to flying visitors with
minimal seed set only
plants with access to ants
set significant seed
Hickman 1974
53
Plant species Ant species Ant pollination Evidence Reference
Retama sphaerocarpa Several Yes Exclusion experiment Gόmez et al 1996
Scleranthus perennis
Several
especially
Formica
rufibarbis
Suggested Ants transferred pollen
analogue to stigmas and
carried pollen on bodies
Svensson 1985
Sedum anglicum Proformica
longiseta
Yes Exclusion experiment Gόmez et al 1996
Trinia glauca Several Yes Exclusion experiment Carvalheiro et al 2008
54
Chapter 2 Ants and ant scent reduce bumblebee pollination of artificial flowers
Cembrowski AR Tan MG Thomson JD and Frederickson ME
In press The American Naturalist
All authors designed the experiment ARC and MGT performed the experiment ARC and MEF
performed statistical analyse
s and ARC wrote the manuscript with input from all authors
Abstract
Ants on flowers can disrupt pollination by consuming rewards or harassing pollinators but it is
difficult to disentangle the effects of these exploitative and interference forms of competition on
pollinator behavior Using highly rewarding and quickly replenishing artificial flowers that
simulate male or female function we allowed bumblebees (Bombus impatiens) to forage (1) on
flowers with or without ants (Myrmica rubra) and (2) on flowers with or without ant scent cues
Bumblebees transferred significantly more pollen analogue both to and from ant free flowers
demonstrating that interference competition with ants is sufficient to modify pollinator foraging
behavior Bees also removed significantly less pollen analogue from ant scented flowers than
from controls making this the first study to show that bees can use ant scent to avoid harassment
at flowers Ant effects on pollinator behavior possibly in addition to their effects on pollen
viability may contribute to the evolution of floral traits minimizing ant visitation
55
Introduction
Trait-mediated indirect interactions arise when a focal species causes phenotypic changes
including behavioral modifications in a second species and these effects cascade to still other
species (Werner and Peacor 2003) Although trait-mediated indirect interactions are often studied
in food webs (eg Werner and Peacor 2003 Preisser et al 2005) they are not limited to trophic
interactions For example the threat of predation can change the behavior of a mutualist thus
affecting its partners (Suttle 2003) In animal-pollinated plants predators can disrupt pollination
directly through density-mediated indirect interactions (Dukas 2005) or via trait-mediated
indirect interactions by changing pollinator behavior When predators are present pollinators
may switch to visiting less rewarding flowers or they may avoid flowers altogether potentially
reducing the amount of pollen donated or received by a flower and thus decreasing plant fitness
(Gonccedilalves-Souza 2008 Ings and Chittka 2009) Similarly competition for floral rewards with
other flower visitors may cause behavioral changes in pollinators (Maloof and Inouye 2000
Ohashi et al 2008) Previous research has shown that bees may spend less time visiting flowers
depleted of rewards (Thomson 1986) and may avoid flowers bearing scent marks that indicate
recent visits by other individuals of the same or different bee species as these flowers are likely
to be depleted (Stout and Goulson 2002)
Whereas competition between pollinators may not be particularly costly for the plant
because both competitors are likely to provide pollination services competition between
pollinators and organisms that do not usually provide a pollination service such as ants has a
greater potential to reduce plant fitness Ants are common floral visitors they are attracted to
flowers for nectar (Lach 2007) and in some cases pollen (Byk and Del Claro 2010) and will
often defend these resources against other flower visitors (Altshuler 1999) However ants rarely
56
contribute much useful pollination antibiotic secretions present on their cuticles kill pollen
grains (Beattie et al 1985 Dutton and Frederickson 2012) and when foraging in flowers ants
sometimes impede female function by damaging stigmas (Galen and Cuba 2001) We will
henceforth consider ants to be non-pollinators and will contrast them to animals like bees that
are pollinators Ants are capable of competing with pollinators in two ways (1) by consuming
floral rewards and reducing their availability to pollinators (ie exploitative competition) and (2)
by directly antagonizing and excluding pollinators from flowers (ie interference competition)
Many plants benefit from the presence of ants numerous plant species recruit ants using
extrafloral nectar food bodies or domatia because ants provide protection against folivores (Heil
and McKey 2003) Similarly some plants have extrafloral nectaries on or near reproductive
tissues that recruit ants that deter florivores (eg Inouye and Taylor 1979) However plants
may incur reproductive costs as a result of their association with ants Some ant species that nest
in myrmecophytes sterilize flowers to shunt plant resources from reproduction to vegetative
growth allowing for greater ant colony growth (Frederickson 2009) Also ants visiting
extrafloral nectaries may be attracted to floral nectaries where they can harass pollinators (Ness
2006) There is growing evidence that many angiosperms have evolved floral traits that prevent
ants from accessing flowers during anthesis (Willmer and Stone 1997 Ballantyne and Willmer
2012) The effects of flower-visiting ants can be substantial Ants can decrease the frequency
duration or species diversity of pollinator visits to flowers (Lach 2008 Hansen and Muumlller 2009
Junker et al 2010 Gonzaacutelvez et al 2012) all of which potentially impact pollen donation and
receipt In some studies changes to pollinator behavior induced by ants have also been linked to
seed set
It is difficult however to disentangle the intertwined effects of exploitative and
interference competition in nature If ants disrupt pollination plants may often evolve traits to
57
deter ants from flowers (Willmer et al 2009) From a practical standpoint this would then limit
our ability to study the effects of ants on pollinator visitation because ants would not visit
flowers Here we used artificial flowers to explore ant-bee interactions in the absence of floral
defenses against ants We used highly rewarding quickly replenishing artificial flowers to
examine how direct harassment (ie interference competition expressed through behavior)
changes bee foraging behavior and how this affects donation and receipt of a pollen analogue
(powdered food dye) while minimizing the effects of exploitative competition We predicted
that flowers visited by ants would both donate and receive less pollen analogue than flowers
without ants because bees would avoid flowers with ants or leave them sooner Because scent
plays a large role in bumblebee communication and flower choice (Stout and Goulson 2002) we
also tested whether the presence or absence of ant scent on artificial flowers would affect pollen
analogue donation because of changes in the foraging behavior of bumblebees
Methods
i) Subjects
Myrmica rubra is an invasive ant in eastern North America with a range and habitat preferences
overlapping that of Bombus impatiens a common bumblebee Because M rubra visits flowers
(A Cembrowski pers obs) the two species likely interact in nature We collected 12 M rubra
colonies in the fall of 2011 and 2012 from Toronto Ontario Canada and the surrounding area
and maintained them in environmental chambers on artificial diet (Dussutour and Simpson 2009)
and a 1410 LD schedule (light 600-2200) We used these colonies as sources of M rubra
workers for experiments
58
Workers from commercially supplied colonies of Bombus impatiens (Biobest Canada
Ltd) foraged on artificial flowers in flight cages (either 24 x 24 x 21 m or 79 x 34 x 20m) at
the University of Toronto We tested a total of five bee colonies four colonies were used in the
ant presence trials and two of these and one additional colony were used in the ant scent trials
Flight cages had overhead fluorescent lights attached to timers In contrast to most previous
studies in which bees have been trained and tested individually the entire worker force of a bee
colony was free to forage at will in our experiments Colonies were trained to forage on artificial
flowers for at least four days before being used in trials After being used for a trial the colony
was not used for at least two days to reduce dye carryover between trials Between trials
bumblebees were fed pollen and given sugar water
ii) Artificial flowers
The flowers (Fig 1 Thomson et al 2012 see also Makino 2008) consisted of glass jars filled
with 30 WV sucrose solution Sugar water travelled by capillary action up a sewing-thread
wick to a hole in a blue-painted lid accumulating in a knot that acts as a nectary Flowers
depleted by visitors were quickly replenished via capillary action and were non-rewarding only
very briefly after visits taking less than a minute to accumulate 05 microl note that bumblebee
visits to a single flower are often separated by several minutes or more (A Cembrowski
unpublished data)
Unlike most previous artificial flower designs these flowers allow for estimation of male
and female fitness because we can measure both the amount and type of dye received by female
flowers (see (d) Dye quantification below) In order to access nectar on a ldquomalerdquo flower a bee
must crawl through a circular ldquoantherrdquo of brush-like weather stripping material dusted in a
59
consistent manner with our pollen analogue powdered food dye (FDampC 5 or 6) which is
transferred to the bee in the process On a ldquofemalerdquo flower a bee crawls through a sticky plastic
reinforcement that functions as a ldquostigmardquo receiving dye from the beersquos body Male flowers
dispensed dye particles over multiple bee visits and could still dispense dye at the end of trials
They resemble many real flowers or flower heads in that pollen is dispensed gradually over time
but less dye is available for transfer with each subsequent bee visit (Harder and Thomson 1989)
iii) Flight cage trials
We conducted 6 ant scent trials between 22 March and 4 June 2012 and the remaining trials (16
ant presence trials and 9 ant scent trials) between 9 November 2012 and 6 January 2013 Ant
presence trials lasted for eight hours and ant scent trials lasted for four hours Artificial flowers
were prepared and placed individually in small plastic containers treated with Fluon (Insect-a-
Slip BioQuip Products Inc) to prevent ants from escaping In ant presence trials we used 32
flowers (16 male and 16 female flowers) arranged in an eight-by-four array with flowers spaced
45 cm apart between rows and 30 cm within rows Ant scent trials used 20 flowers (10 male and
10 female flowers) in a four-by-five array with flowers spaced 30 cm apart between and within
rows In all of the ant presence trials and 9 of the ant scent trials we counted the number of bees
actively foraging after one hour to get a measure of colony activity
a) Ant presence trials
We examined the effects of ants on the amount of dye donated by male flowers and received by
female flowers in sixteen trials testing four bee colonies four times each The containers of eight
randomly chosen male flowers and eight randomly chosen female flowers received 15 M rubra
60
workers each the other 16 flowers remained free of ants Isolated from their colonies M rubra
workers have nowhere to deposit sugar water they collect and therefore may become sated
nonetheless a force of 15 ant workers was enough to maintain visitation to the nectaries where
bees foraged while still rarely having more than one or two ants visit the nectary at any time As
ants attacked bees bees would occasionally carry or throw attacking ants off flowers but despite
this some ants maintained their presence at the nectary throughout the trials Because ants the
size of M rubra consume liquids slowly (~017 ndash 024 microlmin Davidson et al 2004) flowers
replenished nectar more quickly than ants consumed it We used two colors of dye (FDampC 5 and
FDampC 6) to differentiate male flowers with and without ants In two trials for each bee colony
anthers of male flowers with ants were coated with FDampC 5 dye while brushes of male flowers
lacking ants were coated with FDampC 6 dye In the other two trials we reversed dye colors to
control for effects of dye type and color Dye color was assigned in a random order Artificial
flowers were placed in randomly assigned positions in the array before we opened the colony
and allowed the bees to begin foraging
b) Scent trials
We explored the effect of ant scent on dye donation in fifteen trials testing three bee colonies in
six five and four trials each Bees were first exposed to ants by allowing the bees to forage for
eight hours on twenty flowers (ten male and ten female) of which five male flowers had 15 ants
and all others had none No dye was used during this ldquoexposurerdquo day We then collected all but
the five ant-free male flowers in the flight cage leaving these flowers to keep bees foraging
Next we individually stored five new male lids in Fluon-treated containers with ten M rubra
workers Five control male lids were put in identical containers lacking ants The following
61
morning we removed the remaining flowers and set out ten new male and female flowers in a
random spatial arrangement using the lids having or lacking ant scent Thus the ant-scented
flowers were not in the same positions as the ant-visited flowers on which the bees were trained
Only five male flowers had ant scent and the other 15 flowers (five male and ten female) did not
We used the same two dye colors as in i) to differentiate male flowers with and without ant
scent and randomized which color was used for ant-scented flowers between trials We collected
and replaced stigmas from female flowers after one hour and collected the stigmas again after
four hours In the first trial performed we also collected stigmas after two hours but due to the
small amount of dye transferred this was not repeated
iv) Dye quantification
After each trial we quantified the amount of dye transferred to female flowers using a
spectrophotometer We removed the stigmas from female flowers placed them in test tubes
added 51 mL of distilled water to each tube and vortexed each tube thoroughly to ensure the
dye was evenly diluted These were diluted further as needed if the absorbance exceeded the
sensitivity range of the spectrophotometer In the first six scent trials each stigma was analyzed
separately and the dye amounts were summed to obtain a total amount of each dye color
transferred to female flowers in each trial In all the ant presence trials and the other nine scent
trials we opted to treat the experiment as the unit of replication Therefore we combined stigmas
from each treatment type (ant-visited or ant-free) in a test tube and measured the total amount of
each dye color transferred in each treatment in each trial
Because we put different dye colors on male flowers with and without ants we could use
the amount of each dye color donated to all female flowers to measure the reproductive success
62
of ant-visited and ant-free male flowers The total amount of dye (of both colors) received by
female flowers with and without ants was our measure of female reproductive success We
calculated the amount of each dye color in the sample by measuring absorbance at 428 or 486
nm and converting absorbance to micrograms following computational methods for overlapping
spectra (Blanco et al 1989)
v) Statistical analyses
In two trials (one ant presence and one ant scent) most female flowers received no dye because
of low bee activity so we excluded these trials from analyses Because dye reception and
donation values were non-normally distributed we square-root transformed the data before
examining the effects of ant presence or ant scent on dye transfer in ANCOVAs For ant
presence trials we included ant presence on male and female flowers and their interaction as
main effects and the total amount of dye transferred in each trial as a covariate to account for
the large variation in overall dye transfer among trials For ant scent trials we included ant scent
as the main effect time (one or four hours) as a repeated measure the interaction between scent
and time and the total amount of dye transferred as a covariate Covariate by treatment (ant
presence or ant scent) interactions were never significant and so were excluded in final analyses
All analyses were conducted in JMP (v 1000)
Results
Bees usually started foraging within minutes of the beginning of the trial and continued until
flowers were collected from the flight cage or the lights were extinguished An average of 34 plusmn
012 and 40 plusmn 015 (mean plusmn SE) bees were foraging after an hour in the ant presence and ant
63
scent trials respectively Male flowers with ants donated significantly less dye than male flowers
lacking ants (Fig 22a F155 = 4019 p lt 00001) Similarly female flowers with ants received
significantly less dye than female flowers lacking ants (Fig 22a F155 = 461 p = 0036) There
was no significant interaction between ant presence on male flowers and ant presence on female
flowers (F155 = 118 p = 028) Flowers with ant scent donated significantly less dye than
flowers without ant scent (Fig 22b F125 = 11216 p lt 00001) There was no significant effect
of time (F125 = 121 p = 028) or interaction between time and scent (F125 = 111 p = 030) in
the model
Discussion
In this study ants altered bumblebee pollination behavior and exhibited trait-mediated indirect
interactions with (artificial) flowers Through interference competition M rubra workers
significantly affected the pattern of dye transfer by B impatiens causing a preferential flow of
dye from male flowers lacking ants to female flowers lacking ants If manifested in nature such
effects would reduce the reproductive success of plants visited by ants through both male and
female function
Ants could have changed the attractiveness of artificial flowers in at least two ways First
as ants interrupted or altogether prevented bumblebees from foraging at nectaries bumblebees
may have learned to avoid flowers with ants due to the relative inefficiency of foraging at these
flowers something bumblebees take into account (Heinrich 2004) Second bees attempt to
minimize their risk of being attacked during foraging bouts which ants did to visiting bees Ants
often attacked or harassed bees by biting grasping and appearing to sting visiting bees
preventing them from accessing the nectary or reducing their time on flowers (see online video
64
1) and bees sometimes avoided ant-tended flowers entirely This harassment was sometimes
physically traumatic (online video 1) and bees often appeared to have trouble flying after being
attacked by ants Previous research has demonstrated that bumblebees leave or avoid flowers
where they have been harassed (Jones and Dornhaus 2011) and avoid foraging where there is
visual or olfactory evidence of a predator or predation event (Abbott 2006 Goodale and Nieh
2012) Though it is unclear if bumblebees viewed ants as competitors or predators they
responded similarly to flowers having ants as they do to flowers housing predators (Gonccedilalves-
Souza et al 2008)
To avoid artificial flowers with ants bees likely used a combination of visual and
olfactory cues Previous research has shown that bees respond to conspicuous predators or
predator ldquodummiesrdquo on flowers (Suttle 2003 Gonccedilalves-Souza et al 2008) decreasing the
frequency and duration of their visits to these flowers Thus bees may have been able to avoid
flowers harboring ants by sight alone However even in the absence of ants bees still
preferentially visited flowers lacking ant scents (Fig 22b) suggesting that they had learned to
associate ant scent with harassment Bees are adept at associative learning (Wright and Schiestl
2009) and can learn to recognize unique scents left behind by both conspecific and heterospecific
flower visitors (Stout et al 1998) Bees can use these various scents often arising from tarsal
gland deposits (Stout et al 1998) to recognize recently visited flowers that are less likely to be
profitable (Stout and Goulson 2002) Recently Ballantyne and Willmer (2012) demonstrated that
bees learn to associate ant scents with unrewarding artificial flowers and decrease their visitation
to these flowers Our results complement their findings by showing that bees can associate ant
scent with harassment at otherwise rewarding flowers
In our study ant scent caused bees to adjust their foraging strategy and decreased the
amount of pollen analogue that was donated by ant-scented flowers Thus the effects of
65
interference competition with ants on flowers can extend beyond immediate interactions and may
have fitness consequences for plants even when ants are absent Like other olfactory cues these
effects are likely transitory (Stout et al 1998) Although more dye was still donated by male
flowers lacking ant scent than those with ant scent the ratio of dye donated was on average
closer to equality in hours 2-4 than in the first hour (Fig 22b) This effect may be partially
driven by dye depletion of male flowers without ant scent but the lack of a strong corresponding
decrease in flowers with ant scent suggests that visitation patterns became more similar We did
not test whether B impatiensrsquo avoidance of ant scent was an innate or a learned behavior but
previous work has shown that Bombus terrestris does not innately avoid flowers with ant scent
(Ballantyne and Willmer 2012)
Ants may be necessary for plant survival and growth but can be costly for plant
reproduction The evolution of ant attractants such as extrafloral nectaries in some plant lineages
suggests that the costs of ants can be outweighed by their protective abilities However in this
study flowers visited by ants received and donated significantly less dye although they did
retain some sexual function The net benefit of having ants depends on whether ants increase
plant fitness by reducing herbivory more than they decrease plant fitness by disrupting
pollination Alternatively plants may actually benefit from the costs of ants to plant
reproduction ants that castrate flowers may be better defenders increasing plant survival or
vegetative growth when plants are young allowing for increased reproduction later in life when
the plant is colonized by less aggressive non-castrating ants (Frederickson 2009 Palmer et al
2010) In some plant species pollinator harassment by ants may even be beneficial Altshuler
(1999) reported that Ectatomma ants greatly increased fruit set of Psychotria limonensis despite
reductions in pollinator visitation rates presumably due to increases in the rate of pollen
outcrossing Similarly Gonzaacutelvez et al (2012) found that the presence of Oecophylla
66
smaragdina ants on Melastoma malabathricum flowers reduced visitation by less effective
pollinators and increased visitation by more effective Xylocopa bees increasing plant fitness
Cases of ants being beneficial to flowers appear to be the minority and many plant
species have traits that limit ant access to flowers Several studies have detailed floral volatiles
that are thought to mimic ant alarm pheromones and thus repel ants (Junker and Bluumlthgen 2008
Willmer et al 2009) Other plants use structural modifications such as narrow corollas or
slippery stems to limit ant access to flowers (reviewed in Willmer et al 2009) Exploitative
competition between ants and pollinators (Lach 2005 Ballantyne and Willmer 2012) as well as
the lethal effects of ant antibiotic secretions on pollen grains (Beattie et al 1985 Dutton and
Frederickson 2012) may have resulted in selection on plants to reduce ant visits to flowers Our
results suggest that trait-mediated indirect interactions resulting from interference competition
between ants and bees may favor plants that defend their flowers and their pollinators against
ants
Acknowledgements
We thank H Rusnock and S Meadley-Dunphy for assistance J Dix for help with flight cage
construction and Biobest for bumblebee colonies We are grateful to the Thomson and
Frederickson labs for guidance particularly T Makino funded by JSPS for helping to refine
artificial flowers J Ogilvie for help with bees K Prior for M rubra colony collections and K
Turner for study design suggestions The comments of the editor and two anonymous reviewers
prompted improvements in the article especially with regards to study design MEF and JDT
acknowledge NSERC Discovery Grant funding The University of Torontorsquos Faculty of Arts and
Science supported MGRTrsquos participation
67
68
References
Abbott K R 2006 Bumblebees avoid flowers containing evidence of past predation events
Canadian Journal of Zoology 841240-1247
Altshuler D L 1999 Novel interactions of non-pollinating ants with pollinators and fruit
consumers in a tropical forest Oecologia 119600-606
Ballantyne G and Willmer P 2012 Floral visitors and ant scent marks noticed but not used
Ecological Entomology 37402-409
Beattie A J Turnbull C Knox R B and Williams E G 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany
71421-426
Blanco M Runiaga H Maspoch S and Tarin P 1989 A simple method for
spectrophotometric determination of two-components with overlapped spectra Journal of
Chemical Education 66178-180
Byk J amp Del Claro K 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333-38
Davidson D W S C Cook and R R Snelling 2004 Liquid-feeding performances of ants
(Formicidae) ecological and evolutionary implications Oecologia 139255ndash66
Dukas R 2005 Bumble bee predators reduce pollinator density and plant fitness Ecology
861401-1406
Dussutour A and Simpson S J 2009 Communal nutrition in ants Current Biology 19740-
744
69
Dutton E M and Frederickson M E 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561-569
Frederickson ME 2009 Conflict over reproduction in an ant-plant symbiosis why Allomerus
octoarticulatus ants sterilize Cordia nodosa trees American Naturalist 173675-681
Galen C and Cuba J 2001 Down the tube pollinators predators and the evolution of flower
shape in the alpine skypilot Polemonium viscosum Evolution 551963-1971
Gonccedilalves-Souza T Omena P M Souza J C and Romero G Q 2008 Trait-mediated
effects on flowers artificial spiders deceive pollinators and decrease plant fitness Ecology
892407-2413
Gonzaacutelvez F G Santamariacutea L Corlett R T and Rodriacuteguez-Gironeacutes M A 2012 Flowers
attract weaver ants that deter less effective pollinators Journal of Ecology 10178ndash85
Goodale E and Nieh JC 2012 Public use of olfactory information associated with predation
in two species of social bees Animal Behaviour 84919-924
Harder LD and Thomson JD 1989 Evolutionary options for maximizing pollen dispersal of
animal-pollinated plants American Naturalist 133323-344
Heil M and McKey D 2003 Protective ant-plant interactions as model systems in ecological
and evolutionary research Annual Review of Ecology Evolution and Systematics 34425-
453
Heinrich B 2004 Bumblebee economics 2nd
edn Harvard University Press Cambridge
Ings T C and Chittka L 2009 Predator crypsis enhances behaviourally mediated indirect
effects on plants by altering bumblebee foraging preferences Proceedings of the Royal
Society B 2762031-2036
70
Inouye D W and Taylor O R Jr 1979 A temperate region plant-ant-seed predator system
consequences of extra floral nectar secretion by Helianthella quinquenervis Ecology 601-
7
Junker R R and Bluumlthgen N 2008 Floral scents repel potentially nectar-thieving ants
Evolutionary Ecology Research 10295ndash308
Junker R R Bleil R Daehler C C and Bluumlthgen N 2010 Intra-floral resource partitioning
between endemic and invasive flower visitors consequences for pollinator effectiveness
Ecological Entomology 35760ndash767
Jones E I and Dornhaus A 2011 Predation risk makes bees reject rewarding flowers and
reduce foraging activity Behavioral Ecology and Sociobiology 651505-1511
Lach L 2005 Interference and exploitative competition of three nectar-thieving invasive ant
species Insect Sociaux 52257-262
Lach L 2007 A mutualism with a native membracid facilitates pollinator displacement by
Argentine ants Ecology 881994-2004
Lach L 2008 Argentine ants displace floral arthropods in a biodiversity hotspot Diversity and
Distributions 14281-290
Makino T T and Sakai S 2007Experience changes pollinator responses to floral display size
from size-based to reward-based foraging Functional Ecology 21854863
Maloof J E and Inouye D W 2000 Are nectar robbers cheaters or mutualists Ecology
812651ndash2661
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506-514
Ohashi K Leslie A and Thomson JD 2008 Trapline foraging by bumble bees V Effects of
experience and priority on competitive performance Behavioral Ecology 19936-948
71
Palmer T M Doak D F Stanton M L Bronstein J L Kiers E T Young T P Goheen
J R and Pringle R M 2010 Synergy of multiple partners including freeloaders
increases host fitness in a multispecies mutualism Proceedings of the National Academy
of Sciences 10717234-17239
Preisser E L Bolnick D I and Benard M E 2005 Scared to death The effects of
intimidation and consumption in predator-prey interactions Ecology 86501-509
Stout J C Goulson D and Allen J A 1998 Repellent scent-marking of flowers by a guild of
foraging bumblebees (Bombus spp) Behavioral Ecology and Sociobiology 43317-326
Stout J and Goulson D 2002 The influence of nectar secretion rates on the responses of
bumblebees (Bombus spp) to previously visited flowers Behavioral Ecology and
Sociobiology 52239-246
Suttle K B 2003 Pollinators as mediators of top-down effects on plants Ecology Letters
6688-694
Thomson JD 1986 Pollen transport and deposition by bumble bees in Erythronium influences
of floral nectar and bee grooming Journal of Ecology 74329-341
Thomson JD Ogilvie J E Makino T T Arisz A Raju S Rojas-Luengas V and Tan M
G R 2012 Estimating pollination success with novel artificial flowers effects of nectar
concentration Journal of Pollination Ecology 9108-114
Tsuji K Hasyim A and Nakamura K 2004 Asian weaver ants Oecophylla smaragdina and
their repelling of pollinators Ecological Research 19669-673
Ulrich S and Bertsch A 1990 Do foraging bumblebees scent-mark food sources and does it
matter Oecologia 82137-144
Werner E E and Peacor S D 2003 A review of trait-mediated indirect interactions in
ecological communities Ecology 841083-1100
72
Willmer P G Nuttman C V Raine N E Stone G N Pattrick J G Henson K Stillman
P McIlroy L Potts S G and Knudsen J T 2009 Floral volatiles controlling ant
behaviour Functional Ecology 23888-900
Wright G A and Schiestl F P 2009 The evolution of floral scent the influence of olfactory
learning by insect pollinators on the honest signaling of floral rewards Functional Ecology
23841-851
73
Figures
Figure 21 Photograph of artificial flower lids A = anther L = lid N = nectary R = region
where anther or stigma is placed S = stigma T = thread W = weight
74
Figure 22 Dye transferred (mean plusmn 1SE) per trial by B impatiens workers A) from artificial
male and to artificial female flowers in the presence (unfilled circles) or absence (filled circles)
of Myrmica rubra workers and B) from artificial male flowers with (unfilled circles) or without
(filled circles) M rubra scent to ant-free female flowers in the 1st or the 2
nd to 4
th hours of the
trials
75
Chapter 3 Not just for the bees pollen consumption is common among tropical
ants
Cembrowski AR Reurink G and Frederickson ME
Planned submission to Biotropica
ARC and MEF designed the study ARC and GR did laboratory work ARC performed statistical
analyses and wrote the manuscript with input from other authors
Abstract
Although palynivory or pollen consumption is widespread among other hymenopterans there
are few accounts of palynivory in ants To quantify how often ants consume pollen we subjected
adult workers and larvae from 75 species of neo- and paleo-tropical ants to acetolysis a process
that destroys most organic material but leaves behind pollen grains In over half of the species
we examined ants contained a few pollen grains (lt 5) and ants of several Camponotus species
contained abundant pollen grains (gt50) We tested for an association between trophic level and
palynivory using stable nitrogen isotope ratios (δ15
N) but we did not find a significant
correlation we found pollen grains in lsquoherbivorousrsquo omnivorous and carnivorous ants We
suggest that our results indicate sporadic opportunistic pollen consumption by ants in tropical
forests
Introduction
For those organisms that can digest it efficiently pollen can be an excellent food source
Depending on the plant species pollen can contain up to 60 protein as well as lipids
76
carbohydrates and numerous trace nutrients (Roulston and Cane 2000) Pollen feeding
(palynivory) occurs in many taxa including birds mammals and arthropods (Roulston and Cane
2000) but it is best known among the Hymenoptera particularly bees Although adult bees feed
mainly on plant exudates (ie nectar) nearly all bee species rely on pollen as the chief protein
source for developing larvae (Willmer 2011) Closely related to bees ants have similar
nutritional requirements adult workers need carbohydrates whereas larvae are fed a diet rich in
protein (Dussutour and Simpson 2009) Ants also exploit nectar heavily and regularly visit
flowers (eg Herrera et al 1984 Bluumlthgen et al 2004) but unlike bees are not believed to
widely consume pollen Instead ants are thought to receive most of the protein in their diet from
predation or scavenging (eg Floren et al 2002)
However ants may not be as reliant on animal protein sources as commonly assumed In
tropical forests arboreal ants can have similar δ15
N ratios a measure of trophic level as
herbivores suggesting limited carnivory and substantial feeding on plant-derived protein sources
(Davidson et al 2003) One potential protein source is pollen This idea is supported by scattered
accounts of pollen consumption by ants from other ecosystems including deserts (Ness 2006)
boreal forests (Czechowski et al 2009) and tropical savannas (Byk and Del Claro 2010) One
ant genus Cephalotes is widely recognized to consume pollen (Creighton 1967 Baroni Urbani
and de Andrade 1997 Byk and Del Claro 2010) However beyond this genus our knowledge of
pollen consumption by ants has largely been limited to direct observations (but see Czechowski
et al 2011) and it is unknown if palynivory is truly rare among ants or simply under-reported
We borrowed a technique commonly used in palynology acetolysis to assess the frequency of
pollen consumption by ants in 75 neo- and paleo-tropical ant species This technique allowed us
to examine many more ant species than we could have observed directly
77
Methods
Adult worker ants were collected by hand at baits or from leaf litter using Winkler extractors
while larvae along with adults were collected by hand from nests Collections were made by L
Arcila Hernandez and J Sanders in the primary tropical rainforest surrounding the Centro de
Investigaciόn y Capacitaciόn Rio Los Amigos (CICRA 12deg34rsquoS 70deg05rsquoW) in Peru in 2010-
2011 and by E Youngerman from lowland primary and secondary rainforests in Madang
Province Papua New Guinea (PNG) in 2011-2012 We sorted ant samples to morphospecies and
then identified them to genus and when possible to species using existing keys which were
unavailable for PNG ants Ant samples used for acetolysis were whole adult workers guts from
adult workers and whole larvae
We used acetolysis to look for pollen grains inside ants In acetolysis organic material is
dissolved in acetic anhydride and sulfuric acid leaving behind stained pollen exines When
possible we used adult and larval ants of the same species from different colonies or bait
locations in an attempt to sample more representatively We prepared samples by first washing
them in 70 ethanol to remove any pollen present on the samplersquos outer surface before placing
them individually in micro-centrifuge tubes with ~05mL of a 91 mixture of acetic anhydride
sulfuric acid We then heated each sample at 95degC for 20min allowing tissues to dissolve Next
we centrifuged samples at 1000 RPM for 5min and decanted the liquid supernatant before
adding ~05mL of glacial acetic acid to the precipitate centrifuging again as above We decanted
the samples and added a drop of distilled water
78
We examined a 150μL sub-sample of this solution for pollen grains under a light
microscope Larvae gave less than 150μL of solution following acetolysis so we sampled the
entire amount We identified pollen grains by their staining and their surface architecture For
most species we examined at least three adult workers and three larvae but for some species
fewer than three adult workers or larvae were available (Tables 31 and 32)
We investigated the relationship between trophic level as measured by δ15
N ratios and
pollen consumption by ants Stable isotope (SI) data was available for some (n =23) of the
Peruvian species from a separate project in which collections overlapped Samples were stored in
EtOH and dried at 50degC overnight We used heads legs and thoraxes (but not gasters) of ants
for SI analysis Often single individuals did not provide enough material for analysis and we
combined multiple individuals to achieve necessary weights up to 10 for small ant species
Samples were analyzed at the Boston University Stable Isotope Laboratory using a Finnigan
Delta-S analyzer For other Peruvian species for which we had samples but not corresponding
stable isotope data (n =25) we took the median δ15
N ratio value of that speciesrsquo genus and
assigned it to the species in question Using pollen presenceabsence data for these 48 species
we used logistic regression to correlate pollen presence and δ15
N ratio using R (v 2151)
Results
We found pollen grains in 37 of the 197 adult workers 20 of the 102 larvae and 38 of the 75 ant
species we examined (Tables 31 and 32) The proportion of individuals containing pollen did
not differ significantly between larvae and adults (χ2
1 = 002 p = 088) The number of pollen
grains found was often low (median = 3) but highly variable (range 1-468) Also pollen
79
presence was inconsistent among conspecific ant samples of species that contained pollen 31
had pollen in only one of the larvae or adults examined In only three species (two Cephalotes
and one Myrmelachista) did all examined adults contain pollen
Highly lsquoherbivorousrsquo carnivorous and omnivorous ants contained pollen grains (Fig
31) There was no correlation between δ15
N ratio and pollen presence (χ2 = 150 df = 1 p =
022) In fact several carnivorous species contained pollen grains (eg Odontomachus sp 1
Pachycondola sp1)
Discussion
Pollen consumption by ants appears to be more common than suspected The majority of species
(51) had at least one individual that had consumed pollen and in 20 multiple individuals
contained pollen Numbers of grains found tended to be low however with most individuals
containing only a few grains Ants may use pollen as only a small portion of their diet exploiting
a host of other protein-containing foods more heavily including other low- δ15
N foods (eg
honeydew epiphylls Houmllldobler and Wilson 1990 Davidson et al 2003)
There are a few reasons why pollen may not be heavily exploited by ants Pollen requires
much post-consumption processing to extract its nutrients These nutrients are located in the
pollenrsquos cytoplasm which is protected from direct digestion by the exines and intines of the
grains Organisms must either break through these layers or cause the pollen grains to germinate
or pseudo-germinate to access and digest the cytoplasm (Roulston and Cane 2000) This can be
achieved by consuming a sugar-containing solution (such as nectar) along with pollen However
animals that are not adapted to feed on pollen may not be very efficient at this process only
80
extracting nutrients from some of the grains (Herrera and Martiacutenez del Rio 1998) Thus while
ants can likely get some nutrients from pollen other forms of more easily processed protein may
be more attractive
Pollen may also pose handling difficulties for ants Despite the large number of studies
that have recorded ants visiting flowers (Chapter 1) only a few papers detail ants harvesting
pollen directly from anthers Byk and Del Claro (2010) reported that Cephalotes pusillus ants
removed almost all of the available pollen from Ouratea spectabilis flowers and at least two
other studies describe ants consuming pollen along with nectar from flowers (Horskins and
Turner 1999 Ness 2006) Unlike bees which possess both morphological (eg specialized hairs
for holding pollen) and behavioral adaptations (eg stereotyped pollen-packing motions) ants
have no apparent mechanisms to increase the amount of pollen harvested and are likely limited
by the number of grains they can ingest or carry Rather than collect pollen directly from flowers
it appears more likely that ants collect it haphazardly in the environment leading to a smaller
number of pollen grains consumed at any one time It is known that at least Cephalotes ants
engage in such behavior Creighton (1967) observed Cephalotes texanus collecting pollen grains
trapped by Citrus x paradisi leaf hairs
There is a chance that the small numbers of pollen grains we observed came from
secondary pollen consumption (ie consuming a palynivorous organism) or consumption of
liquid containing pollen While we cannot rule this out we believe our results represent actual
targeted pollen consumption by ants Cephalotes ants known palynivores (Creighton 1967
Baroni Urbani and de Andrade 1997) contained similar numbers of pollen grains as many of the
pollen-containing ant species that we examined As well in at least once instance ants that we
found containing pollen grains were in the process of preying on non-pollen consuming
81
organisms (eg a Pachycondola species that was carrying a termite in its mandibles) or were
attracted by sugar baits
Contrary to our expectations there was no relationship between an ant speciesrsquo δ15
N ratio
and whether it had consumed pollen (Fig 32) We expected palynivory to be more common in
herbivorous ants because pollen presents a widespread source of protein a potentially limiting
resource The lack of pattern might reflect how available pollen grains are in the environment
While other forms of food may be preferable ants tend to be opportunistic foragers (Carroll and
Janzen 1973) and there is likely little cost associated with the consumption of scattered pollen
grains during foraging trips beyond the volume they occupy in the gut Some genera were found
to consistently consume pollen In support of previous observations (Baroni Urbani and de
Andrade 1997 Byk and Del Claro 2010) Cephalotes species were among the most common
pollen consumers Camponotus species also consistently contained pollen grains of the eight
species examined five contained pollen and pollen was found in species from both Peru and
PNG Camponotus species also contained the two largest numbers of pollen grains found (468
and 271) Taken together these results suggest that they may be another as yet unrecognized
commonly palynivorous genus
In general larvae did not contain large numbers of pollen grains (ranging from 1-38
grains) This is counter to what is seen in bees where larvae are the main recipients of pollen
(Willmer 2011) This difference may be because ant larvae are continually fed via trophallaxis
by adults throughout their development whereas bee larval cells are provisioned with pollen and
then sealed while larvae complete their development If both adults and larvae are able to extract
nutrients from pollen we would expect to see a preferential flow of nutrients from pollen to
larvae but not necessarily the grains themselves
82
Unlike palynivory by organisms that also provide pollination services pollen
consumption by ants is unlikely to benefit plants Ants rarely act as pollinators (Beattie et al
2004) They have pollen-killing antibiotic secretions on their cuticles (Dutton and Frederickson
2012) and they can compete with other flower visitors reducing flower visitation rates (Ness
2006) The negative impacts of flower-visiting ants are thought to have led to the evolution of
ant-repellent floral volatiles (reviewed in Willmer 2009) These volatiles can pre-empt
competition between ants and pollinators and may often be located within the pollen itself
(Willmer 2009) If floral volatiles truly are a defense against ant visitation our results suggest an
additional evolutionary pressure to not only prevent ant-pollinator interactions but to also
protect plant gametes from occasional consumption by a non-pollinating visitor
Acknowledgements
We would like to thank Jon Sanders Lina Arcila Hernandez and Eric Youngerman for providing
samples the Thomson lab for use of supplies the Pierce lab for funding support during JSrsquo and
EYrsquos collections and the staff of CICRA and the New Guinea Binatang Research Center for
logistical support The Peruvian Ministry of Agriculture provided permits (Nos 394-2009-AG-
DGFFS-DGEFFS and 299-2011-AG-DGFFS-DGEFFS) MEF acknowledges financial support
from an NSERC Discovery Grant a Connaught New Researcher Award an Ontario Ministry of
Economic Development and Innovation Early Researcher Award and the University of Toronto
ARC was supported by an Ontario Graduate Scholarship and Sigma Xi
83
References
Baroni Urbani C and M L de Andrade 1997 Pollen eating storing and spitting by ants
Naturwissenschaften 84256ndash258
Beattie A J C Turnbull R B Knox and E G Williams 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany 71421ndash
426
Bluumlthgen N G Gottsberger and K Fiedler 2004 Sugar and amino acid composition of ant-
attended nectar and honeydew sources from an Australian rainforest Austral Ecology
29418ndash429
Byk J and K Del-Claro 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333ndash38
Carroll C R and D H Janzen 1973 Ecology of foraging ants Annual Review of Ecology and
Systematics 4231ndash257
Creighton W S 1967 Studies on free colonies of Cryptocerus texanus Satschi (Hymenoptera
Formicidae) Psyche 7434ndash42
Czechowski W B Markό and A Radchenko 2009 Rubbish dumps reveal the diet of ant
colonies Myrmica schencki Em and Myrmica rubra (L) (Hymenoptera Formicidae) as
facultative pollen-eaters Polish Journal of Ecology 56737ndash741
Czechowski WB Markoacute K Erős and E Csata 2011 Pollenivory in ants (Hymenoptera
Formicidae) seems to be much more common than it was thought Annales Zoologici
61519ndash525
84
Dussutour A and S J Simpson 2009 Communal nutrition in ants Current Biology 19740ndash4
Dutton E M and M E Frederickson 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561ndash569
Floren A A Biun and K E Linsenmair 2002 Arboreal ants as key predators in tropical
lowland rainforest trees Oecologia 131137ndash144
Grant B R 1996 Pollen digestion by Darwinrsquos finches and its importance for early breeding
Ecology 77489-499
Herrera L G and C Martiacutenez del Rio 1998 Pollen digestion by new world bats effects of
processing time and feeding habits Ecology 792828ndash2838
Houmllldobler B and Wilson E O 1990 The Ants 1st Edition Harvard University Press
Cambridge
Horskins K and Turner V B 1999 Resource use and foraging patterns of honeybees Apis
mellifera and native insects on flowers of Eucalyptus costata Australian Journal of
Ecology 24221-227
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506ndash514
Roulston T H I and J H Cane 2000 Pollen nutritional content and digestibility for animals
Plant Systematics 222187ndash209
Van Tets I G 1997 Extraction of nutrients from Protea pollen by African rodents Belgian
Journal of Zoology 12759-65
Willmer P G 2011 Pollination and Floral Ecology 1st Edition Princeton University Press
New Jersey
85
Willmer P G C V Nuttman N E Raine G N Stone J G Pattrick K Henson P Stillman
et al 2009 Floral volatiles controlling ant behaviour Functional Ecology 23888ndash900
86
Tables
Table 31 Numbers of adult worker and larval ants examined for each species collected in Peru
Numbers of pollen grains found in individual samples are given in parentheses after the sample
size when there were no grains no numbers are given
Genus Species Number of adults analyzed
(numbers of grains found)
Number of larvae analyzed
(numbers of grains found)
Dolichoderinae
Azteca sp 1 3 0
sp 2 2 0
sp 3 0 2
sp 4 3 (1) 0
sp 5 3 (2) 0
Dolichoderus decollatus 3 (66) 0
sp 1 3 0
sp 2 3 1 (2)
sp 3 3 3
sp 4 0 2
Ecitoninae
Labidus sp 1 3 0
Formicinae
Camponotus sp 1 3 3
sp 2 3 (82) 1 (2)
sp 3 3 (3) 0
sp 4 0 2
87
sp 5 8 (11) 0
sp 6 4 (7 16 468) 0
sp 7 5 (12) 0
sp 8 2 (1) 0
Paratrechina sp 1 4 (11) 0
Myrmelachista sp 1 3 (5 11 18) 0
sp 2 0 3
Trachymyrmex sp 1 3 (21) 0
sp 2 3 0
Myrmicinae
Atta sp 1 3 0
Cephalotes atratus 3 (2 2 21) 0
placidus 3 (1 4 21) 0
sp 1 3 0
sp 2 1 0
sp 3 2 0
Crematogaster sp 1 3 1
sp 2 3 (35) 0
sp 3 9 (7) 0
sp 4 2 0
Megalomyrmex sp 1 3 0
sp 2 0 2
sp 3 4 (7) 0
sp 4 3 0
Monomorium sp 1 0 3 (2)
88
Pheidole sp 1 0 3
sp 2 3 0
sp 3 0 3
Procryptocerus sp 1 3 0
Solenopsis sp 1 0 3
sp 2 3 (3 5) 0
Wasmannia sp 1 3 0
Ponerinae
Pachycondola sp 1 3 (3) 0
Odontomachus sp 1 3 (34) 0
Pseudomyrmicinae
Pseudomyrmex sp 1 3 0
sp 2 3 0
sp 3 3 (9) 0
sp 4 0 3
sp 5 0 3
89
Table 32 Numbers of adult worker and larval ants examined for each species collected in Papua
New Guinea Numbers of pollen grains found in individual samples are given in parentheses
after the sample size when there were no grains no numbers are given
Genus Species Number adults analyzed
(grains found)
Number larvae analyzed
(grains found)
Aenictinae
Aenictus sp 1 3 3 (1 1)
Dolichoderinae
Philidris sp 1 3 1 (4)
Tapinoma sp 1 2 3 (38)
Technomyrmex sp 1 0 3 (12 12)
Ectatomminae
Rhytidoponera sp 1 1 3 (2)
Formicinae
Acropyga sp 1 3 3 (2)
sp 2 3 3
sp 3 3 3
Anoplolepsis sp 1 3 (1) 3 (1)
Calomyrmex sp 1 3 3 (1)
Camponotus sp 1 3 (1 271) 3
sp 2 1 3
sp 3 2 0
Nylanderia sp 1 3 3
Opisthopsis sp 1 3 3
Polyrachis sp 1 1 3 (4)
90
Myrmicinae
Crematogaster sp 1 3 3
sp 2 3 (32) 3
Monomorium sp 1 3 (1) 3 (1)
sp 2 3 3 (1 1 1)
Pheidole sp 1 3 3
Pristomyrmex sp 1 3 3 (11)
Pyramica sp 1 3 0
Ponerinae
Odontomachus sp 1 3 3 (4)
91
Figures
Figure 31 Photographs of pollen grains found in two ant species a) Dolichoderus sp 2 and b)
Camponotus sp 6 Scale bars = 50 microm
92
Figure 32 Relationship between trophic level as measured by δ15
N ratio and pollen presence
for 48 neotropical ant species 1 Atta sp 5 Azteca spp 8 Camponotus spp 5 Cephalotes spp 4
Crematogaster spp 5 Dolichoderus spp 1 Labidus sp 3 Megalomyrmex spp 1 Monomorium
sp 2 Myrmelachista spp 1 Odontomachus sp 1 Pachycondola sp 1 Paratrechina sp 3
Pheidole sp 4 Pseudomyrmex spp 2 Solenopsis spp and 1 Wasmannia sp
93
Concluding Remarks
Ants have traditionally been considered to be unwelcome flower visitors they consume floral
rewards ostensibly meant for pollinators while rarely providing pollination services themselves
In this thesis I investigate the validity of this assumption and use an experimental study and an
observational study to examine two ways that ants may impact plant reproduction
In the first chapter I review the literature on ants as flower visitors I show that the common
assumption of ants always being categorically negative for plant reproduction is incorrect while
ants may reduce floral attractiveness to pollinators the net effect of ants on plant fitness
measures is variable ranging from positive to negative to neutral I also discuss ants as
pollinators and ways that plants may minimize ant visitation to flowers before finishing with a
section highlighting questions that still need investigation
In the second chapter I explore how interference competition with ants can structure bumblebee
foraging behavior and how this may alter pollen movement patterns Using artificial flowers with
male or female function I allowed Bombus impatiens bumblebees to forage on flowers that
either did or did not have Myrmica rubra ants on them Ant presence significantly reduced the
amount of pollen analogue donated and received by flowers Building on these results I found
that the presence of ant scent alone elicited similar changes to bumblebee behavior reducing the
amount of pollen analogue donated by male flowers My results show that interference
competition is sufficient to alter bumblebee foraging behavior and that the effects of ants on
pollinators may extend after ant visitation to flowers Further study is needed to determine if bees
are able to differentiate between ant species by visual or olfactory cues Their ability to do so
94
could affect how generalized their response to ant presence is and may help to better predict how
ants will affect plant reproduction
In the final chapter I investigate how common palynivory is amongst tropical ant species I used
acetolysis and light microscopy to examine whole ants larvae and ant guts from Papua New
Guinea and Peru I found that tropical ants consistently contain pollen grains albeit often in
small numbers (lt5) However some genera notably Camponotus and Cephalotes consistently
contained higher numbers providing the first evidence that Camponotus may be a palynivorous
genus The small numbers of pollen grains present may indicate that ants preferentially exploit
other more easily digestible high-protein foods over pollen Future studies should investigate
exactly what role pollen plays in ant diets by supplementing the diet of captive ant colonies with
pollen This would allow us to examine how pollen consumption affects colony demography as
well as how efficiently ants digest pollen and whether ants exhibit pollen preferences
I bring up several pressing and unanswered questions within this thesis and highlight three here
First numerous plant species possess chemical or physical traits assumed to increase plant
fitness by reducing ant visitation However this has rarely been tested and instead research has
focused predominately on documenting ant repellence We should move away from this and
begin examining plant fitness in the presence or absence of these repellent traits to see how
beneficial these traits truly are Second how flower visiting ants impact male fitness has been
almost completely ignored in favor of female fitness Future studies should investigate how ant
visitation affects both pollen donation and pollen movement patterns Lastly research involving
antibiotics present on ant cuticles and their effect on pollen viability has been too small in scope
often limited to demonstrating the pollen killing effects of ant secretions Investigation into
95
whether plants have evolved any resistance to the secretions of the local ant community or why
some plants appear unaffected by their ant partners may yield exciting results
In conclusion ants have the capacity to strongly affect plant reproduction both positively and
negatively The fact that these diametrically opposing forces exist means we cannot approach
flower-visiting ants as either positive or negative a priori Instead we should strive to work
towards a better understanding of what is occurring in the system of interest and what role ants
play within it
96
Copyright Acknowledgments
Chapter 2 ldquoAnts and ant scent reduce bumblebee pollination of artificial flowersrdquo is current in
press in The American Naturalist Request for its inclusion here was sought and approved
vi
Abstract 75
Introduction 75
Methods 77
Results 78
Discussion 79
Acknowledgements 82
References 83
Tables 86
Figures 91
Concluding Remarks 93
Copyright Acknowledgments 96
vii
List of Tables
Table 11 List of studies of flower-visiting ants that consumed floral rewards or interacted with
pollinators and included a measure of fitness39
Table 12 List of studies that have either demonstrated ant pollination through exclusion
experiments or whose results strongly suggest it48
Table 31 Numbers of adult worker and larval ants examined for each species collected in Peru
and numbers of pollen grains found87
Table 32 Numbers of adult worker and larval ants examined for each species collected in Papua
New Guinea and numbers of pollen grains found90
viii
List of Figures
Figure 21 Photograph of artificial flower lids with anther and stigma74
Figure 22 Dye transferred (mean plusmn 1SE) by B impatiens a) in the presence and absence of M
rubra ants and b) with or without M rubra scent75
Figure 31 Photographs of pollen grains found inside ants92
Figure 32 Relationship between trophic level and pollen presence for neotropical
ant species93
1
Chapter 1 Ants as flower visitors and their effects on pollinator behavior and plant
reproduction
Cembrowski AR and Frederickson ME
Planned submission to a journal such as Oikos or Insectes Sociaux
ARC wrote the manuscript with input from MEF
Abstract
Ants often provide a range of benefits to flowering plants particularly by reducing herbivory
However when ants visit flowers they can deter pollinators or damage floral structures
Currently it is unclear how often and in what direction flower-visiting ants affect plant
reproductive success A review of the literature shows that ants can help or hinder pollination
processes but overall flower-visiting ants most commonly have a non-significant net effect on
plant fitness Although ants may directly harm flowers or indirectly affect pollination by
consuming floral nectar or harassing pollinators they can also benefit plants by attacking
florivores or predators of pollinators Ants occasionally act as pollinators themselves though ant
secretions often kill pollen grains There is growing evidence that many plants have traits that
allow them to avoid or minimize the negative effects of flower-visiting ants More research is
needed to determine if these traits provide fitness benefits to plants by preventing ant visitation
or if they function as generalized repellents of floral antagonists
2
Introduction
Ants and angiosperms share a long evolutionary history Ant diversification closely followed that
of flowering plants as ants took advantage of the large prey numbers that angiosperms supported
and the new habitats they provided (Moreau 2006) Subsequently numerous angiosperm
lineages evolved intimate associations with ants that function as an indirect or biotic form of
plant defense Food rewards such as extrafloral nectar or pearl bodies or housing in the form of
domatia attract ants to plants where they reduce herbivory and increase plant fitness (reviewed
in Rosumek et al 2009) However the traits that make ants good plant defenders particularly
their abundance attraction to sugar sources and aggressiveness can also cause problems for
plant reproduction Ants attracted by floral nectar can compete with pollinators for resources
and defend flowers potentially reducing plant fitness (Ness 2006 Junker et al 2007) That ants
negatively affect plant reproduction is an old and pervasive hypothesis Kerner (1878) called ants
ldquomost unwelcome guestsrdquo on flowers and more recent authors have similarly expressed that ant
visitation to flowers reduces plant reproductive success (Ness 2006 Lach 2007 Willmer et al
2009)
Nonetheless studies assessing the impact of flower-visiting ants on plant reproductive
success have produced mixed results Ants can have negative impacts on pollination and flower
function (Galen 1983 Puterbaugh 1998 Ness 2006 Hansen and Muumlller 2009) However other
studies have found no effect or even strong benefits of ant visits to flowers (Rico-Gray and
Thien 1989 Altshuler 1999 Schuumlrch 2000 Ashman and King 2005) This discrepancy between
ants being ldquounwelcome guestsrdquo and their actual effects on plant reproduction requires
clarification Our goal here is to highlight the ways that flower-visiting ants may affect plant
reproduction both positively and negatively and to discern through a review of the literature
3
what impacts ants have on plant reproductive success First we discuss mechanisms by which
flower-visiting ants can directly and indirectly impact plant reproduction both through floral
damage and by mediating changes in pollinator or florivore behavior We end this section by
evaluating published research that collectively shows that flower-visiting ants have
predominantly neutral effects on plant fitness measures Next we discuss pollination by ants
focusing on the ldquoantibiotic hypothesisrdquo before describing how plants may prevent ants from
visiting flowers Lastly we discuss promising new avenues of research and highlight work that
still needs to be performed to give a more complete understanding of ants as flower visitors
1 Ant interactions with flowers and floral visitors
i) Damage to flowers
Ants can directly disrupt plant reproduction by damaging floral structures preventing either the
fertilization of ovules or the presentation of pollen Rarely ants forage on floral tissue itself for
example Puterbaugh (1998) found that Formica neorufibarbus ants chewed through the coronal
ring of Eritrichium aretiodes flowers to get lipids More often but still not commonly foraging
ants chew through reproductive tissue to access floral nectar This can cause sterilization of
whole flowers (Echium plantagineum Kirk 1984) or affect female function only if just styles are
damaged effectively creating male flowers (Polemonium viscosum Galen 1983) Antibiotic
secretions on ant integuments may also directly damage pollen grains but the fitness effects of
this are unclear (see 2 below)
Several species of Crematogaster and Allomerus ants sterilize their host plants by
attacking floral buds (Yu and Pierce 1998 Stanton et al 1999 Gaume et al 2005 Maleacute et al
2012 Table 11) These ants nest in ldquoant-plantsrdquo or ldquomyrmecophytesrdquomdashie plants that host ant
4
colonies in specialized structures (domatia) The sterilizing ants do not appear to consume floral
tissues (Yu and Pierce 1998) and two hypotheses may explain why ants destroy floral buds First
ant colony size is often limited by domatia availability on their host plant By destroying floral
buds ants can cause plants to divert resources from reproductive to vegetative growth increasing
domatia number and allowing for larger ant colonies (Yu amp Pierce 1998 Frederickson 2009)
Second when plant-ants must defend their colony from takeovers by other colonies they may
benefit from limiting access to their host plant By destroying buds that could develop into
flowers branches or leaves ants can prevent their host plant from contacting nearby plants
occupied by enemy ant colonies (Stanton et al 1999)
The impact of sterilization on lifetime plant fitness is unclear Ants may destroy all of
their host plantrsquos floral buds (Yu and Pierce 1998) completely preventing reproduction or only a
portion of them (Maleacute et al 2012) allowing some seed set However counter-intuitively this
sterilization behavior may provide long-term fitness benefits to plants (Frederickson 2009
Palmer et al 2010) Ant-plants often host several different ant species over their lifetimes some
of which are non-sterilizing If hosting a sterilizing species at a certain life stage increases
survival or growth relative to hosting a non-sterilizing species at that life stage plants may
increase lifetime fitness by largely forgoing current reproduction in favor of increasing future
reproduction (Palmer et al 2010) Why plants invest resources in producing flowers that are
quickly destroyed by ants is not known but the timing of flower destruction may be important If
ants attack flowers after anthers dehisce plants may still realize some male fitness via pollen
donation (Yu and Pierce 1998) Additionally if the cost of flower production is relatively minor
and ants sometimes ldquomissrdquo flowers (Edwards and Yu 2008) attempting limited reproduction
may be a better strategy than forgoing it entirely
5
ii) Competition with pollinators
Ant activity on flowers can dramatically alter pollinator behavior Ants can reduce the species
diversity frequency or duration of pollinator visits to flowers (Norment 1988 Tsuji et al 2004
Ness 2006 Junker et al 2007 Lach 2007 2008b Romero et al 2011 and see Chapter 2) These
changes are largely due to either exploitative competition (ie consumption of shared resources)
or interference competition (ie direct aggression) between ants and pollinators
a) Exploitative competition
Pollinators and ants frequently compete for floral nectar Flower-visiting ants are often classified
as nectar thieves (sensu Inouye 1980) because they typically enter flowers through the
ldquolegitimaterdquo opening and consume nectar but transfer no pollen (Wyatt 1980 Ness 2006
Chamberlain and Holland 2008) Ants can also act as primary nectar robbers chewing holes
through corollas to reach nectaries (Willmer and Corbet 1981 Koopowitz and Marchant 1998)
although they are more commonly secondary nectar robbers and use holes made by previous
robbers (Newman and Thomson 2005)
Like other flower visitors foraging ants will often leave nectar behind in flowers This
amount can be less (Bleil et al 2011) more (Fritz and Morse 1982) or similar to (Lach 2008a)
amounts left by other visitors Often ants are morphologically constrained from fully exploiting
nectar Unlike bees and butterflies ants do not have proboscises and must get close to nectar to
consume it Narrow corollas or nectar spurs can prevent ant access to some or all floral nectar
(Schemske 1980 Galen and Cuba 2001 and see below) Smaller ants may be better able to
6
access nectar (Newman and Thomson 2005 Agarwal and Rastogi 2007) but can be limited by
their crop size in the amount of nectar they can remove (Lach 2005) If foraging ants leave
enough nectar behind for a pollinator to recover the cost of visiting a flower ants may have
minimal effects on plant reproductive success (Maloof and Inouye 2000 Turner et al 2012)
Differences among ant species in behavior and diet also affect their consumption of floral
nectar Species vary in how many flowers they visit per inflorescence or per plant and in how
many ants will visit a flower simultaneously (Lach 2005 Junker et al 2007 Lach 2013)
Invasive antsmdashthought to be better at exploiting and defending resources than native ants
(Holway et al 2002)mdashoften recruit to flowers in large numbers sometimes several dozen or
more (Visser et al 1999 Hansen and Muumlller 2009) Overall many factors may influence how
heavily ants exploit floral nectar on plants including ant density plant height the spatial or
temporal distribution of floral nectar and the availability of alternative food resources for ants
(Cushman and Addicott 1991 Lach 2013)
Rarely ants and pollinators may compete for pollen We know of only a few studies that
have recorded ants harvesting pollen directly from anthers (Horskins and Turner 1999 Ness
2006 Byk and Del-Claro 2010) Other studies have found pollen grains in ants (eg Baroni
Urbani and de Andrade 1997 and see Chapter 3) but this may not indicate competition with
pollinators ants sometimes collect stray pollen grains from the environment (Creighton 1967)
One New World genus Cephalotes might be specially adapted for pollen consumption They
possess ldquoproventricular shieldsrdquo structures that may restrict solid food to the crop allowing
nestmates to exchange pollen grains via trophallaxis It is unclear if this structure evolved for this
purpose but Cephalotes are the most frequently documented palynivorous ants (eg Creighton
1967 Baroni Urbani and de Andrade 1997 Byk and Del-Claro 2010) How efficiently ants
7
digest pollen and whether ants like bees feed it to larvae as a protein source is unknown At
least two studies have found that ant larvae consume pollen providing some support for this idea
(Wheeler and Bailey 1920 and Chapter 3)
b) Interference competition
Ants are relatively unique among flower visitors in defending floral resources from competitors
(but see Willmer and Corbett 1981 Roubik 1982) This defense ranges from chance encounters
with ants attacking visitors when flower occupancy overlaps (Ashman and King 2005 Junker et
al 2007) to actively patrolling inflorescences or constructing protective galleries around
nectaries (Horvitz and Schemske 1984 Gaume et al 2005 Hansen and Muumlller 2009)
Interference competition even in the absence of exploitative competition is sufficient to alter
pollinator behavior (Chapter 2)
The strength and outcome of interference competition depends on the identities of the ant
and the pollinator Pollinators might not alter their visitation patterns in response to non-
aggressive ants (Junker et al 2007) but more antagonistic ants may elicit avoidance behavior
before or soon after the visitor lands (Willmer and Corbet 1982 Altshuler 1999 Schuumlrch et al
2000 Table 11) Pollinator size may help predict their reaction to ants because ants may deter
only small-bodied pollinators (Gonzaacutelvez et al 2012) It is not clear whether flower visitors view
ants as aggressive competitors or as potential predators rarely ants prey on pollinators (Schatz
and Wcislo 1999) but in most cases ants merely cause pollinators to relocate
How well pollinators recognize and avoid ant-visited flowers is unresolved Collectively
pollinators have lower visitation rates to flowers visited by ants (Romero et al 2011) This is
8
dependent on pollinator identity however dipterans do not appear to discriminate between ant-
visited and ant-free flowers while bees can identify and avoid ant-visited flowers Bees likely
use a combination of visual and olfactory cues to recognize ant-visited flowers They can
identify previous conspecific and some heterospecific insect visitors by the hydrocarbon
ldquofootprintsrdquo they leave during foraging (Stout and Goulson 2001) Bumblebees can detect ant
hydrocarbons although this has been shown only in the laboratory (Ballantyne and Willmer
2012 Chapter 2) No study has yet examined if bees can use sight alone to detect ants but
several suggest it (Junker et al 2007 Gonzaacutelvez et al 2012) Some bees may discriminate
between ant species when visiting flowers (eg Lach 2008b) which follows if not all ant-
pollinator interactions are equally costly The ability of bees to do so will affect how generalized
their avoidance behaviors will be If they can discriminate between species bees may avoid
specific flowers visited by more ldquocostlyrdquo ants However if they cannot bees might either avoid
all ant-visited flowers or none depending on how negative the average interaction with an ant is
iii) Interactions with flower and pollinator antagonists
Ants can defend the flowers they visit Numerous plant species have circumfloral or bracteal
nectaries (ie extrafloral nectaries around flowers) and ants attracted to these nectaries can
reduce bud ovule or flower predation increasing plant fitness (Inouye and Taylor 1979 Rico-
Gray and Thien 1989 Sugiura et al 2006) Although the presence of EFNs (extrafloral nectaries)
so close to floral nectaries (sometimes less than 1 cm Ness 2006) might increase discovery and
exploitation of floral nectar by ants only one study has investigated this hypothesis finding no
relationship between the proximity of EFNs to floral nectaries and antsrsquo use of floral nectar
(Galen 2005) Holes made by nectar robbers are functionally analogous to circumfloral nectaries
9
and can also attract ants that defend flowers (Newman and Thomson 2005) and nectar-thieving
ants can similarly prevent florivory in some systems (Lach 2007 Bleil et al 2011)
Ants can also reduce the abundance of flower-dwelling predators or parasites of
pollinators Ambush predators such as crab spiders often forage at flowers where they may
reduce pollinator visitation rates and plant fitness (Dukas 2001 Gonccedilalvez-Souza et al 2008)
Ants can decrease spider abundance and richness on plants (Rudgers 2004 Renault et al 2005
Nahas et al 2012) and some evidence suggests that they can do the same on flowers (Lach
2008) In select cases ants may also reduce the abundance of pollinator parasitoids (Holland et
al 2011) Whether ants increase plant reproductive success by removing pollinator antagonists
has not yet been examined but should be If ants create lsquoenemy-freersquo flowers pollinators might
prefer to land on flowers with ants
iv) Net effects
Table 11 compiles studies in which the authors recorded ants consuming floral rewards or
interacting with pollinators and measured some aspect of plant fitness (eg fruit or seed set
pollen deposition) We included only a single study per plant species unless subsequent studies
found conflicting results or measured the fitness of another plant sex Records of ant pollination
are included in a separate table (Table 12)
Table 11 reveals relatively few examples of flower-visiting ants negatively affecting
plant fitness given how costly ant visitation is assumed to be The net effect of ants on plant
reproductive success is actually quite variable with similar numbers of studies finding positive
negative or neutral effects of ant presence Surprisingly when ants do have negative effects it is
10
usually because they directly damage floral structures and not because they prevent pollinators
from visiting flowers and successfully transferring pollen Why are ants not more often negative
for plant reproduction when this is commonly assumed to be true
First ants may compensate for reducing pollinator visitation rates by improving
pollination quality As pollinators move between flowers on a plant they often deposit self-
pollen on stigmas (ie geitonogamy) In some plants with late-acting self-incompatibility ovules
fertilized by self-pollen are subsequently aborted reducing the number of seeds that can be
produced (Barrett 2002 and references therein) Geitonogamy can also reduce offspring viability
or vigor (de Jong et al 1993) and cause pollen discounting a reduction in the amount of pollen
available for outcrossing because of self-pollination (Barrett 2002) By competing with
pollinators ants might decrease rates of geitonogamous pollination if pollinators then visit fewer
sequential flowers within a plant Ants might also cause pollinators to fly further between plants
potentially increasing the genetic diversity of pollen arriving at stigmas Although such effects
have been reported for other floral larcenists (Maloof 2001 Irwin 2003) to our knowledge these
hypotheses have not been investigated in relation to ants
Second ants and pollinators do not always forage for the same resources possibly
allowing them to partition floral rewards If ants forage for nectar but not pollen and flowers can
be pollinated efficiently by visitors looking to gather mostly pollen and little or no nectar then
plant reproduction will not be affected by ants consuming floral nectar Junker et al (2010a)
found that honeybees foraged for both pollen and nectar in the absence of ants but reduced their
foraging on nectar but not pollen when ants could access flowers resulting in no difference in
fruit set between treatments Whether pollinators commonly adjust their foraging behaviors in
response to ants warrants further research
11
Third ants are somewhat unique among floral larcenists in that they often provide direct
benefits to the plants they visit In the studies listed in Table 11 ants sometimes decreased
herbivory on plants (Chamberlain and Holland 2008 Bleil et al 2011) or otherwise aided the
plant (eg ant presence repelling smaller less effective pollinators Gonzaacutelvez et al 2012) If the
positive effects of ants cancel or outweigh the negative effects of flower-visiting ants on
pollinators then ant visitation may be neutral or even positive for plant fitness
Fourth ants may impose fitness costs to plants not captured in the studies in Table 11
Nectar robbers have stronger negative effects when plants are pollen-limited (Burkle et al 2007)
but only one study in Table 11 assessed whether flowers would set more seed if they received
more pollen finding no evidence of pollen limitation (Lach 2008a Table 11) Even if ants
decrease pollinator visitation rates there is limited scope for ants to decrease plant reproductive
success when plants are not pollen-limited Furthermore nearly all the studies in Table 11
measured only female fitness Only three quantified aspects of male fitness (Wyatt 1980 Fritz
and Morse 1981 Galen and Geib 2007) and we know of only one other study in a system in
which ants occasionally pollinate that has also measured male fitness (Ashman 2000) All four
studies measured pollen or pollinia removal rates and none found any effect of flower-visiting
ants on male fitness However whether other measures of male fitness (eg seeds sired pollen
deposition patterns) would reveal similar or different effects needs investigation
Finally although floral defenses against ants (see 3 below) may prevent us from
observing the negative effects of ants on plant reproductive success we consider this unlikely
Several studies have examined interactions between ants and plants that share little or no
coevolutionary history usually in the context of ant invasions Although invasive ants often
recruit to flowers in larger numbers than native ants they do not appear to reduce plant
12
reproductive success (Blancafort and Goacutemez 2005 Lach 2007 Lach 2008a but see Hansen and
Muumlller 2009) And in Hawaii where angiosperms evolved for millions of years in the absence of
ants and are thought to have secondarily lost floral defenses against ants (Junker et al 2011a)
there is no evidence that the preponderance of recently introduced flower-visiting ants negatively
affects plant fitness (Junker et al 2010a Bleil et al 2011)
2 Ants as pollinators
Ants may directly benefit plant reproduction by acting as pollinators but this is a relatively rare
occurrence Ants have long been considered poor pollinators (Kerner 1878) Initially this was
based on morphological and behavioral differences between ants and recognized pollinators
Worker ants are wingless and often relatively hairless making them poor pollen dispersers As
well because many ants exhibit Ortstreue (literally ldquoplace fidelityrdquo) ants were thought to return
to the same branch or flower repeatedly effecting no cross pollination (Houmllldobler and Wilson
1990) Though largely true these traits do not necessarily preclude ants from acting as
pollinators (Table 12) Although they commonly have restricted foraging ranges (Houmllldobler and
Wilson 1990) some ants can effect outcrossing (eg Ashman and King 2005 Carvalheiro 2008)
and many ants are as hairy if not hairier than their pollinating counterparts
In the late 1970s the ldquoantibiotic hypothesisrdquo was put forward to explain the rarity of ant
pollination Ants use antibiotic secretions to protect themselves against the fungal and bacterial
pathogens common in their nests and environs Iwanami and Iwadare (1978) found that one of
these compounds myrmicacin also greatly reduces pollen germination rates Subsequently
Beattie et al (1984 1985) found that short periods of contact (le 30 minutes) with an antrsquos
integument caused pollen grains to fail to germinate Some of these pollen-killing compounds
13
were traced back to the ant metapleural gland which produces hygienic secretions (Houmllldobler
and Wilson 1990) although contact with ants lacking metapleural glands can also reduce pollen
viability (Beattie et al 1985 Dutton and Frederickson 2012) Ant mandibular or anal gland
secretions and potentially ant venom may likewise defend ants against pathogens and have
similar effects on pollen germination (Beattie et al 1985 Graystock and Hughes 2011)
Nonetheless ant pollination has been documented repeatedly There are at least 18 cases
in which ant pollination has been shown experimentally and five others in which observations
strongly suggest it (Table 12) The majority of these plants fit the ldquoant pollination syndromerdquo
originally proposed by Hickman (1974) dense stands of short plants with small flowers and
easily accessible nectaries Typically ant-pollinated plants are associated with hot and dry
climates (principally in the Mediterranean) but dry alpine environments may also favor the
evolution of ant pollination (eg Svensson 1985 Puterbaugh 1998 Schiestl and Glaser 2011) In
most cases ants appear to be the sole or major pollinator of the plant in question (Hickman 1974
Peakall et al 1990 de Vega et al 2009) although ants sometimes make only supplementary
contributions to pollination (Schuumlrch et al 2000 Ashman and King 2005) This suggests that co-
evolution between ants and plants may not be necessary for ants to be useful pollinatorsmdashwhich
is surprising given the potential negative effects of ants on plant reproduction
Ant-pollinated plants cope with ant secretions in a variety of ways Several plant species
may simply tolerate pollen damage (Gόmez and Zamora 1992 de Vega et al 2009) apparently
possessing no overt adaptations to prevent it These plants might receive enough pollen to
overcome reduced pollen viability Other plants may place pollen on ant body parts thought to
have fewer secretions thereby potentially escaping damage (eg on ant legs Ramsey 1995) or
attach pollen to ants with specialized structures avoiding direct contact with the ant integument
14
(Peakall et al 1990) It is not known whether pollen evolves resistance to ant secretions or
whether plants deposit pollen on ants which have less disruptive secretions although these
possibilities have been suggested elsewhere (eg Garciacutea et al 1995 Ramsey 1995)
One key question regarding the effects of ant secretions on pollen grains urgently needs
to be resolved Several studies have suggested that the pollen-killing secretions of ants may have
contributed to the evolution of floral defenses against ants (eg Wagner 2000 Galen and Geib
2007) For this to be true ants must decrease male fitness by damaging pollen grains before they
leave flowers and are deposited on receptive stigmas Only two studies have investigated the
effects of ant secretions on pollen still in anthers (Wagner 2000 Galen and Butchard 2003) and
both constrained ants unnaturally to flowers for significantly longer than ants would normally
stay on flowers Although these studies found strong reductions in pollen vigor it is unclear if
ant secretions decrease male fitness under natural ant visitation conditions
3 Floral defenses against ants
Plants are not passive recipients of flower visitors and have some ability to ldquochooserdquo which
organisms can access floral rewards Flowers resist ants using both structural and chemical traits
More thorough investigation is needed to determine whether these traits evolved to prevent or
minimize the negative effects of ants on plant reproductive success (ie whether these traits can
truly be considered defenses against ants see 4ia below) It is unlikely however that ants
experience reciprocal selection to circumvent floral resistance traits because the fitness benefits
to ants of accessing flowers are probably small Thus there may be little scope for
coevolutionary arms races between floral defenses against ants and ant counter-adaptations
15
i) Structural resistance traits
a) Restricting ant access
Numerous plant species have physical structures preventing ant access to flowers Thin pedicels
can limit antsrsquo gripping abilities and make ants easier to dislodge when flowers are moved by
wind (Gaume et al 2005) and waxy secretions can render stems too slippery for ants to climb
(Harley 1991) Structures surrounding flowers such as water moats or dense hairs may similarly
keep ants from accessing floral rewards (Willmer et al 2009) Many such traits function as broad
filters and prevent not only ants but most crawling insects from reaching flowers Thus it is
difficult to determine whether ants florivores or both acted as selective agents favoring the
evolution of physical barriers to flowers
Flower shape may also reduce ant-pollinator conflict Long narrow corollas or nectar
spurs can prevent all but the smallest ants from accessing floral nectaries (Bluumlthgen et al 2004
Agarwal and Rastogi 2007) while still allowing long-tongued pollinators access to rewards
Selection to exclude nectar-thieving ants in such a manner however may be counteracted by
pollinator preferences for wider corollas that allow easier flower handling (Galen and Cuba
2001) Additionally these types of flowers may be more prone to robbery (Irwin and Maloof
2002) limiting their usefulness in preventing nectar larceny
b) Separation in space or time
Plants may separate floral rewards and ants temporally or spatially to minimize ant impacts on
plant reproduction Several authors have proposed that myrmecophytes have adapted to the
16
sterilization or otherwise detrimental activities of ants by locating domatia or food rewards on
different branches than flowers (Izzo and Vasconcelos 2002 Raine et al 2002 Edwards and Yu
2008) Temporal separation of ant and pollinator activities could likewise reduce conflict though
evidence is limited Some acacia flowers dehisce during periods of low ant activity (Willmer and
Stone 1997 Nicklen and Wagner 2006) but it is unclear if this is in response to ants
temperature or pollinator activity Timing of nectar secretion may similarly reduce ant-pollinator
competition If ants are active prior to pollinators they may deplete floral nectar and reduce
pollinator visitation rates (Schaffer et al 1984) However if nectar is secreted when ants are
inactive pollination may occur unimpeded (Norment 1988) This temporal separation could
favor pollinators that are active when ants are not (eg Wyatt and Morse 1981) but this is
poorly documented Further studies particularly focusing on flowers with the potential for
nocturnal and diurnal pollination may provide more examples
c) Extrafloral nectaries (EFNs)
One hypothesis put forward to explain the evolution of EFNs proposes that EFNs ldquodistractrdquo ants
from consuming floral nectar (Kerner 1878 Wagner and Kay 2002) Wagner and Kay (2002)
tested this hypothesis by constructing model plants using artificial nectaries assigned at random
to represent either floral or extrafloral nectaries They found that the presence of EFNs could
reduce ant exploitation of floral nectar However as highlighted by Galen (2005) this design is
closer to a plant making a surplus of flowers some of which will avoid ant visitation if visitation
rates increase sublinearly with increasing flower number Producing extra flowers to escape ant
visitation appears to be a viable strategy in some systems (Ashman and King 2005 Galen 2005)
However Galen (2005) found that applying honey (simulating extrafloral nectar) to P viscosum
17
leaves had the opposite effect and increased ant visits to flowers Only two studies both in
Pachycereus schottii have found evidence for the distraction hypothesis under field conditions
(Chamberlain and Holland 2008 Holland et al 2011)
There is limited empirical support for the distraction hypothesis but abundant evidence
that EFNs attract ants that defend plants against herbivores (reviewed in Rosumek et al 2009)
To function as attractants the presence of EFNs must increase ant abundance on plants
However because of the increased numbers attracted EFNs would not be expected to reduce the
abundance of ants on nectar rewards or be able to satiate the ants recruited Only if ant
abundance stops increasing at a reward density much lower than that available on the plant
would EFNs serve to distract ants In this case the amount of rewards may overwhelm ants
leaving some flowers underexploited However even in P schottii ants still visited flowers
(Chamberlain and Holland 2008) In cases where ant abundance would continue to increase with
additional rewards it is unlikely that EFNs function as distractions
ii) Chemical resistance traits
a) Toxic nectar
One of the earliest proposed floral defenses against ants was toxic nectar (Janzen 1977)
However studies testing Janzenrsquos (1977) hypothesis uncovered only a very few isolated
examples (eg Catalpa speciosa Stephenson 1981 Crinum erubescens Guerrant and Fiedler
1981) in short most floral nectar is not toxic or repellent to ants (Baker and Baker 1978
Schubart and Anderson 1978) In fact ants sometimes prefer floral nectar over solutions with
equivalent sugar concentrations (Koptur and Truong 1998)
18
In contrast floral nectar can repel other flower visitors (eg birds Johnson et al 2006
lepidopterans Kessler and Baldwin 2007) Repellency is often caused by plant secondary
metabolites which may occur in nectar as a by-product of their production elsewhere in the
plant where they function as defenses against herbivory (Adler 2001) Alternatively toxic or
repellent nectar may be adaptive repellent nectar may promote effective pollination by reducing
the amount of reward any one pollinator takes spreading nectar over several pollinator visits
(Kessler and Baldwin 2007) or it may function as a filter selecting for specific pollinators
(Johnson et al 2006) The latter may explain why repellent nectar is not more common
legitimate pollinators in addition to floral larcenists may be deterred from visiting flowers with
repellent nectar This may be particularly true of nectars that would repel ants which are closely
related to bees nectar distasteful to ants may often also be distasteful to bees and those nectars
acting as a filter for bees may still be palatable to ants For example Prŷs-Jones and Willmer
(1992) found that the ammonia-containing nectar of Lathraea clandestina is readily consumed
by both bees and ants even though it deters birds that act as nectar thieves Although floral
nectar itself rarely repels ants Guerrant and Fielder (1981) found that the presence of macerated
petals within nectar can often keep ants away Alkaloids in petals may be responsible and might
explain why ants seldom act as primary nectar robbers these secondary compounds may prevent
ants from chewing through corollas
b) Floral volatiles
Repellent floral volatiles appear to be a more widespread form of chemical resistance against
ants These were reviewed recently by Willmer et al (2009) and will be discussed here only
briefly Numerous plant taxa produce floral scents repellent to ants including myrmecophilous
19
myrmecophytic and non-ant associated plants in both temperate and tropical ecosystems
(Willmer et al 2009 Junker et al 2011a Ballantyne and Willmer 2012) In three large studies
floral volatiles that repel ants were detected in about a third of all plants tested (Willmer et al
2009 Junker et al 2011a Ballantyne and Willmer 2012) Pollen is often the source of ant-
repellent volatile compounds (Willmer et al 2009 Ballantyne and Willmer 2012) but they can
be emitted from other floral parts including petals That pollen is often the source of repellence
could suggest that preventing ant visitation may be more important in protecting male than
female function Additionally because repellence would decrease as pollen is removed from
anthers ants may return and defend flowers more quickly than if repellent chemicals were in
longer-lasting structures (eg petals) In some cases floral volatiles are thought to mimic ant
alarm pheromones (Willmer et al 2009) However floral scents elicit inconsistent reactions
across ant species ranging from repellence to attraction (Agarwal and Rastogi 2007 Ballantyne
and Willmer 2012)
Producing structural or chemical resistance traits may be costly for plants Native
Hawaiian plants which have little recent coevolutionary history with ants appear to have
secondarily lost floral ant repellents (Junker et al 2011a) Both Willmer et al (2009) and Junker
et al (2011a) found that highly chemical repellent flowers did not have strong physical defenses
and vice versa suggesting trade-offs between the two (but see Ballantyne and Willmer 2012)
Like toxic nectar floral ant repellents might sometimes deter both pollinators and ants For
example Galen et al (2011) found that 2-phenylethanol in P viscosum floral scent repels both
flower-damaging F neorufibarbis ants and the plantrsquos main pollinator Bombus kirbyellus
Interestingly four recent studies have found that floral scents can also attract ants
(Agarwal and Rastogi 2007 Edwards and Yu 2008 Schiestl and Glaser 2011 Gonzaacutelvez et al
20
2012) One study (Schiestl and Glaser 2011) has shown that floral scents attract ants to a
potentially ant-pollinated plant and such systems may provide many more examples
4 Future directions
We have highlighted areas in need of further investigation throughout the review Here we focus
on several questions we consider high priorities for future research
i) Floral defenses against ants
a) Do floral resistance traits increase plant fitness
The existence ant-repellent floral traits is often taken as evidence that ants negatively affect plant
reproductive success However despite the abundance and variety of mechanisms excluding ants
from flowers few studies show that ants reduce plant fitness when they visit flowers (Table 11)
This absence is particularly apparent with repellent floral volatiles which may be the most
widespread floral resistance trait (Willmer et al 2009 Junker et al 2011a Ballantyne and
Willmer 2012) To our knowledge only one study has shown that floral ant repellents benefit
plants with repellent P viscosum flowers avoiding damaging ant visits (Galen et al 2011)
Research is urgently needed to investigate if these traits increase plant fitness by excluding ants
or if ant visitation is inconsequential More studies taking advantage of standing variation in
traits (Galen et al 2011) or creating it artificially (eg Junker et al 2010b) will allow us to
distinguish between the two
21
b) If ant visitation is often not costly what caused the evolution of repellent traits and
what maintains them Similarly are these repellent traits truly defenses against ant
visitation or are they generalized defenses against a range of floral antagonists
Ant-repellent floral traits are widespread and their production may be costly (Willmer et al
2009 Junker et al 2011a) The studies in Table 11 however indicate that flower-visiting ants
often do not disrupt plant reproduction particularly outside of myrmecophytes What then
selected for these traits One possibility is that these repellents are not directed solely at ants
Instead a community of antagonistic flower visitors may exert diffuse selection on plants
thereby selecting for and maintaining these traits Structural modifications may repel crawling
insects while chemical repellents such as floral volatiles may repel both flying and non-flying
insects (Junker et al 2011b) How much overlap there is in floral volatiles that repel both ants and
other antagonists needs investigation Until we examine how specific these defenses are to ants
we risk overstating the importance of ants in their evolution
ii) Do ants alter pollen movement patterns or reduce male fitness
We are aware of only four studies that have measured if ant visitation impacts male fitness
(Wyatt 1980 Fritz and Morse 1981 Ashman 2000 Galen and Geib 2007) While none of these
studies found an effect all measured only amounts of pollen or pollinia removed However this
measure does not adequately encompass male fitness alterations to pollen movement patterns
and eventual deposition location may be more important to male function than changes in
removal rates If as suggested by some studies (Altshuler 1999 Lach 2007) ants increase
pollinator relocation rates they may decrease pollen discounting increasing male fitness
However if this increased relocation reduces overall pollen export rates ant competition with
22
pollinators may depress male function Studies using pollen-tracking methods such as fluorescent
dyes will allow us to see how ants change pollen movement patterns and methods like paternity
analysis will reveal how competition between ants and pollinators alters male fitness
iii) Coevolution between ants and plants do myrmecophytes exhibit stronger more
specific defenses against ants
The floral traits of myrmecophytes are most likely to coevolve with ants because all plant
reproduction occurs in the presence of ants Their floral defenses would thus be expected to be
both stronger and more specific to their resident ants than those other plants visited by an array
of more opportunistic ant species Some evidence supports this Willmer et al (2009) found that
resident ants of acacias were repelled by flowers with more aggressive ant species showing
stronger repellence than less aggressive ants Additionally ants not normally associated with
acacias did not respond to acacia floral volatiles More studies are needed in other systems and
should take advantage of phylogenetic approaches examining floral traits in closely related
myrmecophytic and non-myrmecophytic plant species to determine whether ants commonly
select for floral defenses against ants in myrmecophytes
Acknowledgements
For comments and discussion we thank the Frederickson lab particularly Eric Youngerman
Lina Arcila Hernandez and Kyle Turner as well as Alison Parker and Jordan Pleet MEF
acknowledges financial support from an NSERC Discovery Grant a Connaught New Researcher
23
Award an Ontario Ministry of Economic Development and Innovation Early Researcher Award
and the University of Toronto ARC was supported by an Ontario Graduate Scholarship and
Sigma Xi
24
References
Adler L S 2001 The ecological significance of toxic nectar Oikos 91409ndash420
Agarwal V M and N Rastogi 2007 Role of floral repellents in the regulation of flower visits
of extrafloral nectary-visiting ants in an Indian crop plant Ecological Entomology 3359ndash
65
Altshuler D L 1999 Novel interactions of non-pollinating ants with pollinators and fruit
consumers in a tropical forest Oecologia 119600ndash606
Ashman TL 2000 Pollinator selectivity and its implications for the evolution of dioecy and
sexual dimorphism Ecology 812577-2591
Ashman TL and E A King 2005 Are flower-visiting ants mutualists or antagonists A study
in a gynodioecious wild strawberry American Journal of Botany 92891ndash5
Ballantyne G and P Willmer 2012 Floral visitors and ant scent marks noticed but not used
Ecological Entomology 37402-409
Ballantyne G and P Willmer 2012 Nectar theft and floral ant-repellence a link between
nectar volume and ant-repellent traits PloS One 7e43869
Baker H G and I Baker 1978 Ants and flowers Biotropica 1080
Barrett S C H 2002 Sexual interference of the floral kind Heredity 88154ndash159
Baroni Urbani C and M L de Andrade 1997 Pollen eating storing and spitting by ants
Naturwissenschaften 84256ndash258
25
Beattie A J C Turnbull R B Knox and E G Williams 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany
71421ndash426
Beattie A J C Turnbull T Hough S Jobson and R B Knox 1985 The vulnerability of
pollen and fungal spores to ant secretions evidence and some evolutionary implication
American Journal of Botany 72606ndash614
Blancafort X and C Goacutemez 2005 Consequences of the Argentine ant Linepithema humile
(Mayr) invasion on pollination of Euphorbia characias (L) (Euphorbiaceae) Acta
Oecologica 2849ndash55
Bleil R N Bluumlthgen and R R Junker 2011 Ant-plant mutualism in Hawailsquoi Invasive ants
reduce flower parasitism but also exploit floral nectar of the endemic shrub Vaccinium
reticulatum (Ericaceae) Pacific Science 65291ndash300
Bluumlthgen N G Gottsberger and K Fiedler 2004 Sugar and amino acid composition of ant-
attended nectar and honeydew sources from an Australian rainforest Austral Ecology
29418ndash429
Burkle L A R E Irwin and D A Newman 2007 Predicting the effects of nectar robbing on
plant reproduction implications of pollen limitation and plant mating system American
Journal of Botany 941935ndash1943
Byk J and K Del-Claro 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333ndash38
26
Caballero P C G Ossa W L Gonzaacuteles C Gonzaacutelez-Browne G Astorga M M Muruacutea and
R Medel 2013 Testing non-additive effects of nectar-robbing ants and hummingbird
pollination on the reproductive success of a parasitic plant Plant Ecology 214633ndash640
Carvalheiro L G E R M Barbosa and J Memmott 2008 Pollinator networks alien species
and the conservation of rare plants Trinia glauca as a case study Journal of Applied
Ecology 451419ndash1427
Chamberlain S A and J N Holland 2008 Density-mediated context-dependent consumer-
resource interactions between ants and extrafloral nectar plants Ecology 891364ndash1374
Creighton W S 1967 Studies on free colonies of Cryptocerus texanus Satschi (Hymenoptera
Formicidae) Psyche 7434ndash42
Cursach J and J Rita 2011 Implications of the reproductive biology of the narrow endemic
Naufraga balearica (Apiaceae) for its conservation status Plant Systematics and Evolution
298581ndash596
Cushman JH and Addicott JF In Huxley CR and DF Cutler (eds) 1st edition Ant-plant
interactions Oxford University Press Oxford
de Jong T J N M Waser and P G Klinkhamer 1993 Geitonogamy the neglected side of
selfing Trends in Ecology and Evolution 8321ndash325
de Vega C M Arista P L Oritz C M Herrera and S Talavera 2009 The ant-pollination
system of Cytinus hypocistis (Cytinaceae) a Mediterranean root holoparasite Annals of
Botany 4517ndash8
27
Dukas R 2001 Effects of perceived danger on flower choice by bees Ecology Letters 4327ndash
333
Dutton E M and M E Frederickson 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561ndash569
Edwards D P and D W Yu 2008 Tolerating castration by hiding flowers in plain sight
Behavioral Ecology and Sociobiology 6395ndash102
Frederickson M E 2009 Conflict over reproduction in an ant-plant symbiosis why Allomerus
octoarticulatus ants sterilize Cordia nodosa trees The American Naturalist 173675ndash81
Fritz R S and D H Morse 1981 Nectar parasitism of Asclepias syriaca by ants effect on
nectar levels pollinia insertion pollinaria removal and pod production Oecologia 50316ndash
319
Galen C 1983 The effects of nectar thieving ants on seedset in floral scent morphs of
Polemonium viscosum Oikos 41245ndash249
Galen C 2005 Catching ants with honey an experimental test of distraction and satiation as
alternative modes of escape from flower-damaging ants Oecologia 14480ndash87
Galen C and B Butchart 2003 Ants in your plants effects of nectar-thieves on pollen fertility
and seed-siring capacity in the alpine wildflower Polemonium viscosum Oikos 101521ndash
528
Galen C and J Cuba 2001 Down the tube pollinators predators and the evolution of flower
shape in the alpine skypilot Polemonium viscosum Evolution 551963-1971
28
Galen C and J C Geib 2007 Density-dependent effects of ants on selection for bumble bee
pollination in Polemonium viscosum Ecology 881202ndash1209
Galen C R Kaczorowski S L Todd J Geib and R A Raguso 2011 Dosage-dependent
impacts of a floral volatile compound on pollinators larcenists and the potential for floral
evolution in the alpine skypilot Polemonium viscosum The American Naturalist 177258ndash
272
Garciacutea M B R J Antor and X Espadaler 1995 Ant pollination of the palaeoendemic
dioecious Borderea pyrenaica (Dioscoreaceae) Plant Systematics and Evolution 19817ndash
27
Gaume L M Zacharias and R M Borges 2005 Ant ndash plant conflicts and a novel case of
castration parasitism in a myrmecophyte Evolutionary Ecology Research 7435ndash452
Graystock P and W O H Hughes 2011 Disease resistance in a weaver ant Polyrhachis
dives and the role of antibiotic-producing glands Behavioral Ecology and Sociobiology
652319ndash2327
Gόmez J M 2000 Effectiveness of ants as pollinators of Lobularia maritima effects on main
sequential fitness components of the host plant Oecologia 12290ndash97
Gόmez J M and R Zamora 1992 Pollination by ants consequences of the quantitative effects
on a mutualistic system Oecologia 91410ndash418
Gόmez J M R Zamora J A Hόdar and D Garciacutea 1996 Experimental study of pollination
by ants in Mediterranean high mountain and arid habitats Oecologia 105236ndash242
29
Gonccedilalves-Souza T P M Omena J C Souza and G Q Romero 2008 Trait-mediated effects
on flowers artificial spiders deceive pollinators and decrease plant fitness Ecology
892407ndash2413
Gonzaacutelvez F G L Santamariacutea R T Corlett and M A Rodriacuteguez-Gironeacutes 2012 Flowers
attract weaver ants that deter less effective pollinators Journal of Ecology 10178ndash85
Guerrant E O and P L Fiedler 1981 Flower defenses against nectar-pilferage by ants
Biotropica 1325ndash33
Harley R 1991 The greasy pole syndrome In Huxley CR and DF Cutler (eds) 1st edition
Ant-plant interactions Oxford University Press Oxford
Hansen D M and C B Muumlller 2009 Invasive ants disrupt gecko pollination and seed
dispersal of the endangered plant Roussea simplex in Mauritius Biotropica 41202ndash208
Hickman J C 1974 Pollination by ants a low-energy system Science 1841290ndash1292
Holland J N S a Chamberlain and T E X Miller 2011 Consequences of ants and
extrafloral nectar for a pollinating seed-consuming mutualism ant satiation floral
distraction or plant defense Oikos 120381ndash388
Houmllldobler B and Wilson E O 1990 The Ants 1st Edition Harvard University Press
Cambridge
Holway DA L Lach AV Suarez ND Tsutsui and TJ Case 2002 The causes and
consequences of ant invasions Annual Review of Ecology and Systematics 33181-233
30
Horskins K and Turner VB 1999 Resource use and foraging patterns of honeybees Apis
mellifera and native insects on flowers of Eucalyptus costata Australian Journal of
Ecology 24221-227
Horvitz C C and D W Schemske 1984 Effects of ants and an ant-tended herbivore on seed
production of a neotropical herb Ecology 651369ndash1378
Inouye DW 1980 The terminology of floral larceny Ecology 611251-1253
Inouye DW and O R J Taylor 1979 A temperate region plant-ant-seed predator system
consequences of extra floral nectar secretion by Helianthella quinquenervis Ecology 601ndash
7
Irwin RE 2003 Impact of nectar robbing on estimates of pollen flow conceptual predictions
and empirical outcomes Ecology 84485-495
Irwin RE and Maloof JE 2002 Variation in nectar robbing over time space and species
Oecologia 133525-533
Iwanami Y and T Iwadare 1978 Inhibiting effects of myrmicacin on pollen growth and pollen
tube mitosis Botanical Gazette 13942ndash45
Izzo TJ and Vasconcelos HL 2002 Cheating the cheater domatia loss minimizes the effects
of ant castration in an Amazonian ant plant Oecologia 133200-205
Janzen D H 1977 Why donrsquot ants visit flowers Biotropica 9252
Johnson SD Hargreaves AL and Brown M 2006 Dark bitter-tasting nectar functions as a
filter of flower visitors in a bird-pollinated plant Ecology 872709-2716
31
Junker R A Y C Chung and N Bluumlthgen 2007 Interaction between flowers ants and
pollinators additional evidence for floral repellence against ants Ecological Research
22665ndash670
Junker R R R Bleil C C Daehler and N Bluumlthgen 2010a Intra-floral resource partitioning
between endemic and invasive flower visitors consequences for pollinator effectiveness
Ecological Entomology 35760ndash767
Junker R R and N Bluumlthgen 2010b Floral scents repel facultative flower visitors but attract
obligate ones Annals of botany 105777ndash82
Junker R R C C Daehler S Doetteri A Keller and N Bluumlthgen 2011a Hawaiian ant ndash
flower networks nectar-thieving ants prefer undefended native over introduced plants with
floral defenses Ecological Monographs 81295ndash311
Junker R R J Gershenzon and S B Unsicker 2011b Floral odor bouquet loses its ant
repellent properties after inhibition of terpene biosynthesis Journal of Chemical Ecology
371323ndash31
Kawakita A and M Kato 2002 Floral biology and unique pollination system of root
holoparasites Balanophora kuroiwai and B tobiracola (Balanophraceae) American
Journal of Botany 891164ndash1170
Koopowitz H and TA Marchant 1998 Postpollination nectar reabsorption in the African
epiphyte Aerangis verdickii (Orchidaceae) American Journal of Botany 85508-512
Kirk W D J 1984 Ecological studies on Thrips imaginis Bagnall (Thysanoptera) in flowers of
Echium plantagineum L in Australia Australian Journal of Ecology 99ndash18
32
Kerner A 1878 Flowers and their unbidden guests C Kegan Paul London
Kessler D and I T Baldwin 2007 Making sense of nectar scents the effects of nectar
secondary metabolites on floral visitors of Nicotiana attenuata The Plant Journal 49840ndash
54
Koptur S and N Truong 1998 Facultative ant-plant interactions nectar sugar preferences of
introduced pest species in South Florida Biotropica 30179-189
Lach L 2005 Interference and exploitation competition of three nectar-thieving invasive ant
species Insectes Sociaux 52257ndash262
Lach L 2007 A mutualism with a native membracid facilitates pollinator displacement by
Argentine ants Ecology 881994ndash2004
Lach L 2008a Argentine ants displace floral arthropods in a biodiversity hotspot Diversity and
Distributions 14281ndash290
Lach L 2008b Floral visitation patterns of two invasive ant species and their effects on other
hymenopteran visitors Ecological Entomology 33155ndash160
Lach L 2013 A comparison of floral resource exploitation by native and invasive Argentine
ants Arthropod-Plant Interactions 7177-190
Luo C W K Li X M Chen Z Y Huang and K U N Li 2012 Ants contribute significantly
to the pollination of a biodiesel plant Jatropha curcas Environmental Entomology
411163ndash1168
33
Maleacute PJ G C Leroy A Dejean A Quilichini and J Orivel 2011 An ant symbiont directly
and indirectly limits its host plantrsquos reproductive success Evolutionary Ecology 2655ndash63
Maloof J E 2001 The effects of a bumble bee nectar robber on plant reproductive success and
pollinator behavior American Journal of Botany 881960ndash1965
Maloof J E and D W Inouye 2000 Are nectar robbers cheaters or mutualists Ecology
812651ndash2661
Moreau CS CD Bell R Vila SB Archibald and NE Pierce 2006 Phylogeny of the ants
diversification in the age of the angiosperms Science 312101-104
Nahas L M O Gonzaga and K Del-Claro 2012 Emergent impacts of ant and spider
interactions herbivory reduction in a tropical savanna tree Biotropica 44498ndash505
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506ndash514
Newman D A and J D Thomson 2005 Interactions among nectar robbing floral herbivory
and ant protection in Linaria vulgaris Oikos 110497ndash506
Nicklen E F and D Wagner 2006 Conflict resolution in an ant-plant interaction Acacia
constricta traits reduce ant costs to reproduction Oecologia 14881ndash7
Norment C J 1988 The effect of nectar-thieving ants on the reproductive success of Frasera
speciosa (Gentianaceae) American Midland Naturalist 120331ndash336
34
Orivel J L Lambs P-J G Maleacute C Leroy J Grangier T Otto A Quilichini et al 2011
Dynamics of the association between a long-lived understory myrmecophyte and its
specific associated ants Oecologia 165369ndash76
Palmer T M D F Doak M L Stanton J L Bronstein E T Kiers T P Young J R
Goheen et al 2010 Synergy of multiple partners including freeloaders increases host
fitness in a multispecies mutualism Proceedings of the National Academy of Sciences
10717234ndash9
Peakall R A J Beattie and S H James 1987 Pseudocopulation of an orchid by male ants a
test of two hypotheses accounting for the rarity of ant pollination Oecologia 73522ndash524
Peakall R and A J Beattie 1989 Pollination of the orchid Microtis parviflora R Br by
flightless worker ants Functional Ecology 3515ndash522
Peakall R C J Angus A J Beattie 1990 The significance of ant and plant traits for ant
pollination in Leporella fimbriata pollination Oecologia 84457ndash460
Puterbaugh M N 1998 The roles of ants as flower visitors experimental analysis in three
alpine plant species Oikos 8336ndash46
Prŷs-Jones OE and Willmer PG 1992 The biology of alkaline nectar in purple toothwort
(Lathraea clandenstina) ground level defenses Biological Journal of the Linnean Society
45373-388
Raine NE Willmer P and Stone GN 2002 Spatial structuring and floral avoidance
behaviour present in ant-pollinator conflict in a Mexican ant-acacia Ecology 833086-
3096
35
Ramsey M 1995 Ant pollination of the perennial herb Blandfordia grandiflora (Liliaceae)
Oikos 74265ndash272
Renault CK LM Buffa and Delfino MA 2005 An aphid-ant interaction effects on
different trophic levels Ecological Research 2071-74
Rico-Gray V and L B Thien 1989 Effect of different ant species on reproductive fitness of
Schomburgkia tibicinis (Orchidaceae) Oecologia 81487ndash489
Romero G Q P A P Antiqueira and J Koricheva 2011 A meta-analysis of predation risk
effects on pollinator behaviour PLoS One 6e20689
Rosumek FB FAO Silveira F de S Neves NP de U Barbosa L Diniz Y Oki F Pezzini
et al 2009 Ants on plants a meta-analysis of the role of ants as plant biotic defenses
Oecologia 160537ndash549
Roubik D W 1982 The ecological impact of nectar-robbing bees and pollinating
hummingbirds on a tropical shrub Ecology 63354ndash360
Rudgers J A 2004 Enemies of herbivores can shape plant traits selection in a facultative ant-
plant mutualism Ecology 85192ndash205
Schatz B and WT Wcislo 1999 Ambush predation by the ponerine ant Ectatomma ruidum
Roger (Formicidae) on a sweat bee Lasioglossum umbripenne (Halictidae) in Panama
Journal of Insect Behavior 12641-663
36
Schaffer W M D W Zeh S L Buchmann S Kleinhaus M V Schaffer and J Antrim
1983 Competition for nectar between introduced honey bees and native North American
bees and ants Ecology 64564ndash577
Schemske D W 1980 The evolutionary significance of extrafloral nectar production by Costus
woodsonii (Zingiberaceae) an experimental analysis of ant protection Journal of Ecology
68959ndash967
Schiestl F P and F Glaser 2011 Specific ant-pollination in an alpine orchid and the role of
floral scent in attracting pollinating ants Alpine Botany 1221ndash9
Schubart HOR AB Anderson 1978 Why donrsquot ants visit flowers A response to D H
Janzen Biotropica 10310-311
Schuumlrch S M Pfunder and B A Roy 2000 Effects of ants on the reproductive success of
Euphorbia cyparissias and associated pathogenic rust fungi Oikos 16ndash12
Stanton ML Palmer TM Young TP Evans A and Turner ML 1999 Sterilization and
canopy modification of swollen thorn acacia tree by a plant-ant Nature 401 578-581
Stephenson AG 1981 Toxic nectar deters nectar thieves of Catalpa speciosa American
Midland Naturalist 105381-383
Stout J C and D Goulson 2001 The use of conspecific and interspecific scent marks by
foraging bumblebees and honeybees Animal Behaviour 62183ndash189
Sugiura S T Abe and S Makino 2006 Loss of extrafloral nectary on an oceanic island plant
and its consequences for herbivory American Journal of Botany 93491ndash495
37
Svensson L 1985 An estimate of pollen carryover by ants in a natural population of
Scleranthus perennis L (Caryophyllaceae) Oecologia 66373ndash377
Tsuji K A Hasyim and K Nakamura 2004 Asian weaver ants Oecophylla smaragdina and
their repelling of pollinators Ecological Research 19669ndash673
Turner R C J J Midgley P Barnard R E Simmons and S D Johnson 2012 Experimental
evidence for bird pollination and corolla damage by ants in the short-tubed flowers of
Erica halicacaba (Ericaceae) South African Journal of Botany 7925ndash31
Visser D M G Wright and J H Giliomee 1996 The effect of the Argentine ant
Linepithema humile (Mayr) (Hymenoptera Formicidae) on flower-visiting insects of
Protea nitida Mill (Proteaceae) African Entomology 4285ndash287
Wagner D 2000 Pollen viability reduction as a potential cost of ant association for Acacia
constricta (Fabaceae) American Journal of Botany 87711ndash715
Wagner D and A Kay 2002 Do extrafloral nectaries distract ants from visiting flowers An
experimental test of an overlooked hypothesis Evolutionary Ecology Research 4293ndash305
Wang C Y Luo Y Tai D An and Y Kou 2008 Ants pollinate Neottia listeroides
(Orchidaceae) in Sichuan China 46836ndash846
Wyatt R 1980 The impact of nectar-robbing ants on the pollination system of Asclepias
curassavica Bulletin of the Torrey Botanical Club 10724ndash28
Wheeler WM and IW Bailey 1920 The feeding habits of Pseudomymine and other ants
Transactions of the American Philosophical Society 22235-279
38
Willmer P G and S A Corbet 1981 Temporal and microclimatic partitioning of the floral
resources of Justicia aurea amongst a concourse of pollen vectors and nectar robbers
Oecologia 5167ndash78
Willmer P G and G N Stone 1997 How aggressive ant-guards assist seed-set in Acacia
flowers Nature 388165ndash167
Willmer P G C V Nuttman N E Raine G N Stone J G Pattrick K Henson P Stillman
et al 2009 Floral volatiles controlling ant behaviour Functional Ecology 23888ndash900
Young T P C H Stubblefield and L a Isbell 1997 Ants on swollen-thorn acacias species
coexistence in a simple system Oecologia 10998ndash107
Yu D W and N E Pierce 1998 A castration parasite of an ant-plant mutualism Proceedings
of the Royal Society B Biological Sciences 265375ndash382
39
Tables
Table 11 Studies of ant-flower or ant-pollinator interactions that included a measure of plant fitness Each study system is listed only
once except when results varied between years or when different studies examined male and female fitness Significant effects of ants on
plant fitness are either lsquopositiversquo or lsquonegativersquo non-significant effects are lsquoneutralrsquo When ants significantly affected only one fitness
component but several were measured (eg fruit set and seed production) we still consider the result significant
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Acacia
drepanolobium
Crematogaster
nigriceps
Proportion
fruiting
Negative Destruction of floral buds Young et al 1997
Asclepias exaltata Several Pollinia inserted
removed
Negative for pollinia
insertion neutral for
pollinia removal
Exploitative competition Wyatt 1980 T
40
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Asclepias syriaca Several
Fruit set pollinia
removed
Negative for fruit
set neutral for
pollinia removal
Exploitative competition Fritz and Morse
1981 T
Cordia nodosa Allomerus cf
demerarae
Fruit set Negative Destruction of floral buds Yu and Pierce
1998
Costus woodsonii Wasmannia
auropunctata
Seed set Positive Unknown ant pollination
suggested (but unlikely)
Schemske 1980
Eritrichum aretioides F neorufibarbis
gelida
Seed set Negative Ants damaged flowers Puterbaugh 1998
41
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Euphorbia characias Several Seed set Neutral Plants in Argentine ant-
invaded area set less seed
than those in un-invaded
sites but an ant-exclusion
experiment did not produce
significant results
Blancafort and
Gόmez 2005
Ferocactus wislizeni Solenopsis xyloni
and others
Seed set Unknown negative Interference and exploitative
competition S xyloni
reduced seed set relative to
other ants but the study did
not include a no-ant control
Ness 2006
42
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Frasera speciosa F neorufibarbis
and Formica sp
Seed set Neutral Nocturnal pollination
occurring when ants were
less active may have
compensated for reduced
diurnal pollinator visitation
Norment 1988
Gypsophila struthium Several Seeds per
inflorescence
Neutral No effect despite large
numbers of ants on flowers
Gόmez et al
1996
Hirtella physophora Allomerus
decemarticulatus
Fruit set Negative Destruction of flowers Orivel et al
2011
Humboldtia brunonis Crematogaster
dohrni
Fruit set Negative Destruction of flowers Gaume et al
2005
43
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Lepidium subulatum Several Percentage of
ovules setting
seed
Positive Unknown Gόmez et al
1996
Leucospermum
conocarpodendron
Several Seed set Neutral Site not pollen limited Lach 2008
Linaria vulgaris Several ldquoFemale fitness
indexrdquo
(combination of
number of seeds
fruits flowers
and seed weight)
Positive Reduction in florivory and
seed predation nectar
robbing holes increased ant
visitation
Newman and
Thomson 2005
44
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Melastoma
malabathricum
Oecophylla
smaragdina
Seed set Positive Ants deterred less efficient
pollinators increasing
visitation by the more
efficient pollinator
Gonzaacutelvez et al
2012
Metrosideros
polymorpha
Several Fruit set Neutral Ants reduced nectar
collection rates by foraging
bees but had no effect on
pollen collection rates or
fruit set
Junker et al
2010a
Orexis alpine F neorufibarbis
gelida
Seed set Neutral Autogamy or not pollen
limited
Puterbaugh 1988
45
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Ouratea spectabilis Cephalotes
pusillus and others
Seeds per fruit Neutral positive C pusillus had no effect on
seed number but other ants
collectively increased seed
set
Byk and Del
Claro 2010
Pachycereus schottii Several Seed set Neutral positive In one year ants showed a
positive effect (seed
number 2008) but no effect
with increased sample sizes
Chamberlain and
Holland 2008
Polemonium
viscosum
F neorufibarbis
gelida
Seed set Negative Ants damaged styles Galen 1983
46
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Polemonium
viscosum
F neorufibarbis
gelida
Pollen removal Neutral Ants did not affect pollen
removal even at high ant
densities
Galen and Geib
2007 T
Protea nitida Linepithema
humile and others
Seed set Neutral positive L humile had no effect on
P nitida seed set but native
ants increased it in one year
ants may cause pollinators
to relocate
Lach 2007
Psychotria
limonensis
Ectatomma spp Fruit set Positive Suggested increased
pollinator relocation
Altshuler 1999
47
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Roussea simplex Technomyrmex
albipes
Seed set Negative Interference and exploitative
competition
Hansen and
Muumlller 2009
Tristerix aphyllus Several Fruit set Neutral Nectar robbing ants
minimally reduced nectar
levels and had no effect on
hummingbird visitation
Caballero et al
2013
Vaccinium
reticulatum
Several Fruit set Neutral Ants reduced florivory but
did not affect fruit set
Bleil et al 2011
T Study measured male fitness
Study of sterilizing ants showed short-term decrease in female fitness long-term effects are unclear (see text)
48
Table 12 Studies demonstrating either i) ant pollination through an exclusion experiment or ii) that ants transfer pollen (or pollen
analogue) between flowers strongly suggesting ant pollination
Plant species Ant species Ant pollination Evidence Reference
Alyssum purpureum Several Yes Exclusion experiment Gόmez et al 1996
Arenaria tetraquetra Several Yes Exclusion experiment Gόmez et al 1996
Balanophora kuroiwai Leptothorax sp Suggested Exclusion experiment and
observed pollen transfer
and pollen tube formation
but did not see if plants set
seed
Kawakita and Kato 2002
Blandfordia grandiflora Iridomyrmex sp Yes Exclusion experiment Ramsey 1995
Borderea pyrenaica Several Yes Exclusion experiment Garciacutea et al 1995
49
Plant species Ant species Ant pollination Evidence Reference
Cytinus hypocistis Several Yes Exclusion experiment de Vega et al 2009
Epipactis thunbergii Camponotus
japonicas
Suggested Observed transfer of
pollinia from ants to
orchids
Sugiura et al 2006
Euphorbia cyparissias Several Yes Exclusion experiment Schuumlrch et al 2000
Fragaria virginiana Several Very likely Ant-only treatments set
comparable seed to flying
pollinator-only treatment
seed set increased with
increasing ant visitation
Ashman and King 2005
Frasera speciosa Several Yes Exclusion experiment Norment 1988
Frankenia thymifolia Several Yes Exclusion experiment Gόmez et al 1996
50
Plant species Ant species Ant pollination Evidence Reference
Hormathophylla spinosa Proformica
longiseta
Yes Exclusion experiment Gόmez and Zamora 1992
Jatropha curcas
Several
Tapinoma
melanocephalum
most common
Yes Exclusion experiment Luo et al 2012
Leporella fimbriata Myrmecia urens Yes Observed pollinia transfer
and subsequently
confirmed as ant
pollination
Peakall et al 1987
51
Plant species Ant species Ant pollination Evidence Reference
Lobularia maritima Several
Camponotus
micans most
common
Yes Exclusion experiment Gόmez 2000
Microtis parviflora
Iridomyrmex
gracilis
Yes Observation used
laboratory colonies to
replicate observations
Peakall and Beattie 1989
Naufraga balearica
Several
especially
Plagiolepis
pygmaea and
Lasius grandis
Suggested Observation over three
years of study only ants
were observed visiting
ants carried pollen
Cursach and Rita 2011
52
Plant species Ant species Ant pollination Evidence Reference
Neottia listeroides Leptothoras sp
Paratrechina sp
Suggested Observed transfer of
pollinia from ants to
orchids
Wang et al 2008
Paronychia pulvinata Formica
neorufibarbis
gelida
Yes Exclusion experiment Puterbaugh 1998
Polygonum cascadense Formica argentea
Very likely Grew plants in greenhouse
open to flying visitors with
minimal seed set only
plants with access to ants
set significant seed
Hickman 1974
53
Plant species Ant species Ant pollination Evidence Reference
Retama sphaerocarpa Several Yes Exclusion experiment Gόmez et al 1996
Scleranthus perennis
Several
especially
Formica
rufibarbis
Suggested Ants transferred pollen
analogue to stigmas and
carried pollen on bodies
Svensson 1985
Sedum anglicum Proformica
longiseta
Yes Exclusion experiment Gόmez et al 1996
Trinia glauca Several Yes Exclusion experiment Carvalheiro et al 2008
54
Chapter 2 Ants and ant scent reduce bumblebee pollination of artificial flowers
Cembrowski AR Tan MG Thomson JD and Frederickson ME
In press The American Naturalist
All authors designed the experiment ARC and MGT performed the experiment ARC and MEF
performed statistical analyse
s and ARC wrote the manuscript with input from all authors
Abstract
Ants on flowers can disrupt pollination by consuming rewards or harassing pollinators but it is
difficult to disentangle the effects of these exploitative and interference forms of competition on
pollinator behavior Using highly rewarding and quickly replenishing artificial flowers that
simulate male or female function we allowed bumblebees (Bombus impatiens) to forage (1) on
flowers with or without ants (Myrmica rubra) and (2) on flowers with or without ant scent cues
Bumblebees transferred significantly more pollen analogue both to and from ant free flowers
demonstrating that interference competition with ants is sufficient to modify pollinator foraging
behavior Bees also removed significantly less pollen analogue from ant scented flowers than
from controls making this the first study to show that bees can use ant scent to avoid harassment
at flowers Ant effects on pollinator behavior possibly in addition to their effects on pollen
viability may contribute to the evolution of floral traits minimizing ant visitation
55
Introduction
Trait-mediated indirect interactions arise when a focal species causes phenotypic changes
including behavioral modifications in a second species and these effects cascade to still other
species (Werner and Peacor 2003) Although trait-mediated indirect interactions are often studied
in food webs (eg Werner and Peacor 2003 Preisser et al 2005) they are not limited to trophic
interactions For example the threat of predation can change the behavior of a mutualist thus
affecting its partners (Suttle 2003) In animal-pollinated plants predators can disrupt pollination
directly through density-mediated indirect interactions (Dukas 2005) or via trait-mediated
indirect interactions by changing pollinator behavior When predators are present pollinators
may switch to visiting less rewarding flowers or they may avoid flowers altogether potentially
reducing the amount of pollen donated or received by a flower and thus decreasing plant fitness
(Gonccedilalves-Souza 2008 Ings and Chittka 2009) Similarly competition for floral rewards with
other flower visitors may cause behavioral changes in pollinators (Maloof and Inouye 2000
Ohashi et al 2008) Previous research has shown that bees may spend less time visiting flowers
depleted of rewards (Thomson 1986) and may avoid flowers bearing scent marks that indicate
recent visits by other individuals of the same or different bee species as these flowers are likely
to be depleted (Stout and Goulson 2002)
Whereas competition between pollinators may not be particularly costly for the plant
because both competitors are likely to provide pollination services competition between
pollinators and organisms that do not usually provide a pollination service such as ants has a
greater potential to reduce plant fitness Ants are common floral visitors they are attracted to
flowers for nectar (Lach 2007) and in some cases pollen (Byk and Del Claro 2010) and will
often defend these resources against other flower visitors (Altshuler 1999) However ants rarely
56
contribute much useful pollination antibiotic secretions present on their cuticles kill pollen
grains (Beattie et al 1985 Dutton and Frederickson 2012) and when foraging in flowers ants
sometimes impede female function by damaging stigmas (Galen and Cuba 2001) We will
henceforth consider ants to be non-pollinators and will contrast them to animals like bees that
are pollinators Ants are capable of competing with pollinators in two ways (1) by consuming
floral rewards and reducing their availability to pollinators (ie exploitative competition) and (2)
by directly antagonizing and excluding pollinators from flowers (ie interference competition)
Many plants benefit from the presence of ants numerous plant species recruit ants using
extrafloral nectar food bodies or domatia because ants provide protection against folivores (Heil
and McKey 2003) Similarly some plants have extrafloral nectaries on or near reproductive
tissues that recruit ants that deter florivores (eg Inouye and Taylor 1979) However plants
may incur reproductive costs as a result of their association with ants Some ant species that nest
in myrmecophytes sterilize flowers to shunt plant resources from reproduction to vegetative
growth allowing for greater ant colony growth (Frederickson 2009) Also ants visiting
extrafloral nectaries may be attracted to floral nectaries where they can harass pollinators (Ness
2006) There is growing evidence that many angiosperms have evolved floral traits that prevent
ants from accessing flowers during anthesis (Willmer and Stone 1997 Ballantyne and Willmer
2012) The effects of flower-visiting ants can be substantial Ants can decrease the frequency
duration or species diversity of pollinator visits to flowers (Lach 2008 Hansen and Muumlller 2009
Junker et al 2010 Gonzaacutelvez et al 2012) all of which potentially impact pollen donation and
receipt In some studies changes to pollinator behavior induced by ants have also been linked to
seed set
It is difficult however to disentangle the intertwined effects of exploitative and
interference competition in nature If ants disrupt pollination plants may often evolve traits to
57
deter ants from flowers (Willmer et al 2009) From a practical standpoint this would then limit
our ability to study the effects of ants on pollinator visitation because ants would not visit
flowers Here we used artificial flowers to explore ant-bee interactions in the absence of floral
defenses against ants We used highly rewarding quickly replenishing artificial flowers to
examine how direct harassment (ie interference competition expressed through behavior)
changes bee foraging behavior and how this affects donation and receipt of a pollen analogue
(powdered food dye) while minimizing the effects of exploitative competition We predicted
that flowers visited by ants would both donate and receive less pollen analogue than flowers
without ants because bees would avoid flowers with ants or leave them sooner Because scent
plays a large role in bumblebee communication and flower choice (Stout and Goulson 2002) we
also tested whether the presence or absence of ant scent on artificial flowers would affect pollen
analogue donation because of changes in the foraging behavior of bumblebees
Methods
i) Subjects
Myrmica rubra is an invasive ant in eastern North America with a range and habitat preferences
overlapping that of Bombus impatiens a common bumblebee Because M rubra visits flowers
(A Cembrowski pers obs) the two species likely interact in nature We collected 12 M rubra
colonies in the fall of 2011 and 2012 from Toronto Ontario Canada and the surrounding area
and maintained them in environmental chambers on artificial diet (Dussutour and Simpson 2009)
and a 1410 LD schedule (light 600-2200) We used these colonies as sources of M rubra
workers for experiments
58
Workers from commercially supplied colonies of Bombus impatiens (Biobest Canada
Ltd) foraged on artificial flowers in flight cages (either 24 x 24 x 21 m or 79 x 34 x 20m) at
the University of Toronto We tested a total of five bee colonies four colonies were used in the
ant presence trials and two of these and one additional colony were used in the ant scent trials
Flight cages had overhead fluorescent lights attached to timers In contrast to most previous
studies in which bees have been trained and tested individually the entire worker force of a bee
colony was free to forage at will in our experiments Colonies were trained to forage on artificial
flowers for at least four days before being used in trials After being used for a trial the colony
was not used for at least two days to reduce dye carryover between trials Between trials
bumblebees were fed pollen and given sugar water
ii) Artificial flowers
The flowers (Fig 1 Thomson et al 2012 see also Makino 2008) consisted of glass jars filled
with 30 WV sucrose solution Sugar water travelled by capillary action up a sewing-thread
wick to a hole in a blue-painted lid accumulating in a knot that acts as a nectary Flowers
depleted by visitors were quickly replenished via capillary action and were non-rewarding only
very briefly after visits taking less than a minute to accumulate 05 microl note that bumblebee
visits to a single flower are often separated by several minutes or more (A Cembrowski
unpublished data)
Unlike most previous artificial flower designs these flowers allow for estimation of male
and female fitness because we can measure both the amount and type of dye received by female
flowers (see (d) Dye quantification below) In order to access nectar on a ldquomalerdquo flower a bee
must crawl through a circular ldquoantherrdquo of brush-like weather stripping material dusted in a
59
consistent manner with our pollen analogue powdered food dye (FDampC 5 or 6) which is
transferred to the bee in the process On a ldquofemalerdquo flower a bee crawls through a sticky plastic
reinforcement that functions as a ldquostigmardquo receiving dye from the beersquos body Male flowers
dispensed dye particles over multiple bee visits and could still dispense dye at the end of trials
They resemble many real flowers or flower heads in that pollen is dispensed gradually over time
but less dye is available for transfer with each subsequent bee visit (Harder and Thomson 1989)
iii) Flight cage trials
We conducted 6 ant scent trials between 22 March and 4 June 2012 and the remaining trials (16
ant presence trials and 9 ant scent trials) between 9 November 2012 and 6 January 2013 Ant
presence trials lasted for eight hours and ant scent trials lasted for four hours Artificial flowers
were prepared and placed individually in small plastic containers treated with Fluon (Insect-a-
Slip BioQuip Products Inc) to prevent ants from escaping In ant presence trials we used 32
flowers (16 male and 16 female flowers) arranged in an eight-by-four array with flowers spaced
45 cm apart between rows and 30 cm within rows Ant scent trials used 20 flowers (10 male and
10 female flowers) in a four-by-five array with flowers spaced 30 cm apart between and within
rows In all of the ant presence trials and 9 of the ant scent trials we counted the number of bees
actively foraging after one hour to get a measure of colony activity
a) Ant presence trials
We examined the effects of ants on the amount of dye donated by male flowers and received by
female flowers in sixteen trials testing four bee colonies four times each The containers of eight
randomly chosen male flowers and eight randomly chosen female flowers received 15 M rubra
60
workers each the other 16 flowers remained free of ants Isolated from their colonies M rubra
workers have nowhere to deposit sugar water they collect and therefore may become sated
nonetheless a force of 15 ant workers was enough to maintain visitation to the nectaries where
bees foraged while still rarely having more than one or two ants visit the nectary at any time As
ants attacked bees bees would occasionally carry or throw attacking ants off flowers but despite
this some ants maintained their presence at the nectary throughout the trials Because ants the
size of M rubra consume liquids slowly (~017 ndash 024 microlmin Davidson et al 2004) flowers
replenished nectar more quickly than ants consumed it We used two colors of dye (FDampC 5 and
FDampC 6) to differentiate male flowers with and without ants In two trials for each bee colony
anthers of male flowers with ants were coated with FDampC 5 dye while brushes of male flowers
lacking ants were coated with FDampC 6 dye In the other two trials we reversed dye colors to
control for effects of dye type and color Dye color was assigned in a random order Artificial
flowers were placed in randomly assigned positions in the array before we opened the colony
and allowed the bees to begin foraging
b) Scent trials
We explored the effect of ant scent on dye donation in fifteen trials testing three bee colonies in
six five and four trials each Bees were first exposed to ants by allowing the bees to forage for
eight hours on twenty flowers (ten male and ten female) of which five male flowers had 15 ants
and all others had none No dye was used during this ldquoexposurerdquo day We then collected all but
the five ant-free male flowers in the flight cage leaving these flowers to keep bees foraging
Next we individually stored five new male lids in Fluon-treated containers with ten M rubra
workers Five control male lids were put in identical containers lacking ants The following
61
morning we removed the remaining flowers and set out ten new male and female flowers in a
random spatial arrangement using the lids having or lacking ant scent Thus the ant-scented
flowers were not in the same positions as the ant-visited flowers on which the bees were trained
Only five male flowers had ant scent and the other 15 flowers (five male and ten female) did not
We used the same two dye colors as in i) to differentiate male flowers with and without ant
scent and randomized which color was used for ant-scented flowers between trials We collected
and replaced stigmas from female flowers after one hour and collected the stigmas again after
four hours In the first trial performed we also collected stigmas after two hours but due to the
small amount of dye transferred this was not repeated
iv) Dye quantification
After each trial we quantified the amount of dye transferred to female flowers using a
spectrophotometer We removed the stigmas from female flowers placed them in test tubes
added 51 mL of distilled water to each tube and vortexed each tube thoroughly to ensure the
dye was evenly diluted These were diluted further as needed if the absorbance exceeded the
sensitivity range of the spectrophotometer In the first six scent trials each stigma was analyzed
separately and the dye amounts were summed to obtain a total amount of each dye color
transferred to female flowers in each trial In all the ant presence trials and the other nine scent
trials we opted to treat the experiment as the unit of replication Therefore we combined stigmas
from each treatment type (ant-visited or ant-free) in a test tube and measured the total amount of
each dye color transferred in each treatment in each trial
Because we put different dye colors on male flowers with and without ants we could use
the amount of each dye color donated to all female flowers to measure the reproductive success
62
of ant-visited and ant-free male flowers The total amount of dye (of both colors) received by
female flowers with and without ants was our measure of female reproductive success We
calculated the amount of each dye color in the sample by measuring absorbance at 428 or 486
nm and converting absorbance to micrograms following computational methods for overlapping
spectra (Blanco et al 1989)
v) Statistical analyses
In two trials (one ant presence and one ant scent) most female flowers received no dye because
of low bee activity so we excluded these trials from analyses Because dye reception and
donation values were non-normally distributed we square-root transformed the data before
examining the effects of ant presence or ant scent on dye transfer in ANCOVAs For ant
presence trials we included ant presence on male and female flowers and their interaction as
main effects and the total amount of dye transferred in each trial as a covariate to account for
the large variation in overall dye transfer among trials For ant scent trials we included ant scent
as the main effect time (one or four hours) as a repeated measure the interaction between scent
and time and the total amount of dye transferred as a covariate Covariate by treatment (ant
presence or ant scent) interactions were never significant and so were excluded in final analyses
All analyses were conducted in JMP (v 1000)
Results
Bees usually started foraging within minutes of the beginning of the trial and continued until
flowers were collected from the flight cage or the lights were extinguished An average of 34 plusmn
012 and 40 plusmn 015 (mean plusmn SE) bees were foraging after an hour in the ant presence and ant
63
scent trials respectively Male flowers with ants donated significantly less dye than male flowers
lacking ants (Fig 22a F155 = 4019 p lt 00001) Similarly female flowers with ants received
significantly less dye than female flowers lacking ants (Fig 22a F155 = 461 p = 0036) There
was no significant interaction between ant presence on male flowers and ant presence on female
flowers (F155 = 118 p = 028) Flowers with ant scent donated significantly less dye than
flowers without ant scent (Fig 22b F125 = 11216 p lt 00001) There was no significant effect
of time (F125 = 121 p = 028) or interaction between time and scent (F125 = 111 p = 030) in
the model
Discussion
In this study ants altered bumblebee pollination behavior and exhibited trait-mediated indirect
interactions with (artificial) flowers Through interference competition M rubra workers
significantly affected the pattern of dye transfer by B impatiens causing a preferential flow of
dye from male flowers lacking ants to female flowers lacking ants If manifested in nature such
effects would reduce the reproductive success of plants visited by ants through both male and
female function
Ants could have changed the attractiveness of artificial flowers in at least two ways First
as ants interrupted or altogether prevented bumblebees from foraging at nectaries bumblebees
may have learned to avoid flowers with ants due to the relative inefficiency of foraging at these
flowers something bumblebees take into account (Heinrich 2004) Second bees attempt to
minimize their risk of being attacked during foraging bouts which ants did to visiting bees Ants
often attacked or harassed bees by biting grasping and appearing to sting visiting bees
preventing them from accessing the nectary or reducing their time on flowers (see online video
64
1) and bees sometimes avoided ant-tended flowers entirely This harassment was sometimes
physically traumatic (online video 1) and bees often appeared to have trouble flying after being
attacked by ants Previous research has demonstrated that bumblebees leave or avoid flowers
where they have been harassed (Jones and Dornhaus 2011) and avoid foraging where there is
visual or olfactory evidence of a predator or predation event (Abbott 2006 Goodale and Nieh
2012) Though it is unclear if bumblebees viewed ants as competitors or predators they
responded similarly to flowers having ants as they do to flowers housing predators (Gonccedilalves-
Souza et al 2008)
To avoid artificial flowers with ants bees likely used a combination of visual and
olfactory cues Previous research has shown that bees respond to conspicuous predators or
predator ldquodummiesrdquo on flowers (Suttle 2003 Gonccedilalves-Souza et al 2008) decreasing the
frequency and duration of their visits to these flowers Thus bees may have been able to avoid
flowers harboring ants by sight alone However even in the absence of ants bees still
preferentially visited flowers lacking ant scents (Fig 22b) suggesting that they had learned to
associate ant scent with harassment Bees are adept at associative learning (Wright and Schiestl
2009) and can learn to recognize unique scents left behind by both conspecific and heterospecific
flower visitors (Stout et al 1998) Bees can use these various scents often arising from tarsal
gland deposits (Stout et al 1998) to recognize recently visited flowers that are less likely to be
profitable (Stout and Goulson 2002) Recently Ballantyne and Willmer (2012) demonstrated that
bees learn to associate ant scents with unrewarding artificial flowers and decrease their visitation
to these flowers Our results complement their findings by showing that bees can associate ant
scent with harassment at otherwise rewarding flowers
In our study ant scent caused bees to adjust their foraging strategy and decreased the
amount of pollen analogue that was donated by ant-scented flowers Thus the effects of
65
interference competition with ants on flowers can extend beyond immediate interactions and may
have fitness consequences for plants even when ants are absent Like other olfactory cues these
effects are likely transitory (Stout et al 1998) Although more dye was still donated by male
flowers lacking ant scent than those with ant scent the ratio of dye donated was on average
closer to equality in hours 2-4 than in the first hour (Fig 22b) This effect may be partially
driven by dye depletion of male flowers without ant scent but the lack of a strong corresponding
decrease in flowers with ant scent suggests that visitation patterns became more similar We did
not test whether B impatiensrsquo avoidance of ant scent was an innate or a learned behavior but
previous work has shown that Bombus terrestris does not innately avoid flowers with ant scent
(Ballantyne and Willmer 2012)
Ants may be necessary for plant survival and growth but can be costly for plant
reproduction The evolution of ant attractants such as extrafloral nectaries in some plant lineages
suggests that the costs of ants can be outweighed by their protective abilities However in this
study flowers visited by ants received and donated significantly less dye although they did
retain some sexual function The net benefit of having ants depends on whether ants increase
plant fitness by reducing herbivory more than they decrease plant fitness by disrupting
pollination Alternatively plants may actually benefit from the costs of ants to plant
reproduction ants that castrate flowers may be better defenders increasing plant survival or
vegetative growth when plants are young allowing for increased reproduction later in life when
the plant is colonized by less aggressive non-castrating ants (Frederickson 2009 Palmer et al
2010) In some plant species pollinator harassment by ants may even be beneficial Altshuler
(1999) reported that Ectatomma ants greatly increased fruit set of Psychotria limonensis despite
reductions in pollinator visitation rates presumably due to increases in the rate of pollen
outcrossing Similarly Gonzaacutelvez et al (2012) found that the presence of Oecophylla
66
smaragdina ants on Melastoma malabathricum flowers reduced visitation by less effective
pollinators and increased visitation by more effective Xylocopa bees increasing plant fitness
Cases of ants being beneficial to flowers appear to be the minority and many plant
species have traits that limit ant access to flowers Several studies have detailed floral volatiles
that are thought to mimic ant alarm pheromones and thus repel ants (Junker and Bluumlthgen 2008
Willmer et al 2009) Other plants use structural modifications such as narrow corollas or
slippery stems to limit ant access to flowers (reviewed in Willmer et al 2009) Exploitative
competition between ants and pollinators (Lach 2005 Ballantyne and Willmer 2012) as well as
the lethal effects of ant antibiotic secretions on pollen grains (Beattie et al 1985 Dutton and
Frederickson 2012) may have resulted in selection on plants to reduce ant visits to flowers Our
results suggest that trait-mediated indirect interactions resulting from interference competition
between ants and bees may favor plants that defend their flowers and their pollinators against
ants
Acknowledgements
We thank H Rusnock and S Meadley-Dunphy for assistance J Dix for help with flight cage
construction and Biobest for bumblebee colonies We are grateful to the Thomson and
Frederickson labs for guidance particularly T Makino funded by JSPS for helping to refine
artificial flowers J Ogilvie for help with bees K Prior for M rubra colony collections and K
Turner for study design suggestions The comments of the editor and two anonymous reviewers
prompted improvements in the article especially with regards to study design MEF and JDT
acknowledge NSERC Discovery Grant funding The University of Torontorsquos Faculty of Arts and
Science supported MGRTrsquos participation
67
68
References
Abbott K R 2006 Bumblebees avoid flowers containing evidence of past predation events
Canadian Journal of Zoology 841240-1247
Altshuler D L 1999 Novel interactions of non-pollinating ants with pollinators and fruit
consumers in a tropical forest Oecologia 119600-606
Ballantyne G and Willmer P 2012 Floral visitors and ant scent marks noticed but not used
Ecological Entomology 37402-409
Beattie A J Turnbull C Knox R B and Williams E G 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany
71421-426
Blanco M Runiaga H Maspoch S and Tarin P 1989 A simple method for
spectrophotometric determination of two-components with overlapped spectra Journal of
Chemical Education 66178-180
Byk J amp Del Claro K 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333-38
Davidson D W S C Cook and R R Snelling 2004 Liquid-feeding performances of ants
(Formicidae) ecological and evolutionary implications Oecologia 139255ndash66
Dukas R 2005 Bumble bee predators reduce pollinator density and plant fitness Ecology
861401-1406
Dussutour A and Simpson S J 2009 Communal nutrition in ants Current Biology 19740-
744
69
Dutton E M and Frederickson M E 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561-569
Frederickson ME 2009 Conflict over reproduction in an ant-plant symbiosis why Allomerus
octoarticulatus ants sterilize Cordia nodosa trees American Naturalist 173675-681
Galen C and Cuba J 2001 Down the tube pollinators predators and the evolution of flower
shape in the alpine skypilot Polemonium viscosum Evolution 551963-1971
Gonccedilalves-Souza T Omena P M Souza J C and Romero G Q 2008 Trait-mediated
effects on flowers artificial spiders deceive pollinators and decrease plant fitness Ecology
892407-2413
Gonzaacutelvez F G Santamariacutea L Corlett R T and Rodriacuteguez-Gironeacutes M A 2012 Flowers
attract weaver ants that deter less effective pollinators Journal of Ecology 10178ndash85
Goodale E and Nieh JC 2012 Public use of olfactory information associated with predation
in two species of social bees Animal Behaviour 84919-924
Harder LD and Thomson JD 1989 Evolutionary options for maximizing pollen dispersal of
animal-pollinated plants American Naturalist 133323-344
Heil M and McKey D 2003 Protective ant-plant interactions as model systems in ecological
and evolutionary research Annual Review of Ecology Evolution and Systematics 34425-
453
Heinrich B 2004 Bumblebee economics 2nd
edn Harvard University Press Cambridge
Ings T C and Chittka L 2009 Predator crypsis enhances behaviourally mediated indirect
effects on plants by altering bumblebee foraging preferences Proceedings of the Royal
Society B 2762031-2036
70
Inouye D W and Taylor O R Jr 1979 A temperate region plant-ant-seed predator system
consequences of extra floral nectar secretion by Helianthella quinquenervis Ecology 601-
7
Junker R R and Bluumlthgen N 2008 Floral scents repel potentially nectar-thieving ants
Evolutionary Ecology Research 10295ndash308
Junker R R Bleil R Daehler C C and Bluumlthgen N 2010 Intra-floral resource partitioning
between endemic and invasive flower visitors consequences for pollinator effectiveness
Ecological Entomology 35760ndash767
Jones E I and Dornhaus A 2011 Predation risk makes bees reject rewarding flowers and
reduce foraging activity Behavioral Ecology and Sociobiology 651505-1511
Lach L 2005 Interference and exploitative competition of three nectar-thieving invasive ant
species Insect Sociaux 52257-262
Lach L 2007 A mutualism with a native membracid facilitates pollinator displacement by
Argentine ants Ecology 881994-2004
Lach L 2008 Argentine ants displace floral arthropods in a biodiversity hotspot Diversity and
Distributions 14281-290
Makino T T and Sakai S 2007Experience changes pollinator responses to floral display size
from size-based to reward-based foraging Functional Ecology 21854863
Maloof J E and Inouye D W 2000 Are nectar robbers cheaters or mutualists Ecology
812651ndash2661
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506-514
Ohashi K Leslie A and Thomson JD 2008 Trapline foraging by bumble bees V Effects of
experience and priority on competitive performance Behavioral Ecology 19936-948
71
Palmer T M Doak D F Stanton M L Bronstein J L Kiers E T Young T P Goheen
J R and Pringle R M 2010 Synergy of multiple partners including freeloaders
increases host fitness in a multispecies mutualism Proceedings of the National Academy
of Sciences 10717234-17239
Preisser E L Bolnick D I and Benard M E 2005 Scared to death The effects of
intimidation and consumption in predator-prey interactions Ecology 86501-509
Stout J C Goulson D and Allen J A 1998 Repellent scent-marking of flowers by a guild of
foraging bumblebees (Bombus spp) Behavioral Ecology and Sociobiology 43317-326
Stout J and Goulson D 2002 The influence of nectar secretion rates on the responses of
bumblebees (Bombus spp) to previously visited flowers Behavioral Ecology and
Sociobiology 52239-246
Suttle K B 2003 Pollinators as mediators of top-down effects on plants Ecology Letters
6688-694
Thomson JD 1986 Pollen transport and deposition by bumble bees in Erythronium influences
of floral nectar and bee grooming Journal of Ecology 74329-341
Thomson JD Ogilvie J E Makino T T Arisz A Raju S Rojas-Luengas V and Tan M
G R 2012 Estimating pollination success with novel artificial flowers effects of nectar
concentration Journal of Pollination Ecology 9108-114
Tsuji K Hasyim A and Nakamura K 2004 Asian weaver ants Oecophylla smaragdina and
their repelling of pollinators Ecological Research 19669-673
Ulrich S and Bertsch A 1990 Do foraging bumblebees scent-mark food sources and does it
matter Oecologia 82137-144
Werner E E and Peacor S D 2003 A review of trait-mediated indirect interactions in
ecological communities Ecology 841083-1100
72
Willmer P G Nuttman C V Raine N E Stone G N Pattrick J G Henson K Stillman
P McIlroy L Potts S G and Knudsen J T 2009 Floral volatiles controlling ant
behaviour Functional Ecology 23888-900
Wright G A and Schiestl F P 2009 The evolution of floral scent the influence of olfactory
learning by insect pollinators on the honest signaling of floral rewards Functional Ecology
23841-851
73
Figures
Figure 21 Photograph of artificial flower lids A = anther L = lid N = nectary R = region
where anther or stigma is placed S = stigma T = thread W = weight
74
Figure 22 Dye transferred (mean plusmn 1SE) per trial by B impatiens workers A) from artificial
male and to artificial female flowers in the presence (unfilled circles) or absence (filled circles)
of Myrmica rubra workers and B) from artificial male flowers with (unfilled circles) or without
(filled circles) M rubra scent to ant-free female flowers in the 1st or the 2
nd to 4
th hours of the
trials
75
Chapter 3 Not just for the bees pollen consumption is common among tropical
ants
Cembrowski AR Reurink G and Frederickson ME
Planned submission to Biotropica
ARC and MEF designed the study ARC and GR did laboratory work ARC performed statistical
analyses and wrote the manuscript with input from other authors
Abstract
Although palynivory or pollen consumption is widespread among other hymenopterans there
are few accounts of palynivory in ants To quantify how often ants consume pollen we subjected
adult workers and larvae from 75 species of neo- and paleo-tropical ants to acetolysis a process
that destroys most organic material but leaves behind pollen grains In over half of the species
we examined ants contained a few pollen grains (lt 5) and ants of several Camponotus species
contained abundant pollen grains (gt50) We tested for an association between trophic level and
palynivory using stable nitrogen isotope ratios (δ15
N) but we did not find a significant
correlation we found pollen grains in lsquoherbivorousrsquo omnivorous and carnivorous ants We
suggest that our results indicate sporadic opportunistic pollen consumption by ants in tropical
forests
Introduction
For those organisms that can digest it efficiently pollen can be an excellent food source
Depending on the plant species pollen can contain up to 60 protein as well as lipids
76
carbohydrates and numerous trace nutrients (Roulston and Cane 2000) Pollen feeding
(palynivory) occurs in many taxa including birds mammals and arthropods (Roulston and Cane
2000) but it is best known among the Hymenoptera particularly bees Although adult bees feed
mainly on plant exudates (ie nectar) nearly all bee species rely on pollen as the chief protein
source for developing larvae (Willmer 2011) Closely related to bees ants have similar
nutritional requirements adult workers need carbohydrates whereas larvae are fed a diet rich in
protein (Dussutour and Simpson 2009) Ants also exploit nectar heavily and regularly visit
flowers (eg Herrera et al 1984 Bluumlthgen et al 2004) but unlike bees are not believed to
widely consume pollen Instead ants are thought to receive most of the protein in their diet from
predation or scavenging (eg Floren et al 2002)
However ants may not be as reliant on animal protein sources as commonly assumed In
tropical forests arboreal ants can have similar δ15
N ratios a measure of trophic level as
herbivores suggesting limited carnivory and substantial feeding on plant-derived protein sources
(Davidson et al 2003) One potential protein source is pollen This idea is supported by scattered
accounts of pollen consumption by ants from other ecosystems including deserts (Ness 2006)
boreal forests (Czechowski et al 2009) and tropical savannas (Byk and Del Claro 2010) One
ant genus Cephalotes is widely recognized to consume pollen (Creighton 1967 Baroni Urbani
and de Andrade 1997 Byk and Del Claro 2010) However beyond this genus our knowledge of
pollen consumption by ants has largely been limited to direct observations (but see Czechowski
et al 2011) and it is unknown if palynivory is truly rare among ants or simply under-reported
We borrowed a technique commonly used in palynology acetolysis to assess the frequency of
pollen consumption by ants in 75 neo- and paleo-tropical ant species This technique allowed us
to examine many more ant species than we could have observed directly
77
Methods
Adult worker ants were collected by hand at baits or from leaf litter using Winkler extractors
while larvae along with adults were collected by hand from nests Collections were made by L
Arcila Hernandez and J Sanders in the primary tropical rainforest surrounding the Centro de
Investigaciόn y Capacitaciόn Rio Los Amigos (CICRA 12deg34rsquoS 70deg05rsquoW) in Peru in 2010-
2011 and by E Youngerman from lowland primary and secondary rainforests in Madang
Province Papua New Guinea (PNG) in 2011-2012 We sorted ant samples to morphospecies and
then identified them to genus and when possible to species using existing keys which were
unavailable for PNG ants Ant samples used for acetolysis were whole adult workers guts from
adult workers and whole larvae
We used acetolysis to look for pollen grains inside ants In acetolysis organic material is
dissolved in acetic anhydride and sulfuric acid leaving behind stained pollen exines When
possible we used adult and larval ants of the same species from different colonies or bait
locations in an attempt to sample more representatively We prepared samples by first washing
them in 70 ethanol to remove any pollen present on the samplersquos outer surface before placing
them individually in micro-centrifuge tubes with ~05mL of a 91 mixture of acetic anhydride
sulfuric acid We then heated each sample at 95degC for 20min allowing tissues to dissolve Next
we centrifuged samples at 1000 RPM for 5min and decanted the liquid supernatant before
adding ~05mL of glacial acetic acid to the precipitate centrifuging again as above We decanted
the samples and added a drop of distilled water
78
We examined a 150μL sub-sample of this solution for pollen grains under a light
microscope Larvae gave less than 150μL of solution following acetolysis so we sampled the
entire amount We identified pollen grains by their staining and their surface architecture For
most species we examined at least three adult workers and three larvae but for some species
fewer than three adult workers or larvae were available (Tables 31 and 32)
We investigated the relationship between trophic level as measured by δ15
N ratios and
pollen consumption by ants Stable isotope (SI) data was available for some (n =23) of the
Peruvian species from a separate project in which collections overlapped Samples were stored in
EtOH and dried at 50degC overnight We used heads legs and thoraxes (but not gasters) of ants
for SI analysis Often single individuals did not provide enough material for analysis and we
combined multiple individuals to achieve necessary weights up to 10 for small ant species
Samples were analyzed at the Boston University Stable Isotope Laboratory using a Finnigan
Delta-S analyzer For other Peruvian species for which we had samples but not corresponding
stable isotope data (n =25) we took the median δ15
N ratio value of that speciesrsquo genus and
assigned it to the species in question Using pollen presenceabsence data for these 48 species
we used logistic regression to correlate pollen presence and δ15
N ratio using R (v 2151)
Results
We found pollen grains in 37 of the 197 adult workers 20 of the 102 larvae and 38 of the 75 ant
species we examined (Tables 31 and 32) The proportion of individuals containing pollen did
not differ significantly between larvae and adults (χ2
1 = 002 p = 088) The number of pollen
grains found was often low (median = 3) but highly variable (range 1-468) Also pollen
79
presence was inconsistent among conspecific ant samples of species that contained pollen 31
had pollen in only one of the larvae or adults examined In only three species (two Cephalotes
and one Myrmelachista) did all examined adults contain pollen
Highly lsquoherbivorousrsquo carnivorous and omnivorous ants contained pollen grains (Fig
31) There was no correlation between δ15
N ratio and pollen presence (χ2 = 150 df = 1 p =
022) In fact several carnivorous species contained pollen grains (eg Odontomachus sp 1
Pachycondola sp1)
Discussion
Pollen consumption by ants appears to be more common than suspected The majority of species
(51) had at least one individual that had consumed pollen and in 20 multiple individuals
contained pollen Numbers of grains found tended to be low however with most individuals
containing only a few grains Ants may use pollen as only a small portion of their diet exploiting
a host of other protein-containing foods more heavily including other low- δ15
N foods (eg
honeydew epiphylls Houmllldobler and Wilson 1990 Davidson et al 2003)
There are a few reasons why pollen may not be heavily exploited by ants Pollen requires
much post-consumption processing to extract its nutrients These nutrients are located in the
pollenrsquos cytoplasm which is protected from direct digestion by the exines and intines of the
grains Organisms must either break through these layers or cause the pollen grains to germinate
or pseudo-germinate to access and digest the cytoplasm (Roulston and Cane 2000) This can be
achieved by consuming a sugar-containing solution (such as nectar) along with pollen However
animals that are not adapted to feed on pollen may not be very efficient at this process only
80
extracting nutrients from some of the grains (Herrera and Martiacutenez del Rio 1998) Thus while
ants can likely get some nutrients from pollen other forms of more easily processed protein may
be more attractive
Pollen may also pose handling difficulties for ants Despite the large number of studies
that have recorded ants visiting flowers (Chapter 1) only a few papers detail ants harvesting
pollen directly from anthers Byk and Del Claro (2010) reported that Cephalotes pusillus ants
removed almost all of the available pollen from Ouratea spectabilis flowers and at least two
other studies describe ants consuming pollen along with nectar from flowers (Horskins and
Turner 1999 Ness 2006) Unlike bees which possess both morphological (eg specialized hairs
for holding pollen) and behavioral adaptations (eg stereotyped pollen-packing motions) ants
have no apparent mechanisms to increase the amount of pollen harvested and are likely limited
by the number of grains they can ingest or carry Rather than collect pollen directly from flowers
it appears more likely that ants collect it haphazardly in the environment leading to a smaller
number of pollen grains consumed at any one time It is known that at least Cephalotes ants
engage in such behavior Creighton (1967) observed Cephalotes texanus collecting pollen grains
trapped by Citrus x paradisi leaf hairs
There is a chance that the small numbers of pollen grains we observed came from
secondary pollen consumption (ie consuming a palynivorous organism) or consumption of
liquid containing pollen While we cannot rule this out we believe our results represent actual
targeted pollen consumption by ants Cephalotes ants known palynivores (Creighton 1967
Baroni Urbani and de Andrade 1997) contained similar numbers of pollen grains as many of the
pollen-containing ant species that we examined As well in at least once instance ants that we
found containing pollen grains were in the process of preying on non-pollen consuming
81
organisms (eg a Pachycondola species that was carrying a termite in its mandibles) or were
attracted by sugar baits
Contrary to our expectations there was no relationship between an ant speciesrsquo δ15
N ratio
and whether it had consumed pollen (Fig 32) We expected palynivory to be more common in
herbivorous ants because pollen presents a widespread source of protein a potentially limiting
resource The lack of pattern might reflect how available pollen grains are in the environment
While other forms of food may be preferable ants tend to be opportunistic foragers (Carroll and
Janzen 1973) and there is likely little cost associated with the consumption of scattered pollen
grains during foraging trips beyond the volume they occupy in the gut Some genera were found
to consistently consume pollen In support of previous observations (Baroni Urbani and de
Andrade 1997 Byk and Del Claro 2010) Cephalotes species were among the most common
pollen consumers Camponotus species also consistently contained pollen grains of the eight
species examined five contained pollen and pollen was found in species from both Peru and
PNG Camponotus species also contained the two largest numbers of pollen grains found (468
and 271) Taken together these results suggest that they may be another as yet unrecognized
commonly palynivorous genus
In general larvae did not contain large numbers of pollen grains (ranging from 1-38
grains) This is counter to what is seen in bees where larvae are the main recipients of pollen
(Willmer 2011) This difference may be because ant larvae are continually fed via trophallaxis
by adults throughout their development whereas bee larval cells are provisioned with pollen and
then sealed while larvae complete their development If both adults and larvae are able to extract
nutrients from pollen we would expect to see a preferential flow of nutrients from pollen to
larvae but not necessarily the grains themselves
82
Unlike palynivory by organisms that also provide pollination services pollen
consumption by ants is unlikely to benefit plants Ants rarely act as pollinators (Beattie et al
2004) They have pollen-killing antibiotic secretions on their cuticles (Dutton and Frederickson
2012) and they can compete with other flower visitors reducing flower visitation rates (Ness
2006) The negative impacts of flower-visiting ants are thought to have led to the evolution of
ant-repellent floral volatiles (reviewed in Willmer 2009) These volatiles can pre-empt
competition between ants and pollinators and may often be located within the pollen itself
(Willmer 2009) If floral volatiles truly are a defense against ant visitation our results suggest an
additional evolutionary pressure to not only prevent ant-pollinator interactions but to also
protect plant gametes from occasional consumption by a non-pollinating visitor
Acknowledgements
We would like to thank Jon Sanders Lina Arcila Hernandez and Eric Youngerman for providing
samples the Thomson lab for use of supplies the Pierce lab for funding support during JSrsquo and
EYrsquos collections and the staff of CICRA and the New Guinea Binatang Research Center for
logistical support The Peruvian Ministry of Agriculture provided permits (Nos 394-2009-AG-
DGFFS-DGEFFS and 299-2011-AG-DGFFS-DGEFFS) MEF acknowledges financial support
from an NSERC Discovery Grant a Connaught New Researcher Award an Ontario Ministry of
Economic Development and Innovation Early Researcher Award and the University of Toronto
ARC was supported by an Ontario Graduate Scholarship and Sigma Xi
83
References
Baroni Urbani C and M L de Andrade 1997 Pollen eating storing and spitting by ants
Naturwissenschaften 84256ndash258
Beattie A J C Turnbull R B Knox and E G Williams 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany 71421ndash
426
Bluumlthgen N G Gottsberger and K Fiedler 2004 Sugar and amino acid composition of ant-
attended nectar and honeydew sources from an Australian rainforest Austral Ecology
29418ndash429
Byk J and K Del-Claro 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333ndash38
Carroll C R and D H Janzen 1973 Ecology of foraging ants Annual Review of Ecology and
Systematics 4231ndash257
Creighton W S 1967 Studies on free colonies of Cryptocerus texanus Satschi (Hymenoptera
Formicidae) Psyche 7434ndash42
Czechowski W B Markό and A Radchenko 2009 Rubbish dumps reveal the diet of ant
colonies Myrmica schencki Em and Myrmica rubra (L) (Hymenoptera Formicidae) as
facultative pollen-eaters Polish Journal of Ecology 56737ndash741
Czechowski WB Markoacute K Erős and E Csata 2011 Pollenivory in ants (Hymenoptera
Formicidae) seems to be much more common than it was thought Annales Zoologici
61519ndash525
84
Dussutour A and S J Simpson 2009 Communal nutrition in ants Current Biology 19740ndash4
Dutton E M and M E Frederickson 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561ndash569
Floren A A Biun and K E Linsenmair 2002 Arboreal ants as key predators in tropical
lowland rainforest trees Oecologia 131137ndash144
Grant B R 1996 Pollen digestion by Darwinrsquos finches and its importance for early breeding
Ecology 77489-499
Herrera L G and C Martiacutenez del Rio 1998 Pollen digestion by new world bats effects of
processing time and feeding habits Ecology 792828ndash2838
Houmllldobler B and Wilson E O 1990 The Ants 1st Edition Harvard University Press
Cambridge
Horskins K and Turner V B 1999 Resource use and foraging patterns of honeybees Apis
mellifera and native insects on flowers of Eucalyptus costata Australian Journal of
Ecology 24221-227
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506ndash514
Roulston T H I and J H Cane 2000 Pollen nutritional content and digestibility for animals
Plant Systematics 222187ndash209
Van Tets I G 1997 Extraction of nutrients from Protea pollen by African rodents Belgian
Journal of Zoology 12759-65
Willmer P G 2011 Pollination and Floral Ecology 1st Edition Princeton University Press
New Jersey
85
Willmer P G C V Nuttman N E Raine G N Stone J G Pattrick K Henson P Stillman
et al 2009 Floral volatiles controlling ant behaviour Functional Ecology 23888ndash900
86
Tables
Table 31 Numbers of adult worker and larval ants examined for each species collected in Peru
Numbers of pollen grains found in individual samples are given in parentheses after the sample
size when there were no grains no numbers are given
Genus Species Number of adults analyzed
(numbers of grains found)
Number of larvae analyzed
(numbers of grains found)
Dolichoderinae
Azteca sp 1 3 0
sp 2 2 0
sp 3 0 2
sp 4 3 (1) 0
sp 5 3 (2) 0
Dolichoderus decollatus 3 (66) 0
sp 1 3 0
sp 2 3 1 (2)
sp 3 3 3
sp 4 0 2
Ecitoninae
Labidus sp 1 3 0
Formicinae
Camponotus sp 1 3 3
sp 2 3 (82) 1 (2)
sp 3 3 (3) 0
sp 4 0 2
87
sp 5 8 (11) 0
sp 6 4 (7 16 468) 0
sp 7 5 (12) 0
sp 8 2 (1) 0
Paratrechina sp 1 4 (11) 0
Myrmelachista sp 1 3 (5 11 18) 0
sp 2 0 3
Trachymyrmex sp 1 3 (21) 0
sp 2 3 0
Myrmicinae
Atta sp 1 3 0
Cephalotes atratus 3 (2 2 21) 0
placidus 3 (1 4 21) 0
sp 1 3 0
sp 2 1 0
sp 3 2 0
Crematogaster sp 1 3 1
sp 2 3 (35) 0
sp 3 9 (7) 0
sp 4 2 0
Megalomyrmex sp 1 3 0
sp 2 0 2
sp 3 4 (7) 0
sp 4 3 0
Monomorium sp 1 0 3 (2)
88
Pheidole sp 1 0 3
sp 2 3 0
sp 3 0 3
Procryptocerus sp 1 3 0
Solenopsis sp 1 0 3
sp 2 3 (3 5) 0
Wasmannia sp 1 3 0
Ponerinae
Pachycondola sp 1 3 (3) 0
Odontomachus sp 1 3 (34) 0
Pseudomyrmicinae
Pseudomyrmex sp 1 3 0
sp 2 3 0
sp 3 3 (9) 0
sp 4 0 3
sp 5 0 3
89
Table 32 Numbers of adult worker and larval ants examined for each species collected in Papua
New Guinea Numbers of pollen grains found in individual samples are given in parentheses
after the sample size when there were no grains no numbers are given
Genus Species Number adults analyzed
(grains found)
Number larvae analyzed
(grains found)
Aenictinae
Aenictus sp 1 3 3 (1 1)
Dolichoderinae
Philidris sp 1 3 1 (4)
Tapinoma sp 1 2 3 (38)
Technomyrmex sp 1 0 3 (12 12)
Ectatomminae
Rhytidoponera sp 1 1 3 (2)
Formicinae
Acropyga sp 1 3 3 (2)
sp 2 3 3
sp 3 3 3
Anoplolepsis sp 1 3 (1) 3 (1)
Calomyrmex sp 1 3 3 (1)
Camponotus sp 1 3 (1 271) 3
sp 2 1 3
sp 3 2 0
Nylanderia sp 1 3 3
Opisthopsis sp 1 3 3
Polyrachis sp 1 1 3 (4)
90
Myrmicinae
Crematogaster sp 1 3 3
sp 2 3 (32) 3
Monomorium sp 1 3 (1) 3 (1)
sp 2 3 3 (1 1 1)
Pheidole sp 1 3 3
Pristomyrmex sp 1 3 3 (11)
Pyramica sp 1 3 0
Ponerinae
Odontomachus sp 1 3 3 (4)
91
Figures
Figure 31 Photographs of pollen grains found in two ant species a) Dolichoderus sp 2 and b)
Camponotus sp 6 Scale bars = 50 microm
92
Figure 32 Relationship between trophic level as measured by δ15
N ratio and pollen presence
for 48 neotropical ant species 1 Atta sp 5 Azteca spp 8 Camponotus spp 5 Cephalotes spp 4
Crematogaster spp 5 Dolichoderus spp 1 Labidus sp 3 Megalomyrmex spp 1 Monomorium
sp 2 Myrmelachista spp 1 Odontomachus sp 1 Pachycondola sp 1 Paratrechina sp 3
Pheidole sp 4 Pseudomyrmex spp 2 Solenopsis spp and 1 Wasmannia sp
93
Concluding Remarks
Ants have traditionally been considered to be unwelcome flower visitors they consume floral
rewards ostensibly meant for pollinators while rarely providing pollination services themselves
In this thesis I investigate the validity of this assumption and use an experimental study and an
observational study to examine two ways that ants may impact plant reproduction
In the first chapter I review the literature on ants as flower visitors I show that the common
assumption of ants always being categorically negative for plant reproduction is incorrect while
ants may reduce floral attractiveness to pollinators the net effect of ants on plant fitness
measures is variable ranging from positive to negative to neutral I also discuss ants as
pollinators and ways that plants may minimize ant visitation to flowers before finishing with a
section highlighting questions that still need investigation
In the second chapter I explore how interference competition with ants can structure bumblebee
foraging behavior and how this may alter pollen movement patterns Using artificial flowers with
male or female function I allowed Bombus impatiens bumblebees to forage on flowers that
either did or did not have Myrmica rubra ants on them Ant presence significantly reduced the
amount of pollen analogue donated and received by flowers Building on these results I found
that the presence of ant scent alone elicited similar changes to bumblebee behavior reducing the
amount of pollen analogue donated by male flowers My results show that interference
competition is sufficient to alter bumblebee foraging behavior and that the effects of ants on
pollinators may extend after ant visitation to flowers Further study is needed to determine if bees
are able to differentiate between ant species by visual or olfactory cues Their ability to do so
94
could affect how generalized their response to ant presence is and may help to better predict how
ants will affect plant reproduction
In the final chapter I investigate how common palynivory is amongst tropical ant species I used
acetolysis and light microscopy to examine whole ants larvae and ant guts from Papua New
Guinea and Peru I found that tropical ants consistently contain pollen grains albeit often in
small numbers (lt5) However some genera notably Camponotus and Cephalotes consistently
contained higher numbers providing the first evidence that Camponotus may be a palynivorous
genus The small numbers of pollen grains present may indicate that ants preferentially exploit
other more easily digestible high-protein foods over pollen Future studies should investigate
exactly what role pollen plays in ant diets by supplementing the diet of captive ant colonies with
pollen This would allow us to examine how pollen consumption affects colony demography as
well as how efficiently ants digest pollen and whether ants exhibit pollen preferences
I bring up several pressing and unanswered questions within this thesis and highlight three here
First numerous plant species possess chemical or physical traits assumed to increase plant
fitness by reducing ant visitation However this has rarely been tested and instead research has
focused predominately on documenting ant repellence We should move away from this and
begin examining plant fitness in the presence or absence of these repellent traits to see how
beneficial these traits truly are Second how flower visiting ants impact male fitness has been
almost completely ignored in favor of female fitness Future studies should investigate how ant
visitation affects both pollen donation and pollen movement patterns Lastly research involving
antibiotics present on ant cuticles and their effect on pollen viability has been too small in scope
often limited to demonstrating the pollen killing effects of ant secretions Investigation into
95
whether plants have evolved any resistance to the secretions of the local ant community or why
some plants appear unaffected by their ant partners may yield exciting results
In conclusion ants have the capacity to strongly affect plant reproduction both positively and
negatively The fact that these diametrically opposing forces exist means we cannot approach
flower-visiting ants as either positive or negative a priori Instead we should strive to work
towards a better understanding of what is occurring in the system of interest and what role ants
play within it
96
Copyright Acknowledgments
Chapter 2 ldquoAnts and ant scent reduce bumblebee pollination of artificial flowersrdquo is current in
press in The American Naturalist Request for its inclusion here was sought and approved
vii
List of Tables
Table 11 List of studies of flower-visiting ants that consumed floral rewards or interacted with
pollinators and included a measure of fitness39
Table 12 List of studies that have either demonstrated ant pollination through exclusion
experiments or whose results strongly suggest it48
Table 31 Numbers of adult worker and larval ants examined for each species collected in Peru
and numbers of pollen grains found87
Table 32 Numbers of adult worker and larval ants examined for each species collected in Papua
New Guinea and numbers of pollen grains found90
viii
List of Figures
Figure 21 Photograph of artificial flower lids with anther and stigma74
Figure 22 Dye transferred (mean plusmn 1SE) by B impatiens a) in the presence and absence of M
rubra ants and b) with or without M rubra scent75
Figure 31 Photographs of pollen grains found inside ants92
Figure 32 Relationship between trophic level and pollen presence for neotropical
ant species93
1
Chapter 1 Ants as flower visitors and their effects on pollinator behavior and plant
reproduction
Cembrowski AR and Frederickson ME
Planned submission to a journal such as Oikos or Insectes Sociaux
ARC wrote the manuscript with input from MEF
Abstract
Ants often provide a range of benefits to flowering plants particularly by reducing herbivory
However when ants visit flowers they can deter pollinators or damage floral structures
Currently it is unclear how often and in what direction flower-visiting ants affect plant
reproductive success A review of the literature shows that ants can help or hinder pollination
processes but overall flower-visiting ants most commonly have a non-significant net effect on
plant fitness Although ants may directly harm flowers or indirectly affect pollination by
consuming floral nectar or harassing pollinators they can also benefit plants by attacking
florivores or predators of pollinators Ants occasionally act as pollinators themselves though ant
secretions often kill pollen grains There is growing evidence that many plants have traits that
allow them to avoid or minimize the negative effects of flower-visiting ants More research is
needed to determine if these traits provide fitness benefits to plants by preventing ant visitation
or if they function as generalized repellents of floral antagonists
2
Introduction
Ants and angiosperms share a long evolutionary history Ant diversification closely followed that
of flowering plants as ants took advantage of the large prey numbers that angiosperms supported
and the new habitats they provided (Moreau 2006) Subsequently numerous angiosperm
lineages evolved intimate associations with ants that function as an indirect or biotic form of
plant defense Food rewards such as extrafloral nectar or pearl bodies or housing in the form of
domatia attract ants to plants where they reduce herbivory and increase plant fitness (reviewed
in Rosumek et al 2009) However the traits that make ants good plant defenders particularly
their abundance attraction to sugar sources and aggressiveness can also cause problems for
plant reproduction Ants attracted by floral nectar can compete with pollinators for resources
and defend flowers potentially reducing plant fitness (Ness 2006 Junker et al 2007) That ants
negatively affect plant reproduction is an old and pervasive hypothesis Kerner (1878) called ants
ldquomost unwelcome guestsrdquo on flowers and more recent authors have similarly expressed that ant
visitation to flowers reduces plant reproductive success (Ness 2006 Lach 2007 Willmer et al
2009)
Nonetheless studies assessing the impact of flower-visiting ants on plant reproductive
success have produced mixed results Ants can have negative impacts on pollination and flower
function (Galen 1983 Puterbaugh 1998 Ness 2006 Hansen and Muumlller 2009) However other
studies have found no effect or even strong benefits of ant visits to flowers (Rico-Gray and
Thien 1989 Altshuler 1999 Schuumlrch 2000 Ashman and King 2005) This discrepancy between
ants being ldquounwelcome guestsrdquo and their actual effects on plant reproduction requires
clarification Our goal here is to highlight the ways that flower-visiting ants may affect plant
reproduction both positively and negatively and to discern through a review of the literature
3
what impacts ants have on plant reproductive success First we discuss mechanisms by which
flower-visiting ants can directly and indirectly impact plant reproduction both through floral
damage and by mediating changes in pollinator or florivore behavior We end this section by
evaluating published research that collectively shows that flower-visiting ants have
predominantly neutral effects on plant fitness measures Next we discuss pollination by ants
focusing on the ldquoantibiotic hypothesisrdquo before describing how plants may prevent ants from
visiting flowers Lastly we discuss promising new avenues of research and highlight work that
still needs to be performed to give a more complete understanding of ants as flower visitors
1 Ant interactions with flowers and floral visitors
i) Damage to flowers
Ants can directly disrupt plant reproduction by damaging floral structures preventing either the
fertilization of ovules or the presentation of pollen Rarely ants forage on floral tissue itself for
example Puterbaugh (1998) found that Formica neorufibarbus ants chewed through the coronal
ring of Eritrichium aretiodes flowers to get lipids More often but still not commonly foraging
ants chew through reproductive tissue to access floral nectar This can cause sterilization of
whole flowers (Echium plantagineum Kirk 1984) or affect female function only if just styles are
damaged effectively creating male flowers (Polemonium viscosum Galen 1983) Antibiotic
secretions on ant integuments may also directly damage pollen grains but the fitness effects of
this are unclear (see 2 below)
Several species of Crematogaster and Allomerus ants sterilize their host plants by
attacking floral buds (Yu and Pierce 1998 Stanton et al 1999 Gaume et al 2005 Maleacute et al
2012 Table 11) These ants nest in ldquoant-plantsrdquo or ldquomyrmecophytesrdquomdashie plants that host ant
4
colonies in specialized structures (domatia) The sterilizing ants do not appear to consume floral
tissues (Yu and Pierce 1998) and two hypotheses may explain why ants destroy floral buds First
ant colony size is often limited by domatia availability on their host plant By destroying floral
buds ants can cause plants to divert resources from reproductive to vegetative growth increasing
domatia number and allowing for larger ant colonies (Yu amp Pierce 1998 Frederickson 2009)
Second when plant-ants must defend their colony from takeovers by other colonies they may
benefit from limiting access to their host plant By destroying buds that could develop into
flowers branches or leaves ants can prevent their host plant from contacting nearby plants
occupied by enemy ant colonies (Stanton et al 1999)
The impact of sterilization on lifetime plant fitness is unclear Ants may destroy all of
their host plantrsquos floral buds (Yu and Pierce 1998) completely preventing reproduction or only a
portion of them (Maleacute et al 2012) allowing some seed set However counter-intuitively this
sterilization behavior may provide long-term fitness benefits to plants (Frederickson 2009
Palmer et al 2010) Ant-plants often host several different ant species over their lifetimes some
of which are non-sterilizing If hosting a sterilizing species at a certain life stage increases
survival or growth relative to hosting a non-sterilizing species at that life stage plants may
increase lifetime fitness by largely forgoing current reproduction in favor of increasing future
reproduction (Palmer et al 2010) Why plants invest resources in producing flowers that are
quickly destroyed by ants is not known but the timing of flower destruction may be important If
ants attack flowers after anthers dehisce plants may still realize some male fitness via pollen
donation (Yu and Pierce 1998) Additionally if the cost of flower production is relatively minor
and ants sometimes ldquomissrdquo flowers (Edwards and Yu 2008) attempting limited reproduction
may be a better strategy than forgoing it entirely
5
ii) Competition with pollinators
Ant activity on flowers can dramatically alter pollinator behavior Ants can reduce the species
diversity frequency or duration of pollinator visits to flowers (Norment 1988 Tsuji et al 2004
Ness 2006 Junker et al 2007 Lach 2007 2008b Romero et al 2011 and see Chapter 2) These
changes are largely due to either exploitative competition (ie consumption of shared resources)
or interference competition (ie direct aggression) between ants and pollinators
a) Exploitative competition
Pollinators and ants frequently compete for floral nectar Flower-visiting ants are often classified
as nectar thieves (sensu Inouye 1980) because they typically enter flowers through the
ldquolegitimaterdquo opening and consume nectar but transfer no pollen (Wyatt 1980 Ness 2006
Chamberlain and Holland 2008) Ants can also act as primary nectar robbers chewing holes
through corollas to reach nectaries (Willmer and Corbet 1981 Koopowitz and Marchant 1998)
although they are more commonly secondary nectar robbers and use holes made by previous
robbers (Newman and Thomson 2005)
Like other flower visitors foraging ants will often leave nectar behind in flowers This
amount can be less (Bleil et al 2011) more (Fritz and Morse 1982) or similar to (Lach 2008a)
amounts left by other visitors Often ants are morphologically constrained from fully exploiting
nectar Unlike bees and butterflies ants do not have proboscises and must get close to nectar to
consume it Narrow corollas or nectar spurs can prevent ant access to some or all floral nectar
(Schemske 1980 Galen and Cuba 2001 and see below) Smaller ants may be better able to
6
access nectar (Newman and Thomson 2005 Agarwal and Rastogi 2007) but can be limited by
their crop size in the amount of nectar they can remove (Lach 2005) If foraging ants leave
enough nectar behind for a pollinator to recover the cost of visiting a flower ants may have
minimal effects on plant reproductive success (Maloof and Inouye 2000 Turner et al 2012)
Differences among ant species in behavior and diet also affect their consumption of floral
nectar Species vary in how many flowers they visit per inflorescence or per plant and in how
many ants will visit a flower simultaneously (Lach 2005 Junker et al 2007 Lach 2013)
Invasive antsmdashthought to be better at exploiting and defending resources than native ants
(Holway et al 2002)mdashoften recruit to flowers in large numbers sometimes several dozen or
more (Visser et al 1999 Hansen and Muumlller 2009) Overall many factors may influence how
heavily ants exploit floral nectar on plants including ant density plant height the spatial or
temporal distribution of floral nectar and the availability of alternative food resources for ants
(Cushman and Addicott 1991 Lach 2013)
Rarely ants and pollinators may compete for pollen We know of only a few studies that
have recorded ants harvesting pollen directly from anthers (Horskins and Turner 1999 Ness
2006 Byk and Del-Claro 2010) Other studies have found pollen grains in ants (eg Baroni
Urbani and de Andrade 1997 and see Chapter 3) but this may not indicate competition with
pollinators ants sometimes collect stray pollen grains from the environment (Creighton 1967)
One New World genus Cephalotes might be specially adapted for pollen consumption They
possess ldquoproventricular shieldsrdquo structures that may restrict solid food to the crop allowing
nestmates to exchange pollen grains via trophallaxis It is unclear if this structure evolved for this
purpose but Cephalotes are the most frequently documented palynivorous ants (eg Creighton
1967 Baroni Urbani and de Andrade 1997 Byk and Del-Claro 2010) How efficiently ants
7
digest pollen and whether ants like bees feed it to larvae as a protein source is unknown At
least two studies have found that ant larvae consume pollen providing some support for this idea
(Wheeler and Bailey 1920 and Chapter 3)
b) Interference competition
Ants are relatively unique among flower visitors in defending floral resources from competitors
(but see Willmer and Corbett 1981 Roubik 1982) This defense ranges from chance encounters
with ants attacking visitors when flower occupancy overlaps (Ashman and King 2005 Junker et
al 2007) to actively patrolling inflorescences or constructing protective galleries around
nectaries (Horvitz and Schemske 1984 Gaume et al 2005 Hansen and Muumlller 2009)
Interference competition even in the absence of exploitative competition is sufficient to alter
pollinator behavior (Chapter 2)
The strength and outcome of interference competition depends on the identities of the ant
and the pollinator Pollinators might not alter their visitation patterns in response to non-
aggressive ants (Junker et al 2007) but more antagonistic ants may elicit avoidance behavior
before or soon after the visitor lands (Willmer and Corbet 1982 Altshuler 1999 Schuumlrch et al
2000 Table 11) Pollinator size may help predict their reaction to ants because ants may deter
only small-bodied pollinators (Gonzaacutelvez et al 2012) It is not clear whether flower visitors view
ants as aggressive competitors or as potential predators rarely ants prey on pollinators (Schatz
and Wcislo 1999) but in most cases ants merely cause pollinators to relocate
How well pollinators recognize and avoid ant-visited flowers is unresolved Collectively
pollinators have lower visitation rates to flowers visited by ants (Romero et al 2011) This is
8
dependent on pollinator identity however dipterans do not appear to discriminate between ant-
visited and ant-free flowers while bees can identify and avoid ant-visited flowers Bees likely
use a combination of visual and olfactory cues to recognize ant-visited flowers They can
identify previous conspecific and some heterospecific insect visitors by the hydrocarbon
ldquofootprintsrdquo they leave during foraging (Stout and Goulson 2001) Bumblebees can detect ant
hydrocarbons although this has been shown only in the laboratory (Ballantyne and Willmer
2012 Chapter 2) No study has yet examined if bees can use sight alone to detect ants but
several suggest it (Junker et al 2007 Gonzaacutelvez et al 2012) Some bees may discriminate
between ant species when visiting flowers (eg Lach 2008b) which follows if not all ant-
pollinator interactions are equally costly The ability of bees to do so will affect how generalized
their avoidance behaviors will be If they can discriminate between species bees may avoid
specific flowers visited by more ldquocostlyrdquo ants However if they cannot bees might either avoid
all ant-visited flowers or none depending on how negative the average interaction with an ant is
iii) Interactions with flower and pollinator antagonists
Ants can defend the flowers they visit Numerous plant species have circumfloral or bracteal
nectaries (ie extrafloral nectaries around flowers) and ants attracted to these nectaries can
reduce bud ovule or flower predation increasing plant fitness (Inouye and Taylor 1979 Rico-
Gray and Thien 1989 Sugiura et al 2006) Although the presence of EFNs (extrafloral nectaries)
so close to floral nectaries (sometimes less than 1 cm Ness 2006) might increase discovery and
exploitation of floral nectar by ants only one study has investigated this hypothesis finding no
relationship between the proximity of EFNs to floral nectaries and antsrsquo use of floral nectar
(Galen 2005) Holes made by nectar robbers are functionally analogous to circumfloral nectaries
9
and can also attract ants that defend flowers (Newman and Thomson 2005) and nectar-thieving
ants can similarly prevent florivory in some systems (Lach 2007 Bleil et al 2011)
Ants can also reduce the abundance of flower-dwelling predators or parasites of
pollinators Ambush predators such as crab spiders often forage at flowers where they may
reduce pollinator visitation rates and plant fitness (Dukas 2001 Gonccedilalvez-Souza et al 2008)
Ants can decrease spider abundance and richness on plants (Rudgers 2004 Renault et al 2005
Nahas et al 2012) and some evidence suggests that they can do the same on flowers (Lach
2008) In select cases ants may also reduce the abundance of pollinator parasitoids (Holland et
al 2011) Whether ants increase plant reproductive success by removing pollinator antagonists
has not yet been examined but should be If ants create lsquoenemy-freersquo flowers pollinators might
prefer to land on flowers with ants
iv) Net effects
Table 11 compiles studies in which the authors recorded ants consuming floral rewards or
interacting with pollinators and measured some aspect of plant fitness (eg fruit or seed set
pollen deposition) We included only a single study per plant species unless subsequent studies
found conflicting results or measured the fitness of another plant sex Records of ant pollination
are included in a separate table (Table 12)
Table 11 reveals relatively few examples of flower-visiting ants negatively affecting
plant fitness given how costly ant visitation is assumed to be The net effect of ants on plant
reproductive success is actually quite variable with similar numbers of studies finding positive
negative or neutral effects of ant presence Surprisingly when ants do have negative effects it is
10
usually because they directly damage floral structures and not because they prevent pollinators
from visiting flowers and successfully transferring pollen Why are ants not more often negative
for plant reproduction when this is commonly assumed to be true
First ants may compensate for reducing pollinator visitation rates by improving
pollination quality As pollinators move between flowers on a plant they often deposit self-
pollen on stigmas (ie geitonogamy) In some plants with late-acting self-incompatibility ovules
fertilized by self-pollen are subsequently aborted reducing the number of seeds that can be
produced (Barrett 2002 and references therein) Geitonogamy can also reduce offspring viability
or vigor (de Jong et al 1993) and cause pollen discounting a reduction in the amount of pollen
available for outcrossing because of self-pollination (Barrett 2002) By competing with
pollinators ants might decrease rates of geitonogamous pollination if pollinators then visit fewer
sequential flowers within a plant Ants might also cause pollinators to fly further between plants
potentially increasing the genetic diversity of pollen arriving at stigmas Although such effects
have been reported for other floral larcenists (Maloof 2001 Irwin 2003) to our knowledge these
hypotheses have not been investigated in relation to ants
Second ants and pollinators do not always forage for the same resources possibly
allowing them to partition floral rewards If ants forage for nectar but not pollen and flowers can
be pollinated efficiently by visitors looking to gather mostly pollen and little or no nectar then
plant reproduction will not be affected by ants consuming floral nectar Junker et al (2010a)
found that honeybees foraged for both pollen and nectar in the absence of ants but reduced their
foraging on nectar but not pollen when ants could access flowers resulting in no difference in
fruit set between treatments Whether pollinators commonly adjust their foraging behaviors in
response to ants warrants further research
11
Third ants are somewhat unique among floral larcenists in that they often provide direct
benefits to the plants they visit In the studies listed in Table 11 ants sometimes decreased
herbivory on plants (Chamberlain and Holland 2008 Bleil et al 2011) or otherwise aided the
plant (eg ant presence repelling smaller less effective pollinators Gonzaacutelvez et al 2012) If the
positive effects of ants cancel or outweigh the negative effects of flower-visiting ants on
pollinators then ant visitation may be neutral or even positive for plant fitness
Fourth ants may impose fitness costs to plants not captured in the studies in Table 11
Nectar robbers have stronger negative effects when plants are pollen-limited (Burkle et al 2007)
but only one study in Table 11 assessed whether flowers would set more seed if they received
more pollen finding no evidence of pollen limitation (Lach 2008a Table 11) Even if ants
decrease pollinator visitation rates there is limited scope for ants to decrease plant reproductive
success when plants are not pollen-limited Furthermore nearly all the studies in Table 11
measured only female fitness Only three quantified aspects of male fitness (Wyatt 1980 Fritz
and Morse 1981 Galen and Geib 2007) and we know of only one other study in a system in
which ants occasionally pollinate that has also measured male fitness (Ashman 2000) All four
studies measured pollen or pollinia removal rates and none found any effect of flower-visiting
ants on male fitness However whether other measures of male fitness (eg seeds sired pollen
deposition patterns) would reveal similar or different effects needs investigation
Finally although floral defenses against ants (see 3 below) may prevent us from
observing the negative effects of ants on plant reproductive success we consider this unlikely
Several studies have examined interactions between ants and plants that share little or no
coevolutionary history usually in the context of ant invasions Although invasive ants often
recruit to flowers in larger numbers than native ants they do not appear to reduce plant
12
reproductive success (Blancafort and Goacutemez 2005 Lach 2007 Lach 2008a but see Hansen and
Muumlller 2009) And in Hawaii where angiosperms evolved for millions of years in the absence of
ants and are thought to have secondarily lost floral defenses against ants (Junker et al 2011a)
there is no evidence that the preponderance of recently introduced flower-visiting ants negatively
affects plant fitness (Junker et al 2010a Bleil et al 2011)
2 Ants as pollinators
Ants may directly benefit plant reproduction by acting as pollinators but this is a relatively rare
occurrence Ants have long been considered poor pollinators (Kerner 1878) Initially this was
based on morphological and behavioral differences between ants and recognized pollinators
Worker ants are wingless and often relatively hairless making them poor pollen dispersers As
well because many ants exhibit Ortstreue (literally ldquoplace fidelityrdquo) ants were thought to return
to the same branch or flower repeatedly effecting no cross pollination (Houmllldobler and Wilson
1990) Though largely true these traits do not necessarily preclude ants from acting as
pollinators (Table 12) Although they commonly have restricted foraging ranges (Houmllldobler and
Wilson 1990) some ants can effect outcrossing (eg Ashman and King 2005 Carvalheiro 2008)
and many ants are as hairy if not hairier than their pollinating counterparts
In the late 1970s the ldquoantibiotic hypothesisrdquo was put forward to explain the rarity of ant
pollination Ants use antibiotic secretions to protect themselves against the fungal and bacterial
pathogens common in their nests and environs Iwanami and Iwadare (1978) found that one of
these compounds myrmicacin also greatly reduces pollen germination rates Subsequently
Beattie et al (1984 1985) found that short periods of contact (le 30 minutes) with an antrsquos
integument caused pollen grains to fail to germinate Some of these pollen-killing compounds
13
were traced back to the ant metapleural gland which produces hygienic secretions (Houmllldobler
and Wilson 1990) although contact with ants lacking metapleural glands can also reduce pollen
viability (Beattie et al 1985 Dutton and Frederickson 2012) Ant mandibular or anal gland
secretions and potentially ant venom may likewise defend ants against pathogens and have
similar effects on pollen germination (Beattie et al 1985 Graystock and Hughes 2011)
Nonetheless ant pollination has been documented repeatedly There are at least 18 cases
in which ant pollination has been shown experimentally and five others in which observations
strongly suggest it (Table 12) The majority of these plants fit the ldquoant pollination syndromerdquo
originally proposed by Hickman (1974) dense stands of short plants with small flowers and
easily accessible nectaries Typically ant-pollinated plants are associated with hot and dry
climates (principally in the Mediterranean) but dry alpine environments may also favor the
evolution of ant pollination (eg Svensson 1985 Puterbaugh 1998 Schiestl and Glaser 2011) In
most cases ants appear to be the sole or major pollinator of the plant in question (Hickman 1974
Peakall et al 1990 de Vega et al 2009) although ants sometimes make only supplementary
contributions to pollination (Schuumlrch et al 2000 Ashman and King 2005) This suggests that co-
evolution between ants and plants may not be necessary for ants to be useful pollinatorsmdashwhich
is surprising given the potential negative effects of ants on plant reproduction
Ant-pollinated plants cope with ant secretions in a variety of ways Several plant species
may simply tolerate pollen damage (Gόmez and Zamora 1992 de Vega et al 2009) apparently
possessing no overt adaptations to prevent it These plants might receive enough pollen to
overcome reduced pollen viability Other plants may place pollen on ant body parts thought to
have fewer secretions thereby potentially escaping damage (eg on ant legs Ramsey 1995) or
attach pollen to ants with specialized structures avoiding direct contact with the ant integument
14
(Peakall et al 1990) It is not known whether pollen evolves resistance to ant secretions or
whether plants deposit pollen on ants which have less disruptive secretions although these
possibilities have been suggested elsewhere (eg Garciacutea et al 1995 Ramsey 1995)
One key question regarding the effects of ant secretions on pollen grains urgently needs
to be resolved Several studies have suggested that the pollen-killing secretions of ants may have
contributed to the evolution of floral defenses against ants (eg Wagner 2000 Galen and Geib
2007) For this to be true ants must decrease male fitness by damaging pollen grains before they
leave flowers and are deposited on receptive stigmas Only two studies have investigated the
effects of ant secretions on pollen still in anthers (Wagner 2000 Galen and Butchard 2003) and
both constrained ants unnaturally to flowers for significantly longer than ants would normally
stay on flowers Although these studies found strong reductions in pollen vigor it is unclear if
ant secretions decrease male fitness under natural ant visitation conditions
3 Floral defenses against ants
Plants are not passive recipients of flower visitors and have some ability to ldquochooserdquo which
organisms can access floral rewards Flowers resist ants using both structural and chemical traits
More thorough investigation is needed to determine whether these traits evolved to prevent or
minimize the negative effects of ants on plant reproductive success (ie whether these traits can
truly be considered defenses against ants see 4ia below) It is unlikely however that ants
experience reciprocal selection to circumvent floral resistance traits because the fitness benefits
to ants of accessing flowers are probably small Thus there may be little scope for
coevolutionary arms races between floral defenses against ants and ant counter-adaptations
15
i) Structural resistance traits
a) Restricting ant access
Numerous plant species have physical structures preventing ant access to flowers Thin pedicels
can limit antsrsquo gripping abilities and make ants easier to dislodge when flowers are moved by
wind (Gaume et al 2005) and waxy secretions can render stems too slippery for ants to climb
(Harley 1991) Structures surrounding flowers such as water moats or dense hairs may similarly
keep ants from accessing floral rewards (Willmer et al 2009) Many such traits function as broad
filters and prevent not only ants but most crawling insects from reaching flowers Thus it is
difficult to determine whether ants florivores or both acted as selective agents favoring the
evolution of physical barriers to flowers
Flower shape may also reduce ant-pollinator conflict Long narrow corollas or nectar
spurs can prevent all but the smallest ants from accessing floral nectaries (Bluumlthgen et al 2004
Agarwal and Rastogi 2007) while still allowing long-tongued pollinators access to rewards
Selection to exclude nectar-thieving ants in such a manner however may be counteracted by
pollinator preferences for wider corollas that allow easier flower handling (Galen and Cuba
2001) Additionally these types of flowers may be more prone to robbery (Irwin and Maloof
2002) limiting their usefulness in preventing nectar larceny
b) Separation in space or time
Plants may separate floral rewards and ants temporally or spatially to minimize ant impacts on
plant reproduction Several authors have proposed that myrmecophytes have adapted to the
16
sterilization or otherwise detrimental activities of ants by locating domatia or food rewards on
different branches than flowers (Izzo and Vasconcelos 2002 Raine et al 2002 Edwards and Yu
2008) Temporal separation of ant and pollinator activities could likewise reduce conflict though
evidence is limited Some acacia flowers dehisce during periods of low ant activity (Willmer and
Stone 1997 Nicklen and Wagner 2006) but it is unclear if this is in response to ants
temperature or pollinator activity Timing of nectar secretion may similarly reduce ant-pollinator
competition If ants are active prior to pollinators they may deplete floral nectar and reduce
pollinator visitation rates (Schaffer et al 1984) However if nectar is secreted when ants are
inactive pollination may occur unimpeded (Norment 1988) This temporal separation could
favor pollinators that are active when ants are not (eg Wyatt and Morse 1981) but this is
poorly documented Further studies particularly focusing on flowers with the potential for
nocturnal and diurnal pollination may provide more examples
c) Extrafloral nectaries (EFNs)
One hypothesis put forward to explain the evolution of EFNs proposes that EFNs ldquodistractrdquo ants
from consuming floral nectar (Kerner 1878 Wagner and Kay 2002) Wagner and Kay (2002)
tested this hypothesis by constructing model plants using artificial nectaries assigned at random
to represent either floral or extrafloral nectaries They found that the presence of EFNs could
reduce ant exploitation of floral nectar However as highlighted by Galen (2005) this design is
closer to a plant making a surplus of flowers some of which will avoid ant visitation if visitation
rates increase sublinearly with increasing flower number Producing extra flowers to escape ant
visitation appears to be a viable strategy in some systems (Ashman and King 2005 Galen 2005)
However Galen (2005) found that applying honey (simulating extrafloral nectar) to P viscosum
17
leaves had the opposite effect and increased ant visits to flowers Only two studies both in
Pachycereus schottii have found evidence for the distraction hypothesis under field conditions
(Chamberlain and Holland 2008 Holland et al 2011)
There is limited empirical support for the distraction hypothesis but abundant evidence
that EFNs attract ants that defend plants against herbivores (reviewed in Rosumek et al 2009)
To function as attractants the presence of EFNs must increase ant abundance on plants
However because of the increased numbers attracted EFNs would not be expected to reduce the
abundance of ants on nectar rewards or be able to satiate the ants recruited Only if ant
abundance stops increasing at a reward density much lower than that available on the plant
would EFNs serve to distract ants In this case the amount of rewards may overwhelm ants
leaving some flowers underexploited However even in P schottii ants still visited flowers
(Chamberlain and Holland 2008) In cases where ant abundance would continue to increase with
additional rewards it is unlikely that EFNs function as distractions
ii) Chemical resistance traits
a) Toxic nectar
One of the earliest proposed floral defenses against ants was toxic nectar (Janzen 1977)
However studies testing Janzenrsquos (1977) hypothesis uncovered only a very few isolated
examples (eg Catalpa speciosa Stephenson 1981 Crinum erubescens Guerrant and Fiedler
1981) in short most floral nectar is not toxic or repellent to ants (Baker and Baker 1978
Schubart and Anderson 1978) In fact ants sometimes prefer floral nectar over solutions with
equivalent sugar concentrations (Koptur and Truong 1998)
18
In contrast floral nectar can repel other flower visitors (eg birds Johnson et al 2006
lepidopterans Kessler and Baldwin 2007) Repellency is often caused by plant secondary
metabolites which may occur in nectar as a by-product of their production elsewhere in the
plant where they function as defenses against herbivory (Adler 2001) Alternatively toxic or
repellent nectar may be adaptive repellent nectar may promote effective pollination by reducing
the amount of reward any one pollinator takes spreading nectar over several pollinator visits
(Kessler and Baldwin 2007) or it may function as a filter selecting for specific pollinators
(Johnson et al 2006) The latter may explain why repellent nectar is not more common
legitimate pollinators in addition to floral larcenists may be deterred from visiting flowers with
repellent nectar This may be particularly true of nectars that would repel ants which are closely
related to bees nectar distasteful to ants may often also be distasteful to bees and those nectars
acting as a filter for bees may still be palatable to ants For example Prŷs-Jones and Willmer
(1992) found that the ammonia-containing nectar of Lathraea clandestina is readily consumed
by both bees and ants even though it deters birds that act as nectar thieves Although floral
nectar itself rarely repels ants Guerrant and Fielder (1981) found that the presence of macerated
petals within nectar can often keep ants away Alkaloids in petals may be responsible and might
explain why ants seldom act as primary nectar robbers these secondary compounds may prevent
ants from chewing through corollas
b) Floral volatiles
Repellent floral volatiles appear to be a more widespread form of chemical resistance against
ants These were reviewed recently by Willmer et al (2009) and will be discussed here only
briefly Numerous plant taxa produce floral scents repellent to ants including myrmecophilous
19
myrmecophytic and non-ant associated plants in both temperate and tropical ecosystems
(Willmer et al 2009 Junker et al 2011a Ballantyne and Willmer 2012) In three large studies
floral volatiles that repel ants were detected in about a third of all plants tested (Willmer et al
2009 Junker et al 2011a Ballantyne and Willmer 2012) Pollen is often the source of ant-
repellent volatile compounds (Willmer et al 2009 Ballantyne and Willmer 2012) but they can
be emitted from other floral parts including petals That pollen is often the source of repellence
could suggest that preventing ant visitation may be more important in protecting male than
female function Additionally because repellence would decrease as pollen is removed from
anthers ants may return and defend flowers more quickly than if repellent chemicals were in
longer-lasting structures (eg petals) In some cases floral volatiles are thought to mimic ant
alarm pheromones (Willmer et al 2009) However floral scents elicit inconsistent reactions
across ant species ranging from repellence to attraction (Agarwal and Rastogi 2007 Ballantyne
and Willmer 2012)
Producing structural or chemical resistance traits may be costly for plants Native
Hawaiian plants which have little recent coevolutionary history with ants appear to have
secondarily lost floral ant repellents (Junker et al 2011a) Both Willmer et al (2009) and Junker
et al (2011a) found that highly chemical repellent flowers did not have strong physical defenses
and vice versa suggesting trade-offs between the two (but see Ballantyne and Willmer 2012)
Like toxic nectar floral ant repellents might sometimes deter both pollinators and ants For
example Galen et al (2011) found that 2-phenylethanol in P viscosum floral scent repels both
flower-damaging F neorufibarbis ants and the plantrsquos main pollinator Bombus kirbyellus
Interestingly four recent studies have found that floral scents can also attract ants
(Agarwal and Rastogi 2007 Edwards and Yu 2008 Schiestl and Glaser 2011 Gonzaacutelvez et al
20
2012) One study (Schiestl and Glaser 2011) has shown that floral scents attract ants to a
potentially ant-pollinated plant and such systems may provide many more examples
4 Future directions
We have highlighted areas in need of further investigation throughout the review Here we focus
on several questions we consider high priorities for future research
i) Floral defenses against ants
a) Do floral resistance traits increase plant fitness
The existence ant-repellent floral traits is often taken as evidence that ants negatively affect plant
reproductive success However despite the abundance and variety of mechanisms excluding ants
from flowers few studies show that ants reduce plant fitness when they visit flowers (Table 11)
This absence is particularly apparent with repellent floral volatiles which may be the most
widespread floral resistance trait (Willmer et al 2009 Junker et al 2011a Ballantyne and
Willmer 2012) To our knowledge only one study has shown that floral ant repellents benefit
plants with repellent P viscosum flowers avoiding damaging ant visits (Galen et al 2011)
Research is urgently needed to investigate if these traits increase plant fitness by excluding ants
or if ant visitation is inconsequential More studies taking advantage of standing variation in
traits (Galen et al 2011) or creating it artificially (eg Junker et al 2010b) will allow us to
distinguish between the two
21
b) If ant visitation is often not costly what caused the evolution of repellent traits and
what maintains them Similarly are these repellent traits truly defenses against ant
visitation or are they generalized defenses against a range of floral antagonists
Ant-repellent floral traits are widespread and their production may be costly (Willmer et al
2009 Junker et al 2011a) The studies in Table 11 however indicate that flower-visiting ants
often do not disrupt plant reproduction particularly outside of myrmecophytes What then
selected for these traits One possibility is that these repellents are not directed solely at ants
Instead a community of antagonistic flower visitors may exert diffuse selection on plants
thereby selecting for and maintaining these traits Structural modifications may repel crawling
insects while chemical repellents such as floral volatiles may repel both flying and non-flying
insects (Junker et al 2011b) How much overlap there is in floral volatiles that repel both ants and
other antagonists needs investigation Until we examine how specific these defenses are to ants
we risk overstating the importance of ants in their evolution
ii) Do ants alter pollen movement patterns or reduce male fitness
We are aware of only four studies that have measured if ant visitation impacts male fitness
(Wyatt 1980 Fritz and Morse 1981 Ashman 2000 Galen and Geib 2007) While none of these
studies found an effect all measured only amounts of pollen or pollinia removed However this
measure does not adequately encompass male fitness alterations to pollen movement patterns
and eventual deposition location may be more important to male function than changes in
removal rates If as suggested by some studies (Altshuler 1999 Lach 2007) ants increase
pollinator relocation rates they may decrease pollen discounting increasing male fitness
However if this increased relocation reduces overall pollen export rates ant competition with
22
pollinators may depress male function Studies using pollen-tracking methods such as fluorescent
dyes will allow us to see how ants change pollen movement patterns and methods like paternity
analysis will reveal how competition between ants and pollinators alters male fitness
iii) Coevolution between ants and plants do myrmecophytes exhibit stronger more
specific defenses against ants
The floral traits of myrmecophytes are most likely to coevolve with ants because all plant
reproduction occurs in the presence of ants Their floral defenses would thus be expected to be
both stronger and more specific to their resident ants than those other plants visited by an array
of more opportunistic ant species Some evidence supports this Willmer et al (2009) found that
resident ants of acacias were repelled by flowers with more aggressive ant species showing
stronger repellence than less aggressive ants Additionally ants not normally associated with
acacias did not respond to acacia floral volatiles More studies are needed in other systems and
should take advantage of phylogenetic approaches examining floral traits in closely related
myrmecophytic and non-myrmecophytic plant species to determine whether ants commonly
select for floral defenses against ants in myrmecophytes
Acknowledgements
For comments and discussion we thank the Frederickson lab particularly Eric Youngerman
Lina Arcila Hernandez and Kyle Turner as well as Alison Parker and Jordan Pleet MEF
acknowledges financial support from an NSERC Discovery Grant a Connaught New Researcher
23
Award an Ontario Ministry of Economic Development and Innovation Early Researcher Award
and the University of Toronto ARC was supported by an Ontario Graduate Scholarship and
Sigma Xi
24
References
Adler L S 2001 The ecological significance of toxic nectar Oikos 91409ndash420
Agarwal V M and N Rastogi 2007 Role of floral repellents in the regulation of flower visits
of extrafloral nectary-visiting ants in an Indian crop plant Ecological Entomology 3359ndash
65
Altshuler D L 1999 Novel interactions of non-pollinating ants with pollinators and fruit
consumers in a tropical forest Oecologia 119600ndash606
Ashman TL 2000 Pollinator selectivity and its implications for the evolution of dioecy and
sexual dimorphism Ecology 812577-2591
Ashman TL and E A King 2005 Are flower-visiting ants mutualists or antagonists A study
in a gynodioecious wild strawberry American Journal of Botany 92891ndash5
Ballantyne G and P Willmer 2012 Floral visitors and ant scent marks noticed but not used
Ecological Entomology 37402-409
Ballantyne G and P Willmer 2012 Nectar theft and floral ant-repellence a link between
nectar volume and ant-repellent traits PloS One 7e43869
Baker H G and I Baker 1978 Ants and flowers Biotropica 1080
Barrett S C H 2002 Sexual interference of the floral kind Heredity 88154ndash159
Baroni Urbani C and M L de Andrade 1997 Pollen eating storing and spitting by ants
Naturwissenschaften 84256ndash258
25
Beattie A J C Turnbull R B Knox and E G Williams 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany
71421ndash426
Beattie A J C Turnbull T Hough S Jobson and R B Knox 1985 The vulnerability of
pollen and fungal spores to ant secretions evidence and some evolutionary implication
American Journal of Botany 72606ndash614
Blancafort X and C Goacutemez 2005 Consequences of the Argentine ant Linepithema humile
(Mayr) invasion on pollination of Euphorbia characias (L) (Euphorbiaceae) Acta
Oecologica 2849ndash55
Bleil R N Bluumlthgen and R R Junker 2011 Ant-plant mutualism in Hawailsquoi Invasive ants
reduce flower parasitism but also exploit floral nectar of the endemic shrub Vaccinium
reticulatum (Ericaceae) Pacific Science 65291ndash300
Bluumlthgen N G Gottsberger and K Fiedler 2004 Sugar and amino acid composition of ant-
attended nectar and honeydew sources from an Australian rainforest Austral Ecology
29418ndash429
Burkle L A R E Irwin and D A Newman 2007 Predicting the effects of nectar robbing on
plant reproduction implications of pollen limitation and plant mating system American
Journal of Botany 941935ndash1943
Byk J and K Del-Claro 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333ndash38
26
Caballero P C G Ossa W L Gonzaacuteles C Gonzaacutelez-Browne G Astorga M M Muruacutea and
R Medel 2013 Testing non-additive effects of nectar-robbing ants and hummingbird
pollination on the reproductive success of a parasitic plant Plant Ecology 214633ndash640
Carvalheiro L G E R M Barbosa and J Memmott 2008 Pollinator networks alien species
and the conservation of rare plants Trinia glauca as a case study Journal of Applied
Ecology 451419ndash1427
Chamberlain S A and J N Holland 2008 Density-mediated context-dependent consumer-
resource interactions between ants and extrafloral nectar plants Ecology 891364ndash1374
Creighton W S 1967 Studies on free colonies of Cryptocerus texanus Satschi (Hymenoptera
Formicidae) Psyche 7434ndash42
Cursach J and J Rita 2011 Implications of the reproductive biology of the narrow endemic
Naufraga balearica (Apiaceae) for its conservation status Plant Systematics and Evolution
298581ndash596
Cushman JH and Addicott JF In Huxley CR and DF Cutler (eds) 1st edition Ant-plant
interactions Oxford University Press Oxford
de Jong T J N M Waser and P G Klinkhamer 1993 Geitonogamy the neglected side of
selfing Trends in Ecology and Evolution 8321ndash325
de Vega C M Arista P L Oritz C M Herrera and S Talavera 2009 The ant-pollination
system of Cytinus hypocistis (Cytinaceae) a Mediterranean root holoparasite Annals of
Botany 4517ndash8
27
Dukas R 2001 Effects of perceived danger on flower choice by bees Ecology Letters 4327ndash
333
Dutton E M and M E Frederickson 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561ndash569
Edwards D P and D W Yu 2008 Tolerating castration by hiding flowers in plain sight
Behavioral Ecology and Sociobiology 6395ndash102
Frederickson M E 2009 Conflict over reproduction in an ant-plant symbiosis why Allomerus
octoarticulatus ants sterilize Cordia nodosa trees The American Naturalist 173675ndash81
Fritz R S and D H Morse 1981 Nectar parasitism of Asclepias syriaca by ants effect on
nectar levels pollinia insertion pollinaria removal and pod production Oecologia 50316ndash
319
Galen C 1983 The effects of nectar thieving ants on seedset in floral scent morphs of
Polemonium viscosum Oikos 41245ndash249
Galen C 2005 Catching ants with honey an experimental test of distraction and satiation as
alternative modes of escape from flower-damaging ants Oecologia 14480ndash87
Galen C and B Butchart 2003 Ants in your plants effects of nectar-thieves on pollen fertility
and seed-siring capacity in the alpine wildflower Polemonium viscosum Oikos 101521ndash
528
Galen C and J Cuba 2001 Down the tube pollinators predators and the evolution of flower
shape in the alpine skypilot Polemonium viscosum Evolution 551963-1971
28
Galen C and J C Geib 2007 Density-dependent effects of ants on selection for bumble bee
pollination in Polemonium viscosum Ecology 881202ndash1209
Galen C R Kaczorowski S L Todd J Geib and R A Raguso 2011 Dosage-dependent
impacts of a floral volatile compound on pollinators larcenists and the potential for floral
evolution in the alpine skypilot Polemonium viscosum The American Naturalist 177258ndash
272
Garciacutea M B R J Antor and X Espadaler 1995 Ant pollination of the palaeoendemic
dioecious Borderea pyrenaica (Dioscoreaceae) Plant Systematics and Evolution 19817ndash
27
Gaume L M Zacharias and R M Borges 2005 Ant ndash plant conflicts and a novel case of
castration parasitism in a myrmecophyte Evolutionary Ecology Research 7435ndash452
Graystock P and W O H Hughes 2011 Disease resistance in a weaver ant Polyrhachis
dives and the role of antibiotic-producing glands Behavioral Ecology and Sociobiology
652319ndash2327
Gόmez J M 2000 Effectiveness of ants as pollinators of Lobularia maritima effects on main
sequential fitness components of the host plant Oecologia 12290ndash97
Gόmez J M and R Zamora 1992 Pollination by ants consequences of the quantitative effects
on a mutualistic system Oecologia 91410ndash418
Gόmez J M R Zamora J A Hόdar and D Garciacutea 1996 Experimental study of pollination
by ants in Mediterranean high mountain and arid habitats Oecologia 105236ndash242
29
Gonccedilalves-Souza T P M Omena J C Souza and G Q Romero 2008 Trait-mediated effects
on flowers artificial spiders deceive pollinators and decrease plant fitness Ecology
892407ndash2413
Gonzaacutelvez F G L Santamariacutea R T Corlett and M A Rodriacuteguez-Gironeacutes 2012 Flowers
attract weaver ants that deter less effective pollinators Journal of Ecology 10178ndash85
Guerrant E O and P L Fiedler 1981 Flower defenses against nectar-pilferage by ants
Biotropica 1325ndash33
Harley R 1991 The greasy pole syndrome In Huxley CR and DF Cutler (eds) 1st edition
Ant-plant interactions Oxford University Press Oxford
Hansen D M and C B Muumlller 2009 Invasive ants disrupt gecko pollination and seed
dispersal of the endangered plant Roussea simplex in Mauritius Biotropica 41202ndash208
Hickman J C 1974 Pollination by ants a low-energy system Science 1841290ndash1292
Holland J N S a Chamberlain and T E X Miller 2011 Consequences of ants and
extrafloral nectar for a pollinating seed-consuming mutualism ant satiation floral
distraction or plant defense Oikos 120381ndash388
Houmllldobler B and Wilson E O 1990 The Ants 1st Edition Harvard University Press
Cambridge
Holway DA L Lach AV Suarez ND Tsutsui and TJ Case 2002 The causes and
consequences of ant invasions Annual Review of Ecology and Systematics 33181-233
30
Horskins K and Turner VB 1999 Resource use and foraging patterns of honeybees Apis
mellifera and native insects on flowers of Eucalyptus costata Australian Journal of
Ecology 24221-227
Horvitz C C and D W Schemske 1984 Effects of ants and an ant-tended herbivore on seed
production of a neotropical herb Ecology 651369ndash1378
Inouye DW 1980 The terminology of floral larceny Ecology 611251-1253
Inouye DW and O R J Taylor 1979 A temperate region plant-ant-seed predator system
consequences of extra floral nectar secretion by Helianthella quinquenervis Ecology 601ndash
7
Irwin RE 2003 Impact of nectar robbing on estimates of pollen flow conceptual predictions
and empirical outcomes Ecology 84485-495
Irwin RE and Maloof JE 2002 Variation in nectar robbing over time space and species
Oecologia 133525-533
Iwanami Y and T Iwadare 1978 Inhibiting effects of myrmicacin on pollen growth and pollen
tube mitosis Botanical Gazette 13942ndash45
Izzo TJ and Vasconcelos HL 2002 Cheating the cheater domatia loss minimizes the effects
of ant castration in an Amazonian ant plant Oecologia 133200-205
Janzen D H 1977 Why donrsquot ants visit flowers Biotropica 9252
Johnson SD Hargreaves AL and Brown M 2006 Dark bitter-tasting nectar functions as a
filter of flower visitors in a bird-pollinated plant Ecology 872709-2716
31
Junker R A Y C Chung and N Bluumlthgen 2007 Interaction between flowers ants and
pollinators additional evidence for floral repellence against ants Ecological Research
22665ndash670
Junker R R R Bleil C C Daehler and N Bluumlthgen 2010a Intra-floral resource partitioning
between endemic and invasive flower visitors consequences for pollinator effectiveness
Ecological Entomology 35760ndash767
Junker R R and N Bluumlthgen 2010b Floral scents repel facultative flower visitors but attract
obligate ones Annals of botany 105777ndash82
Junker R R C C Daehler S Doetteri A Keller and N Bluumlthgen 2011a Hawaiian ant ndash
flower networks nectar-thieving ants prefer undefended native over introduced plants with
floral defenses Ecological Monographs 81295ndash311
Junker R R J Gershenzon and S B Unsicker 2011b Floral odor bouquet loses its ant
repellent properties after inhibition of terpene biosynthesis Journal of Chemical Ecology
371323ndash31
Kawakita A and M Kato 2002 Floral biology and unique pollination system of root
holoparasites Balanophora kuroiwai and B tobiracola (Balanophraceae) American
Journal of Botany 891164ndash1170
Koopowitz H and TA Marchant 1998 Postpollination nectar reabsorption in the African
epiphyte Aerangis verdickii (Orchidaceae) American Journal of Botany 85508-512
Kirk W D J 1984 Ecological studies on Thrips imaginis Bagnall (Thysanoptera) in flowers of
Echium plantagineum L in Australia Australian Journal of Ecology 99ndash18
32
Kerner A 1878 Flowers and their unbidden guests C Kegan Paul London
Kessler D and I T Baldwin 2007 Making sense of nectar scents the effects of nectar
secondary metabolites on floral visitors of Nicotiana attenuata The Plant Journal 49840ndash
54
Koptur S and N Truong 1998 Facultative ant-plant interactions nectar sugar preferences of
introduced pest species in South Florida Biotropica 30179-189
Lach L 2005 Interference and exploitation competition of three nectar-thieving invasive ant
species Insectes Sociaux 52257ndash262
Lach L 2007 A mutualism with a native membracid facilitates pollinator displacement by
Argentine ants Ecology 881994ndash2004
Lach L 2008a Argentine ants displace floral arthropods in a biodiversity hotspot Diversity and
Distributions 14281ndash290
Lach L 2008b Floral visitation patterns of two invasive ant species and their effects on other
hymenopteran visitors Ecological Entomology 33155ndash160
Lach L 2013 A comparison of floral resource exploitation by native and invasive Argentine
ants Arthropod-Plant Interactions 7177-190
Luo C W K Li X M Chen Z Y Huang and K U N Li 2012 Ants contribute significantly
to the pollination of a biodiesel plant Jatropha curcas Environmental Entomology
411163ndash1168
33
Maleacute PJ G C Leroy A Dejean A Quilichini and J Orivel 2011 An ant symbiont directly
and indirectly limits its host plantrsquos reproductive success Evolutionary Ecology 2655ndash63
Maloof J E 2001 The effects of a bumble bee nectar robber on plant reproductive success and
pollinator behavior American Journal of Botany 881960ndash1965
Maloof J E and D W Inouye 2000 Are nectar robbers cheaters or mutualists Ecology
812651ndash2661
Moreau CS CD Bell R Vila SB Archibald and NE Pierce 2006 Phylogeny of the ants
diversification in the age of the angiosperms Science 312101-104
Nahas L M O Gonzaga and K Del-Claro 2012 Emergent impacts of ant and spider
interactions herbivory reduction in a tropical savanna tree Biotropica 44498ndash505
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506ndash514
Newman D A and J D Thomson 2005 Interactions among nectar robbing floral herbivory
and ant protection in Linaria vulgaris Oikos 110497ndash506
Nicklen E F and D Wagner 2006 Conflict resolution in an ant-plant interaction Acacia
constricta traits reduce ant costs to reproduction Oecologia 14881ndash7
Norment C J 1988 The effect of nectar-thieving ants on the reproductive success of Frasera
speciosa (Gentianaceae) American Midland Naturalist 120331ndash336
34
Orivel J L Lambs P-J G Maleacute C Leroy J Grangier T Otto A Quilichini et al 2011
Dynamics of the association between a long-lived understory myrmecophyte and its
specific associated ants Oecologia 165369ndash76
Palmer T M D F Doak M L Stanton J L Bronstein E T Kiers T P Young J R
Goheen et al 2010 Synergy of multiple partners including freeloaders increases host
fitness in a multispecies mutualism Proceedings of the National Academy of Sciences
10717234ndash9
Peakall R A J Beattie and S H James 1987 Pseudocopulation of an orchid by male ants a
test of two hypotheses accounting for the rarity of ant pollination Oecologia 73522ndash524
Peakall R and A J Beattie 1989 Pollination of the orchid Microtis parviflora R Br by
flightless worker ants Functional Ecology 3515ndash522
Peakall R C J Angus A J Beattie 1990 The significance of ant and plant traits for ant
pollination in Leporella fimbriata pollination Oecologia 84457ndash460
Puterbaugh M N 1998 The roles of ants as flower visitors experimental analysis in three
alpine plant species Oikos 8336ndash46
Prŷs-Jones OE and Willmer PG 1992 The biology of alkaline nectar in purple toothwort
(Lathraea clandenstina) ground level defenses Biological Journal of the Linnean Society
45373-388
Raine NE Willmer P and Stone GN 2002 Spatial structuring and floral avoidance
behaviour present in ant-pollinator conflict in a Mexican ant-acacia Ecology 833086-
3096
35
Ramsey M 1995 Ant pollination of the perennial herb Blandfordia grandiflora (Liliaceae)
Oikos 74265ndash272
Renault CK LM Buffa and Delfino MA 2005 An aphid-ant interaction effects on
different trophic levels Ecological Research 2071-74
Rico-Gray V and L B Thien 1989 Effect of different ant species on reproductive fitness of
Schomburgkia tibicinis (Orchidaceae) Oecologia 81487ndash489
Romero G Q P A P Antiqueira and J Koricheva 2011 A meta-analysis of predation risk
effects on pollinator behaviour PLoS One 6e20689
Rosumek FB FAO Silveira F de S Neves NP de U Barbosa L Diniz Y Oki F Pezzini
et al 2009 Ants on plants a meta-analysis of the role of ants as plant biotic defenses
Oecologia 160537ndash549
Roubik D W 1982 The ecological impact of nectar-robbing bees and pollinating
hummingbirds on a tropical shrub Ecology 63354ndash360
Rudgers J A 2004 Enemies of herbivores can shape plant traits selection in a facultative ant-
plant mutualism Ecology 85192ndash205
Schatz B and WT Wcislo 1999 Ambush predation by the ponerine ant Ectatomma ruidum
Roger (Formicidae) on a sweat bee Lasioglossum umbripenne (Halictidae) in Panama
Journal of Insect Behavior 12641-663
36
Schaffer W M D W Zeh S L Buchmann S Kleinhaus M V Schaffer and J Antrim
1983 Competition for nectar between introduced honey bees and native North American
bees and ants Ecology 64564ndash577
Schemske D W 1980 The evolutionary significance of extrafloral nectar production by Costus
woodsonii (Zingiberaceae) an experimental analysis of ant protection Journal of Ecology
68959ndash967
Schiestl F P and F Glaser 2011 Specific ant-pollination in an alpine orchid and the role of
floral scent in attracting pollinating ants Alpine Botany 1221ndash9
Schubart HOR AB Anderson 1978 Why donrsquot ants visit flowers A response to D H
Janzen Biotropica 10310-311
Schuumlrch S M Pfunder and B A Roy 2000 Effects of ants on the reproductive success of
Euphorbia cyparissias and associated pathogenic rust fungi Oikos 16ndash12
Stanton ML Palmer TM Young TP Evans A and Turner ML 1999 Sterilization and
canopy modification of swollen thorn acacia tree by a plant-ant Nature 401 578-581
Stephenson AG 1981 Toxic nectar deters nectar thieves of Catalpa speciosa American
Midland Naturalist 105381-383
Stout J C and D Goulson 2001 The use of conspecific and interspecific scent marks by
foraging bumblebees and honeybees Animal Behaviour 62183ndash189
Sugiura S T Abe and S Makino 2006 Loss of extrafloral nectary on an oceanic island plant
and its consequences for herbivory American Journal of Botany 93491ndash495
37
Svensson L 1985 An estimate of pollen carryover by ants in a natural population of
Scleranthus perennis L (Caryophyllaceae) Oecologia 66373ndash377
Tsuji K A Hasyim and K Nakamura 2004 Asian weaver ants Oecophylla smaragdina and
their repelling of pollinators Ecological Research 19669ndash673
Turner R C J J Midgley P Barnard R E Simmons and S D Johnson 2012 Experimental
evidence for bird pollination and corolla damage by ants in the short-tubed flowers of
Erica halicacaba (Ericaceae) South African Journal of Botany 7925ndash31
Visser D M G Wright and J H Giliomee 1996 The effect of the Argentine ant
Linepithema humile (Mayr) (Hymenoptera Formicidae) on flower-visiting insects of
Protea nitida Mill (Proteaceae) African Entomology 4285ndash287
Wagner D 2000 Pollen viability reduction as a potential cost of ant association for Acacia
constricta (Fabaceae) American Journal of Botany 87711ndash715
Wagner D and A Kay 2002 Do extrafloral nectaries distract ants from visiting flowers An
experimental test of an overlooked hypothesis Evolutionary Ecology Research 4293ndash305
Wang C Y Luo Y Tai D An and Y Kou 2008 Ants pollinate Neottia listeroides
(Orchidaceae) in Sichuan China 46836ndash846
Wyatt R 1980 The impact of nectar-robbing ants on the pollination system of Asclepias
curassavica Bulletin of the Torrey Botanical Club 10724ndash28
Wheeler WM and IW Bailey 1920 The feeding habits of Pseudomymine and other ants
Transactions of the American Philosophical Society 22235-279
38
Willmer P G and S A Corbet 1981 Temporal and microclimatic partitioning of the floral
resources of Justicia aurea amongst a concourse of pollen vectors and nectar robbers
Oecologia 5167ndash78
Willmer P G and G N Stone 1997 How aggressive ant-guards assist seed-set in Acacia
flowers Nature 388165ndash167
Willmer P G C V Nuttman N E Raine G N Stone J G Pattrick K Henson P Stillman
et al 2009 Floral volatiles controlling ant behaviour Functional Ecology 23888ndash900
Young T P C H Stubblefield and L a Isbell 1997 Ants on swollen-thorn acacias species
coexistence in a simple system Oecologia 10998ndash107
Yu D W and N E Pierce 1998 A castration parasite of an ant-plant mutualism Proceedings
of the Royal Society B Biological Sciences 265375ndash382
39
Tables
Table 11 Studies of ant-flower or ant-pollinator interactions that included a measure of plant fitness Each study system is listed only
once except when results varied between years or when different studies examined male and female fitness Significant effects of ants on
plant fitness are either lsquopositiversquo or lsquonegativersquo non-significant effects are lsquoneutralrsquo When ants significantly affected only one fitness
component but several were measured (eg fruit set and seed production) we still consider the result significant
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Acacia
drepanolobium
Crematogaster
nigriceps
Proportion
fruiting
Negative Destruction of floral buds Young et al 1997
Asclepias exaltata Several Pollinia inserted
removed
Negative for pollinia
insertion neutral for
pollinia removal
Exploitative competition Wyatt 1980 T
40
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Asclepias syriaca Several
Fruit set pollinia
removed
Negative for fruit
set neutral for
pollinia removal
Exploitative competition Fritz and Morse
1981 T
Cordia nodosa Allomerus cf
demerarae
Fruit set Negative Destruction of floral buds Yu and Pierce
1998
Costus woodsonii Wasmannia
auropunctata
Seed set Positive Unknown ant pollination
suggested (but unlikely)
Schemske 1980
Eritrichum aretioides F neorufibarbis
gelida
Seed set Negative Ants damaged flowers Puterbaugh 1998
41
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Euphorbia characias Several Seed set Neutral Plants in Argentine ant-
invaded area set less seed
than those in un-invaded
sites but an ant-exclusion
experiment did not produce
significant results
Blancafort and
Gόmez 2005
Ferocactus wislizeni Solenopsis xyloni
and others
Seed set Unknown negative Interference and exploitative
competition S xyloni
reduced seed set relative to
other ants but the study did
not include a no-ant control
Ness 2006
42
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Frasera speciosa F neorufibarbis
and Formica sp
Seed set Neutral Nocturnal pollination
occurring when ants were
less active may have
compensated for reduced
diurnal pollinator visitation
Norment 1988
Gypsophila struthium Several Seeds per
inflorescence
Neutral No effect despite large
numbers of ants on flowers
Gόmez et al
1996
Hirtella physophora Allomerus
decemarticulatus
Fruit set Negative Destruction of flowers Orivel et al
2011
Humboldtia brunonis Crematogaster
dohrni
Fruit set Negative Destruction of flowers Gaume et al
2005
43
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Lepidium subulatum Several Percentage of
ovules setting
seed
Positive Unknown Gόmez et al
1996
Leucospermum
conocarpodendron
Several Seed set Neutral Site not pollen limited Lach 2008
Linaria vulgaris Several ldquoFemale fitness
indexrdquo
(combination of
number of seeds
fruits flowers
and seed weight)
Positive Reduction in florivory and
seed predation nectar
robbing holes increased ant
visitation
Newman and
Thomson 2005
44
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Melastoma
malabathricum
Oecophylla
smaragdina
Seed set Positive Ants deterred less efficient
pollinators increasing
visitation by the more
efficient pollinator
Gonzaacutelvez et al
2012
Metrosideros
polymorpha
Several Fruit set Neutral Ants reduced nectar
collection rates by foraging
bees but had no effect on
pollen collection rates or
fruit set
Junker et al
2010a
Orexis alpine F neorufibarbis
gelida
Seed set Neutral Autogamy or not pollen
limited
Puterbaugh 1988
45
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Ouratea spectabilis Cephalotes
pusillus and others
Seeds per fruit Neutral positive C pusillus had no effect on
seed number but other ants
collectively increased seed
set
Byk and Del
Claro 2010
Pachycereus schottii Several Seed set Neutral positive In one year ants showed a
positive effect (seed
number 2008) but no effect
with increased sample sizes
Chamberlain and
Holland 2008
Polemonium
viscosum
F neorufibarbis
gelida
Seed set Negative Ants damaged styles Galen 1983
46
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Polemonium
viscosum
F neorufibarbis
gelida
Pollen removal Neutral Ants did not affect pollen
removal even at high ant
densities
Galen and Geib
2007 T
Protea nitida Linepithema
humile and others
Seed set Neutral positive L humile had no effect on
P nitida seed set but native
ants increased it in one year
ants may cause pollinators
to relocate
Lach 2007
Psychotria
limonensis
Ectatomma spp Fruit set Positive Suggested increased
pollinator relocation
Altshuler 1999
47
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Roussea simplex Technomyrmex
albipes
Seed set Negative Interference and exploitative
competition
Hansen and
Muumlller 2009
Tristerix aphyllus Several Fruit set Neutral Nectar robbing ants
minimally reduced nectar
levels and had no effect on
hummingbird visitation
Caballero et al
2013
Vaccinium
reticulatum
Several Fruit set Neutral Ants reduced florivory but
did not affect fruit set
Bleil et al 2011
T Study measured male fitness
Study of sterilizing ants showed short-term decrease in female fitness long-term effects are unclear (see text)
48
Table 12 Studies demonstrating either i) ant pollination through an exclusion experiment or ii) that ants transfer pollen (or pollen
analogue) between flowers strongly suggesting ant pollination
Plant species Ant species Ant pollination Evidence Reference
Alyssum purpureum Several Yes Exclusion experiment Gόmez et al 1996
Arenaria tetraquetra Several Yes Exclusion experiment Gόmez et al 1996
Balanophora kuroiwai Leptothorax sp Suggested Exclusion experiment and
observed pollen transfer
and pollen tube formation
but did not see if plants set
seed
Kawakita and Kato 2002
Blandfordia grandiflora Iridomyrmex sp Yes Exclusion experiment Ramsey 1995
Borderea pyrenaica Several Yes Exclusion experiment Garciacutea et al 1995
49
Plant species Ant species Ant pollination Evidence Reference
Cytinus hypocistis Several Yes Exclusion experiment de Vega et al 2009
Epipactis thunbergii Camponotus
japonicas
Suggested Observed transfer of
pollinia from ants to
orchids
Sugiura et al 2006
Euphorbia cyparissias Several Yes Exclusion experiment Schuumlrch et al 2000
Fragaria virginiana Several Very likely Ant-only treatments set
comparable seed to flying
pollinator-only treatment
seed set increased with
increasing ant visitation
Ashman and King 2005
Frasera speciosa Several Yes Exclusion experiment Norment 1988
Frankenia thymifolia Several Yes Exclusion experiment Gόmez et al 1996
50
Plant species Ant species Ant pollination Evidence Reference
Hormathophylla spinosa Proformica
longiseta
Yes Exclusion experiment Gόmez and Zamora 1992
Jatropha curcas
Several
Tapinoma
melanocephalum
most common
Yes Exclusion experiment Luo et al 2012
Leporella fimbriata Myrmecia urens Yes Observed pollinia transfer
and subsequently
confirmed as ant
pollination
Peakall et al 1987
51
Plant species Ant species Ant pollination Evidence Reference
Lobularia maritima Several
Camponotus
micans most
common
Yes Exclusion experiment Gόmez 2000
Microtis parviflora
Iridomyrmex
gracilis
Yes Observation used
laboratory colonies to
replicate observations
Peakall and Beattie 1989
Naufraga balearica
Several
especially
Plagiolepis
pygmaea and
Lasius grandis
Suggested Observation over three
years of study only ants
were observed visiting
ants carried pollen
Cursach and Rita 2011
52
Plant species Ant species Ant pollination Evidence Reference
Neottia listeroides Leptothoras sp
Paratrechina sp
Suggested Observed transfer of
pollinia from ants to
orchids
Wang et al 2008
Paronychia pulvinata Formica
neorufibarbis
gelida
Yes Exclusion experiment Puterbaugh 1998
Polygonum cascadense Formica argentea
Very likely Grew plants in greenhouse
open to flying visitors with
minimal seed set only
plants with access to ants
set significant seed
Hickman 1974
53
Plant species Ant species Ant pollination Evidence Reference
Retama sphaerocarpa Several Yes Exclusion experiment Gόmez et al 1996
Scleranthus perennis
Several
especially
Formica
rufibarbis
Suggested Ants transferred pollen
analogue to stigmas and
carried pollen on bodies
Svensson 1985
Sedum anglicum Proformica
longiseta
Yes Exclusion experiment Gόmez et al 1996
Trinia glauca Several Yes Exclusion experiment Carvalheiro et al 2008
54
Chapter 2 Ants and ant scent reduce bumblebee pollination of artificial flowers
Cembrowski AR Tan MG Thomson JD and Frederickson ME
In press The American Naturalist
All authors designed the experiment ARC and MGT performed the experiment ARC and MEF
performed statistical analyse
s and ARC wrote the manuscript with input from all authors
Abstract
Ants on flowers can disrupt pollination by consuming rewards or harassing pollinators but it is
difficult to disentangle the effects of these exploitative and interference forms of competition on
pollinator behavior Using highly rewarding and quickly replenishing artificial flowers that
simulate male or female function we allowed bumblebees (Bombus impatiens) to forage (1) on
flowers with or without ants (Myrmica rubra) and (2) on flowers with or without ant scent cues
Bumblebees transferred significantly more pollen analogue both to and from ant free flowers
demonstrating that interference competition with ants is sufficient to modify pollinator foraging
behavior Bees also removed significantly less pollen analogue from ant scented flowers than
from controls making this the first study to show that bees can use ant scent to avoid harassment
at flowers Ant effects on pollinator behavior possibly in addition to their effects on pollen
viability may contribute to the evolution of floral traits minimizing ant visitation
55
Introduction
Trait-mediated indirect interactions arise when a focal species causes phenotypic changes
including behavioral modifications in a second species and these effects cascade to still other
species (Werner and Peacor 2003) Although trait-mediated indirect interactions are often studied
in food webs (eg Werner and Peacor 2003 Preisser et al 2005) they are not limited to trophic
interactions For example the threat of predation can change the behavior of a mutualist thus
affecting its partners (Suttle 2003) In animal-pollinated plants predators can disrupt pollination
directly through density-mediated indirect interactions (Dukas 2005) or via trait-mediated
indirect interactions by changing pollinator behavior When predators are present pollinators
may switch to visiting less rewarding flowers or they may avoid flowers altogether potentially
reducing the amount of pollen donated or received by a flower and thus decreasing plant fitness
(Gonccedilalves-Souza 2008 Ings and Chittka 2009) Similarly competition for floral rewards with
other flower visitors may cause behavioral changes in pollinators (Maloof and Inouye 2000
Ohashi et al 2008) Previous research has shown that bees may spend less time visiting flowers
depleted of rewards (Thomson 1986) and may avoid flowers bearing scent marks that indicate
recent visits by other individuals of the same or different bee species as these flowers are likely
to be depleted (Stout and Goulson 2002)
Whereas competition between pollinators may not be particularly costly for the plant
because both competitors are likely to provide pollination services competition between
pollinators and organisms that do not usually provide a pollination service such as ants has a
greater potential to reduce plant fitness Ants are common floral visitors they are attracted to
flowers for nectar (Lach 2007) and in some cases pollen (Byk and Del Claro 2010) and will
often defend these resources against other flower visitors (Altshuler 1999) However ants rarely
56
contribute much useful pollination antibiotic secretions present on their cuticles kill pollen
grains (Beattie et al 1985 Dutton and Frederickson 2012) and when foraging in flowers ants
sometimes impede female function by damaging stigmas (Galen and Cuba 2001) We will
henceforth consider ants to be non-pollinators and will contrast them to animals like bees that
are pollinators Ants are capable of competing with pollinators in two ways (1) by consuming
floral rewards and reducing their availability to pollinators (ie exploitative competition) and (2)
by directly antagonizing and excluding pollinators from flowers (ie interference competition)
Many plants benefit from the presence of ants numerous plant species recruit ants using
extrafloral nectar food bodies or domatia because ants provide protection against folivores (Heil
and McKey 2003) Similarly some plants have extrafloral nectaries on or near reproductive
tissues that recruit ants that deter florivores (eg Inouye and Taylor 1979) However plants
may incur reproductive costs as a result of their association with ants Some ant species that nest
in myrmecophytes sterilize flowers to shunt plant resources from reproduction to vegetative
growth allowing for greater ant colony growth (Frederickson 2009) Also ants visiting
extrafloral nectaries may be attracted to floral nectaries where they can harass pollinators (Ness
2006) There is growing evidence that many angiosperms have evolved floral traits that prevent
ants from accessing flowers during anthesis (Willmer and Stone 1997 Ballantyne and Willmer
2012) The effects of flower-visiting ants can be substantial Ants can decrease the frequency
duration or species diversity of pollinator visits to flowers (Lach 2008 Hansen and Muumlller 2009
Junker et al 2010 Gonzaacutelvez et al 2012) all of which potentially impact pollen donation and
receipt In some studies changes to pollinator behavior induced by ants have also been linked to
seed set
It is difficult however to disentangle the intertwined effects of exploitative and
interference competition in nature If ants disrupt pollination plants may often evolve traits to
57
deter ants from flowers (Willmer et al 2009) From a practical standpoint this would then limit
our ability to study the effects of ants on pollinator visitation because ants would not visit
flowers Here we used artificial flowers to explore ant-bee interactions in the absence of floral
defenses against ants We used highly rewarding quickly replenishing artificial flowers to
examine how direct harassment (ie interference competition expressed through behavior)
changes bee foraging behavior and how this affects donation and receipt of a pollen analogue
(powdered food dye) while minimizing the effects of exploitative competition We predicted
that flowers visited by ants would both donate and receive less pollen analogue than flowers
without ants because bees would avoid flowers with ants or leave them sooner Because scent
plays a large role in bumblebee communication and flower choice (Stout and Goulson 2002) we
also tested whether the presence or absence of ant scent on artificial flowers would affect pollen
analogue donation because of changes in the foraging behavior of bumblebees
Methods
i) Subjects
Myrmica rubra is an invasive ant in eastern North America with a range and habitat preferences
overlapping that of Bombus impatiens a common bumblebee Because M rubra visits flowers
(A Cembrowski pers obs) the two species likely interact in nature We collected 12 M rubra
colonies in the fall of 2011 and 2012 from Toronto Ontario Canada and the surrounding area
and maintained them in environmental chambers on artificial diet (Dussutour and Simpson 2009)
and a 1410 LD schedule (light 600-2200) We used these colonies as sources of M rubra
workers for experiments
58
Workers from commercially supplied colonies of Bombus impatiens (Biobest Canada
Ltd) foraged on artificial flowers in flight cages (either 24 x 24 x 21 m or 79 x 34 x 20m) at
the University of Toronto We tested a total of five bee colonies four colonies were used in the
ant presence trials and two of these and one additional colony were used in the ant scent trials
Flight cages had overhead fluorescent lights attached to timers In contrast to most previous
studies in which bees have been trained and tested individually the entire worker force of a bee
colony was free to forage at will in our experiments Colonies were trained to forage on artificial
flowers for at least four days before being used in trials After being used for a trial the colony
was not used for at least two days to reduce dye carryover between trials Between trials
bumblebees were fed pollen and given sugar water
ii) Artificial flowers
The flowers (Fig 1 Thomson et al 2012 see also Makino 2008) consisted of glass jars filled
with 30 WV sucrose solution Sugar water travelled by capillary action up a sewing-thread
wick to a hole in a blue-painted lid accumulating in a knot that acts as a nectary Flowers
depleted by visitors were quickly replenished via capillary action and were non-rewarding only
very briefly after visits taking less than a minute to accumulate 05 microl note that bumblebee
visits to a single flower are often separated by several minutes or more (A Cembrowski
unpublished data)
Unlike most previous artificial flower designs these flowers allow for estimation of male
and female fitness because we can measure both the amount and type of dye received by female
flowers (see (d) Dye quantification below) In order to access nectar on a ldquomalerdquo flower a bee
must crawl through a circular ldquoantherrdquo of brush-like weather stripping material dusted in a
59
consistent manner with our pollen analogue powdered food dye (FDampC 5 or 6) which is
transferred to the bee in the process On a ldquofemalerdquo flower a bee crawls through a sticky plastic
reinforcement that functions as a ldquostigmardquo receiving dye from the beersquos body Male flowers
dispensed dye particles over multiple bee visits and could still dispense dye at the end of trials
They resemble many real flowers or flower heads in that pollen is dispensed gradually over time
but less dye is available for transfer with each subsequent bee visit (Harder and Thomson 1989)
iii) Flight cage trials
We conducted 6 ant scent trials between 22 March and 4 June 2012 and the remaining trials (16
ant presence trials and 9 ant scent trials) between 9 November 2012 and 6 January 2013 Ant
presence trials lasted for eight hours and ant scent trials lasted for four hours Artificial flowers
were prepared and placed individually in small plastic containers treated with Fluon (Insect-a-
Slip BioQuip Products Inc) to prevent ants from escaping In ant presence trials we used 32
flowers (16 male and 16 female flowers) arranged in an eight-by-four array with flowers spaced
45 cm apart between rows and 30 cm within rows Ant scent trials used 20 flowers (10 male and
10 female flowers) in a four-by-five array with flowers spaced 30 cm apart between and within
rows In all of the ant presence trials and 9 of the ant scent trials we counted the number of bees
actively foraging after one hour to get a measure of colony activity
a) Ant presence trials
We examined the effects of ants on the amount of dye donated by male flowers and received by
female flowers in sixteen trials testing four bee colonies four times each The containers of eight
randomly chosen male flowers and eight randomly chosen female flowers received 15 M rubra
60
workers each the other 16 flowers remained free of ants Isolated from their colonies M rubra
workers have nowhere to deposit sugar water they collect and therefore may become sated
nonetheless a force of 15 ant workers was enough to maintain visitation to the nectaries where
bees foraged while still rarely having more than one or two ants visit the nectary at any time As
ants attacked bees bees would occasionally carry or throw attacking ants off flowers but despite
this some ants maintained their presence at the nectary throughout the trials Because ants the
size of M rubra consume liquids slowly (~017 ndash 024 microlmin Davidson et al 2004) flowers
replenished nectar more quickly than ants consumed it We used two colors of dye (FDampC 5 and
FDampC 6) to differentiate male flowers with and without ants In two trials for each bee colony
anthers of male flowers with ants were coated with FDampC 5 dye while brushes of male flowers
lacking ants were coated with FDampC 6 dye In the other two trials we reversed dye colors to
control for effects of dye type and color Dye color was assigned in a random order Artificial
flowers were placed in randomly assigned positions in the array before we opened the colony
and allowed the bees to begin foraging
b) Scent trials
We explored the effect of ant scent on dye donation in fifteen trials testing three bee colonies in
six five and four trials each Bees were first exposed to ants by allowing the bees to forage for
eight hours on twenty flowers (ten male and ten female) of which five male flowers had 15 ants
and all others had none No dye was used during this ldquoexposurerdquo day We then collected all but
the five ant-free male flowers in the flight cage leaving these flowers to keep bees foraging
Next we individually stored five new male lids in Fluon-treated containers with ten M rubra
workers Five control male lids were put in identical containers lacking ants The following
61
morning we removed the remaining flowers and set out ten new male and female flowers in a
random spatial arrangement using the lids having or lacking ant scent Thus the ant-scented
flowers were not in the same positions as the ant-visited flowers on which the bees were trained
Only five male flowers had ant scent and the other 15 flowers (five male and ten female) did not
We used the same two dye colors as in i) to differentiate male flowers with and without ant
scent and randomized which color was used for ant-scented flowers between trials We collected
and replaced stigmas from female flowers after one hour and collected the stigmas again after
four hours In the first trial performed we also collected stigmas after two hours but due to the
small amount of dye transferred this was not repeated
iv) Dye quantification
After each trial we quantified the amount of dye transferred to female flowers using a
spectrophotometer We removed the stigmas from female flowers placed them in test tubes
added 51 mL of distilled water to each tube and vortexed each tube thoroughly to ensure the
dye was evenly diluted These were diluted further as needed if the absorbance exceeded the
sensitivity range of the spectrophotometer In the first six scent trials each stigma was analyzed
separately and the dye amounts were summed to obtain a total amount of each dye color
transferred to female flowers in each trial In all the ant presence trials and the other nine scent
trials we opted to treat the experiment as the unit of replication Therefore we combined stigmas
from each treatment type (ant-visited or ant-free) in a test tube and measured the total amount of
each dye color transferred in each treatment in each trial
Because we put different dye colors on male flowers with and without ants we could use
the amount of each dye color donated to all female flowers to measure the reproductive success
62
of ant-visited and ant-free male flowers The total amount of dye (of both colors) received by
female flowers with and without ants was our measure of female reproductive success We
calculated the amount of each dye color in the sample by measuring absorbance at 428 or 486
nm and converting absorbance to micrograms following computational methods for overlapping
spectra (Blanco et al 1989)
v) Statistical analyses
In two trials (one ant presence and one ant scent) most female flowers received no dye because
of low bee activity so we excluded these trials from analyses Because dye reception and
donation values were non-normally distributed we square-root transformed the data before
examining the effects of ant presence or ant scent on dye transfer in ANCOVAs For ant
presence trials we included ant presence on male and female flowers and their interaction as
main effects and the total amount of dye transferred in each trial as a covariate to account for
the large variation in overall dye transfer among trials For ant scent trials we included ant scent
as the main effect time (one or four hours) as a repeated measure the interaction between scent
and time and the total amount of dye transferred as a covariate Covariate by treatment (ant
presence or ant scent) interactions were never significant and so were excluded in final analyses
All analyses were conducted in JMP (v 1000)
Results
Bees usually started foraging within minutes of the beginning of the trial and continued until
flowers were collected from the flight cage or the lights were extinguished An average of 34 plusmn
012 and 40 plusmn 015 (mean plusmn SE) bees were foraging after an hour in the ant presence and ant
63
scent trials respectively Male flowers with ants donated significantly less dye than male flowers
lacking ants (Fig 22a F155 = 4019 p lt 00001) Similarly female flowers with ants received
significantly less dye than female flowers lacking ants (Fig 22a F155 = 461 p = 0036) There
was no significant interaction between ant presence on male flowers and ant presence on female
flowers (F155 = 118 p = 028) Flowers with ant scent donated significantly less dye than
flowers without ant scent (Fig 22b F125 = 11216 p lt 00001) There was no significant effect
of time (F125 = 121 p = 028) or interaction between time and scent (F125 = 111 p = 030) in
the model
Discussion
In this study ants altered bumblebee pollination behavior and exhibited trait-mediated indirect
interactions with (artificial) flowers Through interference competition M rubra workers
significantly affected the pattern of dye transfer by B impatiens causing a preferential flow of
dye from male flowers lacking ants to female flowers lacking ants If manifested in nature such
effects would reduce the reproductive success of plants visited by ants through both male and
female function
Ants could have changed the attractiveness of artificial flowers in at least two ways First
as ants interrupted or altogether prevented bumblebees from foraging at nectaries bumblebees
may have learned to avoid flowers with ants due to the relative inefficiency of foraging at these
flowers something bumblebees take into account (Heinrich 2004) Second bees attempt to
minimize their risk of being attacked during foraging bouts which ants did to visiting bees Ants
often attacked or harassed bees by biting grasping and appearing to sting visiting bees
preventing them from accessing the nectary or reducing their time on flowers (see online video
64
1) and bees sometimes avoided ant-tended flowers entirely This harassment was sometimes
physically traumatic (online video 1) and bees often appeared to have trouble flying after being
attacked by ants Previous research has demonstrated that bumblebees leave or avoid flowers
where they have been harassed (Jones and Dornhaus 2011) and avoid foraging where there is
visual or olfactory evidence of a predator or predation event (Abbott 2006 Goodale and Nieh
2012) Though it is unclear if bumblebees viewed ants as competitors or predators they
responded similarly to flowers having ants as they do to flowers housing predators (Gonccedilalves-
Souza et al 2008)
To avoid artificial flowers with ants bees likely used a combination of visual and
olfactory cues Previous research has shown that bees respond to conspicuous predators or
predator ldquodummiesrdquo on flowers (Suttle 2003 Gonccedilalves-Souza et al 2008) decreasing the
frequency and duration of their visits to these flowers Thus bees may have been able to avoid
flowers harboring ants by sight alone However even in the absence of ants bees still
preferentially visited flowers lacking ant scents (Fig 22b) suggesting that they had learned to
associate ant scent with harassment Bees are adept at associative learning (Wright and Schiestl
2009) and can learn to recognize unique scents left behind by both conspecific and heterospecific
flower visitors (Stout et al 1998) Bees can use these various scents often arising from tarsal
gland deposits (Stout et al 1998) to recognize recently visited flowers that are less likely to be
profitable (Stout and Goulson 2002) Recently Ballantyne and Willmer (2012) demonstrated that
bees learn to associate ant scents with unrewarding artificial flowers and decrease their visitation
to these flowers Our results complement their findings by showing that bees can associate ant
scent with harassment at otherwise rewarding flowers
In our study ant scent caused bees to adjust their foraging strategy and decreased the
amount of pollen analogue that was donated by ant-scented flowers Thus the effects of
65
interference competition with ants on flowers can extend beyond immediate interactions and may
have fitness consequences for plants even when ants are absent Like other olfactory cues these
effects are likely transitory (Stout et al 1998) Although more dye was still donated by male
flowers lacking ant scent than those with ant scent the ratio of dye donated was on average
closer to equality in hours 2-4 than in the first hour (Fig 22b) This effect may be partially
driven by dye depletion of male flowers without ant scent but the lack of a strong corresponding
decrease in flowers with ant scent suggests that visitation patterns became more similar We did
not test whether B impatiensrsquo avoidance of ant scent was an innate or a learned behavior but
previous work has shown that Bombus terrestris does not innately avoid flowers with ant scent
(Ballantyne and Willmer 2012)
Ants may be necessary for plant survival and growth but can be costly for plant
reproduction The evolution of ant attractants such as extrafloral nectaries in some plant lineages
suggests that the costs of ants can be outweighed by their protective abilities However in this
study flowers visited by ants received and donated significantly less dye although they did
retain some sexual function The net benefit of having ants depends on whether ants increase
plant fitness by reducing herbivory more than they decrease plant fitness by disrupting
pollination Alternatively plants may actually benefit from the costs of ants to plant
reproduction ants that castrate flowers may be better defenders increasing plant survival or
vegetative growth when plants are young allowing for increased reproduction later in life when
the plant is colonized by less aggressive non-castrating ants (Frederickson 2009 Palmer et al
2010) In some plant species pollinator harassment by ants may even be beneficial Altshuler
(1999) reported that Ectatomma ants greatly increased fruit set of Psychotria limonensis despite
reductions in pollinator visitation rates presumably due to increases in the rate of pollen
outcrossing Similarly Gonzaacutelvez et al (2012) found that the presence of Oecophylla
66
smaragdina ants on Melastoma malabathricum flowers reduced visitation by less effective
pollinators and increased visitation by more effective Xylocopa bees increasing plant fitness
Cases of ants being beneficial to flowers appear to be the minority and many plant
species have traits that limit ant access to flowers Several studies have detailed floral volatiles
that are thought to mimic ant alarm pheromones and thus repel ants (Junker and Bluumlthgen 2008
Willmer et al 2009) Other plants use structural modifications such as narrow corollas or
slippery stems to limit ant access to flowers (reviewed in Willmer et al 2009) Exploitative
competition between ants and pollinators (Lach 2005 Ballantyne and Willmer 2012) as well as
the lethal effects of ant antibiotic secretions on pollen grains (Beattie et al 1985 Dutton and
Frederickson 2012) may have resulted in selection on plants to reduce ant visits to flowers Our
results suggest that trait-mediated indirect interactions resulting from interference competition
between ants and bees may favor plants that defend their flowers and their pollinators against
ants
Acknowledgements
We thank H Rusnock and S Meadley-Dunphy for assistance J Dix for help with flight cage
construction and Biobest for bumblebee colonies We are grateful to the Thomson and
Frederickson labs for guidance particularly T Makino funded by JSPS for helping to refine
artificial flowers J Ogilvie for help with bees K Prior for M rubra colony collections and K
Turner for study design suggestions The comments of the editor and two anonymous reviewers
prompted improvements in the article especially with regards to study design MEF and JDT
acknowledge NSERC Discovery Grant funding The University of Torontorsquos Faculty of Arts and
Science supported MGRTrsquos participation
67
68
References
Abbott K R 2006 Bumblebees avoid flowers containing evidence of past predation events
Canadian Journal of Zoology 841240-1247
Altshuler D L 1999 Novel interactions of non-pollinating ants with pollinators and fruit
consumers in a tropical forest Oecologia 119600-606
Ballantyne G and Willmer P 2012 Floral visitors and ant scent marks noticed but not used
Ecological Entomology 37402-409
Beattie A J Turnbull C Knox R B and Williams E G 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany
71421-426
Blanco M Runiaga H Maspoch S and Tarin P 1989 A simple method for
spectrophotometric determination of two-components with overlapped spectra Journal of
Chemical Education 66178-180
Byk J amp Del Claro K 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333-38
Davidson D W S C Cook and R R Snelling 2004 Liquid-feeding performances of ants
(Formicidae) ecological and evolutionary implications Oecologia 139255ndash66
Dukas R 2005 Bumble bee predators reduce pollinator density and plant fitness Ecology
861401-1406
Dussutour A and Simpson S J 2009 Communal nutrition in ants Current Biology 19740-
744
69
Dutton E M and Frederickson M E 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561-569
Frederickson ME 2009 Conflict over reproduction in an ant-plant symbiosis why Allomerus
octoarticulatus ants sterilize Cordia nodosa trees American Naturalist 173675-681
Galen C and Cuba J 2001 Down the tube pollinators predators and the evolution of flower
shape in the alpine skypilot Polemonium viscosum Evolution 551963-1971
Gonccedilalves-Souza T Omena P M Souza J C and Romero G Q 2008 Trait-mediated
effects on flowers artificial spiders deceive pollinators and decrease plant fitness Ecology
892407-2413
Gonzaacutelvez F G Santamariacutea L Corlett R T and Rodriacuteguez-Gironeacutes M A 2012 Flowers
attract weaver ants that deter less effective pollinators Journal of Ecology 10178ndash85
Goodale E and Nieh JC 2012 Public use of olfactory information associated with predation
in two species of social bees Animal Behaviour 84919-924
Harder LD and Thomson JD 1989 Evolutionary options for maximizing pollen dispersal of
animal-pollinated plants American Naturalist 133323-344
Heil M and McKey D 2003 Protective ant-plant interactions as model systems in ecological
and evolutionary research Annual Review of Ecology Evolution and Systematics 34425-
453
Heinrich B 2004 Bumblebee economics 2nd
edn Harvard University Press Cambridge
Ings T C and Chittka L 2009 Predator crypsis enhances behaviourally mediated indirect
effects on plants by altering bumblebee foraging preferences Proceedings of the Royal
Society B 2762031-2036
70
Inouye D W and Taylor O R Jr 1979 A temperate region plant-ant-seed predator system
consequences of extra floral nectar secretion by Helianthella quinquenervis Ecology 601-
7
Junker R R and Bluumlthgen N 2008 Floral scents repel potentially nectar-thieving ants
Evolutionary Ecology Research 10295ndash308
Junker R R Bleil R Daehler C C and Bluumlthgen N 2010 Intra-floral resource partitioning
between endemic and invasive flower visitors consequences for pollinator effectiveness
Ecological Entomology 35760ndash767
Jones E I and Dornhaus A 2011 Predation risk makes bees reject rewarding flowers and
reduce foraging activity Behavioral Ecology and Sociobiology 651505-1511
Lach L 2005 Interference and exploitative competition of three nectar-thieving invasive ant
species Insect Sociaux 52257-262
Lach L 2007 A mutualism with a native membracid facilitates pollinator displacement by
Argentine ants Ecology 881994-2004
Lach L 2008 Argentine ants displace floral arthropods in a biodiversity hotspot Diversity and
Distributions 14281-290
Makino T T and Sakai S 2007Experience changes pollinator responses to floral display size
from size-based to reward-based foraging Functional Ecology 21854863
Maloof J E and Inouye D W 2000 Are nectar robbers cheaters or mutualists Ecology
812651ndash2661
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506-514
Ohashi K Leslie A and Thomson JD 2008 Trapline foraging by bumble bees V Effects of
experience and priority on competitive performance Behavioral Ecology 19936-948
71
Palmer T M Doak D F Stanton M L Bronstein J L Kiers E T Young T P Goheen
J R and Pringle R M 2010 Synergy of multiple partners including freeloaders
increases host fitness in a multispecies mutualism Proceedings of the National Academy
of Sciences 10717234-17239
Preisser E L Bolnick D I and Benard M E 2005 Scared to death The effects of
intimidation and consumption in predator-prey interactions Ecology 86501-509
Stout J C Goulson D and Allen J A 1998 Repellent scent-marking of flowers by a guild of
foraging bumblebees (Bombus spp) Behavioral Ecology and Sociobiology 43317-326
Stout J and Goulson D 2002 The influence of nectar secretion rates on the responses of
bumblebees (Bombus spp) to previously visited flowers Behavioral Ecology and
Sociobiology 52239-246
Suttle K B 2003 Pollinators as mediators of top-down effects on plants Ecology Letters
6688-694
Thomson JD 1986 Pollen transport and deposition by bumble bees in Erythronium influences
of floral nectar and bee grooming Journal of Ecology 74329-341
Thomson JD Ogilvie J E Makino T T Arisz A Raju S Rojas-Luengas V and Tan M
G R 2012 Estimating pollination success with novel artificial flowers effects of nectar
concentration Journal of Pollination Ecology 9108-114
Tsuji K Hasyim A and Nakamura K 2004 Asian weaver ants Oecophylla smaragdina and
their repelling of pollinators Ecological Research 19669-673
Ulrich S and Bertsch A 1990 Do foraging bumblebees scent-mark food sources and does it
matter Oecologia 82137-144
Werner E E and Peacor S D 2003 A review of trait-mediated indirect interactions in
ecological communities Ecology 841083-1100
72
Willmer P G Nuttman C V Raine N E Stone G N Pattrick J G Henson K Stillman
P McIlroy L Potts S G and Knudsen J T 2009 Floral volatiles controlling ant
behaviour Functional Ecology 23888-900
Wright G A and Schiestl F P 2009 The evolution of floral scent the influence of olfactory
learning by insect pollinators on the honest signaling of floral rewards Functional Ecology
23841-851
73
Figures
Figure 21 Photograph of artificial flower lids A = anther L = lid N = nectary R = region
where anther or stigma is placed S = stigma T = thread W = weight
74
Figure 22 Dye transferred (mean plusmn 1SE) per trial by B impatiens workers A) from artificial
male and to artificial female flowers in the presence (unfilled circles) or absence (filled circles)
of Myrmica rubra workers and B) from artificial male flowers with (unfilled circles) or without
(filled circles) M rubra scent to ant-free female flowers in the 1st or the 2
nd to 4
th hours of the
trials
75
Chapter 3 Not just for the bees pollen consumption is common among tropical
ants
Cembrowski AR Reurink G and Frederickson ME
Planned submission to Biotropica
ARC and MEF designed the study ARC and GR did laboratory work ARC performed statistical
analyses and wrote the manuscript with input from other authors
Abstract
Although palynivory or pollen consumption is widespread among other hymenopterans there
are few accounts of palynivory in ants To quantify how often ants consume pollen we subjected
adult workers and larvae from 75 species of neo- and paleo-tropical ants to acetolysis a process
that destroys most organic material but leaves behind pollen grains In over half of the species
we examined ants contained a few pollen grains (lt 5) and ants of several Camponotus species
contained abundant pollen grains (gt50) We tested for an association between trophic level and
palynivory using stable nitrogen isotope ratios (δ15
N) but we did not find a significant
correlation we found pollen grains in lsquoherbivorousrsquo omnivorous and carnivorous ants We
suggest that our results indicate sporadic opportunistic pollen consumption by ants in tropical
forests
Introduction
For those organisms that can digest it efficiently pollen can be an excellent food source
Depending on the plant species pollen can contain up to 60 protein as well as lipids
76
carbohydrates and numerous trace nutrients (Roulston and Cane 2000) Pollen feeding
(palynivory) occurs in many taxa including birds mammals and arthropods (Roulston and Cane
2000) but it is best known among the Hymenoptera particularly bees Although adult bees feed
mainly on plant exudates (ie nectar) nearly all bee species rely on pollen as the chief protein
source for developing larvae (Willmer 2011) Closely related to bees ants have similar
nutritional requirements adult workers need carbohydrates whereas larvae are fed a diet rich in
protein (Dussutour and Simpson 2009) Ants also exploit nectar heavily and regularly visit
flowers (eg Herrera et al 1984 Bluumlthgen et al 2004) but unlike bees are not believed to
widely consume pollen Instead ants are thought to receive most of the protein in their diet from
predation or scavenging (eg Floren et al 2002)
However ants may not be as reliant on animal protein sources as commonly assumed In
tropical forests arboreal ants can have similar δ15
N ratios a measure of trophic level as
herbivores suggesting limited carnivory and substantial feeding on plant-derived protein sources
(Davidson et al 2003) One potential protein source is pollen This idea is supported by scattered
accounts of pollen consumption by ants from other ecosystems including deserts (Ness 2006)
boreal forests (Czechowski et al 2009) and tropical savannas (Byk and Del Claro 2010) One
ant genus Cephalotes is widely recognized to consume pollen (Creighton 1967 Baroni Urbani
and de Andrade 1997 Byk and Del Claro 2010) However beyond this genus our knowledge of
pollen consumption by ants has largely been limited to direct observations (but see Czechowski
et al 2011) and it is unknown if palynivory is truly rare among ants or simply under-reported
We borrowed a technique commonly used in palynology acetolysis to assess the frequency of
pollen consumption by ants in 75 neo- and paleo-tropical ant species This technique allowed us
to examine many more ant species than we could have observed directly
77
Methods
Adult worker ants were collected by hand at baits or from leaf litter using Winkler extractors
while larvae along with adults were collected by hand from nests Collections were made by L
Arcila Hernandez and J Sanders in the primary tropical rainforest surrounding the Centro de
Investigaciόn y Capacitaciόn Rio Los Amigos (CICRA 12deg34rsquoS 70deg05rsquoW) in Peru in 2010-
2011 and by E Youngerman from lowland primary and secondary rainforests in Madang
Province Papua New Guinea (PNG) in 2011-2012 We sorted ant samples to morphospecies and
then identified them to genus and when possible to species using existing keys which were
unavailable for PNG ants Ant samples used for acetolysis were whole adult workers guts from
adult workers and whole larvae
We used acetolysis to look for pollen grains inside ants In acetolysis organic material is
dissolved in acetic anhydride and sulfuric acid leaving behind stained pollen exines When
possible we used adult and larval ants of the same species from different colonies or bait
locations in an attempt to sample more representatively We prepared samples by first washing
them in 70 ethanol to remove any pollen present on the samplersquos outer surface before placing
them individually in micro-centrifuge tubes with ~05mL of a 91 mixture of acetic anhydride
sulfuric acid We then heated each sample at 95degC for 20min allowing tissues to dissolve Next
we centrifuged samples at 1000 RPM for 5min and decanted the liquid supernatant before
adding ~05mL of glacial acetic acid to the precipitate centrifuging again as above We decanted
the samples and added a drop of distilled water
78
We examined a 150μL sub-sample of this solution for pollen grains under a light
microscope Larvae gave less than 150μL of solution following acetolysis so we sampled the
entire amount We identified pollen grains by their staining and their surface architecture For
most species we examined at least three adult workers and three larvae but for some species
fewer than three adult workers or larvae were available (Tables 31 and 32)
We investigated the relationship between trophic level as measured by δ15
N ratios and
pollen consumption by ants Stable isotope (SI) data was available for some (n =23) of the
Peruvian species from a separate project in which collections overlapped Samples were stored in
EtOH and dried at 50degC overnight We used heads legs and thoraxes (but not gasters) of ants
for SI analysis Often single individuals did not provide enough material for analysis and we
combined multiple individuals to achieve necessary weights up to 10 for small ant species
Samples were analyzed at the Boston University Stable Isotope Laboratory using a Finnigan
Delta-S analyzer For other Peruvian species for which we had samples but not corresponding
stable isotope data (n =25) we took the median δ15
N ratio value of that speciesrsquo genus and
assigned it to the species in question Using pollen presenceabsence data for these 48 species
we used logistic regression to correlate pollen presence and δ15
N ratio using R (v 2151)
Results
We found pollen grains in 37 of the 197 adult workers 20 of the 102 larvae and 38 of the 75 ant
species we examined (Tables 31 and 32) The proportion of individuals containing pollen did
not differ significantly between larvae and adults (χ2
1 = 002 p = 088) The number of pollen
grains found was often low (median = 3) but highly variable (range 1-468) Also pollen
79
presence was inconsistent among conspecific ant samples of species that contained pollen 31
had pollen in only one of the larvae or adults examined In only three species (two Cephalotes
and one Myrmelachista) did all examined adults contain pollen
Highly lsquoherbivorousrsquo carnivorous and omnivorous ants contained pollen grains (Fig
31) There was no correlation between δ15
N ratio and pollen presence (χ2 = 150 df = 1 p =
022) In fact several carnivorous species contained pollen grains (eg Odontomachus sp 1
Pachycondola sp1)
Discussion
Pollen consumption by ants appears to be more common than suspected The majority of species
(51) had at least one individual that had consumed pollen and in 20 multiple individuals
contained pollen Numbers of grains found tended to be low however with most individuals
containing only a few grains Ants may use pollen as only a small portion of their diet exploiting
a host of other protein-containing foods more heavily including other low- δ15
N foods (eg
honeydew epiphylls Houmllldobler and Wilson 1990 Davidson et al 2003)
There are a few reasons why pollen may not be heavily exploited by ants Pollen requires
much post-consumption processing to extract its nutrients These nutrients are located in the
pollenrsquos cytoplasm which is protected from direct digestion by the exines and intines of the
grains Organisms must either break through these layers or cause the pollen grains to germinate
or pseudo-germinate to access and digest the cytoplasm (Roulston and Cane 2000) This can be
achieved by consuming a sugar-containing solution (such as nectar) along with pollen However
animals that are not adapted to feed on pollen may not be very efficient at this process only
80
extracting nutrients from some of the grains (Herrera and Martiacutenez del Rio 1998) Thus while
ants can likely get some nutrients from pollen other forms of more easily processed protein may
be more attractive
Pollen may also pose handling difficulties for ants Despite the large number of studies
that have recorded ants visiting flowers (Chapter 1) only a few papers detail ants harvesting
pollen directly from anthers Byk and Del Claro (2010) reported that Cephalotes pusillus ants
removed almost all of the available pollen from Ouratea spectabilis flowers and at least two
other studies describe ants consuming pollen along with nectar from flowers (Horskins and
Turner 1999 Ness 2006) Unlike bees which possess both morphological (eg specialized hairs
for holding pollen) and behavioral adaptations (eg stereotyped pollen-packing motions) ants
have no apparent mechanisms to increase the amount of pollen harvested and are likely limited
by the number of grains they can ingest or carry Rather than collect pollen directly from flowers
it appears more likely that ants collect it haphazardly in the environment leading to a smaller
number of pollen grains consumed at any one time It is known that at least Cephalotes ants
engage in such behavior Creighton (1967) observed Cephalotes texanus collecting pollen grains
trapped by Citrus x paradisi leaf hairs
There is a chance that the small numbers of pollen grains we observed came from
secondary pollen consumption (ie consuming a palynivorous organism) or consumption of
liquid containing pollen While we cannot rule this out we believe our results represent actual
targeted pollen consumption by ants Cephalotes ants known palynivores (Creighton 1967
Baroni Urbani and de Andrade 1997) contained similar numbers of pollen grains as many of the
pollen-containing ant species that we examined As well in at least once instance ants that we
found containing pollen grains were in the process of preying on non-pollen consuming
81
organisms (eg a Pachycondola species that was carrying a termite in its mandibles) or were
attracted by sugar baits
Contrary to our expectations there was no relationship between an ant speciesrsquo δ15
N ratio
and whether it had consumed pollen (Fig 32) We expected palynivory to be more common in
herbivorous ants because pollen presents a widespread source of protein a potentially limiting
resource The lack of pattern might reflect how available pollen grains are in the environment
While other forms of food may be preferable ants tend to be opportunistic foragers (Carroll and
Janzen 1973) and there is likely little cost associated with the consumption of scattered pollen
grains during foraging trips beyond the volume they occupy in the gut Some genera were found
to consistently consume pollen In support of previous observations (Baroni Urbani and de
Andrade 1997 Byk and Del Claro 2010) Cephalotes species were among the most common
pollen consumers Camponotus species also consistently contained pollen grains of the eight
species examined five contained pollen and pollen was found in species from both Peru and
PNG Camponotus species also contained the two largest numbers of pollen grains found (468
and 271) Taken together these results suggest that they may be another as yet unrecognized
commonly palynivorous genus
In general larvae did not contain large numbers of pollen grains (ranging from 1-38
grains) This is counter to what is seen in bees where larvae are the main recipients of pollen
(Willmer 2011) This difference may be because ant larvae are continually fed via trophallaxis
by adults throughout their development whereas bee larval cells are provisioned with pollen and
then sealed while larvae complete their development If both adults and larvae are able to extract
nutrients from pollen we would expect to see a preferential flow of nutrients from pollen to
larvae but not necessarily the grains themselves
82
Unlike palynivory by organisms that also provide pollination services pollen
consumption by ants is unlikely to benefit plants Ants rarely act as pollinators (Beattie et al
2004) They have pollen-killing antibiotic secretions on their cuticles (Dutton and Frederickson
2012) and they can compete with other flower visitors reducing flower visitation rates (Ness
2006) The negative impacts of flower-visiting ants are thought to have led to the evolution of
ant-repellent floral volatiles (reviewed in Willmer 2009) These volatiles can pre-empt
competition between ants and pollinators and may often be located within the pollen itself
(Willmer 2009) If floral volatiles truly are a defense against ant visitation our results suggest an
additional evolutionary pressure to not only prevent ant-pollinator interactions but to also
protect plant gametes from occasional consumption by a non-pollinating visitor
Acknowledgements
We would like to thank Jon Sanders Lina Arcila Hernandez and Eric Youngerman for providing
samples the Thomson lab for use of supplies the Pierce lab for funding support during JSrsquo and
EYrsquos collections and the staff of CICRA and the New Guinea Binatang Research Center for
logistical support The Peruvian Ministry of Agriculture provided permits (Nos 394-2009-AG-
DGFFS-DGEFFS and 299-2011-AG-DGFFS-DGEFFS) MEF acknowledges financial support
from an NSERC Discovery Grant a Connaught New Researcher Award an Ontario Ministry of
Economic Development and Innovation Early Researcher Award and the University of Toronto
ARC was supported by an Ontario Graduate Scholarship and Sigma Xi
83
References
Baroni Urbani C and M L de Andrade 1997 Pollen eating storing and spitting by ants
Naturwissenschaften 84256ndash258
Beattie A J C Turnbull R B Knox and E G Williams 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany 71421ndash
426
Bluumlthgen N G Gottsberger and K Fiedler 2004 Sugar and amino acid composition of ant-
attended nectar and honeydew sources from an Australian rainforest Austral Ecology
29418ndash429
Byk J and K Del-Claro 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333ndash38
Carroll C R and D H Janzen 1973 Ecology of foraging ants Annual Review of Ecology and
Systematics 4231ndash257
Creighton W S 1967 Studies on free colonies of Cryptocerus texanus Satschi (Hymenoptera
Formicidae) Psyche 7434ndash42
Czechowski W B Markό and A Radchenko 2009 Rubbish dumps reveal the diet of ant
colonies Myrmica schencki Em and Myrmica rubra (L) (Hymenoptera Formicidae) as
facultative pollen-eaters Polish Journal of Ecology 56737ndash741
Czechowski WB Markoacute K Erős and E Csata 2011 Pollenivory in ants (Hymenoptera
Formicidae) seems to be much more common than it was thought Annales Zoologici
61519ndash525
84
Dussutour A and S J Simpson 2009 Communal nutrition in ants Current Biology 19740ndash4
Dutton E M and M E Frederickson 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561ndash569
Floren A A Biun and K E Linsenmair 2002 Arboreal ants as key predators in tropical
lowland rainforest trees Oecologia 131137ndash144
Grant B R 1996 Pollen digestion by Darwinrsquos finches and its importance for early breeding
Ecology 77489-499
Herrera L G and C Martiacutenez del Rio 1998 Pollen digestion by new world bats effects of
processing time and feeding habits Ecology 792828ndash2838
Houmllldobler B and Wilson E O 1990 The Ants 1st Edition Harvard University Press
Cambridge
Horskins K and Turner V B 1999 Resource use and foraging patterns of honeybees Apis
mellifera and native insects on flowers of Eucalyptus costata Australian Journal of
Ecology 24221-227
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506ndash514
Roulston T H I and J H Cane 2000 Pollen nutritional content and digestibility for animals
Plant Systematics 222187ndash209
Van Tets I G 1997 Extraction of nutrients from Protea pollen by African rodents Belgian
Journal of Zoology 12759-65
Willmer P G 2011 Pollination and Floral Ecology 1st Edition Princeton University Press
New Jersey
85
Willmer P G C V Nuttman N E Raine G N Stone J G Pattrick K Henson P Stillman
et al 2009 Floral volatiles controlling ant behaviour Functional Ecology 23888ndash900
86
Tables
Table 31 Numbers of adult worker and larval ants examined for each species collected in Peru
Numbers of pollen grains found in individual samples are given in parentheses after the sample
size when there were no grains no numbers are given
Genus Species Number of adults analyzed
(numbers of grains found)
Number of larvae analyzed
(numbers of grains found)
Dolichoderinae
Azteca sp 1 3 0
sp 2 2 0
sp 3 0 2
sp 4 3 (1) 0
sp 5 3 (2) 0
Dolichoderus decollatus 3 (66) 0
sp 1 3 0
sp 2 3 1 (2)
sp 3 3 3
sp 4 0 2
Ecitoninae
Labidus sp 1 3 0
Formicinae
Camponotus sp 1 3 3
sp 2 3 (82) 1 (2)
sp 3 3 (3) 0
sp 4 0 2
87
sp 5 8 (11) 0
sp 6 4 (7 16 468) 0
sp 7 5 (12) 0
sp 8 2 (1) 0
Paratrechina sp 1 4 (11) 0
Myrmelachista sp 1 3 (5 11 18) 0
sp 2 0 3
Trachymyrmex sp 1 3 (21) 0
sp 2 3 0
Myrmicinae
Atta sp 1 3 0
Cephalotes atratus 3 (2 2 21) 0
placidus 3 (1 4 21) 0
sp 1 3 0
sp 2 1 0
sp 3 2 0
Crematogaster sp 1 3 1
sp 2 3 (35) 0
sp 3 9 (7) 0
sp 4 2 0
Megalomyrmex sp 1 3 0
sp 2 0 2
sp 3 4 (7) 0
sp 4 3 0
Monomorium sp 1 0 3 (2)
88
Pheidole sp 1 0 3
sp 2 3 0
sp 3 0 3
Procryptocerus sp 1 3 0
Solenopsis sp 1 0 3
sp 2 3 (3 5) 0
Wasmannia sp 1 3 0
Ponerinae
Pachycondola sp 1 3 (3) 0
Odontomachus sp 1 3 (34) 0
Pseudomyrmicinae
Pseudomyrmex sp 1 3 0
sp 2 3 0
sp 3 3 (9) 0
sp 4 0 3
sp 5 0 3
89
Table 32 Numbers of adult worker and larval ants examined for each species collected in Papua
New Guinea Numbers of pollen grains found in individual samples are given in parentheses
after the sample size when there were no grains no numbers are given
Genus Species Number adults analyzed
(grains found)
Number larvae analyzed
(grains found)
Aenictinae
Aenictus sp 1 3 3 (1 1)
Dolichoderinae
Philidris sp 1 3 1 (4)
Tapinoma sp 1 2 3 (38)
Technomyrmex sp 1 0 3 (12 12)
Ectatomminae
Rhytidoponera sp 1 1 3 (2)
Formicinae
Acropyga sp 1 3 3 (2)
sp 2 3 3
sp 3 3 3
Anoplolepsis sp 1 3 (1) 3 (1)
Calomyrmex sp 1 3 3 (1)
Camponotus sp 1 3 (1 271) 3
sp 2 1 3
sp 3 2 0
Nylanderia sp 1 3 3
Opisthopsis sp 1 3 3
Polyrachis sp 1 1 3 (4)
90
Myrmicinae
Crematogaster sp 1 3 3
sp 2 3 (32) 3
Monomorium sp 1 3 (1) 3 (1)
sp 2 3 3 (1 1 1)
Pheidole sp 1 3 3
Pristomyrmex sp 1 3 3 (11)
Pyramica sp 1 3 0
Ponerinae
Odontomachus sp 1 3 3 (4)
91
Figures
Figure 31 Photographs of pollen grains found in two ant species a) Dolichoderus sp 2 and b)
Camponotus sp 6 Scale bars = 50 microm
92
Figure 32 Relationship between trophic level as measured by δ15
N ratio and pollen presence
for 48 neotropical ant species 1 Atta sp 5 Azteca spp 8 Camponotus spp 5 Cephalotes spp 4
Crematogaster spp 5 Dolichoderus spp 1 Labidus sp 3 Megalomyrmex spp 1 Monomorium
sp 2 Myrmelachista spp 1 Odontomachus sp 1 Pachycondola sp 1 Paratrechina sp 3
Pheidole sp 4 Pseudomyrmex spp 2 Solenopsis spp and 1 Wasmannia sp
93
Concluding Remarks
Ants have traditionally been considered to be unwelcome flower visitors they consume floral
rewards ostensibly meant for pollinators while rarely providing pollination services themselves
In this thesis I investigate the validity of this assumption and use an experimental study and an
observational study to examine two ways that ants may impact plant reproduction
In the first chapter I review the literature on ants as flower visitors I show that the common
assumption of ants always being categorically negative for plant reproduction is incorrect while
ants may reduce floral attractiveness to pollinators the net effect of ants on plant fitness
measures is variable ranging from positive to negative to neutral I also discuss ants as
pollinators and ways that plants may minimize ant visitation to flowers before finishing with a
section highlighting questions that still need investigation
In the second chapter I explore how interference competition with ants can structure bumblebee
foraging behavior and how this may alter pollen movement patterns Using artificial flowers with
male or female function I allowed Bombus impatiens bumblebees to forage on flowers that
either did or did not have Myrmica rubra ants on them Ant presence significantly reduced the
amount of pollen analogue donated and received by flowers Building on these results I found
that the presence of ant scent alone elicited similar changes to bumblebee behavior reducing the
amount of pollen analogue donated by male flowers My results show that interference
competition is sufficient to alter bumblebee foraging behavior and that the effects of ants on
pollinators may extend after ant visitation to flowers Further study is needed to determine if bees
are able to differentiate between ant species by visual or olfactory cues Their ability to do so
94
could affect how generalized their response to ant presence is and may help to better predict how
ants will affect plant reproduction
In the final chapter I investigate how common palynivory is amongst tropical ant species I used
acetolysis and light microscopy to examine whole ants larvae and ant guts from Papua New
Guinea and Peru I found that tropical ants consistently contain pollen grains albeit often in
small numbers (lt5) However some genera notably Camponotus and Cephalotes consistently
contained higher numbers providing the first evidence that Camponotus may be a palynivorous
genus The small numbers of pollen grains present may indicate that ants preferentially exploit
other more easily digestible high-protein foods over pollen Future studies should investigate
exactly what role pollen plays in ant diets by supplementing the diet of captive ant colonies with
pollen This would allow us to examine how pollen consumption affects colony demography as
well as how efficiently ants digest pollen and whether ants exhibit pollen preferences
I bring up several pressing and unanswered questions within this thesis and highlight three here
First numerous plant species possess chemical or physical traits assumed to increase plant
fitness by reducing ant visitation However this has rarely been tested and instead research has
focused predominately on documenting ant repellence We should move away from this and
begin examining plant fitness in the presence or absence of these repellent traits to see how
beneficial these traits truly are Second how flower visiting ants impact male fitness has been
almost completely ignored in favor of female fitness Future studies should investigate how ant
visitation affects both pollen donation and pollen movement patterns Lastly research involving
antibiotics present on ant cuticles and their effect on pollen viability has been too small in scope
often limited to demonstrating the pollen killing effects of ant secretions Investigation into
95
whether plants have evolved any resistance to the secretions of the local ant community or why
some plants appear unaffected by their ant partners may yield exciting results
In conclusion ants have the capacity to strongly affect plant reproduction both positively and
negatively The fact that these diametrically opposing forces exist means we cannot approach
flower-visiting ants as either positive or negative a priori Instead we should strive to work
towards a better understanding of what is occurring in the system of interest and what role ants
play within it
96
Copyright Acknowledgments
Chapter 2 ldquoAnts and ant scent reduce bumblebee pollination of artificial flowersrdquo is current in
press in The American Naturalist Request for its inclusion here was sought and approved
viii
List of Figures
Figure 21 Photograph of artificial flower lids with anther and stigma74
Figure 22 Dye transferred (mean plusmn 1SE) by B impatiens a) in the presence and absence of M
rubra ants and b) with or without M rubra scent75
Figure 31 Photographs of pollen grains found inside ants92
Figure 32 Relationship between trophic level and pollen presence for neotropical
ant species93
1
Chapter 1 Ants as flower visitors and their effects on pollinator behavior and plant
reproduction
Cembrowski AR and Frederickson ME
Planned submission to a journal such as Oikos or Insectes Sociaux
ARC wrote the manuscript with input from MEF
Abstract
Ants often provide a range of benefits to flowering plants particularly by reducing herbivory
However when ants visit flowers they can deter pollinators or damage floral structures
Currently it is unclear how often and in what direction flower-visiting ants affect plant
reproductive success A review of the literature shows that ants can help or hinder pollination
processes but overall flower-visiting ants most commonly have a non-significant net effect on
plant fitness Although ants may directly harm flowers or indirectly affect pollination by
consuming floral nectar or harassing pollinators they can also benefit plants by attacking
florivores or predators of pollinators Ants occasionally act as pollinators themselves though ant
secretions often kill pollen grains There is growing evidence that many plants have traits that
allow them to avoid or minimize the negative effects of flower-visiting ants More research is
needed to determine if these traits provide fitness benefits to plants by preventing ant visitation
or if they function as generalized repellents of floral antagonists
2
Introduction
Ants and angiosperms share a long evolutionary history Ant diversification closely followed that
of flowering plants as ants took advantage of the large prey numbers that angiosperms supported
and the new habitats they provided (Moreau 2006) Subsequently numerous angiosperm
lineages evolved intimate associations with ants that function as an indirect or biotic form of
plant defense Food rewards such as extrafloral nectar or pearl bodies or housing in the form of
domatia attract ants to plants where they reduce herbivory and increase plant fitness (reviewed
in Rosumek et al 2009) However the traits that make ants good plant defenders particularly
their abundance attraction to sugar sources and aggressiveness can also cause problems for
plant reproduction Ants attracted by floral nectar can compete with pollinators for resources
and defend flowers potentially reducing plant fitness (Ness 2006 Junker et al 2007) That ants
negatively affect plant reproduction is an old and pervasive hypothesis Kerner (1878) called ants
ldquomost unwelcome guestsrdquo on flowers and more recent authors have similarly expressed that ant
visitation to flowers reduces plant reproductive success (Ness 2006 Lach 2007 Willmer et al
2009)
Nonetheless studies assessing the impact of flower-visiting ants on plant reproductive
success have produced mixed results Ants can have negative impacts on pollination and flower
function (Galen 1983 Puterbaugh 1998 Ness 2006 Hansen and Muumlller 2009) However other
studies have found no effect or even strong benefits of ant visits to flowers (Rico-Gray and
Thien 1989 Altshuler 1999 Schuumlrch 2000 Ashman and King 2005) This discrepancy between
ants being ldquounwelcome guestsrdquo and their actual effects on plant reproduction requires
clarification Our goal here is to highlight the ways that flower-visiting ants may affect plant
reproduction both positively and negatively and to discern through a review of the literature
3
what impacts ants have on plant reproductive success First we discuss mechanisms by which
flower-visiting ants can directly and indirectly impact plant reproduction both through floral
damage and by mediating changes in pollinator or florivore behavior We end this section by
evaluating published research that collectively shows that flower-visiting ants have
predominantly neutral effects on plant fitness measures Next we discuss pollination by ants
focusing on the ldquoantibiotic hypothesisrdquo before describing how plants may prevent ants from
visiting flowers Lastly we discuss promising new avenues of research and highlight work that
still needs to be performed to give a more complete understanding of ants as flower visitors
1 Ant interactions with flowers and floral visitors
i) Damage to flowers
Ants can directly disrupt plant reproduction by damaging floral structures preventing either the
fertilization of ovules or the presentation of pollen Rarely ants forage on floral tissue itself for
example Puterbaugh (1998) found that Formica neorufibarbus ants chewed through the coronal
ring of Eritrichium aretiodes flowers to get lipids More often but still not commonly foraging
ants chew through reproductive tissue to access floral nectar This can cause sterilization of
whole flowers (Echium plantagineum Kirk 1984) or affect female function only if just styles are
damaged effectively creating male flowers (Polemonium viscosum Galen 1983) Antibiotic
secretions on ant integuments may also directly damage pollen grains but the fitness effects of
this are unclear (see 2 below)
Several species of Crematogaster and Allomerus ants sterilize their host plants by
attacking floral buds (Yu and Pierce 1998 Stanton et al 1999 Gaume et al 2005 Maleacute et al
2012 Table 11) These ants nest in ldquoant-plantsrdquo or ldquomyrmecophytesrdquomdashie plants that host ant
4
colonies in specialized structures (domatia) The sterilizing ants do not appear to consume floral
tissues (Yu and Pierce 1998) and two hypotheses may explain why ants destroy floral buds First
ant colony size is often limited by domatia availability on their host plant By destroying floral
buds ants can cause plants to divert resources from reproductive to vegetative growth increasing
domatia number and allowing for larger ant colonies (Yu amp Pierce 1998 Frederickson 2009)
Second when plant-ants must defend their colony from takeovers by other colonies they may
benefit from limiting access to their host plant By destroying buds that could develop into
flowers branches or leaves ants can prevent their host plant from contacting nearby plants
occupied by enemy ant colonies (Stanton et al 1999)
The impact of sterilization on lifetime plant fitness is unclear Ants may destroy all of
their host plantrsquos floral buds (Yu and Pierce 1998) completely preventing reproduction or only a
portion of them (Maleacute et al 2012) allowing some seed set However counter-intuitively this
sterilization behavior may provide long-term fitness benefits to plants (Frederickson 2009
Palmer et al 2010) Ant-plants often host several different ant species over their lifetimes some
of which are non-sterilizing If hosting a sterilizing species at a certain life stage increases
survival or growth relative to hosting a non-sterilizing species at that life stage plants may
increase lifetime fitness by largely forgoing current reproduction in favor of increasing future
reproduction (Palmer et al 2010) Why plants invest resources in producing flowers that are
quickly destroyed by ants is not known but the timing of flower destruction may be important If
ants attack flowers after anthers dehisce plants may still realize some male fitness via pollen
donation (Yu and Pierce 1998) Additionally if the cost of flower production is relatively minor
and ants sometimes ldquomissrdquo flowers (Edwards and Yu 2008) attempting limited reproduction
may be a better strategy than forgoing it entirely
5
ii) Competition with pollinators
Ant activity on flowers can dramatically alter pollinator behavior Ants can reduce the species
diversity frequency or duration of pollinator visits to flowers (Norment 1988 Tsuji et al 2004
Ness 2006 Junker et al 2007 Lach 2007 2008b Romero et al 2011 and see Chapter 2) These
changes are largely due to either exploitative competition (ie consumption of shared resources)
or interference competition (ie direct aggression) between ants and pollinators
a) Exploitative competition
Pollinators and ants frequently compete for floral nectar Flower-visiting ants are often classified
as nectar thieves (sensu Inouye 1980) because they typically enter flowers through the
ldquolegitimaterdquo opening and consume nectar but transfer no pollen (Wyatt 1980 Ness 2006
Chamberlain and Holland 2008) Ants can also act as primary nectar robbers chewing holes
through corollas to reach nectaries (Willmer and Corbet 1981 Koopowitz and Marchant 1998)
although they are more commonly secondary nectar robbers and use holes made by previous
robbers (Newman and Thomson 2005)
Like other flower visitors foraging ants will often leave nectar behind in flowers This
amount can be less (Bleil et al 2011) more (Fritz and Morse 1982) or similar to (Lach 2008a)
amounts left by other visitors Often ants are morphologically constrained from fully exploiting
nectar Unlike bees and butterflies ants do not have proboscises and must get close to nectar to
consume it Narrow corollas or nectar spurs can prevent ant access to some or all floral nectar
(Schemske 1980 Galen and Cuba 2001 and see below) Smaller ants may be better able to
6
access nectar (Newman and Thomson 2005 Agarwal and Rastogi 2007) but can be limited by
their crop size in the amount of nectar they can remove (Lach 2005) If foraging ants leave
enough nectar behind for a pollinator to recover the cost of visiting a flower ants may have
minimal effects on plant reproductive success (Maloof and Inouye 2000 Turner et al 2012)
Differences among ant species in behavior and diet also affect their consumption of floral
nectar Species vary in how many flowers they visit per inflorescence or per plant and in how
many ants will visit a flower simultaneously (Lach 2005 Junker et al 2007 Lach 2013)
Invasive antsmdashthought to be better at exploiting and defending resources than native ants
(Holway et al 2002)mdashoften recruit to flowers in large numbers sometimes several dozen or
more (Visser et al 1999 Hansen and Muumlller 2009) Overall many factors may influence how
heavily ants exploit floral nectar on plants including ant density plant height the spatial or
temporal distribution of floral nectar and the availability of alternative food resources for ants
(Cushman and Addicott 1991 Lach 2013)
Rarely ants and pollinators may compete for pollen We know of only a few studies that
have recorded ants harvesting pollen directly from anthers (Horskins and Turner 1999 Ness
2006 Byk and Del-Claro 2010) Other studies have found pollen grains in ants (eg Baroni
Urbani and de Andrade 1997 and see Chapter 3) but this may not indicate competition with
pollinators ants sometimes collect stray pollen grains from the environment (Creighton 1967)
One New World genus Cephalotes might be specially adapted for pollen consumption They
possess ldquoproventricular shieldsrdquo structures that may restrict solid food to the crop allowing
nestmates to exchange pollen grains via trophallaxis It is unclear if this structure evolved for this
purpose but Cephalotes are the most frequently documented palynivorous ants (eg Creighton
1967 Baroni Urbani and de Andrade 1997 Byk and Del-Claro 2010) How efficiently ants
7
digest pollen and whether ants like bees feed it to larvae as a protein source is unknown At
least two studies have found that ant larvae consume pollen providing some support for this idea
(Wheeler and Bailey 1920 and Chapter 3)
b) Interference competition
Ants are relatively unique among flower visitors in defending floral resources from competitors
(but see Willmer and Corbett 1981 Roubik 1982) This defense ranges from chance encounters
with ants attacking visitors when flower occupancy overlaps (Ashman and King 2005 Junker et
al 2007) to actively patrolling inflorescences or constructing protective galleries around
nectaries (Horvitz and Schemske 1984 Gaume et al 2005 Hansen and Muumlller 2009)
Interference competition even in the absence of exploitative competition is sufficient to alter
pollinator behavior (Chapter 2)
The strength and outcome of interference competition depends on the identities of the ant
and the pollinator Pollinators might not alter their visitation patterns in response to non-
aggressive ants (Junker et al 2007) but more antagonistic ants may elicit avoidance behavior
before or soon after the visitor lands (Willmer and Corbet 1982 Altshuler 1999 Schuumlrch et al
2000 Table 11) Pollinator size may help predict their reaction to ants because ants may deter
only small-bodied pollinators (Gonzaacutelvez et al 2012) It is not clear whether flower visitors view
ants as aggressive competitors or as potential predators rarely ants prey on pollinators (Schatz
and Wcislo 1999) but in most cases ants merely cause pollinators to relocate
How well pollinators recognize and avoid ant-visited flowers is unresolved Collectively
pollinators have lower visitation rates to flowers visited by ants (Romero et al 2011) This is
8
dependent on pollinator identity however dipterans do not appear to discriminate between ant-
visited and ant-free flowers while bees can identify and avoid ant-visited flowers Bees likely
use a combination of visual and olfactory cues to recognize ant-visited flowers They can
identify previous conspecific and some heterospecific insect visitors by the hydrocarbon
ldquofootprintsrdquo they leave during foraging (Stout and Goulson 2001) Bumblebees can detect ant
hydrocarbons although this has been shown only in the laboratory (Ballantyne and Willmer
2012 Chapter 2) No study has yet examined if bees can use sight alone to detect ants but
several suggest it (Junker et al 2007 Gonzaacutelvez et al 2012) Some bees may discriminate
between ant species when visiting flowers (eg Lach 2008b) which follows if not all ant-
pollinator interactions are equally costly The ability of bees to do so will affect how generalized
their avoidance behaviors will be If they can discriminate between species bees may avoid
specific flowers visited by more ldquocostlyrdquo ants However if they cannot bees might either avoid
all ant-visited flowers or none depending on how negative the average interaction with an ant is
iii) Interactions with flower and pollinator antagonists
Ants can defend the flowers they visit Numerous plant species have circumfloral or bracteal
nectaries (ie extrafloral nectaries around flowers) and ants attracted to these nectaries can
reduce bud ovule or flower predation increasing plant fitness (Inouye and Taylor 1979 Rico-
Gray and Thien 1989 Sugiura et al 2006) Although the presence of EFNs (extrafloral nectaries)
so close to floral nectaries (sometimes less than 1 cm Ness 2006) might increase discovery and
exploitation of floral nectar by ants only one study has investigated this hypothesis finding no
relationship between the proximity of EFNs to floral nectaries and antsrsquo use of floral nectar
(Galen 2005) Holes made by nectar robbers are functionally analogous to circumfloral nectaries
9
and can also attract ants that defend flowers (Newman and Thomson 2005) and nectar-thieving
ants can similarly prevent florivory in some systems (Lach 2007 Bleil et al 2011)
Ants can also reduce the abundance of flower-dwelling predators or parasites of
pollinators Ambush predators such as crab spiders often forage at flowers where they may
reduce pollinator visitation rates and plant fitness (Dukas 2001 Gonccedilalvez-Souza et al 2008)
Ants can decrease spider abundance and richness on plants (Rudgers 2004 Renault et al 2005
Nahas et al 2012) and some evidence suggests that they can do the same on flowers (Lach
2008) In select cases ants may also reduce the abundance of pollinator parasitoids (Holland et
al 2011) Whether ants increase plant reproductive success by removing pollinator antagonists
has not yet been examined but should be If ants create lsquoenemy-freersquo flowers pollinators might
prefer to land on flowers with ants
iv) Net effects
Table 11 compiles studies in which the authors recorded ants consuming floral rewards or
interacting with pollinators and measured some aspect of plant fitness (eg fruit or seed set
pollen deposition) We included only a single study per plant species unless subsequent studies
found conflicting results or measured the fitness of another plant sex Records of ant pollination
are included in a separate table (Table 12)
Table 11 reveals relatively few examples of flower-visiting ants negatively affecting
plant fitness given how costly ant visitation is assumed to be The net effect of ants on plant
reproductive success is actually quite variable with similar numbers of studies finding positive
negative or neutral effects of ant presence Surprisingly when ants do have negative effects it is
10
usually because they directly damage floral structures and not because they prevent pollinators
from visiting flowers and successfully transferring pollen Why are ants not more often negative
for plant reproduction when this is commonly assumed to be true
First ants may compensate for reducing pollinator visitation rates by improving
pollination quality As pollinators move between flowers on a plant they often deposit self-
pollen on stigmas (ie geitonogamy) In some plants with late-acting self-incompatibility ovules
fertilized by self-pollen are subsequently aborted reducing the number of seeds that can be
produced (Barrett 2002 and references therein) Geitonogamy can also reduce offspring viability
or vigor (de Jong et al 1993) and cause pollen discounting a reduction in the amount of pollen
available for outcrossing because of self-pollination (Barrett 2002) By competing with
pollinators ants might decrease rates of geitonogamous pollination if pollinators then visit fewer
sequential flowers within a plant Ants might also cause pollinators to fly further between plants
potentially increasing the genetic diversity of pollen arriving at stigmas Although such effects
have been reported for other floral larcenists (Maloof 2001 Irwin 2003) to our knowledge these
hypotheses have not been investigated in relation to ants
Second ants and pollinators do not always forage for the same resources possibly
allowing them to partition floral rewards If ants forage for nectar but not pollen and flowers can
be pollinated efficiently by visitors looking to gather mostly pollen and little or no nectar then
plant reproduction will not be affected by ants consuming floral nectar Junker et al (2010a)
found that honeybees foraged for both pollen and nectar in the absence of ants but reduced their
foraging on nectar but not pollen when ants could access flowers resulting in no difference in
fruit set between treatments Whether pollinators commonly adjust their foraging behaviors in
response to ants warrants further research
11
Third ants are somewhat unique among floral larcenists in that they often provide direct
benefits to the plants they visit In the studies listed in Table 11 ants sometimes decreased
herbivory on plants (Chamberlain and Holland 2008 Bleil et al 2011) or otherwise aided the
plant (eg ant presence repelling smaller less effective pollinators Gonzaacutelvez et al 2012) If the
positive effects of ants cancel or outweigh the negative effects of flower-visiting ants on
pollinators then ant visitation may be neutral or even positive for plant fitness
Fourth ants may impose fitness costs to plants not captured in the studies in Table 11
Nectar robbers have stronger negative effects when plants are pollen-limited (Burkle et al 2007)
but only one study in Table 11 assessed whether flowers would set more seed if they received
more pollen finding no evidence of pollen limitation (Lach 2008a Table 11) Even if ants
decrease pollinator visitation rates there is limited scope for ants to decrease plant reproductive
success when plants are not pollen-limited Furthermore nearly all the studies in Table 11
measured only female fitness Only three quantified aspects of male fitness (Wyatt 1980 Fritz
and Morse 1981 Galen and Geib 2007) and we know of only one other study in a system in
which ants occasionally pollinate that has also measured male fitness (Ashman 2000) All four
studies measured pollen or pollinia removal rates and none found any effect of flower-visiting
ants on male fitness However whether other measures of male fitness (eg seeds sired pollen
deposition patterns) would reveal similar or different effects needs investigation
Finally although floral defenses against ants (see 3 below) may prevent us from
observing the negative effects of ants on plant reproductive success we consider this unlikely
Several studies have examined interactions between ants and plants that share little or no
coevolutionary history usually in the context of ant invasions Although invasive ants often
recruit to flowers in larger numbers than native ants they do not appear to reduce plant
12
reproductive success (Blancafort and Goacutemez 2005 Lach 2007 Lach 2008a but see Hansen and
Muumlller 2009) And in Hawaii where angiosperms evolved for millions of years in the absence of
ants and are thought to have secondarily lost floral defenses against ants (Junker et al 2011a)
there is no evidence that the preponderance of recently introduced flower-visiting ants negatively
affects plant fitness (Junker et al 2010a Bleil et al 2011)
2 Ants as pollinators
Ants may directly benefit plant reproduction by acting as pollinators but this is a relatively rare
occurrence Ants have long been considered poor pollinators (Kerner 1878) Initially this was
based on morphological and behavioral differences between ants and recognized pollinators
Worker ants are wingless and often relatively hairless making them poor pollen dispersers As
well because many ants exhibit Ortstreue (literally ldquoplace fidelityrdquo) ants were thought to return
to the same branch or flower repeatedly effecting no cross pollination (Houmllldobler and Wilson
1990) Though largely true these traits do not necessarily preclude ants from acting as
pollinators (Table 12) Although they commonly have restricted foraging ranges (Houmllldobler and
Wilson 1990) some ants can effect outcrossing (eg Ashman and King 2005 Carvalheiro 2008)
and many ants are as hairy if not hairier than their pollinating counterparts
In the late 1970s the ldquoantibiotic hypothesisrdquo was put forward to explain the rarity of ant
pollination Ants use antibiotic secretions to protect themselves against the fungal and bacterial
pathogens common in their nests and environs Iwanami and Iwadare (1978) found that one of
these compounds myrmicacin also greatly reduces pollen germination rates Subsequently
Beattie et al (1984 1985) found that short periods of contact (le 30 minutes) with an antrsquos
integument caused pollen grains to fail to germinate Some of these pollen-killing compounds
13
were traced back to the ant metapleural gland which produces hygienic secretions (Houmllldobler
and Wilson 1990) although contact with ants lacking metapleural glands can also reduce pollen
viability (Beattie et al 1985 Dutton and Frederickson 2012) Ant mandibular or anal gland
secretions and potentially ant venom may likewise defend ants against pathogens and have
similar effects on pollen germination (Beattie et al 1985 Graystock and Hughes 2011)
Nonetheless ant pollination has been documented repeatedly There are at least 18 cases
in which ant pollination has been shown experimentally and five others in which observations
strongly suggest it (Table 12) The majority of these plants fit the ldquoant pollination syndromerdquo
originally proposed by Hickman (1974) dense stands of short plants with small flowers and
easily accessible nectaries Typically ant-pollinated plants are associated with hot and dry
climates (principally in the Mediterranean) but dry alpine environments may also favor the
evolution of ant pollination (eg Svensson 1985 Puterbaugh 1998 Schiestl and Glaser 2011) In
most cases ants appear to be the sole or major pollinator of the plant in question (Hickman 1974
Peakall et al 1990 de Vega et al 2009) although ants sometimes make only supplementary
contributions to pollination (Schuumlrch et al 2000 Ashman and King 2005) This suggests that co-
evolution between ants and plants may not be necessary for ants to be useful pollinatorsmdashwhich
is surprising given the potential negative effects of ants on plant reproduction
Ant-pollinated plants cope with ant secretions in a variety of ways Several plant species
may simply tolerate pollen damage (Gόmez and Zamora 1992 de Vega et al 2009) apparently
possessing no overt adaptations to prevent it These plants might receive enough pollen to
overcome reduced pollen viability Other plants may place pollen on ant body parts thought to
have fewer secretions thereby potentially escaping damage (eg on ant legs Ramsey 1995) or
attach pollen to ants with specialized structures avoiding direct contact with the ant integument
14
(Peakall et al 1990) It is not known whether pollen evolves resistance to ant secretions or
whether plants deposit pollen on ants which have less disruptive secretions although these
possibilities have been suggested elsewhere (eg Garciacutea et al 1995 Ramsey 1995)
One key question regarding the effects of ant secretions on pollen grains urgently needs
to be resolved Several studies have suggested that the pollen-killing secretions of ants may have
contributed to the evolution of floral defenses against ants (eg Wagner 2000 Galen and Geib
2007) For this to be true ants must decrease male fitness by damaging pollen grains before they
leave flowers and are deposited on receptive stigmas Only two studies have investigated the
effects of ant secretions on pollen still in anthers (Wagner 2000 Galen and Butchard 2003) and
both constrained ants unnaturally to flowers for significantly longer than ants would normally
stay on flowers Although these studies found strong reductions in pollen vigor it is unclear if
ant secretions decrease male fitness under natural ant visitation conditions
3 Floral defenses against ants
Plants are not passive recipients of flower visitors and have some ability to ldquochooserdquo which
organisms can access floral rewards Flowers resist ants using both structural and chemical traits
More thorough investigation is needed to determine whether these traits evolved to prevent or
minimize the negative effects of ants on plant reproductive success (ie whether these traits can
truly be considered defenses against ants see 4ia below) It is unlikely however that ants
experience reciprocal selection to circumvent floral resistance traits because the fitness benefits
to ants of accessing flowers are probably small Thus there may be little scope for
coevolutionary arms races between floral defenses against ants and ant counter-adaptations
15
i) Structural resistance traits
a) Restricting ant access
Numerous plant species have physical structures preventing ant access to flowers Thin pedicels
can limit antsrsquo gripping abilities and make ants easier to dislodge when flowers are moved by
wind (Gaume et al 2005) and waxy secretions can render stems too slippery for ants to climb
(Harley 1991) Structures surrounding flowers such as water moats or dense hairs may similarly
keep ants from accessing floral rewards (Willmer et al 2009) Many such traits function as broad
filters and prevent not only ants but most crawling insects from reaching flowers Thus it is
difficult to determine whether ants florivores or both acted as selective agents favoring the
evolution of physical barriers to flowers
Flower shape may also reduce ant-pollinator conflict Long narrow corollas or nectar
spurs can prevent all but the smallest ants from accessing floral nectaries (Bluumlthgen et al 2004
Agarwal and Rastogi 2007) while still allowing long-tongued pollinators access to rewards
Selection to exclude nectar-thieving ants in such a manner however may be counteracted by
pollinator preferences for wider corollas that allow easier flower handling (Galen and Cuba
2001) Additionally these types of flowers may be more prone to robbery (Irwin and Maloof
2002) limiting their usefulness in preventing nectar larceny
b) Separation in space or time
Plants may separate floral rewards and ants temporally or spatially to minimize ant impacts on
plant reproduction Several authors have proposed that myrmecophytes have adapted to the
16
sterilization or otherwise detrimental activities of ants by locating domatia or food rewards on
different branches than flowers (Izzo and Vasconcelos 2002 Raine et al 2002 Edwards and Yu
2008) Temporal separation of ant and pollinator activities could likewise reduce conflict though
evidence is limited Some acacia flowers dehisce during periods of low ant activity (Willmer and
Stone 1997 Nicklen and Wagner 2006) but it is unclear if this is in response to ants
temperature or pollinator activity Timing of nectar secretion may similarly reduce ant-pollinator
competition If ants are active prior to pollinators they may deplete floral nectar and reduce
pollinator visitation rates (Schaffer et al 1984) However if nectar is secreted when ants are
inactive pollination may occur unimpeded (Norment 1988) This temporal separation could
favor pollinators that are active when ants are not (eg Wyatt and Morse 1981) but this is
poorly documented Further studies particularly focusing on flowers with the potential for
nocturnal and diurnal pollination may provide more examples
c) Extrafloral nectaries (EFNs)
One hypothesis put forward to explain the evolution of EFNs proposes that EFNs ldquodistractrdquo ants
from consuming floral nectar (Kerner 1878 Wagner and Kay 2002) Wagner and Kay (2002)
tested this hypothesis by constructing model plants using artificial nectaries assigned at random
to represent either floral or extrafloral nectaries They found that the presence of EFNs could
reduce ant exploitation of floral nectar However as highlighted by Galen (2005) this design is
closer to a plant making a surplus of flowers some of which will avoid ant visitation if visitation
rates increase sublinearly with increasing flower number Producing extra flowers to escape ant
visitation appears to be a viable strategy in some systems (Ashman and King 2005 Galen 2005)
However Galen (2005) found that applying honey (simulating extrafloral nectar) to P viscosum
17
leaves had the opposite effect and increased ant visits to flowers Only two studies both in
Pachycereus schottii have found evidence for the distraction hypothesis under field conditions
(Chamberlain and Holland 2008 Holland et al 2011)
There is limited empirical support for the distraction hypothesis but abundant evidence
that EFNs attract ants that defend plants against herbivores (reviewed in Rosumek et al 2009)
To function as attractants the presence of EFNs must increase ant abundance on plants
However because of the increased numbers attracted EFNs would not be expected to reduce the
abundance of ants on nectar rewards or be able to satiate the ants recruited Only if ant
abundance stops increasing at a reward density much lower than that available on the plant
would EFNs serve to distract ants In this case the amount of rewards may overwhelm ants
leaving some flowers underexploited However even in P schottii ants still visited flowers
(Chamberlain and Holland 2008) In cases where ant abundance would continue to increase with
additional rewards it is unlikely that EFNs function as distractions
ii) Chemical resistance traits
a) Toxic nectar
One of the earliest proposed floral defenses against ants was toxic nectar (Janzen 1977)
However studies testing Janzenrsquos (1977) hypothesis uncovered only a very few isolated
examples (eg Catalpa speciosa Stephenson 1981 Crinum erubescens Guerrant and Fiedler
1981) in short most floral nectar is not toxic or repellent to ants (Baker and Baker 1978
Schubart and Anderson 1978) In fact ants sometimes prefer floral nectar over solutions with
equivalent sugar concentrations (Koptur and Truong 1998)
18
In contrast floral nectar can repel other flower visitors (eg birds Johnson et al 2006
lepidopterans Kessler and Baldwin 2007) Repellency is often caused by plant secondary
metabolites which may occur in nectar as a by-product of their production elsewhere in the
plant where they function as defenses against herbivory (Adler 2001) Alternatively toxic or
repellent nectar may be adaptive repellent nectar may promote effective pollination by reducing
the amount of reward any one pollinator takes spreading nectar over several pollinator visits
(Kessler and Baldwin 2007) or it may function as a filter selecting for specific pollinators
(Johnson et al 2006) The latter may explain why repellent nectar is not more common
legitimate pollinators in addition to floral larcenists may be deterred from visiting flowers with
repellent nectar This may be particularly true of nectars that would repel ants which are closely
related to bees nectar distasteful to ants may often also be distasteful to bees and those nectars
acting as a filter for bees may still be palatable to ants For example Prŷs-Jones and Willmer
(1992) found that the ammonia-containing nectar of Lathraea clandestina is readily consumed
by both bees and ants even though it deters birds that act as nectar thieves Although floral
nectar itself rarely repels ants Guerrant and Fielder (1981) found that the presence of macerated
petals within nectar can often keep ants away Alkaloids in petals may be responsible and might
explain why ants seldom act as primary nectar robbers these secondary compounds may prevent
ants from chewing through corollas
b) Floral volatiles
Repellent floral volatiles appear to be a more widespread form of chemical resistance against
ants These were reviewed recently by Willmer et al (2009) and will be discussed here only
briefly Numerous plant taxa produce floral scents repellent to ants including myrmecophilous
19
myrmecophytic and non-ant associated plants in both temperate and tropical ecosystems
(Willmer et al 2009 Junker et al 2011a Ballantyne and Willmer 2012) In three large studies
floral volatiles that repel ants were detected in about a third of all plants tested (Willmer et al
2009 Junker et al 2011a Ballantyne and Willmer 2012) Pollen is often the source of ant-
repellent volatile compounds (Willmer et al 2009 Ballantyne and Willmer 2012) but they can
be emitted from other floral parts including petals That pollen is often the source of repellence
could suggest that preventing ant visitation may be more important in protecting male than
female function Additionally because repellence would decrease as pollen is removed from
anthers ants may return and defend flowers more quickly than if repellent chemicals were in
longer-lasting structures (eg petals) In some cases floral volatiles are thought to mimic ant
alarm pheromones (Willmer et al 2009) However floral scents elicit inconsistent reactions
across ant species ranging from repellence to attraction (Agarwal and Rastogi 2007 Ballantyne
and Willmer 2012)
Producing structural or chemical resistance traits may be costly for plants Native
Hawaiian plants which have little recent coevolutionary history with ants appear to have
secondarily lost floral ant repellents (Junker et al 2011a) Both Willmer et al (2009) and Junker
et al (2011a) found that highly chemical repellent flowers did not have strong physical defenses
and vice versa suggesting trade-offs between the two (but see Ballantyne and Willmer 2012)
Like toxic nectar floral ant repellents might sometimes deter both pollinators and ants For
example Galen et al (2011) found that 2-phenylethanol in P viscosum floral scent repels both
flower-damaging F neorufibarbis ants and the plantrsquos main pollinator Bombus kirbyellus
Interestingly four recent studies have found that floral scents can also attract ants
(Agarwal and Rastogi 2007 Edwards and Yu 2008 Schiestl and Glaser 2011 Gonzaacutelvez et al
20
2012) One study (Schiestl and Glaser 2011) has shown that floral scents attract ants to a
potentially ant-pollinated plant and such systems may provide many more examples
4 Future directions
We have highlighted areas in need of further investigation throughout the review Here we focus
on several questions we consider high priorities for future research
i) Floral defenses against ants
a) Do floral resistance traits increase plant fitness
The existence ant-repellent floral traits is often taken as evidence that ants negatively affect plant
reproductive success However despite the abundance and variety of mechanisms excluding ants
from flowers few studies show that ants reduce plant fitness when they visit flowers (Table 11)
This absence is particularly apparent with repellent floral volatiles which may be the most
widespread floral resistance trait (Willmer et al 2009 Junker et al 2011a Ballantyne and
Willmer 2012) To our knowledge only one study has shown that floral ant repellents benefit
plants with repellent P viscosum flowers avoiding damaging ant visits (Galen et al 2011)
Research is urgently needed to investigate if these traits increase plant fitness by excluding ants
or if ant visitation is inconsequential More studies taking advantage of standing variation in
traits (Galen et al 2011) or creating it artificially (eg Junker et al 2010b) will allow us to
distinguish between the two
21
b) If ant visitation is often not costly what caused the evolution of repellent traits and
what maintains them Similarly are these repellent traits truly defenses against ant
visitation or are they generalized defenses against a range of floral antagonists
Ant-repellent floral traits are widespread and their production may be costly (Willmer et al
2009 Junker et al 2011a) The studies in Table 11 however indicate that flower-visiting ants
often do not disrupt plant reproduction particularly outside of myrmecophytes What then
selected for these traits One possibility is that these repellents are not directed solely at ants
Instead a community of antagonistic flower visitors may exert diffuse selection on plants
thereby selecting for and maintaining these traits Structural modifications may repel crawling
insects while chemical repellents such as floral volatiles may repel both flying and non-flying
insects (Junker et al 2011b) How much overlap there is in floral volatiles that repel both ants and
other antagonists needs investigation Until we examine how specific these defenses are to ants
we risk overstating the importance of ants in their evolution
ii) Do ants alter pollen movement patterns or reduce male fitness
We are aware of only four studies that have measured if ant visitation impacts male fitness
(Wyatt 1980 Fritz and Morse 1981 Ashman 2000 Galen and Geib 2007) While none of these
studies found an effect all measured only amounts of pollen or pollinia removed However this
measure does not adequately encompass male fitness alterations to pollen movement patterns
and eventual deposition location may be more important to male function than changes in
removal rates If as suggested by some studies (Altshuler 1999 Lach 2007) ants increase
pollinator relocation rates they may decrease pollen discounting increasing male fitness
However if this increased relocation reduces overall pollen export rates ant competition with
22
pollinators may depress male function Studies using pollen-tracking methods such as fluorescent
dyes will allow us to see how ants change pollen movement patterns and methods like paternity
analysis will reveal how competition between ants and pollinators alters male fitness
iii) Coevolution between ants and plants do myrmecophytes exhibit stronger more
specific defenses against ants
The floral traits of myrmecophytes are most likely to coevolve with ants because all plant
reproduction occurs in the presence of ants Their floral defenses would thus be expected to be
both stronger and more specific to their resident ants than those other plants visited by an array
of more opportunistic ant species Some evidence supports this Willmer et al (2009) found that
resident ants of acacias were repelled by flowers with more aggressive ant species showing
stronger repellence than less aggressive ants Additionally ants not normally associated with
acacias did not respond to acacia floral volatiles More studies are needed in other systems and
should take advantage of phylogenetic approaches examining floral traits in closely related
myrmecophytic and non-myrmecophytic plant species to determine whether ants commonly
select for floral defenses against ants in myrmecophytes
Acknowledgements
For comments and discussion we thank the Frederickson lab particularly Eric Youngerman
Lina Arcila Hernandez and Kyle Turner as well as Alison Parker and Jordan Pleet MEF
acknowledges financial support from an NSERC Discovery Grant a Connaught New Researcher
23
Award an Ontario Ministry of Economic Development and Innovation Early Researcher Award
and the University of Toronto ARC was supported by an Ontario Graduate Scholarship and
Sigma Xi
24
References
Adler L S 2001 The ecological significance of toxic nectar Oikos 91409ndash420
Agarwal V M and N Rastogi 2007 Role of floral repellents in the regulation of flower visits
of extrafloral nectary-visiting ants in an Indian crop plant Ecological Entomology 3359ndash
65
Altshuler D L 1999 Novel interactions of non-pollinating ants with pollinators and fruit
consumers in a tropical forest Oecologia 119600ndash606
Ashman TL 2000 Pollinator selectivity and its implications for the evolution of dioecy and
sexual dimorphism Ecology 812577-2591
Ashman TL and E A King 2005 Are flower-visiting ants mutualists or antagonists A study
in a gynodioecious wild strawberry American Journal of Botany 92891ndash5
Ballantyne G and P Willmer 2012 Floral visitors and ant scent marks noticed but not used
Ecological Entomology 37402-409
Ballantyne G and P Willmer 2012 Nectar theft and floral ant-repellence a link between
nectar volume and ant-repellent traits PloS One 7e43869
Baker H G and I Baker 1978 Ants and flowers Biotropica 1080
Barrett S C H 2002 Sexual interference of the floral kind Heredity 88154ndash159
Baroni Urbani C and M L de Andrade 1997 Pollen eating storing and spitting by ants
Naturwissenschaften 84256ndash258
25
Beattie A J C Turnbull R B Knox and E G Williams 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany
71421ndash426
Beattie A J C Turnbull T Hough S Jobson and R B Knox 1985 The vulnerability of
pollen and fungal spores to ant secretions evidence and some evolutionary implication
American Journal of Botany 72606ndash614
Blancafort X and C Goacutemez 2005 Consequences of the Argentine ant Linepithema humile
(Mayr) invasion on pollination of Euphorbia characias (L) (Euphorbiaceae) Acta
Oecologica 2849ndash55
Bleil R N Bluumlthgen and R R Junker 2011 Ant-plant mutualism in Hawailsquoi Invasive ants
reduce flower parasitism but also exploit floral nectar of the endemic shrub Vaccinium
reticulatum (Ericaceae) Pacific Science 65291ndash300
Bluumlthgen N G Gottsberger and K Fiedler 2004 Sugar and amino acid composition of ant-
attended nectar and honeydew sources from an Australian rainforest Austral Ecology
29418ndash429
Burkle L A R E Irwin and D A Newman 2007 Predicting the effects of nectar robbing on
plant reproduction implications of pollen limitation and plant mating system American
Journal of Botany 941935ndash1943
Byk J and K Del-Claro 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333ndash38
26
Caballero P C G Ossa W L Gonzaacuteles C Gonzaacutelez-Browne G Astorga M M Muruacutea and
R Medel 2013 Testing non-additive effects of nectar-robbing ants and hummingbird
pollination on the reproductive success of a parasitic plant Plant Ecology 214633ndash640
Carvalheiro L G E R M Barbosa and J Memmott 2008 Pollinator networks alien species
and the conservation of rare plants Trinia glauca as a case study Journal of Applied
Ecology 451419ndash1427
Chamberlain S A and J N Holland 2008 Density-mediated context-dependent consumer-
resource interactions between ants and extrafloral nectar plants Ecology 891364ndash1374
Creighton W S 1967 Studies on free colonies of Cryptocerus texanus Satschi (Hymenoptera
Formicidae) Psyche 7434ndash42
Cursach J and J Rita 2011 Implications of the reproductive biology of the narrow endemic
Naufraga balearica (Apiaceae) for its conservation status Plant Systematics and Evolution
298581ndash596
Cushman JH and Addicott JF In Huxley CR and DF Cutler (eds) 1st edition Ant-plant
interactions Oxford University Press Oxford
de Jong T J N M Waser and P G Klinkhamer 1993 Geitonogamy the neglected side of
selfing Trends in Ecology and Evolution 8321ndash325
de Vega C M Arista P L Oritz C M Herrera and S Talavera 2009 The ant-pollination
system of Cytinus hypocistis (Cytinaceae) a Mediterranean root holoparasite Annals of
Botany 4517ndash8
27
Dukas R 2001 Effects of perceived danger on flower choice by bees Ecology Letters 4327ndash
333
Dutton E M and M E Frederickson 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561ndash569
Edwards D P and D W Yu 2008 Tolerating castration by hiding flowers in plain sight
Behavioral Ecology and Sociobiology 6395ndash102
Frederickson M E 2009 Conflict over reproduction in an ant-plant symbiosis why Allomerus
octoarticulatus ants sterilize Cordia nodosa trees The American Naturalist 173675ndash81
Fritz R S and D H Morse 1981 Nectar parasitism of Asclepias syriaca by ants effect on
nectar levels pollinia insertion pollinaria removal and pod production Oecologia 50316ndash
319
Galen C 1983 The effects of nectar thieving ants on seedset in floral scent morphs of
Polemonium viscosum Oikos 41245ndash249
Galen C 2005 Catching ants with honey an experimental test of distraction and satiation as
alternative modes of escape from flower-damaging ants Oecologia 14480ndash87
Galen C and B Butchart 2003 Ants in your plants effects of nectar-thieves on pollen fertility
and seed-siring capacity in the alpine wildflower Polemonium viscosum Oikos 101521ndash
528
Galen C and J Cuba 2001 Down the tube pollinators predators and the evolution of flower
shape in the alpine skypilot Polemonium viscosum Evolution 551963-1971
28
Galen C and J C Geib 2007 Density-dependent effects of ants on selection for bumble bee
pollination in Polemonium viscosum Ecology 881202ndash1209
Galen C R Kaczorowski S L Todd J Geib and R A Raguso 2011 Dosage-dependent
impacts of a floral volatile compound on pollinators larcenists and the potential for floral
evolution in the alpine skypilot Polemonium viscosum The American Naturalist 177258ndash
272
Garciacutea M B R J Antor and X Espadaler 1995 Ant pollination of the palaeoendemic
dioecious Borderea pyrenaica (Dioscoreaceae) Plant Systematics and Evolution 19817ndash
27
Gaume L M Zacharias and R M Borges 2005 Ant ndash plant conflicts and a novel case of
castration parasitism in a myrmecophyte Evolutionary Ecology Research 7435ndash452
Graystock P and W O H Hughes 2011 Disease resistance in a weaver ant Polyrhachis
dives and the role of antibiotic-producing glands Behavioral Ecology and Sociobiology
652319ndash2327
Gόmez J M 2000 Effectiveness of ants as pollinators of Lobularia maritima effects on main
sequential fitness components of the host plant Oecologia 12290ndash97
Gόmez J M and R Zamora 1992 Pollination by ants consequences of the quantitative effects
on a mutualistic system Oecologia 91410ndash418
Gόmez J M R Zamora J A Hόdar and D Garciacutea 1996 Experimental study of pollination
by ants in Mediterranean high mountain and arid habitats Oecologia 105236ndash242
29
Gonccedilalves-Souza T P M Omena J C Souza and G Q Romero 2008 Trait-mediated effects
on flowers artificial spiders deceive pollinators and decrease plant fitness Ecology
892407ndash2413
Gonzaacutelvez F G L Santamariacutea R T Corlett and M A Rodriacuteguez-Gironeacutes 2012 Flowers
attract weaver ants that deter less effective pollinators Journal of Ecology 10178ndash85
Guerrant E O and P L Fiedler 1981 Flower defenses against nectar-pilferage by ants
Biotropica 1325ndash33
Harley R 1991 The greasy pole syndrome In Huxley CR and DF Cutler (eds) 1st edition
Ant-plant interactions Oxford University Press Oxford
Hansen D M and C B Muumlller 2009 Invasive ants disrupt gecko pollination and seed
dispersal of the endangered plant Roussea simplex in Mauritius Biotropica 41202ndash208
Hickman J C 1974 Pollination by ants a low-energy system Science 1841290ndash1292
Holland J N S a Chamberlain and T E X Miller 2011 Consequences of ants and
extrafloral nectar for a pollinating seed-consuming mutualism ant satiation floral
distraction or plant defense Oikos 120381ndash388
Houmllldobler B and Wilson E O 1990 The Ants 1st Edition Harvard University Press
Cambridge
Holway DA L Lach AV Suarez ND Tsutsui and TJ Case 2002 The causes and
consequences of ant invasions Annual Review of Ecology and Systematics 33181-233
30
Horskins K and Turner VB 1999 Resource use and foraging patterns of honeybees Apis
mellifera and native insects on flowers of Eucalyptus costata Australian Journal of
Ecology 24221-227
Horvitz C C and D W Schemske 1984 Effects of ants and an ant-tended herbivore on seed
production of a neotropical herb Ecology 651369ndash1378
Inouye DW 1980 The terminology of floral larceny Ecology 611251-1253
Inouye DW and O R J Taylor 1979 A temperate region plant-ant-seed predator system
consequences of extra floral nectar secretion by Helianthella quinquenervis Ecology 601ndash
7
Irwin RE 2003 Impact of nectar robbing on estimates of pollen flow conceptual predictions
and empirical outcomes Ecology 84485-495
Irwin RE and Maloof JE 2002 Variation in nectar robbing over time space and species
Oecologia 133525-533
Iwanami Y and T Iwadare 1978 Inhibiting effects of myrmicacin on pollen growth and pollen
tube mitosis Botanical Gazette 13942ndash45
Izzo TJ and Vasconcelos HL 2002 Cheating the cheater domatia loss minimizes the effects
of ant castration in an Amazonian ant plant Oecologia 133200-205
Janzen D H 1977 Why donrsquot ants visit flowers Biotropica 9252
Johnson SD Hargreaves AL and Brown M 2006 Dark bitter-tasting nectar functions as a
filter of flower visitors in a bird-pollinated plant Ecology 872709-2716
31
Junker R A Y C Chung and N Bluumlthgen 2007 Interaction between flowers ants and
pollinators additional evidence for floral repellence against ants Ecological Research
22665ndash670
Junker R R R Bleil C C Daehler and N Bluumlthgen 2010a Intra-floral resource partitioning
between endemic and invasive flower visitors consequences for pollinator effectiveness
Ecological Entomology 35760ndash767
Junker R R and N Bluumlthgen 2010b Floral scents repel facultative flower visitors but attract
obligate ones Annals of botany 105777ndash82
Junker R R C C Daehler S Doetteri A Keller and N Bluumlthgen 2011a Hawaiian ant ndash
flower networks nectar-thieving ants prefer undefended native over introduced plants with
floral defenses Ecological Monographs 81295ndash311
Junker R R J Gershenzon and S B Unsicker 2011b Floral odor bouquet loses its ant
repellent properties after inhibition of terpene biosynthesis Journal of Chemical Ecology
371323ndash31
Kawakita A and M Kato 2002 Floral biology and unique pollination system of root
holoparasites Balanophora kuroiwai and B tobiracola (Balanophraceae) American
Journal of Botany 891164ndash1170
Koopowitz H and TA Marchant 1998 Postpollination nectar reabsorption in the African
epiphyte Aerangis verdickii (Orchidaceae) American Journal of Botany 85508-512
Kirk W D J 1984 Ecological studies on Thrips imaginis Bagnall (Thysanoptera) in flowers of
Echium plantagineum L in Australia Australian Journal of Ecology 99ndash18
32
Kerner A 1878 Flowers and their unbidden guests C Kegan Paul London
Kessler D and I T Baldwin 2007 Making sense of nectar scents the effects of nectar
secondary metabolites on floral visitors of Nicotiana attenuata The Plant Journal 49840ndash
54
Koptur S and N Truong 1998 Facultative ant-plant interactions nectar sugar preferences of
introduced pest species in South Florida Biotropica 30179-189
Lach L 2005 Interference and exploitation competition of three nectar-thieving invasive ant
species Insectes Sociaux 52257ndash262
Lach L 2007 A mutualism with a native membracid facilitates pollinator displacement by
Argentine ants Ecology 881994ndash2004
Lach L 2008a Argentine ants displace floral arthropods in a biodiversity hotspot Diversity and
Distributions 14281ndash290
Lach L 2008b Floral visitation patterns of two invasive ant species and their effects on other
hymenopteran visitors Ecological Entomology 33155ndash160
Lach L 2013 A comparison of floral resource exploitation by native and invasive Argentine
ants Arthropod-Plant Interactions 7177-190
Luo C W K Li X M Chen Z Y Huang and K U N Li 2012 Ants contribute significantly
to the pollination of a biodiesel plant Jatropha curcas Environmental Entomology
411163ndash1168
33
Maleacute PJ G C Leroy A Dejean A Quilichini and J Orivel 2011 An ant symbiont directly
and indirectly limits its host plantrsquos reproductive success Evolutionary Ecology 2655ndash63
Maloof J E 2001 The effects of a bumble bee nectar robber on plant reproductive success and
pollinator behavior American Journal of Botany 881960ndash1965
Maloof J E and D W Inouye 2000 Are nectar robbers cheaters or mutualists Ecology
812651ndash2661
Moreau CS CD Bell R Vila SB Archibald and NE Pierce 2006 Phylogeny of the ants
diversification in the age of the angiosperms Science 312101-104
Nahas L M O Gonzaga and K Del-Claro 2012 Emergent impacts of ant and spider
interactions herbivory reduction in a tropical savanna tree Biotropica 44498ndash505
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506ndash514
Newman D A and J D Thomson 2005 Interactions among nectar robbing floral herbivory
and ant protection in Linaria vulgaris Oikos 110497ndash506
Nicklen E F and D Wagner 2006 Conflict resolution in an ant-plant interaction Acacia
constricta traits reduce ant costs to reproduction Oecologia 14881ndash7
Norment C J 1988 The effect of nectar-thieving ants on the reproductive success of Frasera
speciosa (Gentianaceae) American Midland Naturalist 120331ndash336
34
Orivel J L Lambs P-J G Maleacute C Leroy J Grangier T Otto A Quilichini et al 2011
Dynamics of the association between a long-lived understory myrmecophyte and its
specific associated ants Oecologia 165369ndash76
Palmer T M D F Doak M L Stanton J L Bronstein E T Kiers T P Young J R
Goheen et al 2010 Synergy of multiple partners including freeloaders increases host
fitness in a multispecies mutualism Proceedings of the National Academy of Sciences
10717234ndash9
Peakall R A J Beattie and S H James 1987 Pseudocopulation of an orchid by male ants a
test of two hypotheses accounting for the rarity of ant pollination Oecologia 73522ndash524
Peakall R and A J Beattie 1989 Pollination of the orchid Microtis parviflora R Br by
flightless worker ants Functional Ecology 3515ndash522
Peakall R C J Angus A J Beattie 1990 The significance of ant and plant traits for ant
pollination in Leporella fimbriata pollination Oecologia 84457ndash460
Puterbaugh M N 1998 The roles of ants as flower visitors experimental analysis in three
alpine plant species Oikos 8336ndash46
Prŷs-Jones OE and Willmer PG 1992 The biology of alkaline nectar in purple toothwort
(Lathraea clandenstina) ground level defenses Biological Journal of the Linnean Society
45373-388
Raine NE Willmer P and Stone GN 2002 Spatial structuring and floral avoidance
behaviour present in ant-pollinator conflict in a Mexican ant-acacia Ecology 833086-
3096
35
Ramsey M 1995 Ant pollination of the perennial herb Blandfordia grandiflora (Liliaceae)
Oikos 74265ndash272
Renault CK LM Buffa and Delfino MA 2005 An aphid-ant interaction effects on
different trophic levels Ecological Research 2071-74
Rico-Gray V and L B Thien 1989 Effect of different ant species on reproductive fitness of
Schomburgkia tibicinis (Orchidaceae) Oecologia 81487ndash489
Romero G Q P A P Antiqueira and J Koricheva 2011 A meta-analysis of predation risk
effects on pollinator behaviour PLoS One 6e20689
Rosumek FB FAO Silveira F de S Neves NP de U Barbosa L Diniz Y Oki F Pezzini
et al 2009 Ants on plants a meta-analysis of the role of ants as plant biotic defenses
Oecologia 160537ndash549
Roubik D W 1982 The ecological impact of nectar-robbing bees and pollinating
hummingbirds on a tropical shrub Ecology 63354ndash360
Rudgers J A 2004 Enemies of herbivores can shape plant traits selection in a facultative ant-
plant mutualism Ecology 85192ndash205
Schatz B and WT Wcislo 1999 Ambush predation by the ponerine ant Ectatomma ruidum
Roger (Formicidae) on a sweat bee Lasioglossum umbripenne (Halictidae) in Panama
Journal of Insect Behavior 12641-663
36
Schaffer W M D W Zeh S L Buchmann S Kleinhaus M V Schaffer and J Antrim
1983 Competition for nectar between introduced honey bees and native North American
bees and ants Ecology 64564ndash577
Schemske D W 1980 The evolutionary significance of extrafloral nectar production by Costus
woodsonii (Zingiberaceae) an experimental analysis of ant protection Journal of Ecology
68959ndash967
Schiestl F P and F Glaser 2011 Specific ant-pollination in an alpine orchid and the role of
floral scent in attracting pollinating ants Alpine Botany 1221ndash9
Schubart HOR AB Anderson 1978 Why donrsquot ants visit flowers A response to D H
Janzen Biotropica 10310-311
Schuumlrch S M Pfunder and B A Roy 2000 Effects of ants on the reproductive success of
Euphorbia cyparissias and associated pathogenic rust fungi Oikos 16ndash12
Stanton ML Palmer TM Young TP Evans A and Turner ML 1999 Sterilization and
canopy modification of swollen thorn acacia tree by a plant-ant Nature 401 578-581
Stephenson AG 1981 Toxic nectar deters nectar thieves of Catalpa speciosa American
Midland Naturalist 105381-383
Stout J C and D Goulson 2001 The use of conspecific and interspecific scent marks by
foraging bumblebees and honeybees Animal Behaviour 62183ndash189
Sugiura S T Abe and S Makino 2006 Loss of extrafloral nectary on an oceanic island plant
and its consequences for herbivory American Journal of Botany 93491ndash495
37
Svensson L 1985 An estimate of pollen carryover by ants in a natural population of
Scleranthus perennis L (Caryophyllaceae) Oecologia 66373ndash377
Tsuji K A Hasyim and K Nakamura 2004 Asian weaver ants Oecophylla smaragdina and
their repelling of pollinators Ecological Research 19669ndash673
Turner R C J J Midgley P Barnard R E Simmons and S D Johnson 2012 Experimental
evidence for bird pollination and corolla damage by ants in the short-tubed flowers of
Erica halicacaba (Ericaceae) South African Journal of Botany 7925ndash31
Visser D M G Wright and J H Giliomee 1996 The effect of the Argentine ant
Linepithema humile (Mayr) (Hymenoptera Formicidae) on flower-visiting insects of
Protea nitida Mill (Proteaceae) African Entomology 4285ndash287
Wagner D 2000 Pollen viability reduction as a potential cost of ant association for Acacia
constricta (Fabaceae) American Journal of Botany 87711ndash715
Wagner D and A Kay 2002 Do extrafloral nectaries distract ants from visiting flowers An
experimental test of an overlooked hypothesis Evolutionary Ecology Research 4293ndash305
Wang C Y Luo Y Tai D An and Y Kou 2008 Ants pollinate Neottia listeroides
(Orchidaceae) in Sichuan China 46836ndash846
Wyatt R 1980 The impact of nectar-robbing ants on the pollination system of Asclepias
curassavica Bulletin of the Torrey Botanical Club 10724ndash28
Wheeler WM and IW Bailey 1920 The feeding habits of Pseudomymine and other ants
Transactions of the American Philosophical Society 22235-279
38
Willmer P G and S A Corbet 1981 Temporal and microclimatic partitioning of the floral
resources of Justicia aurea amongst a concourse of pollen vectors and nectar robbers
Oecologia 5167ndash78
Willmer P G and G N Stone 1997 How aggressive ant-guards assist seed-set in Acacia
flowers Nature 388165ndash167
Willmer P G C V Nuttman N E Raine G N Stone J G Pattrick K Henson P Stillman
et al 2009 Floral volatiles controlling ant behaviour Functional Ecology 23888ndash900
Young T P C H Stubblefield and L a Isbell 1997 Ants on swollen-thorn acacias species
coexistence in a simple system Oecologia 10998ndash107
Yu D W and N E Pierce 1998 A castration parasite of an ant-plant mutualism Proceedings
of the Royal Society B Biological Sciences 265375ndash382
39
Tables
Table 11 Studies of ant-flower or ant-pollinator interactions that included a measure of plant fitness Each study system is listed only
once except when results varied between years or when different studies examined male and female fitness Significant effects of ants on
plant fitness are either lsquopositiversquo or lsquonegativersquo non-significant effects are lsquoneutralrsquo When ants significantly affected only one fitness
component but several were measured (eg fruit set and seed production) we still consider the result significant
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Acacia
drepanolobium
Crematogaster
nigriceps
Proportion
fruiting
Negative Destruction of floral buds Young et al 1997
Asclepias exaltata Several Pollinia inserted
removed
Negative for pollinia
insertion neutral for
pollinia removal
Exploitative competition Wyatt 1980 T
40
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Asclepias syriaca Several
Fruit set pollinia
removed
Negative for fruit
set neutral for
pollinia removal
Exploitative competition Fritz and Morse
1981 T
Cordia nodosa Allomerus cf
demerarae
Fruit set Negative Destruction of floral buds Yu and Pierce
1998
Costus woodsonii Wasmannia
auropunctata
Seed set Positive Unknown ant pollination
suggested (but unlikely)
Schemske 1980
Eritrichum aretioides F neorufibarbis
gelida
Seed set Negative Ants damaged flowers Puterbaugh 1998
41
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Euphorbia characias Several Seed set Neutral Plants in Argentine ant-
invaded area set less seed
than those in un-invaded
sites but an ant-exclusion
experiment did not produce
significant results
Blancafort and
Gόmez 2005
Ferocactus wislizeni Solenopsis xyloni
and others
Seed set Unknown negative Interference and exploitative
competition S xyloni
reduced seed set relative to
other ants but the study did
not include a no-ant control
Ness 2006
42
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Frasera speciosa F neorufibarbis
and Formica sp
Seed set Neutral Nocturnal pollination
occurring when ants were
less active may have
compensated for reduced
diurnal pollinator visitation
Norment 1988
Gypsophila struthium Several Seeds per
inflorescence
Neutral No effect despite large
numbers of ants on flowers
Gόmez et al
1996
Hirtella physophora Allomerus
decemarticulatus
Fruit set Negative Destruction of flowers Orivel et al
2011
Humboldtia brunonis Crematogaster
dohrni
Fruit set Negative Destruction of flowers Gaume et al
2005
43
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Lepidium subulatum Several Percentage of
ovules setting
seed
Positive Unknown Gόmez et al
1996
Leucospermum
conocarpodendron
Several Seed set Neutral Site not pollen limited Lach 2008
Linaria vulgaris Several ldquoFemale fitness
indexrdquo
(combination of
number of seeds
fruits flowers
and seed weight)
Positive Reduction in florivory and
seed predation nectar
robbing holes increased ant
visitation
Newman and
Thomson 2005
44
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Melastoma
malabathricum
Oecophylla
smaragdina
Seed set Positive Ants deterred less efficient
pollinators increasing
visitation by the more
efficient pollinator
Gonzaacutelvez et al
2012
Metrosideros
polymorpha
Several Fruit set Neutral Ants reduced nectar
collection rates by foraging
bees but had no effect on
pollen collection rates or
fruit set
Junker et al
2010a
Orexis alpine F neorufibarbis
gelida
Seed set Neutral Autogamy or not pollen
limited
Puterbaugh 1988
45
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Ouratea spectabilis Cephalotes
pusillus and others
Seeds per fruit Neutral positive C pusillus had no effect on
seed number but other ants
collectively increased seed
set
Byk and Del
Claro 2010
Pachycereus schottii Several Seed set Neutral positive In one year ants showed a
positive effect (seed
number 2008) but no effect
with increased sample sizes
Chamberlain and
Holland 2008
Polemonium
viscosum
F neorufibarbis
gelida
Seed set Negative Ants damaged styles Galen 1983
46
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Polemonium
viscosum
F neorufibarbis
gelida
Pollen removal Neutral Ants did not affect pollen
removal even at high ant
densities
Galen and Geib
2007 T
Protea nitida Linepithema
humile and others
Seed set Neutral positive L humile had no effect on
P nitida seed set but native
ants increased it in one year
ants may cause pollinators
to relocate
Lach 2007
Psychotria
limonensis
Ectatomma spp Fruit set Positive Suggested increased
pollinator relocation
Altshuler 1999
47
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Roussea simplex Technomyrmex
albipes
Seed set Negative Interference and exploitative
competition
Hansen and
Muumlller 2009
Tristerix aphyllus Several Fruit set Neutral Nectar robbing ants
minimally reduced nectar
levels and had no effect on
hummingbird visitation
Caballero et al
2013
Vaccinium
reticulatum
Several Fruit set Neutral Ants reduced florivory but
did not affect fruit set
Bleil et al 2011
T Study measured male fitness
Study of sterilizing ants showed short-term decrease in female fitness long-term effects are unclear (see text)
48
Table 12 Studies demonstrating either i) ant pollination through an exclusion experiment or ii) that ants transfer pollen (or pollen
analogue) between flowers strongly suggesting ant pollination
Plant species Ant species Ant pollination Evidence Reference
Alyssum purpureum Several Yes Exclusion experiment Gόmez et al 1996
Arenaria tetraquetra Several Yes Exclusion experiment Gόmez et al 1996
Balanophora kuroiwai Leptothorax sp Suggested Exclusion experiment and
observed pollen transfer
and pollen tube formation
but did not see if plants set
seed
Kawakita and Kato 2002
Blandfordia grandiflora Iridomyrmex sp Yes Exclusion experiment Ramsey 1995
Borderea pyrenaica Several Yes Exclusion experiment Garciacutea et al 1995
49
Plant species Ant species Ant pollination Evidence Reference
Cytinus hypocistis Several Yes Exclusion experiment de Vega et al 2009
Epipactis thunbergii Camponotus
japonicas
Suggested Observed transfer of
pollinia from ants to
orchids
Sugiura et al 2006
Euphorbia cyparissias Several Yes Exclusion experiment Schuumlrch et al 2000
Fragaria virginiana Several Very likely Ant-only treatments set
comparable seed to flying
pollinator-only treatment
seed set increased with
increasing ant visitation
Ashman and King 2005
Frasera speciosa Several Yes Exclusion experiment Norment 1988
Frankenia thymifolia Several Yes Exclusion experiment Gόmez et al 1996
50
Plant species Ant species Ant pollination Evidence Reference
Hormathophylla spinosa Proformica
longiseta
Yes Exclusion experiment Gόmez and Zamora 1992
Jatropha curcas
Several
Tapinoma
melanocephalum
most common
Yes Exclusion experiment Luo et al 2012
Leporella fimbriata Myrmecia urens Yes Observed pollinia transfer
and subsequently
confirmed as ant
pollination
Peakall et al 1987
51
Plant species Ant species Ant pollination Evidence Reference
Lobularia maritima Several
Camponotus
micans most
common
Yes Exclusion experiment Gόmez 2000
Microtis parviflora
Iridomyrmex
gracilis
Yes Observation used
laboratory colonies to
replicate observations
Peakall and Beattie 1989
Naufraga balearica
Several
especially
Plagiolepis
pygmaea and
Lasius grandis
Suggested Observation over three
years of study only ants
were observed visiting
ants carried pollen
Cursach and Rita 2011
52
Plant species Ant species Ant pollination Evidence Reference
Neottia listeroides Leptothoras sp
Paratrechina sp
Suggested Observed transfer of
pollinia from ants to
orchids
Wang et al 2008
Paronychia pulvinata Formica
neorufibarbis
gelida
Yes Exclusion experiment Puterbaugh 1998
Polygonum cascadense Formica argentea
Very likely Grew plants in greenhouse
open to flying visitors with
minimal seed set only
plants with access to ants
set significant seed
Hickman 1974
53
Plant species Ant species Ant pollination Evidence Reference
Retama sphaerocarpa Several Yes Exclusion experiment Gόmez et al 1996
Scleranthus perennis
Several
especially
Formica
rufibarbis
Suggested Ants transferred pollen
analogue to stigmas and
carried pollen on bodies
Svensson 1985
Sedum anglicum Proformica
longiseta
Yes Exclusion experiment Gόmez et al 1996
Trinia glauca Several Yes Exclusion experiment Carvalheiro et al 2008
54
Chapter 2 Ants and ant scent reduce bumblebee pollination of artificial flowers
Cembrowski AR Tan MG Thomson JD and Frederickson ME
In press The American Naturalist
All authors designed the experiment ARC and MGT performed the experiment ARC and MEF
performed statistical analyse
s and ARC wrote the manuscript with input from all authors
Abstract
Ants on flowers can disrupt pollination by consuming rewards or harassing pollinators but it is
difficult to disentangle the effects of these exploitative and interference forms of competition on
pollinator behavior Using highly rewarding and quickly replenishing artificial flowers that
simulate male or female function we allowed bumblebees (Bombus impatiens) to forage (1) on
flowers with or without ants (Myrmica rubra) and (2) on flowers with or without ant scent cues
Bumblebees transferred significantly more pollen analogue both to and from ant free flowers
demonstrating that interference competition with ants is sufficient to modify pollinator foraging
behavior Bees also removed significantly less pollen analogue from ant scented flowers than
from controls making this the first study to show that bees can use ant scent to avoid harassment
at flowers Ant effects on pollinator behavior possibly in addition to their effects on pollen
viability may contribute to the evolution of floral traits minimizing ant visitation
55
Introduction
Trait-mediated indirect interactions arise when a focal species causes phenotypic changes
including behavioral modifications in a second species and these effects cascade to still other
species (Werner and Peacor 2003) Although trait-mediated indirect interactions are often studied
in food webs (eg Werner and Peacor 2003 Preisser et al 2005) they are not limited to trophic
interactions For example the threat of predation can change the behavior of a mutualist thus
affecting its partners (Suttle 2003) In animal-pollinated plants predators can disrupt pollination
directly through density-mediated indirect interactions (Dukas 2005) or via trait-mediated
indirect interactions by changing pollinator behavior When predators are present pollinators
may switch to visiting less rewarding flowers or they may avoid flowers altogether potentially
reducing the amount of pollen donated or received by a flower and thus decreasing plant fitness
(Gonccedilalves-Souza 2008 Ings and Chittka 2009) Similarly competition for floral rewards with
other flower visitors may cause behavioral changes in pollinators (Maloof and Inouye 2000
Ohashi et al 2008) Previous research has shown that bees may spend less time visiting flowers
depleted of rewards (Thomson 1986) and may avoid flowers bearing scent marks that indicate
recent visits by other individuals of the same or different bee species as these flowers are likely
to be depleted (Stout and Goulson 2002)
Whereas competition between pollinators may not be particularly costly for the plant
because both competitors are likely to provide pollination services competition between
pollinators and organisms that do not usually provide a pollination service such as ants has a
greater potential to reduce plant fitness Ants are common floral visitors they are attracted to
flowers for nectar (Lach 2007) and in some cases pollen (Byk and Del Claro 2010) and will
often defend these resources against other flower visitors (Altshuler 1999) However ants rarely
56
contribute much useful pollination antibiotic secretions present on their cuticles kill pollen
grains (Beattie et al 1985 Dutton and Frederickson 2012) and when foraging in flowers ants
sometimes impede female function by damaging stigmas (Galen and Cuba 2001) We will
henceforth consider ants to be non-pollinators and will contrast them to animals like bees that
are pollinators Ants are capable of competing with pollinators in two ways (1) by consuming
floral rewards and reducing their availability to pollinators (ie exploitative competition) and (2)
by directly antagonizing and excluding pollinators from flowers (ie interference competition)
Many plants benefit from the presence of ants numerous plant species recruit ants using
extrafloral nectar food bodies or domatia because ants provide protection against folivores (Heil
and McKey 2003) Similarly some plants have extrafloral nectaries on or near reproductive
tissues that recruit ants that deter florivores (eg Inouye and Taylor 1979) However plants
may incur reproductive costs as a result of their association with ants Some ant species that nest
in myrmecophytes sterilize flowers to shunt plant resources from reproduction to vegetative
growth allowing for greater ant colony growth (Frederickson 2009) Also ants visiting
extrafloral nectaries may be attracted to floral nectaries where they can harass pollinators (Ness
2006) There is growing evidence that many angiosperms have evolved floral traits that prevent
ants from accessing flowers during anthesis (Willmer and Stone 1997 Ballantyne and Willmer
2012) The effects of flower-visiting ants can be substantial Ants can decrease the frequency
duration or species diversity of pollinator visits to flowers (Lach 2008 Hansen and Muumlller 2009
Junker et al 2010 Gonzaacutelvez et al 2012) all of which potentially impact pollen donation and
receipt In some studies changes to pollinator behavior induced by ants have also been linked to
seed set
It is difficult however to disentangle the intertwined effects of exploitative and
interference competition in nature If ants disrupt pollination plants may often evolve traits to
57
deter ants from flowers (Willmer et al 2009) From a practical standpoint this would then limit
our ability to study the effects of ants on pollinator visitation because ants would not visit
flowers Here we used artificial flowers to explore ant-bee interactions in the absence of floral
defenses against ants We used highly rewarding quickly replenishing artificial flowers to
examine how direct harassment (ie interference competition expressed through behavior)
changes bee foraging behavior and how this affects donation and receipt of a pollen analogue
(powdered food dye) while minimizing the effects of exploitative competition We predicted
that flowers visited by ants would both donate and receive less pollen analogue than flowers
without ants because bees would avoid flowers with ants or leave them sooner Because scent
plays a large role in bumblebee communication and flower choice (Stout and Goulson 2002) we
also tested whether the presence or absence of ant scent on artificial flowers would affect pollen
analogue donation because of changes in the foraging behavior of bumblebees
Methods
i) Subjects
Myrmica rubra is an invasive ant in eastern North America with a range and habitat preferences
overlapping that of Bombus impatiens a common bumblebee Because M rubra visits flowers
(A Cembrowski pers obs) the two species likely interact in nature We collected 12 M rubra
colonies in the fall of 2011 and 2012 from Toronto Ontario Canada and the surrounding area
and maintained them in environmental chambers on artificial diet (Dussutour and Simpson 2009)
and a 1410 LD schedule (light 600-2200) We used these colonies as sources of M rubra
workers for experiments
58
Workers from commercially supplied colonies of Bombus impatiens (Biobest Canada
Ltd) foraged on artificial flowers in flight cages (either 24 x 24 x 21 m or 79 x 34 x 20m) at
the University of Toronto We tested a total of five bee colonies four colonies were used in the
ant presence trials and two of these and one additional colony were used in the ant scent trials
Flight cages had overhead fluorescent lights attached to timers In contrast to most previous
studies in which bees have been trained and tested individually the entire worker force of a bee
colony was free to forage at will in our experiments Colonies were trained to forage on artificial
flowers for at least four days before being used in trials After being used for a trial the colony
was not used for at least two days to reduce dye carryover between trials Between trials
bumblebees were fed pollen and given sugar water
ii) Artificial flowers
The flowers (Fig 1 Thomson et al 2012 see also Makino 2008) consisted of glass jars filled
with 30 WV sucrose solution Sugar water travelled by capillary action up a sewing-thread
wick to a hole in a blue-painted lid accumulating in a knot that acts as a nectary Flowers
depleted by visitors were quickly replenished via capillary action and were non-rewarding only
very briefly after visits taking less than a minute to accumulate 05 microl note that bumblebee
visits to a single flower are often separated by several minutes or more (A Cembrowski
unpublished data)
Unlike most previous artificial flower designs these flowers allow for estimation of male
and female fitness because we can measure both the amount and type of dye received by female
flowers (see (d) Dye quantification below) In order to access nectar on a ldquomalerdquo flower a bee
must crawl through a circular ldquoantherrdquo of brush-like weather stripping material dusted in a
59
consistent manner with our pollen analogue powdered food dye (FDampC 5 or 6) which is
transferred to the bee in the process On a ldquofemalerdquo flower a bee crawls through a sticky plastic
reinforcement that functions as a ldquostigmardquo receiving dye from the beersquos body Male flowers
dispensed dye particles over multiple bee visits and could still dispense dye at the end of trials
They resemble many real flowers or flower heads in that pollen is dispensed gradually over time
but less dye is available for transfer with each subsequent bee visit (Harder and Thomson 1989)
iii) Flight cage trials
We conducted 6 ant scent trials between 22 March and 4 June 2012 and the remaining trials (16
ant presence trials and 9 ant scent trials) between 9 November 2012 and 6 January 2013 Ant
presence trials lasted for eight hours and ant scent trials lasted for four hours Artificial flowers
were prepared and placed individually in small plastic containers treated with Fluon (Insect-a-
Slip BioQuip Products Inc) to prevent ants from escaping In ant presence trials we used 32
flowers (16 male and 16 female flowers) arranged in an eight-by-four array with flowers spaced
45 cm apart between rows and 30 cm within rows Ant scent trials used 20 flowers (10 male and
10 female flowers) in a four-by-five array with flowers spaced 30 cm apart between and within
rows In all of the ant presence trials and 9 of the ant scent trials we counted the number of bees
actively foraging after one hour to get a measure of colony activity
a) Ant presence trials
We examined the effects of ants on the amount of dye donated by male flowers and received by
female flowers in sixteen trials testing four bee colonies four times each The containers of eight
randomly chosen male flowers and eight randomly chosen female flowers received 15 M rubra
60
workers each the other 16 flowers remained free of ants Isolated from their colonies M rubra
workers have nowhere to deposit sugar water they collect and therefore may become sated
nonetheless a force of 15 ant workers was enough to maintain visitation to the nectaries where
bees foraged while still rarely having more than one or two ants visit the nectary at any time As
ants attacked bees bees would occasionally carry or throw attacking ants off flowers but despite
this some ants maintained their presence at the nectary throughout the trials Because ants the
size of M rubra consume liquids slowly (~017 ndash 024 microlmin Davidson et al 2004) flowers
replenished nectar more quickly than ants consumed it We used two colors of dye (FDampC 5 and
FDampC 6) to differentiate male flowers with and without ants In two trials for each bee colony
anthers of male flowers with ants were coated with FDampC 5 dye while brushes of male flowers
lacking ants were coated with FDampC 6 dye In the other two trials we reversed dye colors to
control for effects of dye type and color Dye color was assigned in a random order Artificial
flowers were placed in randomly assigned positions in the array before we opened the colony
and allowed the bees to begin foraging
b) Scent trials
We explored the effect of ant scent on dye donation in fifteen trials testing three bee colonies in
six five and four trials each Bees were first exposed to ants by allowing the bees to forage for
eight hours on twenty flowers (ten male and ten female) of which five male flowers had 15 ants
and all others had none No dye was used during this ldquoexposurerdquo day We then collected all but
the five ant-free male flowers in the flight cage leaving these flowers to keep bees foraging
Next we individually stored five new male lids in Fluon-treated containers with ten M rubra
workers Five control male lids were put in identical containers lacking ants The following
61
morning we removed the remaining flowers and set out ten new male and female flowers in a
random spatial arrangement using the lids having or lacking ant scent Thus the ant-scented
flowers were not in the same positions as the ant-visited flowers on which the bees were trained
Only five male flowers had ant scent and the other 15 flowers (five male and ten female) did not
We used the same two dye colors as in i) to differentiate male flowers with and without ant
scent and randomized which color was used for ant-scented flowers between trials We collected
and replaced stigmas from female flowers after one hour and collected the stigmas again after
four hours In the first trial performed we also collected stigmas after two hours but due to the
small amount of dye transferred this was not repeated
iv) Dye quantification
After each trial we quantified the amount of dye transferred to female flowers using a
spectrophotometer We removed the stigmas from female flowers placed them in test tubes
added 51 mL of distilled water to each tube and vortexed each tube thoroughly to ensure the
dye was evenly diluted These were diluted further as needed if the absorbance exceeded the
sensitivity range of the spectrophotometer In the first six scent trials each stigma was analyzed
separately and the dye amounts were summed to obtain a total amount of each dye color
transferred to female flowers in each trial In all the ant presence trials and the other nine scent
trials we opted to treat the experiment as the unit of replication Therefore we combined stigmas
from each treatment type (ant-visited or ant-free) in a test tube and measured the total amount of
each dye color transferred in each treatment in each trial
Because we put different dye colors on male flowers with and without ants we could use
the amount of each dye color donated to all female flowers to measure the reproductive success
62
of ant-visited and ant-free male flowers The total amount of dye (of both colors) received by
female flowers with and without ants was our measure of female reproductive success We
calculated the amount of each dye color in the sample by measuring absorbance at 428 or 486
nm and converting absorbance to micrograms following computational methods for overlapping
spectra (Blanco et al 1989)
v) Statistical analyses
In two trials (one ant presence and one ant scent) most female flowers received no dye because
of low bee activity so we excluded these trials from analyses Because dye reception and
donation values were non-normally distributed we square-root transformed the data before
examining the effects of ant presence or ant scent on dye transfer in ANCOVAs For ant
presence trials we included ant presence on male and female flowers and their interaction as
main effects and the total amount of dye transferred in each trial as a covariate to account for
the large variation in overall dye transfer among trials For ant scent trials we included ant scent
as the main effect time (one or four hours) as a repeated measure the interaction between scent
and time and the total amount of dye transferred as a covariate Covariate by treatment (ant
presence or ant scent) interactions were never significant and so were excluded in final analyses
All analyses were conducted in JMP (v 1000)
Results
Bees usually started foraging within minutes of the beginning of the trial and continued until
flowers were collected from the flight cage or the lights were extinguished An average of 34 plusmn
012 and 40 plusmn 015 (mean plusmn SE) bees were foraging after an hour in the ant presence and ant
63
scent trials respectively Male flowers with ants donated significantly less dye than male flowers
lacking ants (Fig 22a F155 = 4019 p lt 00001) Similarly female flowers with ants received
significantly less dye than female flowers lacking ants (Fig 22a F155 = 461 p = 0036) There
was no significant interaction between ant presence on male flowers and ant presence on female
flowers (F155 = 118 p = 028) Flowers with ant scent donated significantly less dye than
flowers without ant scent (Fig 22b F125 = 11216 p lt 00001) There was no significant effect
of time (F125 = 121 p = 028) or interaction between time and scent (F125 = 111 p = 030) in
the model
Discussion
In this study ants altered bumblebee pollination behavior and exhibited trait-mediated indirect
interactions with (artificial) flowers Through interference competition M rubra workers
significantly affected the pattern of dye transfer by B impatiens causing a preferential flow of
dye from male flowers lacking ants to female flowers lacking ants If manifested in nature such
effects would reduce the reproductive success of plants visited by ants through both male and
female function
Ants could have changed the attractiveness of artificial flowers in at least two ways First
as ants interrupted or altogether prevented bumblebees from foraging at nectaries bumblebees
may have learned to avoid flowers with ants due to the relative inefficiency of foraging at these
flowers something bumblebees take into account (Heinrich 2004) Second bees attempt to
minimize their risk of being attacked during foraging bouts which ants did to visiting bees Ants
often attacked or harassed bees by biting grasping and appearing to sting visiting bees
preventing them from accessing the nectary or reducing their time on flowers (see online video
64
1) and bees sometimes avoided ant-tended flowers entirely This harassment was sometimes
physically traumatic (online video 1) and bees often appeared to have trouble flying after being
attacked by ants Previous research has demonstrated that bumblebees leave or avoid flowers
where they have been harassed (Jones and Dornhaus 2011) and avoid foraging where there is
visual or olfactory evidence of a predator or predation event (Abbott 2006 Goodale and Nieh
2012) Though it is unclear if bumblebees viewed ants as competitors or predators they
responded similarly to flowers having ants as they do to flowers housing predators (Gonccedilalves-
Souza et al 2008)
To avoid artificial flowers with ants bees likely used a combination of visual and
olfactory cues Previous research has shown that bees respond to conspicuous predators or
predator ldquodummiesrdquo on flowers (Suttle 2003 Gonccedilalves-Souza et al 2008) decreasing the
frequency and duration of their visits to these flowers Thus bees may have been able to avoid
flowers harboring ants by sight alone However even in the absence of ants bees still
preferentially visited flowers lacking ant scents (Fig 22b) suggesting that they had learned to
associate ant scent with harassment Bees are adept at associative learning (Wright and Schiestl
2009) and can learn to recognize unique scents left behind by both conspecific and heterospecific
flower visitors (Stout et al 1998) Bees can use these various scents often arising from tarsal
gland deposits (Stout et al 1998) to recognize recently visited flowers that are less likely to be
profitable (Stout and Goulson 2002) Recently Ballantyne and Willmer (2012) demonstrated that
bees learn to associate ant scents with unrewarding artificial flowers and decrease their visitation
to these flowers Our results complement their findings by showing that bees can associate ant
scent with harassment at otherwise rewarding flowers
In our study ant scent caused bees to adjust their foraging strategy and decreased the
amount of pollen analogue that was donated by ant-scented flowers Thus the effects of
65
interference competition with ants on flowers can extend beyond immediate interactions and may
have fitness consequences for plants even when ants are absent Like other olfactory cues these
effects are likely transitory (Stout et al 1998) Although more dye was still donated by male
flowers lacking ant scent than those with ant scent the ratio of dye donated was on average
closer to equality in hours 2-4 than in the first hour (Fig 22b) This effect may be partially
driven by dye depletion of male flowers without ant scent but the lack of a strong corresponding
decrease in flowers with ant scent suggests that visitation patterns became more similar We did
not test whether B impatiensrsquo avoidance of ant scent was an innate or a learned behavior but
previous work has shown that Bombus terrestris does not innately avoid flowers with ant scent
(Ballantyne and Willmer 2012)
Ants may be necessary for plant survival and growth but can be costly for plant
reproduction The evolution of ant attractants such as extrafloral nectaries in some plant lineages
suggests that the costs of ants can be outweighed by their protective abilities However in this
study flowers visited by ants received and donated significantly less dye although they did
retain some sexual function The net benefit of having ants depends on whether ants increase
plant fitness by reducing herbivory more than they decrease plant fitness by disrupting
pollination Alternatively plants may actually benefit from the costs of ants to plant
reproduction ants that castrate flowers may be better defenders increasing plant survival or
vegetative growth when plants are young allowing for increased reproduction later in life when
the plant is colonized by less aggressive non-castrating ants (Frederickson 2009 Palmer et al
2010) In some plant species pollinator harassment by ants may even be beneficial Altshuler
(1999) reported that Ectatomma ants greatly increased fruit set of Psychotria limonensis despite
reductions in pollinator visitation rates presumably due to increases in the rate of pollen
outcrossing Similarly Gonzaacutelvez et al (2012) found that the presence of Oecophylla
66
smaragdina ants on Melastoma malabathricum flowers reduced visitation by less effective
pollinators and increased visitation by more effective Xylocopa bees increasing plant fitness
Cases of ants being beneficial to flowers appear to be the minority and many plant
species have traits that limit ant access to flowers Several studies have detailed floral volatiles
that are thought to mimic ant alarm pheromones and thus repel ants (Junker and Bluumlthgen 2008
Willmer et al 2009) Other plants use structural modifications such as narrow corollas or
slippery stems to limit ant access to flowers (reviewed in Willmer et al 2009) Exploitative
competition between ants and pollinators (Lach 2005 Ballantyne and Willmer 2012) as well as
the lethal effects of ant antibiotic secretions on pollen grains (Beattie et al 1985 Dutton and
Frederickson 2012) may have resulted in selection on plants to reduce ant visits to flowers Our
results suggest that trait-mediated indirect interactions resulting from interference competition
between ants and bees may favor plants that defend their flowers and their pollinators against
ants
Acknowledgements
We thank H Rusnock and S Meadley-Dunphy for assistance J Dix for help with flight cage
construction and Biobest for bumblebee colonies We are grateful to the Thomson and
Frederickson labs for guidance particularly T Makino funded by JSPS for helping to refine
artificial flowers J Ogilvie for help with bees K Prior for M rubra colony collections and K
Turner for study design suggestions The comments of the editor and two anonymous reviewers
prompted improvements in the article especially with regards to study design MEF and JDT
acknowledge NSERC Discovery Grant funding The University of Torontorsquos Faculty of Arts and
Science supported MGRTrsquos participation
67
68
References
Abbott K R 2006 Bumblebees avoid flowers containing evidence of past predation events
Canadian Journal of Zoology 841240-1247
Altshuler D L 1999 Novel interactions of non-pollinating ants with pollinators and fruit
consumers in a tropical forest Oecologia 119600-606
Ballantyne G and Willmer P 2012 Floral visitors and ant scent marks noticed but not used
Ecological Entomology 37402-409
Beattie A J Turnbull C Knox R B and Williams E G 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany
71421-426
Blanco M Runiaga H Maspoch S and Tarin P 1989 A simple method for
spectrophotometric determination of two-components with overlapped spectra Journal of
Chemical Education 66178-180
Byk J amp Del Claro K 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333-38
Davidson D W S C Cook and R R Snelling 2004 Liquid-feeding performances of ants
(Formicidae) ecological and evolutionary implications Oecologia 139255ndash66
Dukas R 2005 Bumble bee predators reduce pollinator density and plant fitness Ecology
861401-1406
Dussutour A and Simpson S J 2009 Communal nutrition in ants Current Biology 19740-
744
69
Dutton E M and Frederickson M E 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561-569
Frederickson ME 2009 Conflict over reproduction in an ant-plant symbiosis why Allomerus
octoarticulatus ants sterilize Cordia nodosa trees American Naturalist 173675-681
Galen C and Cuba J 2001 Down the tube pollinators predators and the evolution of flower
shape in the alpine skypilot Polemonium viscosum Evolution 551963-1971
Gonccedilalves-Souza T Omena P M Souza J C and Romero G Q 2008 Trait-mediated
effects on flowers artificial spiders deceive pollinators and decrease plant fitness Ecology
892407-2413
Gonzaacutelvez F G Santamariacutea L Corlett R T and Rodriacuteguez-Gironeacutes M A 2012 Flowers
attract weaver ants that deter less effective pollinators Journal of Ecology 10178ndash85
Goodale E and Nieh JC 2012 Public use of olfactory information associated with predation
in two species of social bees Animal Behaviour 84919-924
Harder LD and Thomson JD 1989 Evolutionary options for maximizing pollen dispersal of
animal-pollinated plants American Naturalist 133323-344
Heil M and McKey D 2003 Protective ant-plant interactions as model systems in ecological
and evolutionary research Annual Review of Ecology Evolution and Systematics 34425-
453
Heinrich B 2004 Bumblebee economics 2nd
edn Harvard University Press Cambridge
Ings T C and Chittka L 2009 Predator crypsis enhances behaviourally mediated indirect
effects on plants by altering bumblebee foraging preferences Proceedings of the Royal
Society B 2762031-2036
70
Inouye D W and Taylor O R Jr 1979 A temperate region plant-ant-seed predator system
consequences of extra floral nectar secretion by Helianthella quinquenervis Ecology 601-
7
Junker R R and Bluumlthgen N 2008 Floral scents repel potentially nectar-thieving ants
Evolutionary Ecology Research 10295ndash308
Junker R R Bleil R Daehler C C and Bluumlthgen N 2010 Intra-floral resource partitioning
between endemic and invasive flower visitors consequences for pollinator effectiveness
Ecological Entomology 35760ndash767
Jones E I and Dornhaus A 2011 Predation risk makes bees reject rewarding flowers and
reduce foraging activity Behavioral Ecology and Sociobiology 651505-1511
Lach L 2005 Interference and exploitative competition of three nectar-thieving invasive ant
species Insect Sociaux 52257-262
Lach L 2007 A mutualism with a native membracid facilitates pollinator displacement by
Argentine ants Ecology 881994-2004
Lach L 2008 Argentine ants displace floral arthropods in a biodiversity hotspot Diversity and
Distributions 14281-290
Makino T T and Sakai S 2007Experience changes pollinator responses to floral display size
from size-based to reward-based foraging Functional Ecology 21854863
Maloof J E and Inouye D W 2000 Are nectar robbers cheaters or mutualists Ecology
812651ndash2661
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506-514
Ohashi K Leslie A and Thomson JD 2008 Trapline foraging by bumble bees V Effects of
experience and priority on competitive performance Behavioral Ecology 19936-948
71
Palmer T M Doak D F Stanton M L Bronstein J L Kiers E T Young T P Goheen
J R and Pringle R M 2010 Synergy of multiple partners including freeloaders
increases host fitness in a multispecies mutualism Proceedings of the National Academy
of Sciences 10717234-17239
Preisser E L Bolnick D I and Benard M E 2005 Scared to death The effects of
intimidation and consumption in predator-prey interactions Ecology 86501-509
Stout J C Goulson D and Allen J A 1998 Repellent scent-marking of flowers by a guild of
foraging bumblebees (Bombus spp) Behavioral Ecology and Sociobiology 43317-326
Stout J and Goulson D 2002 The influence of nectar secretion rates on the responses of
bumblebees (Bombus spp) to previously visited flowers Behavioral Ecology and
Sociobiology 52239-246
Suttle K B 2003 Pollinators as mediators of top-down effects on plants Ecology Letters
6688-694
Thomson JD 1986 Pollen transport and deposition by bumble bees in Erythronium influences
of floral nectar and bee grooming Journal of Ecology 74329-341
Thomson JD Ogilvie J E Makino T T Arisz A Raju S Rojas-Luengas V and Tan M
G R 2012 Estimating pollination success with novel artificial flowers effects of nectar
concentration Journal of Pollination Ecology 9108-114
Tsuji K Hasyim A and Nakamura K 2004 Asian weaver ants Oecophylla smaragdina and
their repelling of pollinators Ecological Research 19669-673
Ulrich S and Bertsch A 1990 Do foraging bumblebees scent-mark food sources and does it
matter Oecologia 82137-144
Werner E E and Peacor S D 2003 A review of trait-mediated indirect interactions in
ecological communities Ecology 841083-1100
72
Willmer P G Nuttman C V Raine N E Stone G N Pattrick J G Henson K Stillman
P McIlroy L Potts S G and Knudsen J T 2009 Floral volatiles controlling ant
behaviour Functional Ecology 23888-900
Wright G A and Schiestl F P 2009 The evolution of floral scent the influence of olfactory
learning by insect pollinators on the honest signaling of floral rewards Functional Ecology
23841-851
73
Figures
Figure 21 Photograph of artificial flower lids A = anther L = lid N = nectary R = region
where anther or stigma is placed S = stigma T = thread W = weight
74
Figure 22 Dye transferred (mean plusmn 1SE) per trial by B impatiens workers A) from artificial
male and to artificial female flowers in the presence (unfilled circles) or absence (filled circles)
of Myrmica rubra workers and B) from artificial male flowers with (unfilled circles) or without
(filled circles) M rubra scent to ant-free female flowers in the 1st or the 2
nd to 4
th hours of the
trials
75
Chapter 3 Not just for the bees pollen consumption is common among tropical
ants
Cembrowski AR Reurink G and Frederickson ME
Planned submission to Biotropica
ARC and MEF designed the study ARC and GR did laboratory work ARC performed statistical
analyses and wrote the manuscript with input from other authors
Abstract
Although palynivory or pollen consumption is widespread among other hymenopterans there
are few accounts of palynivory in ants To quantify how often ants consume pollen we subjected
adult workers and larvae from 75 species of neo- and paleo-tropical ants to acetolysis a process
that destroys most organic material but leaves behind pollen grains In over half of the species
we examined ants contained a few pollen grains (lt 5) and ants of several Camponotus species
contained abundant pollen grains (gt50) We tested for an association between trophic level and
palynivory using stable nitrogen isotope ratios (δ15
N) but we did not find a significant
correlation we found pollen grains in lsquoherbivorousrsquo omnivorous and carnivorous ants We
suggest that our results indicate sporadic opportunistic pollen consumption by ants in tropical
forests
Introduction
For those organisms that can digest it efficiently pollen can be an excellent food source
Depending on the plant species pollen can contain up to 60 protein as well as lipids
76
carbohydrates and numerous trace nutrients (Roulston and Cane 2000) Pollen feeding
(palynivory) occurs in many taxa including birds mammals and arthropods (Roulston and Cane
2000) but it is best known among the Hymenoptera particularly bees Although adult bees feed
mainly on plant exudates (ie nectar) nearly all bee species rely on pollen as the chief protein
source for developing larvae (Willmer 2011) Closely related to bees ants have similar
nutritional requirements adult workers need carbohydrates whereas larvae are fed a diet rich in
protein (Dussutour and Simpson 2009) Ants also exploit nectar heavily and regularly visit
flowers (eg Herrera et al 1984 Bluumlthgen et al 2004) but unlike bees are not believed to
widely consume pollen Instead ants are thought to receive most of the protein in their diet from
predation or scavenging (eg Floren et al 2002)
However ants may not be as reliant on animal protein sources as commonly assumed In
tropical forests arboreal ants can have similar δ15
N ratios a measure of trophic level as
herbivores suggesting limited carnivory and substantial feeding on plant-derived protein sources
(Davidson et al 2003) One potential protein source is pollen This idea is supported by scattered
accounts of pollen consumption by ants from other ecosystems including deserts (Ness 2006)
boreal forests (Czechowski et al 2009) and tropical savannas (Byk and Del Claro 2010) One
ant genus Cephalotes is widely recognized to consume pollen (Creighton 1967 Baroni Urbani
and de Andrade 1997 Byk and Del Claro 2010) However beyond this genus our knowledge of
pollen consumption by ants has largely been limited to direct observations (but see Czechowski
et al 2011) and it is unknown if palynivory is truly rare among ants or simply under-reported
We borrowed a technique commonly used in palynology acetolysis to assess the frequency of
pollen consumption by ants in 75 neo- and paleo-tropical ant species This technique allowed us
to examine many more ant species than we could have observed directly
77
Methods
Adult worker ants were collected by hand at baits or from leaf litter using Winkler extractors
while larvae along with adults were collected by hand from nests Collections were made by L
Arcila Hernandez and J Sanders in the primary tropical rainforest surrounding the Centro de
Investigaciόn y Capacitaciόn Rio Los Amigos (CICRA 12deg34rsquoS 70deg05rsquoW) in Peru in 2010-
2011 and by E Youngerman from lowland primary and secondary rainforests in Madang
Province Papua New Guinea (PNG) in 2011-2012 We sorted ant samples to morphospecies and
then identified them to genus and when possible to species using existing keys which were
unavailable for PNG ants Ant samples used for acetolysis were whole adult workers guts from
adult workers and whole larvae
We used acetolysis to look for pollen grains inside ants In acetolysis organic material is
dissolved in acetic anhydride and sulfuric acid leaving behind stained pollen exines When
possible we used adult and larval ants of the same species from different colonies or bait
locations in an attempt to sample more representatively We prepared samples by first washing
them in 70 ethanol to remove any pollen present on the samplersquos outer surface before placing
them individually in micro-centrifuge tubes with ~05mL of a 91 mixture of acetic anhydride
sulfuric acid We then heated each sample at 95degC for 20min allowing tissues to dissolve Next
we centrifuged samples at 1000 RPM for 5min and decanted the liquid supernatant before
adding ~05mL of glacial acetic acid to the precipitate centrifuging again as above We decanted
the samples and added a drop of distilled water
78
We examined a 150μL sub-sample of this solution for pollen grains under a light
microscope Larvae gave less than 150μL of solution following acetolysis so we sampled the
entire amount We identified pollen grains by their staining and their surface architecture For
most species we examined at least three adult workers and three larvae but for some species
fewer than three adult workers or larvae were available (Tables 31 and 32)
We investigated the relationship between trophic level as measured by δ15
N ratios and
pollen consumption by ants Stable isotope (SI) data was available for some (n =23) of the
Peruvian species from a separate project in which collections overlapped Samples were stored in
EtOH and dried at 50degC overnight We used heads legs and thoraxes (but not gasters) of ants
for SI analysis Often single individuals did not provide enough material for analysis and we
combined multiple individuals to achieve necessary weights up to 10 for small ant species
Samples were analyzed at the Boston University Stable Isotope Laboratory using a Finnigan
Delta-S analyzer For other Peruvian species for which we had samples but not corresponding
stable isotope data (n =25) we took the median δ15
N ratio value of that speciesrsquo genus and
assigned it to the species in question Using pollen presenceabsence data for these 48 species
we used logistic regression to correlate pollen presence and δ15
N ratio using R (v 2151)
Results
We found pollen grains in 37 of the 197 adult workers 20 of the 102 larvae and 38 of the 75 ant
species we examined (Tables 31 and 32) The proportion of individuals containing pollen did
not differ significantly between larvae and adults (χ2
1 = 002 p = 088) The number of pollen
grains found was often low (median = 3) but highly variable (range 1-468) Also pollen
79
presence was inconsistent among conspecific ant samples of species that contained pollen 31
had pollen in only one of the larvae or adults examined In only three species (two Cephalotes
and one Myrmelachista) did all examined adults contain pollen
Highly lsquoherbivorousrsquo carnivorous and omnivorous ants contained pollen grains (Fig
31) There was no correlation between δ15
N ratio and pollen presence (χ2 = 150 df = 1 p =
022) In fact several carnivorous species contained pollen grains (eg Odontomachus sp 1
Pachycondola sp1)
Discussion
Pollen consumption by ants appears to be more common than suspected The majority of species
(51) had at least one individual that had consumed pollen and in 20 multiple individuals
contained pollen Numbers of grains found tended to be low however with most individuals
containing only a few grains Ants may use pollen as only a small portion of their diet exploiting
a host of other protein-containing foods more heavily including other low- δ15
N foods (eg
honeydew epiphylls Houmllldobler and Wilson 1990 Davidson et al 2003)
There are a few reasons why pollen may not be heavily exploited by ants Pollen requires
much post-consumption processing to extract its nutrients These nutrients are located in the
pollenrsquos cytoplasm which is protected from direct digestion by the exines and intines of the
grains Organisms must either break through these layers or cause the pollen grains to germinate
or pseudo-germinate to access and digest the cytoplasm (Roulston and Cane 2000) This can be
achieved by consuming a sugar-containing solution (such as nectar) along with pollen However
animals that are not adapted to feed on pollen may not be very efficient at this process only
80
extracting nutrients from some of the grains (Herrera and Martiacutenez del Rio 1998) Thus while
ants can likely get some nutrients from pollen other forms of more easily processed protein may
be more attractive
Pollen may also pose handling difficulties for ants Despite the large number of studies
that have recorded ants visiting flowers (Chapter 1) only a few papers detail ants harvesting
pollen directly from anthers Byk and Del Claro (2010) reported that Cephalotes pusillus ants
removed almost all of the available pollen from Ouratea spectabilis flowers and at least two
other studies describe ants consuming pollen along with nectar from flowers (Horskins and
Turner 1999 Ness 2006) Unlike bees which possess both morphological (eg specialized hairs
for holding pollen) and behavioral adaptations (eg stereotyped pollen-packing motions) ants
have no apparent mechanisms to increase the amount of pollen harvested and are likely limited
by the number of grains they can ingest or carry Rather than collect pollen directly from flowers
it appears more likely that ants collect it haphazardly in the environment leading to a smaller
number of pollen grains consumed at any one time It is known that at least Cephalotes ants
engage in such behavior Creighton (1967) observed Cephalotes texanus collecting pollen grains
trapped by Citrus x paradisi leaf hairs
There is a chance that the small numbers of pollen grains we observed came from
secondary pollen consumption (ie consuming a palynivorous organism) or consumption of
liquid containing pollen While we cannot rule this out we believe our results represent actual
targeted pollen consumption by ants Cephalotes ants known palynivores (Creighton 1967
Baroni Urbani and de Andrade 1997) contained similar numbers of pollen grains as many of the
pollen-containing ant species that we examined As well in at least once instance ants that we
found containing pollen grains were in the process of preying on non-pollen consuming
81
organisms (eg a Pachycondola species that was carrying a termite in its mandibles) or were
attracted by sugar baits
Contrary to our expectations there was no relationship between an ant speciesrsquo δ15
N ratio
and whether it had consumed pollen (Fig 32) We expected palynivory to be more common in
herbivorous ants because pollen presents a widespread source of protein a potentially limiting
resource The lack of pattern might reflect how available pollen grains are in the environment
While other forms of food may be preferable ants tend to be opportunistic foragers (Carroll and
Janzen 1973) and there is likely little cost associated with the consumption of scattered pollen
grains during foraging trips beyond the volume they occupy in the gut Some genera were found
to consistently consume pollen In support of previous observations (Baroni Urbani and de
Andrade 1997 Byk and Del Claro 2010) Cephalotes species were among the most common
pollen consumers Camponotus species also consistently contained pollen grains of the eight
species examined five contained pollen and pollen was found in species from both Peru and
PNG Camponotus species also contained the two largest numbers of pollen grains found (468
and 271) Taken together these results suggest that they may be another as yet unrecognized
commonly palynivorous genus
In general larvae did not contain large numbers of pollen grains (ranging from 1-38
grains) This is counter to what is seen in bees where larvae are the main recipients of pollen
(Willmer 2011) This difference may be because ant larvae are continually fed via trophallaxis
by adults throughout their development whereas bee larval cells are provisioned with pollen and
then sealed while larvae complete their development If both adults and larvae are able to extract
nutrients from pollen we would expect to see a preferential flow of nutrients from pollen to
larvae but not necessarily the grains themselves
82
Unlike palynivory by organisms that also provide pollination services pollen
consumption by ants is unlikely to benefit plants Ants rarely act as pollinators (Beattie et al
2004) They have pollen-killing antibiotic secretions on their cuticles (Dutton and Frederickson
2012) and they can compete with other flower visitors reducing flower visitation rates (Ness
2006) The negative impacts of flower-visiting ants are thought to have led to the evolution of
ant-repellent floral volatiles (reviewed in Willmer 2009) These volatiles can pre-empt
competition between ants and pollinators and may often be located within the pollen itself
(Willmer 2009) If floral volatiles truly are a defense against ant visitation our results suggest an
additional evolutionary pressure to not only prevent ant-pollinator interactions but to also
protect plant gametes from occasional consumption by a non-pollinating visitor
Acknowledgements
We would like to thank Jon Sanders Lina Arcila Hernandez and Eric Youngerman for providing
samples the Thomson lab for use of supplies the Pierce lab for funding support during JSrsquo and
EYrsquos collections and the staff of CICRA and the New Guinea Binatang Research Center for
logistical support The Peruvian Ministry of Agriculture provided permits (Nos 394-2009-AG-
DGFFS-DGEFFS and 299-2011-AG-DGFFS-DGEFFS) MEF acknowledges financial support
from an NSERC Discovery Grant a Connaught New Researcher Award an Ontario Ministry of
Economic Development and Innovation Early Researcher Award and the University of Toronto
ARC was supported by an Ontario Graduate Scholarship and Sigma Xi
83
References
Baroni Urbani C and M L de Andrade 1997 Pollen eating storing and spitting by ants
Naturwissenschaften 84256ndash258
Beattie A J C Turnbull R B Knox and E G Williams 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany 71421ndash
426
Bluumlthgen N G Gottsberger and K Fiedler 2004 Sugar and amino acid composition of ant-
attended nectar and honeydew sources from an Australian rainforest Austral Ecology
29418ndash429
Byk J and K Del-Claro 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333ndash38
Carroll C R and D H Janzen 1973 Ecology of foraging ants Annual Review of Ecology and
Systematics 4231ndash257
Creighton W S 1967 Studies on free colonies of Cryptocerus texanus Satschi (Hymenoptera
Formicidae) Psyche 7434ndash42
Czechowski W B Markό and A Radchenko 2009 Rubbish dumps reveal the diet of ant
colonies Myrmica schencki Em and Myrmica rubra (L) (Hymenoptera Formicidae) as
facultative pollen-eaters Polish Journal of Ecology 56737ndash741
Czechowski WB Markoacute K Erős and E Csata 2011 Pollenivory in ants (Hymenoptera
Formicidae) seems to be much more common than it was thought Annales Zoologici
61519ndash525
84
Dussutour A and S J Simpson 2009 Communal nutrition in ants Current Biology 19740ndash4
Dutton E M and M E Frederickson 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561ndash569
Floren A A Biun and K E Linsenmair 2002 Arboreal ants as key predators in tropical
lowland rainforest trees Oecologia 131137ndash144
Grant B R 1996 Pollen digestion by Darwinrsquos finches and its importance for early breeding
Ecology 77489-499
Herrera L G and C Martiacutenez del Rio 1998 Pollen digestion by new world bats effects of
processing time and feeding habits Ecology 792828ndash2838
Houmllldobler B and Wilson E O 1990 The Ants 1st Edition Harvard University Press
Cambridge
Horskins K and Turner V B 1999 Resource use and foraging patterns of honeybees Apis
mellifera and native insects on flowers of Eucalyptus costata Australian Journal of
Ecology 24221-227
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506ndash514
Roulston T H I and J H Cane 2000 Pollen nutritional content and digestibility for animals
Plant Systematics 222187ndash209
Van Tets I G 1997 Extraction of nutrients from Protea pollen by African rodents Belgian
Journal of Zoology 12759-65
Willmer P G 2011 Pollination and Floral Ecology 1st Edition Princeton University Press
New Jersey
85
Willmer P G C V Nuttman N E Raine G N Stone J G Pattrick K Henson P Stillman
et al 2009 Floral volatiles controlling ant behaviour Functional Ecology 23888ndash900
86
Tables
Table 31 Numbers of adult worker and larval ants examined for each species collected in Peru
Numbers of pollen grains found in individual samples are given in parentheses after the sample
size when there were no grains no numbers are given
Genus Species Number of adults analyzed
(numbers of grains found)
Number of larvae analyzed
(numbers of grains found)
Dolichoderinae
Azteca sp 1 3 0
sp 2 2 0
sp 3 0 2
sp 4 3 (1) 0
sp 5 3 (2) 0
Dolichoderus decollatus 3 (66) 0
sp 1 3 0
sp 2 3 1 (2)
sp 3 3 3
sp 4 0 2
Ecitoninae
Labidus sp 1 3 0
Formicinae
Camponotus sp 1 3 3
sp 2 3 (82) 1 (2)
sp 3 3 (3) 0
sp 4 0 2
87
sp 5 8 (11) 0
sp 6 4 (7 16 468) 0
sp 7 5 (12) 0
sp 8 2 (1) 0
Paratrechina sp 1 4 (11) 0
Myrmelachista sp 1 3 (5 11 18) 0
sp 2 0 3
Trachymyrmex sp 1 3 (21) 0
sp 2 3 0
Myrmicinae
Atta sp 1 3 0
Cephalotes atratus 3 (2 2 21) 0
placidus 3 (1 4 21) 0
sp 1 3 0
sp 2 1 0
sp 3 2 0
Crematogaster sp 1 3 1
sp 2 3 (35) 0
sp 3 9 (7) 0
sp 4 2 0
Megalomyrmex sp 1 3 0
sp 2 0 2
sp 3 4 (7) 0
sp 4 3 0
Monomorium sp 1 0 3 (2)
88
Pheidole sp 1 0 3
sp 2 3 0
sp 3 0 3
Procryptocerus sp 1 3 0
Solenopsis sp 1 0 3
sp 2 3 (3 5) 0
Wasmannia sp 1 3 0
Ponerinae
Pachycondola sp 1 3 (3) 0
Odontomachus sp 1 3 (34) 0
Pseudomyrmicinae
Pseudomyrmex sp 1 3 0
sp 2 3 0
sp 3 3 (9) 0
sp 4 0 3
sp 5 0 3
89
Table 32 Numbers of adult worker and larval ants examined for each species collected in Papua
New Guinea Numbers of pollen grains found in individual samples are given in parentheses
after the sample size when there were no grains no numbers are given
Genus Species Number adults analyzed
(grains found)
Number larvae analyzed
(grains found)
Aenictinae
Aenictus sp 1 3 3 (1 1)
Dolichoderinae
Philidris sp 1 3 1 (4)
Tapinoma sp 1 2 3 (38)
Technomyrmex sp 1 0 3 (12 12)
Ectatomminae
Rhytidoponera sp 1 1 3 (2)
Formicinae
Acropyga sp 1 3 3 (2)
sp 2 3 3
sp 3 3 3
Anoplolepsis sp 1 3 (1) 3 (1)
Calomyrmex sp 1 3 3 (1)
Camponotus sp 1 3 (1 271) 3
sp 2 1 3
sp 3 2 0
Nylanderia sp 1 3 3
Opisthopsis sp 1 3 3
Polyrachis sp 1 1 3 (4)
90
Myrmicinae
Crematogaster sp 1 3 3
sp 2 3 (32) 3
Monomorium sp 1 3 (1) 3 (1)
sp 2 3 3 (1 1 1)
Pheidole sp 1 3 3
Pristomyrmex sp 1 3 3 (11)
Pyramica sp 1 3 0
Ponerinae
Odontomachus sp 1 3 3 (4)
91
Figures
Figure 31 Photographs of pollen grains found in two ant species a) Dolichoderus sp 2 and b)
Camponotus sp 6 Scale bars = 50 microm
92
Figure 32 Relationship between trophic level as measured by δ15
N ratio and pollen presence
for 48 neotropical ant species 1 Atta sp 5 Azteca spp 8 Camponotus spp 5 Cephalotes spp 4
Crematogaster spp 5 Dolichoderus spp 1 Labidus sp 3 Megalomyrmex spp 1 Monomorium
sp 2 Myrmelachista spp 1 Odontomachus sp 1 Pachycondola sp 1 Paratrechina sp 3
Pheidole sp 4 Pseudomyrmex spp 2 Solenopsis spp and 1 Wasmannia sp
93
Concluding Remarks
Ants have traditionally been considered to be unwelcome flower visitors they consume floral
rewards ostensibly meant for pollinators while rarely providing pollination services themselves
In this thesis I investigate the validity of this assumption and use an experimental study and an
observational study to examine two ways that ants may impact plant reproduction
In the first chapter I review the literature on ants as flower visitors I show that the common
assumption of ants always being categorically negative for plant reproduction is incorrect while
ants may reduce floral attractiveness to pollinators the net effect of ants on plant fitness
measures is variable ranging from positive to negative to neutral I also discuss ants as
pollinators and ways that plants may minimize ant visitation to flowers before finishing with a
section highlighting questions that still need investigation
In the second chapter I explore how interference competition with ants can structure bumblebee
foraging behavior and how this may alter pollen movement patterns Using artificial flowers with
male or female function I allowed Bombus impatiens bumblebees to forage on flowers that
either did or did not have Myrmica rubra ants on them Ant presence significantly reduced the
amount of pollen analogue donated and received by flowers Building on these results I found
that the presence of ant scent alone elicited similar changes to bumblebee behavior reducing the
amount of pollen analogue donated by male flowers My results show that interference
competition is sufficient to alter bumblebee foraging behavior and that the effects of ants on
pollinators may extend after ant visitation to flowers Further study is needed to determine if bees
are able to differentiate between ant species by visual or olfactory cues Their ability to do so
94
could affect how generalized their response to ant presence is and may help to better predict how
ants will affect plant reproduction
In the final chapter I investigate how common palynivory is amongst tropical ant species I used
acetolysis and light microscopy to examine whole ants larvae and ant guts from Papua New
Guinea and Peru I found that tropical ants consistently contain pollen grains albeit often in
small numbers (lt5) However some genera notably Camponotus and Cephalotes consistently
contained higher numbers providing the first evidence that Camponotus may be a palynivorous
genus The small numbers of pollen grains present may indicate that ants preferentially exploit
other more easily digestible high-protein foods over pollen Future studies should investigate
exactly what role pollen plays in ant diets by supplementing the diet of captive ant colonies with
pollen This would allow us to examine how pollen consumption affects colony demography as
well as how efficiently ants digest pollen and whether ants exhibit pollen preferences
I bring up several pressing and unanswered questions within this thesis and highlight three here
First numerous plant species possess chemical or physical traits assumed to increase plant
fitness by reducing ant visitation However this has rarely been tested and instead research has
focused predominately on documenting ant repellence We should move away from this and
begin examining plant fitness in the presence or absence of these repellent traits to see how
beneficial these traits truly are Second how flower visiting ants impact male fitness has been
almost completely ignored in favor of female fitness Future studies should investigate how ant
visitation affects both pollen donation and pollen movement patterns Lastly research involving
antibiotics present on ant cuticles and their effect on pollen viability has been too small in scope
often limited to demonstrating the pollen killing effects of ant secretions Investigation into
95
whether plants have evolved any resistance to the secretions of the local ant community or why
some plants appear unaffected by their ant partners may yield exciting results
In conclusion ants have the capacity to strongly affect plant reproduction both positively and
negatively The fact that these diametrically opposing forces exist means we cannot approach
flower-visiting ants as either positive or negative a priori Instead we should strive to work
towards a better understanding of what is occurring in the system of interest and what role ants
play within it
96
Copyright Acknowledgments
Chapter 2 ldquoAnts and ant scent reduce bumblebee pollination of artificial flowersrdquo is current in
press in The American Naturalist Request for its inclusion here was sought and approved
1
Chapter 1 Ants as flower visitors and their effects on pollinator behavior and plant
reproduction
Cembrowski AR and Frederickson ME
Planned submission to a journal such as Oikos or Insectes Sociaux
ARC wrote the manuscript with input from MEF
Abstract
Ants often provide a range of benefits to flowering plants particularly by reducing herbivory
However when ants visit flowers they can deter pollinators or damage floral structures
Currently it is unclear how often and in what direction flower-visiting ants affect plant
reproductive success A review of the literature shows that ants can help or hinder pollination
processes but overall flower-visiting ants most commonly have a non-significant net effect on
plant fitness Although ants may directly harm flowers or indirectly affect pollination by
consuming floral nectar or harassing pollinators they can also benefit plants by attacking
florivores or predators of pollinators Ants occasionally act as pollinators themselves though ant
secretions often kill pollen grains There is growing evidence that many plants have traits that
allow them to avoid or minimize the negative effects of flower-visiting ants More research is
needed to determine if these traits provide fitness benefits to plants by preventing ant visitation
or if they function as generalized repellents of floral antagonists
2
Introduction
Ants and angiosperms share a long evolutionary history Ant diversification closely followed that
of flowering plants as ants took advantage of the large prey numbers that angiosperms supported
and the new habitats they provided (Moreau 2006) Subsequently numerous angiosperm
lineages evolved intimate associations with ants that function as an indirect or biotic form of
plant defense Food rewards such as extrafloral nectar or pearl bodies or housing in the form of
domatia attract ants to plants where they reduce herbivory and increase plant fitness (reviewed
in Rosumek et al 2009) However the traits that make ants good plant defenders particularly
their abundance attraction to sugar sources and aggressiveness can also cause problems for
plant reproduction Ants attracted by floral nectar can compete with pollinators for resources
and defend flowers potentially reducing plant fitness (Ness 2006 Junker et al 2007) That ants
negatively affect plant reproduction is an old and pervasive hypothesis Kerner (1878) called ants
ldquomost unwelcome guestsrdquo on flowers and more recent authors have similarly expressed that ant
visitation to flowers reduces plant reproductive success (Ness 2006 Lach 2007 Willmer et al
2009)
Nonetheless studies assessing the impact of flower-visiting ants on plant reproductive
success have produced mixed results Ants can have negative impacts on pollination and flower
function (Galen 1983 Puterbaugh 1998 Ness 2006 Hansen and Muumlller 2009) However other
studies have found no effect or even strong benefits of ant visits to flowers (Rico-Gray and
Thien 1989 Altshuler 1999 Schuumlrch 2000 Ashman and King 2005) This discrepancy between
ants being ldquounwelcome guestsrdquo and their actual effects on plant reproduction requires
clarification Our goal here is to highlight the ways that flower-visiting ants may affect plant
reproduction both positively and negatively and to discern through a review of the literature
3
what impacts ants have on plant reproductive success First we discuss mechanisms by which
flower-visiting ants can directly and indirectly impact plant reproduction both through floral
damage and by mediating changes in pollinator or florivore behavior We end this section by
evaluating published research that collectively shows that flower-visiting ants have
predominantly neutral effects on plant fitness measures Next we discuss pollination by ants
focusing on the ldquoantibiotic hypothesisrdquo before describing how plants may prevent ants from
visiting flowers Lastly we discuss promising new avenues of research and highlight work that
still needs to be performed to give a more complete understanding of ants as flower visitors
1 Ant interactions with flowers and floral visitors
i) Damage to flowers
Ants can directly disrupt plant reproduction by damaging floral structures preventing either the
fertilization of ovules or the presentation of pollen Rarely ants forage on floral tissue itself for
example Puterbaugh (1998) found that Formica neorufibarbus ants chewed through the coronal
ring of Eritrichium aretiodes flowers to get lipids More often but still not commonly foraging
ants chew through reproductive tissue to access floral nectar This can cause sterilization of
whole flowers (Echium plantagineum Kirk 1984) or affect female function only if just styles are
damaged effectively creating male flowers (Polemonium viscosum Galen 1983) Antibiotic
secretions on ant integuments may also directly damage pollen grains but the fitness effects of
this are unclear (see 2 below)
Several species of Crematogaster and Allomerus ants sterilize their host plants by
attacking floral buds (Yu and Pierce 1998 Stanton et al 1999 Gaume et al 2005 Maleacute et al
2012 Table 11) These ants nest in ldquoant-plantsrdquo or ldquomyrmecophytesrdquomdashie plants that host ant
4
colonies in specialized structures (domatia) The sterilizing ants do not appear to consume floral
tissues (Yu and Pierce 1998) and two hypotheses may explain why ants destroy floral buds First
ant colony size is often limited by domatia availability on their host plant By destroying floral
buds ants can cause plants to divert resources from reproductive to vegetative growth increasing
domatia number and allowing for larger ant colonies (Yu amp Pierce 1998 Frederickson 2009)
Second when plant-ants must defend their colony from takeovers by other colonies they may
benefit from limiting access to their host plant By destroying buds that could develop into
flowers branches or leaves ants can prevent their host plant from contacting nearby plants
occupied by enemy ant colonies (Stanton et al 1999)
The impact of sterilization on lifetime plant fitness is unclear Ants may destroy all of
their host plantrsquos floral buds (Yu and Pierce 1998) completely preventing reproduction or only a
portion of them (Maleacute et al 2012) allowing some seed set However counter-intuitively this
sterilization behavior may provide long-term fitness benefits to plants (Frederickson 2009
Palmer et al 2010) Ant-plants often host several different ant species over their lifetimes some
of which are non-sterilizing If hosting a sterilizing species at a certain life stage increases
survival or growth relative to hosting a non-sterilizing species at that life stage plants may
increase lifetime fitness by largely forgoing current reproduction in favor of increasing future
reproduction (Palmer et al 2010) Why plants invest resources in producing flowers that are
quickly destroyed by ants is not known but the timing of flower destruction may be important If
ants attack flowers after anthers dehisce plants may still realize some male fitness via pollen
donation (Yu and Pierce 1998) Additionally if the cost of flower production is relatively minor
and ants sometimes ldquomissrdquo flowers (Edwards and Yu 2008) attempting limited reproduction
may be a better strategy than forgoing it entirely
5
ii) Competition with pollinators
Ant activity on flowers can dramatically alter pollinator behavior Ants can reduce the species
diversity frequency or duration of pollinator visits to flowers (Norment 1988 Tsuji et al 2004
Ness 2006 Junker et al 2007 Lach 2007 2008b Romero et al 2011 and see Chapter 2) These
changes are largely due to either exploitative competition (ie consumption of shared resources)
or interference competition (ie direct aggression) between ants and pollinators
a) Exploitative competition
Pollinators and ants frequently compete for floral nectar Flower-visiting ants are often classified
as nectar thieves (sensu Inouye 1980) because they typically enter flowers through the
ldquolegitimaterdquo opening and consume nectar but transfer no pollen (Wyatt 1980 Ness 2006
Chamberlain and Holland 2008) Ants can also act as primary nectar robbers chewing holes
through corollas to reach nectaries (Willmer and Corbet 1981 Koopowitz and Marchant 1998)
although they are more commonly secondary nectar robbers and use holes made by previous
robbers (Newman and Thomson 2005)
Like other flower visitors foraging ants will often leave nectar behind in flowers This
amount can be less (Bleil et al 2011) more (Fritz and Morse 1982) or similar to (Lach 2008a)
amounts left by other visitors Often ants are morphologically constrained from fully exploiting
nectar Unlike bees and butterflies ants do not have proboscises and must get close to nectar to
consume it Narrow corollas or nectar spurs can prevent ant access to some or all floral nectar
(Schemske 1980 Galen and Cuba 2001 and see below) Smaller ants may be better able to
6
access nectar (Newman and Thomson 2005 Agarwal and Rastogi 2007) but can be limited by
their crop size in the amount of nectar they can remove (Lach 2005) If foraging ants leave
enough nectar behind for a pollinator to recover the cost of visiting a flower ants may have
minimal effects on plant reproductive success (Maloof and Inouye 2000 Turner et al 2012)
Differences among ant species in behavior and diet also affect their consumption of floral
nectar Species vary in how many flowers they visit per inflorescence or per plant and in how
many ants will visit a flower simultaneously (Lach 2005 Junker et al 2007 Lach 2013)
Invasive antsmdashthought to be better at exploiting and defending resources than native ants
(Holway et al 2002)mdashoften recruit to flowers in large numbers sometimes several dozen or
more (Visser et al 1999 Hansen and Muumlller 2009) Overall many factors may influence how
heavily ants exploit floral nectar on plants including ant density plant height the spatial or
temporal distribution of floral nectar and the availability of alternative food resources for ants
(Cushman and Addicott 1991 Lach 2013)
Rarely ants and pollinators may compete for pollen We know of only a few studies that
have recorded ants harvesting pollen directly from anthers (Horskins and Turner 1999 Ness
2006 Byk and Del-Claro 2010) Other studies have found pollen grains in ants (eg Baroni
Urbani and de Andrade 1997 and see Chapter 3) but this may not indicate competition with
pollinators ants sometimes collect stray pollen grains from the environment (Creighton 1967)
One New World genus Cephalotes might be specially adapted for pollen consumption They
possess ldquoproventricular shieldsrdquo structures that may restrict solid food to the crop allowing
nestmates to exchange pollen grains via trophallaxis It is unclear if this structure evolved for this
purpose but Cephalotes are the most frequently documented palynivorous ants (eg Creighton
1967 Baroni Urbani and de Andrade 1997 Byk and Del-Claro 2010) How efficiently ants
7
digest pollen and whether ants like bees feed it to larvae as a protein source is unknown At
least two studies have found that ant larvae consume pollen providing some support for this idea
(Wheeler and Bailey 1920 and Chapter 3)
b) Interference competition
Ants are relatively unique among flower visitors in defending floral resources from competitors
(but see Willmer and Corbett 1981 Roubik 1982) This defense ranges from chance encounters
with ants attacking visitors when flower occupancy overlaps (Ashman and King 2005 Junker et
al 2007) to actively patrolling inflorescences or constructing protective galleries around
nectaries (Horvitz and Schemske 1984 Gaume et al 2005 Hansen and Muumlller 2009)
Interference competition even in the absence of exploitative competition is sufficient to alter
pollinator behavior (Chapter 2)
The strength and outcome of interference competition depends on the identities of the ant
and the pollinator Pollinators might not alter their visitation patterns in response to non-
aggressive ants (Junker et al 2007) but more antagonistic ants may elicit avoidance behavior
before or soon after the visitor lands (Willmer and Corbet 1982 Altshuler 1999 Schuumlrch et al
2000 Table 11) Pollinator size may help predict their reaction to ants because ants may deter
only small-bodied pollinators (Gonzaacutelvez et al 2012) It is not clear whether flower visitors view
ants as aggressive competitors or as potential predators rarely ants prey on pollinators (Schatz
and Wcislo 1999) but in most cases ants merely cause pollinators to relocate
How well pollinators recognize and avoid ant-visited flowers is unresolved Collectively
pollinators have lower visitation rates to flowers visited by ants (Romero et al 2011) This is
8
dependent on pollinator identity however dipterans do not appear to discriminate between ant-
visited and ant-free flowers while bees can identify and avoid ant-visited flowers Bees likely
use a combination of visual and olfactory cues to recognize ant-visited flowers They can
identify previous conspecific and some heterospecific insect visitors by the hydrocarbon
ldquofootprintsrdquo they leave during foraging (Stout and Goulson 2001) Bumblebees can detect ant
hydrocarbons although this has been shown only in the laboratory (Ballantyne and Willmer
2012 Chapter 2) No study has yet examined if bees can use sight alone to detect ants but
several suggest it (Junker et al 2007 Gonzaacutelvez et al 2012) Some bees may discriminate
between ant species when visiting flowers (eg Lach 2008b) which follows if not all ant-
pollinator interactions are equally costly The ability of bees to do so will affect how generalized
their avoidance behaviors will be If they can discriminate between species bees may avoid
specific flowers visited by more ldquocostlyrdquo ants However if they cannot bees might either avoid
all ant-visited flowers or none depending on how negative the average interaction with an ant is
iii) Interactions with flower and pollinator antagonists
Ants can defend the flowers they visit Numerous plant species have circumfloral or bracteal
nectaries (ie extrafloral nectaries around flowers) and ants attracted to these nectaries can
reduce bud ovule or flower predation increasing plant fitness (Inouye and Taylor 1979 Rico-
Gray and Thien 1989 Sugiura et al 2006) Although the presence of EFNs (extrafloral nectaries)
so close to floral nectaries (sometimes less than 1 cm Ness 2006) might increase discovery and
exploitation of floral nectar by ants only one study has investigated this hypothesis finding no
relationship between the proximity of EFNs to floral nectaries and antsrsquo use of floral nectar
(Galen 2005) Holes made by nectar robbers are functionally analogous to circumfloral nectaries
9
and can also attract ants that defend flowers (Newman and Thomson 2005) and nectar-thieving
ants can similarly prevent florivory in some systems (Lach 2007 Bleil et al 2011)
Ants can also reduce the abundance of flower-dwelling predators or parasites of
pollinators Ambush predators such as crab spiders often forage at flowers where they may
reduce pollinator visitation rates and plant fitness (Dukas 2001 Gonccedilalvez-Souza et al 2008)
Ants can decrease spider abundance and richness on plants (Rudgers 2004 Renault et al 2005
Nahas et al 2012) and some evidence suggests that they can do the same on flowers (Lach
2008) In select cases ants may also reduce the abundance of pollinator parasitoids (Holland et
al 2011) Whether ants increase plant reproductive success by removing pollinator antagonists
has not yet been examined but should be If ants create lsquoenemy-freersquo flowers pollinators might
prefer to land on flowers with ants
iv) Net effects
Table 11 compiles studies in which the authors recorded ants consuming floral rewards or
interacting with pollinators and measured some aspect of plant fitness (eg fruit or seed set
pollen deposition) We included only a single study per plant species unless subsequent studies
found conflicting results or measured the fitness of another plant sex Records of ant pollination
are included in a separate table (Table 12)
Table 11 reveals relatively few examples of flower-visiting ants negatively affecting
plant fitness given how costly ant visitation is assumed to be The net effect of ants on plant
reproductive success is actually quite variable with similar numbers of studies finding positive
negative or neutral effects of ant presence Surprisingly when ants do have negative effects it is
10
usually because they directly damage floral structures and not because they prevent pollinators
from visiting flowers and successfully transferring pollen Why are ants not more often negative
for plant reproduction when this is commonly assumed to be true
First ants may compensate for reducing pollinator visitation rates by improving
pollination quality As pollinators move between flowers on a plant they often deposit self-
pollen on stigmas (ie geitonogamy) In some plants with late-acting self-incompatibility ovules
fertilized by self-pollen are subsequently aborted reducing the number of seeds that can be
produced (Barrett 2002 and references therein) Geitonogamy can also reduce offspring viability
or vigor (de Jong et al 1993) and cause pollen discounting a reduction in the amount of pollen
available for outcrossing because of self-pollination (Barrett 2002) By competing with
pollinators ants might decrease rates of geitonogamous pollination if pollinators then visit fewer
sequential flowers within a plant Ants might also cause pollinators to fly further between plants
potentially increasing the genetic diversity of pollen arriving at stigmas Although such effects
have been reported for other floral larcenists (Maloof 2001 Irwin 2003) to our knowledge these
hypotheses have not been investigated in relation to ants
Second ants and pollinators do not always forage for the same resources possibly
allowing them to partition floral rewards If ants forage for nectar but not pollen and flowers can
be pollinated efficiently by visitors looking to gather mostly pollen and little or no nectar then
plant reproduction will not be affected by ants consuming floral nectar Junker et al (2010a)
found that honeybees foraged for both pollen and nectar in the absence of ants but reduced their
foraging on nectar but not pollen when ants could access flowers resulting in no difference in
fruit set between treatments Whether pollinators commonly adjust their foraging behaviors in
response to ants warrants further research
11
Third ants are somewhat unique among floral larcenists in that they often provide direct
benefits to the plants they visit In the studies listed in Table 11 ants sometimes decreased
herbivory on plants (Chamberlain and Holland 2008 Bleil et al 2011) or otherwise aided the
plant (eg ant presence repelling smaller less effective pollinators Gonzaacutelvez et al 2012) If the
positive effects of ants cancel or outweigh the negative effects of flower-visiting ants on
pollinators then ant visitation may be neutral or even positive for plant fitness
Fourth ants may impose fitness costs to plants not captured in the studies in Table 11
Nectar robbers have stronger negative effects when plants are pollen-limited (Burkle et al 2007)
but only one study in Table 11 assessed whether flowers would set more seed if they received
more pollen finding no evidence of pollen limitation (Lach 2008a Table 11) Even if ants
decrease pollinator visitation rates there is limited scope for ants to decrease plant reproductive
success when plants are not pollen-limited Furthermore nearly all the studies in Table 11
measured only female fitness Only three quantified aspects of male fitness (Wyatt 1980 Fritz
and Morse 1981 Galen and Geib 2007) and we know of only one other study in a system in
which ants occasionally pollinate that has also measured male fitness (Ashman 2000) All four
studies measured pollen or pollinia removal rates and none found any effect of flower-visiting
ants on male fitness However whether other measures of male fitness (eg seeds sired pollen
deposition patterns) would reveal similar or different effects needs investigation
Finally although floral defenses against ants (see 3 below) may prevent us from
observing the negative effects of ants on plant reproductive success we consider this unlikely
Several studies have examined interactions between ants and plants that share little or no
coevolutionary history usually in the context of ant invasions Although invasive ants often
recruit to flowers in larger numbers than native ants they do not appear to reduce plant
12
reproductive success (Blancafort and Goacutemez 2005 Lach 2007 Lach 2008a but see Hansen and
Muumlller 2009) And in Hawaii where angiosperms evolved for millions of years in the absence of
ants and are thought to have secondarily lost floral defenses against ants (Junker et al 2011a)
there is no evidence that the preponderance of recently introduced flower-visiting ants negatively
affects plant fitness (Junker et al 2010a Bleil et al 2011)
2 Ants as pollinators
Ants may directly benefit plant reproduction by acting as pollinators but this is a relatively rare
occurrence Ants have long been considered poor pollinators (Kerner 1878) Initially this was
based on morphological and behavioral differences between ants and recognized pollinators
Worker ants are wingless and often relatively hairless making them poor pollen dispersers As
well because many ants exhibit Ortstreue (literally ldquoplace fidelityrdquo) ants were thought to return
to the same branch or flower repeatedly effecting no cross pollination (Houmllldobler and Wilson
1990) Though largely true these traits do not necessarily preclude ants from acting as
pollinators (Table 12) Although they commonly have restricted foraging ranges (Houmllldobler and
Wilson 1990) some ants can effect outcrossing (eg Ashman and King 2005 Carvalheiro 2008)
and many ants are as hairy if not hairier than their pollinating counterparts
In the late 1970s the ldquoantibiotic hypothesisrdquo was put forward to explain the rarity of ant
pollination Ants use antibiotic secretions to protect themselves against the fungal and bacterial
pathogens common in their nests and environs Iwanami and Iwadare (1978) found that one of
these compounds myrmicacin also greatly reduces pollen germination rates Subsequently
Beattie et al (1984 1985) found that short periods of contact (le 30 minutes) with an antrsquos
integument caused pollen grains to fail to germinate Some of these pollen-killing compounds
13
were traced back to the ant metapleural gland which produces hygienic secretions (Houmllldobler
and Wilson 1990) although contact with ants lacking metapleural glands can also reduce pollen
viability (Beattie et al 1985 Dutton and Frederickson 2012) Ant mandibular or anal gland
secretions and potentially ant venom may likewise defend ants against pathogens and have
similar effects on pollen germination (Beattie et al 1985 Graystock and Hughes 2011)
Nonetheless ant pollination has been documented repeatedly There are at least 18 cases
in which ant pollination has been shown experimentally and five others in which observations
strongly suggest it (Table 12) The majority of these plants fit the ldquoant pollination syndromerdquo
originally proposed by Hickman (1974) dense stands of short plants with small flowers and
easily accessible nectaries Typically ant-pollinated plants are associated with hot and dry
climates (principally in the Mediterranean) but dry alpine environments may also favor the
evolution of ant pollination (eg Svensson 1985 Puterbaugh 1998 Schiestl and Glaser 2011) In
most cases ants appear to be the sole or major pollinator of the plant in question (Hickman 1974
Peakall et al 1990 de Vega et al 2009) although ants sometimes make only supplementary
contributions to pollination (Schuumlrch et al 2000 Ashman and King 2005) This suggests that co-
evolution between ants and plants may not be necessary for ants to be useful pollinatorsmdashwhich
is surprising given the potential negative effects of ants on plant reproduction
Ant-pollinated plants cope with ant secretions in a variety of ways Several plant species
may simply tolerate pollen damage (Gόmez and Zamora 1992 de Vega et al 2009) apparently
possessing no overt adaptations to prevent it These plants might receive enough pollen to
overcome reduced pollen viability Other plants may place pollen on ant body parts thought to
have fewer secretions thereby potentially escaping damage (eg on ant legs Ramsey 1995) or
attach pollen to ants with specialized structures avoiding direct contact with the ant integument
14
(Peakall et al 1990) It is not known whether pollen evolves resistance to ant secretions or
whether plants deposit pollen on ants which have less disruptive secretions although these
possibilities have been suggested elsewhere (eg Garciacutea et al 1995 Ramsey 1995)
One key question regarding the effects of ant secretions on pollen grains urgently needs
to be resolved Several studies have suggested that the pollen-killing secretions of ants may have
contributed to the evolution of floral defenses against ants (eg Wagner 2000 Galen and Geib
2007) For this to be true ants must decrease male fitness by damaging pollen grains before they
leave flowers and are deposited on receptive stigmas Only two studies have investigated the
effects of ant secretions on pollen still in anthers (Wagner 2000 Galen and Butchard 2003) and
both constrained ants unnaturally to flowers for significantly longer than ants would normally
stay on flowers Although these studies found strong reductions in pollen vigor it is unclear if
ant secretions decrease male fitness under natural ant visitation conditions
3 Floral defenses against ants
Plants are not passive recipients of flower visitors and have some ability to ldquochooserdquo which
organisms can access floral rewards Flowers resist ants using both structural and chemical traits
More thorough investigation is needed to determine whether these traits evolved to prevent or
minimize the negative effects of ants on plant reproductive success (ie whether these traits can
truly be considered defenses against ants see 4ia below) It is unlikely however that ants
experience reciprocal selection to circumvent floral resistance traits because the fitness benefits
to ants of accessing flowers are probably small Thus there may be little scope for
coevolutionary arms races between floral defenses against ants and ant counter-adaptations
15
i) Structural resistance traits
a) Restricting ant access
Numerous plant species have physical structures preventing ant access to flowers Thin pedicels
can limit antsrsquo gripping abilities and make ants easier to dislodge when flowers are moved by
wind (Gaume et al 2005) and waxy secretions can render stems too slippery for ants to climb
(Harley 1991) Structures surrounding flowers such as water moats or dense hairs may similarly
keep ants from accessing floral rewards (Willmer et al 2009) Many such traits function as broad
filters and prevent not only ants but most crawling insects from reaching flowers Thus it is
difficult to determine whether ants florivores or both acted as selective agents favoring the
evolution of physical barriers to flowers
Flower shape may also reduce ant-pollinator conflict Long narrow corollas or nectar
spurs can prevent all but the smallest ants from accessing floral nectaries (Bluumlthgen et al 2004
Agarwal and Rastogi 2007) while still allowing long-tongued pollinators access to rewards
Selection to exclude nectar-thieving ants in such a manner however may be counteracted by
pollinator preferences for wider corollas that allow easier flower handling (Galen and Cuba
2001) Additionally these types of flowers may be more prone to robbery (Irwin and Maloof
2002) limiting their usefulness in preventing nectar larceny
b) Separation in space or time
Plants may separate floral rewards and ants temporally or spatially to minimize ant impacts on
plant reproduction Several authors have proposed that myrmecophytes have adapted to the
16
sterilization or otherwise detrimental activities of ants by locating domatia or food rewards on
different branches than flowers (Izzo and Vasconcelos 2002 Raine et al 2002 Edwards and Yu
2008) Temporal separation of ant and pollinator activities could likewise reduce conflict though
evidence is limited Some acacia flowers dehisce during periods of low ant activity (Willmer and
Stone 1997 Nicklen and Wagner 2006) but it is unclear if this is in response to ants
temperature or pollinator activity Timing of nectar secretion may similarly reduce ant-pollinator
competition If ants are active prior to pollinators they may deplete floral nectar and reduce
pollinator visitation rates (Schaffer et al 1984) However if nectar is secreted when ants are
inactive pollination may occur unimpeded (Norment 1988) This temporal separation could
favor pollinators that are active when ants are not (eg Wyatt and Morse 1981) but this is
poorly documented Further studies particularly focusing on flowers with the potential for
nocturnal and diurnal pollination may provide more examples
c) Extrafloral nectaries (EFNs)
One hypothesis put forward to explain the evolution of EFNs proposes that EFNs ldquodistractrdquo ants
from consuming floral nectar (Kerner 1878 Wagner and Kay 2002) Wagner and Kay (2002)
tested this hypothesis by constructing model plants using artificial nectaries assigned at random
to represent either floral or extrafloral nectaries They found that the presence of EFNs could
reduce ant exploitation of floral nectar However as highlighted by Galen (2005) this design is
closer to a plant making a surplus of flowers some of which will avoid ant visitation if visitation
rates increase sublinearly with increasing flower number Producing extra flowers to escape ant
visitation appears to be a viable strategy in some systems (Ashman and King 2005 Galen 2005)
However Galen (2005) found that applying honey (simulating extrafloral nectar) to P viscosum
17
leaves had the opposite effect and increased ant visits to flowers Only two studies both in
Pachycereus schottii have found evidence for the distraction hypothesis under field conditions
(Chamberlain and Holland 2008 Holland et al 2011)
There is limited empirical support for the distraction hypothesis but abundant evidence
that EFNs attract ants that defend plants against herbivores (reviewed in Rosumek et al 2009)
To function as attractants the presence of EFNs must increase ant abundance on plants
However because of the increased numbers attracted EFNs would not be expected to reduce the
abundance of ants on nectar rewards or be able to satiate the ants recruited Only if ant
abundance stops increasing at a reward density much lower than that available on the plant
would EFNs serve to distract ants In this case the amount of rewards may overwhelm ants
leaving some flowers underexploited However even in P schottii ants still visited flowers
(Chamberlain and Holland 2008) In cases where ant abundance would continue to increase with
additional rewards it is unlikely that EFNs function as distractions
ii) Chemical resistance traits
a) Toxic nectar
One of the earliest proposed floral defenses against ants was toxic nectar (Janzen 1977)
However studies testing Janzenrsquos (1977) hypothesis uncovered only a very few isolated
examples (eg Catalpa speciosa Stephenson 1981 Crinum erubescens Guerrant and Fiedler
1981) in short most floral nectar is not toxic or repellent to ants (Baker and Baker 1978
Schubart and Anderson 1978) In fact ants sometimes prefer floral nectar over solutions with
equivalent sugar concentrations (Koptur and Truong 1998)
18
In contrast floral nectar can repel other flower visitors (eg birds Johnson et al 2006
lepidopterans Kessler and Baldwin 2007) Repellency is often caused by plant secondary
metabolites which may occur in nectar as a by-product of their production elsewhere in the
plant where they function as defenses against herbivory (Adler 2001) Alternatively toxic or
repellent nectar may be adaptive repellent nectar may promote effective pollination by reducing
the amount of reward any one pollinator takes spreading nectar over several pollinator visits
(Kessler and Baldwin 2007) or it may function as a filter selecting for specific pollinators
(Johnson et al 2006) The latter may explain why repellent nectar is not more common
legitimate pollinators in addition to floral larcenists may be deterred from visiting flowers with
repellent nectar This may be particularly true of nectars that would repel ants which are closely
related to bees nectar distasteful to ants may often also be distasteful to bees and those nectars
acting as a filter for bees may still be palatable to ants For example Prŷs-Jones and Willmer
(1992) found that the ammonia-containing nectar of Lathraea clandestina is readily consumed
by both bees and ants even though it deters birds that act as nectar thieves Although floral
nectar itself rarely repels ants Guerrant and Fielder (1981) found that the presence of macerated
petals within nectar can often keep ants away Alkaloids in petals may be responsible and might
explain why ants seldom act as primary nectar robbers these secondary compounds may prevent
ants from chewing through corollas
b) Floral volatiles
Repellent floral volatiles appear to be a more widespread form of chemical resistance against
ants These were reviewed recently by Willmer et al (2009) and will be discussed here only
briefly Numerous plant taxa produce floral scents repellent to ants including myrmecophilous
19
myrmecophytic and non-ant associated plants in both temperate and tropical ecosystems
(Willmer et al 2009 Junker et al 2011a Ballantyne and Willmer 2012) In three large studies
floral volatiles that repel ants were detected in about a third of all plants tested (Willmer et al
2009 Junker et al 2011a Ballantyne and Willmer 2012) Pollen is often the source of ant-
repellent volatile compounds (Willmer et al 2009 Ballantyne and Willmer 2012) but they can
be emitted from other floral parts including petals That pollen is often the source of repellence
could suggest that preventing ant visitation may be more important in protecting male than
female function Additionally because repellence would decrease as pollen is removed from
anthers ants may return and defend flowers more quickly than if repellent chemicals were in
longer-lasting structures (eg petals) In some cases floral volatiles are thought to mimic ant
alarm pheromones (Willmer et al 2009) However floral scents elicit inconsistent reactions
across ant species ranging from repellence to attraction (Agarwal and Rastogi 2007 Ballantyne
and Willmer 2012)
Producing structural or chemical resistance traits may be costly for plants Native
Hawaiian plants which have little recent coevolutionary history with ants appear to have
secondarily lost floral ant repellents (Junker et al 2011a) Both Willmer et al (2009) and Junker
et al (2011a) found that highly chemical repellent flowers did not have strong physical defenses
and vice versa suggesting trade-offs between the two (but see Ballantyne and Willmer 2012)
Like toxic nectar floral ant repellents might sometimes deter both pollinators and ants For
example Galen et al (2011) found that 2-phenylethanol in P viscosum floral scent repels both
flower-damaging F neorufibarbis ants and the plantrsquos main pollinator Bombus kirbyellus
Interestingly four recent studies have found that floral scents can also attract ants
(Agarwal and Rastogi 2007 Edwards and Yu 2008 Schiestl and Glaser 2011 Gonzaacutelvez et al
20
2012) One study (Schiestl and Glaser 2011) has shown that floral scents attract ants to a
potentially ant-pollinated plant and such systems may provide many more examples
4 Future directions
We have highlighted areas in need of further investigation throughout the review Here we focus
on several questions we consider high priorities for future research
i) Floral defenses against ants
a) Do floral resistance traits increase plant fitness
The existence ant-repellent floral traits is often taken as evidence that ants negatively affect plant
reproductive success However despite the abundance and variety of mechanisms excluding ants
from flowers few studies show that ants reduce plant fitness when they visit flowers (Table 11)
This absence is particularly apparent with repellent floral volatiles which may be the most
widespread floral resistance trait (Willmer et al 2009 Junker et al 2011a Ballantyne and
Willmer 2012) To our knowledge only one study has shown that floral ant repellents benefit
plants with repellent P viscosum flowers avoiding damaging ant visits (Galen et al 2011)
Research is urgently needed to investigate if these traits increase plant fitness by excluding ants
or if ant visitation is inconsequential More studies taking advantage of standing variation in
traits (Galen et al 2011) or creating it artificially (eg Junker et al 2010b) will allow us to
distinguish between the two
21
b) If ant visitation is often not costly what caused the evolution of repellent traits and
what maintains them Similarly are these repellent traits truly defenses against ant
visitation or are they generalized defenses against a range of floral antagonists
Ant-repellent floral traits are widespread and their production may be costly (Willmer et al
2009 Junker et al 2011a) The studies in Table 11 however indicate that flower-visiting ants
often do not disrupt plant reproduction particularly outside of myrmecophytes What then
selected for these traits One possibility is that these repellents are not directed solely at ants
Instead a community of antagonistic flower visitors may exert diffuse selection on plants
thereby selecting for and maintaining these traits Structural modifications may repel crawling
insects while chemical repellents such as floral volatiles may repel both flying and non-flying
insects (Junker et al 2011b) How much overlap there is in floral volatiles that repel both ants and
other antagonists needs investigation Until we examine how specific these defenses are to ants
we risk overstating the importance of ants in their evolution
ii) Do ants alter pollen movement patterns or reduce male fitness
We are aware of only four studies that have measured if ant visitation impacts male fitness
(Wyatt 1980 Fritz and Morse 1981 Ashman 2000 Galen and Geib 2007) While none of these
studies found an effect all measured only amounts of pollen or pollinia removed However this
measure does not adequately encompass male fitness alterations to pollen movement patterns
and eventual deposition location may be more important to male function than changes in
removal rates If as suggested by some studies (Altshuler 1999 Lach 2007) ants increase
pollinator relocation rates they may decrease pollen discounting increasing male fitness
However if this increased relocation reduces overall pollen export rates ant competition with
22
pollinators may depress male function Studies using pollen-tracking methods such as fluorescent
dyes will allow us to see how ants change pollen movement patterns and methods like paternity
analysis will reveal how competition between ants and pollinators alters male fitness
iii) Coevolution between ants and plants do myrmecophytes exhibit stronger more
specific defenses against ants
The floral traits of myrmecophytes are most likely to coevolve with ants because all plant
reproduction occurs in the presence of ants Their floral defenses would thus be expected to be
both stronger and more specific to their resident ants than those other plants visited by an array
of more opportunistic ant species Some evidence supports this Willmer et al (2009) found that
resident ants of acacias were repelled by flowers with more aggressive ant species showing
stronger repellence than less aggressive ants Additionally ants not normally associated with
acacias did not respond to acacia floral volatiles More studies are needed in other systems and
should take advantage of phylogenetic approaches examining floral traits in closely related
myrmecophytic and non-myrmecophytic plant species to determine whether ants commonly
select for floral defenses against ants in myrmecophytes
Acknowledgements
For comments and discussion we thank the Frederickson lab particularly Eric Youngerman
Lina Arcila Hernandez and Kyle Turner as well as Alison Parker and Jordan Pleet MEF
acknowledges financial support from an NSERC Discovery Grant a Connaught New Researcher
23
Award an Ontario Ministry of Economic Development and Innovation Early Researcher Award
and the University of Toronto ARC was supported by an Ontario Graduate Scholarship and
Sigma Xi
24
References
Adler L S 2001 The ecological significance of toxic nectar Oikos 91409ndash420
Agarwal V M and N Rastogi 2007 Role of floral repellents in the regulation of flower visits
of extrafloral nectary-visiting ants in an Indian crop plant Ecological Entomology 3359ndash
65
Altshuler D L 1999 Novel interactions of non-pollinating ants with pollinators and fruit
consumers in a tropical forest Oecologia 119600ndash606
Ashman TL 2000 Pollinator selectivity and its implications for the evolution of dioecy and
sexual dimorphism Ecology 812577-2591
Ashman TL and E A King 2005 Are flower-visiting ants mutualists or antagonists A study
in a gynodioecious wild strawberry American Journal of Botany 92891ndash5
Ballantyne G and P Willmer 2012 Floral visitors and ant scent marks noticed but not used
Ecological Entomology 37402-409
Ballantyne G and P Willmer 2012 Nectar theft and floral ant-repellence a link between
nectar volume and ant-repellent traits PloS One 7e43869
Baker H G and I Baker 1978 Ants and flowers Biotropica 1080
Barrett S C H 2002 Sexual interference of the floral kind Heredity 88154ndash159
Baroni Urbani C and M L de Andrade 1997 Pollen eating storing and spitting by ants
Naturwissenschaften 84256ndash258
25
Beattie A J C Turnbull R B Knox and E G Williams 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany
71421ndash426
Beattie A J C Turnbull T Hough S Jobson and R B Knox 1985 The vulnerability of
pollen and fungal spores to ant secretions evidence and some evolutionary implication
American Journal of Botany 72606ndash614
Blancafort X and C Goacutemez 2005 Consequences of the Argentine ant Linepithema humile
(Mayr) invasion on pollination of Euphorbia characias (L) (Euphorbiaceae) Acta
Oecologica 2849ndash55
Bleil R N Bluumlthgen and R R Junker 2011 Ant-plant mutualism in Hawailsquoi Invasive ants
reduce flower parasitism but also exploit floral nectar of the endemic shrub Vaccinium
reticulatum (Ericaceae) Pacific Science 65291ndash300
Bluumlthgen N G Gottsberger and K Fiedler 2004 Sugar and amino acid composition of ant-
attended nectar and honeydew sources from an Australian rainforest Austral Ecology
29418ndash429
Burkle L A R E Irwin and D A Newman 2007 Predicting the effects of nectar robbing on
plant reproduction implications of pollen limitation and plant mating system American
Journal of Botany 941935ndash1943
Byk J and K Del-Claro 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333ndash38
26
Caballero P C G Ossa W L Gonzaacuteles C Gonzaacutelez-Browne G Astorga M M Muruacutea and
R Medel 2013 Testing non-additive effects of nectar-robbing ants and hummingbird
pollination on the reproductive success of a parasitic plant Plant Ecology 214633ndash640
Carvalheiro L G E R M Barbosa and J Memmott 2008 Pollinator networks alien species
and the conservation of rare plants Trinia glauca as a case study Journal of Applied
Ecology 451419ndash1427
Chamberlain S A and J N Holland 2008 Density-mediated context-dependent consumer-
resource interactions between ants and extrafloral nectar plants Ecology 891364ndash1374
Creighton W S 1967 Studies on free colonies of Cryptocerus texanus Satschi (Hymenoptera
Formicidae) Psyche 7434ndash42
Cursach J and J Rita 2011 Implications of the reproductive biology of the narrow endemic
Naufraga balearica (Apiaceae) for its conservation status Plant Systematics and Evolution
298581ndash596
Cushman JH and Addicott JF In Huxley CR and DF Cutler (eds) 1st edition Ant-plant
interactions Oxford University Press Oxford
de Jong T J N M Waser and P G Klinkhamer 1993 Geitonogamy the neglected side of
selfing Trends in Ecology and Evolution 8321ndash325
de Vega C M Arista P L Oritz C M Herrera and S Talavera 2009 The ant-pollination
system of Cytinus hypocistis (Cytinaceae) a Mediterranean root holoparasite Annals of
Botany 4517ndash8
27
Dukas R 2001 Effects of perceived danger on flower choice by bees Ecology Letters 4327ndash
333
Dutton E M and M E Frederickson 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561ndash569
Edwards D P and D W Yu 2008 Tolerating castration by hiding flowers in plain sight
Behavioral Ecology and Sociobiology 6395ndash102
Frederickson M E 2009 Conflict over reproduction in an ant-plant symbiosis why Allomerus
octoarticulatus ants sterilize Cordia nodosa trees The American Naturalist 173675ndash81
Fritz R S and D H Morse 1981 Nectar parasitism of Asclepias syriaca by ants effect on
nectar levels pollinia insertion pollinaria removal and pod production Oecologia 50316ndash
319
Galen C 1983 The effects of nectar thieving ants on seedset in floral scent morphs of
Polemonium viscosum Oikos 41245ndash249
Galen C 2005 Catching ants with honey an experimental test of distraction and satiation as
alternative modes of escape from flower-damaging ants Oecologia 14480ndash87
Galen C and B Butchart 2003 Ants in your plants effects of nectar-thieves on pollen fertility
and seed-siring capacity in the alpine wildflower Polemonium viscosum Oikos 101521ndash
528
Galen C and J Cuba 2001 Down the tube pollinators predators and the evolution of flower
shape in the alpine skypilot Polemonium viscosum Evolution 551963-1971
28
Galen C and J C Geib 2007 Density-dependent effects of ants on selection for bumble bee
pollination in Polemonium viscosum Ecology 881202ndash1209
Galen C R Kaczorowski S L Todd J Geib and R A Raguso 2011 Dosage-dependent
impacts of a floral volatile compound on pollinators larcenists and the potential for floral
evolution in the alpine skypilot Polemonium viscosum The American Naturalist 177258ndash
272
Garciacutea M B R J Antor and X Espadaler 1995 Ant pollination of the palaeoendemic
dioecious Borderea pyrenaica (Dioscoreaceae) Plant Systematics and Evolution 19817ndash
27
Gaume L M Zacharias and R M Borges 2005 Ant ndash plant conflicts and a novel case of
castration parasitism in a myrmecophyte Evolutionary Ecology Research 7435ndash452
Graystock P and W O H Hughes 2011 Disease resistance in a weaver ant Polyrhachis
dives and the role of antibiotic-producing glands Behavioral Ecology and Sociobiology
652319ndash2327
Gόmez J M 2000 Effectiveness of ants as pollinators of Lobularia maritima effects on main
sequential fitness components of the host plant Oecologia 12290ndash97
Gόmez J M and R Zamora 1992 Pollination by ants consequences of the quantitative effects
on a mutualistic system Oecologia 91410ndash418
Gόmez J M R Zamora J A Hόdar and D Garciacutea 1996 Experimental study of pollination
by ants in Mediterranean high mountain and arid habitats Oecologia 105236ndash242
29
Gonccedilalves-Souza T P M Omena J C Souza and G Q Romero 2008 Trait-mediated effects
on flowers artificial spiders deceive pollinators and decrease plant fitness Ecology
892407ndash2413
Gonzaacutelvez F G L Santamariacutea R T Corlett and M A Rodriacuteguez-Gironeacutes 2012 Flowers
attract weaver ants that deter less effective pollinators Journal of Ecology 10178ndash85
Guerrant E O and P L Fiedler 1981 Flower defenses against nectar-pilferage by ants
Biotropica 1325ndash33
Harley R 1991 The greasy pole syndrome In Huxley CR and DF Cutler (eds) 1st edition
Ant-plant interactions Oxford University Press Oxford
Hansen D M and C B Muumlller 2009 Invasive ants disrupt gecko pollination and seed
dispersal of the endangered plant Roussea simplex in Mauritius Biotropica 41202ndash208
Hickman J C 1974 Pollination by ants a low-energy system Science 1841290ndash1292
Holland J N S a Chamberlain and T E X Miller 2011 Consequences of ants and
extrafloral nectar for a pollinating seed-consuming mutualism ant satiation floral
distraction or plant defense Oikos 120381ndash388
Houmllldobler B and Wilson E O 1990 The Ants 1st Edition Harvard University Press
Cambridge
Holway DA L Lach AV Suarez ND Tsutsui and TJ Case 2002 The causes and
consequences of ant invasions Annual Review of Ecology and Systematics 33181-233
30
Horskins K and Turner VB 1999 Resource use and foraging patterns of honeybees Apis
mellifera and native insects on flowers of Eucalyptus costata Australian Journal of
Ecology 24221-227
Horvitz C C and D W Schemske 1984 Effects of ants and an ant-tended herbivore on seed
production of a neotropical herb Ecology 651369ndash1378
Inouye DW 1980 The terminology of floral larceny Ecology 611251-1253
Inouye DW and O R J Taylor 1979 A temperate region plant-ant-seed predator system
consequences of extra floral nectar secretion by Helianthella quinquenervis Ecology 601ndash
7
Irwin RE 2003 Impact of nectar robbing on estimates of pollen flow conceptual predictions
and empirical outcomes Ecology 84485-495
Irwin RE and Maloof JE 2002 Variation in nectar robbing over time space and species
Oecologia 133525-533
Iwanami Y and T Iwadare 1978 Inhibiting effects of myrmicacin on pollen growth and pollen
tube mitosis Botanical Gazette 13942ndash45
Izzo TJ and Vasconcelos HL 2002 Cheating the cheater domatia loss minimizes the effects
of ant castration in an Amazonian ant plant Oecologia 133200-205
Janzen D H 1977 Why donrsquot ants visit flowers Biotropica 9252
Johnson SD Hargreaves AL and Brown M 2006 Dark bitter-tasting nectar functions as a
filter of flower visitors in a bird-pollinated plant Ecology 872709-2716
31
Junker R A Y C Chung and N Bluumlthgen 2007 Interaction between flowers ants and
pollinators additional evidence for floral repellence against ants Ecological Research
22665ndash670
Junker R R R Bleil C C Daehler and N Bluumlthgen 2010a Intra-floral resource partitioning
between endemic and invasive flower visitors consequences for pollinator effectiveness
Ecological Entomology 35760ndash767
Junker R R and N Bluumlthgen 2010b Floral scents repel facultative flower visitors but attract
obligate ones Annals of botany 105777ndash82
Junker R R C C Daehler S Doetteri A Keller and N Bluumlthgen 2011a Hawaiian ant ndash
flower networks nectar-thieving ants prefer undefended native over introduced plants with
floral defenses Ecological Monographs 81295ndash311
Junker R R J Gershenzon and S B Unsicker 2011b Floral odor bouquet loses its ant
repellent properties after inhibition of terpene biosynthesis Journal of Chemical Ecology
371323ndash31
Kawakita A and M Kato 2002 Floral biology and unique pollination system of root
holoparasites Balanophora kuroiwai and B tobiracola (Balanophraceae) American
Journal of Botany 891164ndash1170
Koopowitz H and TA Marchant 1998 Postpollination nectar reabsorption in the African
epiphyte Aerangis verdickii (Orchidaceae) American Journal of Botany 85508-512
Kirk W D J 1984 Ecological studies on Thrips imaginis Bagnall (Thysanoptera) in flowers of
Echium plantagineum L in Australia Australian Journal of Ecology 99ndash18
32
Kerner A 1878 Flowers and their unbidden guests C Kegan Paul London
Kessler D and I T Baldwin 2007 Making sense of nectar scents the effects of nectar
secondary metabolites on floral visitors of Nicotiana attenuata The Plant Journal 49840ndash
54
Koptur S and N Truong 1998 Facultative ant-plant interactions nectar sugar preferences of
introduced pest species in South Florida Biotropica 30179-189
Lach L 2005 Interference and exploitation competition of three nectar-thieving invasive ant
species Insectes Sociaux 52257ndash262
Lach L 2007 A mutualism with a native membracid facilitates pollinator displacement by
Argentine ants Ecology 881994ndash2004
Lach L 2008a Argentine ants displace floral arthropods in a biodiversity hotspot Diversity and
Distributions 14281ndash290
Lach L 2008b Floral visitation patterns of two invasive ant species and their effects on other
hymenopteran visitors Ecological Entomology 33155ndash160
Lach L 2013 A comparison of floral resource exploitation by native and invasive Argentine
ants Arthropod-Plant Interactions 7177-190
Luo C W K Li X M Chen Z Y Huang and K U N Li 2012 Ants contribute significantly
to the pollination of a biodiesel plant Jatropha curcas Environmental Entomology
411163ndash1168
33
Maleacute PJ G C Leroy A Dejean A Quilichini and J Orivel 2011 An ant symbiont directly
and indirectly limits its host plantrsquos reproductive success Evolutionary Ecology 2655ndash63
Maloof J E 2001 The effects of a bumble bee nectar robber on plant reproductive success and
pollinator behavior American Journal of Botany 881960ndash1965
Maloof J E and D W Inouye 2000 Are nectar robbers cheaters or mutualists Ecology
812651ndash2661
Moreau CS CD Bell R Vila SB Archibald and NE Pierce 2006 Phylogeny of the ants
diversification in the age of the angiosperms Science 312101-104
Nahas L M O Gonzaga and K Del-Claro 2012 Emergent impacts of ant and spider
interactions herbivory reduction in a tropical savanna tree Biotropica 44498ndash505
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506ndash514
Newman D A and J D Thomson 2005 Interactions among nectar robbing floral herbivory
and ant protection in Linaria vulgaris Oikos 110497ndash506
Nicklen E F and D Wagner 2006 Conflict resolution in an ant-plant interaction Acacia
constricta traits reduce ant costs to reproduction Oecologia 14881ndash7
Norment C J 1988 The effect of nectar-thieving ants on the reproductive success of Frasera
speciosa (Gentianaceae) American Midland Naturalist 120331ndash336
34
Orivel J L Lambs P-J G Maleacute C Leroy J Grangier T Otto A Quilichini et al 2011
Dynamics of the association between a long-lived understory myrmecophyte and its
specific associated ants Oecologia 165369ndash76
Palmer T M D F Doak M L Stanton J L Bronstein E T Kiers T P Young J R
Goheen et al 2010 Synergy of multiple partners including freeloaders increases host
fitness in a multispecies mutualism Proceedings of the National Academy of Sciences
10717234ndash9
Peakall R A J Beattie and S H James 1987 Pseudocopulation of an orchid by male ants a
test of two hypotheses accounting for the rarity of ant pollination Oecologia 73522ndash524
Peakall R and A J Beattie 1989 Pollination of the orchid Microtis parviflora R Br by
flightless worker ants Functional Ecology 3515ndash522
Peakall R C J Angus A J Beattie 1990 The significance of ant and plant traits for ant
pollination in Leporella fimbriata pollination Oecologia 84457ndash460
Puterbaugh M N 1998 The roles of ants as flower visitors experimental analysis in three
alpine plant species Oikos 8336ndash46
Prŷs-Jones OE and Willmer PG 1992 The biology of alkaline nectar in purple toothwort
(Lathraea clandenstina) ground level defenses Biological Journal of the Linnean Society
45373-388
Raine NE Willmer P and Stone GN 2002 Spatial structuring and floral avoidance
behaviour present in ant-pollinator conflict in a Mexican ant-acacia Ecology 833086-
3096
35
Ramsey M 1995 Ant pollination of the perennial herb Blandfordia grandiflora (Liliaceae)
Oikos 74265ndash272
Renault CK LM Buffa and Delfino MA 2005 An aphid-ant interaction effects on
different trophic levels Ecological Research 2071-74
Rico-Gray V and L B Thien 1989 Effect of different ant species on reproductive fitness of
Schomburgkia tibicinis (Orchidaceae) Oecologia 81487ndash489
Romero G Q P A P Antiqueira and J Koricheva 2011 A meta-analysis of predation risk
effects on pollinator behaviour PLoS One 6e20689
Rosumek FB FAO Silveira F de S Neves NP de U Barbosa L Diniz Y Oki F Pezzini
et al 2009 Ants on plants a meta-analysis of the role of ants as plant biotic defenses
Oecologia 160537ndash549
Roubik D W 1982 The ecological impact of nectar-robbing bees and pollinating
hummingbirds on a tropical shrub Ecology 63354ndash360
Rudgers J A 2004 Enemies of herbivores can shape plant traits selection in a facultative ant-
plant mutualism Ecology 85192ndash205
Schatz B and WT Wcislo 1999 Ambush predation by the ponerine ant Ectatomma ruidum
Roger (Formicidae) on a sweat bee Lasioglossum umbripenne (Halictidae) in Panama
Journal of Insect Behavior 12641-663
36
Schaffer W M D W Zeh S L Buchmann S Kleinhaus M V Schaffer and J Antrim
1983 Competition for nectar between introduced honey bees and native North American
bees and ants Ecology 64564ndash577
Schemske D W 1980 The evolutionary significance of extrafloral nectar production by Costus
woodsonii (Zingiberaceae) an experimental analysis of ant protection Journal of Ecology
68959ndash967
Schiestl F P and F Glaser 2011 Specific ant-pollination in an alpine orchid and the role of
floral scent in attracting pollinating ants Alpine Botany 1221ndash9
Schubart HOR AB Anderson 1978 Why donrsquot ants visit flowers A response to D H
Janzen Biotropica 10310-311
Schuumlrch S M Pfunder and B A Roy 2000 Effects of ants on the reproductive success of
Euphorbia cyparissias and associated pathogenic rust fungi Oikos 16ndash12
Stanton ML Palmer TM Young TP Evans A and Turner ML 1999 Sterilization and
canopy modification of swollen thorn acacia tree by a plant-ant Nature 401 578-581
Stephenson AG 1981 Toxic nectar deters nectar thieves of Catalpa speciosa American
Midland Naturalist 105381-383
Stout J C and D Goulson 2001 The use of conspecific and interspecific scent marks by
foraging bumblebees and honeybees Animal Behaviour 62183ndash189
Sugiura S T Abe and S Makino 2006 Loss of extrafloral nectary on an oceanic island plant
and its consequences for herbivory American Journal of Botany 93491ndash495
37
Svensson L 1985 An estimate of pollen carryover by ants in a natural population of
Scleranthus perennis L (Caryophyllaceae) Oecologia 66373ndash377
Tsuji K A Hasyim and K Nakamura 2004 Asian weaver ants Oecophylla smaragdina and
their repelling of pollinators Ecological Research 19669ndash673
Turner R C J J Midgley P Barnard R E Simmons and S D Johnson 2012 Experimental
evidence for bird pollination and corolla damage by ants in the short-tubed flowers of
Erica halicacaba (Ericaceae) South African Journal of Botany 7925ndash31
Visser D M G Wright and J H Giliomee 1996 The effect of the Argentine ant
Linepithema humile (Mayr) (Hymenoptera Formicidae) on flower-visiting insects of
Protea nitida Mill (Proteaceae) African Entomology 4285ndash287
Wagner D 2000 Pollen viability reduction as a potential cost of ant association for Acacia
constricta (Fabaceae) American Journal of Botany 87711ndash715
Wagner D and A Kay 2002 Do extrafloral nectaries distract ants from visiting flowers An
experimental test of an overlooked hypothesis Evolutionary Ecology Research 4293ndash305
Wang C Y Luo Y Tai D An and Y Kou 2008 Ants pollinate Neottia listeroides
(Orchidaceae) in Sichuan China 46836ndash846
Wyatt R 1980 The impact of nectar-robbing ants on the pollination system of Asclepias
curassavica Bulletin of the Torrey Botanical Club 10724ndash28
Wheeler WM and IW Bailey 1920 The feeding habits of Pseudomymine and other ants
Transactions of the American Philosophical Society 22235-279
38
Willmer P G and S A Corbet 1981 Temporal and microclimatic partitioning of the floral
resources of Justicia aurea amongst a concourse of pollen vectors and nectar robbers
Oecologia 5167ndash78
Willmer P G and G N Stone 1997 How aggressive ant-guards assist seed-set in Acacia
flowers Nature 388165ndash167
Willmer P G C V Nuttman N E Raine G N Stone J G Pattrick K Henson P Stillman
et al 2009 Floral volatiles controlling ant behaviour Functional Ecology 23888ndash900
Young T P C H Stubblefield and L a Isbell 1997 Ants on swollen-thorn acacias species
coexistence in a simple system Oecologia 10998ndash107
Yu D W and N E Pierce 1998 A castration parasite of an ant-plant mutualism Proceedings
of the Royal Society B Biological Sciences 265375ndash382
39
Tables
Table 11 Studies of ant-flower or ant-pollinator interactions that included a measure of plant fitness Each study system is listed only
once except when results varied between years or when different studies examined male and female fitness Significant effects of ants on
plant fitness are either lsquopositiversquo or lsquonegativersquo non-significant effects are lsquoneutralrsquo When ants significantly affected only one fitness
component but several were measured (eg fruit set and seed production) we still consider the result significant
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Acacia
drepanolobium
Crematogaster
nigriceps
Proportion
fruiting
Negative Destruction of floral buds Young et al 1997
Asclepias exaltata Several Pollinia inserted
removed
Negative for pollinia
insertion neutral for
pollinia removal
Exploitative competition Wyatt 1980 T
40
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Asclepias syriaca Several
Fruit set pollinia
removed
Negative for fruit
set neutral for
pollinia removal
Exploitative competition Fritz and Morse
1981 T
Cordia nodosa Allomerus cf
demerarae
Fruit set Negative Destruction of floral buds Yu and Pierce
1998
Costus woodsonii Wasmannia
auropunctata
Seed set Positive Unknown ant pollination
suggested (but unlikely)
Schemske 1980
Eritrichum aretioides F neorufibarbis
gelida
Seed set Negative Ants damaged flowers Puterbaugh 1998
41
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Euphorbia characias Several Seed set Neutral Plants in Argentine ant-
invaded area set less seed
than those in un-invaded
sites but an ant-exclusion
experiment did not produce
significant results
Blancafort and
Gόmez 2005
Ferocactus wislizeni Solenopsis xyloni
and others
Seed set Unknown negative Interference and exploitative
competition S xyloni
reduced seed set relative to
other ants but the study did
not include a no-ant control
Ness 2006
42
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Frasera speciosa F neorufibarbis
and Formica sp
Seed set Neutral Nocturnal pollination
occurring when ants were
less active may have
compensated for reduced
diurnal pollinator visitation
Norment 1988
Gypsophila struthium Several Seeds per
inflorescence
Neutral No effect despite large
numbers of ants on flowers
Gόmez et al
1996
Hirtella physophora Allomerus
decemarticulatus
Fruit set Negative Destruction of flowers Orivel et al
2011
Humboldtia brunonis Crematogaster
dohrni
Fruit set Negative Destruction of flowers Gaume et al
2005
43
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Lepidium subulatum Several Percentage of
ovules setting
seed
Positive Unknown Gόmez et al
1996
Leucospermum
conocarpodendron
Several Seed set Neutral Site not pollen limited Lach 2008
Linaria vulgaris Several ldquoFemale fitness
indexrdquo
(combination of
number of seeds
fruits flowers
and seed weight)
Positive Reduction in florivory and
seed predation nectar
robbing holes increased ant
visitation
Newman and
Thomson 2005
44
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Melastoma
malabathricum
Oecophylla
smaragdina
Seed set Positive Ants deterred less efficient
pollinators increasing
visitation by the more
efficient pollinator
Gonzaacutelvez et al
2012
Metrosideros
polymorpha
Several Fruit set Neutral Ants reduced nectar
collection rates by foraging
bees but had no effect on
pollen collection rates or
fruit set
Junker et al
2010a
Orexis alpine F neorufibarbis
gelida
Seed set Neutral Autogamy or not pollen
limited
Puterbaugh 1988
45
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Ouratea spectabilis Cephalotes
pusillus and others
Seeds per fruit Neutral positive C pusillus had no effect on
seed number but other ants
collectively increased seed
set
Byk and Del
Claro 2010
Pachycereus schottii Several Seed set Neutral positive In one year ants showed a
positive effect (seed
number 2008) but no effect
with increased sample sizes
Chamberlain and
Holland 2008
Polemonium
viscosum
F neorufibarbis
gelida
Seed set Negative Ants damaged styles Galen 1983
46
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Polemonium
viscosum
F neorufibarbis
gelida
Pollen removal Neutral Ants did not affect pollen
removal even at high ant
densities
Galen and Geib
2007 T
Protea nitida Linepithema
humile and others
Seed set Neutral positive L humile had no effect on
P nitida seed set but native
ants increased it in one year
ants may cause pollinators
to relocate
Lach 2007
Psychotria
limonensis
Ectatomma spp Fruit set Positive Suggested increased
pollinator relocation
Altshuler 1999
47
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Roussea simplex Technomyrmex
albipes
Seed set Negative Interference and exploitative
competition
Hansen and
Muumlller 2009
Tristerix aphyllus Several Fruit set Neutral Nectar robbing ants
minimally reduced nectar
levels and had no effect on
hummingbird visitation
Caballero et al
2013
Vaccinium
reticulatum
Several Fruit set Neutral Ants reduced florivory but
did not affect fruit set
Bleil et al 2011
T Study measured male fitness
Study of sterilizing ants showed short-term decrease in female fitness long-term effects are unclear (see text)
48
Table 12 Studies demonstrating either i) ant pollination through an exclusion experiment or ii) that ants transfer pollen (or pollen
analogue) between flowers strongly suggesting ant pollination
Plant species Ant species Ant pollination Evidence Reference
Alyssum purpureum Several Yes Exclusion experiment Gόmez et al 1996
Arenaria tetraquetra Several Yes Exclusion experiment Gόmez et al 1996
Balanophora kuroiwai Leptothorax sp Suggested Exclusion experiment and
observed pollen transfer
and pollen tube formation
but did not see if plants set
seed
Kawakita and Kato 2002
Blandfordia grandiflora Iridomyrmex sp Yes Exclusion experiment Ramsey 1995
Borderea pyrenaica Several Yes Exclusion experiment Garciacutea et al 1995
49
Plant species Ant species Ant pollination Evidence Reference
Cytinus hypocistis Several Yes Exclusion experiment de Vega et al 2009
Epipactis thunbergii Camponotus
japonicas
Suggested Observed transfer of
pollinia from ants to
orchids
Sugiura et al 2006
Euphorbia cyparissias Several Yes Exclusion experiment Schuumlrch et al 2000
Fragaria virginiana Several Very likely Ant-only treatments set
comparable seed to flying
pollinator-only treatment
seed set increased with
increasing ant visitation
Ashman and King 2005
Frasera speciosa Several Yes Exclusion experiment Norment 1988
Frankenia thymifolia Several Yes Exclusion experiment Gόmez et al 1996
50
Plant species Ant species Ant pollination Evidence Reference
Hormathophylla spinosa Proformica
longiseta
Yes Exclusion experiment Gόmez and Zamora 1992
Jatropha curcas
Several
Tapinoma
melanocephalum
most common
Yes Exclusion experiment Luo et al 2012
Leporella fimbriata Myrmecia urens Yes Observed pollinia transfer
and subsequently
confirmed as ant
pollination
Peakall et al 1987
51
Plant species Ant species Ant pollination Evidence Reference
Lobularia maritima Several
Camponotus
micans most
common
Yes Exclusion experiment Gόmez 2000
Microtis parviflora
Iridomyrmex
gracilis
Yes Observation used
laboratory colonies to
replicate observations
Peakall and Beattie 1989
Naufraga balearica
Several
especially
Plagiolepis
pygmaea and
Lasius grandis
Suggested Observation over three
years of study only ants
were observed visiting
ants carried pollen
Cursach and Rita 2011
52
Plant species Ant species Ant pollination Evidence Reference
Neottia listeroides Leptothoras sp
Paratrechina sp
Suggested Observed transfer of
pollinia from ants to
orchids
Wang et al 2008
Paronychia pulvinata Formica
neorufibarbis
gelida
Yes Exclusion experiment Puterbaugh 1998
Polygonum cascadense Formica argentea
Very likely Grew plants in greenhouse
open to flying visitors with
minimal seed set only
plants with access to ants
set significant seed
Hickman 1974
53
Plant species Ant species Ant pollination Evidence Reference
Retama sphaerocarpa Several Yes Exclusion experiment Gόmez et al 1996
Scleranthus perennis
Several
especially
Formica
rufibarbis
Suggested Ants transferred pollen
analogue to stigmas and
carried pollen on bodies
Svensson 1985
Sedum anglicum Proformica
longiseta
Yes Exclusion experiment Gόmez et al 1996
Trinia glauca Several Yes Exclusion experiment Carvalheiro et al 2008
54
Chapter 2 Ants and ant scent reduce bumblebee pollination of artificial flowers
Cembrowski AR Tan MG Thomson JD and Frederickson ME
In press The American Naturalist
All authors designed the experiment ARC and MGT performed the experiment ARC and MEF
performed statistical analyse
s and ARC wrote the manuscript with input from all authors
Abstract
Ants on flowers can disrupt pollination by consuming rewards or harassing pollinators but it is
difficult to disentangle the effects of these exploitative and interference forms of competition on
pollinator behavior Using highly rewarding and quickly replenishing artificial flowers that
simulate male or female function we allowed bumblebees (Bombus impatiens) to forage (1) on
flowers with or without ants (Myrmica rubra) and (2) on flowers with or without ant scent cues
Bumblebees transferred significantly more pollen analogue both to and from ant free flowers
demonstrating that interference competition with ants is sufficient to modify pollinator foraging
behavior Bees also removed significantly less pollen analogue from ant scented flowers than
from controls making this the first study to show that bees can use ant scent to avoid harassment
at flowers Ant effects on pollinator behavior possibly in addition to their effects on pollen
viability may contribute to the evolution of floral traits minimizing ant visitation
55
Introduction
Trait-mediated indirect interactions arise when a focal species causes phenotypic changes
including behavioral modifications in a second species and these effects cascade to still other
species (Werner and Peacor 2003) Although trait-mediated indirect interactions are often studied
in food webs (eg Werner and Peacor 2003 Preisser et al 2005) they are not limited to trophic
interactions For example the threat of predation can change the behavior of a mutualist thus
affecting its partners (Suttle 2003) In animal-pollinated plants predators can disrupt pollination
directly through density-mediated indirect interactions (Dukas 2005) or via trait-mediated
indirect interactions by changing pollinator behavior When predators are present pollinators
may switch to visiting less rewarding flowers or they may avoid flowers altogether potentially
reducing the amount of pollen donated or received by a flower and thus decreasing plant fitness
(Gonccedilalves-Souza 2008 Ings and Chittka 2009) Similarly competition for floral rewards with
other flower visitors may cause behavioral changes in pollinators (Maloof and Inouye 2000
Ohashi et al 2008) Previous research has shown that bees may spend less time visiting flowers
depleted of rewards (Thomson 1986) and may avoid flowers bearing scent marks that indicate
recent visits by other individuals of the same or different bee species as these flowers are likely
to be depleted (Stout and Goulson 2002)
Whereas competition between pollinators may not be particularly costly for the plant
because both competitors are likely to provide pollination services competition between
pollinators and organisms that do not usually provide a pollination service such as ants has a
greater potential to reduce plant fitness Ants are common floral visitors they are attracted to
flowers for nectar (Lach 2007) and in some cases pollen (Byk and Del Claro 2010) and will
often defend these resources against other flower visitors (Altshuler 1999) However ants rarely
56
contribute much useful pollination antibiotic secretions present on their cuticles kill pollen
grains (Beattie et al 1985 Dutton and Frederickson 2012) and when foraging in flowers ants
sometimes impede female function by damaging stigmas (Galen and Cuba 2001) We will
henceforth consider ants to be non-pollinators and will contrast them to animals like bees that
are pollinators Ants are capable of competing with pollinators in two ways (1) by consuming
floral rewards and reducing their availability to pollinators (ie exploitative competition) and (2)
by directly antagonizing and excluding pollinators from flowers (ie interference competition)
Many plants benefit from the presence of ants numerous plant species recruit ants using
extrafloral nectar food bodies or domatia because ants provide protection against folivores (Heil
and McKey 2003) Similarly some plants have extrafloral nectaries on or near reproductive
tissues that recruit ants that deter florivores (eg Inouye and Taylor 1979) However plants
may incur reproductive costs as a result of their association with ants Some ant species that nest
in myrmecophytes sterilize flowers to shunt plant resources from reproduction to vegetative
growth allowing for greater ant colony growth (Frederickson 2009) Also ants visiting
extrafloral nectaries may be attracted to floral nectaries where they can harass pollinators (Ness
2006) There is growing evidence that many angiosperms have evolved floral traits that prevent
ants from accessing flowers during anthesis (Willmer and Stone 1997 Ballantyne and Willmer
2012) The effects of flower-visiting ants can be substantial Ants can decrease the frequency
duration or species diversity of pollinator visits to flowers (Lach 2008 Hansen and Muumlller 2009
Junker et al 2010 Gonzaacutelvez et al 2012) all of which potentially impact pollen donation and
receipt In some studies changes to pollinator behavior induced by ants have also been linked to
seed set
It is difficult however to disentangle the intertwined effects of exploitative and
interference competition in nature If ants disrupt pollination plants may often evolve traits to
57
deter ants from flowers (Willmer et al 2009) From a practical standpoint this would then limit
our ability to study the effects of ants on pollinator visitation because ants would not visit
flowers Here we used artificial flowers to explore ant-bee interactions in the absence of floral
defenses against ants We used highly rewarding quickly replenishing artificial flowers to
examine how direct harassment (ie interference competition expressed through behavior)
changes bee foraging behavior and how this affects donation and receipt of a pollen analogue
(powdered food dye) while minimizing the effects of exploitative competition We predicted
that flowers visited by ants would both donate and receive less pollen analogue than flowers
without ants because bees would avoid flowers with ants or leave them sooner Because scent
plays a large role in bumblebee communication and flower choice (Stout and Goulson 2002) we
also tested whether the presence or absence of ant scent on artificial flowers would affect pollen
analogue donation because of changes in the foraging behavior of bumblebees
Methods
i) Subjects
Myrmica rubra is an invasive ant in eastern North America with a range and habitat preferences
overlapping that of Bombus impatiens a common bumblebee Because M rubra visits flowers
(A Cembrowski pers obs) the two species likely interact in nature We collected 12 M rubra
colonies in the fall of 2011 and 2012 from Toronto Ontario Canada and the surrounding area
and maintained them in environmental chambers on artificial diet (Dussutour and Simpson 2009)
and a 1410 LD schedule (light 600-2200) We used these colonies as sources of M rubra
workers for experiments
58
Workers from commercially supplied colonies of Bombus impatiens (Biobest Canada
Ltd) foraged on artificial flowers in flight cages (either 24 x 24 x 21 m or 79 x 34 x 20m) at
the University of Toronto We tested a total of five bee colonies four colonies were used in the
ant presence trials and two of these and one additional colony were used in the ant scent trials
Flight cages had overhead fluorescent lights attached to timers In contrast to most previous
studies in which bees have been trained and tested individually the entire worker force of a bee
colony was free to forage at will in our experiments Colonies were trained to forage on artificial
flowers for at least four days before being used in trials After being used for a trial the colony
was not used for at least two days to reduce dye carryover between trials Between trials
bumblebees were fed pollen and given sugar water
ii) Artificial flowers
The flowers (Fig 1 Thomson et al 2012 see also Makino 2008) consisted of glass jars filled
with 30 WV sucrose solution Sugar water travelled by capillary action up a sewing-thread
wick to a hole in a blue-painted lid accumulating in a knot that acts as a nectary Flowers
depleted by visitors were quickly replenished via capillary action and were non-rewarding only
very briefly after visits taking less than a minute to accumulate 05 microl note that bumblebee
visits to a single flower are often separated by several minutes or more (A Cembrowski
unpublished data)
Unlike most previous artificial flower designs these flowers allow for estimation of male
and female fitness because we can measure both the amount and type of dye received by female
flowers (see (d) Dye quantification below) In order to access nectar on a ldquomalerdquo flower a bee
must crawl through a circular ldquoantherrdquo of brush-like weather stripping material dusted in a
59
consistent manner with our pollen analogue powdered food dye (FDampC 5 or 6) which is
transferred to the bee in the process On a ldquofemalerdquo flower a bee crawls through a sticky plastic
reinforcement that functions as a ldquostigmardquo receiving dye from the beersquos body Male flowers
dispensed dye particles over multiple bee visits and could still dispense dye at the end of trials
They resemble many real flowers or flower heads in that pollen is dispensed gradually over time
but less dye is available for transfer with each subsequent bee visit (Harder and Thomson 1989)
iii) Flight cage trials
We conducted 6 ant scent trials between 22 March and 4 June 2012 and the remaining trials (16
ant presence trials and 9 ant scent trials) between 9 November 2012 and 6 January 2013 Ant
presence trials lasted for eight hours and ant scent trials lasted for four hours Artificial flowers
were prepared and placed individually in small plastic containers treated with Fluon (Insect-a-
Slip BioQuip Products Inc) to prevent ants from escaping In ant presence trials we used 32
flowers (16 male and 16 female flowers) arranged in an eight-by-four array with flowers spaced
45 cm apart between rows and 30 cm within rows Ant scent trials used 20 flowers (10 male and
10 female flowers) in a four-by-five array with flowers spaced 30 cm apart between and within
rows In all of the ant presence trials and 9 of the ant scent trials we counted the number of bees
actively foraging after one hour to get a measure of colony activity
a) Ant presence trials
We examined the effects of ants on the amount of dye donated by male flowers and received by
female flowers in sixteen trials testing four bee colonies four times each The containers of eight
randomly chosen male flowers and eight randomly chosen female flowers received 15 M rubra
60
workers each the other 16 flowers remained free of ants Isolated from their colonies M rubra
workers have nowhere to deposit sugar water they collect and therefore may become sated
nonetheless a force of 15 ant workers was enough to maintain visitation to the nectaries where
bees foraged while still rarely having more than one or two ants visit the nectary at any time As
ants attacked bees bees would occasionally carry or throw attacking ants off flowers but despite
this some ants maintained their presence at the nectary throughout the trials Because ants the
size of M rubra consume liquids slowly (~017 ndash 024 microlmin Davidson et al 2004) flowers
replenished nectar more quickly than ants consumed it We used two colors of dye (FDampC 5 and
FDampC 6) to differentiate male flowers with and without ants In two trials for each bee colony
anthers of male flowers with ants were coated with FDampC 5 dye while brushes of male flowers
lacking ants were coated with FDampC 6 dye In the other two trials we reversed dye colors to
control for effects of dye type and color Dye color was assigned in a random order Artificial
flowers were placed in randomly assigned positions in the array before we opened the colony
and allowed the bees to begin foraging
b) Scent trials
We explored the effect of ant scent on dye donation in fifteen trials testing three bee colonies in
six five and four trials each Bees were first exposed to ants by allowing the bees to forage for
eight hours on twenty flowers (ten male and ten female) of which five male flowers had 15 ants
and all others had none No dye was used during this ldquoexposurerdquo day We then collected all but
the five ant-free male flowers in the flight cage leaving these flowers to keep bees foraging
Next we individually stored five new male lids in Fluon-treated containers with ten M rubra
workers Five control male lids were put in identical containers lacking ants The following
61
morning we removed the remaining flowers and set out ten new male and female flowers in a
random spatial arrangement using the lids having or lacking ant scent Thus the ant-scented
flowers were not in the same positions as the ant-visited flowers on which the bees were trained
Only five male flowers had ant scent and the other 15 flowers (five male and ten female) did not
We used the same two dye colors as in i) to differentiate male flowers with and without ant
scent and randomized which color was used for ant-scented flowers between trials We collected
and replaced stigmas from female flowers after one hour and collected the stigmas again after
four hours In the first trial performed we also collected stigmas after two hours but due to the
small amount of dye transferred this was not repeated
iv) Dye quantification
After each trial we quantified the amount of dye transferred to female flowers using a
spectrophotometer We removed the stigmas from female flowers placed them in test tubes
added 51 mL of distilled water to each tube and vortexed each tube thoroughly to ensure the
dye was evenly diluted These were diluted further as needed if the absorbance exceeded the
sensitivity range of the spectrophotometer In the first six scent trials each stigma was analyzed
separately and the dye amounts were summed to obtain a total amount of each dye color
transferred to female flowers in each trial In all the ant presence trials and the other nine scent
trials we opted to treat the experiment as the unit of replication Therefore we combined stigmas
from each treatment type (ant-visited or ant-free) in a test tube and measured the total amount of
each dye color transferred in each treatment in each trial
Because we put different dye colors on male flowers with and without ants we could use
the amount of each dye color donated to all female flowers to measure the reproductive success
62
of ant-visited and ant-free male flowers The total amount of dye (of both colors) received by
female flowers with and without ants was our measure of female reproductive success We
calculated the amount of each dye color in the sample by measuring absorbance at 428 or 486
nm and converting absorbance to micrograms following computational methods for overlapping
spectra (Blanco et al 1989)
v) Statistical analyses
In two trials (one ant presence and one ant scent) most female flowers received no dye because
of low bee activity so we excluded these trials from analyses Because dye reception and
donation values were non-normally distributed we square-root transformed the data before
examining the effects of ant presence or ant scent on dye transfer in ANCOVAs For ant
presence trials we included ant presence on male and female flowers and their interaction as
main effects and the total amount of dye transferred in each trial as a covariate to account for
the large variation in overall dye transfer among trials For ant scent trials we included ant scent
as the main effect time (one or four hours) as a repeated measure the interaction between scent
and time and the total amount of dye transferred as a covariate Covariate by treatment (ant
presence or ant scent) interactions were never significant and so were excluded in final analyses
All analyses were conducted in JMP (v 1000)
Results
Bees usually started foraging within minutes of the beginning of the trial and continued until
flowers were collected from the flight cage or the lights were extinguished An average of 34 plusmn
012 and 40 plusmn 015 (mean plusmn SE) bees were foraging after an hour in the ant presence and ant
63
scent trials respectively Male flowers with ants donated significantly less dye than male flowers
lacking ants (Fig 22a F155 = 4019 p lt 00001) Similarly female flowers with ants received
significantly less dye than female flowers lacking ants (Fig 22a F155 = 461 p = 0036) There
was no significant interaction between ant presence on male flowers and ant presence on female
flowers (F155 = 118 p = 028) Flowers with ant scent donated significantly less dye than
flowers without ant scent (Fig 22b F125 = 11216 p lt 00001) There was no significant effect
of time (F125 = 121 p = 028) or interaction between time and scent (F125 = 111 p = 030) in
the model
Discussion
In this study ants altered bumblebee pollination behavior and exhibited trait-mediated indirect
interactions with (artificial) flowers Through interference competition M rubra workers
significantly affected the pattern of dye transfer by B impatiens causing a preferential flow of
dye from male flowers lacking ants to female flowers lacking ants If manifested in nature such
effects would reduce the reproductive success of plants visited by ants through both male and
female function
Ants could have changed the attractiveness of artificial flowers in at least two ways First
as ants interrupted or altogether prevented bumblebees from foraging at nectaries bumblebees
may have learned to avoid flowers with ants due to the relative inefficiency of foraging at these
flowers something bumblebees take into account (Heinrich 2004) Second bees attempt to
minimize their risk of being attacked during foraging bouts which ants did to visiting bees Ants
often attacked or harassed bees by biting grasping and appearing to sting visiting bees
preventing them from accessing the nectary or reducing their time on flowers (see online video
64
1) and bees sometimes avoided ant-tended flowers entirely This harassment was sometimes
physically traumatic (online video 1) and bees often appeared to have trouble flying after being
attacked by ants Previous research has demonstrated that bumblebees leave or avoid flowers
where they have been harassed (Jones and Dornhaus 2011) and avoid foraging where there is
visual or olfactory evidence of a predator or predation event (Abbott 2006 Goodale and Nieh
2012) Though it is unclear if bumblebees viewed ants as competitors or predators they
responded similarly to flowers having ants as they do to flowers housing predators (Gonccedilalves-
Souza et al 2008)
To avoid artificial flowers with ants bees likely used a combination of visual and
olfactory cues Previous research has shown that bees respond to conspicuous predators or
predator ldquodummiesrdquo on flowers (Suttle 2003 Gonccedilalves-Souza et al 2008) decreasing the
frequency and duration of their visits to these flowers Thus bees may have been able to avoid
flowers harboring ants by sight alone However even in the absence of ants bees still
preferentially visited flowers lacking ant scents (Fig 22b) suggesting that they had learned to
associate ant scent with harassment Bees are adept at associative learning (Wright and Schiestl
2009) and can learn to recognize unique scents left behind by both conspecific and heterospecific
flower visitors (Stout et al 1998) Bees can use these various scents often arising from tarsal
gland deposits (Stout et al 1998) to recognize recently visited flowers that are less likely to be
profitable (Stout and Goulson 2002) Recently Ballantyne and Willmer (2012) demonstrated that
bees learn to associate ant scents with unrewarding artificial flowers and decrease their visitation
to these flowers Our results complement their findings by showing that bees can associate ant
scent with harassment at otherwise rewarding flowers
In our study ant scent caused bees to adjust their foraging strategy and decreased the
amount of pollen analogue that was donated by ant-scented flowers Thus the effects of
65
interference competition with ants on flowers can extend beyond immediate interactions and may
have fitness consequences for plants even when ants are absent Like other olfactory cues these
effects are likely transitory (Stout et al 1998) Although more dye was still donated by male
flowers lacking ant scent than those with ant scent the ratio of dye donated was on average
closer to equality in hours 2-4 than in the first hour (Fig 22b) This effect may be partially
driven by dye depletion of male flowers without ant scent but the lack of a strong corresponding
decrease in flowers with ant scent suggests that visitation patterns became more similar We did
not test whether B impatiensrsquo avoidance of ant scent was an innate or a learned behavior but
previous work has shown that Bombus terrestris does not innately avoid flowers with ant scent
(Ballantyne and Willmer 2012)
Ants may be necessary for plant survival and growth but can be costly for plant
reproduction The evolution of ant attractants such as extrafloral nectaries in some plant lineages
suggests that the costs of ants can be outweighed by their protective abilities However in this
study flowers visited by ants received and donated significantly less dye although they did
retain some sexual function The net benefit of having ants depends on whether ants increase
plant fitness by reducing herbivory more than they decrease plant fitness by disrupting
pollination Alternatively plants may actually benefit from the costs of ants to plant
reproduction ants that castrate flowers may be better defenders increasing plant survival or
vegetative growth when plants are young allowing for increased reproduction later in life when
the plant is colonized by less aggressive non-castrating ants (Frederickson 2009 Palmer et al
2010) In some plant species pollinator harassment by ants may even be beneficial Altshuler
(1999) reported that Ectatomma ants greatly increased fruit set of Psychotria limonensis despite
reductions in pollinator visitation rates presumably due to increases in the rate of pollen
outcrossing Similarly Gonzaacutelvez et al (2012) found that the presence of Oecophylla
66
smaragdina ants on Melastoma malabathricum flowers reduced visitation by less effective
pollinators and increased visitation by more effective Xylocopa bees increasing plant fitness
Cases of ants being beneficial to flowers appear to be the minority and many plant
species have traits that limit ant access to flowers Several studies have detailed floral volatiles
that are thought to mimic ant alarm pheromones and thus repel ants (Junker and Bluumlthgen 2008
Willmer et al 2009) Other plants use structural modifications such as narrow corollas or
slippery stems to limit ant access to flowers (reviewed in Willmer et al 2009) Exploitative
competition between ants and pollinators (Lach 2005 Ballantyne and Willmer 2012) as well as
the lethal effects of ant antibiotic secretions on pollen grains (Beattie et al 1985 Dutton and
Frederickson 2012) may have resulted in selection on plants to reduce ant visits to flowers Our
results suggest that trait-mediated indirect interactions resulting from interference competition
between ants and bees may favor plants that defend their flowers and their pollinators against
ants
Acknowledgements
We thank H Rusnock and S Meadley-Dunphy for assistance J Dix for help with flight cage
construction and Biobest for bumblebee colonies We are grateful to the Thomson and
Frederickson labs for guidance particularly T Makino funded by JSPS for helping to refine
artificial flowers J Ogilvie for help with bees K Prior for M rubra colony collections and K
Turner for study design suggestions The comments of the editor and two anonymous reviewers
prompted improvements in the article especially with regards to study design MEF and JDT
acknowledge NSERC Discovery Grant funding The University of Torontorsquos Faculty of Arts and
Science supported MGRTrsquos participation
67
68
References
Abbott K R 2006 Bumblebees avoid flowers containing evidence of past predation events
Canadian Journal of Zoology 841240-1247
Altshuler D L 1999 Novel interactions of non-pollinating ants with pollinators and fruit
consumers in a tropical forest Oecologia 119600-606
Ballantyne G and Willmer P 2012 Floral visitors and ant scent marks noticed but not used
Ecological Entomology 37402-409
Beattie A J Turnbull C Knox R B and Williams E G 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany
71421-426
Blanco M Runiaga H Maspoch S and Tarin P 1989 A simple method for
spectrophotometric determination of two-components with overlapped spectra Journal of
Chemical Education 66178-180
Byk J amp Del Claro K 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333-38
Davidson D W S C Cook and R R Snelling 2004 Liquid-feeding performances of ants
(Formicidae) ecological and evolutionary implications Oecologia 139255ndash66
Dukas R 2005 Bumble bee predators reduce pollinator density and plant fitness Ecology
861401-1406
Dussutour A and Simpson S J 2009 Communal nutrition in ants Current Biology 19740-
744
69
Dutton E M and Frederickson M E 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561-569
Frederickson ME 2009 Conflict over reproduction in an ant-plant symbiosis why Allomerus
octoarticulatus ants sterilize Cordia nodosa trees American Naturalist 173675-681
Galen C and Cuba J 2001 Down the tube pollinators predators and the evolution of flower
shape in the alpine skypilot Polemonium viscosum Evolution 551963-1971
Gonccedilalves-Souza T Omena P M Souza J C and Romero G Q 2008 Trait-mediated
effects on flowers artificial spiders deceive pollinators and decrease plant fitness Ecology
892407-2413
Gonzaacutelvez F G Santamariacutea L Corlett R T and Rodriacuteguez-Gironeacutes M A 2012 Flowers
attract weaver ants that deter less effective pollinators Journal of Ecology 10178ndash85
Goodale E and Nieh JC 2012 Public use of olfactory information associated with predation
in two species of social bees Animal Behaviour 84919-924
Harder LD and Thomson JD 1989 Evolutionary options for maximizing pollen dispersal of
animal-pollinated plants American Naturalist 133323-344
Heil M and McKey D 2003 Protective ant-plant interactions as model systems in ecological
and evolutionary research Annual Review of Ecology Evolution and Systematics 34425-
453
Heinrich B 2004 Bumblebee economics 2nd
edn Harvard University Press Cambridge
Ings T C and Chittka L 2009 Predator crypsis enhances behaviourally mediated indirect
effects on plants by altering bumblebee foraging preferences Proceedings of the Royal
Society B 2762031-2036
70
Inouye D W and Taylor O R Jr 1979 A temperate region plant-ant-seed predator system
consequences of extra floral nectar secretion by Helianthella quinquenervis Ecology 601-
7
Junker R R and Bluumlthgen N 2008 Floral scents repel potentially nectar-thieving ants
Evolutionary Ecology Research 10295ndash308
Junker R R Bleil R Daehler C C and Bluumlthgen N 2010 Intra-floral resource partitioning
between endemic and invasive flower visitors consequences for pollinator effectiveness
Ecological Entomology 35760ndash767
Jones E I and Dornhaus A 2011 Predation risk makes bees reject rewarding flowers and
reduce foraging activity Behavioral Ecology and Sociobiology 651505-1511
Lach L 2005 Interference and exploitative competition of three nectar-thieving invasive ant
species Insect Sociaux 52257-262
Lach L 2007 A mutualism with a native membracid facilitates pollinator displacement by
Argentine ants Ecology 881994-2004
Lach L 2008 Argentine ants displace floral arthropods in a biodiversity hotspot Diversity and
Distributions 14281-290
Makino T T and Sakai S 2007Experience changes pollinator responses to floral display size
from size-based to reward-based foraging Functional Ecology 21854863
Maloof J E and Inouye D W 2000 Are nectar robbers cheaters or mutualists Ecology
812651ndash2661
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506-514
Ohashi K Leslie A and Thomson JD 2008 Trapline foraging by bumble bees V Effects of
experience and priority on competitive performance Behavioral Ecology 19936-948
71
Palmer T M Doak D F Stanton M L Bronstein J L Kiers E T Young T P Goheen
J R and Pringle R M 2010 Synergy of multiple partners including freeloaders
increases host fitness in a multispecies mutualism Proceedings of the National Academy
of Sciences 10717234-17239
Preisser E L Bolnick D I and Benard M E 2005 Scared to death The effects of
intimidation and consumption in predator-prey interactions Ecology 86501-509
Stout J C Goulson D and Allen J A 1998 Repellent scent-marking of flowers by a guild of
foraging bumblebees (Bombus spp) Behavioral Ecology and Sociobiology 43317-326
Stout J and Goulson D 2002 The influence of nectar secretion rates on the responses of
bumblebees (Bombus spp) to previously visited flowers Behavioral Ecology and
Sociobiology 52239-246
Suttle K B 2003 Pollinators as mediators of top-down effects on plants Ecology Letters
6688-694
Thomson JD 1986 Pollen transport and deposition by bumble bees in Erythronium influences
of floral nectar and bee grooming Journal of Ecology 74329-341
Thomson JD Ogilvie J E Makino T T Arisz A Raju S Rojas-Luengas V and Tan M
G R 2012 Estimating pollination success with novel artificial flowers effects of nectar
concentration Journal of Pollination Ecology 9108-114
Tsuji K Hasyim A and Nakamura K 2004 Asian weaver ants Oecophylla smaragdina and
their repelling of pollinators Ecological Research 19669-673
Ulrich S and Bertsch A 1990 Do foraging bumblebees scent-mark food sources and does it
matter Oecologia 82137-144
Werner E E and Peacor S D 2003 A review of trait-mediated indirect interactions in
ecological communities Ecology 841083-1100
72
Willmer P G Nuttman C V Raine N E Stone G N Pattrick J G Henson K Stillman
P McIlroy L Potts S G and Knudsen J T 2009 Floral volatiles controlling ant
behaviour Functional Ecology 23888-900
Wright G A and Schiestl F P 2009 The evolution of floral scent the influence of olfactory
learning by insect pollinators on the honest signaling of floral rewards Functional Ecology
23841-851
73
Figures
Figure 21 Photograph of artificial flower lids A = anther L = lid N = nectary R = region
where anther or stigma is placed S = stigma T = thread W = weight
74
Figure 22 Dye transferred (mean plusmn 1SE) per trial by B impatiens workers A) from artificial
male and to artificial female flowers in the presence (unfilled circles) or absence (filled circles)
of Myrmica rubra workers and B) from artificial male flowers with (unfilled circles) or without
(filled circles) M rubra scent to ant-free female flowers in the 1st or the 2
nd to 4
th hours of the
trials
75
Chapter 3 Not just for the bees pollen consumption is common among tropical
ants
Cembrowski AR Reurink G and Frederickson ME
Planned submission to Biotropica
ARC and MEF designed the study ARC and GR did laboratory work ARC performed statistical
analyses and wrote the manuscript with input from other authors
Abstract
Although palynivory or pollen consumption is widespread among other hymenopterans there
are few accounts of palynivory in ants To quantify how often ants consume pollen we subjected
adult workers and larvae from 75 species of neo- and paleo-tropical ants to acetolysis a process
that destroys most organic material but leaves behind pollen grains In over half of the species
we examined ants contained a few pollen grains (lt 5) and ants of several Camponotus species
contained abundant pollen grains (gt50) We tested for an association between trophic level and
palynivory using stable nitrogen isotope ratios (δ15
N) but we did not find a significant
correlation we found pollen grains in lsquoherbivorousrsquo omnivorous and carnivorous ants We
suggest that our results indicate sporadic opportunistic pollen consumption by ants in tropical
forests
Introduction
For those organisms that can digest it efficiently pollen can be an excellent food source
Depending on the plant species pollen can contain up to 60 protein as well as lipids
76
carbohydrates and numerous trace nutrients (Roulston and Cane 2000) Pollen feeding
(palynivory) occurs in many taxa including birds mammals and arthropods (Roulston and Cane
2000) but it is best known among the Hymenoptera particularly bees Although adult bees feed
mainly on plant exudates (ie nectar) nearly all bee species rely on pollen as the chief protein
source for developing larvae (Willmer 2011) Closely related to bees ants have similar
nutritional requirements adult workers need carbohydrates whereas larvae are fed a diet rich in
protein (Dussutour and Simpson 2009) Ants also exploit nectar heavily and regularly visit
flowers (eg Herrera et al 1984 Bluumlthgen et al 2004) but unlike bees are not believed to
widely consume pollen Instead ants are thought to receive most of the protein in their diet from
predation or scavenging (eg Floren et al 2002)
However ants may not be as reliant on animal protein sources as commonly assumed In
tropical forests arboreal ants can have similar δ15
N ratios a measure of trophic level as
herbivores suggesting limited carnivory and substantial feeding on plant-derived protein sources
(Davidson et al 2003) One potential protein source is pollen This idea is supported by scattered
accounts of pollen consumption by ants from other ecosystems including deserts (Ness 2006)
boreal forests (Czechowski et al 2009) and tropical savannas (Byk and Del Claro 2010) One
ant genus Cephalotes is widely recognized to consume pollen (Creighton 1967 Baroni Urbani
and de Andrade 1997 Byk and Del Claro 2010) However beyond this genus our knowledge of
pollen consumption by ants has largely been limited to direct observations (but see Czechowski
et al 2011) and it is unknown if palynivory is truly rare among ants or simply under-reported
We borrowed a technique commonly used in palynology acetolysis to assess the frequency of
pollen consumption by ants in 75 neo- and paleo-tropical ant species This technique allowed us
to examine many more ant species than we could have observed directly
77
Methods
Adult worker ants were collected by hand at baits or from leaf litter using Winkler extractors
while larvae along with adults were collected by hand from nests Collections were made by L
Arcila Hernandez and J Sanders in the primary tropical rainforest surrounding the Centro de
Investigaciόn y Capacitaciόn Rio Los Amigos (CICRA 12deg34rsquoS 70deg05rsquoW) in Peru in 2010-
2011 and by E Youngerman from lowland primary and secondary rainforests in Madang
Province Papua New Guinea (PNG) in 2011-2012 We sorted ant samples to morphospecies and
then identified them to genus and when possible to species using existing keys which were
unavailable for PNG ants Ant samples used for acetolysis were whole adult workers guts from
adult workers and whole larvae
We used acetolysis to look for pollen grains inside ants In acetolysis organic material is
dissolved in acetic anhydride and sulfuric acid leaving behind stained pollen exines When
possible we used adult and larval ants of the same species from different colonies or bait
locations in an attempt to sample more representatively We prepared samples by first washing
them in 70 ethanol to remove any pollen present on the samplersquos outer surface before placing
them individually in micro-centrifuge tubes with ~05mL of a 91 mixture of acetic anhydride
sulfuric acid We then heated each sample at 95degC for 20min allowing tissues to dissolve Next
we centrifuged samples at 1000 RPM for 5min and decanted the liquid supernatant before
adding ~05mL of glacial acetic acid to the precipitate centrifuging again as above We decanted
the samples and added a drop of distilled water
78
We examined a 150μL sub-sample of this solution for pollen grains under a light
microscope Larvae gave less than 150μL of solution following acetolysis so we sampled the
entire amount We identified pollen grains by their staining and their surface architecture For
most species we examined at least three adult workers and three larvae but for some species
fewer than three adult workers or larvae were available (Tables 31 and 32)
We investigated the relationship between trophic level as measured by δ15
N ratios and
pollen consumption by ants Stable isotope (SI) data was available for some (n =23) of the
Peruvian species from a separate project in which collections overlapped Samples were stored in
EtOH and dried at 50degC overnight We used heads legs and thoraxes (but not gasters) of ants
for SI analysis Often single individuals did not provide enough material for analysis and we
combined multiple individuals to achieve necessary weights up to 10 for small ant species
Samples were analyzed at the Boston University Stable Isotope Laboratory using a Finnigan
Delta-S analyzer For other Peruvian species for which we had samples but not corresponding
stable isotope data (n =25) we took the median δ15
N ratio value of that speciesrsquo genus and
assigned it to the species in question Using pollen presenceabsence data for these 48 species
we used logistic regression to correlate pollen presence and δ15
N ratio using R (v 2151)
Results
We found pollen grains in 37 of the 197 adult workers 20 of the 102 larvae and 38 of the 75 ant
species we examined (Tables 31 and 32) The proportion of individuals containing pollen did
not differ significantly between larvae and adults (χ2
1 = 002 p = 088) The number of pollen
grains found was often low (median = 3) but highly variable (range 1-468) Also pollen
79
presence was inconsistent among conspecific ant samples of species that contained pollen 31
had pollen in only one of the larvae or adults examined In only three species (two Cephalotes
and one Myrmelachista) did all examined adults contain pollen
Highly lsquoherbivorousrsquo carnivorous and omnivorous ants contained pollen grains (Fig
31) There was no correlation between δ15
N ratio and pollen presence (χ2 = 150 df = 1 p =
022) In fact several carnivorous species contained pollen grains (eg Odontomachus sp 1
Pachycondola sp1)
Discussion
Pollen consumption by ants appears to be more common than suspected The majority of species
(51) had at least one individual that had consumed pollen and in 20 multiple individuals
contained pollen Numbers of grains found tended to be low however with most individuals
containing only a few grains Ants may use pollen as only a small portion of their diet exploiting
a host of other protein-containing foods more heavily including other low- δ15
N foods (eg
honeydew epiphylls Houmllldobler and Wilson 1990 Davidson et al 2003)
There are a few reasons why pollen may not be heavily exploited by ants Pollen requires
much post-consumption processing to extract its nutrients These nutrients are located in the
pollenrsquos cytoplasm which is protected from direct digestion by the exines and intines of the
grains Organisms must either break through these layers or cause the pollen grains to germinate
or pseudo-germinate to access and digest the cytoplasm (Roulston and Cane 2000) This can be
achieved by consuming a sugar-containing solution (such as nectar) along with pollen However
animals that are not adapted to feed on pollen may not be very efficient at this process only
80
extracting nutrients from some of the grains (Herrera and Martiacutenez del Rio 1998) Thus while
ants can likely get some nutrients from pollen other forms of more easily processed protein may
be more attractive
Pollen may also pose handling difficulties for ants Despite the large number of studies
that have recorded ants visiting flowers (Chapter 1) only a few papers detail ants harvesting
pollen directly from anthers Byk and Del Claro (2010) reported that Cephalotes pusillus ants
removed almost all of the available pollen from Ouratea spectabilis flowers and at least two
other studies describe ants consuming pollen along with nectar from flowers (Horskins and
Turner 1999 Ness 2006) Unlike bees which possess both morphological (eg specialized hairs
for holding pollen) and behavioral adaptations (eg stereotyped pollen-packing motions) ants
have no apparent mechanisms to increase the amount of pollen harvested and are likely limited
by the number of grains they can ingest or carry Rather than collect pollen directly from flowers
it appears more likely that ants collect it haphazardly in the environment leading to a smaller
number of pollen grains consumed at any one time It is known that at least Cephalotes ants
engage in such behavior Creighton (1967) observed Cephalotes texanus collecting pollen grains
trapped by Citrus x paradisi leaf hairs
There is a chance that the small numbers of pollen grains we observed came from
secondary pollen consumption (ie consuming a palynivorous organism) or consumption of
liquid containing pollen While we cannot rule this out we believe our results represent actual
targeted pollen consumption by ants Cephalotes ants known palynivores (Creighton 1967
Baroni Urbani and de Andrade 1997) contained similar numbers of pollen grains as many of the
pollen-containing ant species that we examined As well in at least once instance ants that we
found containing pollen grains were in the process of preying on non-pollen consuming
81
organisms (eg a Pachycondola species that was carrying a termite in its mandibles) or were
attracted by sugar baits
Contrary to our expectations there was no relationship between an ant speciesrsquo δ15
N ratio
and whether it had consumed pollen (Fig 32) We expected palynivory to be more common in
herbivorous ants because pollen presents a widespread source of protein a potentially limiting
resource The lack of pattern might reflect how available pollen grains are in the environment
While other forms of food may be preferable ants tend to be opportunistic foragers (Carroll and
Janzen 1973) and there is likely little cost associated with the consumption of scattered pollen
grains during foraging trips beyond the volume they occupy in the gut Some genera were found
to consistently consume pollen In support of previous observations (Baroni Urbani and de
Andrade 1997 Byk and Del Claro 2010) Cephalotes species were among the most common
pollen consumers Camponotus species also consistently contained pollen grains of the eight
species examined five contained pollen and pollen was found in species from both Peru and
PNG Camponotus species also contained the two largest numbers of pollen grains found (468
and 271) Taken together these results suggest that they may be another as yet unrecognized
commonly palynivorous genus
In general larvae did not contain large numbers of pollen grains (ranging from 1-38
grains) This is counter to what is seen in bees where larvae are the main recipients of pollen
(Willmer 2011) This difference may be because ant larvae are continually fed via trophallaxis
by adults throughout their development whereas bee larval cells are provisioned with pollen and
then sealed while larvae complete their development If both adults and larvae are able to extract
nutrients from pollen we would expect to see a preferential flow of nutrients from pollen to
larvae but not necessarily the grains themselves
82
Unlike palynivory by organisms that also provide pollination services pollen
consumption by ants is unlikely to benefit plants Ants rarely act as pollinators (Beattie et al
2004) They have pollen-killing antibiotic secretions on their cuticles (Dutton and Frederickson
2012) and they can compete with other flower visitors reducing flower visitation rates (Ness
2006) The negative impacts of flower-visiting ants are thought to have led to the evolution of
ant-repellent floral volatiles (reviewed in Willmer 2009) These volatiles can pre-empt
competition between ants and pollinators and may often be located within the pollen itself
(Willmer 2009) If floral volatiles truly are a defense against ant visitation our results suggest an
additional evolutionary pressure to not only prevent ant-pollinator interactions but to also
protect plant gametes from occasional consumption by a non-pollinating visitor
Acknowledgements
We would like to thank Jon Sanders Lina Arcila Hernandez and Eric Youngerman for providing
samples the Thomson lab for use of supplies the Pierce lab for funding support during JSrsquo and
EYrsquos collections and the staff of CICRA and the New Guinea Binatang Research Center for
logistical support The Peruvian Ministry of Agriculture provided permits (Nos 394-2009-AG-
DGFFS-DGEFFS and 299-2011-AG-DGFFS-DGEFFS) MEF acknowledges financial support
from an NSERC Discovery Grant a Connaught New Researcher Award an Ontario Ministry of
Economic Development and Innovation Early Researcher Award and the University of Toronto
ARC was supported by an Ontario Graduate Scholarship and Sigma Xi
83
References
Baroni Urbani C and M L de Andrade 1997 Pollen eating storing and spitting by ants
Naturwissenschaften 84256ndash258
Beattie A J C Turnbull R B Knox and E G Williams 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany 71421ndash
426
Bluumlthgen N G Gottsberger and K Fiedler 2004 Sugar and amino acid composition of ant-
attended nectar and honeydew sources from an Australian rainforest Austral Ecology
29418ndash429
Byk J and K Del-Claro 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333ndash38
Carroll C R and D H Janzen 1973 Ecology of foraging ants Annual Review of Ecology and
Systematics 4231ndash257
Creighton W S 1967 Studies on free colonies of Cryptocerus texanus Satschi (Hymenoptera
Formicidae) Psyche 7434ndash42
Czechowski W B Markό and A Radchenko 2009 Rubbish dumps reveal the diet of ant
colonies Myrmica schencki Em and Myrmica rubra (L) (Hymenoptera Formicidae) as
facultative pollen-eaters Polish Journal of Ecology 56737ndash741
Czechowski WB Markoacute K Erős and E Csata 2011 Pollenivory in ants (Hymenoptera
Formicidae) seems to be much more common than it was thought Annales Zoologici
61519ndash525
84
Dussutour A and S J Simpson 2009 Communal nutrition in ants Current Biology 19740ndash4
Dutton E M and M E Frederickson 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561ndash569
Floren A A Biun and K E Linsenmair 2002 Arboreal ants as key predators in tropical
lowland rainforest trees Oecologia 131137ndash144
Grant B R 1996 Pollen digestion by Darwinrsquos finches and its importance for early breeding
Ecology 77489-499
Herrera L G and C Martiacutenez del Rio 1998 Pollen digestion by new world bats effects of
processing time and feeding habits Ecology 792828ndash2838
Houmllldobler B and Wilson E O 1990 The Ants 1st Edition Harvard University Press
Cambridge
Horskins K and Turner V B 1999 Resource use and foraging patterns of honeybees Apis
mellifera and native insects on flowers of Eucalyptus costata Australian Journal of
Ecology 24221-227
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506ndash514
Roulston T H I and J H Cane 2000 Pollen nutritional content and digestibility for animals
Plant Systematics 222187ndash209
Van Tets I G 1997 Extraction of nutrients from Protea pollen by African rodents Belgian
Journal of Zoology 12759-65
Willmer P G 2011 Pollination and Floral Ecology 1st Edition Princeton University Press
New Jersey
85
Willmer P G C V Nuttman N E Raine G N Stone J G Pattrick K Henson P Stillman
et al 2009 Floral volatiles controlling ant behaviour Functional Ecology 23888ndash900
86
Tables
Table 31 Numbers of adult worker and larval ants examined for each species collected in Peru
Numbers of pollen grains found in individual samples are given in parentheses after the sample
size when there were no grains no numbers are given
Genus Species Number of adults analyzed
(numbers of grains found)
Number of larvae analyzed
(numbers of grains found)
Dolichoderinae
Azteca sp 1 3 0
sp 2 2 0
sp 3 0 2
sp 4 3 (1) 0
sp 5 3 (2) 0
Dolichoderus decollatus 3 (66) 0
sp 1 3 0
sp 2 3 1 (2)
sp 3 3 3
sp 4 0 2
Ecitoninae
Labidus sp 1 3 0
Formicinae
Camponotus sp 1 3 3
sp 2 3 (82) 1 (2)
sp 3 3 (3) 0
sp 4 0 2
87
sp 5 8 (11) 0
sp 6 4 (7 16 468) 0
sp 7 5 (12) 0
sp 8 2 (1) 0
Paratrechina sp 1 4 (11) 0
Myrmelachista sp 1 3 (5 11 18) 0
sp 2 0 3
Trachymyrmex sp 1 3 (21) 0
sp 2 3 0
Myrmicinae
Atta sp 1 3 0
Cephalotes atratus 3 (2 2 21) 0
placidus 3 (1 4 21) 0
sp 1 3 0
sp 2 1 0
sp 3 2 0
Crematogaster sp 1 3 1
sp 2 3 (35) 0
sp 3 9 (7) 0
sp 4 2 0
Megalomyrmex sp 1 3 0
sp 2 0 2
sp 3 4 (7) 0
sp 4 3 0
Monomorium sp 1 0 3 (2)
88
Pheidole sp 1 0 3
sp 2 3 0
sp 3 0 3
Procryptocerus sp 1 3 0
Solenopsis sp 1 0 3
sp 2 3 (3 5) 0
Wasmannia sp 1 3 0
Ponerinae
Pachycondola sp 1 3 (3) 0
Odontomachus sp 1 3 (34) 0
Pseudomyrmicinae
Pseudomyrmex sp 1 3 0
sp 2 3 0
sp 3 3 (9) 0
sp 4 0 3
sp 5 0 3
89
Table 32 Numbers of adult worker and larval ants examined for each species collected in Papua
New Guinea Numbers of pollen grains found in individual samples are given in parentheses
after the sample size when there were no grains no numbers are given
Genus Species Number adults analyzed
(grains found)
Number larvae analyzed
(grains found)
Aenictinae
Aenictus sp 1 3 3 (1 1)
Dolichoderinae
Philidris sp 1 3 1 (4)
Tapinoma sp 1 2 3 (38)
Technomyrmex sp 1 0 3 (12 12)
Ectatomminae
Rhytidoponera sp 1 1 3 (2)
Formicinae
Acropyga sp 1 3 3 (2)
sp 2 3 3
sp 3 3 3
Anoplolepsis sp 1 3 (1) 3 (1)
Calomyrmex sp 1 3 3 (1)
Camponotus sp 1 3 (1 271) 3
sp 2 1 3
sp 3 2 0
Nylanderia sp 1 3 3
Opisthopsis sp 1 3 3
Polyrachis sp 1 1 3 (4)
90
Myrmicinae
Crematogaster sp 1 3 3
sp 2 3 (32) 3
Monomorium sp 1 3 (1) 3 (1)
sp 2 3 3 (1 1 1)
Pheidole sp 1 3 3
Pristomyrmex sp 1 3 3 (11)
Pyramica sp 1 3 0
Ponerinae
Odontomachus sp 1 3 3 (4)
91
Figures
Figure 31 Photographs of pollen grains found in two ant species a) Dolichoderus sp 2 and b)
Camponotus sp 6 Scale bars = 50 microm
92
Figure 32 Relationship between trophic level as measured by δ15
N ratio and pollen presence
for 48 neotropical ant species 1 Atta sp 5 Azteca spp 8 Camponotus spp 5 Cephalotes spp 4
Crematogaster spp 5 Dolichoderus spp 1 Labidus sp 3 Megalomyrmex spp 1 Monomorium
sp 2 Myrmelachista spp 1 Odontomachus sp 1 Pachycondola sp 1 Paratrechina sp 3
Pheidole sp 4 Pseudomyrmex spp 2 Solenopsis spp and 1 Wasmannia sp
93
Concluding Remarks
Ants have traditionally been considered to be unwelcome flower visitors they consume floral
rewards ostensibly meant for pollinators while rarely providing pollination services themselves
In this thesis I investigate the validity of this assumption and use an experimental study and an
observational study to examine two ways that ants may impact plant reproduction
In the first chapter I review the literature on ants as flower visitors I show that the common
assumption of ants always being categorically negative for plant reproduction is incorrect while
ants may reduce floral attractiveness to pollinators the net effect of ants on plant fitness
measures is variable ranging from positive to negative to neutral I also discuss ants as
pollinators and ways that plants may minimize ant visitation to flowers before finishing with a
section highlighting questions that still need investigation
In the second chapter I explore how interference competition with ants can structure bumblebee
foraging behavior and how this may alter pollen movement patterns Using artificial flowers with
male or female function I allowed Bombus impatiens bumblebees to forage on flowers that
either did or did not have Myrmica rubra ants on them Ant presence significantly reduced the
amount of pollen analogue donated and received by flowers Building on these results I found
that the presence of ant scent alone elicited similar changes to bumblebee behavior reducing the
amount of pollen analogue donated by male flowers My results show that interference
competition is sufficient to alter bumblebee foraging behavior and that the effects of ants on
pollinators may extend after ant visitation to flowers Further study is needed to determine if bees
are able to differentiate between ant species by visual or olfactory cues Their ability to do so
94
could affect how generalized their response to ant presence is and may help to better predict how
ants will affect plant reproduction
In the final chapter I investigate how common palynivory is amongst tropical ant species I used
acetolysis and light microscopy to examine whole ants larvae and ant guts from Papua New
Guinea and Peru I found that tropical ants consistently contain pollen grains albeit often in
small numbers (lt5) However some genera notably Camponotus and Cephalotes consistently
contained higher numbers providing the first evidence that Camponotus may be a palynivorous
genus The small numbers of pollen grains present may indicate that ants preferentially exploit
other more easily digestible high-protein foods over pollen Future studies should investigate
exactly what role pollen plays in ant diets by supplementing the diet of captive ant colonies with
pollen This would allow us to examine how pollen consumption affects colony demography as
well as how efficiently ants digest pollen and whether ants exhibit pollen preferences
I bring up several pressing and unanswered questions within this thesis and highlight three here
First numerous plant species possess chemical or physical traits assumed to increase plant
fitness by reducing ant visitation However this has rarely been tested and instead research has
focused predominately on documenting ant repellence We should move away from this and
begin examining plant fitness in the presence or absence of these repellent traits to see how
beneficial these traits truly are Second how flower visiting ants impact male fitness has been
almost completely ignored in favor of female fitness Future studies should investigate how ant
visitation affects both pollen donation and pollen movement patterns Lastly research involving
antibiotics present on ant cuticles and their effect on pollen viability has been too small in scope
often limited to demonstrating the pollen killing effects of ant secretions Investigation into
95
whether plants have evolved any resistance to the secretions of the local ant community or why
some plants appear unaffected by their ant partners may yield exciting results
In conclusion ants have the capacity to strongly affect plant reproduction both positively and
negatively The fact that these diametrically opposing forces exist means we cannot approach
flower-visiting ants as either positive or negative a priori Instead we should strive to work
towards a better understanding of what is occurring in the system of interest and what role ants
play within it
96
Copyright Acknowledgments
Chapter 2 ldquoAnts and ant scent reduce bumblebee pollination of artificial flowersrdquo is current in
press in The American Naturalist Request for its inclusion here was sought and approved
2
Introduction
Ants and angiosperms share a long evolutionary history Ant diversification closely followed that
of flowering plants as ants took advantage of the large prey numbers that angiosperms supported
and the new habitats they provided (Moreau 2006) Subsequently numerous angiosperm
lineages evolved intimate associations with ants that function as an indirect or biotic form of
plant defense Food rewards such as extrafloral nectar or pearl bodies or housing in the form of
domatia attract ants to plants where they reduce herbivory and increase plant fitness (reviewed
in Rosumek et al 2009) However the traits that make ants good plant defenders particularly
their abundance attraction to sugar sources and aggressiveness can also cause problems for
plant reproduction Ants attracted by floral nectar can compete with pollinators for resources
and defend flowers potentially reducing plant fitness (Ness 2006 Junker et al 2007) That ants
negatively affect plant reproduction is an old and pervasive hypothesis Kerner (1878) called ants
ldquomost unwelcome guestsrdquo on flowers and more recent authors have similarly expressed that ant
visitation to flowers reduces plant reproductive success (Ness 2006 Lach 2007 Willmer et al
2009)
Nonetheless studies assessing the impact of flower-visiting ants on plant reproductive
success have produced mixed results Ants can have negative impacts on pollination and flower
function (Galen 1983 Puterbaugh 1998 Ness 2006 Hansen and Muumlller 2009) However other
studies have found no effect or even strong benefits of ant visits to flowers (Rico-Gray and
Thien 1989 Altshuler 1999 Schuumlrch 2000 Ashman and King 2005) This discrepancy between
ants being ldquounwelcome guestsrdquo and their actual effects on plant reproduction requires
clarification Our goal here is to highlight the ways that flower-visiting ants may affect plant
reproduction both positively and negatively and to discern through a review of the literature
3
what impacts ants have on plant reproductive success First we discuss mechanisms by which
flower-visiting ants can directly and indirectly impact plant reproduction both through floral
damage and by mediating changes in pollinator or florivore behavior We end this section by
evaluating published research that collectively shows that flower-visiting ants have
predominantly neutral effects on plant fitness measures Next we discuss pollination by ants
focusing on the ldquoantibiotic hypothesisrdquo before describing how plants may prevent ants from
visiting flowers Lastly we discuss promising new avenues of research and highlight work that
still needs to be performed to give a more complete understanding of ants as flower visitors
1 Ant interactions with flowers and floral visitors
i) Damage to flowers
Ants can directly disrupt plant reproduction by damaging floral structures preventing either the
fertilization of ovules or the presentation of pollen Rarely ants forage on floral tissue itself for
example Puterbaugh (1998) found that Formica neorufibarbus ants chewed through the coronal
ring of Eritrichium aretiodes flowers to get lipids More often but still not commonly foraging
ants chew through reproductive tissue to access floral nectar This can cause sterilization of
whole flowers (Echium plantagineum Kirk 1984) or affect female function only if just styles are
damaged effectively creating male flowers (Polemonium viscosum Galen 1983) Antibiotic
secretions on ant integuments may also directly damage pollen grains but the fitness effects of
this are unclear (see 2 below)
Several species of Crematogaster and Allomerus ants sterilize their host plants by
attacking floral buds (Yu and Pierce 1998 Stanton et al 1999 Gaume et al 2005 Maleacute et al
2012 Table 11) These ants nest in ldquoant-plantsrdquo or ldquomyrmecophytesrdquomdashie plants that host ant
4
colonies in specialized structures (domatia) The sterilizing ants do not appear to consume floral
tissues (Yu and Pierce 1998) and two hypotheses may explain why ants destroy floral buds First
ant colony size is often limited by domatia availability on their host plant By destroying floral
buds ants can cause plants to divert resources from reproductive to vegetative growth increasing
domatia number and allowing for larger ant colonies (Yu amp Pierce 1998 Frederickson 2009)
Second when plant-ants must defend their colony from takeovers by other colonies they may
benefit from limiting access to their host plant By destroying buds that could develop into
flowers branches or leaves ants can prevent their host plant from contacting nearby plants
occupied by enemy ant colonies (Stanton et al 1999)
The impact of sterilization on lifetime plant fitness is unclear Ants may destroy all of
their host plantrsquos floral buds (Yu and Pierce 1998) completely preventing reproduction or only a
portion of them (Maleacute et al 2012) allowing some seed set However counter-intuitively this
sterilization behavior may provide long-term fitness benefits to plants (Frederickson 2009
Palmer et al 2010) Ant-plants often host several different ant species over their lifetimes some
of which are non-sterilizing If hosting a sterilizing species at a certain life stage increases
survival or growth relative to hosting a non-sterilizing species at that life stage plants may
increase lifetime fitness by largely forgoing current reproduction in favor of increasing future
reproduction (Palmer et al 2010) Why plants invest resources in producing flowers that are
quickly destroyed by ants is not known but the timing of flower destruction may be important If
ants attack flowers after anthers dehisce plants may still realize some male fitness via pollen
donation (Yu and Pierce 1998) Additionally if the cost of flower production is relatively minor
and ants sometimes ldquomissrdquo flowers (Edwards and Yu 2008) attempting limited reproduction
may be a better strategy than forgoing it entirely
5
ii) Competition with pollinators
Ant activity on flowers can dramatically alter pollinator behavior Ants can reduce the species
diversity frequency or duration of pollinator visits to flowers (Norment 1988 Tsuji et al 2004
Ness 2006 Junker et al 2007 Lach 2007 2008b Romero et al 2011 and see Chapter 2) These
changes are largely due to either exploitative competition (ie consumption of shared resources)
or interference competition (ie direct aggression) between ants and pollinators
a) Exploitative competition
Pollinators and ants frequently compete for floral nectar Flower-visiting ants are often classified
as nectar thieves (sensu Inouye 1980) because they typically enter flowers through the
ldquolegitimaterdquo opening and consume nectar but transfer no pollen (Wyatt 1980 Ness 2006
Chamberlain and Holland 2008) Ants can also act as primary nectar robbers chewing holes
through corollas to reach nectaries (Willmer and Corbet 1981 Koopowitz and Marchant 1998)
although they are more commonly secondary nectar robbers and use holes made by previous
robbers (Newman and Thomson 2005)
Like other flower visitors foraging ants will often leave nectar behind in flowers This
amount can be less (Bleil et al 2011) more (Fritz and Morse 1982) or similar to (Lach 2008a)
amounts left by other visitors Often ants are morphologically constrained from fully exploiting
nectar Unlike bees and butterflies ants do not have proboscises and must get close to nectar to
consume it Narrow corollas or nectar spurs can prevent ant access to some or all floral nectar
(Schemske 1980 Galen and Cuba 2001 and see below) Smaller ants may be better able to
6
access nectar (Newman and Thomson 2005 Agarwal and Rastogi 2007) but can be limited by
their crop size in the amount of nectar they can remove (Lach 2005) If foraging ants leave
enough nectar behind for a pollinator to recover the cost of visiting a flower ants may have
minimal effects on plant reproductive success (Maloof and Inouye 2000 Turner et al 2012)
Differences among ant species in behavior and diet also affect their consumption of floral
nectar Species vary in how many flowers they visit per inflorescence or per plant and in how
many ants will visit a flower simultaneously (Lach 2005 Junker et al 2007 Lach 2013)
Invasive antsmdashthought to be better at exploiting and defending resources than native ants
(Holway et al 2002)mdashoften recruit to flowers in large numbers sometimes several dozen or
more (Visser et al 1999 Hansen and Muumlller 2009) Overall many factors may influence how
heavily ants exploit floral nectar on plants including ant density plant height the spatial or
temporal distribution of floral nectar and the availability of alternative food resources for ants
(Cushman and Addicott 1991 Lach 2013)
Rarely ants and pollinators may compete for pollen We know of only a few studies that
have recorded ants harvesting pollen directly from anthers (Horskins and Turner 1999 Ness
2006 Byk and Del-Claro 2010) Other studies have found pollen grains in ants (eg Baroni
Urbani and de Andrade 1997 and see Chapter 3) but this may not indicate competition with
pollinators ants sometimes collect stray pollen grains from the environment (Creighton 1967)
One New World genus Cephalotes might be specially adapted for pollen consumption They
possess ldquoproventricular shieldsrdquo structures that may restrict solid food to the crop allowing
nestmates to exchange pollen grains via trophallaxis It is unclear if this structure evolved for this
purpose but Cephalotes are the most frequently documented palynivorous ants (eg Creighton
1967 Baroni Urbani and de Andrade 1997 Byk and Del-Claro 2010) How efficiently ants
7
digest pollen and whether ants like bees feed it to larvae as a protein source is unknown At
least two studies have found that ant larvae consume pollen providing some support for this idea
(Wheeler and Bailey 1920 and Chapter 3)
b) Interference competition
Ants are relatively unique among flower visitors in defending floral resources from competitors
(but see Willmer and Corbett 1981 Roubik 1982) This defense ranges from chance encounters
with ants attacking visitors when flower occupancy overlaps (Ashman and King 2005 Junker et
al 2007) to actively patrolling inflorescences or constructing protective galleries around
nectaries (Horvitz and Schemske 1984 Gaume et al 2005 Hansen and Muumlller 2009)
Interference competition even in the absence of exploitative competition is sufficient to alter
pollinator behavior (Chapter 2)
The strength and outcome of interference competition depends on the identities of the ant
and the pollinator Pollinators might not alter their visitation patterns in response to non-
aggressive ants (Junker et al 2007) but more antagonistic ants may elicit avoidance behavior
before or soon after the visitor lands (Willmer and Corbet 1982 Altshuler 1999 Schuumlrch et al
2000 Table 11) Pollinator size may help predict their reaction to ants because ants may deter
only small-bodied pollinators (Gonzaacutelvez et al 2012) It is not clear whether flower visitors view
ants as aggressive competitors or as potential predators rarely ants prey on pollinators (Schatz
and Wcislo 1999) but in most cases ants merely cause pollinators to relocate
How well pollinators recognize and avoid ant-visited flowers is unresolved Collectively
pollinators have lower visitation rates to flowers visited by ants (Romero et al 2011) This is
8
dependent on pollinator identity however dipterans do not appear to discriminate between ant-
visited and ant-free flowers while bees can identify and avoid ant-visited flowers Bees likely
use a combination of visual and olfactory cues to recognize ant-visited flowers They can
identify previous conspecific and some heterospecific insect visitors by the hydrocarbon
ldquofootprintsrdquo they leave during foraging (Stout and Goulson 2001) Bumblebees can detect ant
hydrocarbons although this has been shown only in the laboratory (Ballantyne and Willmer
2012 Chapter 2) No study has yet examined if bees can use sight alone to detect ants but
several suggest it (Junker et al 2007 Gonzaacutelvez et al 2012) Some bees may discriminate
between ant species when visiting flowers (eg Lach 2008b) which follows if not all ant-
pollinator interactions are equally costly The ability of bees to do so will affect how generalized
their avoidance behaviors will be If they can discriminate between species bees may avoid
specific flowers visited by more ldquocostlyrdquo ants However if they cannot bees might either avoid
all ant-visited flowers or none depending on how negative the average interaction with an ant is
iii) Interactions with flower and pollinator antagonists
Ants can defend the flowers they visit Numerous plant species have circumfloral or bracteal
nectaries (ie extrafloral nectaries around flowers) and ants attracted to these nectaries can
reduce bud ovule or flower predation increasing plant fitness (Inouye and Taylor 1979 Rico-
Gray and Thien 1989 Sugiura et al 2006) Although the presence of EFNs (extrafloral nectaries)
so close to floral nectaries (sometimes less than 1 cm Ness 2006) might increase discovery and
exploitation of floral nectar by ants only one study has investigated this hypothesis finding no
relationship between the proximity of EFNs to floral nectaries and antsrsquo use of floral nectar
(Galen 2005) Holes made by nectar robbers are functionally analogous to circumfloral nectaries
9
and can also attract ants that defend flowers (Newman and Thomson 2005) and nectar-thieving
ants can similarly prevent florivory in some systems (Lach 2007 Bleil et al 2011)
Ants can also reduce the abundance of flower-dwelling predators or parasites of
pollinators Ambush predators such as crab spiders often forage at flowers where they may
reduce pollinator visitation rates and plant fitness (Dukas 2001 Gonccedilalvez-Souza et al 2008)
Ants can decrease spider abundance and richness on plants (Rudgers 2004 Renault et al 2005
Nahas et al 2012) and some evidence suggests that they can do the same on flowers (Lach
2008) In select cases ants may also reduce the abundance of pollinator parasitoids (Holland et
al 2011) Whether ants increase plant reproductive success by removing pollinator antagonists
has not yet been examined but should be If ants create lsquoenemy-freersquo flowers pollinators might
prefer to land on flowers with ants
iv) Net effects
Table 11 compiles studies in which the authors recorded ants consuming floral rewards or
interacting with pollinators and measured some aspect of plant fitness (eg fruit or seed set
pollen deposition) We included only a single study per plant species unless subsequent studies
found conflicting results or measured the fitness of another plant sex Records of ant pollination
are included in a separate table (Table 12)
Table 11 reveals relatively few examples of flower-visiting ants negatively affecting
plant fitness given how costly ant visitation is assumed to be The net effect of ants on plant
reproductive success is actually quite variable with similar numbers of studies finding positive
negative or neutral effects of ant presence Surprisingly when ants do have negative effects it is
10
usually because they directly damage floral structures and not because they prevent pollinators
from visiting flowers and successfully transferring pollen Why are ants not more often negative
for plant reproduction when this is commonly assumed to be true
First ants may compensate for reducing pollinator visitation rates by improving
pollination quality As pollinators move between flowers on a plant they often deposit self-
pollen on stigmas (ie geitonogamy) In some plants with late-acting self-incompatibility ovules
fertilized by self-pollen are subsequently aborted reducing the number of seeds that can be
produced (Barrett 2002 and references therein) Geitonogamy can also reduce offspring viability
or vigor (de Jong et al 1993) and cause pollen discounting a reduction in the amount of pollen
available for outcrossing because of self-pollination (Barrett 2002) By competing with
pollinators ants might decrease rates of geitonogamous pollination if pollinators then visit fewer
sequential flowers within a plant Ants might also cause pollinators to fly further between plants
potentially increasing the genetic diversity of pollen arriving at stigmas Although such effects
have been reported for other floral larcenists (Maloof 2001 Irwin 2003) to our knowledge these
hypotheses have not been investigated in relation to ants
Second ants and pollinators do not always forage for the same resources possibly
allowing them to partition floral rewards If ants forage for nectar but not pollen and flowers can
be pollinated efficiently by visitors looking to gather mostly pollen and little or no nectar then
plant reproduction will not be affected by ants consuming floral nectar Junker et al (2010a)
found that honeybees foraged for both pollen and nectar in the absence of ants but reduced their
foraging on nectar but not pollen when ants could access flowers resulting in no difference in
fruit set between treatments Whether pollinators commonly adjust their foraging behaviors in
response to ants warrants further research
11
Third ants are somewhat unique among floral larcenists in that they often provide direct
benefits to the plants they visit In the studies listed in Table 11 ants sometimes decreased
herbivory on plants (Chamberlain and Holland 2008 Bleil et al 2011) or otherwise aided the
plant (eg ant presence repelling smaller less effective pollinators Gonzaacutelvez et al 2012) If the
positive effects of ants cancel or outweigh the negative effects of flower-visiting ants on
pollinators then ant visitation may be neutral or even positive for plant fitness
Fourth ants may impose fitness costs to plants not captured in the studies in Table 11
Nectar robbers have stronger negative effects when plants are pollen-limited (Burkle et al 2007)
but only one study in Table 11 assessed whether flowers would set more seed if they received
more pollen finding no evidence of pollen limitation (Lach 2008a Table 11) Even if ants
decrease pollinator visitation rates there is limited scope for ants to decrease plant reproductive
success when plants are not pollen-limited Furthermore nearly all the studies in Table 11
measured only female fitness Only three quantified aspects of male fitness (Wyatt 1980 Fritz
and Morse 1981 Galen and Geib 2007) and we know of only one other study in a system in
which ants occasionally pollinate that has also measured male fitness (Ashman 2000) All four
studies measured pollen or pollinia removal rates and none found any effect of flower-visiting
ants on male fitness However whether other measures of male fitness (eg seeds sired pollen
deposition patterns) would reveal similar or different effects needs investigation
Finally although floral defenses against ants (see 3 below) may prevent us from
observing the negative effects of ants on plant reproductive success we consider this unlikely
Several studies have examined interactions between ants and plants that share little or no
coevolutionary history usually in the context of ant invasions Although invasive ants often
recruit to flowers in larger numbers than native ants they do not appear to reduce plant
12
reproductive success (Blancafort and Goacutemez 2005 Lach 2007 Lach 2008a but see Hansen and
Muumlller 2009) And in Hawaii where angiosperms evolved for millions of years in the absence of
ants and are thought to have secondarily lost floral defenses against ants (Junker et al 2011a)
there is no evidence that the preponderance of recently introduced flower-visiting ants negatively
affects plant fitness (Junker et al 2010a Bleil et al 2011)
2 Ants as pollinators
Ants may directly benefit plant reproduction by acting as pollinators but this is a relatively rare
occurrence Ants have long been considered poor pollinators (Kerner 1878) Initially this was
based on morphological and behavioral differences between ants and recognized pollinators
Worker ants are wingless and often relatively hairless making them poor pollen dispersers As
well because many ants exhibit Ortstreue (literally ldquoplace fidelityrdquo) ants were thought to return
to the same branch or flower repeatedly effecting no cross pollination (Houmllldobler and Wilson
1990) Though largely true these traits do not necessarily preclude ants from acting as
pollinators (Table 12) Although they commonly have restricted foraging ranges (Houmllldobler and
Wilson 1990) some ants can effect outcrossing (eg Ashman and King 2005 Carvalheiro 2008)
and many ants are as hairy if not hairier than their pollinating counterparts
In the late 1970s the ldquoantibiotic hypothesisrdquo was put forward to explain the rarity of ant
pollination Ants use antibiotic secretions to protect themselves against the fungal and bacterial
pathogens common in their nests and environs Iwanami and Iwadare (1978) found that one of
these compounds myrmicacin also greatly reduces pollen germination rates Subsequently
Beattie et al (1984 1985) found that short periods of contact (le 30 minutes) with an antrsquos
integument caused pollen grains to fail to germinate Some of these pollen-killing compounds
13
were traced back to the ant metapleural gland which produces hygienic secretions (Houmllldobler
and Wilson 1990) although contact with ants lacking metapleural glands can also reduce pollen
viability (Beattie et al 1985 Dutton and Frederickson 2012) Ant mandibular or anal gland
secretions and potentially ant venom may likewise defend ants against pathogens and have
similar effects on pollen germination (Beattie et al 1985 Graystock and Hughes 2011)
Nonetheless ant pollination has been documented repeatedly There are at least 18 cases
in which ant pollination has been shown experimentally and five others in which observations
strongly suggest it (Table 12) The majority of these plants fit the ldquoant pollination syndromerdquo
originally proposed by Hickman (1974) dense stands of short plants with small flowers and
easily accessible nectaries Typically ant-pollinated plants are associated with hot and dry
climates (principally in the Mediterranean) but dry alpine environments may also favor the
evolution of ant pollination (eg Svensson 1985 Puterbaugh 1998 Schiestl and Glaser 2011) In
most cases ants appear to be the sole or major pollinator of the plant in question (Hickman 1974
Peakall et al 1990 de Vega et al 2009) although ants sometimes make only supplementary
contributions to pollination (Schuumlrch et al 2000 Ashman and King 2005) This suggests that co-
evolution between ants and plants may not be necessary for ants to be useful pollinatorsmdashwhich
is surprising given the potential negative effects of ants on plant reproduction
Ant-pollinated plants cope with ant secretions in a variety of ways Several plant species
may simply tolerate pollen damage (Gόmez and Zamora 1992 de Vega et al 2009) apparently
possessing no overt adaptations to prevent it These plants might receive enough pollen to
overcome reduced pollen viability Other plants may place pollen on ant body parts thought to
have fewer secretions thereby potentially escaping damage (eg on ant legs Ramsey 1995) or
attach pollen to ants with specialized structures avoiding direct contact with the ant integument
14
(Peakall et al 1990) It is not known whether pollen evolves resistance to ant secretions or
whether plants deposit pollen on ants which have less disruptive secretions although these
possibilities have been suggested elsewhere (eg Garciacutea et al 1995 Ramsey 1995)
One key question regarding the effects of ant secretions on pollen grains urgently needs
to be resolved Several studies have suggested that the pollen-killing secretions of ants may have
contributed to the evolution of floral defenses against ants (eg Wagner 2000 Galen and Geib
2007) For this to be true ants must decrease male fitness by damaging pollen grains before they
leave flowers and are deposited on receptive stigmas Only two studies have investigated the
effects of ant secretions on pollen still in anthers (Wagner 2000 Galen and Butchard 2003) and
both constrained ants unnaturally to flowers for significantly longer than ants would normally
stay on flowers Although these studies found strong reductions in pollen vigor it is unclear if
ant secretions decrease male fitness under natural ant visitation conditions
3 Floral defenses against ants
Plants are not passive recipients of flower visitors and have some ability to ldquochooserdquo which
organisms can access floral rewards Flowers resist ants using both structural and chemical traits
More thorough investigation is needed to determine whether these traits evolved to prevent or
minimize the negative effects of ants on plant reproductive success (ie whether these traits can
truly be considered defenses against ants see 4ia below) It is unlikely however that ants
experience reciprocal selection to circumvent floral resistance traits because the fitness benefits
to ants of accessing flowers are probably small Thus there may be little scope for
coevolutionary arms races between floral defenses against ants and ant counter-adaptations
15
i) Structural resistance traits
a) Restricting ant access
Numerous plant species have physical structures preventing ant access to flowers Thin pedicels
can limit antsrsquo gripping abilities and make ants easier to dislodge when flowers are moved by
wind (Gaume et al 2005) and waxy secretions can render stems too slippery for ants to climb
(Harley 1991) Structures surrounding flowers such as water moats or dense hairs may similarly
keep ants from accessing floral rewards (Willmer et al 2009) Many such traits function as broad
filters and prevent not only ants but most crawling insects from reaching flowers Thus it is
difficult to determine whether ants florivores or both acted as selective agents favoring the
evolution of physical barriers to flowers
Flower shape may also reduce ant-pollinator conflict Long narrow corollas or nectar
spurs can prevent all but the smallest ants from accessing floral nectaries (Bluumlthgen et al 2004
Agarwal and Rastogi 2007) while still allowing long-tongued pollinators access to rewards
Selection to exclude nectar-thieving ants in such a manner however may be counteracted by
pollinator preferences for wider corollas that allow easier flower handling (Galen and Cuba
2001) Additionally these types of flowers may be more prone to robbery (Irwin and Maloof
2002) limiting their usefulness in preventing nectar larceny
b) Separation in space or time
Plants may separate floral rewards and ants temporally or spatially to minimize ant impacts on
plant reproduction Several authors have proposed that myrmecophytes have adapted to the
16
sterilization or otherwise detrimental activities of ants by locating domatia or food rewards on
different branches than flowers (Izzo and Vasconcelos 2002 Raine et al 2002 Edwards and Yu
2008) Temporal separation of ant and pollinator activities could likewise reduce conflict though
evidence is limited Some acacia flowers dehisce during periods of low ant activity (Willmer and
Stone 1997 Nicklen and Wagner 2006) but it is unclear if this is in response to ants
temperature or pollinator activity Timing of nectar secretion may similarly reduce ant-pollinator
competition If ants are active prior to pollinators they may deplete floral nectar and reduce
pollinator visitation rates (Schaffer et al 1984) However if nectar is secreted when ants are
inactive pollination may occur unimpeded (Norment 1988) This temporal separation could
favor pollinators that are active when ants are not (eg Wyatt and Morse 1981) but this is
poorly documented Further studies particularly focusing on flowers with the potential for
nocturnal and diurnal pollination may provide more examples
c) Extrafloral nectaries (EFNs)
One hypothesis put forward to explain the evolution of EFNs proposes that EFNs ldquodistractrdquo ants
from consuming floral nectar (Kerner 1878 Wagner and Kay 2002) Wagner and Kay (2002)
tested this hypothesis by constructing model plants using artificial nectaries assigned at random
to represent either floral or extrafloral nectaries They found that the presence of EFNs could
reduce ant exploitation of floral nectar However as highlighted by Galen (2005) this design is
closer to a plant making a surplus of flowers some of which will avoid ant visitation if visitation
rates increase sublinearly with increasing flower number Producing extra flowers to escape ant
visitation appears to be a viable strategy in some systems (Ashman and King 2005 Galen 2005)
However Galen (2005) found that applying honey (simulating extrafloral nectar) to P viscosum
17
leaves had the opposite effect and increased ant visits to flowers Only two studies both in
Pachycereus schottii have found evidence for the distraction hypothesis under field conditions
(Chamberlain and Holland 2008 Holland et al 2011)
There is limited empirical support for the distraction hypothesis but abundant evidence
that EFNs attract ants that defend plants against herbivores (reviewed in Rosumek et al 2009)
To function as attractants the presence of EFNs must increase ant abundance on plants
However because of the increased numbers attracted EFNs would not be expected to reduce the
abundance of ants on nectar rewards or be able to satiate the ants recruited Only if ant
abundance stops increasing at a reward density much lower than that available on the plant
would EFNs serve to distract ants In this case the amount of rewards may overwhelm ants
leaving some flowers underexploited However even in P schottii ants still visited flowers
(Chamberlain and Holland 2008) In cases where ant abundance would continue to increase with
additional rewards it is unlikely that EFNs function as distractions
ii) Chemical resistance traits
a) Toxic nectar
One of the earliest proposed floral defenses against ants was toxic nectar (Janzen 1977)
However studies testing Janzenrsquos (1977) hypothesis uncovered only a very few isolated
examples (eg Catalpa speciosa Stephenson 1981 Crinum erubescens Guerrant and Fiedler
1981) in short most floral nectar is not toxic or repellent to ants (Baker and Baker 1978
Schubart and Anderson 1978) In fact ants sometimes prefer floral nectar over solutions with
equivalent sugar concentrations (Koptur and Truong 1998)
18
In contrast floral nectar can repel other flower visitors (eg birds Johnson et al 2006
lepidopterans Kessler and Baldwin 2007) Repellency is often caused by plant secondary
metabolites which may occur in nectar as a by-product of their production elsewhere in the
plant where they function as defenses against herbivory (Adler 2001) Alternatively toxic or
repellent nectar may be adaptive repellent nectar may promote effective pollination by reducing
the amount of reward any one pollinator takes spreading nectar over several pollinator visits
(Kessler and Baldwin 2007) or it may function as a filter selecting for specific pollinators
(Johnson et al 2006) The latter may explain why repellent nectar is not more common
legitimate pollinators in addition to floral larcenists may be deterred from visiting flowers with
repellent nectar This may be particularly true of nectars that would repel ants which are closely
related to bees nectar distasteful to ants may often also be distasteful to bees and those nectars
acting as a filter for bees may still be palatable to ants For example Prŷs-Jones and Willmer
(1992) found that the ammonia-containing nectar of Lathraea clandestina is readily consumed
by both bees and ants even though it deters birds that act as nectar thieves Although floral
nectar itself rarely repels ants Guerrant and Fielder (1981) found that the presence of macerated
petals within nectar can often keep ants away Alkaloids in petals may be responsible and might
explain why ants seldom act as primary nectar robbers these secondary compounds may prevent
ants from chewing through corollas
b) Floral volatiles
Repellent floral volatiles appear to be a more widespread form of chemical resistance against
ants These were reviewed recently by Willmer et al (2009) and will be discussed here only
briefly Numerous plant taxa produce floral scents repellent to ants including myrmecophilous
19
myrmecophytic and non-ant associated plants in both temperate and tropical ecosystems
(Willmer et al 2009 Junker et al 2011a Ballantyne and Willmer 2012) In three large studies
floral volatiles that repel ants were detected in about a third of all plants tested (Willmer et al
2009 Junker et al 2011a Ballantyne and Willmer 2012) Pollen is often the source of ant-
repellent volatile compounds (Willmer et al 2009 Ballantyne and Willmer 2012) but they can
be emitted from other floral parts including petals That pollen is often the source of repellence
could suggest that preventing ant visitation may be more important in protecting male than
female function Additionally because repellence would decrease as pollen is removed from
anthers ants may return and defend flowers more quickly than if repellent chemicals were in
longer-lasting structures (eg petals) In some cases floral volatiles are thought to mimic ant
alarm pheromones (Willmer et al 2009) However floral scents elicit inconsistent reactions
across ant species ranging from repellence to attraction (Agarwal and Rastogi 2007 Ballantyne
and Willmer 2012)
Producing structural or chemical resistance traits may be costly for plants Native
Hawaiian plants which have little recent coevolutionary history with ants appear to have
secondarily lost floral ant repellents (Junker et al 2011a) Both Willmer et al (2009) and Junker
et al (2011a) found that highly chemical repellent flowers did not have strong physical defenses
and vice versa suggesting trade-offs between the two (but see Ballantyne and Willmer 2012)
Like toxic nectar floral ant repellents might sometimes deter both pollinators and ants For
example Galen et al (2011) found that 2-phenylethanol in P viscosum floral scent repels both
flower-damaging F neorufibarbis ants and the plantrsquos main pollinator Bombus kirbyellus
Interestingly four recent studies have found that floral scents can also attract ants
(Agarwal and Rastogi 2007 Edwards and Yu 2008 Schiestl and Glaser 2011 Gonzaacutelvez et al
20
2012) One study (Schiestl and Glaser 2011) has shown that floral scents attract ants to a
potentially ant-pollinated plant and such systems may provide many more examples
4 Future directions
We have highlighted areas in need of further investigation throughout the review Here we focus
on several questions we consider high priorities for future research
i) Floral defenses against ants
a) Do floral resistance traits increase plant fitness
The existence ant-repellent floral traits is often taken as evidence that ants negatively affect plant
reproductive success However despite the abundance and variety of mechanisms excluding ants
from flowers few studies show that ants reduce plant fitness when they visit flowers (Table 11)
This absence is particularly apparent with repellent floral volatiles which may be the most
widespread floral resistance trait (Willmer et al 2009 Junker et al 2011a Ballantyne and
Willmer 2012) To our knowledge only one study has shown that floral ant repellents benefit
plants with repellent P viscosum flowers avoiding damaging ant visits (Galen et al 2011)
Research is urgently needed to investigate if these traits increase plant fitness by excluding ants
or if ant visitation is inconsequential More studies taking advantage of standing variation in
traits (Galen et al 2011) or creating it artificially (eg Junker et al 2010b) will allow us to
distinguish between the two
21
b) If ant visitation is often not costly what caused the evolution of repellent traits and
what maintains them Similarly are these repellent traits truly defenses against ant
visitation or are they generalized defenses against a range of floral antagonists
Ant-repellent floral traits are widespread and their production may be costly (Willmer et al
2009 Junker et al 2011a) The studies in Table 11 however indicate that flower-visiting ants
often do not disrupt plant reproduction particularly outside of myrmecophytes What then
selected for these traits One possibility is that these repellents are not directed solely at ants
Instead a community of antagonistic flower visitors may exert diffuse selection on plants
thereby selecting for and maintaining these traits Structural modifications may repel crawling
insects while chemical repellents such as floral volatiles may repel both flying and non-flying
insects (Junker et al 2011b) How much overlap there is in floral volatiles that repel both ants and
other antagonists needs investigation Until we examine how specific these defenses are to ants
we risk overstating the importance of ants in their evolution
ii) Do ants alter pollen movement patterns or reduce male fitness
We are aware of only four studies that have measured if ant visitation impacts male fitness
(Wyatt 1980 Fritz and Morse 1981 Ashman 2000 Galen and Geib 2007) While none of these
studies found an effect all measured only amounts of pollen or pollinia removed However this
measure does not adequately encompass male fitness alterations to pollen movement patterns
and eventual deposition location may be more important to male function than changes in
removal rates If as suggested by some studies (Altshuler 1999 Lach 2007) ants increase
pollinator relocation rates they may decrease pollen discounting increasing male fitness
However if this increased relocation reduces overall pollen export rates ant competition with
22
pollinators may depress male function Studies using pollen-tracking methods such as fluorescent
dyes will allow us to see how ants change pollen movement patterns and methods like paternity
analysis will reveal how competition between ants and pollinators alters male fitness
iii) Coevolution between ants and plants do myrmecophytes exhibit stronger more
specific defenses against ants
The floral traits of myrmecophytes are most likely to coevolve with ants because all plant
reproduction occurs in the presence of ants Their floral defenses would thus be expected to be
both stronger and more specific to their resident ants than those other plants visited by an array
of more opportunistic ant species Some evidence supports this Willmer et al (2009) found that
resident ants of acacias were repelled by flowers with more aggressive ant species showing
stronger repellence than less aggressive ants Additionally ants not normally associated with
acacias did not respond to acacia floral volatiles More studies are needed in other systems and
should take advantage of phylogenetic approaches examining floral traits in closely related
myrmecophytic and non-myrmecophytic plant species to determine whether ants commonly
select for floral defenses against ants in myrmecophytes
Acknowledgements
For comments and discussion we thank the Frederickson lab particularly Eric Youngerman
Lina Arcila Hernandez and Kyle Turner as well as Alison Parker and Jordan Pleet MEF
acknowledges financial support from an NSERC Discovery Grant a Connaught New Researcher
23
Award an Ontario Ministry of Economic Development and Innovation Early Researcher Award
and the University of Toronto ARC was supported by an Ontario Graduate Scholarship and
Sigma Xi
24
References
Adler L S 2001 The ecological significance of toxic nectar Oikos 91409ndash420
Agarwal V M and N Rastogi 2007 Role of floral repellents in the regulation of flower visits
of extrafloral nectary-visiting ants in an Indian crop plant Ecological Entomology 3359ndash
65
Altshuler D L 1999 Novel interactions of non-pollinating ants with pollinators and fruit
consumers in a tropical forest Oecologia 119600ndash606
Ashman TL 2000 Pollinator selectivity and its implications for the evolution of dioecy and
sexual dimorphism Ecology 812577-2591
Ashman TL and E A King 2005 Are flower-visiting ants mutualists or antagonists A study
in a gynodioecious wild strawberry American Journal of Botany 92891ndash5
Ballantyne G and P Willmer 2012 Floral visitors and ant scent marks noticed but not used
Ecological Entomology 37402-409
Ballantyne G and P Willmer 2012 Nectar theft and floral ant-repellence a link between
nectar volume and ant-repellent traits PloS One 7e43869
Baker H G and I Baker 1978 Ants and flowers Biotropica 1080
Barrett S C H 2002 Sexual interference of the floral kind Heredity 88154ndash159
Baroni Urbani C and M L de Andrade 1997 Pollen eating storing and spitting by ants
Naturwissenschaften 84256ndash258
25
Beattie A J C Turnbull R B Knox and E G Williams 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany
71421ndash426
Beattie A J C Turnbull T Hough S Jobson and R B Knox 1985 The vulnerability of
pollen and fungal spores to ant secretions evidence and some evolutionary implication
American Journal of Botany 72606ndash614
Blancafort X and C Goacutemez 2005 Consequences of the Argentine ant Linepithema humile
(Mayr) invasion on pollination of Euphorbia characias (L) (Euphorbiaceae) Acta
Oecologica 2849ndash55
Bleil R N Bluumlthgen and R R Junker 2011 Ant-plant mutualism in Hawailsquoi Invasive ants
reduce flower parasitism but also exploit floral nectar of the endemic shrub Vaccinium
reticulatum (Ericaceae) Pacific Science 65291ndash300
Bluumlthgen N G Gottsberger and K Fiedler 2004 Sugar and amino acid composition of ant-
attended nectar and honeydew sources from an Australian rainforest Austral Ecology
29418ndash429
Burkle L A R E Irwin and D A Newman 2007 Predicting the effects of nectar robbing on
plant reproduction implications of pollen limitation and plant mating system American
Journal of Botany 941935ndash1943
Byk J and K Del-Claro 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333ndash38
26
Caballero P C G Ossa W L Gonzaacuteles C Gonzaacutelez-Browne G Astorga M M Muruacutea and
R Medel 2013 Testing non-additive effects of nectar-robbing ants and hummingbird
pollination on the reproductive success of a parasitic plant Plant Ecology 214633ndash640
Carvalheiro L G E R M Barbosa and J Memmott 2008 Pollinator networks alien species
and the conservation of rare plants Trinia glauca as a case study Journal of Applied
Ecology 451419ndash1427
Chamberlain S A and J N Holland 2008 Density-mediated context-dependent consumer-
resource interactions between ants and extrafloral nectar plants Ecology 891364ndash1374
Creighton W S 1967 Studies on free colonies of Cryptocerus texanus Satschi (Hymenoptera
Formicidae) Psyche 7434ndash42
Cursach J and J Rita 2011 Implications of the reproductive biology of the narrow endemic
Naufraga balearica (Apiaceae) for its conservation status Plant Systematics and Evolution
298581ndash596
Cushman JH and Addicott JF In Huxley CR and DF Cutler (eds) 1st edition Ant-plant
interactions Oxford University Press Oxford
de Jong T J N M Waser and P G Klinkhamer 1993 Geitonogamy the neglected side of
selfing Trends in Ecology and Evolution 8321ndash325
de Vega C M Arista P L Oritz C M Herrera and S Talavera 2009 The ant-pollination
system of Cytinus hypocistis (Cytinaceae) a Mediterranean root holoparasite Annals of
Botany 4517ndash8
27
Dukas R 2001 Effects of perceived danger on flower choice by bees Ecology Letters 4327ndash
333
Dutton E M and M E Frederickson 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561ndash569
Edwards D P and D W Yu 2008 Tolerating castration by hiding flowers in plain sight
Behavioral Ecology and Sociobiology 6395ndash102
Frederickson M E 2009 Conflict over reproduction in an ant-plant symbiosis why Allomerus
octoarticulatus ants sterilize Cordia nodosa trees The American Naturalist 173675ndash81
Fritz R S and D H Morse 1981 Nectar parasitism of Asclepias syriaca by ants effect on
nectar levels pollinia insertion pollinaria removal and pod production Oecologia 50316ndash
319
Galen C 1983 The effects of nectar thieving ants on seedset in floral scent morphs of
Polemonium viscosum Oikos 41245ndash249
Galen C 2005 Catching ants with honey an experimental test of distraction and satiation as
alternative modes of escape from flower-damaging ants Oecologia 14480ndash87
Galen C and B Butchart 2003 Ants in your plants effects of nectar-thieves on pollen fertility
and seed-siring capacity in the alpine wildflower Polemonium viscosum Oikos 101521ndash
528
Galen C and J Cuba 2001 Down the tube pollinators predators and the evolution of flower
shape in the alpine skypilot Polemonium viscosum Evolution 551963-1971
28
Galen C and J C Geib 2007 Density-dependent effects of ants on selection for bumble bee
pollination in Polemonium viscosum Ecology 881202ndash1209
Galen C R Kaczorowski S L Todd J Geib and R A Raguso 2011 Dosage-dependent
impacts of a floral volatile compound on pollinators larcenists and the potential for floral
evolution in the alpine skypilot Polemonium viscosum The American Naturalist 177258ndash
272
Garciacutea M B R J Antor and X Espadaler 1995 Ant pollination of the palaeoendemic
dioecious Borderea pyrenaica (Dioscoreaceae) Plant Systematics and Evolution 19817ndash
27
Gaume L M Zacharias and R M Borges 2005 Ant ndash plant conflicts and a novel case of
castration parasitism in a myrmecophyte Evolutionary Ecology Research 7435ndash452
Graystock P and W O H Hughes 2011 Disease resistance in a weaver ant Polyrhachis
dives and the role of antibiotic-producing glands Behavioral Ecology and Sociobiology
652319ndash2327
Gόmez J M 2000 Effectiveness of ants as pollinators of Lobularia maritima effects on main
sequential fitness components of the host plant Oecologia 12290ndash97
Gόmez J M and R Zamora 1992 Pollination by ants consequences of the quantitative effects
on a mutualistic system Oecologia 91410ndash418
Gόmez J M R Zamora J A Hόdar and D Garciacutea 1996 Experimental study of pollination
by ants in Mediterranean high mountain and arid habitats Oecologia 105236ndash242
29
Gonccedilalves-Souza T P M Omena J C Souza and G Q Romero 2008 Trait-mediated effects
on flowers artificial spiders deceive pollinators and decrease plant fitness Ecology
892407ndash2413
Gonzaacutelvez F G L Santamariacutea R T Corlett and M A Rodriacuteguez-Gironeacutes 2012 Flowers
attract weaver ants that deter less effective pollinators Journal of Ecology 10178ndash85
Guerrant E O and P L Fiedler 1981 Flower defenses against nectar-pilferage by ants
Biotropica 1325ndash33
Harley R 1991 The greasy pole syndrome In Huxley CR and DF Cutler (eds) 1st edition
Ant-plant interactions Oxford University Press Oxford
Hansen D M and C B Muumlller 2009 Invasive ants disrupt gecko pollination and seed
dispersal of the endangered plant Roussea simplex in Mauritius Biotropica 41202ndash208
Hickman J C 1974 Pollination by ants a low-energy system Science 1841290ndash1292
Holland J N S a Chamberlain and T E X Miller 2011 Consequences of ants and
extrafloral nectar for a pollinating seed-consuming mutualism ant satiation floral
distraction or plant defense Oikos 120381ndash388
Houmllldobler B and Wilson E O 1990 The Ants 1st Edition Harvard University Press
Cambridge
Holway DA L Lach AV Suarez ND Tsutsui and TJ Case 2002 The causes and
consequences of ant invasions Annual Review of Ecology and Systematics 33181-233
30
Horskins K and Turner VB 1999 Resource use and foraging patterns of honeybees Apis
mellifera and native insects on flowers of Eucalyptus costata Australian Journal of
Ecology 24221-227
Horvitz C C and D W Schemske 1984 Effects of ants and an ant-tended herbivore on seed
production of a neotropical herb Ecology 651369ndash1378
Inouye DW 1980 The terminology of floral larceny Ecology 611251-1253
Inouye DW and O R J Taylor 1979 A temperate region plant-ant-seed predator system
consequences of extra floral nectar secretion by Helianthella quinquenervis Ecology 601ndash
7
Irwin RE 2003 Impact of nectar robbing on estimates of pollen flow conceptual predictions
and empirical outcomes Ecology 84485-495
Irwin RE and Maloof JE 2002 Variation in nectar robbing over time space and species
Oecologia 133525-533
Iwanami Y and T Iwadare 1978 Inhibiting effects of myrmicacin on pollen growth and pollen
tube mitosis Botanical Gazette 13942ndash45
Izzo TJ and Vasconcelos HL 2002 Cheating the cheater domatia loss minimizes the effects
of ant castration in an Amazonian ant plant Oecologia 133200-205
Janzen D H 1977 Why donrsquot ants visit flowers Biotropica 9252
Johnson SD Hargreaves AL and Brown M 2006 Dark bitter-tasting nectar functions as a
filter of flower visitors in a bird-pollinated plant Ecology 872709-2716
31
Junker R A Y C Chung and N Bluumlthgen 2007 Interaction between flowers ants and
pollinators additional evidence for floral repellence against ants Ecological Research
22665ndash670
Junker R R R Bleil C C Daehler and N Bluumlthgen 2010a Intra-floral resource partitioning
between endemic and invasive flower visitors consequences for pollinator effectiveness
Ecological Entomology 35760ndash767
Junker R R and N Bluumlthgen 2010b Floral scents repel facultative flower visitors but attract
obligate ones Annals of botany 105777ndash82
Junker R R C C Daehler S Doetteri A Keller and N Bluumlthgen 2011a Hawaiian ant ndash
flower networks nectar-thieving ants prefer undefended native over introduced plants with
floral defenses Ecological Monographs 81295ndash311
Junker R R J Gershenzon and S B Unsicker 2011b Floral odor bouquet loses its ant
repellent properties after inhibition of terpene biosynthesis Journal of Chemical Ecology
371323ndash31
Kawakita A and M Kato 2002 Floral biology and unique pollination system of root
holoparasites Balanophora kuroiwai and B tobiracola (Balanophraceae) American
Journal of Botany 891164ndash1170
Koopowitz H and TA Marchant 1998 Postpollination nectar reabsorption in the African
epiphyte Aerangis verdickii (Orchidaceae) American Journal of Botany 85508-512
Kirk W D J 1984 Ecological studies on Thrips imaginis Bagnall (Thysanoptera) in flowers of
Echium plantagineum L in Australia Australian Journal of Ecology 99ndash18
32
Kerner A 1878 Flowers and their unbidden guests C Kegan Paul London
Kessler D and I T Baldwin 2007 Making sense of nectar scents the effects of nectar
secondary metabolites on floral visitors of Nicotiana attenuata The Plant Journal 49840ndash
54
Koptur S and N Truong 1998 Facultative ant-plant interactions nectar sugar preferences of
introduced pest species in South Florida Biotropica 30179-189
Lach L 2005 Interference and exploitation competition of three nectar-thieving invasive ant
species Insectes Sociaux 52257ndash262
Lach L 2007 A mutualism with a native membracid facilitates pollinator displacement by
Argentine ants Ecology 881994ndash2004
Lach L 2008a Argentine ants displace floral arthropods in a biodiversity hotspot Diversity and
Distributions 14281ndash290
Lach L 2008b Floral visitation patterns of two invasive ant species and their effects on other
hymenopteran visitors Ecological Entomology 33155ndash160
Lach L 2013 A comparison of floral resource exploitation by native and invasive Argentine
ants Arthropod-Plant Interactions 7177-190
Luo C W K Li X M Chen Z Y Huang and K U N Li 2012 Ants contribute significantly
to the pollination of a biodiesel plant Jatropha curcas Environmental Entomology
411163ndash1168
33
Maleacute PJ G C Leroy A Dejean A Quilichini and J Orivel 2011 An ant symbiont directly
and indirectly limits its host plantrsquos reproductive success Evolutionary Ecology 2655ndash63
Maloof J E 2001 The effects of a bumble bee nectar robber on plant reproductive success and
pollinator behavior American Journal of Botany 881960ndash1965
Maloof J E and D W Inouye 2000 Are nectar robbers cheaters or mutualists Ecology
812651ndash2661
Moreau CS CD Bell R Vila SB Archibald and NE Pierce 2006 Phylogeny of the ants
diversification in the age of the angiosperms Science 312101-104
Nahas L M O Gonzaga and K Del-Claro 2012 Emergent impacts of ant and spider
interactions herbivory reduction in a tropical savanna tree Biotropica 44498ndash505
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506ndash514
Newman D A and J D Thomson 2005 Interactions among nectar robbing floral herbivory
and ant protection in Linaria vulgaris Oikos 110497ndash506
Nicklen E F and D Wagner 2006 Conflict resolution in an ant-plant interaction Acacia
constricta traits reduce ant costs to reproduction Oecologia 14881ndash7
Norment C J 1988 The effect of nectar-thieving ants on the reproductive success of Frasera
speciosa (Gentianaceae) American Midland Naturalist 120331ndash336
34
Orivel J L Lambs P-J G Maleacute C Leroy J Grangier T Otto A Quilichini et al 2011
Dynamics of the association between a long-lived understory myrmecophyte and its
specific associated ants Oecologia 165369ndash76
Palmer T M D F Doak M L Stanton J L Bronstein E T Kiers T P Young J R
Goheen et al 2010 Synergy of multiple partners including freeloaders increases host
fitness in a multispecies mutualism Proceedings of the National Academy of Sciences
10717234ndash9
Peakall R A J Beattie and S H James 1987 Pseudocopulation of an orchid by male ants a
test of two hypotheses accounting for the rarity of ant pollination Oecologia 73522ndash524
Peakall R and A J Beattie 1989 Pollination of the orchid Microtis parviflora R Br by
flightless worker ants Functional Ecology 3515ndash522
Peakall R C J Angus A J Beattie 1990 The significance of ant and plant traits for ant
pollination in Leporella fimbriata pollination Oecologia 84457ndash460
Puterbaugh M N 1998 The roles of ants as flower visitors experimental analysis in three
alpine plant species Oikos 8336ndash46
Prŷs-Jones OE and Willmer PG 1992 The biology of alkaline nectar in purple toothwort
(Lathraea clandenstina) ground level defenses Biological Journal of the Linnean Society
45373-388
Raine NE Willmer P and Stone GN 2002 Spatial structuring and floral avoidance
behaviour present in ant-pollinator conflict in a Mexican ant-acacia Ecology 833086-
3096
35
Ramsey M 1995 Ant pollination of the perennial herb Blandfordia grandiflora (Liliaceae)
Oikos 74265ndash272
Renault CK LM Buffa and Delfino MA 2005 An aphid-ant interaction effects on
different trophic levels Ecological Research 2071-74
Rico-Gray V and L B Thien 1989 Effect of different ant species on reproductive fitness of
Schomburgkia tibicinis (Orchidaceae) Oecologia 81487ndash489
Romero G Q P A P Antiqueira and J Koricheva 2011 A meta-analysis of predation risk
effects on pollinator behaviour PLoS One 6e20689
Rosumek FB FAO Silveira F de S Neves NP de U Barbosa L Diniz Y Oki F Pezzini
et al 2009 Ants on plants a meta-analysis of the role of ants as plant biotic defenses
Oecologia 160537ndash549
Roubik D W 1982 The ecological impact of nectar-robbing bees and pollinating
hummingbirds on a tropical shrub Ecology 63354ndash360
Rudgers J A 2004 Enemies of herbivores can shape plant traits selection in a facultative ant-
plant mutualism Ecology 85192ndash205
Schatz B and WT Wcislo 1999 Ambush predation by the ponerine ant Ectatomma ruidum
Roger (Formicidae) on a sweat bee Lasioglossum umbripenne (Halictidae) in Panama
Journal of Insect Behavior 12641-663
36
Schaffer W M D W Zeh S L Buchmann S Kleinhaus M V Schaffer and J Antrim
1983 Competition for nectar between introduced honey bees and native North American
bees and ants Ecology 64564ndash577
Schemske D W 1980 The evolutionary significance of extrafloral nectar production by Costus
woodsonii (Zingiberaceae) an experimental analysis of ant protection Journal of Ecology
68959ndash967
Schiestl F P and F Glaser 2011 Specific ant-pollination in an alpine orchid and the role of
floral scent in attracting pollinating ants Alpine Botany 1221ndash9
Schubart HOR AB Anderson 1978 Why donrsquot ants visit flowers A response to D H
Janzen Biotropica 10310-311
Schuumlrch S M Pfunder and B A Roy 2000 Effects of ants on the reproductive success of
Euphorbia cyparissias and associated pathogenic rust fungi Oikos 16ndash12
Stanton ML Palmer TM Young TP Evans A and Turner ML 1999 Sterilization and
canopy modification of swollen thorn acacia tree by a plant-ant Nature 401 578-581
Stephenson AG 1981 Toxic nectar deters nectar thieves of Catalpa speciosa American
Midland Naturalist 105381-383
Stout J C and D Goulson 2001 The use of conspecific and interspecific scent marks by
foraging bumblebees and honeybees Animal Behaviour 62183ndash189
Sugiura S T Abe and S Makino 2006 Loss of extrafloral nectary on an oceanic island plant
and its consequences for herbivory American Journal of Botany 93491ndash495
37
Svensson L 1985 An estimate of pollen carryover by ants in a natural population of
Scleranthus perennis L (Caryophyllaceae) Oecologia 66373ndash377
Tsuji K A Hasyim and K Nakamura 2004 Asian weaver ants Oecophylla smaragdina and
their repelling of pollinators Ecological Research 19669ndash673
Turner R C J J Midgley P Barnard R E Simmons and S D Johnson 2012 Experimental
evidence for bird pollination and corolla damage by ants in the short-tubed flowers of
Erica halicacaba (Ericaceae) South African Journal of Botany 7925ndash31
Visser D M G Wright and J H Giliomee 1996 The effect of the Argentine ant
Linepithema humile (Mayr) (Hymenoptera Formicidae) on flower-visiting insects of
Protea nitida Mill (Proteaceae) African Entomology 4285ndash287
Wagner D 2000 Pollen viability reduction as a potential cost of ant association for Acacia
constricta (Fabaceae) American Journal of Botany 87711ndash715
Wagner D and A Kay 2002 Do extrafloral nectaries distract ants from visiting flowers An
experimental test of an overlooked hypothesis Evolutionary Ecology Research 4293ndash305
Wang C Y Luo Y Tai D An and Y Kou 2008 Ants pollinate Neottia listeroides
(Orchidaceae) in Sichuan China 46836ndash846
Wyatt R 1980 The impact of nectar-robbing ants on the pollination system of Asclepias
curassavica Bulletin of the Torrey Botanical Club 10724ndash28
Wheeler WM and IW Bailey 1920 The feeding habits of Pseudomymine and other ants
Transactions of the American Philosophical Society 22235-279
38
Willmer P G and S A Corbet 1981 Temporal and microclimatic partitioning of the floral
resources of Justicia aurea amongst a concourse of pollen vectors and nectar robbers
Oecologia 5167ndash78
Willmer P G and G N Stone 1997 How aggressive ant-guards assist seed-set in Acacia
flowers Nature 388165ndash167
Willmer P G C V Nuttman N E Raine G N Stone J G Pattrick K Henson P Stillman
et al 2009 Floral volatiles controlling ant behaviour Functional Ecology 23888ndash900
Young T P C H Stubblefield and L a Isbell 1997 Ants on swollen-thorn acacias species
coexistence in a simple system Oecologia 10998ndash107
Yu D W and N E Pierce 1998 A castration parasite of an ant-plant mutualism Proceedings
of the Royal Society B Biological Sciences 265375ndash382
39
Tables
Table 11 Studies of ant-flower or ant-pollinator interactions that included a measure of plant fitness Each study system is listed only
once except when results varied between years or when different studies examined male and female fitness Significant effects of ants on
plant fitness are either lsquopositiversquo or lsquonegativersquo non-significant effects are lsquoneutralrsquo When ants significantly affected only one fitness
component but several were measured (eg fruit set and seed production) we still consider the result significant
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Acacia
drepanolobium
Crematogaster
nigriceps
Proportion
fruiting
Negative Destruction of floral buds Young et al 1997
Asclepias exaltata Several Pollinia inserted
removed
Negative for pollinia
insertion neutral for
pollinia removal
Exploitative competition Wyatt 1980 T
40
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Asclepias syriaca Several
Fruit set pollinia
removed
Negative for fruit
set neutral for
pollinia removal
Exploitative competition Fritz and Morse
1981 T
Cordia nodosa Allomerus cf
demerarae
Fruit set Negative Destruction of floral buds Yu and Pierce
1998
Costus woodsonii Wasmannia
auropunctata
Seed set Positive Unknown ant pollination
suggested (but unlikely)
Schemske 1980
Eritrichum aretioides F neorufibarbis
gelida
Seed set Negative Ants damaged flowers Puterbaugh 1998
41
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Euphorbia characias Several Seed set Neutral Plants in Argentine ant-
invaded area set less seed
than those in un-invaded
sites but an ant-exclusion
experiment did not produce
significant results
Blancafort and
Gόmez 2005
Ferocactus wislizeni Solenopsis xyloni
and others
Seed set Unknown negative Interference and exploitative
competition S xyloni
reduced seed set relative to
other ants but the study did
not include a no-ant control
Ness 2006
42
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Frasera speciosa F neorufibarbis
and Formica sp
Seed set Neutral Nocturnal pollination
occurring when ants were
less active may have
compensated for reduced
diurnal pollinator visitation
Norment 1988
Gypsophila struthium Several Seeds per
inflorescence
Neutral No effect despite large
numbers of ants on flowers
Gόmez et al
1996
Hirtella physophora Allomerus
decemarticulatus
Fruit set Negative Destruction of flowers Orivel et al
2011
Humboldtia brunonis Crematogaster
dohrni
Fruit set Negative Destruction of flowers Gaume et al
2005
43
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Lepidium subulatum Several Percentage of
ovules setting
seed
Positive Unknown Gόmez et al
1996
Leucospermum
conocarpodendron
Several Seed set Neutral Site not pollen limited Lach 2008
Linaria vulgaris Several ldquoFemale fitness
indexrdquo
(combination of
number of seeds
fruits flowers
and seed weight)
Positive Reduction in florivory and
seed predation nectar
robbing holes increased ant
visitation
Newman and
Thomson 2005
44
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Melastoma
malabathricum
Oecophylla
smaragdina
Seed set Positive Ants deterred less efficient
pollinators increasing
visitation by the more
efficient pollinator
Gonzaacutelvez et al
2012
Metrosideros
polymorpha
Several Fruit set Neutral Ants reduced nectar
collection rates by foraging
bees but had no effect on
pollen collection rates or
fruit set
Junker et al
2010a
Orexis alpine F neorufibarbis
gelida
Seed set Neutral Autogamy or not pollen
limited
Puterbaugh 1988
45
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Ouratea spectabilis Cephalotes
pusillus and others
Seeds per fruit Neutral positive C pusillus had no effect on
seed number but other ants
collectively increased seed
set
Byk and Del
Claro 2010
Pachycereus schottii Several Seed set Neutral positive In one year ants showed a
positive effect (seed
number 2008) but no effect
with increased sample sizes
Chamberlain and
Holland 2008
Polemonium
viscosum
F neorufibarbis
gelida
Seed set Negative Ants damaged styles Galen 1983
46
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Polemonium
viscosum
F neorufibarbis
gelida
Pollen removal Neutral Ants did not affect pollen
removal even at high ant
densities
Galen and Geib
2007 T
Protea nitida Linepithema
humile and others
Seed set Neutral positive L humile had no effect on
P nitida seed set but native
ants increased it in one year
ants may cause pollinators
to relocate
Lach 2007
Psychotria
limonensis
Ectatomma spp Fruit set Positive Suggested increased
pollinator relocation
Altshuler 1999
47
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Roussea simplex Technomyrmex
albipes
Seed set Negative Interference and exploitative
competition
Hansen and
Muumlller 2009
Tristerix aphyllus Several Fruit set Neutral Nectar robbing ants
minimally reduced nectar
levels and had no effect on
hummingbird visitation
Caballero et al
2013
Vaccinium
reticulatum
Several Fruit set Neutral Ants reduced florivory but
did not affect fruit set
Bleil et al 2011
T Study measured male fitness
Study of sterilizing ants showed short-term decrease in female fitness long-term effects are unclear (see text)
48
Table 12 Studies demonstrating either i) ant pollination through an exclusion experiment or ii) that ants transfer pollen (or pollen
analogue) between flowers strongly suggesting ant pollination
Plant species Ant species Ant pollination Evidence Reference
Alyssum purpureum Several Yes Exclusion experiment Gόmez et al 1996
Arenaria tetraquetra Several Yes Exclusion experiment Gόmez et al 1996
Balanophora kuroiwai Leptothorax sp Suggested Exclusion experiment and
observed pollen transfer
and pollen tube formation
but did not see if plants set
seed
Kawakita and Kato 2002
Blandfordia grandiflora Iridomyrmex sp Yes Exclusion experiment Ramsey 1995
Borderea pyrenaica Several Yes Exclusion experiment Garciacutea et al 1995
49
Plant species Ant species Ant pollination Evidence Reference
Cytinus hypocistis Several Yes Exclusion experiment de Vega et al 2009
Epipactis thunbergii Camponotus
japonicas
Suggested Observed transfer of
pollinia from ants to
orchids
Sugiura et al 2006
Euphorbia cyparissias Several Yes Exclusion experiment Schuumlrch et al 2000
Fragaria virginiana Several Very likely Ant-only treatments set
comparable seed to flying
pollinator-only treatment
seed set increased with
increasing ant visitation
Ashman and King 2005
Frasera speciosa Several Yes Exclusion experiment Norment 1988
Frankenia thymifolia Several Yes Exclusion experiment Gόmez et al 1996
50
Plant species Ant species Ant pollination Evidence Reference
Hormathophylla spinosa Proformica
longiseta
Yes Exclusion experiment Gόmez and Zamora 1992
Jatropha curcas
Several
Tapinoma
melanocephalum
most common
Yes Exclusion experiment Luo et al 2012
Leporella fimbriata Myrmecia urens Yes Observed pollinia transfer
and subsequently
confirmed as ant
pollination
Peakall et al 1987
51
Plant species Ant species Ant pollination Evidence Reference
Lobularia maritima Several
Camponotus
micans most
common
Yes Exclusion experiment Gόmez 2000
Microtis parviflora
Iridomyrmex
gracilis
Yes Observation used
laboratory colonies to
replicate observations
Peakall and Beattie 1989
Naufraga balearica
Several
especially
Plagiolepis
pygmaea and
Lasius grandis
Suggested Observation over three
years of study only ants
were observed visiting
ants carried pollen
Cursach and Rita 2011
52
Plant species Ant species Ant pollination Evidence Reference
Neottia listeroides Leptothoras sp
Paratrechina sp
Suggested Observed transfer of
pollinia from ants to
orchids
Wang et al 2008
Paronychia pulvinata Formica
neorufibarbis
gelida
Yes Exclusion experiment Puterbaugh 1998
Polygonum cascadense Formica argentea
Very likely Grew plants in greenhouse
open to flying visitors with
minimal seed set only
plants with access to ants
set significant seed
Hickman 1974
53
Plant species Ant species Ant pollination Evidence Reference
Retama sphaerocarpa Several Yes Exclusion experiment Gόmez et al 1996
Scleranthus perennis
Several
especially
Formica
rufibarbis
Suggested Ants transferred pollen
analogue to stigmas and
carried pollen on bodies
Svensson 1985
Sedum anglicum Proformica
longiseta
Yes Exclusion experiment Gόmez et al 1996
Trinia glauca Several Yes Exclusion experiment Carvalheiro et al 2008
54
Chapter 2 Ants and ant scent reduce bumblebee pollination of artificial flowers
Cembrowski AR Tan MG Thomson JD and Frederickson ME
In press The American Naturalist
All authors designed the experiment ARC and MGT performed the experiment ARC and MEF
performed statistical analyse
s and ARC wrote the manuscript with input from all authors
Abstract
Ants on flowers can disrupt pollination by consuming rewards or harassing pollinators but it is
difficult to disentangle the effects of these exploitative and interference forms of competition on
pollinator behavior Using highly rewarding and quickly replenishing artificial flowers that
simulate male or female function we allowed bumblebees (Bombus impatiens) to forage (1) on
flowers with or without ants (Myrmica rubra) and (2) on flowers with or without ant scent cues
Bumblebees transferred significantly more pollen analogue both to and from ant free flowers
demonstrating that interference competition with ants is sufficient to modify pollinator foraging
behavior Bees also removed significantly less pollen analogue from ant scented flowers than
from controls making this the first study to show that bees can use ant scent to avoid harassment
at flowers Ant effects on pollinator behavior possibly in addition to their effects on pollen
viability may contribute to the evolution of floral traits minimizing ant visitation
55
Introduction
Trait-mediated indirect interactions arise when a focal species causes phenotypic changes
including behavioral modifications in a second species and these effects cascade to still other
species (Werner and Peacor 2003) Although trait-mediated indirect interactions are often studied
in food webs (eg Werner and Peacor 2003 Preisser et al 2005) they are not limited to trophic
interactions For example the threat of predation can change the behavior of a mutualist thus
affecting its partners (Suttle 2003) In animal-pollinated plants predators can disrupt pollination
directly through density-mediated indirect interactions (Dukas 2005) or via trait-mediated
indirect interactions by changing pollinator behavior When predators are present pollinators
may switch to visiting less rewarding flowers or they may avoid flowers altogether potentially
reducing the amount of pollen donated or received by a flower and thus decreasing plant fitness
(Gonccedilalves-Souza 2008 Ings and Chittka 2009) Similarly competition for floral rewards with
other flower visitors may cause behavioral changes in pollinators (Maloof and Inouye 2000
Ohashi et al 2008) Previous research has shown that bees may spend less time visiting flowers
depleted of rewards (Thomson 1986) and may avoid flowers bearing scent marks that indicate
recent visits by other individuals of the same or different bee species as these flowers are likely
to be depleted (Stout and Goulson 2002)
Whereas competition between pollinators may not be particularly costly for the plant
because both competitors are likely to provide pollination services competition between
pollinators and organisms that do not usually provide a pollination service such as ants has a
greater potential to reduce plant fitness Ants are common floral visitors they are attracted to
flowers for nectar (Lach 2007) and in some cases pollen (Byk and Del Claro 2010) and will
often defend these resources against other flower visitors (Altshuler 1999) However ants rarely
56
contribute much useful pollination antibiotic secretions present on their cuticles kill pollen
grains (Beattie et al 1985 Dutton and Frederickson 2012) and when foraging in flowers ants
sometimes impede female function by damaging stigmas (Galen and Cuba 2001) We will
henceforth consider ants to be non-pollinators and will contrast them to animals like bees that
are pollinators Ants are capable of competing with pollinators in two ways (1) by consuming
floral rewards and reducing their availability to pollinators (ie exploitative competition) and (2)
by directly antagonizing and excluding pollinators from flowers (ie interference competition)
Many plants benefit from the presence of ants numerous plant species recruit ants using
extrafloral nectar food bodies or domatia because ants provide protection against folivores (Heil
and McKey 2003) Similarly some plants have extrafloral nectaries on or near reproductive
tissues that recruit ants that deter florivores (eg Inouye and Taylor 1979) However plants
may incur reproductive costs as a result of their association with ants Some ant species that nest
in myrmecophytes sterilize flowers to shunt plant resources from reproduction to vegetative
growth allowing for greater ant colony growth (Frederickson 2009) Also ants visiting
extrafloral nectaries may be attracted to floral nectaries where they can harass pollinators (Ness
2006) There is growing evidence that many angiosperms have evolved floral traits that prevent
ants from accessing flowers during anthesis (Willmer and Stone 1997 Ballantyne and Willmer
2012) The effects of flower-visiting ants can be substantial Ants can decrease the frequency
duration or species diversity of pollinator visits to flowers (Lach 2008 Hansen and Muumlller 2009
Junker et al 2010 Gonzaacutelvez et al 2012) all of which potentially impact pollen donation and
receipt In some studies changes to pollinator behavior induced by ants have also been linked to
seed set
It is difficult however to disentangle the intertwined effects of exploitative and
interference competition in nature If ants disrupt pollination plants may often evolve traits to
57
deter ants from flowers (Willmer et al 2009) From a practical standpoint this would then limit
our ability to study the effects of ants on pollinator visitation because ants would not visit
flowers Here we used artificial flowers to explore ant-bee interactions in the absence of floral
defenses against ants We used highly rewarding quickly replenishing artificial flowers to
examine how direct harassment (ie interference competition expressed through behavior)
changes bee foraging behavior and how this affects donation and receipt of a pollen analogue
(powdered food dye) while minimizing the effects of exploitative competition We predicted
that flowers visited by ants would both donate and receive less pollen analogue than flowers
without ants because bees would avoid flowers with ants or leave them sooner Because scent
plays a large role in bumblebee communication and flower choice (Stout and Goulson 2002) we
also tested whether the presence or absence of ant scent on artificial flowers would affect pollen
analogue donation because of changes in the foraging behavior of bumblebees
Methods
i) Subjects
Myrmica rubra is an invasive ant in eastern North America with a range and habitat preferences
overlapping that of Bombus impatiens a common bumblebee Because M rubra visits flowers
(A Cembrowski pers obs) the two species likely interact in nature We collected 12 M rubra
colonies in the fall of 2011 and 2012 from Toronto Ontario Canada and the surrounding area
and maintained them in environmental chambers on artificial diet (Dussutour and Simpson 2009)
and a 1410 LD schedule (light 600-2200) We used these colonies as sources of M rubra
workers for experiments
58
Workers from commercially supplied colonies of Bombus impatiens (Biobest Canada
Ltd) foraged on artificial flowers in flight cages (either 24 x 24 x 21 m or 79 x 34 x 20m) at
the University of Toronto We tested a total of five bee colonies four colonies were used in the
ant presence trials and two of these and one additional colony were used in the ant scent trials
Flight cages had overhead fluorescent lights attached to timers In contrast to most previous
studies in which bees have been trained and tested individually the entire worker force of a bee
colony was free to forage at will in our experiments Colonies were trained to forage on artificial
flowers for at least four days before being used in trials After being used for a trial the colony
was not used for at least two days to reduce dye carryover between trials Between trials
bumblebees were fed pollen and given sugar water
ii) Artificial flowers
The flowers (Fig 1 Thomson et al 2012 see also Makino 2008) consisted of glass jars filled
with 30 WV sucrose solution Sugar water travelled by capillary action up a sewing-thread
wick to a hole in a blue-painted lid accumulating in a knot that acts as a nectary Flowers
depleted by visitors were quickly replenished via capillary action and were non-rewarding only
very briefly after visits taking less than a minute to accumulate 05 microl note that bumblebee
visits to a single flower are often separated by several minutes or more (A Cembrowski
unpublished data)
Unlike most previous artificial flower designs these flowers allow for estimation of male
and female fitness because we can measure both the amount and type of dye received by female
flowers (see (d) Dye quantification below) In order to access nectar on a ldquomalerdquo flower a bee
must crawl through a circular ldquoantherrdquo of brush-like weather stripping material dusted in a
59
consistent manner with our pollen analogue powdered food dye (FDampC 5 or 6) which is
transferred to the bee in the process On a ldquofemalerdquo flower a bee crawls through a sticky plastic
reinforcement that functions as a ldquostigmardquo receiving dye from the beersquos body Male flowers
dispensed dye particles over multiple bee visits and could still dispense dye at the end of trials
They resemble many real flowers or flower heads in that pollen is dispensed gradually over time
but less dye is available for transfer with each subsequent bee visit (Harder and Thomson 1989)
iii) Flight cage trials
We conducted 6 ant scent trials between 22 March and 4 June 2012 and the remaining trials (16
ant presence trials and 9 ant scent trials) between 9 November 2012 and 6 January 2013 Ant
presence trials lasted for eight hours and ant scent trials lasted for four hours Artificial flowers
were prepared and placed individually in small plastic containers treated with Fluon (Insect-a-
Slip BioQuip Products Inc) to prevent ants from escaping In ant presence trials we used 32
flowers (16 male and 16 female flowers) arranged in an eight-by-four array with flowers spaced
45 cm apart between rows and 30 cm within rows Ant scent trials used 20 flowers (10 male and
10 female flowers) in a four-by-five array with flowers spaced 30 cm apart between and within
rows In all of the ant presence trials and 9 of the ant scent trials we counted the number of bees
actively foraging after one hour to get a measure of colony activity
a) Ant presence trials
We examined the effects of ants on the amount of dye donated by male flowers and received by
female flowers in sixteen trials testing four bee colonies four times each The containers of eight
randomly chosen male flowers and eight randomly chosen female flowers received 15 M rubra
60
workers each the other 16 flowers remained free of ants Isolated from their colonies M rubra
workers have nowhere to deposit sugar water they collect and therefore may become sated
nonetheless a force of 15 ant workers was enough to maintain visitation to the nectaries where
bees foraged while still rarely having more than one or two ants visit the nectary at any time As
ants attacked bees bees would occasionally carry or throw attacking ants off flowers but despite
this some ants maintained their presence at the nectary throughout the trials Because ants the
size of M rubra consume liquids slowly (~017 ndash 024 microlmin Davidson et al 2004) flowers
replenished nectar more quickly than ants consumed it We used two colors of dye (FDampC 5 and
FDampC 6) to differentiate male flowers with and without ants In two trials for each bee colony
anthers of male flowers with ants were coated with FDampC 5 dye while brushes of male flowers
lacking ants were coated with FDampC 6 dye In the other two trials we reversed dye colors to
control for effects of dye type and color Dye color was assigned in a random order Artificial
flowers were placed in randomly assigned positions in the array before we opened the colony
and allowed the bees to begin foraging
b) Scent trials
We explored the effect of ant scent on dye donation in fifteen trials testing three bee colonies in
six five and four trials each Bees were first exposed to ants by allowing the bees to forage for
eight hours on twenty flowers (ten male and ten female) of which five male flowers had 15 ants
and all others had none No dye was used during this ldquoexposurerdquo day We then collected all but
the five ant-free male flowers in the flight cage leaving these flowers to keep bees foraging
Next we individually stored five new male lids in Fluon-treated containers with ten M rubra
workers Five control male lids were put in identical containers lacking ants The following
61
morning we removed the remaining flowers and set out ten new male and female flowers in a
random spatial arrangement using the lids having or lacking ant scent Thus the ant-scented
flowers were not in the same positions as the ant-visited flowers on which the bees were trained
Only five male flowers had ant scent and the other 15 flowers (five male and ten female) did not
We used the same two dye colors as in i) to differentiate male flowers with and without ant
scent and randomized which color was used for ant-scented flowers between trials We collected
and replaced stigmas from female flowers after one hour and collected the stigmas again after
four hours In the first trial performed we also collected stigmas after two hours but due to the
small amount of dye transferred this was not repeated
iv) Dye quantification
After each trial we quantified the amount of dye transferred to female flowers using a
spectrophotometer We removed the stigmas from female flowers placed them in test tubes
added 51 mL of distilled water to each tube and vortexed each tube thoroughly to ensure the
dye was evenly diluted These were diluted further as needed if the absorbance exceeded the
sensitivity range of the spectrophotometer In the first six scent trials each stigma was analyzed
separately and the dye amounts were summed to obtain a total amount of each dye color
transferred to female flowers in each trial In all the ant presence trials and the other nine scent
trials we opted to treat the experiment as the unit of replication Therefore we combined stigmas
from each treatment type (ant-visited or ant-free) in a test tube and measured the total amount of
each dye color transferred in each treatment in each trial
Because we put different dye colors on male flowers with and without ants we could use
the amount of each dye color donated to all female flowers to measure the reproductive success
62
of ant-visited and ant-free male flowers The total amount of dye (of both colors) received by
female flowers with and without ants was our measure of female reproductive success We
calculated the amount of each dye color in the sample by measuring absorbance at 428 or 486
nm and converting absorbance to micrograms following computational methods for overlapping
spectra (Blanco et al 1989)
v) Statistical analyses
In two trials (one ant presence and one ant scent) most female flowers received no dye because
of low bee activity so we excluded these trials from analyses Because dye reception and
donation values were non-normally distributed we square-root transformed the data before
examining the effects of ant presence or ant scent on dye transfer in ANCOVAs For ant
presence trials we included ant presence on male and female flowers and their interaction as
main effects and the total amount of dye transferred in each trial as a covariate to account for
the large variation in overall dye transfer among trials For ant scent trials we included ant scent
as the main effect time (one or four hours) as a repeated measure the interaction between scent
and time and the total amount of dye transferred as a covariate Covariate by treatment (ant
presence or ant scent) interactions were never significant and so were excluded in final analyses
All analyses were conducted in JMP (v 1000)
Results
Bees usually started foraging within minutes of the beginning of the trial and continued until
flowers were collected from the flight cage or the lights were extinguished An average of 34 plusmn
012 and 40 plusmn 015 (mean plusmn SE) bees were foraging after an hour in the ant presence and ant
63
scent trials respectively Male flowers with ants donated significantly less dye than male flowers
lacking ants (Fig 22a F155 = 4019 p lt 00001) Similarly female flowers with ants received
significantly less dye than female flowers lacking ants (Fig 22a F155 = 461 p = 0036) There
was no significant interaction between ant presence on male flowers and ant presence on female
flowers (F155 = 118 p = 028) Flowers with ant scent donated significantly less dye than
flowers without ant scent (Fig 22b F125 = 11216 p lt 00001) There was no significant effect
of time (F125 = 121 p = 028) or interaction between time and scent (F125 = 111 p = 030) in
the model
Discussion
In this study ants altered bumblebee pollination behavior and exhibited trait-mediated indirect
interactions with (artificial) flowers Through interference competition M rubra workers
significantly affected the pattern of dye transfer by B impatiens causing a preferential flow of
dye from male flowers lacking ants to female flowers lacking ants If manifested in nature such
effects would reduce the reproductive success of plants visited by ants through both male and
female function
Ants could have changed the attractiveness of artificial flowers in at least two ways First
as ants interrupted or altogether prevented bumblebees from foraging at nectaries bumblebees
may have learned to avoid flowers with ants due to the relative inefficiency of foraging at these
flowers something bumblebees take into account (Heinrich 2004) Second bees attempt to
minimize their risk of being attacked during foraging bouts which ants did to visiting bees Ants
often attacked or harassed bees by biting grasping and appearing to sting visiting bees
preventing them from accessing the nectary or reducing their time on flowers (see online video
64
1) and bees sometimes avoided ant-tended flowers entirely This harassment was sometimes
physically traumatic (online video 1) and bees often appeared to have trouble flying after being
attacked by ants Previous research has demonstrated that bumblebees leave or avoid flowers
where they have been harassed (Jones and Dornhaus 2011) and avoid foraging where there is
visual or olfactory evidence of a predator or predation event (Abbott 2006 Goodale and Nieh
2012) Though it is unclear if bumblebees viewed ants as competitors or predators they
responded similarly to flowers having ants as they do to flowers housing predators (Gonccedilalves-
Souza et al 2008)
To avoid artificial flowers with ants bees likely used a combination of visual and
olfactory cues Previous research has shown that bees respond to conspicuous predators or
predator ldquodummiesrdquo on flowers (Suttle 2003 Gonccedilalves-Souza et al 2008) decreasing the
frequency and duration of their visits to these flowers Thus bees may have been able to avoid
flowers harboring ants by sight alone However even in the absence of ants bees still
preferentially visited flowers lacking ant scents (Fig 22b) suggesting that they had learned to
associate ant scent with harassment Bees are adept at associative learning (Wright and Schiestl
2009) and can learn to recognize unique scents left behind by both conspecific and heterospecific
flower visitors (Stout et al 1998) Bees can use these various scents often arising from tarsal
gland deposits (Stout et al 1998) to recognize recently visited flowers that are less likely to be
profitable (Stout and Goulson 2002) Recently Ballantyne and Willmer (2012) demonstrated that
bees learn to associate ant scents with unrewarding artificial flowers and decrease their visitation
to these flowers Our results complement their findings by showing that bees can associate ant
scent with harassment at otherwise rewarding flowers
In our study ant scent caused bees to adjust their foraging strategy and decreased the
amount of pollen analogue that was donated by ant-scented flowers Thus the effects of
65
interference competition with ants on flowers can extend beyond immediate interactions and may
have fitness consequences for plants even when ants are absent Like other olfactory cues these
effects are likely transitory (Stout et al 1998) Although more dye was still donated by male
flowers lacking ant scent than those with ant scent the ratio of dye donated was on average
closer to equality in hours 2-4 than in the first hour (Fig 22b) This effect may be partially
driven by dye depletion of male flowers without ant scent but the lack of a strong corresponding
decrease in flowers with ant scent suggests that visitation patterns became more similar We did
not test whether B impatiensrsquo avoidance of ant scent was an innate or a learned behavior but
previous work has shown that Bombus terrestris does not innately avoid flowers with ant scent
(Ballantyne and Willmer 2012)
Ants may be necessary for plant survival and growth but can be costly for plant
reproduction The evolution of ant attractants such as extrafloral nectaries in some plant lineages
suggests that the costs of ants can be outweighed by their protective abilities However in this
study flowers visited by ants received and donated significantly less dye although they did
retain some sexual function The net benefit of having ants depends on whether ants increase
plant fitness by reducing herbivory more than they decrease plant fitness by disrupting
pollination Alternatively plants may actually benefit from the costs of ants to plant
reproduction ants that castrate flowers may be better defenders increasing plant survival or
vegetative growth when plants are young allowing for increased reproduction later in life when
the plant is colonized by less aggressive non-castrating ants (Frederickson 2009 Palmer et al
2010) In some plant species pollinator harassment by ants may even be beneficial Altshuler
(1999) reported that Ectatomma ants greatly increased fruit set of Psychotria limonensis despite
reductions in pollinator visitation rates presumably due to increases in the rate of pollen
outcrossing Similarly Gonzaacutelvez et al (2012) found that the presence of Oecophylla
66
smaragdina ants on Melastoma malabathricum flowers reduced visitation by less effective
pollinators and increased visitation by more effective Xylocopa bees increasing plant fitness
Cases of ants being beneficial to flowers appear to be the minority and many plant
species have traits that limit ant access to flowers Several studies have detailed floral volatiles
that are thought to mimic ant alarm pheromones and thus repel ants (Junker and Bluumlthgen 2008
Willmer et al 2009) Other plants use structural modifications such as narrow corollas or
slippery stems to limit ant access to flowers (reviewed in Willmer et al 2009) Exploitative
competition between ants and pollinators (Lach 2005 Ballantyne and Willmer 2012) as well as
the lethal effects of ant antibiotic secretions on pollen grains (Beattie et al 1985 Dutton and
Frederickson 2012) may have resulted in selection on plants to reduce ant visits to flowers Our
results suggest that trait-mediated indirect interactions resulting from interference competition
between ants and bees may favor plants that defend their flowers and their pollinators against
ants
Acknowledgements
We thank H Rusnock and S Meadley-Dunphy for assistance J Dix for help with flight cage
construction and Biobest for bumblebee colonies We are grateful to the Thomson and
Frederickson labs for guidance particularly T Makino funded by JSPS for helping to refine
artificial flowers J Ogilvie for help with bees K Prior for M rubra colony collections and K
Turner for study design suggestions The comments of the editor and two anonymous reviewers
prompted improvements in the article especially with regards to study design MEF and JDT
acknowledge NSERC Discovery Grant funding The University of Torontorsquos Faculty of Arts and
Science supported MGRTrsquos participation
67
68
References
Abbott K R 2006 Bumblebees avoid flowers containing evidence of past predation events
Canadian Journal of Zoology 841240-1247
Altshuler D L 1999 Novel interactions of non-pollinating ants with pollinators and fruit
consumers in a tropical forest Oecologia 119600-606
Ballantyne G and Willmer P 2012 Floral visitors and ant scent marks noticed but not used
Ecological Entomology 37402-409
Beattie A J Turnbull C Knox R B and Williams E G 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany
71421-426
Blanco M Runiaga H Maspoch S and Tarin P 1989 A simple method for
spectrophotometric determination of two-components with overlapped spectra Journal of
Chemical Education 66178-180
Byk J amp Del Claro K 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333-38
Davidson D W S C Cook and R R Snelling 2004 Liquid-feeding performances of ants
(Formicidae) ecological and evolutionary implications Oecologia 139255ndash66
Dukas R 2005 Bumble bee predators reduce pollinator density and plant fitness Ecology
861401-1406
Dussutour A and Simpson S J 2009 Communal nutrition in ants Current Biology 19740-
744
69
Dutton E M and Frederickson M E 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561-569
Frederickson ME 2009 Conflict over reproduction in an ant-plant symbiosis why Allomerus
octoarticulatus ants sterilize Cordia nodosa trees American Naturalist 173675-681
Galen C and Cuba J 2001 Down the tube pollinators predators and the evolution of flower
shape in the alpine skypilot Polemonium viscosum Evolution 551963-1971
Gonccedilalves-Souza T Omena P M Souza J C and Romero G Q 2008 Trait-mediated
effects on flowers artificial spiders deceive pollinators and decrease plant fitness Ecology
892407-2413
Gonzaacutelvez F G Santamariacutea L Corlett R T and Rodriacuteguez-Gironeacutes M A 2012 Flowers
attract weaver ants that deter less effective pollinators Journal of Ecology 10178ndash85
Goodale E and Nieh JC 2012 Public use of olfactory information associated with predation
in two species of social bees Animal Behaviour 84919-924
Harder LD and Thomson JD 1989 Evolutionary options for maximizing pollen dispersal of
animal-pollinated plants American Naturalist 133323-344
Heil M and McKey D 2003 Protective ant-plant interactions as model systems in ecological
and evolutionary research Annual Review of Ecology Evolution and Systematics 34425-
453
Heinrich B 2004 Bumblebee economics 2nd
edn Harvard University Press Cambridge
Ings T C and Chittka L 2009 Predator crypsis enhances behaviourally mediated indirect
effects on plants by altering bumblebee foraging preferences Proceedings of the Royal
Society B 2762031-2036
70
Inouye D W and Taylor O R Jr 1979 A temperate region plant-ant-seed predator system
consequences of extra floral nectar secretion by Helianthella quinquenervis Ecology 601-
7
Junker R R and Bluumlthgen N 2008 Floral scents repel potentially nectar-thieving ants
Evolutionary Ecology Research 10295ndash308
Junker R R Bleil R Daehler C C and Bluumlthgen N 2010 Intra-floral resource partitioning
between endemic and invasive flower visitors consequences for pollinator effectiveness
Ecological Entomology 35760ndash767
Jones E I and Dornhaus A 2011 Predation risk makes bees reject rewarding flowers and
reduce foraging activity Behavioral Ecology and Sociobiology 651505-1511
Lach L 2005 Interference and exploitative competition of three nectar-thieving invasive ant
species Insect Sociaux 52257-262
Lach L 2007 A mutualism with a native membracid facilitates pollinator displacement by
Argentine ants Ecology 881994-2004
Lach L 2008 Argentine ants displace floral arthropods in a biodiversity hotspot Diversity and
Distributions 14281-290
Makino T T and Sakai S 2007Experience changes pollinator responses to floral display size
from size-based to reward-based foraging Functional Ecology 21854863
Maloof J E and Inouye D W 2000 Are nectar robbers cheaters or mutualists Ecology
812651ndash2661
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506-514
Ohashi K Leslie A and Thomson JD 2008 Trapline foraging by bumble bees V Effects of
experience and priority on competitive performance Behavioral Ecology 19936-948
71
Palmer T M Doak D F Stanton M L Bronstein J L Kiers E T Young T P Goheen
J R and Pringle R M 2010 Synergy of multiple partners including freeloaders
increases host fitness in a multispecies mutualism Proceedings of the National Academy
of Sciences 10717234-17239
Preisser E L Bolnick D I and Benard M E 2005 Scared to death The effects of
intimidation and consumption in predator-prey interactions Ecology 86501-509
Stout J C Goulson D and Allen J A 1998 Repellent scent-marking of flowers by a guild of
foraging bumblebees (Bombus spp) Behavioral Ecology and Sociobiology 43317-326
Stout J and Goulson D 2002 The influence of nectar secretion rates on the responses of
bumblebees (Bombus spp) to previously visited flowers Behavioral Ecology and
Sociobiology 52239-246
Suttle K B 2003 Pollinators as mediators of top-down effects on plants Ecology Letters
6688-694
Thomson JD 1986 Pollen transport and deposition by bumble bees in Erythronium influences
of floral nectar and bee grooming Journal of Ecology 74329-341
Thomson JD Ogilvie J E Makino T T Arisz A Raju S Rojas-Luengas V and Tan M
G R 2012 Estimating pollination success with novel artificial flowers effects of nectar
concentration Journal of Pollination Ecology 9108-114
Tsuji K Hasyim A and Nakamura K 2004 Asian weaver ants Oecophylla smaragdina and
their repelling of pollinators Ecological Research 19669-673
Ulrich S and Bertsch A 1990 Do foraging bumblebees scent-mark food sources and does it
matter Oecologia 82137-144
Werner E E and Peacor S D 2003 A review of trait-mediated indirect interactions in
ecological communities Ecology 841083-1100
72
Willmer P G Nuttman C V Raine N E Stone G N Pattrick J G Henson K Stillman
P McIlroy L Potts S G and Knudsen J T 2009 Floral volatiles controlling ant
behaviour Functional Ecology 23888-900
Wright G A and Schiestl F P 2009 The evolution of floral scent the influence of olfactory
learning by insect pollinators on the honest signaling of floral rewards Functional Ecology
23841-851
73
Figures
Figure 21 Photograph of artificial flower lids A = anther L = lid N = nectary R = region
where anther or stigma is placed S = stigma T = thread W = weight
74
Figure 22 Dye transferred (mean plusmn 1SE) per trial by B impatiens workers A) from artificial
male and to artificial female flowers in the presence (unfilled circles) or absence (filled circles)
of Myrmica rubra workers and B) from artificial male flowers with (unfilled circles) or without
(filled circles) M rubra scent to ant-free female flowers in the 1st or the 2
nd to 4
th hours of the
trials
75
Chapter 3 Not just for the bees pollen consumption is common among tropical
ants
Cembrowski AR Reurink G and Frederickson ME
Planned submission to Biotropica
ARC and MEF designed the study ARC and GR did laboratory work ARC performed statistical
analyses and wrote the manuscript with input from other authors
Abstract
Although palynivory or pollen consumption is widespread among other hymenopterans there
are few accounts of palynivory in ants To quantify how often ants consume pollen we subjected
adult workers and larvae from 75 species of neo- and paleo-tropical ants to acetolysis a process
that destroys most organic material but leaves behind pollen grains In over half of the species
we examined ants contained a few pollen grains (lt 5) and ants of several Camponotus species
contained abundant pollen grains (gt50) We tested for an association between trophic level and
palynivory using stable nitrogen isotope ratios (δ15
N) but we did not find a significant
correlation we found pollen grains in lsquoherbivorousrsquo omnivorous and carnivorous ants We
suggest that our results indicate sporadic opportunistic pollen consumption by ants in tropical
forests
Introduction
For those organisms that can digest it efficiently pollen can be an excellent food source
Depending on the plant species pollen can contain up to 60 protein as well as lipids
76
carbohydrates and numerous trace nutrients (Roulston and Cane 2000) Pollen feeding
(palynivory) occurs in many taxa including birds mammals and arthropods (Roulston and Cane
2000) but it is best known among the Hymenoptera particularly bees Although adult bees feed
mainly on plant exudates (ie nectar) nearly all bee species rely on pollen as the chief protein
source for developing larvae (Willmer 2011) Closely related to bees ants have similar
nutritional requirements adult workers need carbohydrates whereas larvae are fed a diet rich in
protein (Dussutour and Simpson 2009) Ants also exploit nectar heavily and regularly visit
flowers (eg Herrera et al 1984 Bluumlthgen et al 2004) but unlike bees are not believed to
widely consume pollen Instead ants are thought to receive most of the protein in their diet from
predation or scavenging (eg Floren et al 2002)
However ants may not be as reliant on animal protein sources as commonly assumed In
tropical forests arboreal ants can have similar δ15
N ratios a measure of trophic level as
herbivores suggesting limited carnivory and substantial feeding on plant-derived protein sources
(Davidson et al 2003) One potential protein source is pollen This idea is supported by scattered
accounts of pollen consumption by ants from other ecosystems including deserts (Ness 2006)
boreal forests (Czechowski et al 2009) and tropical savannas (Byk and Del Claro 2010) One
ant genus Cephalotes is widely recognized to consume pollen (Creighton 1967 Baroni Urbani
and de Andrade 1997 Byk and Del Claro 2010) However beyond this genus our knowledge of
pollen consumption by ants has largely been limited to direct observations (but see Czechowski
et al 2011) and it is unknown if palynivory is truly rare among ants or simply under-reported
We borrowed a technique commonly used in palynology acetolysis to assess the frequency of
pollen consumption by ants in 75 neo- and paleo-tropical ant species This technique allowed us
to examine many more ant species than we could have observed directly
77
Methods
Adult worker ants were collected by hand at baits or from leaf litter using Winkler extractors
while larvae along with adults were collected by hand from nests Collections were made by L
Arcila Hernandez and J Sanders in the primary tropical rainforest surrounding the Centro de
Investigaciόn y Capacitaciόn Rio Los Amigos (CICRA 12deg34rsquoS 70deg05rsquoW) in Peru in 2010-
2011 and by E Youngerman from lowland primary and secondary rainforests in Madang
Province Papua New Guinea (PNG) in 2011-2012 We sorted ant samples to morphospecies and
then identified them to genus and when possible to species using existing keys which were
unavailable for PNG ants Ant samples used for acetolysis were whole adult workers guts from
adult workers and whole larvae
We used acetolysis to look for pollen grains inside ants In acetolysis organic material is
dissolved in acetic anhydride and sulfuric acid leaving behind stained pollen exines When
possible we used adult and larval ants of the same species from different colonies or bait
locations in an attempt to sample more representatively We prepared samples by first washing
them in 70 ethanol to remove any pollen present on the samplersquos outer surface before placing
them individually in micro-centrifuge tubes with ~05mL of a 91 mixture of acetic anhydride
sulfuric acid We then heated each sample at 95degC for 20min allowing tissues to dissolve Next
we centrifuged samples at 1000 RPM for 5min and decanted the liquid supernatant before
adding ~05mL of glacial acetic acid to the precipitate centrifuging again as above We decanted
the samples and added a drop of distilled water
78
We examined a 150μL sub-sample of this solution for pollen grains under a light
microscope Larvae gave less than 150μL of solution following acetolysis so we sampled the
entire amount We identified pollen grains by their staining and their surface architecture For
most species we examined at least three adult workers and three larvae but for some species
fewer than three adult workers or larvae were available (Tables 31 and 32)
We investigated the relationship between trophic level as measured by δ15
N ratios and
pollen consumption by ants Stable isotope (SI) data was available for some (n =23) of the
Peruvian species from a separate project in which collections overlapped Samples were stored in
EtOH and dried at 50degC overnight We used heads legs and thoraxes (but not gasters) of ants
for SI analysis Often single individuals did not provide enough material for analysis and we
combined multiple individuals to achieve necessary weights up to 10 for small ant species
Samples were analyzed at the Boston University Stable Isotope Laboratory using a Finnigan
Delta-S analyzer For other Peruvian species for which we had samples but not corresponding
stable isotope data (n =25) we took the median δ15
N ratio value of that speciesrsquo genus and
assigned it to the species in question Using pollen presenceabsence data for these 48 species
we used logistic regression to correlate pollen presence and δ15
N ratio using R (v 2151)
Results
We found pollen grains in 37 of the 197 adult workers 20 of the 102 larvae and 38 of the 75 ant
species we examined (Tables 31 and 32) The proportion of individuals containing pollen did
not differ significantly between larvae and adults (χ2
1 = 002 p = 088) The number of pollen
grains found was often low (median = 3) but highly variable (range 1-468) Also pollen
79
presence was inconsistent among conspecific ant samples of species that contained pollen 31
had pollen in only one of the larvae or adults examined In only three species (two Cephalotes
and one Myrmelachista) did all examined adults contain pollen
Highly lsquoherbivorousrsquo carnivorous and omnivorous ants contained pollen grains (Fig
31) There was no correlation between δ15
N ratio and pollen presence (χ2 = 150 df = 1 p =
022) In fact several carnivorous species contained pollen grains (eg Odontomachus sp 1
Pachycondola sp1)
Discussion
Pollen consumption by ants appears to be more common than suspected The majority of species
(51) had at least one individual that had consumed pollen and in 20 multiple individuals
contained pollen Numbers of grains found tended to be low however with most individuals
containing only a few grains Ants may use pollen as only a small portion of their diet exploiting
a host of other protein-containing foods more heavily including other low- δ15
N foods (eg
honeydew epiphylls Houmllldobler and Wilson 1990 Davidson et al 2003)
There are a few reasons why pollen may not be heavily exploited by ants Pollen requires
much post-consumption processing to extract its nutrients These nutrients are located in the
pollenrsquos cytoplasm which is protected from direct digestion by the exines and intines of the
grains Organisms must either break through these layers or cause the pollen grains to germinate
or pseudo-germinate to access and digest the cytoplasm (Roulston and Cane 2000) This can be
achieved by consuming a sugar-containing solution (such as nectar) along with pollen However
animals that are not adapted to feed on pollen may not be very efficient at this process only
80
extracting nutrients from some of the grains (Herrera and Martiacutenez del Rio 1998) Thus while
ants can likely get some nutrients from pollen other forms of more easily processed protein may
be more attractive
Pollen may also pose handling difficulties for ants Despite the large number of studies
that have recorded ants visiting flowers (Chapter 1) only a few papers detail ants harvesting
pollen directly from anthers Byk and Del Claro (2010) reported that Cephalotes pusillus ants
removed almost all of the available pollen from Ouratea spectabilis flowers and at least two
other studies describe ants consuming pollen along with nectar from flowers (Horskins and
Turner 1999 Ness 2006) Unlike bees which possess both morphological (eg specialized hairs
for holding pollen) and behavioral adaptations (eg stereotyped pollen-packing motions) ants
have no apparent mechanisms to increase the amount of pollen harvested and are likely limited
by the number of grains they can ingest or carry Rather than collect pollen directly from flowers
it appears more likely that ants collect it haphazardly in the environment leading to a smaller
number of pollen grains consumed at any one time It is known that at least Cephalotes ants
engage in such behavior Creighton (1967) observed Cephalotes texanus collecting pollen grains
trapped by Citrus x paradisi leaf hairs
There is a chance that the small numbers of pollen grains we observed came from
secondary pollen consumption (ie consuming a palynivorous organism) or consumption of
liquid containing pollen While we cannot rule this out we believe our results represent actual
targeted pollen consumption by ants Cephalotes ants known palynivores (Creighton 1967
Baroni Urbani and de Andrade 1997) contained similar numbers of pollen grains as many of the
pollen-containing ant species that we examined As well in at least once instance ants that we
found containing pollen grains were in the process of preying on non-pollen consuming
81
organisms (eg a Pachycondola species that was carrying a termite in its mandibles) or were
attracted by sugar baits
Contrary to our expectations there was no relationship between an ant speciesrsquo δ15
N ratio
and whether it had consumed pollen (Fig 32) We expected palynivory to be more common in
herbivorous ants because pollen presents a widespread source of protein a potentially limiting
resource The lack of pattern might reflect how available pollen grains are in the environment
While other forms of food may be preferable ants tend to be opportunistic foragers (Carroll and
Janzen 1973) and there is likely little cost associated with the consumption of scattered pollen
grains during foraging trips beyond the volume they occupy in the gut Some genera were found
to consistently consume pollen In support of previous observations (Baroni Urbani and de
Andrade 1997 Byk and Del Claro 2010) Cephalotes species were among the most common
pollen consumers Camponotus species also consistently contained pollen grains of the eight
species examined five contained pollen and pollen was found in species from both Peru and
PNG Camponotus species also contained the two largest numbers of pollen grains found (468
and 271) Taken together these results suggest that they may be another as yet unrecognized
commonly palynivorous genus
In general larvae did not contain large numbers of pollen grains (ranging from 1-38
grains) This is counter to what is seen in bees where larvae are the main recipients of pollen
(Willmer 2011) This difference may be because ant larvae are continually fed via trophallaxis
by adults throughout their development whereas bee larval cells are provisioned with pollen and
then sealed while larvae complete their development If both adults and larvae are able to extract
nutrients from pollen we would expect to see a preferential flow of nutrients from pollen to
larvae but not necessarily the grains themselves
82
Unlike palynivory by organisms that also provide pollination services pollen
consumption by ants is unlikely to benefit plants Ants rarely act as pollinators (Beattie et al
2004) They have pollen-killing antibiotic secretions on their cuticles (Dutton and Frederickson
2012) and they can compete with other flower visitors reducing flower visitation rates (Ness
2006) The negative impacts of flower-visiting ants are thought to have led to the evolution of
ant-repellent floral volatiles (reviewed in Willmer 2009) These volatiles can pre-empt
competition between ants and pollinators and may often be located within the pollen itself
(Willmer 2009) If floral volatiles truly are a defense against ant visitation our results suggest an
additional evolutionary pressure to not only prevent ant-pollinator interactions but to also
protect plant gametes from occasional consumption by a non-pollinating visitor
Acknowledgements
We would like to thank Jon Sanders Lina Arcila Hernandez and Eric Youngerman for providing
samples the Thomson lab for use of supplies the Pierce lab for funding support during JSrsquo and
EYrsquos collections and the staff of CICRA and the New Guinea Binatang Research Center for
logistical support The Peruvian Ministry of Agriculture provided permits (Nos 394-2009-AG-
DGFFS-DGEFFS and 299-2011-AG-DGFFS-DGEFFS) MEF acknowledges financial support
from an NSERC Discovery Grant a Connaught New Researcher Award an Ontario Ministry of
Economic Development and Innovation Early Researcher Award and the University of Toronto
ARC was supported by an Ontario Graduate Scholarship and Sigma Xi
83
References
Baroni Urbani C and M L de Andrade 1997 Pollen eating storing and spitting by ants
Naturwissenschaften 84256ndash258
Beattie A J C Turnbull R B Knox and E G Williams 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany 71421ndash
426
Bluumlthgen N G Gottsberger and K Fiedler 2004 Sugar and amino acid composition of ant-
attended nectar and honeydew sources from an Australian rainforest Austral Ecology
29418ndash429
Byk J and K Del-Claro 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333ndash38
Carroll C R and D H Janzen 1973 Ecology of foraging ants Annual Review of Ecology and
Systematics 4231ndash257
Creighton W S 1967 Studies on free colonies of Cryptocerus texanus Satschi (Hymenoptera
Formicidae) Psyche 7434ndash42
Czechowski W B Markό and A Radchenko 2009 Rubbish dumps reveal the diet of ant
colonies Myrmica schencki Em and Myrmica rubra (L) (Hymenoptera Formicidae) as
facultative pollen-eaters Polish Journal of Ecology 56737ndash741
Czechowski WB Markoacute K Erős and E Csata 2011 Pollenivory in ants (Hymenoptera
Formicidae) seems to be much more common than it was thought Annales Zoologici
61519ndash525
84
Dussutour A and S J Simpson 2009 Communal nutrition in ants Current Biology 19740ndash4
Dutton E M and M E Frederickson 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561ndash569
Floren A A Biun and K E Linsenmair 2002 Arboreal ants as key predators in tropical
lowland rainforest trees Oecologia 131137ndash144
Grant B R 1996 Pollen digestion by Darwinrsquos finches and its importance for early breeding
Ecology 77489-499
Herrera L G and C Martiacutenez del Rio 1998 Pollen digestion by new world bats effects of
processing time and feeding habits Ecology 792828ndash2838
Houmllldobler B and Wilson E O 1990 The Ants 1st Edition Harvard University Press
Cambridge
Horskins K and Turner V B 1999 Resource use and foraging patterns of honeybees Apis
mellifera and native insects on flowers of Eucalyptus costata Australian Journal of
Ecology 24221-227
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506ndash514
Roulston T H I and J H Cane 2000 Pollen nutritional content and digestibility for animals
Plant Systematics 222187ndash209
Van Tets I G 1997 Extraction of nutrients from Protea pollen by African rodents Belgian
Journal of Zoology 12759-65
Willmer P G 2011 Pollination and Floral Ecology 1st Edition Princeton University Press
New Jersey
85
Willmer P G C V Nuttman N E Raine G N Stone J G Pattrick K Henson P Stillman
et al 2009 Floral volatiles controlling ant behaviour Functional Ecology 23888ndash900
86
Tables
Table 31 Numbers of adult worker and larval ants examined for each species collected in Peru
Numbers of pollen grains found in individual samples are given in parentheses after the sample
size when there were no grains no numbers are given
Genus Species Number of adults analyzed
(numbers of grains found)
Number of larvae analyzed
(numbers of grains found)
Dolichoderinae
Azteca sp 1 3 0
sp 2 2 0
sp 3 0 2
sp 4 3 (1) 0
sp 5 3 (2) 0
Dolichoderus decollatus 3 (66) 0
sp 1 3 0
sp 2 3 1 (2)
sp 3 3 3
sp 4 0 2
Ecitoninae
Labidus sp 1 3 0
Formicinae
Camponotus sp 1 3 3
sp 2 3 (82) 1 (2)
sp 3 3 (3) 0
sp 4 0 2
87
sp 5 8 (11) 0
sp 6 4 (7 16 468) 0
sp 7 5 (12) 0
sp 8 2 (1) 0
Paratrechina sp 1 4 (11) 0
Myrmelachista sp 1 3 (5 11 18) 0
sp 2 0 3
Trachymyrmex sp 1 3 (21) 0
sp 2 3 0
Myrmicinae
Atta sp 1 3 0
Cephalotes atratus 3 (2 2 21) 0
placidus 3 (1 4 21) 0
sp 1 3 0
sp 2 1 0
sp 3 2 0
Crematogaster sp 1 3 1
sp 2 3 (35) 0
sp 3 9 (7) 0
sp 4 2 0
Megalomyrmex sp 1 3 0
sp 2 0 2
sp 3 4 (7) 0
sp 4 3 0
Monomorium sp 1 0 3 (2)
88
Pheidole sp 1 0 3
sp 2 3 0
sp 3 0 3
Procryptocerus sp 1 3 0
Solenopsis sp 1 0 3
sp 2 3 (3 5) 0
Wasmannia sp 1 3 0
Ponerinae
Pachycondola sp 1 3 (3) 0
Odontomachus sp 1 3 (34) 0
Pseudomyrmicinae
Pseudomyrmex sp 1 3 0
sp 2 3 0
sp 3 3 (9) 0
sp 4 0 3
sp 5 0 3
89
Table 32 Numbers of adult worker and larval ants examined for each species collected in Papua
New Guinea Numbers of pollen grains found in individual samples are given in parentheses
after the sample size when there were no grains no numbers are given
Genus Species Number adults analyzed
(grains found)
Number larvae analyzed
(grains found)
Aenictinae
Aenictus sp 1 3 3 (1 1)
Dolichoderinae
Philidris sp 1 3 1 (4)
Tapinoma sp 1 2 3 (38)
Technomyrmex sp 1 0 3 (12 12)
Ectatomminae
Rhytidoponera sp 1 1 3 (2)
Formicinae
Acropyga sp 1 3 3 (2)
sp 2 3 3
sp 3 3 3
Anoplolepsis sp 1 3 (1) 3 (1)
Calomyrmex sp 1 3 3 (1)
Camponotus sp 1 3 (1 271) 3
sp 2 1 3
sp 3 2 0
Nylanderia sp 1 3 3
Opisthopsis sp 1 3 3
Polyrachis sp 1 1 3 (4)
90
Myrmicinae
Crematogaster sp 1 3 3
sp 2 3 (32) 3
Monomorium sp 1 3 (1) 3 (1)
sp 2 3 3 (1 1 1)
Pheidole sp 1 3 3
Pristomyrmex sp 1 3 3 (11)
Pyramica sp 1 3 0
Ponerinae
Odontomachus sp 1 3 3 (4)
91
Figures
Figure 31 Photographs of pollen grains found in two ant species a) Dolichoderus sp 2 and b)
Camponotus sp 6 Scale bars = 50 microm
92
Figure 32 Relationship between trophic level as measured by δ15
N ratio and pollen presence
for 48 neotropical ant species 1 Atta sp 5 Azteca spp 8 Camponotus spp 5 Cephalotes spp 4
Crematogaster spp 5 Dolichoderus spp 1 Labidus sp 3 Megalomyrmex spp 1 Monomorium
sp 2 Myrmelachista spp 1 Odontomachus sp 1 Pachycondola sp 1 Paratrechina sp 3
Pheidole sp 4 Pseudomyrmex spp 2 Solenopsis spp and 1 Wasmannia sp
93
Concluding Remarks
Ants have traditionally been considered to be unwelcome flower visitors they consume floral
rewards ostensibly meant for pollinators while rarely providing pollination services themselves
In this thesis I investigate the validity of this assumption and use an experimental study and an
observational study to examine two ways that ants may impact plant reproduction
In the first chapter I review the literature on ants as flower visitors I show that the common
assumption of ants always being categorically negative for plant reproduction is incorrect while
ants may reduce floral attractiveness to pollinators the net effect of ants on plant fitness
measures is variable ranging from positive to negative to neutral I also discuss ants as
pollinators and ways that plants may minimize ant visitation to flowers before finishing with a
section highlighting questions that still need investigation
In the second chapter I explore how interference competition with ants can structure bumblebee
foraging behavior and how this may alter pollen movement patterns Using artificial flowers with
male or female function I allowed Bombus impatiens bumblebees to forage on flowers that
either did or did not have Myrmica rubra ants on them Ant presence significantly reduced the
amount of pollen analogue donated and received by flowers Building on these results I found
that the presence of ant scent alone elicited similar changes to bumblebee behavior reducing the
amount of pollen analogue donated by male flowers My results show that interference
competition is sufficient to alter bumblebee foraging behavior and that the effects of ants on
pollinators may extend after ant visitation to flowers Further study is needed to determine if bees
are able to differentiate between ant species by visual or olfactory cues Their ability to do so
94
could affect how generalized their response to ant presence is and may help to better predict how
ants will affect plant reproduction
In the final chapter I investigate how common palynivory is amongst tropical ant species I used
acetolysis and light microscopy to examine whole ants larvae and ant guts from Papua New
Guinea and Peru I found that tropical ants consistently contain pollen grains albeit often in
small numbers (lt5) However some genera notably Camponotus and Cephalotes consistently
contained higher numbers providing the first evidence that Camponotus may be a palynivorous
genus The small numbers of pollen grains present may indicate that ants preferentially exploit
other more easily digestible high-protein foods over pollen Future studies should investigate
exactly what role pollen plays in ant diets by supplementing the diet of captive ant colonies with
pollen This would allow us to examine how pollen consumption affects colony demography as
well as how efficiently ants digest pollen and whether ants exhibit pollen preferences
I bring up several pressing and unanswered questions within this thesis and highlight three here
First numerous plant species possess chemical or physical traits assumed to increase plant
fitness by reducing ant visitation However this has rarely been tested and instead research has
focused predominately on documenting ant repellence We should move away from this and
begin examining plant fitness in the presence or absence of these repellent traits to see how
beneficial these traits truly are Second how flower visiting ants impact male fitness has been
almost completely ignored in favor of female fitness Future studies should investigate how ant
visitation affects both pollen donation and pollen movement patterns Lastly research involving
antibiotics present on ant cuticles and their effect on pollen viability has been too small in scope
often limited to demonstrating the pollen killing effects of ant secretions Investigation into
95
whether plants have evolved any resistance to the secretions of the local ant community or why
some plants appear unaffected by their ant partners may yield exciting results
In conclusion ants have the capacity to strongly affect plant reproduction both positively and
negatively The fact that these diametrically opposing forces exist means we cannot approach
flower-visiting ants as either positive or negative a priori Instead we should strive to work
towards a better understanding of what is occurring in the system of interest and what role ants
play within it
96
Copyright Acknowledgments
Chapter 2 ldquoAnts and ant scent reduce bumblebee pollination of artificial flowersrdquo is current in
press in The American Naturalist Request for its inclusion here was sought and approved
3
what impacts ants have on plant reproductive success First we discuss mechanisms by which
flower-visiting ants can directly and indirectly impact plant reproduction both through floral
damage and by mediating changes in pollinator or florivore behavior We end this section by
evaluating published research that collectively shows that flower-visiting ants have
predominantly neutral effects on plant fitness measures Next we discuss pollination by ants
focusing on the ldquoantibiotic hypothesisrdquo before describing how plants may prevent ants from
visiting flowers Lastly we discuss promising new avenues of research and highlight work that
still needs to be performed to give a more complete understanding of ants as flower visitors
1 Ant interactions with flowers and floral visitors
i) Damage to flowers
Ants can directly disrupt plant reproduction by damaging floral structures preventing either the
fertilization of ovules or the presentation of pollen Rarely ants forage on floral tissue itself for
example Puterbaugh (1998) found that Formica neorufibarbus ants chewed through the coronal
ring of Eritrichium aretiodes flowers to get lipids More often but still not commonly foraging
ants chew through reproductive tissue to access floral nectar This can cause sterilization of
whole flowers (Echium plantagineum Kirk 1984) or affect female function only if just styles are
damaged effectively creating male flowers (Polemonium viscosum Galen 1983) Antibiotic
secretions on ant integuments may also directly damage pollen grains but the fitness effects of
this are unclear (see 2 below)
Several species of Crematogaster and Allomerus ants sterilize their host plants by
attacking floral buds (Yu and Pierce 1998 Stanton et al 1999 Gaume et al 2005 Maleacute et al
2012 Table 11) These ants nest in ldquoant-plantsrdquo or ldquomyrmecophytesrdquomdashie plants that host ant
4
colonies in specialized structures (domatia) The sterilizing ants do not appear to consume floral
tissues (Yu and Pierce 1998) and two hypotheses may explain why ants destroy floral buds First
ant colony size is often limited by domatia availability on their host plant By destroying floral
buds ants can cause plants to divert resources from reproductive to vegetative growth increasing
domatia number and allowing for larger ant colonies (Yu amp Pierce 1998 Frederickson 2009)
Second when plant-ants must defend their colony from takeovers by other colonies they may
benefit from limiting access to their host plant By destroying buds that could develop into
flowers branches or leaves ants can prevent their host plant from contacting nearby plants
occupied by enemy ant colonies (Stanton et al 1999)
The impact of sterilization on lifetime plant fitness is unclear Ants may destroy all of
their host plantrsquos floral buds (Yu and Pierce 1998) completely preventing reproduction or only a
portion of them (Maleacute et al 2012) allowing some seed set However counter-intuitively this
sterilization behavior may provide long-term fitness benefits to plants (Frederickson 2009
Palmer et al 2010) Ant-plants often host several different ant species over their lifetimes some
of which are non-sterilizing If hosting a sterilizing species at a certain life stage increases
survival or growth relative to hosting a non-sterilizing species at that life stage plants may
increase lifetime fitness by largely forgoing current reproduction in favor of increasing future
reproduction (Palmer et al 2010) Why plants invest resources in producing flowers that are
quickly destroyed by ants is not known but the timing of flower destruction may be important If
ants attack flowers after anthers dehisce plants may still realize some male fitness via pollen
donation (Yu and Pierce 1998) Additionally if the cost of flower production is relatively minor
and ants sometimes ldquomissrdquo flowers (Edwards and Yu 2008) attempting limited reproduction
may be a better strategy than forgoing it entirely
5
ii) Competition with pollinators
Ant activity on flowers can dramatically alter pollinator behavior Ants can reduce the species
diversity frequency or duration of pollinator visits to flowers (Norment 1988 Tsuji et al 2004
Ness 2006 Junker et al 2007 Lach 2007 2008b Romero et al 2011 and see Chapter 2) These
changes are largely due to either exploitative competition (ie consumption of shared resources)
or interference competition (ie direct aggression) between ants and pollinators
a) Exploitative competition
Pollinators and ants frequently compete for floral nectar Flower-visiting ants are often classified
as nectar thieves (sensu Inouye 1980) because they typically enter flowers through the
ldquolegitimaterdquo opening and consume nectar but transfer no pollen (Wyatt 1980 Ness 2006
Chamberlain and Holland 2008) Ants can also act as primary nectar robbers chewing holes
through corollas to reach nectaries (Willmer and Corbet 1981 Koopowitz and Marchant 1998)
although they are more commonly secondary nectar robbers and use holes made by previous
robbers (Newman and Thomson 2005)
Like other flower visitors foraging ants will often leave nectar behind in flowers This
amount can be less (Bleil et al 2011) more (Fritz and Morse 1982) or similar to (Lach 2008a)
amounts left by other visitors Often ants are morphologically constrained from fully exploiting
nectar Unlike bees and butterflies ants do not have proboscises and must get close to nectar to
consume it Narrow corollas or nectar spurs can prevent ant access to some or all floral nectar
(Schemske 1980 Galen and Cuba 2001 and see below) Smaller ants may be better able to
6
access nectar (Newman and Thomson 2005 Agarwal and Rastogi 2007) but can be limited by
their crop size in the amount of nectar they can remove (Lach 2005) If foraging ants leave
enough nectar behind for a pollinator to recover the cost of visiting a flower ants may have
minimal effects on plant reproductive success (Maloof and Inouye 2000 Turner et al 2012)
Differences among ant species in behavior and diet also affect their consumption of floral
nectar Species vary in how many flowers they visit per inflorescence or per plant and in how
many ants will visit a flower simultaneously (Lach 2005 Junker et al 2007 Lach 2013)
Invasive antsmdashthought to be better at exploiting and defending resources than native ants
(Holway et al 2002)mdashoften recruit to flowers in large numbers sometimes several dozen or
more (Visser et al 1999 Hansen and Muumlller 2009) Overall many factors may influence how
heavily ants exploit floral nectar on plants including ant density plant height the spatial or
temporal distribution of floral nectar and the availability of alternative food resources for ants
(Cushman and Addicott 1991 Lach 2013)
Rarely ants and pollinators may compete for pollen We know of only a few studies that
have recorded ants harvesting pollen directly from anthers (Horskins and Turner 1999 Ness
2006 Byk and Del-Claro 2010) Other studies have found pollen grains in ants (eg Baroni
Urbani and de Andrade 1997 and see Chapter 3) but this may not indicate competition with
pollinators ants sometimes collect stray pollen grains from the environment (Creighton 1967)
One New World genus Cephalotes might be specially adapted for pollen consumption They
possess ldquoproventricular shieldsrdquo structures that may restrict solid food to the crop allowing
nestmates to exchange pollen grains via trophallaxis It is unclear if this structure evolved for this
purpose but Cephalotes are the most frequently documented palynivorous ants (eg Creighton
1967 Baroni Urbani and de Andrade 1997 Byk and Del-Claro 2010) How efficiently ants
7
digest pollen and whether ants like bees feed it to larvae as a protein source is unknown At
least two studies have found that ant larvae consume pollen providing some support for this idea
(Wheeler and Bailey 1920 and Chapter 3)
b) Interference competition
Ants are relatively unique among flower visitors in defending floral resources from competitors
(but see Willmer and Corbett 1981 Roubik 1982) This defense ranges from chance encounters
with ants attacking visitors when flower occupancy overlaps (Ashman and King 2005 Junker et
al 2007) to actively patrolling inflorescences or constructing protective galleries around
nectaries (Horvitz and Schemske 1984 Gaume et al 2005 Hansen and Muumlller 2009)
Interference competition even in the absence of exploitative competition is sufficient to alter
pollinator behavior (Chapter 2)
The strength and outcome of interference competition depends on the identities of the ant
and the pollinator Pollinators might not alter their visitation patterns in response to non-
aggressive ants (Junker et al 2007) but more antagonistic ants may elicit avoidance behavior
before or soon after the visitor lands (Willmer and Corbet 1982 Altshuler 1999 Schuumlrch et al
2000 Table 11) Pollinator size may help predict their reaction to ants because ants may deter
only small-bodied pollinators (Gonzaacutelvez et al 2012) It is not clear whether flower visitors view
ants as aggressive competitors or as potential predators rarely ants prey on pollinators (Schatz
and Wcislo 1999) but in most cases ants merely cause pollinators to relocate
How well pollinators recognize and avoid ant-visited flowers is unresolved Collectively
pollinators have lower visitation rates to flowers visited by ants (Romero et al 2011) This is
8
dependent on pollinator identity however dipterans do not appear to discriminate between ant-
visited and ant-free flowers while bees can identify and avoid ant-visited flowers Bees likely
use a combination of visual and olfactory cues to recognize ant-visited flowers They can
identify previous conspecific and some heterospecific insect visitors by the hydrocarbon
ldquofootprintsrdquo they leave during foraging (Stout and Goulson 2001) Bumblebees can detect ant
hydrocarbons although this has been shown only in the laboratory (Ballantyne and Willmer
2012 Chapter 2) No study has yet examined if bees can use sight alone to detect ants but
several suggest it (Junker et al 2007 Gonzaacutelvez et al 2012) Some bees may discriminate
between ant species when visiting flowers (eg Lach 2008b) which follows if not all ant-
pollinator interactions are equally costly The ability of bees to do so will affect how generalized
their avoidance behaviors will be If they can discriminate between species bees may avoid
specific flowers visited by more ldquocostlyrdquo ants However if they cannot bees might either avoid
all ant-visited flowers or none depending on how negative the average interaction with an ant is
iii) Interactions with flower and pollinator antagonists
Ants can defend the flowers they visit Numerous plant species have circumfloral or bracteal
nectaries (ie extrafloral nectaries around flowers) and ants attracted to these nectaries can
reduce bud ovule or flower predation increasing plant fitness (Inouye and Taylor 1979 Rico-
Gray and Thien 1989 Sugiura et al 2006) Although the presence of EFNs (extrafloral nectaries)
so close to floral nectaries (sometimes less than 1 cm Ness 2006) might increase discovery and
exploitation of floral nectar by ants only one study has investigated this hypothesis finding no
relationship between the proximity of EFNs to floral nectaries and antsrsquo use of floral nectar
(Galen 2005) Holes made by nectar robbers are functionally analogous to circumfloral nectaries
9
and can also attract ants that defend flowers (Newman and Thomson 2005) and nectar-thieving
ants can similarly prevent florivory in some systems (Lach 2007 Bleil et al 2011)
Ants can also reduce the abundance of flower-dwelling predators or parasites of
pollinators Ambush predators such as crab spiders often forage at flowers where they may
reduce pollinator visitation rates and plant fitness (Dukas 2001 Gonccedilalvez-Souza et al 2008)
Ants can decrease spider abundance and richness on plants (Rudgers 2004 Renault et al 2005
Nahas et al 2012) and some evidence suggests that they can do the same on flowers (Lach
2008) In select cases ants may also reduce the abundance of pollinator parasitoids (Holland et
al 2011) Whether ants increase plant reproductive success by removing pollinator antagonists
has not yet been examined but should be If ants create lsquoenemy-freersquo flowers pollinators might
prefer to land on flowers with ants
iv) Net effects
Table 11 compiles studies in which the authors recorded ants consuming floral rewards or
interacting with pollinators and measured some aspect of plant fitness (eg fruit or seed set
pollen deposition) We included only a single study per plant species unless subsequent studies
found conflicting results or measured the fitness of another plant sex Records of ant pollination
are included in a separate table (Table 12)
Table 11 reveals relatively few examples of flower-visiting ants negatively affecting
plant fitness given how costly ant visitation is assumed to be The net effect of ants on plant
reproductive success is actually quite variable with similar numbers of studies finding positive
negative or neutral effects of ant presence Surprisingly when ants do have negative effects it is
10
usually because they directly damage floral structures and not because they prevent pollinators
from visiting flowers and successfully transferring pollen Why are ants not more often negative
for plant reproduction when this is commonly assumed to be true
First ants may compensate for reducing pollinator visitation rates by improving
pollination quality As pollinators move between flowers on a plant they often deposit self-
pollen on stigmas (ie geitonogamy) In some plants with late-acting self-incompatibility ovules
fertilized by self-pollen are subsequently aborted reducing the number of seeds that can be
produced (Barrett 2002 and references therein) Geitonogamy can also reduce offspring viability
or vigor (de Jong et al 1993) and cause pollen discounting a reduction in the amount of pollen
available for outcrossing because of self-pollination (Barrett 2002) By competing with
pollinators ants might decrease rates of geitonogamous pollination if pollinators then visit fewer
sequential flowers within a plant Ants might also cause pollinators to fly further between plants
potentially increasing the genetic diversity of pollen arriving at stigmas Although such effects
have been reported for other floral larcenists (Maloof 2001 Irwin 2003) to our knowledge these
hypotheses have not been investigated in relation to ants
Second ants and pollinators do not always forage for the same resources possibly
allowing them to partition floral rewards If ants forage for nectar but not pollen and flowers can
be pollinated efficiently by visitors looking to gather mostly pollen and little or no nectar then
plant reproduction will not be affected by ants consuming floral nectar Junker et al (2010a)
found that honeybees foraged for both pollen and nectar in the absence of ants but reduced their
foraging on nectar but not pollen when ants could access flowers resulting in no difference in
fruit set between treatments Whether pollinators commonly adjust their foraging behaviors in
response to ants warrants further research
11
Third ants are somewhat unique among floral larcenists in that they often provide direct
benefits to the plants they visit In the studies listed in Table 11 ants sometimes decreased
herbivory on plants (Chamberlain and Holland 2008 Bleil et al 2011) or otherwise aided the
plant (eg ant presence repelling smaller less effective pollinators Gonzaacutelvez et al 2012) If the
positive effects of ants cancel or outweigh the negative effects of flower-visiting ants on
pollinators then ant visitation may be neutral or even positive for plant fitness
Fourth ants may impose fitness costs to plants not captured in the studies in Table 11
Nectar robbers have stronger negative effects when plants are pollen-limited (Burkle et al 2007)
but only one study in Table 11 assessed whether flowers would set more seed if they received
more pollen finding no evidence of pollen limitation (Lach 2008a Table 11) Even if ants
decrease pollinator visitation rates there is limited scope for ants to decrease plant reproductive
success when plants are not pollen-limited Furthermore nearly all the studies in Table 11
measured only female fitness Only three quantified aspects of male fitness (Wyatt 1980 Fritz
and Morse 1981 Galen and Geib 2007) and we know of only one other study in a system in
which ants occasionally pollinate that has also measured male fitness (Ashman 2000) All four
studies measured pollen or pollinia removal rates and none found any effect of flower-visiting
ants on male fitness However whether other measures of male fitness (eg seeds sired pollen
deposition patterns) would reveal similar or different effects needs investigation
Finally although floral defenses against ants (see 3 below) may prevent us from
observing the negative effects of ants on plant reproductive success we consider this unlikely
Several studies have examined interactions between ants and plants that share little or no
coevolutionary history usually in the context of ant invasions Although invasive ants often
recruit to flowers in larger numbers than native ants they do not appear to reduce plant
12
reproductive success (Blancafort and Goacutemez 2005 Lach 2007 Lach 2008a but see Hansen and
Muumlller 2009) And in Hawaii where angiosperms evolved for millions of years in the absence of
ants and are thought to have secondarily lost floral defenses against ants (Junker et al 2011a)
there is no evidence that the preponderance of recently introduced flower-visiting ants negatively
affects plant fitness (Junker et al 2010a Bleil et al 2011)
2 Ants as pollinators
Ants may directly benefit plant reproduction by acting as pollinators but this is a relatively rare
occurrence Ants have long been considered poor pollinators (Kerner 1878) Initially this was
based on morphological and behavioral differences between ants and recognized pollinators
Worker ants are wingless and often relatively hairless making them poor pollen dispersers As
well because many ants exhibit Ortstreue (literally ldquoplace fidelityrdquo) ants were thought to return
to the same branch or flower repeatedly effecting no cross pollination (Houmllldobler and Wilson
1990) Though largely true these traits do not necessarily preclude ants from acting as
pollinators (Table 12) Although they commonly have restricted foraging ranges (Houmllldobler and
Wilson 1990) some ants can effect outcrossing (eg Ashman and King 2005 Carvalheiro 2008)
and many ants are as hairy if not hairier than their pollinating counterparts
In the late 1970s the ldquoantibiotic hypothesisrdquo was put forward to explain the rarity of ant
pollination Ants use antibiotic secretions to protect themselves against the fungal and bacterial
pathogens common in their nests and environs Iwanami and Iwadare (1978) found that one of
these compounds myrmicacin also greatly reduces pollen germination rates Subsequently
Beattie et al (1984 1985) found that short periods of contact (le 30 minutes) with an antrsquos
integument caused pollen grains to fail to germinate Some of these pollen-killing compounds
13
were traced back to the ant metapleural gland which produces hygienic secretions (Houmllldobler
and Wilson 1990) although contact with ants lacking metapleural glands can also reduce pollen
viability (Beattie et al 1985 Dutton and Frederickson 2012) Ant mandibular or anal gland
secretions and potentially ant venom may likewise defend ants against pathogens and have
similar effects on pollen germination (Beattie et al 1985 Graystock and Hughes 2011)
Nonetheless ant pollination has been documented repeatedly There are at least 18 cases
in which ant pollination has been shown experimentally and five others in which observations
strongly suggest it (Table 12) The majority of these plants fit the ldquoant pollination syndromerdquo
originally proposed by Hickman (1974) dense stands of short plants with small flowers and
easily accessible nectaries Typically ant-pollinated plants are associated with hot and dry
climates (principally in the Mediterranean) but dry alpine environments may also favor the
evolution of ant pollination (eg Svensson 1985 Puterbaugh 1998 Schiestl and Glaser 2011) In
most cases ants appear to be the sole or major pollinator of the plant in question (Hickman 1974
Peakall et al 1990 de Vega et al 2009) although ants sometimes make only supplementary
contributions to pollination (Schuumlrch et al 2000 Ashman and King 2005) This suggests that co-
evolution between ants and plants may not be necessary for ants to be useful pollinatorsmdashwhich
is surprising given the potential negative effects of ants on plant reproduction
Ant-pollinated plants cope with ant secretions in a variety of ways Several plant species
may simply tolerate pollen damage (Gόmez and Zamora 1992 de Vega et al 2009) apparently
possessing no overt adaptations to prevent it These plants might receive enough pollen to
overcome reduced pollen viability Other plants may place pollen on ant body parts thought to
have fewer secretions thereby potentially escaping damage (eg on ant legs Ramsey 1995) or
attach pollen to ants with specialized structures avoiding direct contact with the ant integument
14
(Peakall et al 1990) It is not known whether pollen evolves resistance to ant secretions or
whether plants deposit pollen on ants which have less disruptive secretions although these
possibilities have been suggested elsewhere (eg Garciacutea et al 1995 Ramsey 1995)
One key question regarding the effects of ant secretions on pollen grains urgently needs
to be resolved Several studies have suggested that the pollen-killing secretions of ants may have
contributed to the evolution of floral defenses against ants (eg Wagner 2000 Galen and Geib
2007) For this to be true ants must decrease male fitness by damaging pollen grains before they
leave flowers and are deposited on receptive stigmas Only two studies have investigated the
effects of ant secretions on pollen still in anthers (Wagner 2000 Galen and Butchard 2003) and
both constrained ants unnaturally to flowers for significantly longer than ants would normally
stay on flowers Although these studies found strong reductions in pollen vigor it is unclear if
ant secretions decrease male fitness under natural ant visitation conditions
3 Floral defenses against ants
Plants are not passive recipients of flower visitors and have some ability to ldquochooserdquo which
organisms can access floral rewards Flowers resist ants using both structural and chemical traits
More thorough investigation is needed to determine whether these traits evolved to prevent or
minimize the negative effects of ants on plant reproductive success (ie whether these traits can
truly be considered defenses against ants see 4ia below) It is unlikely however that ants
experience reciprocal selection to circumvent floral resistance traits because the fitness benefits
to ants of accessing flowers are probably small Thus there may be little scope for
coevolutionary arms races between floral defenses against ants and ant counter-adaptations
15
i) Structural resistance traits
a) Restricting ant access
Numerous plant species have physical structures preventing ant access to flowers Thin pedicels
can limit antsrsquo gripping abilities and make ants easier to dislodge when flowers are moved by
wind (Gaume et al 2005) and waxy secretions can render stems too slippery for ants to climb
(Harley 1991) Structures surrounding flowers such as water moats or dense hairs may similarly
keep ants from accessing floral rewards (Willmer et al 2009) Many such traits function as broad
filters and prevent not only ants but most crawling insects from reaching flowers Thus it is
difficult to determine whether ants florivores or both acted as selective agents favoring the
evolution of physical barriers to flowers
Flower shape may also reduce ant-pollinator conflict Long narrow corollas or nectar
spurs can prevent all but the smallest ants from accessing floral nectaries (Bluumlthgen et al 2004
Agarwal and Rastogi 2007) while still allowing long-tongued pollinators access to rewards
Selection to exclude nectar-thieving ants in such a manner however may be counteracted by
pollinator preferences for wider corollas that allow easier flower handling (Galen and Cuba
2001) Additionally these types of flowers may be more prone to robbery (Irwin and Maloof
2002) limiting their usefulness in preventing nectar larceny
b) Separation in space or time
Plants may separate floral rewards and ants temporally or spatially to minimize ant impacts on
plant reproduction Several authors have proposed that myrmecophytes have adapted to the
16
sterilization or otherwise detrimental activities of ants by locating domatia or food rewards on
different branches than flowers (Izzo and Vasconcelos 2002 Raine et al 2002 Edwards and Yu
2008) Temporal separation of ant and pollinator activities could likewise reduce conflict though
evidence is limited Some acacia flowers dehisce during periods of low ant activity (Willmer and
Stone 1997 Nicklen and Wagner 2006) but it is unclear if this is in response to ants
temperature or pollinator activity Timing of nectar secretion may similarly reduce ant-pollinator
competition If ants are active prior to pollinators they may deplete floral nectar and reduce
pollinator visitation rates (Schaffer et al 1984) However if nectar is secreted when ants are
inactive pollination may occur unimpeded (Norment 1988) This temporal separation could
favor pollinators that are active when ants are not (eg Wyatt and Morse 1981) but this is
poorly documented Further studies particularly focusing on flowers with the potential for
nocturnal and diurnal pollination may provide more examples
c) Extrafloral nectaries (EFNs)
One hypothesis put forward to explain the evolution of EFNs proposes that EFNs ldquodistractrdquo ants
from consuming floral nectar (Kerner 1878 Wagner and Kay 2002) Wagner and Kay (2002)
tested this hypothesis by constructing model plants using artificial nectaries assigned at random
to represent either floral or extrafloral nectaries They found that the presence of EFNs could
reduce ant exploitation of floral nectar However as highlighted by Galen (2005) this design is
closer to a plant making a surplus of flowers some of which will avoid ant visitation if visitation
rates increase sublinearly with increasing flower number Producing extra flowers to escape ant
visitation appears to be a viable strategy in some systems (Ashman and King 2005 Galen 2005)
However Galen (2005) found that applying honey (simulating extrafloral nectar) to P viscosum
17
leaves had the opposite effect and increased ant visits to flowers Only two studies both in
Pachycereus schottii have found evidence for the distraction hypothesis under field conditions
(Chamberlain and Holland 2008 Holland et al 2011)
There is limited empirical support for the distraction hypothesis but abundant evidence
that EFNs attract ants that defend plants against herbivores (reviewed in Rosumek et al 2009)
To function as attractants the presence of EFNs must increase ant abundance on plants
However because of the increased numbers attracted EFNs would not be expected to reduce the
abundance of ants on nectar rewards or be able to satiate the ants recruited Only if ant
abundance stops increasing at a reward density much lower than that available on the plant
would EFNs serve to distract ants In this case the amount of rewards may overwhelm ants
leaving some flowers underexploited However even in P schottii ants still visited flowers
(Chamberlain and Holland 2008) In cases where ant abundance would continue to increase with
additional rewards it is unlikely that EFNs function as distractions
ii) Chemical resistance traits
a) Toxic nectar
One of the earliest proposed floral defenses against ants was toxic nectar (Janzen 1977)
However studies testing Janzenrsquos (1977) hypothesis uncovered only a very few isolated
examples (eg Catalpa speciosa Stephenson 1981 Crinum erubescens Guerrant and Fiedler
1981) in short most floral nectar is not toxic or repellent to ants (Baker and Baker 1978
Schubart and Anderson 1978) In fact ants sometimes prefer floral nectar over solutions with
equivalent sugar concentrations (Koptur and Truong 1998)
18
In contrast floral nectar can repel other flower visitors (eg birds Johnson et al 2006
lepidopterans Kessler and Baldwin 2007) Repellency is often caused by plant secondary
metabolites which may occur in nectar as a by-product of their production elsewhere in the
plant where they function as defenses against herbivory (Adler 2001) Alternatively toxic or
repellent nectar may be adaptive repellent nectar may promote effective pollination by reducing
the amount of reward any one pollinator takes spreading nectar over several pollinator visits
(Kessler and Baldwin 2007) or it may function as a filter selecting for specific pollinators
(Johnson et al 2006) The latter may explain why repellent nectar is not more common
legitimate pollinators in addition to floral larcenists may be deterred from visiting flowers with
repellent nectar This may be particularly true of nectars that would repel ants which are closely
related to bees nectar distasteful to ants may often also be distasteful to bees and those nectars
acting as a filter for bees may still be palatable to ants For example Prŷs-Jones and Willmer
(1992) found that the ammonia-containing nectar of Lathraea clandestina is readily consumed
by both bees and ants even though it deters birds that act as nectar thieves Although floral
nectar itself rarely repels ants Guerrant and Fielder (1981) found that the presence of macerated
petals within nectar can often keep ants away Alkaloids in petals may be responsible and might
explain why ants seldom act as primary nectar robbers these secondary compounds may prevent
ants from chewing through corollas
b) Floral volatiles
Repellent floral volatiles appear to be a more widespread form of chemical resistance against
ants These were reviewed recently by Willmer et al (2009) and will be discussed here only
briefly Numerous plant taxa produce floral scents repellent to ants including myrmecophilous
19
myrmecophytic and non-ant associated plants in both temperate and tropical ecosystems
(Willmer et al 2009 Junker et al 2011a Ballantyne and Willmer 2012) In three large studies
floral volatiles that repel ants were detected in about a third of all plants tested (Willmer et al
2009 Junker et al 2011a Ballantyne and Willmer 2012) Pollen is often the source of ant-
repellent volatile compounds (Willmer et al 2009 Ballantyne and Willmer 2012) but they can
be emitted from other floral parts including petals That pollen is often the source of repellence
could suggest that preventing ant visitation may be more important in protecting male than
female function Additionally because repellence would decrease as pollen is removed from
anthers ants may return and defend flowers more quickly than if repellent chemicals were in
longer-lasting structures (eg petals) In some cases floral volatiles are thought to mimic ant
alarm pheromones (Willmer et al 2009) However floral scents elicit inconsistent reactions
across ant species ranging from repellence to attraction (Agarwal and Rastogi 2007 Ballantyne
and Willmer 2012)
Producing structural or chemical resistance traits may be costly for plants Native
Hawaiian plants which have little recent coevolutionary history with ants appear to have
secondarily lost floral ant repellents (Junker et al 2011a) Both Willmer et al (2009) and Junker
et al (2011a) found that highly chemical repellent flowers did not have strong physical defenses
and vice versa suggesting trade-offs between the two (but see Ballantyne and Willmer 2012)
Like toxic nectar floral ant repellents might sometimes deter both pollinators and ants For
example Galen et al (2011) found that 2-phenylethanol in P viscosum floral scent repels both
flower-damaging F neorufibarbis ants and the plantrsquos main pollinator Bombus kirbyellus
Interestingly four recent studies have found that floral scents can also attract ants
(Agarwal and Rastogi 2007 Edwards and Yu 2008 Schiestl and Glaser 2011 Gonzaacutelvez et al
20
2012) One study (Schiestl and Glaser 2011) has shown that floral scents attract ants to a
potentially ant-pollinated plant and such systems may provide many more examples
4 Future directions
We have highlighted areas in need of further investigation throughout the review Here we focus
on several questions we consider high priorities for future research
i) Floral defenses against ants
a) Do floral resistance traits increase plant fitness
The existence ant-repellent floral traits is often taken as evidence that ants negatively affect plant
reproductive success However despite the abundance and variety of mechanisms excluding ants
from flowers few studies show that ants reduce plant fitness when they visit flowers (Table 11)
This absence is particularly apparent with repellent floral volatiles which may be the most
widespread floral resistance trait (Willmer et al 2009 Junker et al 2011a Ballantyne and
Willmer 2012) To our knowledge only one study has shown that floral ant repellents benefit
plants with repellent P viscosum flowers avoiding damaging ant visits (Galen et al 2011)
Research is urgently needed to investigate if these traits increase plant fitness by excluding ants
or if ant visitation is inconsequential More studies taking advantage of standing variation in
traits (Galen et al 2011) or creating it artificially (eg Junker et al 2010b) will allow us to
distinguish between the two
21
b) If ant visitation is often not costly what caused the evolution of repellent traits and
what maintains them Similarly are these repellent traits truly defenses against ant
visitation or are they generalized defenses against a range of floral antagonists
Ant-repellent floral traits are widespread and their production may be costly (Willmer et al
2009 Junker et al 2011a) The studies in Table 11 however indicate that flower-visiting ants
often do not disrupt plant reproduction particularly outside of myrmecophytes What then
selected for these traits One possibility is that these repellents are not directed solely at ants
Instead a community of antagonistic flower visitors may exert diffuse selection on plants
thereby selecting for and maintaining these traits Structural modifications may repel crawling
insects while chemical repellents such as floral volatiles may repel both flying and non-flying
insects (Junker et al 2011b) How much overlap there is in floral volatiles that repel both ants and
other antagonists needs investigation Until we examine how specific these defenses are to ants
we risk overstating the importance of ants in their evolution
ii) Do ants alter pollen movement patterns or reduce male fitness
We are aware of only four studies that have measured if ant visitation impacts male fitness
(Wyatt 1980 Fritz and Morse 1981 Ashman 2000 Galen and Geib 2007) While none of these
studies found an effect all measured only amounts of pollen or pollinia removed However this
measure does not adequately encompass male fitness alterations to pollen movement patterns
and eventual deposition location may be more important to male function than changes in
removal rates If as suggested by some studies (Altshuler 1999 Lach 2007) ants increase
pollinator relocation rates they may decrease pollen discounting increasing male fitness
However if this increased relocation reduces overall pollen export rates ant competition with
22
pollinators may depress male function Studies using pollen-tracking methods such as fluorescent
dyes will allow us to see how ants change pollen movement patterns and methods like paternity
analysis will reveal how competition between ants and pollinators alters male fitness
iii) Coevolution between ants and plants do myrmecophytes exhibit stronger more
specific defenses against ants
The floral traits of myrmecophytes are most likely to coevolve with ants because all plant
reproduction occurs in the presence of ants Their floral defenses would thus be expected to be
both stronger and more specific to their resident ants than those other plants visited by an array
of more opportunistic ant species Some evidence supports this Willmer et al (2009) found that
resident ants of acacias were repelled by flowers with more aggressive ant species showing
stronger repellence than less aggressive ants Additionally ants not normally associated with
acacias did not respond to acacia floral volatiles More studies are needed in other systems and
should take advantage of phylogenetic approaches examining floral traits in closely related
myrmecophytic and non-myrmecophytic plant species to determine whether ants commonly
select for floral defenses against ants in myrmecophytes
Acknowledgements
For comments and discussion we thank the Frederickson lab particularly Eric Youngerman
Lina Arcila Hernandez and Kyle Turner as well as Alison Parker and Jordan Pleet MEF
acknowledges financial support from an NSERC Discovery Grant a Connaught New Researcher
23
Award an Ontario Ministry of Economic Development and Innovation Early Researcher Award
and the University of Toronto ARC was supported by an Ontario Graduate Scholarship and
Sigma Xi
24
References
Adler L S 2001 The ecological significance of toxic nectar Oikos 91409ndash420
Agarwal V M and N Rastogi 2007 Role of floral repellents in the regulation of flower visits
of extrafloral nectary-visiting ants in an Indian crop plant Ecological Entomology 3359ndash
65
Altshuler D L 1999 Novel interactions of non-pollinating ants with pollinators and fruit
consumers in a tropical forest Oecologia 119600ndash606
Ashman TL 2000 Pollinator selectivity and its implications for the evolution of dioecy and
sexual dimorphism Ecology 812577-2591
Ashman TL and E A King 2005 Are flower-visiting ants mutualists or antagonists A study
in a gynodioecious wild strawberry American Journal of Botany 92891ndash5
Ballantyne G and P Willmer 2012 Floral visitors and ant scent marks noticed but not used
Ecological Entomology 37402-409
Ballantyne G and P Willmer 2012 Nectar theft and floral ant-repellence a link between
nectar volume and ant-repellent traits PloS One 7e43869
Baker H G and I Baker 1978 Ants and flowers Biotropica 1080
Barrett S C H 2002 Sexual interference of the floral kind Heredity 88154ndash159
Baroni Urbani C and M L de Andrade 1997 Pollen eating storing and spitting by ants
Naturwissenschaften 84256ndash258
25
Beattie A J C Turnbull R B Knox and E G Williams 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany
71421ndash426
Beattie A J C Turnbull T Hough S Jobson and R B Knox 1985 The vulnerability of
pollen and fungal spores to ant secretions evidence and some evolutionary implication
American Journal of Botany 72606ndash614
Blancafort X and C Goacutemez 2005 Consequences of the Argentine ant Linepithema humile
(Mayr) invasion on pollination of Euphorbia characias (L) (Euphorbiaceae) Acta
Oecologica 2849ndash55
Bleil R N Bluumlthgen and R R Junker 2011 Ant-plant mutualism in Hawailsquoi Invasive ants
reduce flower parasitism but also exploit floral nectar of the endemic shrub Vaccinium
reticulatum (Ericaceae) Pacific Science 65291ndash300
Bluumlthgen N G Gottsberger and K Fiedler 2004 Sugar and amino acid composition of ant-
attended nectar and honeydew sources from an Australian rainforest Austral Ecology
29418ndash429
Burkle L A R E Irwin and D A Newman 2007 Predicting the effects of nectar robbing on
plant reproduction implications of pollen limitation and plant mating system American
Journal of Botany 941935ndash1943
Byk J and K Del-Claro 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333ndash38
26
Caballero P C G Ossa W L Gonzaacuteles C Gonzaacutelez-Browne G Astorga M M Muruacutea and
R Medel 2013 Testing non-additive effects of nectar-robbing ants and hummingbird
pollination on the reproductive success of a parasitic plant Plant Ecology 214633ndash640
Carvalheiro L G E R M Barbosa and J Memmott 2008 Pollinator networks alien species
and the conservation of rare plants Trinia glauca as a case study Journal of Applied
Ecology 451419ndash1427
Chamberlain S A and J N Holland 2008 Density-mediated context-dependent consumer-
resource interactions between ants and extrafloral nectar plants Ecology 891364ndash1374
Creighton W S 1967 Studies on free colonies of Cryptocerus texanus Satschi (Hymenoptera
Formicidae) Psyche 7434ndash42
Cursach J and J Rita 2011 Implications of the reproductive biology of the narrow endemic
Naufraga balearica (Apiaceae) for its conservation status Plant Systematics and Evolution
298581ndash596
Cushman JH and Addicott JF In Huxley CR and DF Cutler (eds) 1st edition Ant-plant
interactions Oxford University Press Oxford
de Jong T J N M Waser and P G Klinkhamer 1993 Geitonogamy the neglected side of
selfing Trends in Ecology and Evolution 8321ndash325
de Vega C M Arista P L Oritz C M Herrera and S Talavera 2009 The ant-pollination
system of Cytinus hypocistis (Cytinaceae) a Mediterranean root holoparasite Annals of
Botany 4517ndash8
27
Dukas R 2001 Effects of perceived danger on flower choice by bees Ecology Letters 4327ndash
333
Dutton E M and M E Frederickson 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561ndash569
Edwards D P and D W Yu 2008 Tolerating castration by hiding flowers in plain sight
Behavioral Ecology and Sociobiology 6395ndash102
Frederickson M E 2009 Conflict over reproduction in an ant-plant symbiosis why Allomerus
octoarticulatus ants sterilize Cordia nodosa trees The American Naturalist 173675ndash81
Fritz R S and D H Morse 1981 Nectar parasitism of Asclepias syriaca by ants effect on
nectar levels pollinia insertion pollinaria removal and pod production Oecologia 50316ndash
319
Galen C 1983 The effects of nectar thieving ants on seedset in floral scent morphs of
Polemonium viscosum Oikos 41245ndash249
Galen C 2005 Catching ants with honey an experimental test of distraction and satiation as
alternative modes of escape from flower-damaging ants Oecologia 14480ndash87
Galen C and B Butchart 2003 Ants in your plants effects of nectar-thieves on pollen fertility
and seed-siring capacity in the alpine wildflower Polemonium viscosum Oikos 101521ndash
528
Galen C and J Cuba 2001 Down the tube pollinators predators and the evolution of flower
shape in the alpine skypilot Polemonium viscosum Evolution 551963-1971
28
Galen C and J C Geib 2007 Density-dependent effects of ants on selection for bumble bee
pollination in Polemonium viscosum Ecology 881202ndash1209
Galen C R Kaczorowski S L Todd J Geib and R A Raguso 2011 Dosage-dependent
impacts of a floral volatile compound on pollinators larcenists and the potential for floral
evolution in the alpine skypilot Polemonium viscosum The American Naturalist 177258ndash
272
Garciacutea M B R J Antor and X Espadaler 1995 Ant pollination of the palaeoendemic
dioecious Borderea pyrenaica (Dioscoreaceae) Plant Systematics and Evolution 19817ndash
27
Gaume L M Zacharias and R M Borges 2005 Ant ndash plant conflicts and a novel case of
castration parasitism in a myrmecophyte Evolutionary Ecology Research 7435ndash452
Graystock P and W O H Hughes 2011 Disease resistance in a weaver ant Polyrhachis
dives and the role of antibiotic-producing glands Behavioral Ecology and Sociobiology
652319ndash2327
Gόmez J M 2000 Effectiveness of ants as pollinators of Lobularia maritima effects on main
sequential fitness components of the host plant Oecologia 12290ndash97
Gόmez J M and R Zamora 1992 Pollination by ants consequences of the quantitative effects
on a mutualistic system Oecologia 91410ndash418
Gόmez J M R Zamora J A Hόdar and D Garciacutea 1996 Experimental study of pollination
by ants in Mediterranean high mountain and arid habitats Oecologia 105236ndash242
29
Gonccedilalves-Souza T P M Omena J C Souza and G Q Romero 2008 Trait-mediated effects
on flowers artificial spiders deceive pollinators and decrease plant fitness Ecology
892407ndash2413
Gonzaacutelvez F G L Santamariacutea R T Corlett and M A Rodriacuteguez-Gironeacutes 2012 Flowers
attract weaver ants that deter less effective pollinators Journal of Ecology 10178ndash85
Guerrant E O and P L Fiedler 1981 Flower defenses against nectar-pilferage by ants
Biotropica 1325ndash33
Harley R 1991 The greasy pole syndrome In Huxley CR and DF Cutler (eds) 1st edition
Ant-plant interactions Oxford University Press Oxford
Hansen D M and C B Muumlller 2009 Invasive ants disrupt gecko pollination and seed
dispersal of the endangered plant Roussea simplex in Mauritius Biotropica 41202ndash208
Hickman J C 1974 Pollination by ants a low-energy system Science 1841290ndash1292
Holland J N S a Chamberlain and T E X Miller 2011 Consequences of ants and
extrafloral nectar for a pollinating seed-consuming mutualism ant satiation floral
distraction or plant defense Oikos 120381ndash388
Houmllldobler B and Wilson E O 1990 The Ants 1st Edition Harvard University Press
Cambridge
Holway DA L Lach AV Suarez ND Tsutsui and TJ Case 2002 The causes and
consequences of ant invasions Annual Review of Ecology and Systematics 33181-233
30
Horskins K and Turner VB 1999 Resource use and foraging patterns of honeybees Apis
mellifera and native insects on flowers of Eucalyptus costata Australian Journal of
Ecology 24221-227
Horvitz C C and D W Schemske 1984 Effects of ants and an ant-tended herbivore on seed
production of a neotropical herb Ecology 651369ndash1378
Inouye DW 1980 The terminology of floral larceny Ecology 611251-1253
Inouye DW and O R J Taylor 1979 A temperate region plant-ant-seed predator system
consequences of extra floral nectar secretion by Helianthella quinquenervis Ecology 601ndash
7
Irwin RE 2003 Impact of nectar robbing on estimates of pollen flow conceptual predictions
and empirical outcomes Ecology 84485-495
Irwin RE and Maloof JE 2002 Variation in nectar robbing over time space and species
Oecologia 133525-533
Iwanami Y and T Iwadare 1978 Inhibiting effects of myrmicacin on pollen growth and pollen
tube mitosis Botanical Gazette 13942ndash45
Izzo TJ and Vasconcelos HL 2002 Cheating the cheater domatia loss minimizes the effects
of ant castration in an Amazonian ant plant Oecologia 133200-205
Janzen D H 1977 Why donrsquot ants visit flowers Biotropica 9252
Johnson SD Hargreaves AL and Brown M 2006 Dark bitter-tasting nectar functions as a
filter of flower visitors in a bird-pollinated plant Ecology 872709-2716
31
Junker R A Y C Chung and N Bluumlthgen 2007 Interaction between flowers ants and
pollinators additional evidence for floral repellence against ants Ecological Research
22665ndash670
Junker R R R Bleil C C Daehler and N Bluumlthgen 2010a Intra-floral resource partitioning
between endemic and invasive flower visitors consequences for pollinator effectiveness
Ecological Entomology 35760ndash767
Junker R R and N Bluumlthgen 2010b Floral scents repel facultative flower visitors but attract
obligate ones Annals of botany 105777ndash82
Junker R R C C Daehler S Doetteri A Keller and N Bluumlthgen 2011a Hawaiian ant ndash
flower networks nectar-thieving ants prefer undefended native over introduced plants with
floral defenses Ecological Monographs 81295ndash311
Junker R R J Gershenzon and S B Unsicker 2011b Floral odor bouquet loses its ant
repellent properties after inhibition of terpene biosynthesis Journal of Chemical Ecology
371323ndash31
Kawakita A and M Kato 2002 Floral biology and unique pollination system of root
holoparasites Balanophora kuroiwai and B tobiracola (Balanophraceae) American
Journal of Botany 891164ndash1170
Koopowitz H and TA Marchant 1998 Postpollination nectar reabsorption in the African
epiphyte Aerangis verdickii (Orchidaceae) American Journal of Botany 85508-512
Kirk W D J 1984 Ecological studies on Thrips imaginis Bagnall (Thysanoptera) in flowers of
Echium plantagineum L in Australia Australian Journal of Ecology 99ndash18
32
Kerner A 1878 Flowers and their unbidden guests C Kegan Paul London
Kessler D and I T Baldwin 2007 Making sense of nectar scents the effects of nectar
secondary metabolites on floral visitors of Nicotiana attenuata The Plant Journal 49840ndash
54
Koptur S and N Truong 1998 Facultative ant-plant interactions nectar sugar preferences of
introduced pest species in South Florida Biotropica 30179-189
Lach L 2005 Interference and exploitation competition of three nectar-thieving invasive ant
species Insectes Sociaux 52257ndash262
Lach L 2007 A mutualism with a native membracid facilitates pollinator displacement by
Argentine ants Ecology 881994ndash2004
Lach L 2008a Argentine ants displace floral arthropods in a biodiversity hotspot Diversity and
Distributions 14281ndash290
Lach L 2008b Floral visitation patterns of two invasive ant species and their effects on other
hymenopteran visitors Ecological Entomology 33155ndash160
Lach L 2013 A comparison of floral resource exploitation by native and invasive Argentine
ants Arthropod-Plant Interactions 7177-190
Luo C W K Li X M Chen Z Y Huang and K U N Li 2012 Ants contribute significantly
to the pollination of a biodiesel plant Jatropha curcas Environmental Entomology
411163ndash1168
33
Maleacute PJ G C Leroy A Dejean A Quilichini and J Orivel 2011 An ant symbiont directly
and indirectly limits its host plantrsquos reproductive success Evolutionary Ecology 2655ndash63
Maloof J E 2001 The effects of a bumble bee nectar robber on plant reproductive success and
pollinator behavior American Journal of Botany 881960ndash1965
Maloof J E and D W Inouye 2000 Are nectar robbers cheaters or mutualists Ecology
812651ndash2661
Moreau CS CD Bell R Vila SB Archibald and NE Pierce 2006 Phylogeny of the ants
diversification in the age of the angiosperms Science 312101-104
Nahas L M O Gonzaga and K Del-Claro 2012 Emergent impacts of ant and spider
interactions herbivory reduction in a tropical savanna tree Biotropica 44498ndash505
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506ndash514
Newman D A and J D Thomson 2005 Interactions among nectar robbing floral herbivory
and ant protection in Linaria vulgaris Oikos 110497ndash506
Nicklen E F and D Wagner 2006 Conflict resolution in an ant-plant interaction Acacia
constricta traits reduce ant costs to reproduction Oecologia 14881ndash7
Norment C J 1988 The effect of nectar-thieving ants on the reproductive success of Frasera
speciosa (Gentianaceae) American Midland Naturalist 120331ndash336
34
Orivel J L Lambs P-J G Maleacute C Leroy J Grangier T Otto A Quilichini et al 2011
Dynamics of the association between a long-lived understory myrmecophyte and its
specific associated ants Oecologia 165369ndash76
Palmer T M D F Doak M L Stanton J L Bronstein E T Kiers T P Young J R
Goheen et al 2010 Synergy of multiple partners including freeloaders increases host
fitness in a multispecies mutualism Proceedings of the National Academy of Sciences
10717234ndash9
Peakall R A J Beattie and S H James 1987 Pseudocopulation of an orchid by male ants a
test of two hypotheses accounting for the rarity of ant pollination Oecologia 73522ndash524
Peakall R and A J Beattie 1989 Pollination of the orchid Microtis parviflora R Br by
flightless worker ants Functional Ecology 3515ndash522
Peakall R C J Angus A J Beattie 1990 The significance of ant and plant traits for ant
pollination in Leporella fimbriata pollination Oecologia 84457ndash460
Puterbaugh M N 1998 The roles of ants as flower visitors experimental analysis in three
alpine plant species Oikos 8336ndash46
Prŷs-Jones OE and Willmer PG 1992 The biology of alkaline nectar in purple toothwort
(Lathraea clandenstina) ground level defenses Biological Journal of the Linnean Society
45373-388
Raine NE Willmer P and Stone GN 2002 Spatial structuring and floral avoidance
behaviour present in ant-pollinator conflict in a Mexican ant-acacia Ecology 833086-
3096
35
Ramsey M 1995 Ant pollination of the perennial herb Blandfordia grandiflora (Liliaceae)
Oikos 74265ndash272
Renault CK LM Buffa and Delfino MA 2005 An aphid-ant interaction effects on
different trophic levels Ecological Research 2071-74
Rico-Gray V and L B Thien 1989 Effect of different ant species on reproductive fitness of
Schomburgkia tibicinis (Orchidaceae) Oecologia 81487ndash489
Romero G Q P A P Antiqueira and J Koricheva 2011 A meta-analysis of predation risk
effects on pollinator behaviour PLoS One 6e20689
Rosumek FB FAO Silveira F de S Neves NP de U Barbosa L Diniz Y Oki F Pezzini
et al 2009 Ants on plants a meta-analysis of the role of ants as plant biotic defenses
Oecologia 160537ndash549
Roubik D W 1982 The ecological impact of nectar-robbing bees and pollinating
hummingbirds on a tropical shrub Ecology 63354ndash360
Rudgers J A 2004 Enemies of herbivores can shape plant traits selection in a facultative ant-
plant mutualism Ecology 85192ndash205
Schatz B and WT Wcislo 1999 Ambush predation by the ponerine ant Ectatomma ruidum
Roger (Formicidae) on a sweat bee Lasioglossum umbripenne (Halictidae) in Panama
Journal of Insect Behavior 12641-663
36
Schaffer W M D W Zeh S L Buchmann S Kleinhaus M V Schaffer and J Antrim
1983 Competition for nectar between introduced honey bees and native North American
bees and ants Ecology 64564ndash577
Schemske D W 1980 The evolutionary significance of extrafloral nectar production by Costus
woodsonii (Zingiberaceae) an experimental analysis of ant protection Journal of Ecology
68959ndash967
Schiestl F P and F Glaser 2011 Specific ant-pollination in an alpine orchid and the role of
floral scent in attracting pollinating ants Alpine Botany 1221ndash9
Schubart HOR AB Anderson 1978 Why donrsquot ants visit flowers A response to D H
Janzen Biotropica 10310-311
Schuumlrch S M Pfunder and B A Roy 2000 Effects of ants on the reproductive success of
Euphorbia cyparissias and associated pathogenic rust fungi Oikos 16ndash12
Stanton ML Palmer TM Young TP Evans A and Turner ML 1999 Sterilization and
canopy modification of swollen thorn acacia tree by a plant-ant Nature 401 578-581
Stephenson AG 1981 Toxic nectar deters nectar thieves of Catalpa speciosa American
Midland Naturalist 105381-383
Stout J C and D Goulson 2001 The use of conspecific and interspecific scent marks by
foraging bumblebees and honeybees Animal Behaviour 62183ndash189
Sugiura S T Abe and S Makino 2006 Loss of extrafloral nectary on an oceanic island plant
and its consequences for herbivory American Journal of Botany 93491ndash495
37
Svensson L 1985 An estimate of pollen carryover by ants in a natural population of
Scleranthus perennis L (Caryophyllaceae) Oecologia 66373ndash377
Tsuji K A Hasyim and K Nakamura 2004 Asian weaver ants Oecophylla smaragdina and
their repelling of pollinators Ecological Research 19669ndash673
Turner R C J J Midgley P Barnard R E Simmons and S D Johnson 2012 Experimental
evidence for bird pollination and corolla damage by ants in the short-tubed flowers of
Erica halicacaba (Ericaceae) South African Journal of Botany 7925ndash31
Visser D M G Wright and J H Giliomee 1996 The effect of the Argentine ant
Linepithema humile (Mayr) (Hymenoptera Formicidae) on flower-visiting insects of
Protea nitida Mill (Proteaceae) African Entomology 4285ndash287
Wagner D 2000 Pollen viability reduction as a potential cost of ant association for Acacia
constricta (Fabaceae) American Journal of Botany 87711ndash715
Wagner D and A Kay 2002 Do extrafloral nectaries distract ants from visiting flowers An
experimental test of an overlooked hypothesis Evolutionary Ecology Research 4293ndash305
Wang C Y Luo Y Tai D An and Y Kou 2008 Ants pollinate Neottia listeroides
(Orchidaceae) in Sichuan China 46836ndash846
Wyatt R 1980 The impact of nectar-robbing ants on the pollination system of Asclepias
curassavica Bulletin of the Torrey Botanical Club 10724ndash28
Wheeler WM and IW Bailey 1920 The feeding habits of Pseudomymine and other ants
Transactions of the American Philosophical Society 22235-279
38
Willmer P G and S A Corbet 1981 Temporal and microclimatic partitioning of the floral
resources of Justicia aurea amongst a concourse of pollen vectors and nectar robbers
Oecologia 5167ndash78
Willmer P G and G N Stone 1997 How aggressive ant-guards assist seed-set in Acacia
flowers Nature 388165ndash167
Willmer P G C V Nuttman N E Raine G N Stone J G Pattrick K Henson P Stillman
et al 2009 Floral volatiles controlling ant behaviour Functional Ecology 23888ndash900
Young T P C H Stubblefield and L a Isbell 1997 Ants on swollen-thorn acacias species
coexistence in a simple system Oecologia 10998ndash107
Yu D W and N E Pierce 1998 A castration parasite of an ant-plant mutualism Proceedings
of the Royal Society B Biological Sciences 265375ndash382
39
Tables
Table 11 Studies of ant-flower or ant-pollinator interactions that included a measure of plant fitness Each study system is listed only
once except when results varied between years or when different studies examined male and female fitness Significant effects of ants on
plant fitness are either lsquopositiversquo or lsquonegativersquo non-significant effects are lsquoneutralrsquo When ants significantly affected only one fitness
component but several were measured (eg fruit set and seed production) we still consider the result significant
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Acacia
drepanolobium
Crematogaster
nigriceps
Proportion
fruiting
Negative Destruction of floral buds Young et al 1997
Asclepias exaltata Several Pollinia inserted
removed
Negative for pollinia
insertion neutral for
pollinia removal
Exploitative competition Wyatt 1980 T
40
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Asclepias syriaca Several
Fruit set pollinia
removed
Negative for fruit
set neutral for
pollinia removal
Exploitative competition Fritz and Morse
1981 T
Cordia nodosa Allomerus cf
demerarae
Fruit set Negative Destruction of floral buds Yu and Pierce
1998
Costus woodsonii Wasmannia
auropunctata
Seed set Positive Unknown ant pollination
suggested (but unlikely)
Schemske 1980
Eritrichum aretioides F neorufibarbis
gelida
Seed set Negative Ants damaged flowers Puterbaugh 1998
41
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Euphorbia characias Several Seed set Neutral Plants in Argentine ant-
invaded area set less seed
than those in un-invaded
sites but an ant-exclusion
experiment did not produce
significant results
Blancafort and
Gόmez 2005
Ferocactus wislizeni Solenopsis xyloni
and others
Seed set Unknown negative Interference and exploitative
competition S xyloni
reduced seed set relative to
other ants but the study did
not include a no-ant control
Ness 2006
42
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Frasera speciosa F neorufibarbis
and Formica sp
Seed set Neutral Nocturnal pollination
occurring when ants were
less active may have
compensated for reduced
diurnal pollinator visitation
Norment 1988
Gypsophila struthium Several Seeds per
inflorescence
Neutral No effect despite large
numbers of ants on flowers
Gόmez et al
1996
Hirtella physophora Allomerus
decemarticulatus
Fruit set Negative Destruction of flowers Orivel et al
2011
Humboldtia brunonis Crematogaster
dohrni
Fruit set Negative Destruction of flowers Gaume et al
2005
43
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Lepidium subulatum Several Percentage of
ovules setting
seed
Positive Unknown Gόmez et al
1996
Leucospermum
conocarpodendron
Several Seed set Neutral Site not pollen limited Lach 2008
Linaria vulgaris Several ldquoFemale fitness
indexrdquo
(combination of
number of seeds
fruits flowers
and seed weight)
Positive Reduction in florivory and
seed predation nectar
robbing holes increased ant
visitation
Newman and
Thomson 2005
44
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Melastoma
malabathricum
Oecophylla
smaragdina
Seed set Positive Ants deterred less efficient
pollinators increasing
visitation by the more
efficient pollinator
Gonzaacutelvez et al
2012
Metrosideros
polymorpha
Several Fruit set Neutral Ants reduced nectar
collection rates by foraging
bees but had no effect on
pollen collection rates or
fruit set
Junker et al
2010a
Orexis alpine F neorufibarbis
gelida
Seed set Neutral Autogamy or not pollen
limited
Puterbaugh 1988
45
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Ouratea spectabilis Cephalotes
pusillus and others
Seeds per fruit Neutral positive C pusillus had no effect on
seed number but other ants
collectively increased seed
set
Byk and Del
Claro 2010
Pachycereus schottii Several Seed set Neutral positive In one year ants showed a
positive effect (seed
number 2008) but no effect
with increased sample sizes
Chamberlain and
Holland 2008
Polemonium
viscosum
F neorufibarbis
gelida
Seed set Negative Ants damaged styles Galen 1983
46
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Polemonium
viscosum
F neorufibarbis
gelida
Pollen removal Neutral Ants did not affect pollen
removal even at high ant
densities
Galen and Geib
2007 T
Protea nitida Linepithema
humile and others
Seed set Neutral positive L humile had no effect on
P nitida seed set but native
ants increased it in one year
ants may cause pollinators
to relocate
Lach 2007
Psychotria
limonensis
Ectatomma spp Fruit set Positive Suggested increased
pollinator relocation
Altshuler 1999
47
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Roussea simplex Technomyrmex
albipes
Seed set Negative Interference and exploitative
competition
Hansen and
Muumlller 2009
Tristerix aphyllus Several Fruit set Neutral Nectar robbing ants
minimally reduced nectar
levels and had no effect on
hummingbird visitation
Caballero et al
2013
Vaccinium
reticulatum
Several Fruit set Neutral Ants reduced florivory but
did not affect fruit set
Bleil et al 2011
T Study measured male fitness
Study of sterilizing ants showed short-term decrease in female fitness long-term effects are unclear (see text)
48
Table 12 Studies demonstrating either i) ant pollination through an exclusion experiment or ii) that ants transfer pollen (or pollen
analogue) between flowers strongly suggesting ant pollination
Plant species Ant species Ant pollination Evidence Reference
Alyssum purpureum Several Yes Exclusion experiment Gόmez et al 1996
Arenaria tetraquetra Several Yes Exclusion experiment Gόmez et al 1996
Balanophora kuroiwai Leptothorax sp Suggested Exclusion experiment and
observed pollen transfer
and pollen tube formation
but did not see if plants set
seed
Kawakita and Kato 2002
Blandfordia grandiflora Iridomyrmex sp Yes Exclusion experiment Ramsey 1995
Borderea pyrenaica Several Yes Exclusion experiment Garciacutea et al 1995
49
Plant species Ant species Ant pollination Evidence Reference
Cytinus hypocistis Several Yes Exclusion experiment de Vega et al 2009
Epipactis thunbergii Camponotus
japonicas
Suggested Observed transfer of
pollinia from ants to
orchids
Sugiura et al 2006
Euphorbia cyparissias Several Yes Exclusion experiment Schuumlrch et al 2000
Fragaria virginiana Several Very likely Ant-only treatments set
comparable seed to flying
pollinator-only treatment
seed set increased with
increasing ant visitation
Ashman and King 2005
Frasera speciosa Several Yes Exclusion experiment Norment 1988
Frankenia thymifolia Several Yes Exclusion experiment Gόmez et al 1996
50
Plant species Ant species Ant pollination Evidence Reference
Hormathophylla spinosa Proformica
longiseta
Yes Exclusion experiment Gόmez and Zamora 1992
Jatropha curcas
Several
Tapinoma
melanocephalum
most common
Yes Exclusion experiment Luo et al 2012
Leporella fimbriata Myrmecia urens Yes Observed pollinia transfer
and subsequently
confirmed as ant
pollination
Peakall et al 1987
51
Plant species Ant species Ant pollination Evidence Reference
Lobularia maritima Several
Camponotus
micans most
common
Yes Exclusion experiment Gόmez 2000
Microtis parviflora
Iridomyrmex
gracilis
Yes Observation used
laboratory colonies to
replicate observations
Peakall and Beattie 1989
Naufraga balearica
Several
especially
Plagiolepis
pygmaea and
Lasius grandis
Suggested Observation over three
years of study only ants
were observed visiting
ants carried pollen
Cursach and Rita 2011
52
Plant species Ant species Ant pollination Evidence Reference
Neottia listeroides Leptothoras sp
Paratrechina sp
Suggested Observed transfer of
pollinia from ants to
orchids
Wang et al 2008
Paronychia pulvinata Formica
neorufibarbis
gelida
Yes Exclusion experiment Puterbaugh 1998
Polygonum cascadense Formica argentea
Very likely Grew plants in greenhouse
open to flying visitors with
minimal seed set only
plants with access to ants
set significant seed
Hickman 1974
53
Plant species Ant species Ant pollination Evidence Reference
Retama sphaerocarpa Several Yes Exclusion experiment Gόmez et al 1996
Scleranthus perennis
Several
especially
Formica
rufibarbis
Suggested Ants transferred pollen
analogue to stigmas and
carried pollen on bodies
Svensson 1985
Sedum anglicum Proformica
longiseta
Yes Exclusion experiment Gόmez et al 1996
Trinia glauca Several Yes Exclusion experiment Carvalheiro et al 2008
54
Chapter 2 Ants and ant scent reduce bumblebee pollination of artificial flowers
Cembrowski AR Tan MG Thomson JD and Frederickson ME
In press The American Naturalist
All authors designed the experiment ARC and MGT performed the experiment ARC and MEF
performed statistical analyse
s and ARC wrote the manuscript with input from all authors
Abstract
Ants on flowers can disrupt pollination by consuming rewards or harassing pollinators but it is
difficult to disentangle the effects of these exploitative and interference forms of competition on
pollinator behavior Using highly rewarding and quickly replenishing artificial flowers that
simulate male or female function we allowed bumblebees (Bombus impatiens) to forage (1) on
flowers with or without ants (Myrmica rubra) and (2) on flowers with or without ant scent cues
Bumblebees transferred significantly more pollen analogue both to and from ant free flowers
demonstrating that interference competition with ants is sufficient to modify pollinator foraging
behavior Bees also removed significantly less pollen analogue from ant scented flowers than
from controls making this the first study to show that bees can use ant scent to avoid harassment
at flowers Ant effects on pollinator behavior possibly in addition to their effects on pollen
viability may contribute to the evolution of floral traits minimizing ant visitation
55
Introduction
Trait-mediated indirect interactions arise when a focal species causes phenotypic changes
including behavioral modifications in a second species and these effects cascade to still other
species (Werner and Peacor 2003) Although trait-mediated indirect interactions are often studied
in food webs (eg Werner and Peacor 2003 Preisser et al 2005) they are not limited to trophic
interactions For example the threat of predation can change the behavior of a mutualist thus
affecting its partners (Suttle 2003) In animal-pollinated plants predators can disrupt pollination
directly through density-mediated indirect interactions (Dukas 2005) or via trait-mediated
indirect interactions by changing pollinator behavior When predators are present pollinators
may switch to visiting less rewarding flowers or they may avoid flowers altogether potentially
reducing the amount of pollen donated or received by a flower and thus decreasing plant fitness
(Gonccedilalves-Souza 2008 Ings and Chittka 2009) Similarly competition for floral rewards with
other flower visitors may cause behavioral changes in pollinators (Maloof and Inouye 2000
Ohashi et al 2008) Previous research has shown that bees may spend less time visiting flowers
depleted of rewards (Thomson 1986) and may avoid flowers bearing scent marks that indicate
recent visits by other individuals of the same or different bee species as these flowers are likely
to be depleted (Stout and Goulson 2002)
Whereas competition between pollinators may not be particularly costly for the plant
because both competitors are likely to provide pollination services competition between
pollinators and organisms that do not usually provide a pollination service such as ants has a
greater potential to reduce plant fitness Ants are common floral visitors they are attracted to
flowers for nectar (Lach 2007) and in some cases pollen (Byk and Del Claro 2010) and will
often defend these resources against other flower visitors (Altshuler 1999) However ants rarely
56
contribute much useful pollination antibiotic secretions present on their cuticles kill pollen
grains (Beattie et al 1985 Dutton and Frederickson 2012) and when foraging in flowers ants
sometimes impede female function by damaging stigmas (Galen and Cuba 2001) We will
henceforth consider ants to be non-pollinators and will contrast them to animals like bees that
are pollinators Ants are capable of competing with pollinators in two ways (1) by consuming
floral rewards and reducing their availability to pollinators (ie exploitative competition) and (2)
by directly antagonizing and excluding pollinators from flowers (ie interference competition)
Many plants benefit from the presence of ants numerous plant species recruit ants using
extrafloral nectar food bodies or domatia because ants provide protection against folivores (Heil
and McKey 2003) Similarly some plants have extrafloral nectaries on or near reproductive
tissues that recruit ants that deter florivores (eg Inouye and Taylor 1979) However plants
may incur reproductive costs as a result of their association with ants Some ant species that nest
in myrmecophytes sterilize flowers to shunt plant resources from reproduction to vegetative
growth allowing for greater ant colony growth (Frederickson 2009) Also ants visiting
extrafloral nectaries may be attracted to floral nectaries where they can harass pollinators (Ness
2006) There is growing evidence that many angiosperms have evolved floral traits that prevent
ants from accessing flowers during anthesis (Willmer and Stone 1997 Ballantyne and Willmer
2012) The effects of flower-visiting ants can be substantial Ants can decrease the frequency
duration or species diversity of pollinator visits to flowers (Lach 2008 Hansen and Muumlller 2009
Junker et al 2010 Gonzaacutelvez et al 2012) all of which potentially impact pollen donation and
receipt In some studies changes to pollinator behavior induced by ants have also been linked to
seed set
It is difficult however to disentangle the intertwined effects of exploitative and
interference competition in nature If ants disrupt pollination plants may often evolve traits to
57
deter ants from flowers (Willmer et al 2009) From a practical standpoint this would then limit
our ability to study the effects of ants on pollinator visitation because ants would not visit
flowers Here we used artificial flowers to explore ant-bee interactions in the absence of floral
defenses against ants We used highly rewarding quickly replenishing artificial flowers to
examine how direct harassment (ie interference competition expressed through behavior)
changes bee foraging behavior and how this affects donation and receipt of a pollen analogue
(powdered food dye) while minimizing the effects of exploitative competition We predicted
that flowers visited by ants would both donate and receive less pollen analogue than flowers
without ants because bees would avoid flowers with ants or leave them sooner Because scent
plays a large role in bumblebee communication and flower choice (Stout and Goulson 2002) we
also tested whether the presence or absence of ant scent on artificial flowers would affect pollen
analogue donation because of changes in the foraging behavior of bumblebees
Methods
i) Subjects
Myrmica rubra is an invasive ant in eastern North America with a range and habitat preferences
overlapping that of Bombus impatiens a common bumblebee Because M rubra visits flowers
(A Cembrowski pers obs) the two species likely interact in nature We collected 12 M rubra
colonies in the fall of 2011 and 2012 from Toronto Ontario Canada and the surrounding area
and maintained them in environmental chambers on artificial diet (Dussutour and Simpson 2009)
and a 1410 LD schedule (light 600-2200) We used these colonies as sources of M rubra
workers for experiments
58
Workers from commercially supplied colonies of Bombus impatiens (Biobest Canada
Ltd) foraged on artificial flowers in flight cages (either 24 x 24 x 21 m or 79 x 34 x 20m) at
the University of Toronto We tested a total of five bee colonies four colonies were used in the
ant presence trials and two of these and one additional colony were used in the ant scent trials
Flight cages had overhead fluorescent lights attached to timers In contrast to most previous
studies in which bees have been trained and tested individually the entire worker force of a bee
colony was free to forage at will in our experiments Colonies were trained to forage on artificial
flowers for at least four days before being used in trials After being used for a trial the colony
was not used for at least two days to reduce dye carryover between trials Between trials
bumblebees were fed pollen and given sugar water
ii) Artificial flowers
The flowers (Fig 1 Thomson et al 2012 see also Makino 2008) consisted of glass jars filled
with 30 WV sucrose solution Sugar water travelled by capillary action up a sewing-thread
wick to a hole in a blue-painted lid accumulating in a knot that acts as a nectary Flowers
depleted by visitors were quickly replenished via capillary action and were non-rewarding only
very briefly after visits taking less than a minute to accumulate 05 microl note that bumblebee
visits to a single flower are often separated by several minutes or more (A Cembrowski
unpublished data)
Unlike most previous artificial flower designs these flowers allow for estimation of male
and female fitness because we can measure both the amount and type of dye received by female
flowers (see (d) Dye quantification below) In order to access nectar on a ldquomalerdquo flower a bee
must crawl through a circular ldquoantherrdquo of brush-like weather stripping material dusted in a
59
consistent manner with our pollen analogue powdered food dye (FDampC 5 or 6) which is
transferred to the bee in the process On a ldquofemalerdquo flower a bee crawls through a sticky plastic
reinforcement that functions as a ldquostigmardquo receiving dye from the beersquos body Male flowers
dispensed dye particles over multiple bee visits and could still dispense dye at the end of trials
They resemble many real flowers or flower heads in that pollen is dispensed gradually over time
but less dye is available for transfer with each subsequent bee visit (Harder and Thomson 1989)
iii) Flight cage trials
We conducted 6 ant scent trials between 22 March and 4 June 2012 and the remaining trials (16
ant presence trials and 9 ant scent trials) between 9 November 2012 and 6 January 2013 Ant
presence trials lasted for eight hours and ant scent trials lasted for four hours Artificial flowers
were prepared and placed individually in small plastic containers treated with Fluon (Insect-a-
Slip BioQuip Products Inc) to prevent ants from escaping In ant presence trials we used 32
flowers (16 male and 16 female flowers) arranged in an eight-by-four array with flowers spaced
45 cm apart between rows and 30 cm within rows Ant scent trials used 20 flowers (10 male and
10 female flowers) in a four-by-five array with flowers spaced 30 cm apart between and within
rows In all of the ant presence trials and 9 of the ant scent trials we counted the number of bees
actively foraging after one hour to get a measure of colony activity
a) Ant presence trials
We examined the effects of ants on the amount of dye donated by male flowers and received by
female flowers in sixteen trials testing four bee colonies four times each The containers of eight
randomly chosen male flowers and eight randomly chosen female flowers received 15 M rubra
60
workers each the other 16 flowers remained free of ants Isolated from their colonies M rubra
workers have nowhere to deposit sugar water they collect and therefore may become sated
nonetheless a force of 15 ant workers was enough to maintain visitation to the nectaries where
bees foraged while still rarely having more than one or two ants visit the nectary at any time As
ants attacked bees bees would occasionally carry or throw attacking ants off flowers but despite
this some ants maintained their presence at the nectary throughout the trials Because ants the
size of M rubra consume liquids slowly (~017 ndash 024 microlmin Davidson et al 2004) flowers
replenished nectar more quickly than ants consumed it We used two colors of dye (FDampC 5 and
FDampC 6) to differentiate male flowers with and without ants In two trials for each bee colony
anthers of male flowers with ants were coated with FDampC 5 dye while brushes of male flowers
lacking ants were coated with FDampC 6 dye In the other two trials we reversed dye colors to
control for effects of dye type and color Dye color was assigned in a random order Artificial
flowers were placed in randomly assigned positions in the array before we opened the colony
and allowed the bees to begin foraging
b) Scent trials
We explored the effect of ant scent on dye donation in fifteen trials testing three bee colonies in
six five and four trials each Bees were first exposed to ants by allowing the bees to forage for
eight hours on twenty flowers (ten male and ten female) of which five male flowers had 15 ants
and all others had none No dye was used during this ldquoexposurerdquo day We then collected all but
the five ant-free male flowers in the flight cage leaving these flowers to keep bees foraging
Next we individually stored five new male lids in Fluon-treated containers with ten M rubra
workers Five control male lids were put in identical containers lacking ants The following
61
morning we removed the remaining flowers and set out ten new male and female flowers in a
random spatial arrangement using the lids having or lacking ant scent Thus the ant-scented
flowers were not in the same positions as the ant-visited flowers on which the bees were trained
Only five male flowers had ant scent and the other 15 flowers (five male and ten female) did not
We used the same two dye colors as in i) to differentiate male flowers with and without ant
scent and randomized which color was used for ant-scented flowers between trials We collected
and replaced stigmas from female flowers after one hour and collected the stigmas again after
four hours In the first trial performed we also collected stigmas after two hours but due to the
small amount of dye transferred this was not repeated
iv) Dye quantification
After each trial we quantified the amount of dye transferred to female flowers using a
spectrophotometer We removed the stigmas from female flowers placed them in test tubes
added 51 mL of distilled water to each tube and vortexed each tube thoroughly to ensure the
dye was evenly diluted These were diluted further as needed if the absorbance exceeded the
sensitivity range of the spectrophotometer In the first six scent trials each stigma was analyzed
separately and the dye amounts were summed to obtain a total amount of each dye color
transferred to female flowers in each trial In all the ant presence trials and the other nine scent
trials we opted to treat the experiment as the unit of replication Therefore we combined stigmas
from each treatment type (ant-visited or ant-free) in a test tube and measured the total amount of
each dye color transferred in each treatment in each trial
Because we put different dye colors on male flowers with and without ants we could use
the amount of each dye color donated to all female flowers to measure the reproductive success
62
of ant-visited and ant-free male flowers The total amount of dye (of both colors) received by
female flowers with and without ants was our measure of female reproductive success We
calculated the amount of each dye color in the sample by measuring absorbance at 428 or 486
nm and converting absorbance to micrograms following computational methods for overlapping
spectra (Blanco et al 1989)
v) Statistical analyses
In two trials (one ant presence and one ant scent) most female flowers received no dye because
of low bee activity so we excluded these trials from analyses Because dye reception and
donation values were non-normally distributed we square-root transformed the data before
examining the effects of ant presence or ant scent on dye transfer in ANCOVAs For ant
presence trials we included ant presence on male and female flowers and their interaction as
main effects and the total amount of dye transferred in each trial as a covariate to account for
the large variation in overall dye transfer among trials For ant scent trials we included ant scent
as the main effect time (one or four hours) as a repeated measure the interaction between scent
and time and the total amount of dye transferred as a covariate Covariate by treatment (ant
presence or ant scent) interactions were never significant and so were excluded in final analyses
All analyses were conducted in JMP (v 1000)
Results
Bees usually started foraging within minutes of the beginning of the trial and continued until
flowers were collected from the flight cage or the lights were extinguished An average of 34 plusmn
012 and 40 plusmn 015 (mean plusmn SE) bees were foraging after an hour in the ant presence and ant
63
scent trials respectively Male flowers with ants donated significantly less dye than male flowers
lacking ants (Fig 22a F155 = 4019 p lt 00001) Similarly female flowers with ants received
significantly less dye than female flowers lacking ants (Fig 22a F155 = 461 p = 0036) There
was no significant interaction between ant presence on male flowers and ant presence on female
flowers (F155 = 118 p = 028) Flowers with ant scent donated significantly less dye than
flowers without ant scent (Fig 22b F125 = 11216 p lt 00001) There was no significant effect
of time (F125 = 121 p = 028) or interaction between time and scent (F125 = 111 p = 030) in
the model
Discussion
In this study ants altered bumblebee pollination behavior and exhibited trait-mediated indirect
interactions with (artificial) flowers Through interference competition M rubra workers
significantly affected the pattern of dye transfer by B impatiens causing a preferential flow of
dye from male flowers lacking ants to female flowers lacking ants If manifested in nature such
effects would reduce the reproductive success of plants visited by ants through both male and
female function
Ants could have changed the attractiveness of artificial flowers in at least two ways First
as ants interrupted or altogether prevented bumblebees from foraging at nectaries bumblebees
may have learned to avoid flowers with ants due to the relative inefficiency of foraging at these
flowers something bumblebees take into account (Heinrich 2004) Second bees attempt to
minimize their risk of being attacked during foraging bouts which ants did to visiting bees Ants
often attacked or harassed bees by biting grasping and appearing to sting visiting bees
preventing them from accessing the nectary or reducing their time on flowers (see online video
64
1) and bees sometimes avoided ant-tended flowers entirely This harassment was sometimes
physically traumatic (online video 1) and bees often appeared to have trouble flying after being
attacked by ants Previous research has demonstrated that bumblebees leave or avoid flowers
where they have been harassed (Jones and Dornhaus 2011) and avoid foraging where there is
visual or olfactory evidence of a predator or predation event (Abbott 2006 Goodale and Nieh
2012) Though it is unclear if bumblebees viewed ants as competitors or predators they
responded similarly to flowers having ants as they do to flowers housing predators (Gonccedilalves-
Souza et al 2008)
To avoid artificial flowers with ants bees likely used a combination of visual and
olfactory cues Previous research has shown that bees respond to conspicuous predators or
predator ldquodummiesrdquo on flowers (Suttle 2003 Gonccedilalves-Souza et al 2008) decreasing the
frequency and duration of their visits to these flowers Thus bees may have been able to avoid
flowers harboring ants by sight alone However even in the absence of ants bees still
preferentially visited flowers lacking ant scents (Fig 22b) suggesting that they had learned to
associate ant scent with harassment Bees are adept at associative learning (Wright and Schiestl
2009) and can learn to recognize unique scents left behind by both conspecific and heterospecific
flower visitors (Stout et al 1998) Bees can use these various scents often arising from tarsal
gland deposits (Stout et al 1998) to recognize recently visited flowers that are less likely to be
profitable (Stout and Goulson 2002) Recently Ballantyne and Willmer (2012) demonstrated that
bees learn to associate ant scents with unrewarding artificial flowers and decrease their visitation
to these flowers Our results complement their findings by showing that bees can associate ant
scent with harassment at otherwise rewarding flowers
In our study ant scent caused bees to adjust their foraging strategy and decreased the
amount of pollen analogue that was donated by ant-scented flowers Thus the effects of
65
interference competition with ants on flowers can extend beyond immediate interactions and may
have fitness consequences for plants even when ants are absent Like other olfactory cues these
effects are likely transitory (Stout et al 1998) Although more dye was still donated by male
flowers lacking ant scent than those with ant scent the ratio of dye donated was on average
closer to equality in hours 2-4 than in the first hour (Fig 22b) This effect may be partially
driven by dye depletion of male flowers without ant scent but the lack of a strong corresponding
decrease in flowers with ant scent suggests that visitation patterns became more similar We did
not test whether B impatiensrsquo avoidance of ant scent was an innate or a learned behavior but
previous work has shown that Bombus terrestris does not innately avoid flowers with ant scent
(Ballantyne and Willmer 2012)
Ants may be necessary for plant survival and growth but can be costly for plant
reproduction The evolution of ant attractants such as extrafloral nectaries in some plant lineages
suggests that the costs of ants can be outweighed by their protective abilities However in this
study flowers visited by ants received and donated significantly less dye although they did
retain some sexual function The net benefit of having ants depends on whether ants increase
plant fitness by reducing herbivory more than they decrease plant fitness by disrupting
pollination Alternatively plants may actually benefit from the costs of ants to plant
reproduction ants that castrate flowers may be better defenders increasing plant survival or
vegetative growth when plants are young allowing for increased reproduction later in life when
the plant is colonized by less aggressive non-castrating ants (Frederickson 2009 Palmer et al
2010) In some plant species pollinator harassment by ants may even be beneficial Altshuler
(1999) reported that Ectatomma ants greatly increased fruit set of Psychotria limonensis despite
reductions in pollinator visitation rates presumably due to increases in the rate of pollen
outcrossing Similarly Gonzaacutelvez et al (2012) found that the presence of Oecophylla
66
smaragdina ants on Melastoma malabathricum flowers reduced visitation by less effective
pollinators and increased visitation by more effective Xylocopa bees increasing plant fitness
Cases of ants being beneficial to flowers appear to be the minority and many plant
species have traits that limit ant access to flowers Several studies have detailed floral volatiles
that are thought to mimic ant alarm pheromones and thus repel ants (Junker and Bluumlthgen 2008
Willmer et al 2009) Other plants use structural modifications such as narrow corollas or
slippery stems to limit ant access to flowers (reviewed in Willmer et al 2009) Exploitative
competition between ants and pollinators (Lach 2005 Ballantyne and Willmer 2012) as well as
the lethal effects of ant antibiotic secretions on pollen grains (Beattie et al 1985 Dutton and
Frederickson 2012) may have resulted in selection on plants to reduce ant visits to flowers Our
results suggest that trait-mediated indirect interactions resulting from interference competition
between ants and bees may favor plants that defend their flowers and their pollinators against
ants
Acknowledgements
We thank H Rusnock and S Meadley-Dunphy for assistance J Dix for help with flight cage
construction and Biobest for bumblebee colonies We are grateful to the Thomson and
Frederickson labs for guidance particularly T Makino funded by JSPS for helping to refine
artificial flowers J Ogilvie for help with bees K Prior for M rubra colony collections and K
Turner for study design suggestions The comments of the editor and two anonymous reviewers
prompted improvements in the article especially with regards to study design MEF and JDT
acknowledge NSERC Discovery Grant funding The University of Torontorsquos Faculty of Arts and
Science supported MGRTrsquos participation
67
68
References
Abbott K R 2006 Bumblebees avoid flowers containing evidence of past predation events
Canadian Journal of Zoology 841240-1247
Altshuler D L 1999 Novel interactions of non-pollinating ants with pollinators and fruit
consumers in a tropical forest Oecologia 119600-606
Ballantyne G and Willmer P 2012 Floral visitors and ant scent marks noticed but not used
Ecological Entomology 37402-409
Beattie A J Turnbull C Knox R B and Williams E G 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany
71421-426
Blanco M Runiaga H Maspoch S and Tarin P 1989 A simple method for
spectrophotometric determination of two-components with overlapped spectra Journal of
Chemical Education 66178-180
Byk J amp Del Claro K 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333-38
Davidson D W S C Cook and R R Snelling 2004 Liquid-feeding performances of ants
(Formicidae) ecological and evolutionary implications Oecologia 139255ndash66
Dukas R 2005 Bumble bee predators reduce pollinator density and plant fitness Ecology
861401-1406
Dussutour A and Simpson S J 2009 Communal nutrition in ants Current Biology 19740-
744
69
Dutton E M and Frederickson M E 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561-569
Frederickson ME 2009 Conflict over reproduction in an ant-plant symbiosis why Allomerus
octoarticulatus ants sterilize Cordia nodosa trees American Naturalist 173675-681
Galen C and Cuba J 2001 Down the tube pollinators predators and the evolution of flower
shape in the alpine skypilot Polemonium viscosum Evolution 551963-1971
Gonccedilalves-Souza T Omena P M Souza J C and Romero G Q 2008 Trait-mediated
effects on flowers artificial spiders deceive pollinators and decrease plant fitness Ecology
892407-2413
Gonzaacutelvez F G Santamariacutea L Corlett R T and Rodriacuteguez-Gironeacutes M A 2012 Flowers
attract weaver ants that deter less effective pollinators Journal of Ecology 10178ndash85
Goodale E and Nieh JC 2012 Public use of olfactory information associated with predation
in two species of social bees Animal Behaviour 84919-924
Harder LD and Thomson JD 1989 Evolutionary options for maximizing pollen dispersal of
animal-pollinated plants American Naturalist 133323-344
Heil M and McKey D 2003 Protective ant-plant interactions as model systems in ecological
and evolutionary research Annual Review of Ecology Evolution and Systematics 34425-
453
Heinrich B 2004 Bumblebee economics 2nd
edn Harvard University Press Cambridge
Ings T C and Chittka L 2009 Predator crypsis enhances behaviourally mediated indirect
effects on plants by altering bumblebee foraging preferences Proceedings of the Royal
Society B 2762031-2036
70
Inouye D W and Taylor O R Jr 1979 A temperate region plant-ant-seed predator system
consequences of extra floral nectar secretion by Helianthella quinquenervis Ecology 601-
7
Junker R R and Bluumlthgen N 2008 Floral scents repel potentially nectar-thieving ants
Evolutionary Ecology Research 10295ndash308
Junker R R Bleil R Daehler C C and Bluumlthgen N 2010 Intra-floral resource partitioning
between endemic and invasive flower visitors consequences for pollinator effectiveness
Ecological Entomology 35760ndash767
Jones E I and Dornhaus A 2011 Predation risk makes bees reject rewarding flowers and
reduce foraging activity Behavioral Ecology and Sociobiology 651505-1511
Lach L 2005 Interference and exploitative competition of three nectar-thieving invasive ant
species Insect Sociaux 52257-262
Lach L 2007 A mutualism with a native membracid facilitates pollinator displacement by
Argentine ants Ecology 881994-2004
Lach L 2008 Argentine ants displace floral arthropods in a biodiversity hotspot Diversity and
Distributions 14281-290
Makino T T and Sakai S 2007Experience changes pollinator responses to floral display size
from size-based to reward-based foraging Functional Ecology 21854863
Maloof J E and Inouye D W 2000 Are nectar robbers cheaters or mutualists Ecology
812651ndash2661
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506-514
Ohashi K Leslie A and Thomson JD 2008 Trapline foraging by bumble bees V Effects of
experience and priority on competitive performance Behavioral Ecology 19936-948
71
Palmer T M Doak D F Stanton M L Bronstein J L Kiers E T Young T P Goheen
J R and Pringle R M 2010 Synergy of multiple partners including freeloaders
increases host fitness in a multispecies mutualism Proceedings of the National Academy
of Sciences 10717234-17239
Preisser E L Bolnick D I and Benard M E 2005 Scared to death The effects of
intimidation and consumption in predator-prey interactions Ecology 86501-509
Stout J C Goulson D and Allen J A 1998 Repellent scent-marking of flowers by a guild of
foraging bumblebees (Bombus spp) Behavioral Ecology and Sociobiology 43317-326
Stout J and Goulson D 2002 The influence of nectar secretion rates on the responses of
bumblebees (Bombus spp) to previously visited flowers Behavioral Ecology and
Sociobiology 52239-246
Suttle K B 2003 Pollinators as mediators of top-down effects on plants Ecology Letters
6688-694
Thomson JD 1986 Pollen transport and deposition by bumble bees in Erythronium influences
of floral nectar and bee grooming Journal of Ecology 74329-341
Thomson JD Ogilvie J E Makino T T Arisz A Raju S Rojas-Luengas V and Tan M
G R 2012 Estimating pollination success with novel artificial flowers effects of nectar
concentration Journal of Pollination Ecology 9108-114
Tsuji K Hasyim A and Nakamura K 2004 Asian weaver ants Oecophylla smaragdina and
their repelling of pollinators Ecological Research 19669-673
Ulrich S and Bertsch A 1990 Do foraging bumblebees scent-mark food sources and does it
matter Oecologia 82137-144
Werner E E and Peacor S D 2003 A review of trait-mediated indirect interactions in
ecological communities Ecology 841083-1100
72
Willmer P G Nuttman C V Raine N E Stone G N Pattrick J G Henson K Stillman
P McIlroy L Potts S G and Knudsen J T 2009 Floral volatiles controlling ant
behaviour Functional Ecology 23888-900
Wright G A and Schiestl F P 2009 The evolution of floral scent the influence of olfactory
learning by insect pollinators on the honest signaling of floral rewards Functional Ecology
23841-851
73
Figures
Figure 21 Photograph of artificial flower lids A = anther L = lid N = nectary R = region
where anther or stigma is placed S = stigma T = thread W = weight
74
Figure 22 Dye transferred (mean plusmn 1SE) per trial by B impatiens workers A) from artificial
male and to artificial female flowers in the presence (unfilled circles) or absence (filled circles)
of Myrmica rubra workers and B) from artificial male flowers with (unfilled circles) or without
(filled circles) M rubra scent to ant-free female flowers in the 1st or the 2
nd to 4
th hours of the
trials
75
Chapter 3 Not just for the bees pollen consumption is common among tropical
ants
Cembrowski AR Reurink G and Frederickson ME
Planned submission to Biotropica
ARC and MEF designed the study ARC and GR did laboratory work ARC performed statistical
analyses and wrote the manuscript with input from other authors
Abstract
Although palynivory or pollen consumption is widespread among other hymenopterans there
are few accounts of palynivory in ants To quantify how often ants consume pollen we subjected
adult workers and larvae from 75 species of neo- and paleo-tropical ants to acetolysis a process
that destroys most organic material but leaves behind pollen grains In over half of the species
we examined ants contained a few pollen grains (lt 5) and ants of several Camponotus species
contained abundant pollen grains (gt50) We tested for an association between trophic level and
palynivory using stable nitrogen isotope ratios (δ15
N) but we did not find a significant
correlation we found pollen grains in lsquoherbivorousrsquo omnivorous and carnivorous ants We
suggest that our results indicate sporadic opportunistic pollen consumption by ants in tropical
forests
Introduction
For those organisms that can digest it efficiently pollen can be an excellent food source
Depending on the plant species pollen can contain up to 60 protein as well as lipids
76
carbohydrates and numerous trace nutrients (Roulston and Cane 2000) Pollen feeding
(palynivory) occurs in many taxa including birds mammals and arthropods (Roulston and Cane
2000) but it is best known among the Hymenoptera particularly bees Although adult bees feed
mainly on plant exudates (ie nectar) nearly all bee species rely on pollen as the chief protein
source for developing larvae (Willmer 2011) Closely related to bees ants have similar
nutritional requirements adult workers need carbohydrates whereas larvae are fed a diet rich in
protein (Dussutour and Simpson 2009) Ants also exploit nectar heavily and regularly visit
flowers (eg Herrera et al 1984 Bluumlthgen et al 2004) but unlike bees are not believed to
widely consume pollen Instead ants are thought to receive most of the protein in their diet from
predation or scavenging (eg Floren et al 2002)
However ants may not be as reliant on animal protein sources as commonly assumed In
tropical forests arboreal ants can have similar δ15
N ratios a measure of trophic level as
herbivores suggesting limited carnivory and substantial feeding on plant-derived protein sources
(Davidson et al 2003) One potential protein source is pollen This idea is supported by scattered
accounts of pollen consumption by ants from other ecosystems including deserts (Ness 2006)
boreal forests (Czechowski et al 2009) and tropical savannas (Byk and Del Claro 2010) One
ant genus Cephalotes is widely recognized to consume pollen (Creighton 1967 Baroni Urbani
and de Andrade 1997 Byk and Del Claro 2010) However beyond this genus our knowledge of
pollen consumption by ants has largely been limited to direct observations (but see Czechowski
et al 2011) and it is unknown if palynivory is truly rare among ants or simply under-reported
We borrowed a technique commonly used in palynology acetolysis to assess the frequency of
pollen consumption by ants in 75 neo- and paleo-tropical ant species This technique allowed us
to examine many more ant species than we could have observed directly
77
Methods
Adult worker ants were collected by hand at baits or from leaf litter using Winkler extractors
while larvae along with adults were collected by hand from nests Collections were made by L
Arcila Hernandez and J Sanders in the primary tropical rainforest surrounding the Centro de
Investigaciόn y Capacitaciόn Rio Los Amigos (CICRA 12deg34rsquoS 70deg05rsquoW) in Peru in 2010-
2011 and by E Youngerman from lowland primary and secondary rainforests in Madang
Province Papua New Guinea (PNG) in 2011-2012 We sorted ant samples to morphospecies and
then identified them to genus and when possible to species using existing keys which were
unavailable for PNG ants Ant samples used for acetolysis were whole adult workers guts from
adult workers and whole larvae
We used acetolysis to look for pollen grains inside ants In acetolysis organic material is
dissolved in acetic anhydride and sulfuric acid leaving behind stained pollen exines When
possible we used adult and larval ants of the same species from different colonies or bait
locations in an attempt to sample more representatively We prepared samples by first washing
them in 70 ethanol to remove any pollen present on the samplersquos outer surface before placing
them individually in micro-centrifuge tubes with ~05mL of a 91 mixture of acetic anhydride
sulfuric acid We then heated each sample at 95degC for 20min allowing tissues to dissolve Next
we centrifuged samples at 1000 RPM for 5min and decanted the liquid supernatant before
adding ~05mL of glacial acetic acid to the precipitate centrifuging again as above We decanted
the samples and added a drop of distilled water
78
We examined a 150μL sub-sample of this solution for pollen grains under a light
microscope Larvae gave less than 150μL of solution following acetolysis so we sampled the
entire amount We identified pollen grains by their staining and their surface architecture For
most species we examined at least three adult workers and three larvae but for some species
fewer than three adult workers or larvae were available (Tables 31 and 32)
We investigated the relationship between trophic level as measured by δ15
N ratios and
pollen consumption by ants Stable isotope (SI) data was available for some (n =23) of the
Peruvian species from a separate project in which collections overlapped Samples were stored in
EtOH and dried at 50degC overnight We used heads legs and thoraxes (but not gasters) of ants
for SI analysis Often single individuals did not provide enough material for analysis and we
combined multiple individuals to achieve necessary weights up to 10 for small ant species
Samples were analyzed at the Boston University Stable Isotope Laboratory using a Finnigan
Delta-S analyzer For other Peruvian species for which we had samples but not corresponding
stable isotope data (n =25) we took the median δ15
N ratio value of that speciesrsquo genus and
assigned it to the species in question Using pollen presenceabsence data for these 48 species
we used logistic regression to correlate pollen presence and δ15
N ratio using R (v 2151)
Results
We found pollen grains in 37 of the 197 adult workers 20 of the 102 larvae and 38 of the 75 ant
species we examined (Tables 31 and 32) The proportion of individuals containing pollen did
not differ significantly between larvae and adults (χ2
1 = 002 p = 088) The number of pollen
grains found was often low (median = 3) but highly variable (range 1-468) Also pollen
79
presence was inconsistent among conspecific ant samples of species that contained pollen 31
had pollen in only one of the larvae or adults examined In only three species (two Cephalotes
and one Myrmelachista) did all examined adults contain pollen
Highly lsquoherbivorousrsquo carnivorous and omnivorous ants contained pollen grains (Fig
31) There was no correlation between δ15
N ratio and pollen presence (χ2 = 150 df = 1 p =
022) In fact several carnivorous species contained pollen grains (eg Odontomachus sp 1
Pachycondola sp1)
Discussion
Pollen consumption by ants appears to be more common than suspected The majority of species
(51) had at least one individual that had consumed pollen and in 20 multiple individuals
contained pollen Numbers of grains found tended to be low however with most individuals
containing only a few grains Ants may use pollen as only a small portion of their diet exploiting
a host of other protein-containing foods more heavily including other low- δ15
N foods (eg
honeydew epiphylls Houmllldobler and Wilson 1990 Davidson et al 2003)
There are a few reasons why pollen may not be heavily exploited by ants Pollen requires
much post-consumption processing to extract its nutrients These nutrients are located in the
pollenrsquos cytoplasm which is protected from direct digestion by the exines and intines of the
grains Organisms must either break through these layers or cause the pollen grains to germinate
or pseudo-germinate to access and digest the cytoplasm (Roulston and Cane 2000) This can be
achieved by consuming a sugar-containing solution (such as nectar) along with pollen However
animals that are not adapted to feed on pollen may not be very efficient at this process only
80
extracting nutrients from some of the grains (Herrera and Martiacutenez del Rio 1998) Thus while
ants can likely get some nutrients from pollen other forms of more easily processed protein may
be more attractive
Pollen may also pose handling difficulties for ants Despite the large number of studies
that have recorded ants visiting flowers (Chapter 1) only a few papers detail ants harvesting
pollen directly from anthers Byk and Del Claro (2010) reported that Cephalotes pusillus ants
removed almost all of the available pollen from Ouratea spectabilis flowers and at least two
other studies describe ants consuming pollen along with nectar from flowers (Horskins and
Turner 1999 Ness 2006) Unlike bees which possess both morphological (eg specialized hairs
for holding pollen) and behavioral adaptations (eg stereotyped pollen-packing motions) ants
have no apparent mechanisms to increase the amount of pollen harvested and are likely limited
by the number of grains they can ingest or carry Rather than collect pollen directly from flowers
it appears more likely that ants collect it haphazardly in the environment leading to a smaller
number of pollen grains consumed at any one time It is known that at least Cephalotes ants
engage in such behavior Creighton (1967) observed Cephalotes texanus collecting pollen grains
trapped by Citrus x paradisi leaf hairs
There is a chance that the small numbers of pollen grains we observed came from
secondary pollen consumption (ie consuming a palynivorous organism) or consumption of
liquid containing pollen While we cannot rule this out we believe our results represent actual
targeted pollen consumption by ants Cephalotes ants known palynivores (Creighton 1967
Baroni Urbani and de Andrade 1997) contained similar numbers of pollen grains as many of the
pollen-containing ant species that we examined As well in at least once instance ants that we
found containing pollen grains were in the process of preying on non-pollen consuming
81
organisms (eg a Pachycondola species that was carrying a termite in its mandibles) or were
attracted by sugar baits
Contrary to our expectations there was no relationship between an ant speciesrsquo δ15
N ratio
and whether it had consumed pollen (Fig 32) We expected palynivory to be more common in
herbivorous ants because pollen presents a widespread source of protein a potentially limiting
resource The lack of pattern might reflect how available pollen grains are in the environment
While other forms of food may be preferable ants tend to be opportunistic foragers (Carroll and
Janzen 1973) and there is likely little cost associated with the consumption of scattered pollen
grains during foraging trips beyond the volume they occupy in the gut Some genera were found
to consistently consume pollen In support of previous observations (Baroni Urbani and de
Andrade 1997 Byk and Del Claro 2010) Cephalotes species were among the most common
pollen consumers Camponotus species also consistently contained pollen grains of the eight
species examined five contained pollen and pollen was found in species from both Peru and
PNG Camponotus species also contained the two largest numbers of pollen grains found (468
and 271) Taken together these results suggest that they may be another as yet unrecognized
commonly palynivorous genus
In general larvae did not contain large numbers of pollen grains (ranging from 1-38
grains) This is counter to what is seen in bees where larvae are the main recipients of pollen
(Willmer 2011) This difference may be because ant larvae are continually fed via trophallaxis
by adults throughout their development whereas bee larval cells are provisioned with pollen and
then sealed while larvae complete their development If both adults and larvae are able to extract
nutrients from pollen we would expect to see a preferential flow of nutrients from pollen to
larvae but not necessarily the grains themselves
82
Unlike palynivory by organisms that also provide pollination services pollen
consumption by ants is unlikely to benefit plants Ants rarely act as pollinators (Beattie et al
2004) They have pollen-killing antibiotic secretions on their cuticles (Dutton and Frederickson
2012) and they can compete with other flower visitors reducing flower visitation rates (Ness
2006) The negative impacts of flower-visiting ants are thought to have led to the evolution of
ant-repellent floral volatiles (reviewed in Willmer 2009) These volatiles can pre-empt
competition between ants and pollinators and may often be located within the pollen itself
(Willmer 2009) If floral volatiles truly are a defense against ant visitation our results suggest an
additional evolutionary pressure to not only prevent ant-pollinator interactions but to also
protect plant gametes from occasional consumption by a non-pollinating visitor
Acknowledgements
We would like to thank Jon Sanders Lina Arcila Hernandez and Eric Youngerman for providing
samples the Thomson lab for use of supplies the Pierce lab for funding support during JSrsquo and
EYrsquos collections and the staff of CICRA and the New Guinea Binatang Research Center for
logistical support The Peruvian Ministry of Agriculture provided permits (Nos 394-2009-AG-
DGFFS-DGEFFS and 299-2011-AG-DGFFS-DGEFFS) MEF acknowledges financial support
from an NSERC Discovery Grant a Connaught New Researcher Award an Ontario Ministry of
Economic Development and Innovation Early Researcher Award and the University of Toronto
ARC was supported by an Ontario Graduate Scholarship and Sigma Xi
83
References
Baroni Urbani C and M L de Andrade 1997 Pollen eating storing and spitting by ants
Naturwissenschaften 84256ndash258
Beattie A J C Turnbull R B Knox and E G Williams 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany 71421ndash
426
Bluumlthgen N G Gottsberger and K Fiedler 2004 Sugar and amino acid composition of ant-
attended nectar and honeydew sources from an Australian rainforest Austral Ecology
29418ndash429
Byk J and K Del-Claro 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333ndash38
Carroll C R and D H Janzen 1973 Ecology of foraging ants Annual Review of Ecology and
Systematics 4231ndash257
Creighton W S 1967 Studies on free colonies of Cryptocerus texanus Satschi (Hymenoptera
Formicidae) Psyche 7434ndash42
Czechowski W B Markό and A Radchenko 2009 Rubbish dumps reveal the diet of ant
colonies Myrmica schencki Em and Myrmica rubra (L) (Hymenoptera Formicidae) as
facultative pollen-eaters Polish Journal of Ecology 56737ndash741
Czechowski WB Markoacute K Erős and E Csata 2011 Pollenivory in ants (Hymenoptera
Formicidae) seems to be much more common than it was thought Annales Zoologici
61519ndash525
84
Dussutour A and S J Simpson 2009 Communal nutrition in ants Current Biology 19740ndash4
Dutton E M and M E Frederickson 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561ndash569
Floren A A Biun and K E Linsenmair 2002 Arboreal ants as key predators in tropical
lowland rainforest trees Oecologia 131137ndash144
Grant B R 1996 Pollen digestion by Darwinrsquos finches and its importance for early breeding
Ecology 77489-499
Herrera L G and C Martiacutenez del Rio 1998 Pollen digestion by new world bats effects of
processing time and feeding habits Ecology 792828ndash2838
Houmllldobler B and Wilson E O 1990 The Ants 1st Edition Harvard University Press
Cambridge
Horskins K and Turner V B 1999 Resource use and foraging patterns of honeybees Apis
mellifera and native insects on flowers of Eucalyptus costata Australian Journal of
Ecology 24221-227
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506ndash514
Roulston T H I and J H Cane 2000 Pollen nutritional content and digestibility for animals
Plant Systematics 222187ndash209
Van Tets I G 1997 Extraction of nutrients from Protea pollen by African rodents Belgian
Journal of Zoology 12759-65
Willmer P G 2011 Pollination and Floral Ecology 1st Edition Princeton University Press
New Jersey
85
Willmer P G C V Nuttman N E Raine G N Stone J G Pattrick K Henson P Stillman
et al 2009 Floral volatiles controlling ant behaviour Functional Ecology 23888ndash900
86
Tables
Table 31 Numbers of adult worker and larval ants examined for each species collected in Peru
Numbers of pollen grains found in individual samples are given in parentheses after the sample
size when there were no grains no numbers are given
Genus Species Number of adults analyzed
(numbers of grains found)
Number of larvae analyzed
(numbers of grains found)
Dolichoderinae
Azteca sp 1 3 0
sp 2 2 0
sp 3 0 2
sp 4 3 (1) 0
sp 5 3 (2) 0
Dolichoderus decollatus 3 (66) 0
sp 1 3 0
sp 2 3 1 (2)
sp 3 3 3
sp 4 0 2
Ecitoninae
Labidus sp 1 3 0
Formicinae
Camponotus sp 1 3 3
sp 2 3 (82) 1 (2)
sp 3 3 (3) 0
sp 4 0 2
87
sp 5 8 (11) 0
sp 6 4 (7 16 468) 0
sp 7 5 (12) 0
sp 8 2 (1) 0
Paratrechina sp 1 4 (11) 0
Myrmelachista sp 1 3 (5 11 18) 0
sp 2 0 3
Trachymyrmex sp 1 3 (21) 0
sp 2 3 0
Myrmicinae
Atta sp 1 3 0
Cephalotes atratus 3 (2 2 21) 0
placidus 3 (1 4 21) 0
sp 1 3 0
sp 2 1 0
sp 3 2 0
Crematogaster sp 1 3 1
sp 2 3 (35) 0
sp 3 9 (7) 0
sp 4 2 0
Megalomyrmex sp 1 3 0
sp 2 0 2
sp 3 4 (7) 0
sp 4 3 0
Monomorium sp 1 0 3 (2)
88
Pheidole sp 1 0 3
sp 2 3 0
sp 3 0 3
Procryptocerus sp 1 3 0
Solenopsis sp 1 0 3
sp 2 3 (3 5) 0
Wasmannia sp 1 3 0
Ponerinae
Pachycondola sp 1 3 (3) 0
Odontomachus sp 1 3 (34) 0
Pseudomyrmicinae
Pseudomyrmex sp 1 3 0
sp 2 3 0
sp 3 3 (9) 0
sp 4 0 3
sp 5 0 3
89
Table 32 Numbers of adult worker and larval ants examined for each species collected in Papua
New Guinea Numbers of pollen grains found in individual samples are given in parentheses
after the sample size when there were no grains no numbers are given
Genus Species Number adults analyzed
(grains found)
Number larvae analyzed
(grains found)
Aenictinae
Aenictus sp 1 3 3 (1 1)
Dolichoderinae
Philidris sp 1 3 1 (4)
Tapinoma sp 1 2 3 (38)
Technomyrmex sp 1 0 3 (12 12)
Ectatomminae
Rhytidoponera sp 1 1 3 (2)
Formicinae
Acropyga sp 1 3 3 (2)
sp 2 3 3
sp 3 3 3
Anoplolepsis sp 1 3 (1) 3 (1)
Calomyrmex sp 1 3 3 (1)
Camponotus sp 1 3 (1 271) 3
sp 2 1 3
sp 3 2 0
Nylanderia sp 1 3 3
Opisthopsis sp 1 3 3
Polyrachis sp 1 1 3 (4)
90
Myrmicinae
Crematogaster sp 1 3 3
sp 2 3 (32) 3
Monomorium sp 1 3 (1) 3 (1)
sp 2 3 3 (1 1 1)
Pheidole sp 1 3 3
Pristomyrmex sp 1 3 3 (11)
Pyramica sp 1 3 0
Ponerinae
Odontomachus sp 1 3 3 (4)
91
Figures
Figure 31 Photographs of pollen grains found in two ant species a) Dolichoderus sp 2 and b)
Camponotus sp 6 Scale bars = 50 microm
92
Figure 32 Relationship between trophic level as measured by δ15
N ratio and pollen presence
for 48 neotropical ant species 1 Atta sp 5 Azteca spp 8 Camponotus spp 5 Cephalotes spp 4
Crematogaster spp 5 Dolichoderus spp 1 Labidus sp 3 Megalomyrmex spp 1 Monomorium
sp 2 Myrmelachista spp 1 Odontomachus sp 1 Pachycondola sp 1 Paratrechina sp 3
Pheidole sp 4 Pseudomyrmex spp 2 Solenopsis spp and 1 Wasmannia sp
93
Concluding Remarks
Ants have traditionally been considered to be unwelcome flower visitors they consume floral
rewards ostensibly meant for pollinators while rarely providing pollination services themselves
In this thesis I investigate the validity of this assumption and use an experimental study and an
observational study to examine two ways that ants may impact plant reproduction
In the first chapter I review the literature on ants as flower visitors I show that the common
assumption of ants always being categorically negative for plant reproduction is incorrect while
ants may reduce floral attractiveness to pollinators the net effect of ants on plant fitness
measures is variable ranging from positive to negative to neutral I also discuss ants as
pollinators and ways that plants may minimize ant visitation to flowers before finishing with a
section highlighting questions that still need investigation
In the second chapter I explore how interference competition with ants can structure bumblebee
foraging behavior and how this may alter pollen movement patterns Using artificial flowers with
male or female function I allowed Bombus impatiens bumblebees to forage on flowers that
either did or did not have Myrmica rubra ants on them Ant presence significantly reduced the
amount of pollen analogue donated and received by flowers Building on these results I found
that the presence of ant scent alone elicited similar changes to bumblebee behavior reducing the
amount of pollen analogue donated by male flowers My results show that interference
competition is sufficient to alter bumblebee foraging behavior and that the effects of ants on
pollinators may extend after ant visitation to flowers Further study is needed to determine if bees
are able to differentiate between ant species by visual or olfactory cues Their ability to do so
94
could affect how generalized their response to ant presence is and may help to better predict how
ants will affect plant reproduction
In the final chapter I investigate how common palynivory is amongst tropical ant species I used
acetolysis and light microscopy to examine whole ants larvae and ant guts from Papua New
Guinea and Peru I found that tropical ants consistently contain pollen grains albeit often in
small numbers (lt5) However some genera notably Camponotus and Cephalotes consistently
contained higher numbers providing the first evidence that Camponotus may be a palynivorous
genus The small numbers of pollen grains present may indicate that ants preferentially exploit
other more easily digestible high-protein foods over pollen Future studies should investigate
exactly what role pollen plays in ant diets by supplementing the diet of captive ant colonies with
pollen This would allow us to examine how pollen consumption affects colony demography as
well as how efficiently ants digest pollen and whether ants exhibit pollen preferences
I bring up several pressing and unanswered questions within this thesis and highlight three here
First numerous plant species possess chemical or physical traits assumed to increase plant
fitness by reducing ant visitation However this has rarely been tested and instead research has
focused predominately on documenting ant repellence We should move away from this and
begin examining plant fitness in the presence or absence of these repellent traits to see how
beneficial these traits truly are Second how flower visiting ants impact male fitness has been
almost completely ignored in favor of female fitness Future studies should investigate how ant
visitation affects both pollen donation and pollen movement patterns Lastly research involving
antibiotics present on ant cuticles and their effect on pollen viability has been too small in scope
often limited to demonstrating the pollen killing effects of ant secretions Investigation into
95
whether plants have evolved any resistance to the secretions of the local ant community or why
some plants appear unaffected by their ant partners may yield exciting results
In conclusion ants have the capacity to strongly affect plant reproduction both positively and
negatively The fact that these diametrically opposing forces exist means we cannot approach
flower-visiting ants as either positive or negative a priori Instead we should strive to work
towards a better understanding of what is occurring in the system of interest and what role ants
play within it
96
Copyright Acknowledgments
Chapter 2 ldquoAnts and ant scent reduce bumblebee pollination of artificial flowersrdquo is current in
press in The American Naturalist Request for its inclusion here was sought and approved
4
colonies in specialized structures (domatia) The sterilizing ants do not appear to consume floral
tissues (Yu and Pierce 1998) and two hypotheses may explain why ants destroy floral buds First
ant colony size is often limited by domatia availability on their host plant By destroying floral
buds ants can cause plants to divert resources from reproductive to vegetative growth increasing
domatia number and allowing for larger ant colonies (Yu amp Pierce 1998 Frederickson 2009)
Second when plant-ants must defend their colony from takeovers by other colonies they may
benefit from limiting access to their host plant By destroying buds that could develop into
flowers branches or leaves ants can prevent their host plant from contacting nearby plants
occupied by enemy ant colonies (Stanton et al 1999)
The impact of sterilization on lifetime plant fitness is unclear Ants may destroy all of
their host plantrsquos floral buds (Yu and Pierce 1998) completely preventing reproduction or only a
portion of them (Maleacute et al 2012) allowing some seed set However counter-intuitively this
sterilization behavior may provide long-term fitness benefits to plants (Frederickson 2009
Palmer et al 2010) Ant-plants often host several different ant species over their lifetimes some
of which are non-sterilizing If hosting a sterilizing species at a certain life stage increases
survival or growth relative to hosting a non-sterilizing species at that life stage plants may
increase lifetime fitness by largely forgoing current reproduction in favor of increasing future
reproduction (Palmer et al 2010) Why plants invest resources in producing flowers that are
quickly destroyed by ants is not known but the timing of flower destruction may be important If
ants attack flowers after anthers dehisce plants may still realize some male fitness via pollen
donation (Yu and Pierce 1998) Additionally if the cost of flower production is relatively minor
and ants sometimes ldquomissrdquo flowers (Edwards and Yu 2008) attempting limited reproduction
may be a better strategy than forgoing it entirely
5
ii) Competition with pollinators
Ant activity on flowers can dramatically alter pollinator behavior Ants can reduce the species
diversity frequency or duration of pollinator visits to flowers (Norment 1988 Tsuji et al 2004
Ness 2006 Junker et al 2007 Lach 2007 2008b Romero et al 2011 and see Chapter 2) These
changes are largely due to either exploitative competition (ie consumption of shared resources)
or interference competition (ie direct aggression) between ants and pollinators
a) Exploitative competition
Pollinators and ants frequently compete for floral nectar Flower-visiting ants are often classified
as nectar thieves (sensu Inouye 1980) because they typically enter flowers through the
ldquolegitimaterdquo opening and consume nectar but transfer no pollen (Wyatt 1980 Ness 2006
Chamberlain and Holland 2008) Ants can also act as primary nectar robbers chewing holes
through corollas to reach nectaries (Willmer and Corbet 1981 Koopowitz and Marchant 1998)
although they are more commonly secondary nectar robbers and use holes made by previous
robbers (Newman and Thomson 2005)
Like other flower visitors foraging ants will often leave nectar behind in flowers This
amount can be less (Bleil et al 2011) more (Fritz and Morse 1982) or similar to (Lach 2008a)
amounts left by other visitors Often ants are morphologically constrained from fully exploiting
nectar Unlike bees and butterflies ants do not have proboscises and must get close to nectar to
consume it Narrow corollas or nectar spurs can prevent ant access to some or all floral nectar
(Schemske 1980 Galen and Cuba 2001 and see below) Smaller ants may be better able to
6
access nectar (Newman and Thomson 2005 Agarwal and Rastogi 2007) but can be limited by
their crop size in the amount of nectar they can remove (Lach 2005) If foraging ants leave
enough nectar behind for a pollinator to recover the cost of visiting a flower ants may have
minimal effects on plant reproductive success (Maloof and Inouye 2000 Turner et al 2012)
Differences among ant species in behavior and diet also affect their consumption of floral
nectar Species vary in how many flowers they visit per inflorescence or per plant and in how
many ants will visit a flower simultaneously (Lach 2005 Junker et al 2007 Lach 2013)
Invasive antsmdashthought to be better at exploiting and defending resources than native ants
(Holway et al 2002)mdashoften recruit to flowers in large numbers sometimes several dozen or
more (Visser et al 1999 Hansen and Muumlller 2009) Overall many factors may influence how
heavily ants exploit floral nectar on plants including ant density plant height the spatial or
temporal distribution of floral nectar and the availability of alternative food resources for ants
(Cushman and Addicott 1991 Lach 2013)
Rarely ants and pollinators may compete for pollen We know of only a few studies that
have recorded ants harvesting pollen directly from anthers (Horskins and Turner 1999 Ness
2006 Byk and Del-Claro 2010) Other studies have found pollen grains in ants (eg Baroni
Urbani and de Andrade 1997 and see Chapter 3) but this may not indicate competition with
pollinators ants sometimes collect stray pollen grains from the environment (Creighton 1967)
One New World genus Cephalotes might be specially adapted for pollen consumption They
possess ldquoproventricular shieldsrdquo structures that may restrict solid food to the crop allowing
nestmates to exchange pollen grains via trophallaxis It is unclear if this structure evolved for this
purpose but Cephalotes are the most frequently documented palynivorous ants (eg Creighton
1967 Baroni Urbani and de Andrade 1997 Byk and Del-Claro 2010) How efficiently ants
7
digest pollen and whether ants like bees feed it to larvae as a protein source is unknown At
least two studies have found that ant larvae consume pollen providing some support for this idea
(Wheeler and Bailey 1920 and Chapter 3)
b) Interference competition
Ants are relatively unique among flower visitors in defending floral resources from competitors
(but see Willmer and Corbett 1981 Roubik 1982) This defense ranges from chance encounters
with ants attacking visitors when flower occupancy overlaps (Ashman and King 2005 Junker et
al 2007) to actively patrolling inflorescences or constructing protective galleries around
nectaries (Horvitz and Schemske 1984 Gaume et al 2005 Hansen and Muumlller 2009)
Interference competition even in the absence of exploitative competition is sufficient to alter
pollinator behavior (Chapter 2)
The strength and outcome of interference competition depends on the identities of the ant
and the pollinator Pollinators might not alter their visitation patterns in response to non-
aggressive ants (Junker et al 2007) but more antagonistic ants may elicit avoidance behavior
before or soon after the visitor lands (Willmer and Corbet 1982 Altshuler 1999 Schuumlrch et al
2000 Table 11) Pollinator size may help predict their reaction to ants because ants may deter
only small-bodied pollinators (Gonzaacutelvez et al 2012) It is not clear whether flower visitors view
ants as aggressive competitors or as potential predators rarely ants prey on pollinators (Schatz
and Wcislo 1999) but in most cases ants merely cause pollinators to relocate
How well pollinators recognize and avoid ant-visited flowers is unresolved Collectively
pollinators have lower visitation rates to flowers visited by ants (Romero et al 2011) This is
8
dependent on pollinator identity however dipterans do not appear to discriminate between ant-
visited and ant-free flowers while bees can identify and avoid ant-visited flowers Bees likely
use a combination of visual and olfactory cues to recognize ant-visited flowers They can
identify previous conspecific and some heterospecific insect visitors by the hydrocarbon
ldquofootprintsrdquo they leave during foraging (Stout and Goulson 2001) Bumblebees can detect ant
hydrocarbons although this has been shown only in the laboratory (Ballantyne and Willmer
2012 Chapter 2) No study has yet examined if bees can use sight alone to detect ants but
several suggest it (Junker et al 2007 Gonzaacutelvez et al 2012) Some bees may discriminate
between ant species when visiting flowers (eg Lach 2008b) which follows if not all ant-
pollinator interactions are equally costly The ability of bees to do so will affect how generalized
their avoidance behaviors will be If they can discriminate between species bees may avoid
specific flowers visited by more ldquocostlyrdquo ants However if they cannot bees might either avoid
all ant-visited flowers or none depending on how negative the average interaction with an ant is
iii) Interactions with flower and pollinator antagonists
Ants can defend the flowers they visit Numerous plant species have circumfloral or bracteal
nectaries (ie extrafloral nectaries around flowers) and ants attracted to these nectaries can
reduce bud ovule or flower predation increasing plant fitness (Inouye and Taylor 1979 Rico-
Gray and Thien 1989 Sugiura et al 2006) Although the presence of EFNs (extrafloral nectaries)
so close to floral nectaries (sometimes less than 1 cm Ness 2006) might increase discovery and
exploitation of floral nectar by ants only one study has investigated this hypothesis finding no
relationship between the proximity of EFNs to floral nectaries and antsrsquo use of floral nectar
(Galen 2005) Holes made by nectar robbers are functionally analogous to circumfloral nectaries
9
and can also attract ants that defend flowers (Newman and Thomson 2005) and nectar-thieving
ants can similarly prevent florivory in some systems (Lach 2007 Bleil et al 2011)
Ants can also reduce the abundance of flower-dwelling predators or parasites of
pollinators Ambush predators such as crab spiders often forage at flowers where they may
reduce pollinator visitation rates and plant fitness (Dukas 2001 Gonccedilalvez-Souza et al 2008)
Ants can decrease spider abundance and richness on plants (Rudgers 2004 Renault et al 2005
Nahas et al 2012) and some evidence suggests that they can do the same on flowers (Lach
2008) In select cases ants may also reduce the abundance of pollinator parasitoids (Holland et
al 2011) Whether ants increase plant reproductive success by removing pollinator antagonists
has not yet been examined but should be If ants create lsquoenemy-freersquo flowers pollinators might
prefer to land on flowers with ants
iv) Net effects
Table 11 compiles studies in which the authors recorded ants consuming floral rewards or
interacting with pollinators and measured some aspect of plant fitness (eg fruit or seed set
pollen deposition) We included only a single study per plant species unless subsequent studies
found conflicting results or measured the fitness of another plant sex Records of ant pollination
are included in a separate table (Table 12)
Table 11 reveals relatively few examples of flower-visiting ants negatively affecting
plant fitness given how costly ant visitation is assumed to be The net effect of ants on plant
reproductive success is actually quite variable with similar numbers of studies finding positive
negative or neutral effects of ant presence Surprisingly when ants do have negative effects it is
10
usually because they directly damage floral structures and not because they prevent pollinators
from visiting flowers and successfully transferring pollen Why are ants not more often negative
for plant reproduction when this is commonly assumed to be true
First ants may compensate for reducing pollinator visitation rates by improving
pollination quality As pollinators move between flowers on a plant they often deposit self-
pollen on stigmas (ie geitonogamy) In some plants with late-acting self-incompatibility ovules
fertilized by self-pollen are subsequently aborted reducing the number of seeds that can be
produced (Barrett 2002 and references therein) Geitonogamy can also reduce offspring viability
or vigor (de Jong et al 1993) and cause pollen discounting a reduction in the amount of pollen
available for outcrossing because of self-pollination (Barrett 2002) By competing with
pollinators ants might decrease rates of geitonogamous pollination if pollinators then visit fewer
sequential flowers within a plant Ants might also cause pollinators to fly further between plants
potentially increasing the genetic diversity of pollen arriving at stigmas Although such effects
have been reported for other floral larcenists (Maloof 2001 Irwin 2003) to our knowledge these
hypotheses have not been investigated in relation to ants
Second ants and pollinators do not always forage for the same resources possibly
allowing them to partition floral rewards If ants forage for nectar but not pollen and flowers can
be pollinated efficiently by visitors looking to gather mostly pollen and little or no nectar then
plant reproduction will not be affected by ants consuming floral nectar Junker et al (2010a)
found that honeybees foraged for both pollen and nectar in the absence of ants but reduced their
foraging on nectar but not pollen when ants could access flowers resulting in no difference in
fruit set between treatments Whether pollinators commonly adjust their foraging behaviors in
response to ants warrants further research
11
Third ants are somewhat unique among floral larcenists in that they often provide direct
benefits to the plants they visit In the studies listed in Table 11 ants sometimes decreased
herbivory on plants (Chamberlain and Holland 2008 Bleil et al 2011) or otherwise aided the
plant (eg ant presence repelling smaller less effective pollinators Gonzaacutelvez et al 2012) If the
positive effects of ants cancel or outweigh the negative effects of flower-visiting ants on
pollinators then ant visitation may be neutral or even positive for plant fitness
Fourth ants may impose fitness costs to plants not captured in the studies in Table 11
Nectar robbers have stronger negative effects when plants are pollen-limited (Burkle et al 2007)
but only one study in Table 11 assessed whether flowers would set more seed if they received
more pollen finding no evidence of pollen limitation (Lach 2008a Table 11) Even if ants
decrease pollinator visitation rates there is limited scope for ants to decrease plant reproductive
success when plants are not pollen-limited Furthermore nearly all the studies in Table 11
measured only female fitness Only three quantified aspects of male fitness (Wyatt 1980 Fritz
and Morse 1981 Galen and Geib 2007) and we know of only one other study in a system in
which ants occasionally pollinate that has also measured male fitness (Ashman 2000) All four
studies measured pollen or pollinia removal rates and none found any effect of flower-visiting
ants on male fitness However whether other measures of male fitness (eg seeds sired pollen
deposition patterns) would reveal similar or different effects needs investigation
Finally although floral defenses against ants (see 3 below) may prevent us from
observing the negative effects of ants on plant reproductive success we consider this unlikely
Several studies have examined interactions between ants and plants that share little or no
coevolutionary history usually in the context of ant invasions Although invasive ants often
recruit to flowers in larger numbers than native ants they do not appear to reduce plant
12
reproductive success (Blancafort and Goacutemez 2005 Lach 2007 Lach 2008a but see Hansen and
Muumlller 2009) And in Hawaii where angiosperms evolved for millions of years in the absence of
ants and are thought to have secondarily lost floral defenses against ants (Junker et al 2011a)
there is no evidence that the preponderance of recently introduced flower-visiting ants negatively
affects plant fitness (Junker et al 2010a Bleil et al 2011)
2 Ants as pollinators
Ants may directly benefit plant reproduction by acting as pollinators but this is a relatively rare
occurrence Ants have long been considered poor pollinators (Kerner 1878) Initially this was
based on morphological and behavioral differences between ants and recognized pollinators
Worker ants are wingless and often relatively hairless making them poor pollen dispersers As
well because many ants exhibit Ortstreue (literally ldquoplace fidelityrdquo) ants were thought to return
to the same branch or flower repeatedly effecting no cross pollination (Houmllldobler and Wilson
1990) Though largely true these traits do not necessarily preclude ants from acting as
pollinators (Table 12) Although they commonly have restricted foraging ranges (Houmllldobler and
Wilson 1990) some ants can effect outcrossing (eg Ashman and King 2005 Carvalheiro 2008)
and many ants are as hairy if not hairier than their pollinating counterparts
In the late 1970s the ldquoantibiotic hypothesisrdquo was put forward to explain the rarity of ant
pollination Ants use antibiotic secretions to protect themselves against the fungal and bacterial
pathogens common in their nests and environs Iwanami and Iwadare (1978) found that one of
these compounds myrmicacin also greatly reduces pollen germination rates Subsequently
Beattie et al (1984 1985) found that short periods of contact (le 30 minutes) with an antrsquos
integument caused pollen grains to fail to germinate Some of these pollen-killing compounds
13
were traced back to the ant metapleural gland which produces hygienic secretions (Houmllldobler
and Wilson 1990) although contact with ants lacking metapleural glands can also reduce pollen
viability (Beattie et al 1985 Dutton and Frederickson 2012) Ant mandibular or anal gland
secretions and potentially ant venom may likewise defend ants against pathogens and have
similar effects on pollen germination (Beattie et al 1985 Graystock and Hughes 2011)
Nonetheless ant pollination has been documented repeatedly There are at least 18 cases
in which ant pollination has been shown experimentally and five others in which observations
strongly suggest it (Table 12) The majority of these plants fit the ldquoant pollination syndromerdquo
originally proposed by Hickman (1974) dense stands of short plants with small flowers and
easily accessible nectaries Typically ant-pollinated plants are associated with hot and dry
climates (principally in the Mediterranean) but dry alpine environments may also favor the
evolution of ant pollination (eg Svensson 1985 Puterbaugh 1998 Schiestl and Glaser 2011) In
most cases ants appear to be the sole or major pollinator of the plant in question (Hickman 1974
Peakall et al 1990 de Vega et al 2009) although ants sometimes make only supplementary
contributions to pollination (Schuumlrch et al 2000 Ashman and King 2005) This suggests that co-
evolution between ants and plants may not be necessary for ants to be useful pollinatorsmdashwhich
is surprising given the potential negative effects of ants on plant reproduction
Ant-pollinated plants cope with ant secretions in a variety of ways Several plant species
may simply tolerate pollen damage (Gόmez and Zamora 1992 de Vega et al 2009) apparently
possessing no overt adaptations to prevent it These plants might receive enough pollen to
overcome reduced pollen viability Other plants may place pollen on ant body parts thought to
have fewer secretions thereby potentially escaping damage (eg on ant legs Ramsey 1995) or
attach pollen to ants with specialized structures avoiding direct contact with the ant integument
14
(Peakall et al 1990) It is not known whether pollen evolves resistance to ant secretions or
whether plants deposit pollen on ants which have less disruptive secretions although these
possibilities have been suggested elsewhere (eg Garciacutea et al 1995 Ramsey 1995)
One key question regarding the effects of ant secretions on pollen grains urgently needs
to be resolved Several studies have suggested that the pollen-killing secretions of ants may have
contributed to the evolution of floral defenses against ants (eg Wagner 2000 Galen and Geib
2007) For this to be true ants must decrease male fitness by damaging pollen grains before they
leave flowers and are deposited on receptive stigmas Only two studies have investigated the
effects of ant secretions on pollen still in anthers (Wagner 2000 Galen and Butchard 2003) and
both constrained ants unnaturally to flowers for significantly longer than ants would normally
stay on flowers Although these studies found strong reductions in pollen vigor it is unclear if
ant secretions decrease male fitness under natural ant visitation conditions
3 Floral defenses against ants
Plants are not passive recipients of flower visitors and have some ability to ldquochooserdquo which
organisms can access floral rewards Flowers resist ants using both structural and chemical traits
More thorough investigation is needed to determine whether these traits evolved to prevent or
minimize the negative effects of ants on plant reproductive success (ie whether these traits can
truly be considered defenses against ants see 4ia below) It is unlikely however that ants
experience reciprocal selection to circumvent floral resistance traits because the fitness benefits
to ants of accessing flowers are probably small Thus there may be little scope for
coevolutionary arms races between floral defenses against ants and ant counter-adaptations
15
i) Structural resistance traits
a) Restricting ant access
Numerous plant species have physical structures preventing ant access to flowers Thin pedicels
can limit antsrsquo gripping abilities and make ants easier to dislodge when flowers are moved by
wind (Gaume et al 2005) and waxy secretions can render stems too slippery for ants to climb
(Harley 1991) Structures surrounding flowers such as water moats or dense hairs may similarly
keep ants from accessing floral rewards (Willmer et al 2009) Many such traits function as broad
filters and prevent not only ants but most crawling insects from reaching flowers Thus it is
difficult to determine whether ants florivores or both acted as selective agents favoring the
evolution of physical barriers to flowers
Flower shape may also reduce ant-pollinator conflict Long narrow corollas or nectar
spurs can prevent all but the smallest ants from accessing floral nectaries (Bluumlthgen et al 2004
Agarwal and Rastogi 2007) while still allowing long-tongued pollinators access to rewards
Selection to exclude nectar-thieving ants in such a manner however may be counteracted by
pollinator preferences for wider corollas that allow easier flower handling (Galen and Cuba
2001) Additionally these types of flowers may be more prone to robbery (Irwin and Maloof
2002) limiting their usefulness in preventing nectar larceny
b) Separation in space or time
Plants may separate floral rewards and ants temporally or spatially to minimize ant impacts on
plant reproduction Several authors have proposed that myrmecophytes have adapted to the
16
sterilization or otherwise detrimental activities of ants by locating domatia or food rewards on
different branches than flowers (Izzo and Vasconcelos 2002 Raine et al 2002 Edwards and Yu
2008) Temporal separation of ant and pollinator activities could likewise reduce conflict though
evidence is limited Some acacia flowers dehisce during periods of low ant activity (Willmer and
Stone 1997 Nicklen and Wagner 2006) but it is unclear if this is in response to ants
temperature or pollinator activity Timing of nectar secretion may similarly reduce ant-pollinator
competition If ants are active prior to pollinators they may deplete floral nectar and reduce
pollinator visitation rates (Schaffer et al 1984) However if nectar is secreted when ants are
inactive pollination may occur unimpeded (Norment 1988) This temporal separation could
favor pollinators that are active when ants are not (eg Wyatt and Morse 1981) but this is
poorly documented Further studies particularly focusing on flowers with the potential for
nocturnal and diurnal pollination may provide more examples
c) Extrafloral nectaries (EFNs)
One hypothesis put forward to explain the evolution of EFNs proposes that EFNs ldquodistractrdquo ants
from consuming floral nectar (Kerner 1878 Wagner and Kay 2002) Wagner and Kay (2002)
tested this hypothesis by constructing model plants using artificial nectaries assigned at random
to represent either floral or extrafloral nectaries They found that the presence of EFNs could
reduce ant exploitation of floral nectar However as highlighted by Galen (2005) this design is
closer to a plant making a surplus of flowers some of which will avoid ant visitation if visitation
rates increase sublinearly with increasing flower number Producing extra flowers to escape ant
visitation appears to be a viable strategy in some systems (Ashman and King 2005 Galen 2005)
However Galen (2005) found that applying honey (simulating extrafloral nectar) to P viscosum
17
leaves had the opposite effect and increased ant visits to flowers Only two studies both in
Pachycereus schottii have found evidence for the distraction hypothesis under field conditions
(Chamberlain and Holland 2008 Holland et al 2011)
There is limited empirical support for the distraction hypothesis but abundant evidence
that EFNs attract ants that defend plants against herbivores (reviewed in Rosumek et al 2009)
To function as attractants the presence of EFNs must increase ant abundance on plants
However because of the increased numbers attracted EFNs would not be expected to reduce the
abundance of ants on nectar rewards or be able to satiate the ants recruited Only if ant
abundance stops increasing at a reward density much lower than that available on the plant
would EFNs serve to distract ants In this case the amount of rewards may overwhelm ants
leaving some flowers underexploited However even in P schottii ants still visited flowers
(Chamberlain and Holland 2008) In cases where ant abundance would continue to increase with
additional rewards it is unlikely that EFNs function as distractions
ii) Chemical resistance traits
a) Toxic nectar
One of the earliest proposed floral defenses against ants was toxic nectar (Janzen 1977)
However studies testing Janzenrsquos (1977) hypothesis uncovered only a very few isolated
examples (eg Catalpa speciosa Stephenson 1981 Crinum erubescens Guerrant and Fiedler
1981) in short most floral nectar is not toxic or repellent to ants (Baker and Baker 1978
Schubart and Anderson 1978) In fact ants sometimes prefer floral nectar over solutions with
equivalent sugar concentrations (Koptur and Truong 1998)
18
In contrast floral nectar can repel other flower visitors (eg birds Johnson et al 2006
lepidopterans Kessler and Baldwin 2007) Repellency is often caused by plant secondary
metabolites which may occur in nectar as a by-product of their production elsewhere in the
plant where they function as defenses against herbivory (Adler 2001) Alternatively toxic or
repellent nectar may be adaptive repellent nectar may promote effective pollination by reducing
the amount of reward any one pollinator takes spreading nectar over several pollinator visits
(Kessler and Baldwin 2007) or it may function as a filter selecting for specific pollinators
(Johnson et al 2006) The latter may explain why repellent nectar is not more common
legitimate pollinators in addition to floral larcenists may be deterred from visiting flowers with
repellent nectar This may be particularly true of nectars that would repel ants which are closely
related to bees nectar distasteful to ants may often also be distasteful to bees and those nectars
acting as a filter for bees may still be palatable to ants For example Prŷs-Jones and Willmer
(1992) found that the ammonia-containing nectar of Lathraea clandestina is readily consumed
by both bees and ants even though it deters birds that act as nectar thieves Although floral
nectar itself rarely repels ants Guerrant and Fielder (1981) found that the presence of macerated
petals within nectar can often keep ants away Alkaloids in petals may be responsible and might
explain why ants seldom act as primary nectar robbers these secondary compounds may prevent
ants from chewing through corollas
b) Floral volatiles
Repellent floral volatiles appear to be a more widespread form of chemical resistance against
ants These were reviewed recently by Willmer et al (2009) and will be discussed here only
briefly Numerous plant taxa produce floral scents repellent to ants including myrmecophilous
19
myrmecophytic and non-ant associated plants in both temperate and tropical ecosystems
(Willmer et al 2009 Junker et al 2011a Ballantyne and Willmer 2012) In three large studies
floral volatiles that repel ants were detected in about a third of all plants tested (Willmer et al
2009 Junker et al 2011a Ballantyne and Willmer 2012) Pollen is often the source of ant-
repellent volatile compounds (Willmer et al 2009 Ballantyne and Willmer 2012) but they can
be emitted from other floral parts including petals That pollen is often the source of repellence
could suggest that preventing ant visitation may be more important in protecting male than
female function Additionally because repellence would decrease as pollen is removed from
anthers ants may return and defend flowers more quickly than if repellent chemicals were in
longer-lasting structures (eg petals) In some cases floral volatiles are thought to mimic ant
alarm pheromones (Willmer et al 2009) However floral scents elicit inconsistent reactions
across ant species ranging from repellence to attraction (Agarwal and Rastogi 2007 Ballantyne
and Willmer 2012)
Producing structural or chemical resistance traits may be costly for plants Native
Hawaiian plants which have little recent coevolutionary history with ants appear to have
secondarily lost floral ant repellents (Junker et al 2011a) Both Willmer et al (2009) and Junker
et al (2011a) found that highly chemical repellent flowers did not have strong physical defenses
and vice versa suggesting trade-offs between the two (but see Ballantyne and Willmer 2012)
Like toxic nectar floral ant repellents might sometimes deter both pollinators and ants For
example Galen et al (2011) found that 2-phenylethanol in P viscosum floral scent repels both
flower-damaging F neorufibarbis ants and the plantrsquos main pollinator Bombus kirbyellus
Interestingly four recent studies have found that floral scents can also attract ants
(Agarwal and Rastogi 2007 Edwards and Yu 2008 Schiestl and Glaser 2011 Gonzaacutelvez et al
20
2012) One study (Schiestl and Glaser 2011) has shown that floral scents attract ants to a
potentially ant-pollinated plant and such systems may provide many more examples
4 Future directions
We have highlighted areas in need of further investigation throughout the review Here we focus
on several questions we consider high priorities for future research
i) Floral defenses against ants
a) Do floral resistance traits increase plant fitness
The existence ant-repellent floral traits is often taken as evidence that ants negatively affect plant
reproductive success However despite the abundance and variety of mechanisms excluding ants
from flowers few studies show that ants reduce plant fitness when they visit flowers (Table 11)
This absence is particularly apparent with repellent floral volatiles which may be the most
widespread floral resistance trait (Willmer et al 2009 Junker et al 2011a Ballantyne and
Willmer 2012) To our knowledge only one study has shown that floral ant repellents benefit
plants with repellent P viscosum flowers avoiding damaging ant visits (Galen et al 2011)
Research is urgently needed to investigate if these traits increase plant fitness by excluding ants
or if ant visitation is inconsequential More studies taking advantage of standing variation in
traits (Galen et al 2011) or creating it artificially (eg Junker et al 2010b) will allow us to
distinguish between the two
21
b) If ant visitation is often not costly what caused the evolution of repellent traits and
what maintains them Similarly are these repellent traits truly defenses against ant
visitation or are they generalized defenses against a range of floral antagonists
Ant-repellent floral traits are widespread and their production may be costly (Willmer et al
2009 Junker et al 2011a) The studies in Table 11 however indicate that flower-visiting ants
often do not disrupt plant reproduction particularly outside of myrmecophytes What then
selected for these traits One possibility is that these repellents are not directed solely at ants
Instead a community of antagonistic flower visitors may exert diffuse selection on plants
thereby selecting for and maintaining these traits Structural modifications may repel crawling
insects while chemical repellents such as floral volatiles may repel both flying and non-flying
insects (Junker et al 2011b) How much overlap there is in floral volatiles that repel both ants and
other antagonists needs investigation Until we examine how specific these defenses are to ants
we risk overstating the importance of ants in their evolution
ii) Do ants alter pollen movement patterns or reduce male fitness
We are aware of only four studies that have measured if ant visitation impacts male fitness
(Wyatt 1980 Fritz and Morse 1981 Ashman 2000 Galen and Geib 2007) While none of these
studies found an effect all measured only amounts of pollen or pollinia removed However this
measure does not adequately encompass male fitness alterations to pollen movement patterns
and eventual deposition location may be more important to male function than changes in
removal rates If as suggested by some studies (Altshuler 1999 Lach 2007) ants increase
pollinator relocation rates they may decrease pollen discounting increasing male fitness
However if this increased relocation reduces overall pollen export rates ant competition with
22
pollinators may depress male function Studies using pollen-tracking methods such as fluorescent
dyes will allow us to see how ants change pollen movement patterns and methods like paternity
analysis will reveal how competition between ants and pollinators alters male fitness
iii) Coevolution between ants and plants do myrmecophytes exhibit stronger more
specific defenses against ants
The floral traits of myrmecophytes are most likely to coevolve with ants because all plant
reproduction occurs in the presence of ants Their floral defenses would thus be expected to be
both stronger and more specific to their resident ants than those other plants visited by an array
of more opportunistic ant species Some evidence supports this Willmer et al (2009) found that
resident ants of acacias were repelled by flowers with more aggressive ant species showing
stronger repellence than less aggressive ants Additionally ants not normally associated with
acacias did not respond to acacia floral volatiles More studies are needed in other systems and
should take advantage of phylogenetic approaches examining floral traits in closely related
myrmecophytic and non-myrmecophytic plant species to determine whether ants commonly
select for floral defenses against ants in myrmecophytes
Acknowledgements
For comments and discussion we thank the Frederickson lab particularly Eric Youngerman
Lina Arcila Hernandez and Kyle Turner as well as Alison Parker and Jordan Pleet MEF
acknowledges financial support from an NSERC Discovery Grant a Connaught New Researcher
23
Award an Ontario Ministry of Economic Development and Innovation Early Researcher Award
and the University of Toronto ARC was supported by an Ontario Graduate Scholarship and
Sigma Xi
24
References
Adler L S 2001 The ecological significance of toxic nectar Oikos 91409ndash420
Agarwal V M and N Rastogi 2007 Role of floral repellents in the regulation of flower visits
of extrafloral nectary-visiting ants in an Indian crop plant Ecological Entomology 3359ndash
65
Altshuler D L 1999 Novel interactions of non-pollinating ants with pollinators and fruit
consumers in a tropical forest Oecologia 119600ndash606
Ashman TL 2000 Pollinator selectivity and its implications for the evolution of dioecy and
sexual dimorphism Ecology 812577-2591
Ashman TL and E A King 2005 Are flower-visiting ants mutualists or antagonists A study
in a gynodioecious wild strawberry American Journal of Botany 92891ndash5
Ballantyne G and P Willmer 2012 Floral visitors and ant scent marks noticed but not used
Ecological Entomology 37402-409
Ballantyne G and P Willmer 2012 Nectar theft and floral ant-repellence a link between
nectar volume and ant-repellent traits PloS One 7e43869
Baker H G and I Baker 1978 Ants and flowers Biotropica 1080
Barrett S C H 2002 Sexual interference of the floral kind Heredity 88154ndash159
Baroni Urbani C and M L de Andrade 1997 Pollen eating storing and spitting by ants
Naturwissenschaften 84256ndash258
25
Beattie A J C Turnbull R B Knox and E G Williams 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany
71421ndash426
Beattie A J C Turnbull T Hough S Jobson and R B Knox 1985 The vulnerability of
pollen and fungal spores to ant secretions evidence and some evolutionary implication
American Journal of Botany 72606ndash614
Blancafort X and C Goacutemez 2005 Consequences of the Argentine ant Linepithema humile
(Mayr) invasion on pollination of Euphorbia characias (L) (Euphorbiaceae) Acta
Oecologica 2849ndash55
Bleil R N Bluumlthgen and R R Junker 2011 Ant-plant mutualism in Hawailsquoi Invasive ants
reduce flower parasitism but also exploit floral nectar of the endemic shrub Vaccinium
reticulatum (Ericaceae) Pacific Science 65291ndash300
Bluumlthgen N G Gottsberger and K Fiedler 2004 Sugar and amino acid composition of ant-
attended nectar and honeydew sources from an Australian rainforest Austral Ecology
29418ndash429
Burkle L A R E Irwin and D A Newman 2007 Predicting the effects of nectar robbing on
plant reproduction implications of pollen limitation and plant mating system American
Journal of Botany 941935ndash1943
Byk J and K Del-Claro 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333ndash38
26
Caballero P C G Ossa W L Gonzaacuteles C Gonzaacutelez-Browne G Astorga M M Muruacutea and
R Medel 2013 Testing non-additive effects of nectar-robbing ants and hummingbird
pollination on the reproductive success of a parasitic plant Plant Ecology 214633ndash640
Carvalheiro L G E R M Barbosa and J Memmott 2008 Pollinator networks alien species
and the conservation of rare plants Trinia glauca as a case study Journal of Applied
Ecology 451419ndash1427
Chamberlain S A and J N Holland 2008 Density-mediated context-dependent consumer-
resource interactions between ants and extrafloral nectar plants Ecology 891364ndash1374
Creighton W S 1967 Studies on free colonies of Cryptocerus texanus Satschi (Hymenoptera
Formicidae) Psyche 7434ndash42
Cursach J and J Rita 2011 Implications of the reproductive biology of the narrow endemic
Naufraga balearica (Apiaceae) for its conservation status Plant Systematics and Evolution
298581ndash596
Cushman JH and Addicott JF In Huxley CR and DF Cutler (eds) 1st edition Ant-plant
interactions Oxford University Press Oxford
de Jong T J N M Waser and P G Klinkhamer 1993 Geitonogamy the neglected side of
selfing Trends in Ecology and Evolution 8321ndash325
de Vega C M Arista P L Oritz C M Herrera and S Talavera 2009 The ant-pollination
system of Cytinus hypocistis (Cytinaceae) a Mediterranean root holoparasite Annals of
Botany 4517ndash8
27
Dukas R 2001 Effects of perceived danger on flower choice by bees Ecology Letters 4327ndash
333
Dutton E M and M E Frederickson 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561ndash569
Edwards D P and D W Yu 2008 Tolerating castration by hiding flowers in plain sight
Behavioral Ecology and Sociobiology 6395ndash102
Frederickson M E 2009 Conflict over reproduction in an ant-plant symbiosis why Allomerus
octoarticulatus ants sterilize Cordia nodosa trees The American Naturalist 173675ndash81
Fritz R S and D H Morse 1981 Nectar parasitism of Asclepias syriaca by ants effect on
nectar levels pollinia insertion pollinaria removal and pod production Oecologia 50316ndash
319
Galen C 1983 The effects of nectar thieving ants on seedset in floral scent morphs of
Polemonium viscosum Oikos 41245ndash249
Galen C 2005 Catching ants with honey an experimental test of distraction and satiation as
alternative modes of escape from flower-damaging ants Oecologia 14480ndash87
Galen C and B Butchart 2003 Ants in your plants effects of nectar-thieves on pollen fertility
and seed-siring capacity in the alpine wildflower Polemonium viscosum Oikos 101521ndash
528
Galen C and J Cuba 2001 Down the tube pollinators predators and the evolution of flower
shape in the alpine skypilot Polemonium viscosum Evolution 551963-1971
28
Galen C and J C Geib 2007 Density-dependent effects of ants on selection for bumble bee
pollination in Polemonium viscosum Ecology 881202ndash1209
Galen C R Kaczorowski S L Todd J Geib and R A Raguso 2011 Dosage-dependent
impacts of a floral volatile compound on pollinators larcenists and the potential for floral
evolution in the alpine skypilot Polemonium viscosum The American Naturalist 177258ndash
272
Garciacutea M B R J Antor and X Espadaler 1995 Ant pollination of the palaeoendemic
dioecious Borderea pyrenaica (Dioscoreaceae) Plant Systematics and Evolution 19817ndash
27
Gaume L M Zacharias and R M Borges 2005 Ant ndash plant conflicts and a novel case of
castration parasitism in a myrmecophyte Evolutionary Ecology Research 7435ndash452
Graystock P and W O H Hughes 2011 Disease resistance in a weaver ant Polyrhachis
dives and the role of antibiotic-producing glands Behavioral Ecology and Sociobiology
652319ndash2327
Gόmez J M 2000 Effectiveness of ants as pollinators of Lobularia maritima effects on main
sequential fitness components of the host plant Oecologia 12290ndash97
Gόmez J M and R Zamora 1992 Pollination by ants consequences of the quantitative effects
on a mutualistic system Oecologia 91410ndash418
Gόmez J M R Zamora J A Hόdar and D Garciacutea 1996 Experimental study of pollination
by ants in Mediterranean high mountain and arid habitats Oecologia 105236ndash242
29
Gonccedilalves-Souza T P M Omena J C Souza and G Q Romero 2008 Trait-mediated effects
on flowers artificial spiders deceive pollinators and decrease plant fitness Ecology
892407ndash2413
Gonzaacutelvez F G L Santamariacutea R T Corlett and M A Rodriacuteguez-Gironeacutes 2012 Flowers
attract weaver ants that deter less effective pollinators Journal of Ecology 10178ndash85
Guerrant E O and P L Fiedler 1981 Flower defenses against nectar-pilferage by ants
Biotropica 1325ndash33
Harley R 1991 The greasy pole syndrome In Huxley CR and DF Cutler (eds) 1st edition
Ant-plant interactions Oxford University Press Oxford
Hansen D M and C B Muumlller 2009 Invasive ants disrupt gecko pollination and seed
dispersal of the endangered plant Roussea simplex in Mauritius Biotropica 41202ndash208
Hickman J C 1974 Pollination by ants a low-energy system Science 1841290ndash1292
Holland J N S a Chamberlain and T E X Miller 2011 Consequences of ants and
extrafloral nectar for a pollinating seed-consuming mutualism ant satiation floral
distraction or plant defense Oikos 120381ndash388
Houmllldobler B and Wilson E O 1990 The Ants 1st Edition Harvard University Press
Cambridge
Holway DA L Lach AV Suarez ND Tsutsui and TJ Case 2002 The causes and
consequences of ant invasions Annual Review of Ecology and Systematics 33181-233
30
Horskins K and Turner VB 1999 Resource use and foraging patterns of honeybees Apis
mellifera and native insects on flowers of Eucalyptus costata Australian Journal of
Ecology 24221-227
Horvitz C C and D W Schemske 1984 Effects of ants and an ant-tended herbivore on seed
production of a neotropical herb Ecology 651369ndash1378
Inouye DW 1980 The terminology of floral larceny Ecology 611251-1253
Inouye DW and O R J Taylor 1979 A temperate region plant-ant-seed predator system
consequences of extra floral nectar secretion by Helianthella quinquenervis Ecology 601ndash
7
Irwin RE 2003 Impact of nectar robbing on estimates of pollen flow conceptual predictions
and empirical outcomes Ecology 84485-495
Irwin RE and Maloof JE 2002 Variation in nectar robbing over time space and species
Oecologia 133525-533
Iwanami Y and T Iwadare 1978 Inhibiting effects of myrmicacin on pollen growth and pollen
tube mitosis Botanical Gazette 13942ndash45
Izzo TJ and Vasconcelos HL 2002 Cheating the cheater domatia loss minimizes the effects
of ant castration in an Amazonian ant plant Oecologia 133200-205
Janzen D H 1977 Why donrsquot ants visit flowers Biotropica 9252
Johnson SD Hargreaves AL and Brown M 2006 Dark bitter-tasting nectar functions as a
filter of flower visitors in a bird-pollinated plant Ecology 872709-2716
31
Junker R A Y C Chung and N Bluumlthgen 2007 Interaction between flowers ants and
pollinators additional evidence for floral repellence against ants Ecological Research
22665ndash670
Junker R R R Bleil C C Daehler and N Bluumlthgen 2010a Intra-floral resource partitioning
between endemic and invasive flower visitors consequences for pollinator effectiveness
Ecological Entomology 35760ndash767
Junker R R and N Bluumlthgen 2010b Floral scents repel facultative flower visitors but attract
obligate ones Annals of botany 105777ndash82
Junker R R C C Daehler S Doetteri A Keller and N Bluumlthgen 2011a Hawaiian ant ndash
flower networks nectar-thieving ants prefer undefended native over introduced plants with
floral defenses Ecological Monographs 81295ndash311
Junker R R J Gershenzon and S B Unsicker 2011b Floral odor bouquet loses its ant
repellent properties after inhibition of terpene biosynthesis Journal of Chemical Ecology
371323ndash31
Kawakita A and M Kato 2002 Floral biology and unique pollination system of root
holoparasites Balanophora kuroiwai and B tobiracola (Balanophraceae) American
Journal of Botany 891164ndash1170
Koopowitz H and TA Marchant 1998 Postpollination nectar reabsorption in the African
epiphyte Aerangis verdickii (Orchidaceae) American Journal of Botany 85508-512
Kirk W D J 1984 Ecological studies on Thrips imaginis Bagnall (Thysanoptera) in flowers of
Echium plantagineum L in Australia Australian Journal of Ecology 99ndash18
32
Kerner A 1878 Flowers and their unbidden guests C Kegan Paul London
Kessler D and I T Baldwin 2007 Making sense of nectar scents the effects of nectar
secondary metabolites on floral visitors of Nicotiana attenuata The Plant Journal 49840ndash
54
Koptur S and N Truong 1998 Facultative ant-plant interactions nectar sugar preferences of
introduced pest species in South Florida Biotropica 30179-189
Lach L 2005 Interference and exploitation competition of three nectar-thieving invasive ant
species Insectes Sociaux 52257ndash262
Lach L 2007 A mutualism with a native membracid facilitates pollinator displacement by
Argentine ants Ecology 881994ndash2004
Lach L 2008a Argentine ants displace floral arthropods in a biodiversity hotspot Diversity and
Distributions 14281ndash290
Lach L 2008b Floral visitation patterns of two invasive ant species and their effects on other
hymenopteran visitors Ecological Entomology 33155ndash160
Lach L 2013 A comparison of floral resource exploitation by native and invasive Argentine
ants Arthropod-Plant Interactions 7177-190
Luo C W K Li X M Chen Z Y Huang and K U N Li 2012 Ants contribute significantly
to the pollination of a biodiesel plant Jatropha curcas Environmental Entomology
411163ndash1168
33
Maleacute PJ G C Leroy A Dejean A Quilichini and J Orivel 2011 An ant symbiont directly
and indirectly limits its host plantrsquos reproductive success Evolutionary Ecology 2655ndash63
Maloof J E 2001 The effects of a bumble bee nectar robber on plant reproductive success and
pollinator behavior American Journal of Botany 881960ndash1965
Maloof J E and D W Inouye 2000 Are nectar robbers cheaters or mutualists Ecology
812651ndash2661
Moreau CS CD Bell R Vila SB Archibald and NE Pierce 2006 Phylogeny of the ants
diversification in the age of the angiosperms Science 312101-104
Nahas L M O Gonzaga and K Del-Claro 2012 Emergent impacts of ant and spider
interactions herbivory reduction in a tropical savanna tree Biotropica 44498ndash505
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506ndash514
Newman D A and J D Thomson 2005 Interactions among nectar robbing floral herbivory
and ant protection in Linaria vulgaris Oikos 110497ndash506
Nicklen E F and D Wagner 2006 Conflict resolution in an ant-plant interaction Acacia
constricta traits reduce ant costs to reproduction Oecologia 14881ndash7
Norment C J 1988 The effect of nectar-thieving ants on the reproductive success of Frasera
speciosa (Gentianaceae) American Midland Naturalist 120331ndash336
34
Orivel J L Lambs P-J G Maleacute C Leroy J Grangier T Otto A Quilichini et al 2011
Dynamics of the association between a long-lived understory myrmecophyte and its
specific associated ants Oecologia 165369ndash76
Palmer T M D F Doak M L Stanton J L Bronstein E T Kiers T P Young J R
Goheen et al 2010 Synergy of multiple partners including freeloaders increases host
fitness in a multispecies mutualism Proceedings of the National Academy of Sciences
10717234ndash9
Peakall R A J Beattie and S H James 1987 Pseudocopulation of an orchid by male ants a
test of two hypotheses accounting for the rarity of ant pollination Oecologia 73522ndash524
Peakall R and A J Beattie 1989 Pollination of the orchid Microtis parviflora R Br by
flightless worker ants Functional Ecology 3515ndash522
Peakall R C J Angus A J Beattie 1990 The significance of ant and plant traits for ant
pollination in Leporella fimbriata pollination Oecologia 84457ndash460
Puterbaugh M N 1998 The roles of ants as flower visitors experimental analysis in three
alpine plant species Oikos 8336ndash46
Prŷs-Jones OE and Willmer PG 1992 The biology of alkaline nectar in purple toothwort
(Lathraea clandenstina) ground level defenses Biological Journal of the Linnean Society
45373-388
Raine NE Willmer P and Stone GN 2002 Spatial structuring and floral avoidance
behaviour present in ant-pollinator conflict in a Mexican ant-acacia Ecology 833086-
3096
35
Ramsey M 1995 Ant pollination of the perennial herb Blandfordia grandiflora (Liliaceae)
Oikos 74265ndash272
Renault CK LM Buffa and Delfino MA 2005 An aphid-ant interaction effects on
different trophic levels Ecological Research 2071-74
Rico-Gray V and L B Thien 1989 Effect of different ant species on reproductive fitness of
Schomburgkia tibicinis (Orchidaceae) Oecologia 81487ndash489
Romero G Q P A P Antiqueira and J Koricheva 2011 A meta-analysis of predation risk
effects on pollinator behaviour PLoS One 6e20689
Rosumek FB FAO Silveira F de S Neves NP de U Barbosa L Diniz Y Oki F Pezzini
et al 2009 Ants on plants a meta-analysis of the role of ants as plant biotic defenses
Oecologia 160537ndash549
Roubik D W 1982 The ecological impact of nectar-robbing bees and pollinating
hummingbirds on a tropical shrub Ecology 63354ndash360
Rudgers J A 2004 Enemies of herbivores can shape plant traits selection in a facultative ant-
plant mutualism Ecology 85192ndash205
Schatz B and WT Wcislo 1999 Ambush predation by the ponerine ant Ectatomma ruidum
Roger (Formicidae) on a sweat bee Lasioglossum umbripenne (Halictidae) in Panama
Journal of Insect Behavior 12641-663
36
Schaffer W M D W Zeh S L Buchmann S Kleinhaus M V Schaffer and J Antrim
1983 Competition for nectar between introduced honey bees and native North American
bees and ants Ecology 64564ndash577
Schemske D W 1980 The evolutionary significance of extrafloral nectar production by Costus
woodsonii (Zingiberaceae) an experimental analysis of ant protection Journal of Ecology
68959ndash967
Schiestl F P and F Glaser 2011 Specific ant-pollination in an alpine orchid and the role of
floral scent in attracting pollinating ants Alpine Botany 1221ndash9
Schubart HOR AB Anderson 1978 Why donrsquot ants visit flowers A response to D H
Janzen Biotropica 10310-311
Schuumlrch S M Pfunder and B A Roy 2000 Effects of ants on the reproductive success of
Euphorbia cyparissias and associated pathogenic rust fungi Oikos 16ndash12
Stanton ML Palmer TM Young TP Evans A and Turner ML 1999 Sterilization and
canopy modification of swollen thorn acacia tree by a plant-ant Nature 401 578-581
Stephenson AG 1981 Toxic nectar deters nectar thieves of Catalpa speciosa American
Midland Naturalist 105381-383
Stout J C and D Goulson 2001 The use of conspecific and interspecific scent marks by
foraging bumblebees and honeybees Animal Behaviour 62183ndash189
Sugiura S T Abe and S Makino 2006 Loss of extrafloral nectary on an oceanic island plant
and its consequences for herbivory American Journal of Botany 93491ndash495
37
Svensson L 1985 An estimate of pollen carryover by ants in a natural population of
Scleranthus perennis L (Caryophyllaceae) Oecologia 66373ndash377
Tsuji K A Hasyim and K Nakamura 2004 Asian weaver ants Oecophylla smaragdina and
their repelling of pollinators Ecological Research 19669ndash673
Turner R C J J Midgley P Barnard R E Simmons and S D Johnson 2012 Experimental
evidence for bird pollination and corolla damage by ants in the short-tubed flowers of
Erica halicacaba (Ericaceae) South African Journal of Botany 7925ndash31
Visser D M G Wright and J H Giliomee 1996 The effect of the Argentine ant
Linepithema humile (Mayr) (Hymenoptera Formicidae) on flower-visiting insects of
Protea nitida Mill (Proteaceae) African Entomology 4285ndash287
Wagner D 2000 Pollen viability reduction as a potential cost of ant association for Acacia
constricta (Fabaceae) American Journal of Botany 87711ndash715
Wagner D and A Kay 2002 Do extrafloral nectaries distract ants from visiting flowers An
experimental test of an overlooked hypothesis Evolutionary Ecology Research 4293ndash305
Wang C Y Luo Y Tai D An and Y Kou 2008 Ants pollinate Neottia listeroides
(Orchidaceae) in Sichuan China 46836ndash846
Wyatt R 1980 The impact of nectar-robbing ants on the pollination system of Asclepias
curassavica Bulletin of the Torrey Botanical Club 10724ndash28
Wheeler WM and IW Bailey 1920 The feeding habits of Pseudomymine and other ants
Transactions of the American Philosophical Society 22235-279
38
Willmer P G and S A Corbet 1981 Temporal and microclimatic partitioning of the floral
resources of Justicia aurea amongst a concourse of pollen vectors and nectar robbers
Oecologia 5167ndash78
Willmer P G and G N Stone 1997 How aggressive ant-guards assist seed-set in Acacia
flowers Nature 388165ndash167
Willmer P G C V Nuttman N E Raine G N Stone J G Pattrick K Henson P Stillman
et al 2009 Floral volatiles controlling ant behaviour Functional Ecology 23888ndash900
Young T P C H Stubblefield and L a Isbell 1997 Ants on swollen-thorn acacias species
coexistence in a simple system Oecologia 10998ndash107
Yu D W and N E Pierce 1998 A castration parasite of an ant-plant mutualism Proceedings
of the Royal Society B Biological Sciences 265375ndash382
39
Tables
Table 11 Studies of ant-flower or ant-pollinator interactions that included a measure of plant fitness Each study system is listed only
once except when results varied between years or when different studies examined male and female fitness Significant effects of ants on
plant fitness are either lsquopositiversquo or lsquonegativersquo non-significant effects are lsquoneutralrsquo When ants significantly affected only one fitness
component but several were measured (eg fruit set and seed production) we still consider the result significant
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Acacia
drepanolobium
Crematogaster
nigriceps
Proportion
fruiting
Negative Destruction of floral buds Young et al 1997
Asclepias exaltata Several Pollinia inserted
removed
Negative for pollinia
insertion neutral for
pollinia removal
Exploitative competition Wyatt 1980 T
40
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Asclepias syriaca Several
Fruit set pollinia
removed
Negative for fruit
set neutral for
pollinia removal
Exploitative competition Fritz and Morse
1981 T
Cordia nodosa Allomerus cf
demerarae
Fruit set Negative Destruction of floral buds Yu and Pierce
1998
Costus woodsonii Wasmannia
auropunctata
Seed set Positive Unknown ant pollination
suggested (but unlikely)
Schemske 1980
Eritrichum aretioides F neorufibarbis
gelida
Seed set Negative Ants damaged flowers Puterbaugh 1998
41
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Euphorbia characias Several Seed set Neutral Plants in Argentine ant-
invaded area set less seed
than those in un-invaded
sites but an ant-exclusion
experiment did not produce
significant results
Blancafort and
Gόmez 2005
Ferocactus wislizeni Solenopsis xyloni
and others
Seed set Unknown negative Interference and exploitative
competition S xyloni
reduced seed set relative to
other ants but the study did
not include a no-ant control
Ness 2006
42
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Frasera speciosa F neorufibarbis
and Formica sp
Seed set Neutral Nocturnal pollination
occurring when ants were
less active may have
compensated for reduced
diurnal pollinator visitation
Norment 1988
Gypsophila struthium Several Seeds per
inflorescence
Neutral No effect despite large
numbers of ants on flowers
Gόmez et al
1996
Hirtella physophora Allomerus
decemarticulatus
Fruit set Negative Destruction of flowers Orivel et al
2011
Humboldtia brunonis Crematogaster
dohrni
Fruit set Negative Destruction of flowers Gaume et al
2005
43
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Lepidium subulatum Several Percentage of
ovules setting
seed
Positive Unknown Gόmez et al
1996
Leucospermum
conocarpodendron
Several Seed set Neutral Site not pollen limited Lach 2008
Linaria vulgaris Several ldquoFemale fitness
indexrdquo
(combination of
number of seeds
fruits flowers
and seed weight)
Positive Reduction in florivory and
seed predation nectar
robbing holes increased ant
visitation
Newman and
Thomson 2005
44
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Melastoma
malabathricum
Oecophylla
smaragdina
Seed set Positive Ants deterred less efficient
pollinators increasing
visitation by the more
efficient pollinator
Gonzaacutelvez et al
2012
Metrosideros
polymorpha
Several Fruit set Neutral Ants reduced nectar
collection rates by foraging
bees but had no effect on
pollen collection rates or
fruit set
Junker et al
2010a
Orexis alpine F neorufibarbis
gelida
Seed set Neutral Autogamy or not pollen
limited
Puterbaugh 1988
45
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Ouratea spectabilis Cephalotes
pusillus and others
Seeds per fruit Neutral positive C pusillus had no effect on
seed number but other ants
collectively increased seed
set
Byk and Del
Claro 2010
Pachycereus schottii Several Seed set Neutral positive In one year ants showed a
positive effect (seed
number 2008) but no effect
with increased sample sizes
Chamberlain and
Holland 2008
Polemonium
viscosum
F neorufibarbis
gelida
Seed set Negative Ants damaged styles Galen 1983
46
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Polemonium
viscosum
F neorufibarbis
gelida
Pollen removal Neutral Ants did not affect pollen
removal even at high ant
densities
Galen and Geib
2007 T
Protea nitida Linepithema
humile and others
Seed set Neutral positive L humile had no effect on
P nitida seed set but native
ants increased it in one year
ants may cause pollinators
to relocate
Lach 2007
Psychotria
limonensis
Ectatomma spp Fruit set Positive Suggested increased
pollinator relocation
Altshuler 1999
47
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Roussea simplex Technomyrmex
albipes
Seed set Negative Interference and exploitative
competition
Hansen and
Muumlller 2009
Tristerix aphyllus Several Fruit set Neutral Nectar robbing ants
minimally reduced nectar
levels and had no effect on
hummingbird visitation
Caballero et al
2013
Vaccinium
reticulatum
Several Fruit set Neutral Ants reduced florivory but
did not affect fruit set
Bleil et al 2011
T Study measured male fitness
Study of sterilizing ants showed short-term decrease in female fitness long-term effects are unclear (see text)
48
Table 12 Studies demonstrating either i) ant pollination through an exclusion experiment or ii) that ants transfer pollen (or pollen
analogue) between flowers strongly suggesting ant pollination
Plant species Ant species Ant pollination Evidence Reference
Alyssum purpureum Several Yes Exclusion experiment Gόmez et al 1996
Arenaria tetraquetra Several Yes Exclusion experiment Gόmez et al 1996
Balanophora kuroiwai Leptothorax sp Suggested Exclusion experiment and
observed pollen transfer
and pollen tube formation
but did not see if plants set
seed
Kawakita and Kato 2002
Blandfordia grandiflora Iridomyrmex sp Yes Exclusion experiment Ramsey 1995
Borderea pyrenaica Several Yes Exclusion experiment Garciacutea et al 1995
49
Plant species Ant species Ant pollination Evidence Reference
Cytinus hypocistis Several Yes Exclusion experiment de Vega et al 2009
Epipactis thunbergii Camponotus
japonicas
Suggested Observed transfer of
pollinia from ants to
orchids
Sugiura et al 2006
Euphorbia cyparissias Several Yes Exclusion experiment Schuumlrch et al 2000
Fragaria virginiana Several Very likely Ant-only treatments set
comparable seed to flying
pollinator-only treatment
seed set increased with
increasing ant visitation
Ashman and King 2005
Frasera speciosa Several Yes Exclusion experiment Norment 1988
Frankenia thymifolia Several Yes Exclusion experiment Gόmez et al 1996
50
Plant species Ant species Ant pollination Evidence Reference
Hormathophylla spinosa Proformica
longiseta
Yes Exclusion experiment Gόmez and Zamora 1992
Jatropha curcas
Several
Tapinoma
melanocephalum
most common
Yes Exclusion experiment Luo et al 2012
Leporella fimbriata Myrmecia urens Yes Observed pollinia transfer
and subsequently
confirmed as ant
pollination
Peakall et al 1987
51
Plant species Ant species Ant pollination Evidence Reference
Lobularia maritima Several
Camponotus
micans most
common
Yes Exclusion experiment Gόmez 2000
Microtis parviflora
Iridomyrmex
gracilis
Yes Observation used
laboratory colonies to
replicate observations
Peakall and Beattie 1989
Naufraga balearica
Several
especially
Plagiolepis
pygmaea and
Lasius grandis
Suggested Observation over three
years of study only ants
were observed visiting
ants carried pollen
Cursach and Rita 2011
52
Plant species Ant species Ant pollination Evidence Reference
Neottia listeroides Leptothoras sp
Paratrechina sp
Suggested Observed transfer of
pollinia from ants to
orchids
Wang et al 2008
Paronychia pulvinata Formica
neorufibarbis
gelida
Yes Exclusion experiment Puterbaugh 1998
Polygonum cascadense Formica argentea
Very likely Grew plants in greenhouse
open to flying visitors with
minimal seed set only
plants with access to ants
set significant seed
Hickman 1974
53
Plant species Ant species Ant pollination Evidence Reference
Retama sphaerocarpa Several Yes Exclusion experiment Gόmez et al 1996
Scleranthus perennis
Several
especially
Formica
rufibarbis
Suggested Ants transferred pollen
analogue to stigmas and
carried pollen on bodies
Svensson 1985
Sedum anglicum Proformica
longiseta
Yes Exclusion experiment Gόmez et al 1996
Trinia glauca Several Yes Exclusion experiment Carvalheiro et al 2008
54
Chapter 2 Ants and ant scent reduce bumblebee pollination of artificial flowers
Cembrowski AR Tan MG Thomson JD and Frederickson ME
In press The American Naturalist
All authors designed the experiment ARC and MGT performed the experiment ARC and MEF
performed statistical analyse
s and ARC wrote the manuscript with input from all authors
Abstract
Ants on flowers can disrupt pollination by consuming rewards or harassing pollinators but it is
difficult to disentangle the effects of these exploitative and interference forms of competition on
pollinator behavior Using highly rewarding and quickly replenishing artificial flowers that
simulate male or female function we allowed bumblebees (Bombus impatiens) to forage (1) on
flowers with or without ants (Myrmica rubra) and (2) on flowers with or without ant scent cues
Bumblebees transferred significantly more pollen analogue both to and from ant free flowers
demonstrating that interference competition with ants is sufficient to modify pollinator foraging
behavior Bees also removed significantly less pollen analogue from ant scented flowers than
from controls making this the first study to show that bees can use ant scent to avoid harassment
at flowers Ant effects on pollinator behavior possibly in addition to their effects on pollen
viability may contribute to the evolution of floral traits minimizing ant visitation
55
Introduction
Trait-mediated indirect interactions arise when a focal species causes phenotypic changes
including behavioral modifications in a second species and these effects cascade to still other
species (Werner and Peacor 2003) Although trait-mediated indirect interactions are often studied
in food webs (eg Werner and Peacor 2003 Preisser et al 2005) they are not limited to trophic
interactions For example the threat of predation can change the behavior of a mutualist thus
affecting its partners (Suttle 2003) In animal-pollinated plants predators can disrupt pollination
directly through density-mediated indirect interactions (Dukas 2005) or via trait-mediated
indirect interactions by changing pollinator behavior When predators are present pollinators
may switch to visiting less rewarding flowers or they may avoid flowers altogether potentially
reducing the amount of pollen donated or received by a flower and thus decreasing plant fitness
(Gonccedilalves-Souza 2008 Ings and Chittka 2009) Similarly competition for floral rewards with
other flower visitors may cause behavioral changes in pollinators (Maloof and Inouye 2000
Ohashi et al 2008) Previous research has shown that bees may spend less time visiting flowers
depleted of rewards (Thomson 1986) and may avoid flowers bearing scent marks that indicate
recent visits by other individuals of the same or different bee species as these flowers are likely
to be depleted (Stout and Goulson 2002)
Whereas competition between pollinators may not be particularly costly for the plant
because both competitors are likely to provide pollination services competition between
pollinators and organisms that do not usually provide a pollination service such as ants has a
greater potential to reduce plant fitness Ants are common floral visitors they are attracted to
flowers for nectar (Lach 2007) and in some cases pollen (Byk and Del Claro 2010) and will
often defend these resources against other flower visitors (Altshuler 1999) However ants rarely
56
contribute much useful pollination antibiotic secretions present on their cuticles kill pollen
grains (Beattie et al 1985 Dutton and Frederickson 2012) and when foraging in flowers ants
sometimes impede female function by damaging stigmas (Galen and Cuba 2001) We will
henceforth consider ants to be non-pollinators and will contrast them to animals like bees that
are pollinators Ants are capable of competing with pollinators in two ways (1) by consuming
floral rewards and reducing their availability to pollinators (ie exploitative competition) and (2)
by directly antagonizing and excluding pollinators from flowers (ie interference competition)
Many plants benefit from the presence of ants numerous plant species recruit ants using
extrafloral nectar food bodies or domatia because ants provide protection against folivores (Heil
and McKey 2003) Similarly some plants have extrafloral nectaries on or near reproductive
tissues that recruit ants that deter florivores (eg Inouye and Taylor 1979) However plants
may incur reproductive costs as a result of their association with ants Some ant species that nest
in myrmecophytes sterilize flowers to shunt plant resources from reproduction to vegetative
growth allowing for greater ant colony growth (Frederickson 2009) Also ants visiting
extrafloral nectaries may be attracted to floral nectaries where they can harass pollinators (Ness
2006) There is growing evidence that many angiosperms have evolved floral traits that prevent
ants from accessing flowers during anthesis (Willmer and Stone 1997 Ballantyne and Willmer
2012) The effects of flower-visiting ants can be substantial Ants can decrease the frequency
duration or species diversity of pollinator visits to flowers (Lach 2008 Hansen and Muumlller 2009
Junker et al 2010 Gonzaacutelvez et al 2012) all of which potentially impact pollen donation and
receipt In some studies changes to pollinator behavior induced by ants have also been linked to
seed set
It is difficult however to disentangle the intertwined effects of exploitative and
interference competition in nature If ants disrupt pollination plants may often evolve traits to
57
deter ants from flowers (Willmer et al 2009) From a practical standpoint this would then limit
our ability to study the effects of ants on pollinator visitation because ants would not visit
flowers Here we used artificial flowers to explore ant-bee interactions in the absence of floral
defenses against ants We used highly rewarding quickly replenishing artificial flowers to
examine how direct harassment (ie interference competition expressed through behavior)
changes bee foraging behavior and how this affects donation and receipt of a pollen analogue
(powdered food dye) while minimizing the effects of exploitative competition We predicted
that flowers visited by ants would both donate and receive less pollen analogue than flowers
without ants because bees would avoid flowers with ants or leave them sooner Because scent
plays a large role in bumblebee communication and flower choice (Stout and Goulson 2002) we
also tested whether the presence or absence of ant scent on artificial flowers would affect pollen
analogue donation because of changes in the foraging behavior of bumblebees
Methods
i) Subjects
Myrmica rubra is an invasive ant in eastern North America with a range and habitat preferences
overlapping that of Bombus impatiens a common bumblebee Because M rubra visits flowers
(A Cembrowski pers obs) the two species likely interact in nature We collected 12 M rubra
colonies in the fall of 2011 and 2012 from Toronto Ontario Canada and the surrounding area
and maintained them in environmental chambers on artificial diet (Dussutour and Simpson 2009)
and a 1410 LD schedule (light 600-2200) We used these colonies as sources of M rubra
workers for experiments
58
Workers from commercially supplied colonies of Bombus impatiens (Biobest Canada
Ltd) foraged on artificial flowers in flight cages (either 24 x 24 x 21 m or 79 x 34 x 20m) at
the University of Toronto We tested a total of five bee colonies four colonies were used in the
ant presence trials and two of these and one additional colony were used in the ant scent trials
Flight cages had overhead fluorescent lights attached to timers In contrast to most previous
studies in which bees have been trained and tested individually the entire worker force of a bee
colony was free to forage at will in our experiments Colonies were trained to forage on artificial
flowers for at least four days before being used in trials After being used for a trial the colony
was not used for at least two days to reduce dye carryover between trials Between trials
bumblebees were fed pollen and given sugar water
ii) Artificial flowers
The flowers (Fig 1 Thomson et al 2012 see also Makino 2008) consisted of glass jars filled
with 30 WV sucrose solution Sugar water travelled by capillary action up a sewing-thread
wick to a hole in a blue-painted lid accumulating in a knot that acts as a nectary Flowers
depleted by visitors were quickly replenished via capillary action and were non-rewarding only
very briefly after visits taking less than a minute to accumulate 05 microl note that bumblebee
visits to a single flower are often separated by several minutes or more (A Cembrowski
unpublished data)
Unlike most previous artificial flower designs these flowers allow for estimation of male
and female fitness because we can measure both the amount and type of dye received by female
flowers (see (d) Dye quantification below) In order to access nectar on a ldquomalerdquo flower a bee
must crawl through a circular ldquoantherrdquo of brush-like weather stripping material dusted in a
59
consistent manner with our pollen analogue powdered food dye (FDampC 5 or 6) which is
transferred to the bee in the process On a ldquofemalerdquo flower a bee crawls through a sticky plastic
reinforcement that functions as a ldquostigmardquo receiving dye from the beersquos body Male flowers
dispensed dye particles over multiple bee visits and could still dispense dye at the end of trials
They resemble many real flowers or flower heads in that pollen is dispensed gradually over time
but less dye is available for transfer with each subsequent bee visit (Harder and Thomson 1989)
iii) Flight cage trials
We conducted 6 ant scent trials between 22 March and 4 June 2012 and the remaining trials (16
ant presence trials and 9 ant scent trials) between 9 November 2012 and 6 January 2013 Ant
presence trials lasted for eight hours and ant scent trials lasted for four hours Artificial flowers
were prepared and placed individually in small plastic containers treated with Fluon (Insect-a-
Slip BioQuip Products Inc) to prevent ants from escaping In ant presence trials we used 32
flowers (16 male and 16 female flowers) arranged in an eight-by-four array with flowers spaced
45 cm apart between rows and 30 cm within rows Ant scent trials used 20 flowers (10 male and
10 female flowers) in a four-by-five array with flowers spaced 30 cm apart between and within
rows In all of the ant presence trials and 9 of the ant scent trials we counted the number of bees
actively foraging after one hour to get a measure of colony activity
a) Ant presence trials
We examined the effects of ants on the amount of dye donated by male flowers and received by
female flowers in sixteen trials testing four bee colonies four times each The containers of eight
randomly chosen male flowers and eight randomly chosen female flowers received 15 M rubra
60
workers each the other 16 flowers remained free of ants Isolated from their colonies M rubra
workers have nowhere to deposit sugar water they collect and therefore may become sated
nonetheless a force of 15 ant workers was enough to maintain visitation to the nectaries where
bees foraged while still rarely having more than one or two ants visit the nectary at any time As
ants attacked bees bees would occasionally carry or throw attacking ants off flowers but despite
this some ants maintained their presence at the nectary throughout the trials Because ants the
size of M rubra consume liquids slowly (~017 ndash 024 microlmin Davidson et al 2004) flowers
replenished nectar more quickly than ants consumed it We used two colors of dye (FDampC 5 and
FDampC 6) to differentiate male flowers with and without ants In two trials for each bee colony
anthers of male flowers with ants were coated with FDampC 5 dye while brushes of male flowers
lacking ants were coated with FDampC 6 dye In the other two trials we reversed dye colors to
control for effects of dye type and color Dye color was assigned in a random order Artificial
flowers were placed in randomly assigned positions in the array before we opened the colony
and allowed the bees to begin foraging
b) Scent trials
We explored the effect of ant scent on dye donation in fifteen trials testing three bee colonies in
six five and four trials each Bees were first exposed to ants by allowing the bees to forage for
eight hours on twenty flowers (ten male and ten female) of which five male flowers had 15 ants
and all others had none No dye was used during this ldquoexposurerdquo day We then collected all but
the five ant-free male flowers in the flight cage leaving these flowers to keep bees foraging
Next we individually stored five new male lids in Fluon-treated containers with ten M rubra
workers Five control male lids were put in identical containers lacking ants The following
61
morning we removed the remaining flowers and set out ten new male and female flowers in a
random spatial arrangement using the lids having or lacking ant scent Thus the ant-scented
flowers were not in the same positions as the ant-visited flowers on which the bees were trained
Only five male flowers had ant scent and the other 15 flowers (five male and ten female) did not
We used the same two dye colors as in i) to differentiate male flowers with and without ant
scent and randomized which color was used for ant-scented flowers between trials We collected
and replaced stigmas from female flowers after one hour and collected the stigmas again after
four hours In the first trial performed we also collected stigmas after two hours but due to the
small amount of dye transferred this was not repeated
iv) Dye quantification
After each trial we quantified the amount of dye transferred to female flowers using a
spectrophotometer We removed the stigmas from female flowers placed them in test tubes
added 51 mL of distilled water to each tube and vortexed each tube thoroughly to ensure the
dye was evenly diluted These were diluted further as needed if the absorbance exceeded the
sensitivity range of the spectrophotometer In the first six scent trials each stigma was analyzed
separately and the dye amounts were summed to obtain a total amount of each dye color
transferred to female flowers in each trial In all the ant presence trials and the other nine scent
trials we opted to treat the experiment as the unit of replication Therefore we combined stigmas
from each treatment type (ant-visited or ant-free) in a test tube and measured the total amount of
each dye color transferred in each treatment in each trial
Because we put different dye colors on male flowers with and without ants we could use
the amount of each dye color donated to all female flowers to measure the reproductive success
62
of ant-visited and ant-free male flowers The total amount of dye (of both colors) received by
female flowers with and without ants was our measure of female reproductive success We
calculated the amount of each dye color in the sample by measuring absorbance at 428 or 486
nm and converting absorbance to micrograms following computational methods for overlapping
spectra (Blanco et al 1989)
v) Statistical analyses
In two trials (one ant presence and one ant scent) most female flowers received no dye because
of low bee activity so we excluded these trials from analyses Because dye reception and
donation values were non-normally distributed we square-root transformed the data before
examining the effects of ant presence or ant scent on dye transfer in ANCOVAs For ant
presence trials we included ant presence on male and female flowers and their interaction as
main effects and the total amount of dye transferred in each trial as a covariate to account for
the large variation in overall dye transfer among trials For ant scent trials we included ant scent
as the main effect time (one or four hours) as a repeated measure the interaction between scent
and time and the total amount of dye transferred as a covariate Covariate by treatment (ant
presence or ant scent) interactions were never significant and so were excluded in final analyses
All analyses were conducted in JMP (v 1000)
Results
Bees usually started foraging within minutes of the beginning of the trial and continued until
flowers were collected from the flight cage or the lights were extinguished An average of 34 plusmn
012 and 40 plusmn 015 (mean plusmn SE) bees were foraging after an hour in the ant presence and ant
63
scent trials respectively Male flowers with ants donated significantly less dye than male flowers
lacking ants (Fig 22a F155 = 4019 p lt 00001) Similarly female flowers with ants received
significantly less dye than female flowers lacking ants (Fig 22a F155 = 461 p = 0036) There
was no significant interaction between ant presence on male flowers and ant presence on female
flowers (F155 = 118 p = 028) Flowers with ant scent donated significantly less dye than
flowers without ant scent (Fig 22b F125 = 11216 p lt 00001) There was no significant effect
of time (F125 = 121 p = 028) or interaction between time and scent (F125 = 111 p = 030) in
the model
Discussion
In this study ants altered bumblebee pollination behavior and exhibited trait-mediated indirect
interactions with (artificial) flowers Through interference competition M rubra workers
significantly affected the pattern of dye transfer by B impatiens causing a preferential flow of
dye from male flowers lacking ants to female flowers lacking ants If manifested in nature such
effects would reduce the reproductive success of plants visited by ants through both male and
female function
Ants could have changed the attractiveness of artificial flowers in at least two ways First
as ants interrupted or altogether prevented bumblebees from foraging at nectaries bumblebees
may have learned to avoid flowers with ants due to the relative inefficiency of foraging at these
flowers something bumblebees take into account (Heinrich 2004) Second bees attempt to
minimize their risk of being attacked during foraging bouts which ants did to visiting bees Ants
often attacked or harassed bees by biting grasping and appearing to sting visiting bees
preventing them from accessing the nectary or reducing their time on flowers (see online video
64
1) and bees sometimes avoided ant-tended flowers entirely This harassment was sometimes
physically traumatic (online video 1) and bees often appeared to have trouble flying after being
attacked by ants Previous research has demonstrated that bumblebees leave or avoid flowers
where they have been harassed (Jones and Dornhaus 2011) and avoid foraging where there is
visual or olfactory evidence of a predator or predation event (Abbott 2006 Goodale and Nieh
2012) Though it is unclear if bumblebees viewed ants as competitors or predators they
responded similarly to flowers having ants as they do to flowers housing predators (Gonccedilalves-
Souza et al 2008)
To avoid artificial flowers with ants bees likely used a combination of visual and
olfactory cues Previous research has shown that bees respond to conspicuous predators or
predator ldquodummiesrdquo on flowers (Suttle 2003 Gonccedilalves-Souza et al 2008) decreasing the
frequency and duration of their visits to these flowers Thus bees may have been able to avoid
flowers harboring ants by sight alone However even in the absence of ants bees still
preferentially visited flowers lacking ant scents (Fig 22b) suggesting that they had learned to
associate ant scent with harassment Bees are adept at associative learning (Wright and Schiestl
2009) and can learn to recognize unique scents left behind by both conspecific and heterospecific
flower visitors (Stout et al 1998) Bees can use these various scents often arising from tarsal
gland deposits (Stout et al 1998) to recognize recently visited flowers that are less likely to be
profitable (Stout and Goulson 2002) Recently Ballantyne and Willmer (2012) demonstrated that
bees learn to associate ant scents with unrewarding artificial flowers and decrease their visitation
to these flowers Our results complement their findings by showing that bees can associate ant
scent with harassment at otherwise rewarding flowers
In our study ant scent caused bees to adjust their foraging strategy and decreased the
amount of pollen analogue that was donated by ant-scented flowers Thus the effects of
65
interference competition with ants on flowers can extend beyond immediate interactions and may
have fitness consequences for plants even when ants are absent Like other olfactory cues these
effects are likely transitory (Stout et al 1998) Although more dye was still donated by male
flowers lacking ant scent than those with ant scent the ratio of dye donated was on average
closer to equality in hours 2-4 than in the first hour (Fig 22b) This effect may be partially
driven by dye depletion of male flowers without ant scent but the lack of a strong corresponding
decrease in flowers with ant scent suggests that visitation patterns became more similar We did
not test whether B impatiensrsquo avoidance of ant scent was an innate or a learned behavior but
previous work has shown that Bombus terrestris does not innately avoid flowers with ant scent
(Ballantyne and Willmer 2012)
Ants may be necessary for plant survival and growth but can be costly for plant
reproduction The evolution of ant attractants such as extrafloral nectaries in some plant lineages
suggests that the costs of ants can be outweighed by their protective abilities However in this
study flowers visited by ants received and donated significantly less dye although they did
retain some sexual function The net benefit of having ants depends on whether ants increase
plant fitness by reducing herbivory more than they decrease plant fitness by disrupting
pollination Alternatively plants may actually benefit from the costs of ants to plant
reproduction ants that castrate flowers may be better defenders increasing plant survival or
vegetative growth when plants are young allowing for increased reproduction later in life when
the plant is colonized by less aggressive non-castrating ants (Frederickson 2009 Palmer et al
2010) In some plant species pollinator harassment by ants may even be beneficial Altshuler
(1999) reported that Ectatomma ants greatly increased fruit set of Psychotria limonensis despite
reductions in pollinator visitation rates presumably due to increases in the rate of pollen
outcrossing Similarly Gonzaacutelvez et al (2012) found that the presence of Oecophylla
66
smaragdina ants on Melastoma malabathricum flowers reduced visitation by less effective
pollinators and increased visitation by more effective Xylocopa bees increasing plant fitness
Cases of ants being beneficial to flowers appear to be the minority and many plant
species have traits that limit ant access to flowers Several studies have detailed floral volatiles
that are thought to mimic ant alarm pheromones and thus repel ants (Junker and Bluumlthgen 2008
Willmer et al 2009) Other plants use structural modifications such as narrow corollas or
slippery stems to limit ant access to flowers (reviewed in Willmer et al 2009) Exploitative
competition between ants and pollinators (Lach 2005 Ballantyne and Willmer 2012) as well as
the lethal effects of ant antibiotic secretions on pollen grains (Beattie et al 1985 Dutton and
Frederickson 2012) may have resulted in selection on plants to reduce ant visits to flowers Our
results suggest that trait-mediated indirect interactions resulting from interference competition
between ants and bees may favor plants that defend their flowers and their pollinators against
ants
Acknowledgements
We thank H Rusnock and S Meadley-Dunphy for assistance J Dix for help with flight cage
construction and Biobest for bumblebee colonies We are grateful to the Thomson and
Frederickson labs for guidance particularly T Makino funded by JSPS for helping to refine
artificial flowers J Ogilvie for help with bees K Prior for M rubra colony collections and K
Turner for study design suggestions The comments of the editor and two anonymous reviewers
prompted improvements in the article especially with regards to study design MEF and JDT
acknowledge NSERC Discovery Grant funding The University of Torontorsquos Faculty of Arts and
Science supported MGRTrsquos participation
67
68
References
Abbott K R 2006 Bumblebees avoid flowers containing evidence of past predation events
Canadian Journal of Zoology 841240-1247
Altshuler D L 1999 Novel interactions of non-pollinating ants with pollinators and fruit
consumers in a tropical forest Oecologia 119600-606
Ballantyne G and Willmer P 2012 Floral visitors and ant scent marks noticed but not used
Ecological Entomology 37402-409
Beattie A J Turnbull C Knox R B and Williams E G 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany
71421-426
Blanco M Runiaga H Maspoch S and Tarin P 1989 A simple method for
spectrophotometric determination of two-components with overlapped spectra Journal of
Chemical Education 66178-180
Byk J amp Del Claro K 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333-38
Davidson D W S C Cook and R R Snelling 2004 Liquid-feeding performances of ants
(Formicidae) ecological and evolutionary implications Oecologia 139255ndash66
Dukas R 2005 Bumble bee predators reduce pollinator density and plant fitness Ecology
861401-1406
Dussutour A and Simpson S J 2009 Communal nutrition in ants Current Biology 19740-
744
69
Dutton E M and Frederickson M E 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561-569
Frederickson ME 2009 Conflict over reproduction in an ant-plant symbiosis why Allomerus
octoarticulatus ants sterilize Cordia nodosa trees American Naturalist 173675-681
Galen C and Cuba J 2001 Down the tube pollinators predators and the evolution of flower
shape in the alpine skypilot Polemonium viscosum Evolution 551963-1971
Gonccedilalves-Souza T Omena P M Souza J C and Romero G Q 2008 Trait-mediated
effects on flowers artificial spiders deceive pollinators and decrease plant fitness Ecology
892407-2413
Gonzaacutelvez F G Santamariacutea L Corlett R T and Rodriacuteguez-Gironeacutes M A 2012 Flowers
attract weaver ants that deter less effective pollinators Journal of Ecology 10178ndash85
Goodale E and Nieh JC 2012 Public use of olfactory information associated with predation
in two species of social bees Animal Behaviour 84919-924
Harder LD and Thomson JD 1989 Evolutionary options for maximizing pollen dispersal of
animal-pollinated plants American Naturalist 133323-344
Heil M and McKey D 2003 Protective ant-plant interactions as model systems in ecological
and evolutionary research Annual Review of Ecology Evolution and Systematics 34425-
453
Heinrich B 2004 Bumblebee economics 2nd
edn Harvard University Press Cambridge
Ings T C and Chittka L 2009 Predator crypsis enhances behaviourally mediated indirect
effects on plants by altering bumblebee foraging preferences Proceedings of the Royal
Society B 2762031-2036
70
Inouye D W and Taylor O R Jr 1979 A temperate region plant-ant-seed predator system
consequences of extra floral nectar secretion by Helianthella quinquenervis Ecology 601-
7
Junker R R and Bluumlthgen N 2008 Floral scents repel potentially nectar-thieving ants
Evolutionary Ecology Research 10295ndash308
Junker R R Bleil R Daehler C C and Bluumlthgen N 2010 Intra-floral resource partitioning
between endemic and invasive flower visitors consequences for pollinator effectiveness
Ecological Entomology 35760ndash767
Jones E I and Dornhaus A 2011 Predation risk makes bees reject rewarding flowers and
reduce foraging activity Behavioral Ecology and Sociobiology 651505-1511
Lach L 2005 Interference and exploitative competition of three nectar-thieving invasive ant
species Insect Sociaux 52257-262
Lach L 2007 A mutualism with a native membracid facilitates pollinator displacement by
Argentine ants Ecology 881994-2004
Lach L 2008 Argentine ants displace floral arthropods in a biodiversity hotspot Diversity and
Distributions 14281-290
Makino T T and Sakai S 2007Experience changes pollinator responses to floral display size
from size-based to reward-based foraging Functional Ecology 21854863
Maloof J E and Inouye D W 2000 Are nectar robbers cheaters or mutualists Ecology
812651ndash2661
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506-514
Ohashi K Leslie A and Thomson JD 2008 Trapline foraging by bumble bees V Effects of
experience and priority on competitive performance Behavioral Ecology 19936-948
71
Palmer T M Doak D F Stanton M L Bronstein J L Kiers E T Young T P Goheen
J R and Pringle R M 2010 Synergy of multiple partners including freeloaders
increases host fitness in a multispecies mutualism Proceedings of the National Academy
of Sciences 10717234-17239
Preisser E L Bolnick D I and Benard M E 2005 Scared to death The effects of
intimidation and consumption in predator-prey interactions Ecology 86501-509
Stout J C Goulson D and Allen J A 1998 Repellent scent-marking of flowers by a guild of
foraging bumblebees (Bombus spp) Behavioral Ecology and Sociobiology 43317-326
Stout J and Goulson D 2002 The influence of nectar secretion rates on the responses of
bumblebees (Bombus spp) to previously visited flowers Behavioral Ecology and
Sociobiology 52239-246
Suttle K B 2003 Pollinators as mediators of top-down effects on plants Ecology Letters
6688-694
Thomson JD 1986 Pollen transport and deposition by bumble bees in Erythronium influences
of floral nectar and bee grooming Journal of Ecology 74329-341
Thomson JD Ogilvie J E Makino T T Arisz A Raju S Rojas-Luengas V and Tan M
G R 2012 Estimating pollination success with novel artificial flowers effects of nectar
concentration Journal of Pollination Ecology 9108-114
Tsuji K Hasyim A and Nakamura K 2004 Asian weaver ants Oecophylla smaragdina and
their repelling of pollinators Ecological Research 19669-673
Ulrich S and Bertsch A 1990 Do foraging bumblebees scent-mark food sources and does it
matter Oecologia 82137-144
Werner E E and Peacor S D 2003 A review of trait-mediated indirect interactions in
ecological communities Ecology 841083-1100
72
Willmer P G Nuttman C V Raine N E Stone G N Pattrick J G Henson K Stillman
P McIlroy L Potts S G and Knudsen J T 2009 Floral volatiles controlling ant
behaviour Functional Ecology 23888-900
Wright G A and Schiestl F P 2009 The evolution of floral scent the influence of olfactory
learning by insect pollinators on the honest signaling of floral rewards Functional Ecology
23841-851
73
Figures
Figure 21 Photograph of artificial flower lids A = anther L = lid N = nectary R = region
where anther or stigma is placed S = stigma T = thread W = weight
74
Figure 22 Dye transferred (mean plusmn 1SE) per trial by B impatiens workers A) from artificial
male and to artificial female flowers in the presence (unfilled circles) or absence (filled circles)
of Myrmica rubra workers and B) from artificial male flowers with (unfilled circles) or without
(filled circles) M rubra scent to ant-free female flowers in the 1st or the 2
nd to 4
th hours of the
trials
75
Chapter 3 Not just for the bees pollen consumption is common among tropical
ants
Cembrowski AR Reurink G and Frederickson ME
Planned submission to Biotropica
ARC and MEF designed the study ARC and GR did laboratory work ARC performed statistical
analyses and wrote the manuscript with input from other authors
Abstract
Although palynivory or pollen consumption is widespread among other hymenopterans there
are few accounts of palynivory in ants To quantify how often ants consume pollen we subjected
adult workers and larvae from 75 species of neo- and paleo-tropical ants to acetolysis a process
that destroys most organic material but leaves behind pollen grains In over half of the species
we examined ants contained a few pollen grains (lt 5) and ants of several Camponotus species
contained abundant pollen grains (gt50) We tested for an association between trophic level and
palynivory using stable nitrogen isotope ratios (δ15
N) but we did not find a significant
correlation we found pollen grains in lsquoherbivorousrsquo omnivorous and carnivorous ants We
suggest that our results indicate sporadic opportunistic pollen consumption by ants in tropical
forests
Introduction
For those organisms that can digest it efficiently pollen can be an excellent food source
Depending on the plant species pollen can contain up to 60 protein as well as lipids
76
carbohydrates and numerous trace nutrients (Roulston and Cane 2000) Pollen feeding
(palynivory) occurs in many taxa including birds mammals and arthropods (Roulston and Cane
2000) but it is best known among the Hymenoptera particularly bees Although adult bees feed
mainly on plant exudates (ie nectar) nearly all bee species rely on pollen as the chief protein
source for developing larvae (Willmer 2011) Closely related to bees ants have similar
nutritional requirements adult workers need carbohydrates whereas larvae are fed a diet rich in
protein (Dussutour and Simpson 2009) Ants also exploit nectar heavily and regularly visit
flowers (eg Herrera et al 1984 Bluumlthgen et al 2004) but unlike bees are not believed to
widely consume pollen Instead ants are thought to receive most of the protein in their diet from
predation or scavenging (eg Floren et al 2002)
However ants may not be as reliant on animal protein sources as commonly assumed In
tropical forests arboreal ants can have similar δ15
N ratios a measure of trophic level as
herbivores suggesting limited carnivory and substantial feeding on plant-derived protein sources
(Davidson et al 2003) One potential protein source is pollen This idea is supported by scattered
accounts of pollen consumption by ants from other ecosystems including deserts (Ness 2006)
boreal forests (Czechowski et al 2009) and tropical savannas (Byk and Del Claro 2010) One
ant genus Cephalotes is widely recognized to consume pollen (Creighton 1967 Baroni Urbani
and de Andrade 1997 Byk and Del Claro 2010) However beyond this genus our knowledge of
pollen consumption by ants has largely been limited to direct observations (but see Czechowski
et al 2011) and it is unknown if palynivory is truly rare among ants or simply under-reported
We borrowed a technique commonly used in palynology acetolysis to assess the frequency of
pollen consumption by ants in 75 neo- and paleo-tropical ant species This technique allowed us
to examine many more ant species than we could have observed directly
77
Methods
Adult worker ants were collected by hand at baits or from leaf litter using Winkler extractors
while larvae along with adults were collected by hand from nests Collections were made by L
Arcila Hernandez and J Sanders in the primary tropical rainforest surrounding the Centro de
Investigaciόn y Capacitaciόn Rio Los Amigos (CICRA 12deg34rsquoS 70deg05rsquoW) in Peru in 2010-
2011 and by E Youngerman from lowland primary and secondary rainforests in Madang
Province Papua New Guinea (PNG) in 2011-2012 We sorted ant samples to morphospecies and
then identified them to genus and when possible to species using existing keys which were
unavailable for PNG ants Ant samples used for acetolysis were whole adult workers guts from
adult workers and whole larvae
We used acetolysis to look for pollen grains inside ants In acetolysis organic material is
dissolved in acetic anhydride and sulfuric acid leaving behind stained pollen exines When
possible we used adult and larval ants of the same species from different colonies or bait
locations in an attempt to sample more representatively We prepared samples by first washing
them in 70 ethanol to remove any pollen present on the samplersquos outer surface before placing
them individually in micro-centrifuge tubes with ~05mL of a 91 mixture of acetic anhydride
sulfuric acid We then heated each sample at 95degC for 20min allowing tissues to dissolve Next
we centrifuged samples at 1000 RPM for 5min and decanted the liquid supernatant before
adding ~05mL of glacial acetic acid to the precipitate centrifuging again as above We decanted
the samples and added a drop of distilled water
78
We examined a 150μL sub-sample of this solution for pollen grains under a light
microscope Larvae gave less than 150μL of solution following acetolysis so we sampled the
entire amount We identified pollen grains by their staining and their surface architecture For
most species we examined at least three adult workers and three larvae but for some species
fewer than three adult workers or larvae were available (Tables 31 and 32)
We investigated the relationship between trophic level as measured by δ15
N ratios and
pollen consumption by ants Stable isotope (SI) data was available for some (n =23) of the
Peruvian species from a separate project in which collections overlapped Samples were stored in
EtOH and dried at 50degC overnight We used heads legs and thoraxes (but not gasters) of ants
for SI analysis Often single individuals did not provide enough material for analysis and we
combined multiple individuals to achieve necessary weights up to 10 for small ant species
Samples were analyzed at the Boston University Stable Isotope Laboratory using a Finnigan
Delta-S analyzer For other Peruvian species for which we had samples but not corresponding
stable isotope data (n =25) we took the median δ15
N ratio value of that speciesrsquo genus and
assigned it to the species in question Using pollen presenceabsence data for these 48 species
we used logistic regression to correlate pollen presence and δ15
N ratio using R (v 2151)
Results
We found pollen grains in 37 of the 197 adult workers 20 of the 102 larvae and 38 of the 75 ant
species we examined (Tables 31 and 32) The proportion of individuals containing pollen did
not differ significantly between larvae and adults (χ2
1 = 002 p = 088) The number of pollen
grains found was often low (median = 3) but highly variable (range 1-468) Also pollen
79
presence was inconsistent among conspecific ant samples of species that contained pollen 31
had pollen in only one of the larvae or adults examined In only three species (two Cephalotes
and one Myrmelachista) did all examined adults contain pollen
Highly lsquoherbivorousrsquo carnivorous and omnivorous ants contained pollen grains (Fig
31) There was no correlation between δ15
N ratio and pollen presence (χ2 = 150 df = 1 p =
022) In fact several carnivorous species contained pollen grains (eg Odontomachus sp 1
Pachycondola sp1)
Discussion
Pollen consumption by ants appears to be more common than suspected The majority of species
(51) had at least one individual that had consumed pollen and in 20 multiple individuals
contained pollen Numbers of grains found tended to be low however with most individuals
containing only a few grains Ants may use pollen as only a small portion of their diet exploiting
a host of other protein-containing foods more heavily including other low- δ15
N foods (eg
honeydew epiphylls Houmllldobler and Wilson 1990 Davidson et al 2003)
There are a few reasons why pollen may not be heavily exploited by ants Pollen requires
much post-consumption processing to extract its nutrients These nutrients are located in the
pollenrsquos cytoplasm which is protected from direct digestion by the exines and intines of the
grains Organisms must either break through these layers or cause the pollen grains to germinate
or pseudo-germinate to access and digest the cytoplasm (Roulston and Cane 2000) This can be
achieved by consuming a sugar-containing solution (such as nectar) along with pollen However
animals that are not adapted to feed on pollen may not be very efficient at this process only
80
extracting nutrients from some of the grains (Herrera and Martiacutenez del Rio 1998) Thus while
ants can likely get some nutrients from pollen other forms of more easily processed protein may
be more attractive
Pollen may also pose handling difficulties for ants Despite the large number of studies
that have recorded ants visiting flowers (Chapter 1) only a few papers detail ants harvesting
pollen directly from anthers Byk and Del Claro (2010) reported that Cephalotes pusillus ants
removed almost all of the available pollen from Ouratea spectabilis flowers and at least two
other studies describe ants consuming pollen along with nectar from flowers (Horskins and
Turner 1999 Ness 2006) Unlike bees which possess both morphological (eg specialized hairs
for holding pollen) and behavioral adaptations (eg stereotyped pollen-packing motions) ants
have no apparent mechanisms to increase the amount of pollen harvested and are likely limited
by the number of grains they can ingest or carry Rather than collect pollen directly from flowers
it appears more likely that ants collect it haphazardly in the environment leading to a smaller
number of pollen grains consumed at any one time It is known that at least Cephalotes ants
engage in such behavior Creighton (1967) observed Cephalotes texanus collecting pollen grains
trapped by Citrus x paradisi leaf hairs
There is a chance that the small numbers of pollen grains we observed came from
secondary pollen consumption (ie consuming a palynivorous organism) or consumption of
liquid containing pollen While we cannot rule this out we believe our results represent actual
targeted pollen consumption by ants Cephalotes ants known palynivores (Creighton 1967
Baroni Urbani and de Andrade 1997) contained similar numbers of pollen grains as many of the
pollen-containing ant species that we examined As well in at least once instance ants that we
found containing pollen grains were in the process of preying on non-pollen consuming
81
organisms (eg a Pachycondola species that was carrying a termite in its mandibles) or were
attracted by sugar baits
Contrary to our expectations there was no relationship between an ant speciesrsquo δ15
N ratio
and whether it had consumed pollen (Fig 32) We expected palynivory to be more common in
herbivorous ants because pollen presents a widespread source of protein a potentially limiting
resource The lack of pattern might reflect how available pollen grains are in the environment
While other forms of food may be preferable ants tend to be opportunistic foragers (Carroll and
Janzen 1973) and there is likely little cost associated with the consumption of scattered pollen
grains during foraging trips beyond the volume they occupy in the gut Some genera were found
to consistently consume pollen In support of previous observations (Baroni Urbani and de
Andrade 1997 Byk and Del Claro 2010) Cephalotes species were among the most common
pollen consumers Camponotus species also consistently contained pollen grains of the eight
species examined five contained pollen and pollen was found in species from both Peru and
PNG Camponotus species also contained the two largest numbers of pollen grains found (468
and 271) Taken together these results suggest that they may be another as yet unrecognized
commonly palynivorous genus
In general larvae did not contain large numbers of pollen grains (ranging from 1-38
grains) This is counter to what is seen in bees where larvae are the main recipients of pollen
(Willmer 2011) This difference may be because ant larvae are continually fed via trophallaxis
by adults throughout their development whereas bee larval cells are provisioned with pollen and
then sealed while larvae complete their development If both adults and larvae are able to extract
nutrients from pollen we would expect to see a preferential flow of nutrients from pollen to
larvae but not necessarily the grains themselves
82
Unlike palynivory by organisms that also provide pollination services pollen
consumption by ants is unlikely to benefit plants Ants rarely act as pollinators (Beattie et al
2004) They have pollen-killing antibiotic secretions on their cuticles (Dutton and Frederickson
2012) and they can compete with other flower visitors reducing flower visitation rates (Ness
2006) The negative impacts of flower-visiting ants are thought to have led to the evolution of
ant-repellent floral volatiles (reviewed in Willmer 2009) These volatiles can pre-empt
competition between ants and pollinators and may often be located within the pollen itself
(Willmer 2009) If floral volatiles truly are a defense against ant visitation our results suggest an
additional evolutionary pressure to not only prevent ant-pollinator interactions but to also
protect plant gametes from occasional consumption by a non-pollinating visitor
Acknowledgements
We would like to thank Jon Sanders Lina Arcila Hernandez and Eric Youngerman for providing
samples the Thomson lab for use of supplies the Pierce lab for funding support during JSrsquo and
EYrsquos collections and the staff of CICRA and the New Guinea Binatang Research Center for
logistical support The Peruvian Ministry of Agriculture provided permits (Nos 394-2009-AG-
DGFFS-DGEFFS and 299-2011-AG-DGFFS-DGEFFS) MEF acknowledges financial support
from an NSERC Discovery Grant a Connaught New Researcher Award an Ontario Ministry of
Economic Development and Innovation Early Researcher Award and the University of Toronto
ARC was supported by an Ontario Graduate Scholarship and Sigma Xi
83
References
Baroni Urbani C and M L de Andrade 1997 Pollen eating storing and spitting by ants
Naturwissenschaften 84256ndash258
Beattie A J C Turnbull R B Knox and E G Williams 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany 71421ndash
426
Bluumlthgen N G Gottsberger and K Fiedler 2004 Sugar and amino acid composition of ant-
attended nectar and honeydew sources from an Australian rainforest Austral Ecology
29418ndash429
Byk J and K Del-Claro 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333ndash38
Carroll C R and D H Janzen 1973 Ecology of foraging ants Annual Review of Ecology and
Systematics 4231ndash257
Creighton W S 1967 Studies on free colonies of Cryptocerus texanus Satschi (Hymenoptera
Formicidae) Psyche 7434ndash42
Czechowski W B Markό and A Radchenko 2009 Rubbish dumps reveal the diet of ant
colonies Myrmica schencki Em and Myrmica rubra (L) (Hymenoptera Formicidae) as
facultative pollen-eaters Polish Journal of Ecology 56737ndash741
Czechowski WB Markoacute K Erős and E Csata 2011 Pollenivory in ants (Hymenoptera
Formicidae) seems to be much more common than it was thought Annales Zoologici
61519ndash525
84
Dussutour A and S J Simpson 2009 Communal nutrition in ants Current Biology 19740ndash4
Dutton E M and M E Frederickson 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561ndash569
Floren A A Biun and K E Linsenmair 2002 Arboreal ants as key predators in tropical
lowland rainforest trees Oecologia 131137ndash144
Grant B R 1996 Pollen digestion by Darwinrsquos finches and its importance for early breeding
Ecology 77489-499
Herrera L G and C Martiacutenez del Rio 1998 Pollen digestion by new world bats effects of
processing time and feeding habits Ecology 792828ndash2838
Houmllldobler B and Wilson E O 1990 The Ants 1st Edition Harvard University Press
Cambridge
Horskins K and Turner V B 1999 Resource use and foraging patterns of honeybees Apis
mellifera and native insects on flowers of Eucalyptus costata Australian Journal of
Ecology 24221-227
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506ndash514
Roulston T H I and J H Cane 2000 Pollen nutritional content and digestibility for animals
Plant Systematics 222187ndash209
Van Tets I G 1997 Extraction of nutrients from Protea pollen by African rodents Belgian
Journal of Zoology 12759-65
Willmer P G 2011 Pollination and Floral Ecology 1st Edition Princeton University Press
New Jersey
85
Willmer P G C V Nuttman N E Raine G N Stone J G Pattrick K Henson P Stillman
et al 2009 Floral volatiles controlling ant behaviour Functional Ecology 23888ndash900
86
Tables
Table 31 Numbers of adult worker and larval ants examined for each species collected in Peru
Numbers of pollen grains found in individual samples are given in parentheses after the sample
size when there were no grains no numbers are given
Genus Species Number of adults analyzed
(numbers of grains found)
Number of larvae analyzed
(numbers of grains found)
Dolichoderinae
Azteca sp 1 3 0
sp 2 2 0
sp 3 0 2
sp 4 3 (1) 0
sp 5 3 (2) 0
Dolichoderus decollatus 3 (66) 0
sp 1 3 0
sp 2 3 1 (2)
sp 3 3 3
sp 4 0 2
Ecitoninae
Labidus sp 1 3 0
Formicinae
Camponotus sp 1 3 3
sp 2 3 (82) 1 (2)
sp 3 3 (3) 0
sp 4 0 2
87
sp 5 8 (11) 0
sp 6 4 (7 16 468) 0
sp 7 5 (12) 0
sp 8 2 (1) 0
Paratrechina sp 1 4 (11) 0
Myrmelachista sp 1 3 (5 11 18) 0
sp 2 0 3
Trachymyrmex sp 1 3 (21) 0
sp 2 3 0
Myrmicinae
Atta sp 1 3 0
Cephalotes atratus 3 (2 2 21) 0
placidus 3 (1 4 21) 0
sp 1 3 0
sp 2 1 0
sp 3 2 0
Crematogaster sp 1 3 1
sp 2 3 (35) 0
sp 3 9 (7) 0
sp 4 2 0
Megalomyrmex sp 1 3 0
sp 2 0 2
sp 3 4 (7) 0
sp 4 3 0
Monomorium sp 1 0 3 (2)
88
Pheidole sp 1 0 3
sp 2 3 0
sp 3 0 3
Procryptocerus sp 1 3 0
Solenopsis sp 1 0 3
sp 2 3 (3 5) 0
Wasmannia sp 1 3 0
Ponerinae
Pachycondola sp 1 3 (3) 0
Odontomachus sp 1 3 (34) 0
Pseudomyrmicinae
Pseudomyrmex sp 1 3 0
sp 2 3 0
sp 3 3 (9) 0
sp 4 0 3
sp 5 0 3
89
Table 32 Numbers of adult worker and larval ants examined for each species collected in Papua
New Guinea Numbers of pollen grains found in individual samples are given in parentheses
after the sample size when there were no grains no numbers are given
Genus Species Number adults analyzed
(grains found)
Number larvae analyzed
(grains found)
Aenictinae
Aenictus sp 1 3 3 (1 1)
Dolichoderinae
Philidris sp 1 3 1 (4)
Tapinoma sp 1 2 3 (38)
Technomyrmex sp 1 0 3 (12 12)
Ectatomminae
Rhytidoponera sp 1 1 3 (2)
Formicinae
Acropyga sp 1 3 3 (2)
sp 2 3 3
sp 3 3 3
Anoplolepsis sp 1 3 (1) 3 (1)
Calomyrmex sp 1 3 3 (1)
Camponotus sp 1 3 (1 271) 3
sp 2 1 3
sp 3 2 0
Nylanderia sp 1 3 3
Opisthopsis sp 1 3 3
Polyrachis sp 1 1 3 (4)
90
Myrmicinae
Crematogaster sp 1 3 3
sp 2 3 (32) 3
Monomorium sp 1 3 (1) 3 (1)
sp 2 3 3 (1 1 1)
Pheidole sp 1 3 3
Pristomyrmex sp 1 3 3 (11)
Pyramica sp 1 3 0
Ponerinae
Odontomachus sp 1 3 3 (4)
91
Figures
Figure 31 Photographs of pollen grains found in two ant species a) Dolichoderus sp 2 and b)
Camponotus sp 6 Scale bars = 50 microm
92
Figure 32 Relationship between trophic level as measured by δ15
N ratio and pollen presence
for 48 neotropical ant species 1 Atta sp 5 Azteca spp 8 Camponotus spp 5 Cephalotes spp 4
Crematogaster spp 5 Dolichoderus spp 1 Labidus sp 3 Megalomyrmex spp 1 Monomorium
sp 2 Myrmelachista spp 1 Odontomachus sp 1 Pachycondola sp 1 Paratrechina sp 3
Pheidole sp 4 Pseudomyrmex spp 2 Solenopsis spp and 1 Wasmannia sp
93
Concluding Remarks
Ants have traditionally been considered to be unwelcome flower visitors they consume floral
rewards ostensibly meant for pollinators while rarely providing pollination services themselves
In this thesis I investigate the validity of this assumption and use an experimental study and an
observational study to examine two ways that ants may impact plant reproduction
In the first chapter I review the literature on ants as flower visitors I show that the common
assumption of ants always being categorically negative for plant reproduction is incorrect while
ants may reduce floral attractiveness to pollinators the net effect of ants on plant fitness
measures is variable ranging from positive to negative to neutral I also discuss ants as
pollinators and ways that plants may minimize ant visitation to flowers before finishing with a
section highlighting questions that still need investigation
In the second chapter I explore how interference competition with ants can structure bumblebee
foraging behavior and how this may alter pollen movement patterns Using artificial flowers with
male or female function I allowed Bombus impatiens bumblebees to forage on flowers that
either did or did not have Myrmica rubra ants on them Ant presence significantly reduced the
amount of pollen analogue donated and received by flowers Building on these results I found
that the presence of ant scent alone elicited similar changes to bumblebee behavior reducing the
amount of pollen analogue donated by male flowers My results show that interference
competition is sufficient to alter bumblebee foraging behavior and that the effects of ants on
pollinators may extend after ant visitation to flowers Further study is needed to determine if bees
are able to differentiate between ant species by visual or olfactory cues Their ability to do so
94
could affect how generalized their response to ant presence is and may help to better predict how
ants will affect plant reproduction
In the final chapter I investigate how common palynivory is amongst tropical ant species I used
acetolysis and light microscopy to examine whole ants larvae and ant guts from Papua New
Guinea and Peru I found that tropical ants consistently contain pollen grains albeit often in
small numbers (lt5) However some genera notably Camponotus and Cephalotes consistently
contained higher numbers providing the first evidence that Camponotus may be a palynivorous
genus The small numbers of pollen grains present may indicate that ants preferentially exploit
other more easily digestible high-protein foods over pollen Future studies should investigate
exactly what role pollen plays in ant diets by supplementing the diet of captive ant colonies with
pollen This would allow us to examine how pollen consumption affects colony demography as
well as how efficiently ants digest pollen and whether ants exhibit pollen preferences
I bring up several pressing and unanswered questions within this thesis and highlight three here
First numerous plant species possess chemical or physical traits assumed to increase plant
fitness by reducing ant visitation However this has rarely been tested and instead research has
focused predominately on documenting ant repellence We should move away from this and
begin examining plant fitness in the presence or absence of these repellent traits to see how
beneficial these traits truly are Second how flower visiting ants impact male fitness has been
almost completely ignored in favor of female fitness Future studies should investigate how ant
visitation affects both pollen donation and pollen movement patterns Lastly research involving
antibiotics present on ant cuticles and their effect on pollen viability has been too small in scope
often limited to demonstrating the pollen killing effects of ant secretions Investigation into
95
whether plants have evolved any resistance to the secretions of the local ant community or why
some plants appear unaffected by their ant partners may yield exciting results
In conclusion ants have the capacity to strongly affect plant reproduction both positively and
negatively The fact that these diametrically opposing forces exist means we cannot approach
flower-visiting ants as either positive or negative a priori Instead we should strive to work
towards a better understanding of what is occurring in the system of interest and what role ants
play within it
96
Copyright Acknowledgments
Chapter 2 ldquoAnts and ant scent reduce bumblebee pollination of artificial flowersrdquo is current in
press in The American Naturalist Request for its inclusion here was sought and approved
5
ii) Competition with pollinators
Ant activity on flowers can dramatically alter pollinator behavior Ants can reduce the species
diversity frequency or duration of pollinator visits to flowers (Norment 1988 Tsuji et al 2004
Ness 2006 Junker et al 2007 Lach 2007 2008b Romero et al 2011 and see Chapter 2) These
changes are largely due to either exploitative competition (ie consumption of shared resources)
or interference competition (ie direct aggression) between ants and pollinators
a) Exploitative competition
Pollinators and ants frequently compete for floral nectar Flower-visiting ants are often classified
as nectar thieves (sensu Inouye 1980) because they typically enter flowers through the
ldquolegitimaterdquo opening and consume nectar but transfer no pollen (Wyatt 1980 Ness 2006
Chamberlain and Holland 2008) Ants can also act as primary nectar robbers chewing holes
through corollas to reach nectaries (Willmer and Corbet 1981 Koopowitz and Marchant 1998)
although they are more commonly secondary nectar robbers and use holes made by previous
robbers (Newman and Thomson 2005)
Like other flower visitors foraging ants will often leave nectar behind in flowers This
amount can be less (Bleil et al 2011) more (Fritz and Morse 1982) or similar to (Lach 2008a)
amounts left by other visitors Often ants are morphologically constrained from fully exploiting
nectar Unlike bees and butterflies ants do not have proboscises and must get close to nectar to
consume it Narrow corollas or nectar spurs can prevent ant access to some or all floral nectar
(Schemske 1980 Galen and Cuba 2001 and see below) Smaller ants may be better able to
6
access nectar (Newman and Thomson 2005 Agarwal and Rastogi 2007) but can be limited by
their crop size in the amount of nectar they can remove (Lach 2005) If foraging ants leave
enough nectar behind for a pollinator to recover the cost of visiting a flower ants may have
minimal effects on plant reproductive success (Maloof and Inouye 2000 Turner et al 2012)
Differences among ant species in behavior and diet also affect their consumption of floral
nectar Species vary in how many flowers they visit per inflorescence or per plant and in how
many ants will visit a flower simultaneously (Lach 2005 Junker et al 2007 Lach 2013)
Invasive antsmdashthought to be better at exploiting and defending resources than native ants
(Holway et al 2002)mdashoften recruit to flowers in large numbers sometimes several dozen or
more (Visser et al 1999 Hansen and Muumlller 2009) Overall many factors may influence how
heavily ants exploit floral nectar on plants including ant density plant height the spatial or
temporal distribution of floral nectar and the availability of alternative food resources for ants
(Cushman and Addicott 1991 Lach 2013)
Rarely ants and pollinators may compete for pollen We know of only a few studies that
have recorded ants harvesting pollen directly from anthers (Horskins and Turner 1999 Ness
2006 Byk and Del-Claro 2010) Other studies have found pollen grains in ants (eg Baroni
Urbani and de Andrade 1997 and see Chapter 3) but this may not indicate competition with
pollinators ants sometimes collect stray pollen grains from the environment (Creighton 1967)
One New World genus Cephalotes might be specially adapted for pollen consumption They
possess ldquoproventricular shieldsrdquo structures that may restrict solid food to the crop allowing
nestmates to exchange pollen grains via trophallaxis It is unclear if this structure evolved for this
purpose but Cephalotes are the most frequently documented palynivorous ants (eg Creighton
1967 Baroni Urbani and de Andrade 1997 Byk and Del-Claro 2010) How efficiently ants
7
digest pollen and whether ants like bees feed it to larvae as a protein source is unknown At
least two studies have found that ant larvae consume pollen providing some support for this idea
(Wheeler and Bailey 1920 and Chapter 3)
b) Interference competition
Ants are relatively unique among flower visitors in defending floral resources from competitors
(but see Willmer and Corbett 1981 Roubik 1982) This defense ranges from chance encounters
with ants attacking visitors when flower occupancy overlaps (Ashman and King 2005 Junker et
al 2007) to actively patrolling inflorescences or constructing protective galleries around
nectaries (Horvitz and Schemske 1984 Gaume et al 2005 Hansen and Muumlller 2009)
Interference competition even in the absence of exploitative competition is sufficient to alter
pollinator behavior (Chapter 2)
The strength and outcome of interference competition depends on the identities of the ant
and the pollinator Pollinators might not alter their visitation patterns in response to non-
aggressive ants (Junker et al 2007) but more antagonistic ants may elicit avoidance behavior
before or soon after the visitor lands (Willmer and Corbet 1982 Altshuler 1999 Schuumlrch et al
2000 Table 11) Pollinator size may help predict their reaction to ants because ants may deter
only small-bodied pollinators (Gonzaacutelvez et al 2012) It is not clear whether flower visitors view
ants as aggressive competitors or as potential predators rarely ants prey on pollinators (Schatz
and Wcislo 1999) but in most cases ants merely cause pollinators to relocate
How well pollinators recognize and avoid ant-visited flowers is unresolved Collectively
pollinators have lower visitation rates to flowers visited by ants (Romero et al 2011) This is
8
dependent on pollinator identity however dipterans do not appear to discriminate between ant-
visited and ant-free flowers while bees can identify and avoid ant-visited flowers Bees likely
use a combination of visual and olfactory cues to recognize ant-visited flowers They can
identify previous conspecific and some heterospecific insect visitors by the hydrocarbon
ldquofootprintsrdquo they leave during foraging (Stout and Goulson 2001) Bumblebees can detect ant
hydrocarbons although this has been shown only in the laboratory (Ballantyne and Willmer
2012 Chapter 2) No study has yet examined if bees can use sight alone to detect ants but
several suggest it (Junker et al 2007 Gonzaacutelvez et al 2012) Some bees may discriminate
between ant species when visiting flowers (eg Lach 2008b) which follows if not all ant-
pollinator interactions are equally costly The ability of bees to do so will affect how generalized
their avoidance behaviors will be If they can discriminate between species bees may avoid
specific flowers visited by more ldquocostlyrdquo ants However if they cannot bees might either avoid
all ant-visited flowers or none depending on how negative the average interaction with an ant is
iii) Interactions with flower and pollinator antagonists
Ants can defend the flowers they visit Numerous plant species have circumfloral or bracteal
nectaries (ie extrafloral nectaries around flowers) and ants attracted to these nectaries can
reduce bud ovule or flower predation increasing plant fitness (Inouye and Taylor 1979 Rico-
Gray and Thien 1989 Sugiura et al 2006) Although the presence of EFNs (extrafloral nectaries)
so close to floral nectaries (sometimes less than 1 cm Ness 2006) might increase discovery and
exploitation of floral nectar by ants only one study has investigated this hypothesis finding no
relationship between the proximity of EFNs to floral nectaries and antsrsquo use of floral nectar
(Galen 2005) Holes made by nectar robbers are functionally analogous to circumfloral nectaries
9
and can also attract ants that defend flowers (Newman and Thomson 2005) and nectar-thieving
ants can similarly prevent florivory in some systems (Lach 2007 Bleil et al 2011)
Ants can also reduce the abundance of flower-dwelling predators or parasites of
pollinators Ambush predators such as crab spiders often forage at flowers where they may
reduce pollinator visitation rates and plant fitness (Dukas 2001 Gonccedilalvez-Souza et al 2008)
Ants can decrease spider abundance and richness on plants (Rudgers 2004 Renault et al 2005
Nahas et al 2012) and some evidence suggests that they can do the same on flowers (Lach
2008) In select cases ants may also reduce the abundance of pollinator parasitoids (Holland et
al 2011) Whether ants increase plant reproductive success by removing pollinator antagonists
has not yet been examined but should be If ants create lsquoenemy-freersquo flowers pollinators might
prefer to land on flowers with ants
iv) Net effects
Table 11 compiles studies in which the authors recorded ants consuming floral rewards or
interacting with pollinators and measured some aspect of plant fitness (eg fruit or seed set
pollen deposition) We included only a single study per plant species unless subsequent studies
found conflicting results or measured the fitness of another plant sex Records of ant pollination
are included in a separate table (Table 12)
Table 11 reveals relatively few examples of flower-visiting ants negatively affecting
plant fitness given how costly ant visitation is assumed to be The net effect of ants on plant
reproductive success is actually quite variable with similar numbers of studies finding positive
negative or neutral effects of ant presence Surprisingly when ants do have negative effects it is
10
usually because they directly damage floral structures and not because they prevent pollinators
from visiting flowers and successfully transferring pollen Why are ants not more often negative
for plant reproduction when this is commonly assumed to be true
First ants may compensate for reducing pollinator visitation rates by improving
pollination quality As pollinators move between flowers on a plant they often deposit self-
pollen on stigmas (ie geitonogamy) In some plants with late-acting self-incompatibility ovules
fertilized by self-pollen are subsequently aborted reducing the number of seeds that can be
produced (Barrett 2002 and references therein) Geitonogamy can also reduce offspring viability
or vigor (de Jong et al 1993) and cause pollen discounting a reduction in the amount of pollen
available for outcrossing because of self-pollination (Barrett 2002) By competing with
pollinators ants might decrease rates of geitonogamous pollination if pollinators then visit fewer
sequential flowers within a plant Ants might also cause pollinators to fly further between plants
potentially increasing the genetic diversity of pollen arriving at stigmas Although such effects
have been reported for other floral larcenists (Maloof 2001 Irwin 2003) to our knowledge these
hypotheses have not been investigated in relation to ants
Second ants and pollinators do not always forage for the same resources possibly
allowing them to partition floral rewards If ants forage for nectar but not pollen and flowers can
be pollinated efficiently by visitors looking to gather mostly pollen and little or no nectar then
plant reproduction will not be affected by ants consuming floral nectar Junker et al (2010a)
found that honeybees foraged for both pollen and nectar in the absence of ants but reduced their
foraging on nectar but not pollen when ants could access flowers resulting in no difference in
fruit set between treatments Whether pollinators commonly adjust their foraging behaviors in
response to ants warrants further research
11
Third ants are somewhat unique among floral larcenists in that they often provide direct
benefits to the plants they visit In the studies listed in Table 11 ants sometimes decreased
herbivory on plants (Chamberlain and Holland 2008 Bleil et al 2011) or otherwise aided the
plant (eg ant presence repelling smaller less effective pollinators Gonzaacutelvez et al 2012) If the
positive effects of ants cancel or outweigh the negative effects of flower-visiting ants on
pollinators then ant visitation may be neutral or even positive for plant fitness
Fourth ants may impose fitness costs to plants not captured in the studies in Table 11
Nectar robbers have stronger negative effects when plants are pollen-limited (Burkle et al 2007)
but only one study in Table 11 assessed whether flowers would set more seed if they received
more pollen finding no evidence of pollen limitation (Lach 2008a Table 11) Even if ants
decrease pollinator visitation rates there is limited scope for ants to decrease plant reproductive
success when plants are not pollen-limited Furthermore nearly all the studies in Table 11
measured only female fitness Only three quantified aspects of male fitness (Wyatt 1980 Fritz
and Morse 1981 Galen and Geib 2007) and we know of only one other study in a system in
which ants occasionally pollinate that has also measured male fitness (Ashman 2000) All four
studies measured pollen or pollinia removal rates and none found any effect of flower-visiting
ants on male fitness However whether other measures of male fitness (eg seeds sired pollen
deposition patterns) would reveal similar or different effects needs investigation
Finally although floral defenses against ants (see 3 below) may prevent us from
observing the negative effects of ants on plant reproductive success we consider this unlikely
Several studies have examined interactions between ants and plants that share little or no
coevolutionary history usually in the context of ant invasions Although invasive ants often
recruit to flowers in larger numbers than native ants they do not appear to reduce plant
12
reproductive success (Blancafort and Goacutemez 2005 Lach 2007 Lach 2008a but see Hansen and
Muumlller 2009) And in Hawaii where angiosperms evolved for millions of years in the absence of
ants and are thought to have secondarily lost floral defenses against ants (Junker et al 2011a)
there is no evidence that the preponderance of recently introduced flower-visiting ants negatively
affects plant fitness (Junker et al 2010a Bleil et al 2011)
2 Ants as pollinators
Ants may directly benefit plant reproduction by acting as pollinators but this is a relatively rare
occurrence Ants have long been considered poor pollinators (Kerner 1878) Initially this was
based on morphological and behavioral differences between ants and recognized pollinators
Worker ants are wingless and often relatively hairless making them poor pollen dispersers As
well because many ants exhibit Ortstreue (literally ldquoplace fidelityrdquo) ants were thought to return
to the same branch or flower repeatedly effecting no cross pollination (Houmllldobler and Wilson
1990) Though largely true these traits do not necessarily preclude ants from acting as
pollinators (Table 12) Although they commonly have restricted foraging ranges (Houmllldobler and
Wilson 1990) some ants can effect outcrossing (eg Ashman and King 2005 Carvalheiro 2008)
and many ants are as hairy if not hairier than their pollinating counterparts
In the late 1970s the ldquoantibiotic hypothesisrdquo was put forward to explain the rarity of ant
pollination Ants use antibiotic secretions to protect themselves against the fungal and bacterial
pathogens common in their nests and environs Iwanami and Iwadare (1978) found that one of
these compounds myrmicacin also greatly reduces pollen germination rates Subsequently
Beattie et al (1984 1985) found that short periods of contact (le 30 minutes) with an antrsquos
integument caused pollen grains to fail to germinate Some of these pollen-killing compounds
13
were traced back to the ant metapleural gland which produces hygienic secretions (Houmllldobler
and Wilson 1990) although contact with ants lacking metapleural glands can also reduce pollen
viability (Beattie et al 1985 Dutton and Frederickson 2012) Ant mandibular or anal gland
secretions and potentially ant venom may likewise defend ants against pathogens and have
similar effects on pollen germination (Beattie et al 1985 Graystock and Hughes 2011)
Nonetheless ant pollination has been documented repeatedly There are at least 18 cases
in which ant pollination has been shown experimentally and five others in which observations
strongly suggest it (Table 12) The majority of these plants fit the ldquoant pollination syndromerdquo
originally proposed by Hickman (1974) dense stands of short plants with small flowers and
easily accessible nectaries Typically ant-pollinated plants are associated with hot and dry
climates (principally in the Mediterranean) but dry alpine environments may also favor the
evolution of ant pollination (eg Svensson 1985 Puterbaugh 1998 Schiestl and Glaser 2011) In
most cases ants appear to be the sole or major pollinator of the plant in question (Hickman 1974
Peakall et al 1990 de Vega et al 2009) although ants sometimes make only supplementary
contributions to pollination (Schuumlrch et al 2000 Ashman and King 2005) This suggests that co-
evolution between ants and plants may not be necessary for ants to be useful pollinatorsmdashwhich
is surprising given the potential negative effects of ants on plant reproduction
Ant-pollinated plants cope with ant secretions in a variety of ways Several plant species
may simply tolerate pollen damage (Gόmez and Zamora 1992 de Vega et al 2009) apparently
possessing no overt adaptations to prevent it These plants might receive enough pollen to
overcome reduced pollen viability Other plants may place pollen on ant body parts thought to
have fewer secretions thereby potentially escaping damage (eg on ant legs Ramsey 1995) or
attach pollen to ants with specialized structures avoiding direct contact with the ant integument
14
(Peakall et al 1990) It is not known whether pollen evolves resistance to ant secretions or
whether plants deposit pollen on ants which have less disruptive secretions although these
possibilities have been suggested elsewhere (eg Garciacutea et al 1995 Ramsey 1995)
One key question regarding the effects of ant secretions on pollen grains urgently needs
to be resolved Several studies have suggested that the pollen-killing secretions of ants may have
contributed to the evolution of floral defenses against ants (eg Wagner 2000 Galen and Geib
2007) For this to be true ants must decrease male fitness by damaging pollen grains before they
leave flowers and are deposited on receptive stigmas Only two studies have investigated the
effects of ant secretions on pollen still in anthers (Wagner 2000 Galen and Butchard 2003) and
both constrained ants unnaturally to flowers for significantly longer than ants would normally
stay on flowers Although these studies found strong reductions in pollen vigor it is unclear if
ant secretions decrease male fitness under natural ant visitation conditions
3 Floral defenses against ants
Plants are not passive recipients of flower visitors and have some ability to ldquochooserdquo which
organisms can access floral rewards Flowers resist ants using both structural and chemical traits
More thorough investigation is needed to determine whether these traits evolved to prevent or
minimize the negative effects of ants on plant reproductive success (ie whether these traits can
truly be considered defenses against ants see 4ia below) It is unlikely however that ants
experience reciprocal selection to circumvent floral resistance traits because the fitness benefits
to ants of accessing flowers are probably small Thus there may be little scope for
coevolutionary arms races between floral defenses against ants and ant counter-adaptations
15
i) Structural resistance traits
a) Restricting ant access
Numerous plant species have physical structures preventing ant access to flowers Thin pedicels
can limit antsrsquo gripping abilities and make ants easier to dislodge when flowers are moved by
wind (Gaume et al 2005) and waxy secretions can render stems too slippery for ants to climb
(Harley 1991) Structures surrounding flowers such as water moats or dense hairs may similarly
keep ants from accessing floral rewards (Willmer et al 2009) Many such traits function as broad
filters and prevent not only ants but most crawling insects from reaching flowers Thus it is
difficult to determine whether ants florivores or both acted as selective agents favoring the
evolution of physical barriers to flowers
Flower shape may also reduce ant-pollinator conflict Long narrow corollas or nectar
spurs can prevent all but the smallest ants from accessing floral nectaries (Bluumlthgen et al 2004
Agarwal and Rastogi 2007) while still allowing long-tongued pollinators access to rewards
Selection to exclude nectar-thieving ants in such a manner however may be counteracted by
pollinator preferences for wider corollas that allow easier flower handling (Galen and Cuba
2001) Additionally these types of flowers may be more prone to robbery (Irwin and Maloof
2002) limiting their usefulness in preventing nectar larceny
b) Separation in space or time
Plants may separate floral rewards and ants temporally or spatially to minimize ant impacts on
plant reproduction Several authors have proposed that myrmecophytes have adapted to the
16
sterilization or otherwise detrimental activities of ants by locating domatia or food rewards on
different branches than flowers (Izzo and Vasconcelos 2002 Raine et al 2002 Edwards and Yu
2008) Temporal separation of ant and pollinator activities could likewise reduce conflict though
evidence is limited Some acacia flowers dehisce during periods of low ant activity (Willmer and
Stone 1997 Nicklen and Wagner 2006) but it is unclear if this is in response to ants
temperature or pollinator activity Timing of nectar secretion may similarly reduce ant-pollinator
competition If ants are active prior to pollinators they may deplete floral nectar and reduce
pollinator visitation rates (Schaffer et al 1984) However if nectar is secreted when ants are
inactive pollination may occur unimpeded (Norment 1988) This temporal separation could
favor pollinators that are active when ants are not (eg Wyatt and Morse 1981) but this is
poorly documented Further studies particularly focusing on flowers with the potential for
nocturnal and diurnal pollination may provide more examples
c) Extrafloral nectaries (EFNs)
One hypothesis put forward to explain the evolution of EFNs proposes that EFNs ldquodistractrdquo ants
from consuming floral nectar (Kerner 1878 Wagner and Kay 2002) Wagner and Kay (2002)
tested this hypothesis by constructing model plants using artificial nectaries assigned at random
to represent either floral or extrafloral nectaries They found that the presence of EFNs could
reduce ant exploitation of floral nectar However as highlighted by Galen (2005) this design is
closer to a plant making a surplus of flowers some of which will avoid ant visitation if visitation
rates increase sublinearly with increasing flower number Producing extra flowers to escape ant
visitation appears to be a viable strategy in some systems (Ashman and King 2005 Galen 2005)
However Galen (2005) found that applying honey (simulating extrafloral nectar) to P viscosum
17
leaves had the opposite effect and increased ant visits to flowers Only two studies both in
Pachycereus schottii have found evidence for the distraction hypothesis under field conditions
(Chamberlain and Holland 2008 Holland et al 2011)
There is limited empirical support for the distraction hypothesis but abundant evidence
that EFNs attract ants that defend plants against herbivores (reviewed in Rosumek et al 2009)
To function as attractants the presence of EFNs must increase ant abundance on plants
However because of the increased numbers attracted EFNs would not be expected to reduce the
abundance of ants on nectar rewards or be able to satiate the ants recruited Only if ant
abundance stops increasing at a reward density much lower than that available on the plant
would EFNs serve to distract ants In this case the amount of rewards may overwhelm ants
leaving some flowers underexploited However even in P schottii ants still visited flowers
(Chamberlain and Holland 2008) In cases where ant abundance would continue to increase with
additional rewards it is unlikely that EFNs function as distractions
ii) Chemical resistance traits
a) Toxic nectar
One of the earliest proposed floral defenses against ants was toxic nectar (Janzen 1977)
However studies testing Janzenrsquos (1977) hypothesis uncovered only a very few isolated
examples (eg Catalpa speciosa Stephenson 1981 Crinum erubescens Guerrant and Fiedler
1981) in short most floral nectar is not toxic or repellent to ants (Baker and Baker 1978
Schubart and Anderson 1978) In fact ants sometimes prefer floral nectar over solutions with
equivalent sugar concentrations (Koptur and Truong 1998)
18
In contrast floral nectar can repel other flower visitors (eg birds Johnson et al 2006
lepidopterans Kessler and Baldwin 2007) Repellency is often caused by plant secondary
metabolites which may occur in nectar as a by-product of their production elsewhere in the
plant where they function as defenses against herbivory (Adler 2001) Alternatively toxic or
repellent nectar may be adaptive repellent nectar may promote effective pollination by reducing
the amount of reward any one pollinator takes spreading nectar over several pollinator visits
(Kessler and Baldwin 2007) or it may function as a filter selecting for specific pollinators
(Johnson et al 2006) The latter may explain why repellent nectar is not more common
legitimate pollinators in addition to floral larcenists may be deterred from visiting flowers with
repellent nectar This may be particularly true of nectars that would repel ants which are closely
related to bees nectar distasteful to ants may often also be distasteful to bees and those nectars
acting as a filter for bees may still be palatable to ants For example Prŷs-Jones and Willmer
(1992) found that the ammonia-containing nectar of Lathraea clandestina is readily consumed
by both bees and ants even though it deters birds that act as nectar thieves Although floral
nectar itself rarely repels ants Guerrant and Fielder (1981) found that the presence of macerated
petals within nectar can often keep ants away Alkaloids in petals may be responsible and might
explain why ants seldom act as primary nectar robbers these secondary compounds may prevent
ants from chewing through corollas
b) Floral volatiles
Repellent floral volatiles appear to be a more widespread form of chemical resistance against
ants These were reviewed recently by Willmer et al (2009) and will be discussed here only
briefly Numerous plant taxa produce floral scents repellent to ants including myrmecophilous
19
myrmecophytic and non-ant associated plants in both temperate and tropical ecosystems
(Willmer et al 2009 Junker et al 2011a Ballantyne and Willmer 2012) In three large studies
floral volatiles that repel ants were detected in about a third of all plants tested (Willmer et al
2009 Junker et al 2011a Ballantyne and Willmer 2012) Pollen is often the source of ant-
repellent volatile compounds (Willmer et al 2009 Ballantyne and Willmer 2012) but they can
be emitted from other floral parts including petals That pollen is often the source of repellence
could suggest that preventing ant visitation may be more important in protecting male than
female function Additionally because repellence would decrease as pollen is removed from
anthers ants may return and defend flowers more quickly than if repellent chemicals were in
longer-lasting structures (eg petals) In some cases floral volatiles are thought to mimic ant
alarm pheromones (Willmer et al 2009) However floral scents elicit inconsistent reactions
across ant species ranging from repellence to attraction (Agarwal and Rastogi 2007 Ballantyne
and Willmer 2012)
Producing structural or chemical resistance traits may be costly for plants Native
Hawaiian plants which have little recent coevolutionary history with ants appear to have
secondarily lost floral ant repellents (Junker et al 2011a) Both Willmer et al (2009) and Junker
et al (2011a) found that highly chemical repellent flowers did not have strong physical defenses
and vice versa suggesting trade-offs between the two (but see Ballantyne and Willmer 2012)
Like toxic nectar floral ant repellents might sometimes deter both pollinators and ants For
example Galen et al (2011) found that 2-phenylethanol in P viscosum floral scent repels both
flower-damaging F neorufibarbis ants and the plantrsquos main pollinator Bombus kirbyellus
Interestingly four recent studies have found that floral scents can also attract ants
(Agarwal and Rastogi 2007 Edwards and Yu 2008 Schiestl and Glaser 2011 Gonzaacutelvez et al
20
2012) One study (Schiestl and Glaser 2011) has shown that floral scents attract ants to a
potentially ant-pollinated plant and such systems may provide many more examples
4 Future directions
We have highlighted areas in need of further investigation throughout the review Here we focus
on several questions we consider high priorities for future research
i) Floral defenses against ants
a) Do floral resistance traits increase plant fitness
The existence ant-repellent floral traits is often taken as evidence that ants negatively affect plant
reproductive success However despite the abundance and variety of mechanisms excluding ants
from flowers few studies show that ants reduce plant fitness when they visit flowers (Table 11)
This absence is particularly apparent with repellent floral volatiles which may be the most
widespread floral resistance trait (Willmer et al 2009 Junker et al 2011a Ballantyne and
Willmer 2012) To our knowledge only one study has shown that floral ant repellents benefit
plants with repellent P viscosum flowers avoiding damaging ant visits (Galen et al 2011)
Research is urgently needed to investigate if these traits increase plant fitness by excluding ants
or if ant visitation is inconsequential More studies taking advantage of standing variation in
traits (Galen et al 2011) or creating it artificially (eg Junker et al 2010b) will allow us to
distinguish between the two
21
b) If ant visitation is often not costly what caused the evolution of repellent traits and
what maintains them Similarly are these repellent traits truly defenses against ant
visitation or are they generalized defenses against a range of floral antagonists
Ant-repellent floral traits are widespread and their production may be costly (Willmer et al
2009 Junker et al 2011a) The studies in Table 11 however indicate that flower-visiting ants
often do not disrupt plant reproduction particularly outside of myrmecophytes What then
selected for these traits One possibility is that these repellents are not directed solely at ants
Instead a community of antagonistic flower visitors may exert diffuse selection on plants
thereby selecting for and maintaining these traits Structural modifications may repel crawling
insects while chemical repellents such as floral volatiles may repel both flying and non-flying
insects (Junker et al 2011b) How much overlap there is in floral volatiles that repel both ants and
other antagonists needs investigation Until we examine how specific these defenses are to ants
we risk overstating the importance of ants in their evolution
ii) Do ants alter pollen movement patterns or reduce male fitness
We are aware of only four studies that have measured if ant visitation impacts male fitness
(Wyatt 1980 Fritz and Morse 1981 Ashman 2000 Galen and Geib 2007) While none of these
studies found an effect all measured only amounts of pollen or pollinia removed However this
measure does not adequately encompass male fitness alterations to pollen movement patterns
and eventual deposition location may be more important to male function than changes in
removal rates If as suggested by some studies (Altshuler 1999 Lach 2007) ants increase
pollinator relocation rates they may decrease pollen discounting increasing male fitness
However if this increased relocation reduces overall pollen export rates ant competition with
22
pollinators may depress male function Studies using pollen-tracking methods such as fluorescent
dyes will allow us to see how ants change pollen movement patterns and methods like paternity
analysis will reveal how competition between ants and pollinators alters male fitness
iii) Coevolution between ants and plants do myrmecophytes exhibit stronger more
specific defenses against ants
The floral traits of myrmecophytes are most likely to coevolve with ants because all plant
reproduction occurs in the presence of ants Their floral defenses would thus be expected to be
both stronger and more specific to their resident ants than those other plants visited by an array
of more opportunistic ant species Some evidence supports this Willmer et al (2009) found that
resident ants of acacias were repelled by flowers with more aggressive ant species showing
stronger repellence than less aggressive ants Additionally ants not normally associated with
acacias did not respond to acacia floral volatiles More studies are needed in other systems and
should take advantage of phylogenetic approaches examining floral traits in closely related
myrmecophytic and non-myrmecophytic plant species to determine whether ants commonly
select for floral defenses against ants in myrmecophytes
Acknowledgements
For comments and discussion we thank the Frederickson lab particularly Eric Youngerman
Lina Arcila Hernandez and Kyle Turner as well as Alison Parker and Jordan Pleet MEF
acknowledges financial support from an NSERC Discovery Grant a Connaught New Researcher
23
Award an Ontario Ministry of Economic Development and Innovation Early Researcher Award
and the University of Toronto ARC was supported by an Ontario Graduate Scholarship and
Sigma Xi
24
References
Adler L S 2001 The ecological significance of toxic nectar Oikos 91409ndash420
Agarwal V M and N Rastogi 2007 Role of floral repellents in the regulation of flower visits
of extrafloral nectary-visiting ants in an Indian crop plant Ecological Entomology 3359ndash
65
Altshuler D L 1999 Novel interactions of non-pollinating ants with pollinators and fruit
consumers in a tropical forest Oecologia 119600ndash606
Ashman TL 2000 Pollinator selectivity and its implications for the evolution of dioecy and
sexual dimorphism Ecology 812577-2591
Ashman TL and E A King 2005 Are flower-visiting ants mutualists or antagonists A study
in a gynodioecious wild strawberry American Journal of Botany 92891ndash5
Ballantyne G and P Willmer 2012 Floral visitors and ant scent marks noticed but not used
Ecological Entomology 37402-409
Ballantyne G and P Willmer 2012 Nectar theft and floral ant-repellence a link between
nectar volume and ant-repellent traits PloS One 7e43869
Baker H G and I Baker 1978 Ants and flowers Biotropica 1080
Barrett S C H 2002 Sexual interference of the floral kind Heredity 88154ndash159
Baroni Urbani C and M L de Andrade 1997 Pollen eating storing and spitting by ants
Naturwissenschaften 84256ndash258
25
Beattie A J C Turnbull R B Knox and E G Williams 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany
71421ndash426
Beattie A J C Turnbull T Hough S Jobson and R B Knox 1985 The vulnerability of
pollen and fungal spores to ant secretions evidence and some evolutionary implication
American Journal of Botany 72606ndash614
Blancafort X and C Goacutemez 2005 Consequences of the Argentine ant Linepithema humile
(Mayr) invasion on pollination of Euphorbia characias (L) (Euphorbiaceae) Acta
Oecologica 2849ndash55
Bleil R N Bluumlthgen and R R Junker 2011 Ant-plant mutualism in Hawailsquoi Invasive ants
reduce flower parasitism but also exploit floral nectar of the endemic shrub Vaccinium
reticulatum (Ericaceae) Pacific Science 65291ndash300
Bluumlthgen N G Gottsberger and K Fiedler 2004 Sugar and amino acid composition of ant-
attended nectar and honeydew sources from an Australian rainforest Austral Ecology
29418ndash429
Burkle L A R E Irwin and D A Newman 2007 Predicting the effects of nectar robbing on
plant reproduction implications of pollen limitation and plant mating system American
Journal of Botany 941935ndash1943
Byk J and K Del-Claro 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333ndash38
26
Caballero P C G Ossa W L Gonzaacuteles C Gonzaacutelez-Browne G Astorga M M Muruacutea and
R Medel 2013 Testing non-additive effects of nectar-robbing ants and hummingbird
pollination on the reproductive success of a parasitic plant Plant Ecology 214633ndash640
Carvalheiro L G E R M Barbosa and J Memmott 2008 Pollinator networks alien species
and the conservation of rare plants Trinia glauca as a case study Journal of Applied
Ecology 451419ndash1427
Chamberlain S A and J N Holland 2008 Density-mediated context-dependent consumer-
resource interactions between ants and extrafloral nectar plants Ecology 891364ndash1374
Creighton W S 1967 Studies on free colonies of Cryptocerus texanus Satschi (Hymenoptera
Formicidae) Psyche 7434ndash42
Cursach J and J Rita 2011 Implications of the reproductive biology of the narrow endemic
Naufraga balearica (Apiaceae) for its conservation status Plant Systematics and Evolution
298581ndash596
Cushman JH and Addicott JF In Huxley CR and DF Cutler (eds) 1st edition Ant-plant
interactions Oxford University Press Oxford
de Jong T J N M Waser and P G Klinkhamer 1993 Geitonogamy the neglected side of
selfing Trends in Ecology and Evolution 8321ndash325
de Vega C M Arista P L Oritz C M Herrera and S Talavera 2009 The ant-pollination
system of Cytinus hypocistis (Cytinaceae) a Mediterranean root holoparasite Annals of
Botany 4517ndash8
27
Dukas R 2001 Effects of perceived danger on flower choice by bees Ecology Letters 4327ndash
333
Dutton E M and M E Frederickson 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561ndash569
Edwards D P and D W Yu 2008 Tolerating castration by hiding flowers in plain sight
Behavioral Ecology and Sociobiology 6395ndash102
Frederickson M E 2009 Conflict over reproduction in an ant-plant symbiosis why Allomerus
octoarticulatus ants sterilize Cordia nodosa trees The American Naturalist 173675ndash81
Fritz R S and D H Morse 1981 Nectar parasitism of Asclepias syriaca by ants effect on
nectar levels pollinia insertion pollinaria removal and pod production Oecologia 50316ndash
319
Galen C 1983 The effects of nectar thieving ants on seedset in floral scent morphs of
Polemonium viscosum Oikos 41245ndash249
Galen C 2005 Catching ants with honey an experimental test of distraction and satiation as
alternative modes of escape from flower-damaging ants Oecologia 14480ndash87
Galen C and B Butchart 2003 Ants in your plants effects of nectar-thieves on pollen fertility
and seed-siring capacity in the alpine wildflower Polemonium viscosum Oikos 101521ndash
528
Galen C and J Cuba 2001 Down the tube pollinators predators and the evolution of flower
shape in the alpine skypilot Polemonium viscosum Evolution 551963-1971
28
Galen C and J C Geib 2007 Density-dependent effects of ants on selection for bumble bee
pollination in Polemonium viscosum Ecology 881202ndash1209
Galen C R Kaczorowski S L Todd J Geib and R A Raguso 2011 Dosage-dependent
impacts of a floral volatile compound on pollinators larcenists and the potential for floral
evolution in the alpine skypilot Polemonium viscosum The American Naturalist 177258ndash
272
Garciacutea M B R J Antor and X Espadaler 1995 Ant pollination of the palaeoendemic
dioecious Borderea pyrenaica (Dioscoreaceae) Plant Systematics and Evolution 19817ndash
27
Gaume L M Zacharias and R M Borges 2005 Ant ndash plant conflicts and a novel case of
castration parasitism in a myrmecophyte Evolutionary Ecology Research 7435ndash452
Graystock P and W O H Hughes 2011 Disease resistance in a weaver ant Polyrhachis
dives and the role of antibiotic-producing glands Behavioral Ecology and Sociobiology
652319ndash2327
Gόmez J M 2000 Effectiveness of ants as pollinators of Lobularia maritima effects on main
sequential fitness components of the host plant Oecologia 12290ndash97
Gόmez J M and R Zamora 1992 Pollination by ants consequences of the quantitative effects
on a mutualistic system Oecologia 91410ndash418
Gόmez J M R Zamora J A Hόdar and D Garciacutea 1996 Experimental study of pollination
by ants in Mediterranean high mountain and arid habitats Oecologia 105236ndash242
29
Gonccedilalves-Souza T P M Omena J C Souza and G Q Romero 2008 Trait-mediated effects
on flowers artificial spiders deceive pollinators and decrease plant fitness Ecology
892407ndash2413
Gonzaacutelvez F G L Santamariacutea R T Corlett and M A Rodriacuteguez-Gironeacutes 2012 Flowers
attract weaver ants that deter less effective pollinators Journal of Ecology 10178ndash85
Guerrant E O and P L Fiedler 1981 Flower defenses against nectar-pilferage by ants
Biotropica 1325ndash33
Harley R 1991 The greasy pole syndrome In Huxley CR and DF Cutler (eds) 1st edition
Ant-plant interactions Oxford University Press Oxford
Hansen D M and C B Muumlller 2009 Invasive ants disrupt gecko pollination and seed
dispersal of the endangered plant Roussea simplex in Mauritius Biotropica 41202ndash208
Hickman J C 1974 Pollination by ants a low-energy system Science 1841290ndash1292
Holland J N S a Chamberlain and T E X Miller 2011 Consequences of ants and
extrafloral nectar for a pollinating seed-consuming mutualism ant satiation floral
distraction or plant defense Oikos 120381ndash388
Houmllldobler B and Wilson E O 1990 The Ants 1st Edition Harvard University Press
Cambridge
Holway DA L Lach AV Suarez ND Tsutsui and TJ Case 2002 The causes and
consequences of ant invasions Annual Review of Ecology and Systematics 33181-233
30
Horskins K and Turner VB 1999 Resource use and foraging patterns of honeybees Apis
mellifera and native insects on flowers of Eucalyptus costata Australian Journal of
Ecology 24221-227
Horvitz C C and D W Schemske 1984 Effects of ants and an ant-tended herbivore on seed
production of a neotropical herb Ecology 651369ndash1378
Inouye DW 1980 The terminology of floral larceny Ecology 611251-1253
Inouye DW and O R J Taylor 1979 A temperate region plant-ant-seed predator system
consequences of extra floral nectar secretion by Helianthella quinquenervis Ecology 601ndash
7
Irwin RE 2003 Impact of nectar robbing on estimates of pollen flow conceptual predictions
and empirical outcomes Ecology 84485-495
Irwin RE and Maloof JE 2002 Variation in nectar robbing over time space and species
Oecologia 133525-533
Iwanami Y and T Iwadare 1978 Inhibiting effects of myrmicacin on pollen growth and pollen
tube mitosis Botanical Gazette 13942ndash45
Izzo TJ and Vasconcelos HL 2002 Cheating the cheater domatia loss minimizes the effects
of ant castration in an Amazonian ant plant Oecologia 133200-205
Janzen D H 1977 Why donrsquot ants visit flowers Biotropica 9252
Johnson SD Hargreaves AL and Brown M 2006 Dark bitter-tasting nectar functions as a
filter of flower visitors in a bird-pollinated plant Ecology 872709-2716
31
Junker R A Y C Chung and N Bluumlthgen 2007 Interaction between flowers ants and
pollinators additional evidence for floral repellence against ants Ecological Research
22665ndash670
Junker R R R Bleil C C Daehler and N Bluumlthgen 2010a Intra-floral resource partitioning
between endemic and invasive flower visitors consequences for pollinator effectiveness
Ecological Entomology 35760ndash767
Junker R R and N Bluumlthgen 2010b Floral scents repel facultative flower visitors but attract
obligate ones Annals of botany 105777ndash82
Junker R R C C Daehler S Doetteri A Keller and N Bluumlthgen 2011a Hawaiian ant ndash
flower networks nectar-thieving ants prefer undefended native over introduced plants with
floral defenses Ecological Monographs 81295ndash311
Junker R R J Gershenzon and S B Unsicker 2011b Floral odor bouquet loses its ant
repellent properties after inhibition of terpene biosynthesis Journal of Chemical Ecology
371323ndash31
Kawakita A and M Kato 2002 Floral biology and unique pollination system of root
holoparasites Balanophora kuroiwai and B tobiracola (Balanophraceae) American
Journal of Botany 891164ndash1170
Koopowitz H and TA Marchant 1998 Postpollination nectar reabsorption in the African
epiphyte Aerangis verdickii (Orchidaceae) American Journal of Botany 85508-512
Kirk W D J 1984 Ecological studies on Thrips imaginis Bagnall (Thysanoptera) in flowers of
Echium plantagineum L in Australia Australian Journal of Ecology 99ndash18
32
Kerner A 1878 Flowers and their unbidden guests C Kegan Paul London
Kessler D and I T Baldwin 2007 Making sense of nectar scents the effects of nectar
secondary metabolites on floral visitors of Nicotiana attenuata The Plant Journal 49840ndash
54
Koptur S and N Truong 1998 Facultative ant-plant interactions nectar sugar preferences of
introduced pest species in South Florida Biotropica 30179-189
Lach L 2005 Interference and exploitation competition of three nectar-thieving invasive ant
species Insectes Sociaux 52257ndash262
Lach L 2007 A mutualism with a native membracid facilitates pollinator displacement by
Argentine ants Ecology 881994ndash2004
Lach L 2008a Argentine ants displace floral arthropods in a biodiversity hotspot Diversity and
Distributions 14281ndash290
Lach L 2008b Floral visitation patterns of two invasive ant species and their effects on other
hymenopteran visitors Ecological Entomology 33155ndash160
Lach L 2013 A comparison of floral resource exploitation by native and invasive Argentine
ants Arthropod-Plant Interactions 7177-190
Luo C W K Li X M Chen Z Y Huang and K U N Li 2012 Ants contribute significantly
to the pollination of a biodiesel plant Jatropha curcas Environmental Entomology
411163ndash1168
33
Maleacute PJ G C Leroy A Dejean A Quilichini and J Orivel 2011 An ant symbiont directly
and indirectly limits its host plantrsquos reproductive success Evolutionary Ecology 2655ndash63
Maloof J E 2001 The effects of a bumble bee nectar robber on plant reproductive success and
pollinator behavior American Journal of Botany 881960ndash1965
Maloof J E and D W Inouye 2000 Are nectar robbers cheaters or mutualists Ecology
812651ndash2661
Moreau CS CD Bell R Vila SB Archibald and NE Pierce 2006 Phylogeny of the ants
diversification in the age of the angiosperms Science 312101-104
Nahas L M O Gonzaga and K Del-Claro 2012 Emergent impacts of ant and spider
interactions herbivory reduction in a tropical savanna tree Biotropica 44498ndash505
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506ndash514
Newman D A and J D Thomson 2005 Interactions among nectar robbing floral herbivory
and ant protection in Linaria vulgaris Oikos 110497ndash506
Nicklen E F and D Wagner 2006 Conflict resolution in an ant-plant interaction Acacia
constricta traits reduce ant costs to reproduction Oecologia 14881ndash7
Norment C J 1988 The effect of nectar-thieving ants on the reproductive success of Frasera
speciosa (Gentianaceae) American Midland Naturalist 120331ndash336
34
Orivel J L Lambs P-J G Maleacute C Leroy J Grangier T Otto A Quilichini et al 2011
Dynamics of the association between a long-lived understory myrmecophyte and its
specific associated ants Oecologia 165369ndash76
Palmer T M D F Doak M L Stanton J L Bronstein E T Kiers T P Young J R
Goheen et al 2010 Synergy of multiple partners including freeloaders increases host
fitness in a multispecies mutualism Proceedings of the National Academy of Sciences
10717234ndash9
Peakall R A J Beattie and S H James 1987 Pseudocopulation of an orchid by male ants a
test of two hypotheses accounting for the rarity of ant pollination Oecologia 73522ndash524
Peakall R and A J Beattie 1989 Pollination of the orchid Microtis parviflora R Br by
flightless worker ants Functional Ecology 3515ndash522
Peakall R C J Angus A J Beattie 1990 The significance of ant and plant traits for ant
pollination in Leporella fimbriata pollination Oecologia 84457ndash460
Puterbaugh M N 1998 The roles of ants as flower visitors experimental analysis in three
alpine plant species Oikos 8336ndash46
Prŷs-Jones OE and Willmer PG 1992 The biology of alkaline nectar in purple toothwort
(Lathraea clandenstina) ground level defenses Biological Journal of the Linnean Society
45373-388
Raine NE Willmer P and Stone GN 2002 Spatial structuring and floral avoidance
behaviour present in ant-pollinator conflict in a Mexican ant-acacia Ecology 833086-
3096
35
Ramsey M 1995 Ant pollination of the perennial herb Blandfordia grandiflora (Liliaceae)
Oikos 74265ndash272
Renault CK LM Buffa and Delfino MA 2005 An aphid-ant interaction effects on
different trophic levels Ecological Research 2071-74
Rico-Gray V and L B Thien 1989 Effect of different ant species on reproductive fitness of
Schomburgkia tibicinis (Orchidaceae) Oecologia 81487ndash489
Romero G Q P A P Antiqueira and J Koricheva 2011 A meta-analysis of predation risk
effects on pollinator behaviour PLoS One 6e20689
Rosumek FB FAO Silveira F de S Neves NP de U Barbosa L Diniz Y Oki F Pezzini
et al 2009 Ants on plants a meta-analysis of the role of ants as plant biotic defenses
Oecologia 160537ndash549
Roubik D W 1982 The ecological impact of nectar-robbing bees and pollinating
hummingbirds on a tropical shrub Ecology 63354ndash360
Rudgers J A 2004 Enemies of herbivores can shape plant traits selection in a facultative ant-
plant mutualism Ecology 85192ndash205
Schatz B and WT Wcislo 1999 Ambush predation by the ponerine ant Ectatomma ruidum
Roger (Formicidae) on a sweat bee Lasioglossum umbripenne (Halictidae) in Panama
Journal of Insect Behavior 12641-663
36
Schaffer W M D W Zeh S L Buchmann S Kleinhaus M V Schaffer and J Antrim
1983 Competition for nectar between introduced honey bees and native North American
bees and ants Ecology 64564ndash577
Schemske D W 1980 The evolutionary significance of extrafloral nectar production by Costus
woodsonii (Zingiberaceae) an experimental analysis of ant protection Journal of Ecology
68959ndash967
Schiestl F P and F Glaser 2011 Specific ant-pollination in an alpine orchid and the role of
floral scent in attracting pollinating ants Alpine Botany 1221ndash9
Schubart HOR AB Anderson 1978 Why donrsquot ants visit flowers A response to D H
Janzen Biotropica 10310-311
Schuumlrch S M Pfunder and B A Roy 2000 Effects of ants on the reproductive success of
Euphorbia cyparissias and associated pathogenic rust fungi Oikos 16ndash12
Stanton ML Palmer TM Young TP Evans A and Turner ML 1999 Sterilization and
canopy modification of swollen thorn acacia tree by a plant-ant Nature 401 578-581
Stephenson AG 1981 Toxic nectar deters nectar thieves of Catalpa speciosa American
Midland Naturalist 105381-383
Stout J C and D Goulson 2001 The use of conspecific and interspecific scent marks by
foraging bumblebees and honeybees Animal Behaviour 62183ndash189
Sugiura S T Abe and S Makino 2006 Loss of extrafloral nectary on an oceanic island plant
and its consequences for herbivory American Journal of Botany 93491ndash495
37
Svensson L 1985 An estimate of pollen carryover by ants in a natural population of
Scleranthus perennis L (Caryophyllaceae) Oecologia 66373ndash377
Tsuji K A Hasyim and K Nakamura 2004 Asian weaver ants Oecophylla smaragdina and
their repelling of pollinators Ecological Research 19669ndash673
Turner R C J J Midgley P Barnard R E Simmons and S D Johnson 2012 Experimental
evidence for bird pollination and corolla damage by ants in the short-tubed flowers of
Erica halicacaba (Ericaceae) South African Journal of Botany 7925ndash31
Visser D M G Wright and J H Giliomee 1996 The effect of the Argentine ant
Linepithema humile (Mayr) (Hymenoptera Formicidae) on flower-visiting insects of
Protea nitida Mill (Proteaceae) African Entomology 4285ndash287
Wagner D 2000 Pollen viability reduction as a potential cost of ant association for Acacia
constricta (Fabaceae) American Journal of Botany 87711ndash715
Wagner D and A Kay 2002 Do extrafloral nectaries distract ants from visiting flowers An
experimental test of an overlooked hypothesis Evolutionary Ecology Research 4293ndash305
Wang C Y Luo Y Tai D An and Y Kou 2008 Ants pollinate Neottia listeroides
(Orchidaceae) in Sichuan China 46836ndash846
Wyatt R 1980 The impact of nectar-robbing ants on the pollination system of Asclepias
curassavica Bulletin of the Torrey Botanical Club 10724ndash28
Wheeler WM and IW Bailey 1920 The feeding habits of Pseudomymine and other ants
Transactions of the American Philosophical Society 22235-279
38
Willmer P G and S A Corbet 1981 Temporal and microclimatic partitioning of the floral
resources of Justicia aurea amongst a concourse of pollen vectors and nectar robbers
Oecologia 5167ndash78
Willmer P G and G N Stone 1997 How aggressive ant-guards assist seed-set in Acacia
flowers Nature 388165ndash167
Willmer P G C V Nuttman N E Raine G N Stone J G Pattrick K Henson P Stillman
et al 2009 Floral volatiles controlling ant behaviour Functional Ecology 23888ndash900
Young T P C H Stubblefield and L a Isbell 1997 Ants on swollen-thorn acacias species
coexistence in a simple system Oecologia 10998ndash107
Yu D W and N E Pierce 1998 A castration parasite of an ant-plant mutualism Proceedings
of the Royal Society B Biological Sciences 265375ndash382
39
Tables
Table 11 Studies of ant-flower or ant-pollinator interactions that included a measure of plant fitness Each study system is listed only
once except when results varied between years or when different studies examined male and female fitness Significant effects of ants on
plant fitness are either lsquopositiversquo or lsquonegativersquo non-significant effects are lsquoneutralrsquo When ants significantly affected only one fitness
component but several were measured (eg fruit set and seed production) we still consider the result significant
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Acacia
drepanolobium
Crematogaster
nigriceps
Proportion
fruiting
Negative Destruction of floral buds Young et al 1997
Asclepias exaltata Several Pollinia inserted
removed
Negative for pollinia
insertion neutral for
pollinia removal
Exploitative competition Wyatt 1980 T
40
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Asclepias syriaca Several
Fruit set pollinia
removed
Negative for fruit
set neutral for
pollinia removal
Exploitative competition Fritz and Morse
1981 T
Cordia nodosa Allomerus cf
demerarae
Fruit set Negative Destruction of floral buds Yu and Pierce
1998
Costus woodsonii Wasmannia
auropunctata
Seed set Positive Unknown ant pollination
suggested (but unlikely)
Schemske 1980
Eritrichum aretioides F neorufibarbis
gelida
Seed set Negative Ants damaged flowers Puterbaugh 1998
41
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Euphorbia characias Several Seed set Neutral Plants in Argentine ant-
invaded area set less seed
than those in un-invaded
sites but an ant-exclusion
experiment did not produce
significant results
Blancafort and
Gόmez 2005
Ferocactus wislizeni Solenopsis xyloni
and others
Seed set Unknown negative Interference and exploitative
competition S xyloni
reduced seed set relative to
other ants but the study did
not include a no-ant control
Ness 2006
42
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Frasera speciosa F neorufibarbis
and Formica sp
Seed set Neutral Nocturnal pollination
occurring when ants were
less active may have
compensated for reduced
diurnal pollinator visitation
Norment 1988
Gypsophila struthium Several Seeds per
inflorescence
Neutral No effect despite large
numbers of ants on flowers
Gόmez et al
1996
Hirtella physophora Allomerus
decemarticulatus
Fruit set Negative Destruction of flowers Orivel et al
2011
Humboldtia brunonis Crematogaster
dohrni
Fruit set Negative Destruction of flowers Gaume et al
2005
43
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Lepidium subulatum Several Percentage of
ovules setting
seed
Positive Unknown Gόmez et al
1996
Leucospermum
conocarpodendron
Several Seed set Neutral Site not pollen limited Lach 2008
Linaria vulgaris Several ldquoFemale fitness
indexrdquo
(combination of
number of seeds
fruits flowers
and seed weight)
Positive Reduction in florivory and
seed predation nectar
robbing holes increased ant
visitation
Newman and
Thomson 2005
44
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Melastoma
malabathricum
Oecophylla
smaragdina
Seed set Positive Ants deterred less efficient
pollinators increasing
visitation by the more
efficient pollinator
Gonzaacutelvez et al
2012
Metrosideros
polymorpha
Several Fruit set Neutral Ants reduced nectar
collection rates by foraging
bees but had no effect on
pollen collection rates or
fruit set
Junker et al
2010a
Orexis alpine F neorufibarbis
gelida
Seed set Neutral Autogamy or not pollen
limited
Puterbaugh 1988
45
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Ouratea spectabilis Cephalotes
pusillus and others
Seeds per fruit Neutral positive C pusillus had no effect on
seed number but other ants
collectively increased seed
set
Byk and Del
Claro 2010
Pachycereus schottii Several Seed set Neutral positive In one year ants showed a
positive effect (seed
number 2008) but no effect
with increased sample sizes
Chamberlain and
Holland 2008
Polemonium
viscosum
F neorufibarbis
gelida
Seed set Negative Ants damaged styles Galen 1983
46
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Polemonium
viscosum
F neorufibarbis
gelida
Pollen removal Neutral Ants did not affect pollen
removal even at high ant
densities
Galen and Geib
2007 T
Protea nitida Linepithema
humile and others
Seed set Neutral positive L humile had no effect on
P nitida seed set but native
ants increased it in one year
ants may cause pollinators
to relocate
Lach 2007
Psychotria
limonensis
Ectatomma spp Fruit set Positive Suggested increased
pollinator relocation
Altshuler 1999
47
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Roussea simplex Technomyrmex
albipes
Seed set Negative Interference and exploitative
competition
Hansen and
Muumlller 2009
Tristerix aphyllus Several Fruit set Neutral Nectar robbing ants
minimally reduced nectar
levels and had no effect on
hummingbird visitation
Caballero et al
2013
Vaccinium
reticulatum
Several Fruit set Neutral Ants reduced florivory but
did not affect fruit set
Bleil et al 2011
T Study measured male fitness
Study of sterilizing ants showed short-term decrease in female fitness long-term effects are unclear (see text)
48
Table 12 Studies demonstrating either i) ant pollination through an exclusion experiment or ii) that ants transfer pollen (or pollen
analogue) between flowers strongly suggesting ant pollination
Plant species Ant species Ant pollination Evidence Reference
Alyssum purpureum Several Yes Exclusion experiment Gόmez et al 1996
Arenaria tetraquetra Several Yes Exclusion experiment Gόmez et al 1996
Balanophora kuroiwai Leptothorax sp Suggested Exclusion experiment and
observed pollen transfer
and pollen tube formation
but did not see if plants set
seed
Kawakita and Kato 2002
Blandfordia grandiflora Iridomyrmex sp Yes Exclusion experiment Ramsey 1995
Borderea pyrenaica Several Yes Exclusion experiment Garciacutea et al 1995
49
Plant species Ant species Ant pollination Evidence Reference
Cytinus hypocistis Several Yes Exclusion experiment de Vega et al 2009
Epipactis thunbergii Camponotus
japonicas
Suggested Observed transfer of
pollinia from ants to
orchids
Sugiura et al 2006
Euphorbia cyparissias Several Yes Exclusion experiment Schuumlrch et al 2000
Fragaria virginiana Several Very likely Ant-only treatments set
comparable seed to flying
pollinator-only treatment
seed set increased with
increasing ant visitation
Ashman and King 2005
Frasera speciosa Several Yes Exclusion experiment Norment 1988
Frankenia thymifolia Several Yes Exclusion experiment Gόmez et al 1996
50
Plant species Ant species Ant pollination Evidence Reference
Hormathophylla spinosa Proformica
longiseta
Yes Exclusion experiment Gόmez and Zamora 1992
Jatropha curcas
Several
Tapinoma
melanocephalum
most common
Yes Exclusion experiment Luo et al 2012
Leporella fimbriata Myrmecia urens Yes Observed pollinia transfer
and subsequently
confirmed as ant
pollination
Peakall et al 1987
51
Plant species Ant species Ant pollination Evidence Reference
Lobularia maritima Several
Camponotus
micans most
common
Yes Exclusion experiment Gόmez 2000
Microtis parviflora
Iridomyrmex
gracilis
Yes Observation used
laboratory colonies to
replicate observations
Peakall and Beattie 1989
Naufraga balearica
Several
especially
Plagiolepis
pygmaea and
Lasius grandis
Suggested Observation over three
years of study only ants
were observed visiting
ants carried pollen
Cursach and Rita 2011
52
Plant species Ant species Ant pollination Evidence Reference
Neottia listeroides Leptothoras sp
Paratrechina sp
Suggested Observed transfer of
pollinia from ants to
orchids
Wang et al 2008
Paronychia pulvinata Formica
neorufibarbis
gelida
Yes Exclusion experiment Puterbaugh 1998
Polygonum cascadense Formica argentea
Very likely Grew plants in greenhouse
open to flying visitors with
minimal seed set only
plants with access to ants
set significant seed
Hickman 1974
53
Plant species Ant species Ant pollination Evidence Reference
Retama sphaerocarpa Several Yes Exclusion experiment Gόmez et al 1996
Scleranthus perennis
Several
especially
Formica
rufibarbis
Suggested Ants transferred pollen
analogue to stigmas and
carried pollen on bodies
Svensson 1985
Sedum anglicum Proformica
longiseta
Yes Exclusion experiment Gόmez et al 1996
Trinia glauca Several Yes Exclusion experiment Carvalheiro et al 2008
54
Chapter 2 Ants and ant scent reduce bumblebee pollination of artificial flowers
Cembrowski AR Tan MG Thomson JD and Frederickson ME
In press The American Naturalist
All authors designed the experiment ARC and MGT performed the experiment ARC and MEF
performed statistical analyse
s and ARC wrote the manuscript with input from all authors
Abstract
Ants on flowers can disrupt pollination by consuming rewards or harassing pollinators but it is
difficult to disentangle the effects of these exploitative and interference forms of competition on
pollinator behavior Using highly rewarding and quickly replenishing artificial flowers that
simulate male or female function we allowed bumblebees (Bombus impatiens) to forage (1) on
flowers with or without ants (Myrmica rubra) and (2) on flowers with or without ant scent cues
Bumblebees transferred significantly more pollen analogue both to and from ant free flowers
demonstrating that interference competition with ants is sufficient to modify pollinator foraging
behavior Bees also removed significantly less pollen analogue from ant scented flowers than
from controls making this the first study to show that bees can use ant scent to avoid harassment
at flowers Ant effects on pollinator behavior possibly in addition to their effects on pollen
viability may contribute to the evolution of floral traits minimizing ant visitation
55
Introduction
Trait-mediated indirect interactions arise when a focal species causes phenotypic changes
including behavioral modifications in a second species and these effects cascade to still other
species (Werner and Peacor 2003) Although trait-mediated indirect interactions are often studied
in food webs (eg Werner and Peacor 2003 Preisser et al 2005) they are not limited to trophic
interactions For example the threat of predation can change the behavior of a mutualist thus
affecting its partners (Suttle 2003) In animal-pollinated plants predators can disrupt pollination
directly through density-mediated indirect interactions (Dukas 2005) or via trait-mediated
indirect interactions by changing pollinator behavior When predators are present pollinators
may switch to visiting less rewarding flowers or they may avoid flowers altogether potentially
reducing the amount of pollen donated or received by a flower and thus decreasing plant fitness
(Gonccedilalves-Souza 2008 Ings and Chittka 2009) Similarly competition for floral rewards with
other flower visitors may cause behavioral changes in pollinators (Maloof and Inouye 2000
Ohashi et al 2008) Previous research has shown that bees may spend less time visiting flowers
depleted of rewards (Thomson 1986) and may avoid flowers bearing scent marks that indicate
recent visits by other individuals of the same or different bee species as these flowers are likely
to be depleted (Stout and Goulson 2002)
Whereas competition between pollinators may not be particularly costly for the plant
because both competitors are likely to provide pollination services competition between
pollinators and organisms that do not usually provide a pollination service such as ants has a
greater potential to reduce plant fitness Ants are common floral visitors they are attracted to
flowers for nectar (Lach 2007) and in some cases pollen (Byk and Del Claro 2010) and will
often defend these resources against other flower visitors (Altshuler 1999) However ants rarely
56
contribute much useful pollination antibiotic secretions present on their cuticles kill pollen
grains (Beattie et al 1985 Dutton and Frederickson 2012) and when foraging in flowers ants
sometimes impede female function by damaging stigmas (Galen and Cuba 2001) We will
henceforth consider ants to be non-pollinators and will contrast them to animals like bees that
are pollinators Ants are capable of competing with pollinators in two ways (1) by consuming
floral rewards and reducing their availability to pollinators (ie exploitative competition) and (2)
by directly antagonizing and excluding pollinators from flowers (ie interference competition)
Many plants benefit from the presence of ants numerous plant species recruit ants using
extrafloral nectar food bodies or domatia because ants provide protection against folivores (Heil
and McKey 2003) Similarly some plants have extrafloral nectaries on or near reproductive
tissues that recruit ants that deter florivores (eg Inouye and Taylor 1979) However plants
may incur reproductive costs as a result of their association with ants Some ant species that nest
in myrmecophytes sterilize flowers to shunt plant resources from reproduction to vegetative
growth allowing for greater ant colony growth (Frederickson 2009) Also ants visiting
extrafloral nectaries may be attracted to floral nectaries where they can harass pollinators (Ness
2006) There is growing evidence that many angiosperms have evolved floral traits that prevent
ants from accessing flowers during anthesis (Willmer and Stone 1997 Ballantyne and Willmer
2012) The effects of flower-visiting ants can be substantial Ants can decrease the frequency
duration or species diversity of pollinator visits to flowers (Lach 2008 Hansen and Muumlller 2009
Junker et al 2010 Gonzaacutelvez et al 2012) all of which potentially impact pollen donation and
receipt In some studies changes to pollinator behavior induced by ants have also been linked to
seed set
It is difficult however to disentangle the intertwined effects of exploitative and
interference competition in nature If ants disrupt pollination plants may often evolve traits to
57
deter ants from flowers (Willmer et al 2009) From a practical standpoint this would then limit
our ability to study the effects of ants on pollinator visitation because ants would not visit
flowers Here we used artificial flowers to explore ant-bee interactions in the absence of floral
defenses against ants We used highly rewarding quickly replenishing artificial flowers to
examine how direct harassment (ie interference competition expressed through behavior)
changes bee foraging behavior and how this affects donation and receipt of a pollen analogue
(powdered food dye) while minimizing the effects of exploitative competition We predicted
that flowers visited by ants would both donate and receive less pollen analogue than flowers
without ants because bees would avoid flowers with ants or leave them sooner Because scent
plays a large role in bumblebee communication and flower choice (Stout and Goulson 2002) we
also tested whether the presence or absence of ant scent on artificial flowers would affect pollen
analogue donation because of changes in the foraging behavior of bumblebees
Methods
i) Subjects
Myrmica rubra is an invasive ant in eastern North America with a range and habitat preferences
overlapping that of Bombus impatiens a common bumblebee Because M rubra visits flowers
(A Cembrowski pers obs) the two species likely interact in nature We collected 12 M rubra
colonies in the fall of 2011 and 2012 from Toronto Ontario Canada and the surrounding area
and maintained them in environmental chambers on artificial diet (Dussutour and Simpson 2009)
and a 1410 LD schedule (light 600-2200) We used these colonies as sources of M rubra
workers for experiments
58
Workers from commercially supplied colonies of Bombus impatiens (Biobest Canada
Ltd) foraged on artificial flowers in flight cages (either 24 x 24 x 21 m or 79 x 34 x 20m) at
the University of Toronto We tested a total of five bee colonies four colonies were used in the
ant presence trials and two of these and one additional colony were used in the ant scent trials
Flight cages had overhead fluorescent lights attached to timers In contrast to most previous
studies in which bees have been trained and tested individually the entire worker force of a bee
colony was free to forage at will in our experiments Colonies were trained to forage on artificial
flowers for at least four days before being used in trials After being used for a trial the colony
was not used for at least two days to reduce dye carryover between trials Between trials
bumblebees were fed pollen and given sugar water
ii) Artificial flowers
The flowers (Fig 1 Thomson et al 2012 see also Makino 2008) consisted of glass jars filled
with 30 WV sucrose solution Sugar water travelled by capillary action up a sewing-thread
wick to a hole in a blue-painted lid accumulating in a knot that acts as a nectary Flowers
depleted by visitors were quickly replenished via capillary action and were non-rewarding only
very briefly after visits taking less than a minute to accumulate 05 microl note that bumblebee
visits to a single flower are often separated by several minutes or more (A Cembrowski
unpublished data)
Unlike most previous artificial flower designs these flowers allow for estimation of male
and female fitness because we can measure both the amount and type of dye received by female
flowers (see (d) Dye quantification below) In order to access nectar on a ldquomalerdquo flower a bee
must crawl through a circular ldquoantherrdquo of brush-like weather stripping material dusted in a
59
consistent manner with our pollen analogue powdered food dye (FDampC 5 or 6) which is
transferred to the bee in the process On a ldquofemalerdquo flower a bee crawls through a sticky plastic
reinforcement that functions as a ldquostigmardquo receiving dye from the beersquos body Male flowers
dispensed dye particles over multiple bee visits and could still dispense dye at the end of trials
They resemble many real flowers or flower heads in that pollen is dispensed gradually over time
but less dye is available for transfer with each subsequent bee visit (Harder and Thomson 1989)
iii) Flight cage trials
We conducted 6 ant scent trials between 22 March and 4 June 2012 and the remaining trials (16
ant presence trials and 9 ant scent trials) between 9 November 2012 and 6 January 2013 Ant
presence trials lasted for eight hours and ant scent trials lasted for four hours Artificial flowers
were prepared and placed individually in small plastic containers treated with Fluon (Insect-a-
Slip BioQuip Products Inc) to prevent ants from escaping In ant presence trials we used 32
flowers (16 male and 16 female flowers) arranged in an eight-by-four array with flowers spaced
45 cm apart between rows and 30 cm within rows Ant scent trials used 20 flowers (10 male and
10 female flowers) in a four-by-five array with flowers spaced 30 cm apart between and within
rows In all of the ant presence trials and 9 of the ant scent trials we counted the number of bees
actively foraging after one hour to get a measure of colony activity
a) Ant presence trials
We examined the effects of ants on the amount of dye donated by male flowers and received by
female flowers in sixteen trials testing four bee colonies four times each The containers of eight
randomly chosen male flowers and eight randomly chosen female flowers received 15 M rubra
60
workers each the other 16 flowers remained free of ants Isolated from their colonies M rubra
workers have nowhere to deposit sugar water they collect and therefore may become sated
nonetheless a force of 15 ant workers was enough to maintain visitation to the nectaries where
bees foraged while still rarely having more than one or two ants visit the nectary at any time As
ants attacked bees bees would occasionally carry or throw attacking ants off flowers but despite
this some ants maintained their presence at the nectary throughout the trials Because ants the
size of M rubra consume liquids slowly (~017 ndash 024 microlmin Davidson et al 2004) flowers
replenished nectar more quickly than ants consumed it We used two colors of dye (FDampC 5 and
FDampC 6) to differentiate male flowers with and without ants In two trials for each bee colony
anthers of male flowers with ants were coated with FDampC 5 dye while brushes of male flowers
lacking ants were coated with FDampC 6 dye In the other two trials we reversed dye colors to
control for effects of dye type and color Dye color was assigned in a random order Artificial
flowers were placed in randomly assigned positions in the array before we opened the colony
and allowed the bees to begin foraging
b) Scent trials
We explored the effect of ant scent on dye donation in fifteen trials testing three bee colonies in
six five and four trials each Bees were first exposed to ants by allowing the bees to forage for
eight hours on twenty flowers (ten male and ten female) of which five male flowers had 15 ants
and all others had none No dye was used during this ldquoexposurerdquo day We then collected all but
the five ant-free male flowers in the flight cage leaving these flowers to keep bees foraging
Next we individually stored five new male lids in Fluon-treated containers with ten M rubra
workers Five control male lids were put in identical containers lacking ants The following
61
morning we removed the remaining flowers and set out ten new male and female flowers in a
random spatial arrangement using the lids having or lacking ant scent Thus the ant-scented
flowers were not in the same positions as the ant-visited flowers on which the bees were trained
Only five male flowers had ant scent and the other 15 flowers (five male and ten female) did not
We used the same two dye colors as in i) to differentiate male flowers with and without ant
scent and randomized which color was used for ant-scented flowers between trials We collected
and replaced stigmas from female flowers after one hour and collected the stigmas again after
four hours In the first trial performed we also collected stigmas after two hours but due to the
small amount of dye transferred this was not repeated
iv) Dye quantification
After each trial we quantified the amount of dye transferred to female flowers using a
spectrophotometer We removed the stigmas from female flowers placed them in test tubes
added 51 mL of distilled water to each tube and vortexed each tube thoroughly to ensure the
dye was evenly diluted These were diluted further as needed if the absorbance exceeded the
sensitivity range of the spectrophotometer In the first six scent trials each stigma was analyzed
separately and the dye amounts were summed to obtain a total amount of each dye color
transferred to female flowers in each trial In all the ant presence trials and the other nine scent
trials we opted to treat the experiment as the unit of replication Therefore we combined stigmas
from each treatment type (ant-visited or ant-free) in a test tube and measured the total amount of
each dye color transferred in each treatment in each trial
Because we put different dye colors on male flowers with and without ants we could use
the amount of each dye color donated to all female flowers to measure the reproductive success
62
of ant-visited and ant-free male flowers The total amount of dye (of both colors) received by
female flowers with and without ants was our measure of female reproductive success We
calculated the amount of each dye color in the sample by measuring absorbance at 428 or 486
nm and converting absorbance to micrograms following computational methods for overlapping
spectra (Blanco et al 1989)
v) Statistical analyses
In two trials (one ant presence and one ant scent) most female flowers received no dye because
of low bee activity so we excluded these trials from analyses Because dye reception and
donation values were non-normally distributed we square-root transformed the data before
examining the effects of ant presence or ant scent on dye transfer in ANCOVAs For ant
presence trials we included ant presence on male and female flowers and their interaction as
main effects and the total amount of dye transferred in each trial as a covariate to account for
the large variation in overall dye transfer among trials For ant scent trials we included ant scent
as the main effect time (one or four hours) as a repeated measure the interaction between scent
and time and the total amount of dye transferred as a covariate Covariate by treatment (ant
presence or ant scent) interactions were never significant and so were excluded in final analyses
All analyses were conducted in JMP (v 1000)
Results
Bees usually started foraging within minutes of the beginning of the trial and continued until
flowers were collected from the flight cage or the lights were extinguished An average of 34 plusmn
012 and 40 plusmn 015 (mean plusmn SE) bees were foraging after an hour in the ant presence and ant
63
scent trials respectively Male flowers with ants donated significantly less dye than male flowers
lacking ants (Fig 22a F155 = 4019 p lt 00001) Similarly female flowers with ants received
significantly less dye than female flowers lacking ants (Fig 22a F155 = 461 p = 0036) There
was no significant interaction between ant presence on male flowers and ant presence on female
flowers (F155 = 118 p = 028) Flowers with ant scent donated significantly less dye than
flowers without ant scent (Fig 22b F125 = 11216 p lt 00001) There was no significant effect
of time (F125 = 121 p = 028) or interaction between time and scent (F125 = 111 p = 030) in
the model
Discussion
In this study ants altered bumblebee pollination behavior and exhibited trait-mediated indirect
interactions with (artificial) flowers Through interference competition M rubra workers
significantly affected the pattern of dye transfer by B impatiens causing a preferential flow of
dye from male flowers lacking ants to female flowers lacking ants If manifested in nature such
effects would reduce the reproductive success of plants visited by ants through both male and
female function
Ants could have changed the attractiveness of artificial flowers in at least two ways First
as ants interrupted or altogether prevented bumblebees from foraging at nectaries bumblebees
may have learned to avoid flowers with ants due to the relative inefficiency of foraging at these
flowers something bumblebees take into account (Heinrich 2004) Second bees attempt to
minimize their risk of being attacked during foraging bouts which ants did to visiting bees Ants
often attacked or harassed bees by biting grasping and appearing to sting visiting bees
preventing them from accessing the nectary or reducing their time on flowers (see online video
64
1) and bees sometimes avoided ant-tended flowers entirely This harassment was sometimes
physically traumatic (online video 1) and bees often appeared to have trouble flying after being
attacked by ants Previous research has demonstrated that bumblebees leave or avoid flowers
where they have been harassed (Jones and Dornhaus 2011) and avoid foraging where there is
visual or olfactory evidence of a predator or predation event (Abbott 2006 Goodale and Nieh
2012) Though it is unclear if bumblebees viewed ants as competitors or predators they
responded similarly to flowers having ants as they do to flowers housing predators (Gonccedilalves-
Souza et al 2008)
To avoid artificial flowers with ants bees likely used a combination of visual and
olfactory cues Previous research has shown that bees respond to conspicuous predators or
predator ldquodummiesrdquo on flowers (Suttle 2003 Gonccedilalves-Souza et al 2008) decreasing the
frequency and duration of their visits to these flowers Thus bees may have been able to avoid
flowers harboring ants by sight alone However even in the absence of ants bees still
preferentially visited flowers lacking ant scents (Fig 22b) suggesting that they had learned to
associate ant scent with harassment Bees are adept at associative learning (Wright and Schiestl
2009) and can learn to recognize unique scents left behind by both conspecific and heterospecific
flower visitors (Stout et al 1998) Bees can use these various scents often arising from tarsal
gland deposits (Stout et al 1998) to recognize recently visited flowers that are less likely to be
profitable (Stout and Goulson 2002) Recently Ballantyne and Willmer (2012) demonstrated that
bees learn to associate ant scents with unrewarding artificial flowers and decrease their visitation
to these flowers Our results complement their findings by showing that bees can associate ant
scent with harassment at otherwise rewarding flowers
In our study ant scent caused bees to adjust their foraging strategy and decreased the
amount of pollen analogue that was donated by ant-scented flowers Thus the effects of
65
interference competition with ants on flowers can extend beyond immediate interactions and may
have fitness consequences for plants even when ants are absent Like other olfactory cues these
effects are likely transitory (Stout et al 1998) Although more dye was still donated by male
flowers lacking ant scent than those with ant scent the ratio of dye donated was on average
closer to equality in hours 2-4 than in the first hour (Fig 22b) This effect may be partially
driven by dye depletion of male flowers without ant scent but the lack of a strong corresponding
decrease in flowers with ant scent suggests that visitation patterns became more similar We did
not test whether B impatiensrsquo avoidance of ant scent was an innate or a learned behavior but
previous work has shown that Bombus terrestris does not innately avoid flowers with ant scent
(Ballantyne and Willmer 2012)
Ants may be necessary for plant survival and growth but can be costly for plant
reproduction The evolution of ant attractants such as extrafloral nectaries in some plant lineages
suggests that the costs of ants can be outweighed by their protective abilities However in this
study flowers visited by ants received and donated significantly less dye although they did
retain some sexual function The net benefit of having ants depends on whether ants increase
plant fitness by reducing herbivory more than they decrease plant fitness by disrupting
pollination Alternatively plants may actually benefit from the costs of ants to plant
reproduction ants that castrate flowers may be better defenders increasing plant survival or
vegetative growth when plants are young allowing for increased reproduction later in life when
the plant is colonized by less aggressive non-castrating ants (Frederickson 2009 Palmer et al
2010) In some plant species pollinator harassment by ants may even be beneficial Altshuler
(1999) reported that Ectatomma ants greatly increased fruit set of Psychotria limonensis despite
reductions in pollinator visitation rates presumably due to increases in the rate of pollen
outcrossing Similarly Gonzaacutelvez et al (2012) found that the presence of Oecophylla
66
smaragdina ants on Melastoma malabathricum flowers reduced visitation by less effective
pollinators and increased visitation by more effective Xylocopa bees increasing plant fitness
Cases of ants being beneficial to flowers appear to be the minority and many plant
species have traits that limit ant access to flowers Several studies have detailed floral volatiles
that are thought to mimic ant alarm pheromones and thus repel ants (Junker and Bluumlthgen 2008
Willmer et al 2009) Other plants use structural modifications such as narrow corollas or
slippery stems to limit ant access to flowers (reviewed in Willmer et al 2009) Exploitative
competition between ants and pollinators (Lach 2005 Ballantyne and Willmer 2012) as well as
the lethal effects of ant antibiotic secretions on pollen grains (Beattie et al 1985 Dutton and
Frederickson 2012) may have resulted in selection on plants to reduce ant visits to flowers Our
results suggest that trait-mediated indirect interactions resulting from interference competition
between ants and bees may favor plants that defend their flowers and their pollinators against
ants
Acknowledgements
We thank H Rusnock and S Meadley-Dunphy for assistance J Dix for help with flight cage
construction and Biobest for bumblebee colonies We are grateful to the Thomson and
Frederickson labs for guidance particularly T Makino funded by JSPS for helping to refine
artificial flowers J Ogilvie for help with bees K Prior for M rubra colony collections and K
Turner for study design suggestions The comments of the editor and two anonymous reviewers
prompted improvements in the article especially with regards to study design MEF and JDT
acknowledge NSERC Discovery Grant funding The University of Torontorsquos Faculty of Arts and
Science supported MGRTrsquos participation
67
68
References
Abbott K R 2006 Bumblebees avoid flowers containing evidence of past predation events
Canadian Journal of Zoology 841240-1247
Altshuler D L 1999 Novel interactions of non-pollinating ants with pollinators and fruit
consumers in a tropical forest Oecologia 119600-606
Ballantyne G and Willmer P 2012 Floral visitors and ant scent marks noticed but not used
Ecological Entomology 37402-409
Beattie A J Turnbull C Knox R B and Williams E G 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany
71421-426
Blanco M Runiaga H Maspoch S and Tarin P 1989 A simple method for
spectrophotometric determination of two-components with overlapped spectra Journal of
Chemical Education 66178-180
Byk J amp Del Claro K 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333-38
Davidson D W S C Cook and R R Snelling 2004 Liquid-feeding performances of ants
(Formicidae) ecological and evolutionary implications Oecologia 139255ndash66
Dukas R 2005 Bumble bee predators reduce pollinator density and plant fitness Ecology
861401-1406
Dussutour A and Simpson S J 2009 Communal nutrition in ants Current Biology 19740-
744
69
Dutton E M and Frederickson M E 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561-569
Frederickson ME 2009 Conflict over reproduction in an ant-plant symbiosis why Allomerus
octoarticulatus ants sterilize Cordia nodosa trees American Naturalist 173675-681
Galen C and Cuba J 2001 Down the tube pollinators predators and the evolution of flower
shape in the alpine skypilot Polemonium viscosum Evolution 551963-1971
Gonccedilalves-Souza T Omena P M Souza J C and Romero G Q 2008 Trait-mediated
effects on flowers artificial spiders deceive pollinators and decrease plant fitness Ecology
892407-2413
Gonzaacutelvez F G Santamariacutea L Corlett R T and Rodriacuteguez-Gironeacutes M A 2012 Flowers
attract weaver ants that deter less effective pollinators Journal of Ecology 10178ndash85
Goodale E and Nieh JC 2012 Public use of olfactory information associated with predation
in two species of social bees Animal Behaviour 84919-924
Harder LD and Thomson JD 1989 Evolutionary options for maximizing pollen dispersal of
animal-pollinated plants American Naturalist 133323-344
Heil M and McKey D 2003 Protective ant-plant interactions as model systems in ecological
and evolutionary research Annual Review of Ecology Evolution and Systematics 34425-
453
Heinrich B 2004 Bumblebee economics 2nd
edn Harvard University Press Cambridge
Ings T C and Chittka L 2009 Predator crypsis enhances behaviourally mediated indirect
effects on plants by altering bumblebee foraging preferences Proceedings of the Royal
Society B 2762031-2036
70
Inouye D W and Taylor O R Jr 1979 A temperate region plant-ant-seed predator system
consequences of extra floral nectar secretion by Helianthella quinquenervis Ecology 601-
7
Junker R R and Bluumlthgen N 2008 Floral scents repel potentially nectar-thieving ants
Evolutionary Ecology Research 10295ndash308
Junker R R Bleil R Daehler C C and Bluumlthgen N 2010 Intra-floral resource partitioning
between endemic and invasive flower visitors consequences for pollinator effectiveness
Ecological Entomology 35760ndash767
Jones E I and Dornhaus A 2011 Predation risk makes bees reject rewarding flowers and
reduce foraging activity Behavioral Ecology and Sociobiology 651505-1511
Lach L 2005 Interference and exploitative competition of three nectar-thieving invasive ant
species Insect Sociaux 52257-262
Lach L 2007 A mutualism with a native membracid facilitates pollinator displacement by
Argentine ants Ecology 881994-2004
Lach L 2008 Argentine ants displace floral arthropods in a biodiversity hotspot Diversity and
Distributions 14281-290
Makino T T and Sakai S 2007Experience changes pollinator responses to floral display size
from size-based to reward-based foraging Functional Ecology 21854863
Maloof J E and Inouye D W 2000 Are nectar robbers cheaters or mutualists Ecology
812651ndash2661
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506-514
Ohashi K Leslie A and Thomson JD 2008 Trapline foraging by bumble bees V Effects of
experience and priority on competitive performance Behavioral Ecology 19936-948
71
Palmer T M Doak D F Stanton M L Bronstein J L Kiers E T Young T P Goheen
J R and Pringle R M 2010 Synergy of multiple partners including freeloaders
increases host fitness in a multispecies mutualism Proceedings of the National Academy
of Sciences 10717234-17239
Preisser E L Bolnick D I and Benard M E 2005 Scared to death The effects of
intimidation and consumption in predator-prey interactions Ecology 86501-509
Stout J C Goulson D and Allen J A 1998 Repellent scent-marking of flowers by a guild of
foraging bumblebees (Bombus spp) Behavioral Ecology and Sociobiology 43317-326
Stout J and Goulson D 2002 The influence of nectar secretion rates on the responses of
bumblebees (Bombus spp) to previously visited flowers Behavioral Ecology and
Sociobiology 52239-246
Suttle K B 2003 Pollinators as mediators of top-down effects on plants Ecology Letters
6688-694
Thomson JD 1986 Pollen transport and deposition by bumble bees in Erythronium influences
of floral nectar and bee grooming Journal of Ecology 74329-341
Thomson JD Ogilvie J E Makino T T Arisz A Raju S Rojas-Luengas V and Tan M
G R 2012 Estimating pollination success with novel artificial flowers effects of nectar
concentration Journal of Pollination Ecology 9108-114
Tsuji K Hasyim A and Nakamura K 2004 Asian weaver ants Oecophylla smaragdina and
their repelling of pollinators Ecological Research 19669-673
Ulrich S and Bertsch A 1990 Do foraging bumblebees scent-mark food sources and does it
matter Oecologia 82137-144
Werner E E and Peacor S D 2003 A review of trait-mediated indirect interactions in
ecological communities Ecology 841083-1100
72
Willmer P G Nuttman C V Raine N E Stone G N Pattrick J G Henson K Stillman
P McIlroy L Potts S G and Knudsen J T 2009 Floral volatiles controlling ant
behaviour Functional Ecology 23888-900
Wright G A and Schiestl F P 2009 The evolution of floral scent the influence of olfactory
learning by insect pollinators on the honest signaling of floral rewards Functional Ecology
23841-851
73
Figures
Figure 21 Photograph of artificial flower lids A = anther L = lid N = nectary R = region
where anther or stigma is placed S = stigma T = thread W = weight
74
Figure 22 Dye transferred (mean plusmn 1SE) per trial by B impatiens workers A) from artificial
male and to artificial female flowers in the presence (unfilled circles) or absence (filled circles)
of Myrmica rubra workers and B) from artificial male flowers with (unfilled circles) or without
(filled circles) M rubra scent to ant-free female flowers in the 1st or the 2
nd to 4
th hours of the
trials
75
Chapter 3 Not just for the bees pollen consumption is common among tropical
ants
Cembrowski AR Reurink G and Frederickson ME
Planned submission to Biotropica
ARC and MEF designed the study ARC and GR did laboratory work ARC performed statistical
analyses and wrote the manuscript with input from other authors
Abstract
Although palynivory or pollen consumption is widespread among other hymenopterans there
are few accounts of palynivory in ants To quantify how often ants consume pollen we subjected
adult workers and larvae from 75 species of neo- and paleo-tropical ants to acetolysis a process
that destroys most organic material but leaves behind pollen grains In over half of the species
we examined ants contained a few pollen grains (lt 5) and ants of several Camponotus species
contained abundant pollen grains (gt50) We tested for an association between trophic level and
palynivory using stable nitrogen isotope ratios (δ15
N) but we did not find a significant
correlation we found pollen grains in lsquoherbivorousrsquo omnivorous and carnivorous ants We
suggest that our results indicate sporadic opportunistic pollen consumption by ants in tropical
forests
Introduction
For those organisms that can digest it efficiently pollen can be an excellent food source
Depending on the plant species pollen can contain up to 60 protein as well as lipids
76
carbohydrates and numerous trace nutrients (Roulston and Cane 2000) Pollen feeding
(palynivory) occurs in many taxa including birds mammals and arthropods (Roulston and Cane
2000) but it is best known among the Hymenoptera particularly bees Although adult bees feed
mainly on plant exudates (ie nectar) nearly all bee species rely on pollen as the chief protein
source for developing larvae (Willmer 2011) Closely related to bees ants have similar
nutritional requirements adult workers need carbohydrates whereas larvae are fed a diet rich in
protein (Dussutour and Simpson 2009) Ants also exploit nectar heavily and regularly visit
flowers (eg Herrera et al 1984 Bluumlthgen et al 2004) but unlike bees are not believed to
widely consume pollen Instead ants are thought to receive most of the protein in their diet from
predation or scavenging (eg Floren et al 2002)
However ants may not be as reliant on animal protein sources as commonly assumed In
tropical forests arboreal ants can have similar δ15
N ratios a measure of trophic level as
herbivores suggesting limited carnivory and substantial feeding on plant-derived protein sources
(Davidson et al 2003) One potential protein source is pollen This idea is supported by scattered
accounts of pollen consumption by ants from other ecosystems including deserts (Ness 2006)
boreal forests (Czechowski et al 2009) and tropical savannas (Byk and Del Claro 2010) One
ant genus Cephalotes is widely recognized to consume pollen (Creighton 1967 Baroni Urbani
and de Andrade 1997 Byk and Del Claro 2010) However beyond this genus our knowledge of
pollen consumption by ants has largely been limited to direct observations (but see Czechowski
et al 2011) and it is unknown if palynivory is truly rare among ants or simply under-reported
We borrowed a technique commonly used in palynology acetolysis to assess the frequency of
pollen consumption by ants in 75 neo- and paleo-tropical ant species This technique allowed us
to examine many more ant species than we could have observed directly
77
Methods
Adult worker ants were collected by hand at baits or from leaf litter using Winkler extractors
while larvae along with adults were collected by hand from nests Collections were made by L
Arcila Hernandez and J Sanders in the primary tropical rainforest surrounding the Centro de
Investigaciόn y Capacitaciόn Rio Los Amigos (CICRA 12deg34rsquoS 70deg05rsquoW) in Peru in 2010-
2011 and by E Youngerman from lowland primary and secondary rainforests in Madang
Province Papua New Guinea (PNG) in 2011-2012 We sorted ant samples to morphospecies and
then identified them to genus and when possible to species using existing keys which were
unavailable for PNG ants Ant samples used for acetolysis were whole adult workers guts from
adult workers and whole larvae
We used acetolysis to look for pollen grains inside ants In acetolysis organic material is
dissolved in acetic anhydride and sulfuric acid leaving behind stained pollen exines When
possible we used adult and larval ants of the same species from different colonies or bait
locations in an attempt to sample more representatively We prepared samples by first washing
them in 70 ethanol to remove any pollen present on the samplersquos outer surface before placing
them individually in micro-centrifuge tubes with ~05mL of a 91 mixture of acetic anhydride
sulfuric acid We then heated each sample at 95degC for 20min allowing tissues to dissolve Next
we centrifuged samples at 1000 RPM for 5min and decanted the liquid supernatant before
adding ~05mL of glacial acetic acid to the precipitate centrifuging again as above We decanted
the samples and added a drop of distilled water
78
We examined a 150μL sub-sample of this solution for pollen grains under a light
microscope Larvae gave less than 150μL of solution following acetolysis so we sampled the
entire amount We identified pollen grains by their staining and their surface architecture For
most species we examined at least three adult workers and three larvae but for some species
fewer than three adult workers or larvae were available (Tables 31 and 32)
We investigated the relationship between trophic level as measured by δ15
N ratios and
pollen consumption by ants Stable isotope (SI) data was available for some (n =23) of the
Peruvian species from a separate project in which collections overlapped Samples were stored in
EtOH and dried at 50degC overnight We used heads legs and thoraxes (but not gasters) of ants
for SI analysis Often single individuals did not provide enough material for analysis and we
combined multiple individuals to achieve necessary weights up to 10 for small ant species
Samples were analyzed at the Boston University Stable Isotope Laboratory using a Finnigan
Delta-S analyzer For other Peruvian species for which we had samples but not corresponding
stable isotope data (n =25) we took the median δ15
N ratio value of that speciesrsquo genus and
assigned it to the species in question Using pollen presenceabsence data for these 48 species
we used logistic regression to correlate pollen presence and δ15
N ratio using R (v 2151)
Results
We found pollen grains in 37 of the 197 adult workers 20 of the 102 larvae and 38 of the 75 ant
species we examined (Tables 31 and 32) The proportion of individuals containing pollen did
not differ significantly between larvae and adults (χ2
1 = 002 p = 088) The number of pollen
grains found was often low (median = 3) but highly variable (range 1-468) Also pollen
79
presence was inconsistent among conspecific ant samples of species that contained pollen 31
had pollen in only one of the larvae or adults examined In only three species (two Cephalotes
and one Myrmelachista) did all examined adults contain pollen
Highly lsquoherbivorousrsquo carnivorous and omnivorous ants contained pollen grains (Fig
31) There was no correlation between δ15
N ratio and pollen presence (χ2 = 150 df = 1 p =
022) In fact several carnivorous species contained pollen grains (eg Odontomachus sp 1
Pachycondola sp1)
Discussion
Pollen consumption by ants appears to be more common than suspected The majority of species
(51) had at least one individual that had consumed pollen and in 20 multiple individuals
contained pollen Numbers of grains found tended to be low however with most individuals
containing only a few grains Ants may use pollen as only a small portion of their diet exploiting
a host of other protein-containing foods more heavily including other low- δ15
N foods (eg
honeydew epiphylls Houmllldobler and Wilson 1990 Davidson et al 2003)
There are a few reasons why pollen may not be heavily exploited by ants Pollen requires
much post-consumption processing to extract its nutrients These nutrients are located in the
pollenrsquos cytoplasm which is protected from direct digestion by the exines and intines of the
grains Organisms must either break through these layers or cause the pollen grains to germinate
or pseudo-germinate to access and digest the cytoplasm (Roulston and Cane 2000) This can be
achieved by consuming a sugar-containing solution (such as nectar) along with pollen However
animals that are not adapted to feed on pollen may not be very efficient at this process only
80
extracting nutrients from some of the grains (Herrera and Martiacutenez del Rio 1998) Thus while
ants can likely get some nutrients from pollen other forms of more easily processed protein may
be more attractive
Pollen may also pose handling difficulties for ants Despite the large number of studies
that have recorded ants visiting flowers (Chapter 1) only a few papers detail ants harvesting
pollen directly from anthers Byk and Del Claro (2010) reported that Cephalotes pusillus ants
removed almost all of the available pollen from Ouratea spectabilis flowers and at least two
other studies describe ants consuming pollen along with nectar from flowers (Horskins and
Turner 1999 Ness 2006) Unlike bees which possess both morphological (eg specialized hairs
for holding pollen) and behavioral adaptations (eg stereotyped pollen-packing motions) ants
have no apparent mechanisms to increase the amount of pollen harvested and are likely limited
by the number of grains they can ingest or carry Rather than collect pollen directly from flowers
it appears more likely that ants collect it haphazardly in the environment leading to a smaller
number of pollen grains consumed at any one time It is known that at least Cephalotes ants
engage in such behavior Creighton (1967) observed Cephalotes texanus collecting pollen grains
trapped by Citrus x paradisi leaf hairs
There is a chance that the small numbers of pollen grains we observed came from
secondary pollen consumption (ie consuming a palynivorous organism) or consumption of
liquid containing pollen While we cannot rule this out we believe our results represent actual
targeted pollen consumption by ants Cephalotes ants known palynivores (Creighton 1967
Baroni Urbani and de Andrade 1997) contained similar numbers of pollen grains as many of the
pollen-containing ant species that we examined As well in at least once instance ants that we
found containing pollen grains were in the process of preying on non-pollen consuming
81
organisms (eg a Pachycondola species that was carrying a termite in its mandibles) or were
attracted by sugar baits
Contrary to our expectations there was no relationship between an ant speciesrsquo δ15
N ratio
and whether it had consumed pollen (Fig 32) We expected palynivory to be more common in
herbivorous ants because pollen presents a widespread source of protein a potentially limiting
resource The lack of pattern might reflect how available pollen grains are in the environment
While other forms of food may be preferable ants tend to be opportunistic foragers (Carroll and
Janzen 1973) and there is likely little cost associated with the consumption of scattered pollen
grains during foraging trips beyond the volume they occupy in the gut Some genera were found
to consistently consume pollen In support of previous observations (Baroni Urbani and de
Andrade 1997 Byk and Del Claro 2010) Cephalotes species were among the most common
pollen consumers Camponotus species also consistently contained pollen grains of the eight
species examined five contained pollen and pollen was found in species from both Peru and
PNG Camponotus species also contained the two largest numbers of pollen grains found (468
and 271) Taken together these results suggest that they may be another as yet unrecognized
commonly palynivorous genus
In general larvae did not contain large numbers of pollen grains (ranging from 1-38
grains) This is counter to what is seen in bees where larvae are the main recipients of pollen
(Willmer 2011) This difference may be because ant larvae are continually fed via trophallaxis
by adults throughout their development whereas bee larval cells are provisioned with pollen and
then sealed while larvae complete their development If both adults and larvae are able to extract
nutrients from pollen we would expect to see a preferential flow of nutrients from pollen to
larvae but not necessarily the grains themselves
82
Unlike palynivory by organisms that also provide pollination services pollen
consumption by ants is unlikely to benefit plants Ants rarely act as pollinators (Beattie et al
2004) They have pollen-killing antibiotic secretions on their cuticles (Dutton and Frederickson
2012) and they can compete with other flower visitors reducing flower visitation rates (Ness
2006) The negative impacts of flower-visiting ants are thought to have led to the evolution of
ant-repellent floral volatiles (reviewed in Willmer 2009) These volatiles can pre-empt
competition between ants and pollinators and may often be located within the pollen itself
(Willmer 2009) If floral volatiles truly are a defense against ant visitation our results suggest an
additional evolutionary pressure to not only prevent ant-pollinator interactions but to also
protect plant gametes from occasional consumption by a non-pollinating visitor
Acknowledgements
We would like to thank Jon Sanders Lina Arcila Hernandez and Eric Youngerman for providing
samples the Thomson lab for use of supplies the Pierce lab for funding support during JSrsquo and
EYrsquos collections and the staff of CICRA and the New Guinea Binatang Research Center for
logistical support The Peruvian Ministry of Agriculture provided permits (Nos 394-2009-AG-
DGFFS-DGEFFS and 299-2011-AG-DGFFS-DGEFFS) MEF acknowledges financial support
from an NSERC Discovery Grant a Connaught New Researcher Award an Ontario Ministry of
Economic Development and Innovation Early Researcher Award and the University of Toronto
ARC was supported by an Ontario Graduate Scholarship and Sigma Xi
83
References
Baroni Urbani C and M L de Andrade 1997 Pollen eating storing and spitting by ants
Naturwissenschaften 84256ndash258
Beattie A J C Turnbull R B Knox and E G Williams 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany 71421ndash
426
Bluumlthgen N G Gottsberger and K Fiedler 2004 Sugar and amino acid composition of ant-
attended nectar and honeydew sources from an Australian rainforest Austral Ecology
29418ndash429
Byk J and K Del-Claro 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333ndash38
Carroll C R and D H Janzen 1973 Ecology of foraging ants Annual Review of Ecology and
Systematics 4231ndash257
Creighton W S 1967 Studies on free colonies of Cryptocerus texanus Satschi (Hymenoptera
Formicidae) Psyche 7434ndash42
Czechowski W B Markό and A Radchenko 2009 Rubbish dumps reveal the diet of ant
colonies Myrmica schencki Em and Myrmica rubra (L) (Hymenoptera Formicidae) as
facultative pollen-eaters Polish Journal of Ecology 56737ndash741
Czechowski WB Markoacute K Erős and E Csata 2011 Pollenivory in ants (Hymenoptera
Formicidae) seems to be much more common than it was thought Annales Zoologici
61519ndash525
84
Dussutour A and S J Simpson 2009 Communal nutrition in ants Current Biology 19740ndash4
Dutton E M and M E Frederickson 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561ndash569
Floren A A Biun and K E Linsenmair 2002 Arboreal ants as key predators in tropical
lowland rainforest trees Oecologia 131137ndash144
Grant B R 1996 Pollen digestion by Darwinrsquos finches and its importance for early breeding
Ecology 77489-499
Herrera L G and C Martiacutenez del Rio 1998 Pollen digestion by new world bats effects of
processing time and feeding habits Ecology 792828ndash2838
Houmllldobler B and Wilson E O 1990 The Ants 1st Edition Harvard University Press
Cambridge
Horskins K and Turner V B 1999 Resource use and foraging patterns of honeybees Apis
mellifera and native insects on flowers of Eucalyptus costata Australian Journal of
Ecology 24221-227
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506ndash514
Roulston T H I and J H Cane 2000 Pollen nutritional content and digestibility for animals
Plant Systematics 222187ndash209
Van Tets I G 1997 Extraction of nutrients from Protea pollen by African rodents Belgian
Journal of Zoology 12759-65
Willmer P G 2011 Pollination and Floral Ecology 1st Edition Princeton University Press
New Jersey
85
Willmer P G C V Nuttman N E Raine G N Stone J G Pattrick K Henson P Stillman
et al 2009 Floral volatiles controlling ant behaviour Functional Ecology 23888ndash900
86
Tables
Table 31 Numbers of adult worker and larval ants examined for each species collected in Peru
Numbers of pollen grains found in individual samples are given in parentheses after the sample
size when there were no grains no numbers are given
Genus Species Number of adults analyzed
(numbers of grains found)
Number of larvae analyzed
(numbers of grains found)
Dolichoderinae
Azteca sp 1 3 0
sp 2 2 0
sp 3 0 2
sp 4 3 (1) 0
sp 5 3 (2) 0
Dolichoderus decollatus 3 (66) 0
sp 1 3 0
sp 2 3 1 (2)
sp 3 3 3
sp 4 0 2
Ecitoninae
Labidus sp 1 3 0
Formicinae
Camponotus sp 1 3 3
sp 2 3 (82) 1 (2)
sp 3 3 (3) 0
sp 4 0 2
87
sp 5 8 (11) 0
sp 6 4 (7 16 468) 0
sp 7 5 (12) 0
sp 8 2 (1) 0
Paratrechina sp 1 4 (11) 0
Myrmelachista sp 1 3 (5 11 18) 0
sp 2 0 3
Trachymyrmex sp 1 3 (21) 0
sp 2 3 0
Myrmicinae
Atta sp 1 3 0
Cephalotes atratus 3 (2 2 21) 0
placidus 3 (1 4 21) 0
sp 1 3 0
sp 2 1 0
sp 3 2 0
Crematogaster sp 1 3 1
sp 2 3 (35) 0
sp 3 9 (7) 0
sp 4 2 0
Megalomyrmex sp 1 3 0
sp 2 0 2
sp 3 4 (7) 0
sp 4 3 0
Monomorium sp 1 0 3 (2)
88
Pheidole sp 1 0 3
sp 2 3 0
sp 3 0 3
Procryptocerus sp 1 3 0
Solenopsis sp 1 0 3
sp 2 3 (3 5) 0
Wasmannia sp 1 3 0
Ponerinae
Pachycondola sp 1 3 (3) 0
Odontomachus sp 1 3 (34) 0
Pseudomyrmicinae
Pseudomyrmex sp 1 3 0
sp 2 3 0
sp 3 3 (9) 0
sp 4 0 3
sp 5 0 3
89
Table 32 Numbers of adult worker and larval ants examined for each species collected in Papua
New Guinea Numbers of pollen grains found in individual samples are given in parentheses
after the sample size when there were no grains no numbers are given
Genus Species Number adults analyzed
(grains found)
Number larvae analyzed
(grains found)
Aenictinae
Aenictus sp 1 3 3 (1 1)
Dolichoderinae
Philidris sp 1 3 1 (4)
Tapinoma sp 1 2 3 (38)
Technomyrmex sp 1 0 3 (12 12)
Ectatomminae
Rhytidoponera sp 1 1 3 (2)
Formicinae
Acropyga sp 1 3 3 (2)
sp 2 3 3
sp 3 3 3
Anoplolepsis sp 1 3 (1) 3 (1)
Calomyrmex sp 1 3 3 (1)
Camponotus sp 1 3 (1 271) 3
sp 2 1 3
sp 3 2 0
Nylanderia sp 1 3 3
Opisthopsis sp 1 3 3
Polyrachis sp 1 1 3 (4)
90
Myrmicinae
Crematogaster sp 1 3 3
sp 2 3 (32) 3
Monomorium sp 1 3 (1) 3 (1)
sp 2 3 3 (1 1 1)
Pheidole sp 1 3 3
Pristomyrmex sp 1 3 3 (11)
Pyramica sp 1 3 0
Ponerinae
Odontomachus sp 1 3 3 (4)
91
Figures
Figure 31 Photographs of pollen grains found in two ant species a) Dolichoderus sp 2 and b)
Camponotus sp 6 Scale bars = 50 microm
92
Figure 32 Relationship between trophic level as measured by δ15
N ratio and pollen presence
for 48 neotropical ant species 1 Atta sp 5 Azteca spp 8 Camponotus spp 5 Cephalotes spp 4
Crematogaster spp 5 Dolichoderus spp 1 Labidus sp 3 Megalomyrmex spp 1 Monomorium
sp 2 Myrmelachista spp 1 Odontomachus sp 1 Pachycondola sp 1 Paratrechina sp 3
Pheidole sp 4 Pseudomyrmex spp 2 Solenopsis spp and 1 Wasmannia sp
93
Concluding Remarks
Ants have traditionally been considered to be unwelcome flower visitors they consume floral
rewards ostensibly meant for pollinators while rarely providing pollination services themselves
In this thesis I investigate the validity of this assumption and use an experimental study and an
observational study to examine two ways that ants may impact plant reproduction
In the first chapter I review the literature on ants as flower visitors I show that the common
assumption of ants always being categorically negative for plant reproduction is incorrect while
ants may reduce floral attractiveness to pollinators the net effect of ants on plant fitness
measures is variable ranging from positive to negative to neutral I also discuss ants as
pollinators and ways that plants may minimize ant visitation to flowers before finishing with a
section highlighting questions that still need investigation
In the second chapter I explore how interference competition with ants can structure bumblebee
foraging behavior and how this may alter pollen movement patterns Using artificial flowers with
male or female function I allowed Bombus impatiens bumblebees to forage on flowers that
either did or did not have Myrmica rubra ants on them Ant presence significantly reduced the
amount of pollen analogue donated and received by flowers Building on these results I found
that the presence of ant scent alone elicited similar changes to bumblebee behavior reducing the
amount of pollen analogue donated by male flowers My results show that interference
competition is sufficient to alter bumblebee foraging behavior and that the effects of ants on
pollinators may extend after ant visitation to flowers Further study is needed to determine if bees
are able to differentiate between ant species by visual or olfactory cues Their ability to do so
94
could affect how generalized their response to ant presence is and may help to better predict how
ants will affect plant reproduction
In the final chapter I investigate how common palynivory is amongst tropical ant species I used
acetolysis and light microscopy to examine whole ants larvae and ant guts from Papua New
Guinea and Peru I found that tropical ants consistently contain pollen grains albeit often in
small numbers (lt5) However some genera notably Camponotus and Cephalotes consistently
contained higher numbers providing the first evidence that Camponotus may be a palynivorous
genus The small numbers of pollen grains present may indicate that ants preferentially exploit
other more easily digestible high-protein foods over pollen Future studies should investigate
exactly what role pollen plays in ant diets by supplementing the diet of captive ant colonies with
pollen This would allow us to examine how pollen consumption affects colony demography as
well as how efficiently ants digest pollen and whether ants exhibit pollen preferences
I bring up several pressing and unanswered questions within this thesis and highlight three here
First numerous plant species possess chemical or physical traits assumed to increase plant
fitness by reducing ant visitation However this has rarely been tested and instead research has
focused predominately on documenting ant repellence We should move away from this and
begin examining plant fitness in the presence or absence of these repellent traits to see how
beneficial these traits truly are Second how flower visiting ants impact male fitness has been
almost completely ignored in favor of female fitness Future studies should investigate how ant
visitation affects both pollen donation and pollen movement patterns Lastly research involving
antibiotics present on ant cuticles and their effect on pollen viability has been too small in scope
often limited to demonstrating the pollen killing effects of ant secretions Investigation into
95
whether plants have evolved any resistance to the secretions of the local ant community or why
some plants appear unaffected by their ant partners may yield exciting results
In conclusion ants have the capacity to strongly affect plant reproduction both positively and
negatively The fact that these diametrically opposing forces exist means we cannot approach
flower-visiting ants as either positive or negative a priori Instead we should strive to work
towards a better understanding of what is occurring in the system of interest and what role ants
play within it
96
Copyright Acknowledgments
Chapter 2 ldquoAnts and ant scent reduce bumblebee pollination of artificial flowersrdquo is current in
press in The American Naturalist Request for its inclusion here was sought and approved
6
access nectar (Newman and Thomson 2005 Agarwal and Rastogi 2007) but can be limited by
their crop size in the amount of nectar they can remove (Lach 2005) If foraging ants leave
enough nectar behind for a pollinator to recover the cost of visiting a flower ants may have
minimal effects on plant reproductive success (Maloof and Inouye 2000 Turner et al 2012)
Differences among ant species in behavior and diet also affect their consumption of floral
nectar Species vary in how many flowers they visit per inflorescence or per plant and in how
many ants will visit a flower simultaneously (Lach 2005 Junker et al 2007 Lach 2013)
Invasive antsmdashthought to be better at exploiting and defending resources than native ants
(Holway et al 2002)mdashoften recruit to flowers in large numbers sometimes several dozen or
more (Visser et al 1999 Hansen and Muumlller 2009) Overall many factors may influence how
heavily ants exploit floral nectar on plants including ant density plant height the spatial or
temporal distribution of floral nectar and the availability of alternative food resources for ants
(Cushman and Addicott 1991 Lach 2013)
Rarely ants and pollinators may compete for pollen We know of only a few studies that
have recorded ants harvesting pollen directly from anthers (Horskins and Turner 1999 Ness
2006 Byk and Del-Claro 2010) Other studies have found pollen grains in ants (eg Baroni
Urbani and de Andrade 1997 and see Chapter 3) but this may not indicate competition with
pollinators ants sometimes collect stray pollen grains from the environment (Creighton 1967)
One New World genus Cephalotes might be specially adapted for pollen consumption They
possess ldquoproventricular shieldsrdquo structures that may restrict solid food to the crop allowing
nestmates to exchange pollen grains via trophallaxis It is unclear if this structure evolved for this
purpose but Cephalotes are the most frequently documented palynivorous ants (eg Creighton
1967 Baroni Urbani and de Andrade 1997 Byk and Del-Claro 2010) How efficiently ants
7
digest pollen and whether ants like bees feed it to larvae as a protein source is unknown At
least two studies have found that ant larvae consume pollen providing some support for this idea
(Wheeler and Bailey 1920 and Chapter 3)
b) Interference competition
Ants are relatively unique among flower visitors in defending floral resources from competitors
(but see Willmer and Corbett 1981 Roubik 1982) This defense ranges from chance encounters
with ants attacking visitors when flower occupancy overlaps (Ashman and King 2005 Junker et
al 2007) to actively patrolling inflorescences or constructing protective galleries around
nectaries (Horvitz and Schemske 1984 Gaume et al 2005 Hansen and Muumlller 2009)
Interference competition even in the absence of exploitative competition is sufficient to alter
pollinator behavior (Chapter 2)
The strength and outcome of interference competition depends on the identities of the ant
and the pollinator Pollinators might not alter their visitation patterns in response to non-
aggressive ants (Junker et al 2007) but more antagonistic ants may elicit avoidance behavior
before or soon after the visitor lands (Willmer and Corbet 1982 Altshuler 1999 Schuumlrch et al
2000 Table 11) Pollinator size may help predict their reaction to ants because ants may deter
only small-bodied pollinators (Gonzaacutelvez et al 2012) It is not clear whether flower visitors view
ants as aggressive competitors or as potential predators rarely ants prey on pollinators (Schatz
and Wcislo 1999) but in most cases ants merely cause pollinators to relocate
How well pollinators recognize and avoid ant-visited flowers is unresolved Collectively
pollinators have lower visitation rates to flowers visited by ants (Romero et al 2011) This is
8
dependent on pollinator identity however dipterans do not appear to discriminate between ant-
visited and ant-free flowers while bees can identify and avoid ant-visited flowers Bees likely
use a combination of visual and olfactory cues to recognize ant-visited flowers They can
identify previous conspecific and some heterospecific insect visitors by the hydrocarbon
ldquofootprintsrdquo they leave during foraging (Stout and Goulson 2001) Bumblebees can detect ant
hydrocarbons although this has been shown only in the laboratory (Ballantyne and Willmer
2012 Chapter 2) No study has yet examined if bees can use sight alone to detect ants but
several suggest it (Junker et al 2007 Gonzaacutelvez et al 2012) Some bees may discriminate
between ant species when visiting flowers (eg Lach 2008b) which follows if not all ant-
pollinator interactions are equally costly The ability of bees to do so will affect how generalized
their avoidance behaviors will be If they can discriminate between species bees may avoid
specific flowers visited by more ldquocostlyrdquo ants However if they cannot bees might either avoid
all ant-visited flowers or none depending on how negative the average interaction with an ant is
iii) Interactions with flower and pollinator antagonists
Ants can defend the flowers they visit Numerous plant species have circumfloral or bracteal
nectaries (ie extrafloral nectaries around flowers) and ants attracted to these nectaries can
reduce bud ovule or flower predation increasing plant fitness (Inouye and Taylor 1979 Rico-
Gray and Thien 1989 Sugiura et al 2006) Although the presence of EFNs (extrafloral nectaries)
so close to floral nectaries (sometimes less than 1 cm Ness 2006) might increase discovery and
exploitation of floral nectar by ants only one study has investigated this hypothesis finding no
relationship between the proximity of EFNs to floral nectaries and antsrsquo use of floral nectar
(Galen 2005) Holes made by nectar robbers are functionally analogous to circumfloral nectaries
9
and can also attract ants that defend flowers (Newman and Thomson 2005) and nectar-thieving
ants can similarly prevent florivory in some systems (Lach 2007 Bleil et al 2011)
Ants can also reduce the abundance of flower-dwelling predators or parasites of
pollinators Ambush predators such as crab spiders often forage at flowers where they may
reduce pollinator visitation rates and plant fitness (Dukas 2001 Gonccedilalvez-Souza et al 2008)
Ants can decrease spider abundance and richness on plants (Rudgers 2004 Renault et al 2005
Nahas et al 2012) and some evidence suggests that they can do the same on flowers (Lach
2008) In select cases ants may also reduce the abundance of pollinator parasitoids (Holland et
al 2011) Whether ants increase plant reproductive success by removing pollinator antagonists
has not yet been examined but should be If ants create lsquoenemy-freersquo flowers pollinators might
prefer to land on flowers with ants
iv) Net effects
Table 11 compiles studies in which the authors recorded ants consuming floral rewards or
interacting with pollinators and measured some aspect of plant fitness (eg fruit or seed set
pollen deposition) We included only a single study per plant species unless subsequent studies
found conflicting results or measured the fitness of another plant sex Records of ant pollination
are included in a separate table (Table 12)
Table 11 reveals relatively few examples of flower-visiting ants negatively affecting
plant fitness given how costly ant visitation is assumed to be The net effect of ants on plant
reproductive success is actually quite variable with similar numbers of studies finding positive
negative or neutral effects of ant presence Surprisingly when ants do have negative effects it is
10
usually because they directly damage floral structures and not because they prevent pollinators
from visiting flowers and successfully transferring pollen Why are ants not more often negative
for plant reproduction when this is commonly assumed to be true
First ants may compensate for reducing pollinator visitation rates by improving
pollination quality As pollinators move between flowers on a plant they often deposit self-
pollen on stigmas (ie geitonogamy) In some plants with late-acting self-incompatibility ovules
fertilized by self-pollen are subsequently aborted reducing the number of seeds that can be
produced (Barrett 2002 and references therein) Geitonogamy can also reduce offspring viability
or vigor (de Jong et al 1993) and cause pollen discounting a reduction in the amount of pollen
available for outcrossing because of self-pollination (Barrett 2002) By competing with
pollinators ants might decrease rates of geitonogamous pollination if pollinators then visit fewer
sequential flowers within a plant Ants might also cause pollinators to fly further between plants
potentially increasing the genetic diversity of pollen arriving at stigmas Although such effects
have been reported for other floral larcenists (Maloof 2001 Irwin 2003) to our knowledge these
hypotheses have not been investigated in relation to ants
Second ants and pollinators do not always forage for the same resources possibly
allowing them to partition floral rewards If ants forage for nectar but not pollen and flowers can
be pollinated efficiently by visitors looking to gather mostly pollen and little or no nectar then
plant reproduction will not be affected by ants consuming floral nectar Junker et al (2010a)
found that honeybees foraged for both pollen and nectar in the absence of ants but reduced their
foraging on nectar but not pollen when ants could access flowers resulting in no difference in
fruit set between treatments Whether pollinators commonly adjust their foraging behaviors in
response to ants warrants further research
11
Third ants are somewhat unique among floral larcenists in that they often provide direct
benefits to the plants they visit In the studies listed in Table 11 ants sometimes decreased
herbivory on plants (Chamberlain and Holland 2008 Bleil et al 2011) or otherwise aided the
plant (eg ant presence repelling smaller less effective pollinators Gonzaacutelvez et al 2012) If the
positive effects of ants cancel or outweigh the negative effects of flower-visiting ants on
pollinators then ant visitation may be neutral or even positive for plant fitness
Fourth ants may impose fitness costs to plants not captured in the studies in Table 11
Nectar robbers have stronger negative effects when plants are pollen-limited (Burkle et al 2007)
but only one study in Table 11 assessed whether flowers would set more seed if they received
more pollen finding no evidence of pollen limitation (Lach 2008a Table 11) Even if ants
decrease pollinator visitation rates there is limited scope for ants to decrease plant reproductive
success when plants are not pollen-limited Furthermore nearly all the studies in Table 11
measured only female fitness Only three quantified aspects of male fitness (Wyatt 1980 Fritz
and Morse 1981 Galen and Geib 2007) and we know of only one other study in a system in
which ants occasionally pollinate that has also measured male fitness (Ashman 2000) All four
studies measured pollen or pollinia removal rates and none found any effect of flower-visiting
ants on male fitness However whether other measures of male fitness (eg seeds sired pollen
deposition patterns) would reveal similar or different effects needs investigation
Finally although floral defenses against ants (see 3 below) may prevent us from
observing the negative effects of ants on plant reproductive success we consider this unlikely
Several studies have examined interactions between ants and plants that share little or no
coevolutionary history usually in the context of ant invasions Although invasive ants often
recruit to flowers in larger numbers than native ants they do not appear to reduce plant
12
reproductive success (Blancafort and Goacutemez 2005 Lach 2007 Lach 2008a but see Hansen and
Muumlller 2009) And in Hawaii where angiosperms evolved for millions of years in the absence of
ants and are thought to have secondarily lost floral defenses against ants (Junker et al 2011a)
there is no evidence that the preponderance of recently introduced flower-visiting ants negatively
affects plant fitness (Junker et al 2010a Bleil et al 2011)
2 Ants as pollinators
Ants may directly benefit plant reproduction by acting as pollinators but this is a relatively rare
occurrence Ants have long been considered poor pollinators (Kerner 1878) Initially this was
based on morphological and behavioral differences between ants and recognized pollinators
Worker ants are wingless and often relatively hairless making them poor pollen dispersers As
well because many ants exhibit Ortstreue (literally ldquoplace fidelityrdquo) ants were thought to return
to the same branch or flower repeatedly effecting no cross pollination (Houmllldobler and Wilson
1990) Though largely true these traits do not necessarily preclude ants from acting as
pollinators (Table 12) Although they commonly have restricted foraging ranges (Houmllldobler and
Wilson 1990) some ants can effect outcrossing (eg Ashman and King 2005 Carvalheiro 2008)
and many ants are as hairy if not hairier than their pollinating counterparts
In the late 1970s the ldquoantibiotic hypothesisrdquo was put forward to explain the rarity of ant
pollination Ants use antibiotic secretions to protect themselves against the fungal and bacterial
pathogens common in their nests and environs Iwanami and Iwadare (1978) found that one of
these compounds myrmicacin also greatly reduces pollen germination rates Subsequently
Beattie et al (1984 1985) found that short periods of contact (le 30 minutes) with an antrsquos
integument caused pollen grains to fail to germinate Some of these pollen-killing compounds
13
were traced back to the ant metapleural gland which produces hygienic secretions (Houmllldobler
and Wilson 1990) although contact with ants lacking metapleural glands can also reduce pollen
viability (Beattie et al 1985 Dutton and Frederickson 2012) Ant mandibular or anal gland
secretions and potentially ant venom may likewise defend ants against pathogens and have
similar effects on pollen germination (Beattie et al 1985 Graystock and Hughes 2011)
Nonetheless ant pollination has been documented repeatedly There are at least 18 cases
in which ant pollination has been shown experimentally and five others in which observations
strongly suggest it (Table 12) The majority of these plants fit the ldquoant pollination syndromerdquo
originally proposed by Hickman (1974) dense stands of short plants with small flowers and
easily accessible nectaries Typically ant-pollinated plants are associated with hot and dry
climates (principally in the Mediterranean) but dry alpine environments may also favor the
evolution of ant pollination (eg Svensson 1985 Puterbaugh 1998 Schiestl and Glaser 2011) In
most cases ants appear to be the sole or major pollinator of the plant in question (Hickman 1974
Peakall et al 1990 de Vega et al 2009) although ants sometimes make only supplementary
contributions to pollination (Schuumlrch et al 2000 Ashman and King 2005) This suggests that co-
evolution between ants and plants may not be necessary for ants to be useful pollinatorsmdashwhich
is surprising given the potential negative effects of ants on plant reproduction
Ant-pollinated plants cope with ant secretions in a variety of ways Several plant species
may simply tolerate pollen damage (Gόmez and Zamora 1992 de Vega et al 2009) apparently
possessing no overt adaptations to prevent it These plants might receive enough pollen to
overcome reduced pollen viability Other plants may place pollen on ant body parts thought to
have fewer secretions thereby potentially escaping damage (eg on ant legs Ramsey 1995) or
attach pollen to ants with specialized structures avoiding direct contact with the ant integument
14
(Peakall et al 1990) It is not known whether pollen evolves resistance to ant secretions or
whether plants deposit pollen on ants which have less disruptive secretions although these
possibilities have been suggested elsewhere (eg Garciacutea et al 1995 Ramsey 1995)
One key question regarding the effects of ant secretions on pollen grains urgently needs
to be resolved Several studies have suggested that the pollen-killing secretions of ants may have
contributed to the evolution of floral defenses against ants (eg Wagner 2000 Galen and Geib
2007) For this to be true ants must decrease male fitness by damaging pollen grains before they
leave flowers and are deposited on receptive stigmas Only two studies have investigated the
effects of ant secretions on pollen still in anthers (Wagner 2000 Galen and Butchard 2003) and
both constrained ants unnaturally to flowers for significantly longer than ants would normally
stay on flowers Although these studies found strong reductions in pollen vigor it is unclear if
ant secretions decrease male fitness under natural ant visitation conditions
3 Floral defenses against ants
Plants are not passive recipients of flower visitors and have some ability to ldquochooserdquo which
organisms can access floral rewards Flowers resist ants using both structural and chemical traits
More thorough investigation is needed to determine whether these traits evolved to prevent or
minimize the negative effects of ants on plant reproductive success (ie whether these traits can
truly be considered defenses against ants see 4ia below) It is unlikely however that ants
experience reciprocal selection to circumvent floral resistance traits because the fitness benefits
to ants of accessing flowers are probably small Thus there may be little scope for
coevolutionary arms races between floral defenses against ants and ant counter-adaptations
15
i) Structural resistance traits
a) Restricting ant access
Numerous plant species have physical structures preventing ant access to flowers Thin pedicels
can limit antsrsquo gripping abilities and make ants easier to dislodge when flowers are moved by
wind (Gaume et al 2005) and waxy secretions can render stems too slippery for ants to climb
(Harley 1991) Structures surrounding flowers such as water moats or dense hairs may similarly
keep ants from accessing floral rewards (Willmer et al 2009) Many such traits function as broad
filters and prevent not only ants but most crawling insects from reaching flowers Thus it is
difficult to determine whether ants florivores or both acted as selective agents favoring the
evolution of physical barriers to flowers
Flower shape may also reduce ant-pollinator conflict Long narrow corollas or nectar
spurs can prevent all but the smallest ants from accessing floral nectaries (Bluumlthgen et al 2004
Agarwal and Rastogi 2007) while still allowing long-tongued pollinators access to rewards
Selection to exclude nectar-thieving ants in such a manner however may be counteracted by
pollinator preferences for wider corollas that allow easier flower handling (Galen and Cuba
2001) Additionally these types of flowers may be more prone to robbery (Irwin and Maloof
2002) limiting their usefulness in preventing nectar larceny
b) Separation in space or time
Plants may separate floral rewards and ants temporally or spatially to minimize ant impacts on
plant reproduction Several authors have proposed that myrmecophytes have adapted to the
16
sterilization or otherwise detrimental activities of ants by locating domatia or food rewards on
different branches than flowers (Izzo and Vasconcelos 2002 Raine et al 2002 Edwards and Yu
2008) Temporal separation of ant and pollinator activities could likewise reduce conflict though
evidence is limited Some acacia flowers dehisce during periods of low ant activity (Willmer and
Stone 1997 Nicklen and Wagner 2006) but it is unclear if this is in response to ants
temperature or pollinator activity Timing of nectar secretion may similarly reduce ant-pollinator
competition If ants are active prior to pollinators they may deplete floral nectar and reduce
pollinator visitation rates (Schaffer et al 1984) However if nectar is secreted when ants are
inactive pollination may occur unimpeded (Norment 1988) This temporal separation could
favor pollinators that are active when ants are not (eg Wyatt and Morse 1981) but this is
poorly documented Further studies particularly focusing on flowers with the potential for
nocturnal and diurnal pollination may provide more examples
c) Extrafloral nectaries (EFNs)
One hypothesis put forward to explain the evolution of EFNs proposes that EFNs ldquodistractrdquo ants
from consuming floral nectar (Kerner 1878 Wagner and Kay 2002) Wagner and Kay (2002)
tested this hypothesis by constructing model plants using artificial nectaries assigned at random
to represent either floral or extrafloral nectaries They found that the presence of EFNs could
reduce ant exploitation of floral nectar However as highlighted by Galen (2005) this design is
closer to a plant making a surplus of flowers some of which will avoid ant visitation if visitation
rates increase sublinearly with increasing flower number Producing extra flowers to escape ant
visitation appears to be a viable strategy in some systems (Ashman and King 2005 Galen 2005)
However Galen (2005) found that applying honey (simulating extrafloral nectar) to P viscosum
17
leaves had the opposite effect and increased ant visits to flowers Only two studies both in
Pachycereus schottii have found evidence for the distraction hypothesis under field conditions
(Chamberlain and Holland 2008 Holland et al 2011)
There is limited empirical support for the distraction hypothesis but abundant evidence
that EFNs attract ants that defend plants against herbivores (reviewed in Rosumek et al 2009)
To function as attractants the presence of EFNs must increase ant abundance on plants
However because of the increased numbers attracted EFNs would not be expected to reduce the
abundance of ants on nectar rewards or be able to satiate the ants recruited Only if ant
abundance stops increasing at a reward density much lower than that available on the plant
would EFNs serve to distract ants In this case the amount of rewards may overwhelm ants
leaving some flowers underexploited However even in P schottii ants still visited flowers
(Chamberlain and Holland 2008) In cases where ant abundance would continue to increase with
additional rewards it is unlikely that EFNs function as distractions
ii) Chemical resistance traits
a) Toxic nectar
One of the earliest proposed floral defenses against ants was toxic nectar (Janzen 1977)
However studies testing Janzenrsquos (1977) hypothesis uncovered only a very few isolated
examples (eg Catalpa speciosa Stephenson 1981 Crinum erubescens Guerrant and Fiedler
1981) in short most floral nectar is not toxic or repellent to ants (Baker and Baker 1978
Schubart and Anderson 1978) In fact ants sometimes prefer floral nectar over solutions with
equivalent sugar concentrations (Koptur and Truong 1998)
18
In contrast floral nectar can repel other flower visitors (eg birds Johnson et al 2006
lepidopterans Kessler and Baldwin 2007) Repellency is often caused by plant secondary
metabolites which may occur in nectar as a by-product of their production elsewhere in the
plant where they function as defenses against herbivory (Adler 2001) Alternatively toxic or
repellent nectar may be adaptive repellent nectar may promote effective pollination by reducing
the amount of reward any one pollinator takes spreading nectar over several pollinator visits
(Kessler and Baldwin 2007) or it may function as a filter selecting for specific pollinators
(Johnson et al 2006) The latter may explain why repellent nectar is not more common
legitimate pollinators in addition to floral larcenists may be deterred from visiting flowers with
repellent nectar This may be particularly true of nectars that would repel ants which are closely
related to bees nectar distasteful to ants may often also be distasteful to bees and those nectars
acting as a filter for bees may still be palatable to ants For example Prŷs-Jones and Willmer
(1992) found that the ammonia-containing nectar of Lathraea clandestina is readily consumed
by both bees and ants even though it deters birds that act as nectar thieves Although floral
nectar itself rarely repels ants Guerrant and Fielder (1981) found that the presence of macerated
petals within nectar can often keep ants away Alkaloids in petals may be responsible and might
explain why ants seldom act as primary nectar robbers these secondary compounds may prevent
ants from chewing through corollas
b) Floral volatiles
Repellent floral volatiles appear to be a more widespread form of chemical resistance against
ants These were reviewed recently by Willmer et al (2009) and will be discussed here only
briefly Numerous plant taxa produce floral scents repellent to ants including myrmecophilous
19
myrmecophytic and non-ant associated plants in both temperate and tropical ecosystems
(Willmer et al 2009 Junker et al 2011a Ballantyne and Willmer 2012) In three large studies
floral volatiles that repel ants were detected in about a third of all plants tested (Willmer et al
2009 Junker et al 2011a Ballantyne and Willmer 2012) Pollen is often the source of ant-
repellent volatile compounds (Willmer et al 2009 Ballantyne and Willmer 2012) but they can
be emitted from other floral parts including petals That pollen is often the source of repellence
could suggest that preventing ant visitation may be more important in protecting male than
female function Additionally because repellence would decrease as pollen is removed from
anthers ants may return and defend flowers more quickly than if repellent chemicals were in
longer-lasting structures (eg petals) In some cases floral volatiles are thought to mimic ant
alarm pheromones (Willmer et al 2009) However floral scents elicit inconsistent reactions
across ant species ranging from repellence to attraction (Agarwal and Rastogi 2007 Ballantyne
and Willmer 2012)
Producing structural or chemical resistance traits may be costly for plants Native
Hawaiian plants which have little recent coevolutionary history with ants appear to have
secondarily lost floral ant repellents (Junker et al 2011a) Both Willmer et al (2009) and Junker
et al (2011a) found that highly chemical repellent flowers did not have strong physical defenses
and vice versa suggesting trade-offs between the two (but see Ballantyne and Willmer 2012)
Like toxic nectar floral ant repellents might sometimes deter both pollinators and ants For
example Galen et al (2011) found that 2-phenylethanol in P viscosum floral scent repels both
flower-damaging F neorufibarbis ants and the plantrsquos main pollinator Bombus kirbyellus
Interestingly four recent studies have found that floral scents can also attract ants
(Agarwal and Rastogi 2007 Edwards and Yu 2008 Schiestl and Glaser 2011 Gonzaacutelvez et al
20
2012) One study (Schiestl and Glaser 2011) has shown that floral scents attract ants to a
potentially ant-pollinated plant and such systems may provide many more examples
4 Future directions
We have highlighted areas in need of further investigation throughout the review Here we focus
on several questions we consider high priorities for future research
i) Floral defenses against ants
a) Do floral resistance traits increase plant fitness
The existence ant-repellent floral traits is often taken as evidence that ants negatively affect plant
reproductive success However despite the abundance and variety of mechanisms excluding ants
from flowers few studies show that ants reduce plant fitness when they visit flowers (Table 11)
This absence is particularly apparent with repellent floral volatiles which may be the most
widespread floral resistance trait (Willmer et al 2009 Junker et al 2011a Ballantyne and
Willmer 2012) To our knowledge only one study has shown that floral ant repellents benefit
plants with repellent P viscosum flowers avoiding damaging ant visits (Galen et al 2011)
Research is urgently needed to investigate if these traits increase plant fitness by excluding ants
or if ant visitation is inconsequential More studies taking advantage of standing variation in
traits (Galen et al 2011) or creating it artificially (eg Junker et al 2010b) will allow us to
distinguish between the two
21
b) If ant visitation is often not costly what caused the evolution of repellent traits and
what maintains them Similarly are these repellent traits truly defenses against ant
visitation or are they generalized defenses against a range of floral antagonists
Ant-repellent floral traits are widespread and their production may be costly (Willmer et al
2009 Junker et al 2011a) The studies in Table 11 however indicate that flower-visiting ants
often do not disrupt plant reproduction particularly outside of myrmecophytes What then
selected for these traits One possibility is that these repellents are not directed solely at ants
Instead a community of antagonistic flower visitors may exert diffuse selection on plants
thereby selecting for and maintaining these traits Structural modifications may repel crawling
insects while chemical repellents such as floral volatiles may repel both flying and non-flying
insects (Junker et al 2011b) How much overlap there is in floral volatiles that repel both ants and
other antagonists needs investigation Until we examine how specific these defenses are to ants
we risk overstating the importance of ants in their evolution
ii) Do ants alter pollen movement patterns or reduce male fitness
We are aware of only four studies that have measured if ant visitation impacts male fitness
(Wyatt 1980 Fritz and Morse 1981 Ashman 2000 Galen and Geib 2007) While none of these
studies found an effect all measured only amounts of pollen or pollinia removed However this
measure does not adequately encompass male fitness alterations to pollen movement patterns
and eventual deposition location may be more important to male function than changes in
removal rates If as suggested by some studies (Altshuler 1999 Lach 2007) ants increase
pollinator relocation rates they may decrease pollen discounting increasing male fitness
However if this increased relocation reduces overall pollen export rates ant competition with
22
pollinators may depress male function Studies using pollen-tracking methods such as fluorescent
dyes will allow us to see how ants change pollen movement patterns and methods like paternity
analysis will reveal how competition between ants and pollinators alters male fitness
iii) Coevolution between ants and plants do myrmecophytes exhibit stronger more
specific defenses against ants
The floral traits of myrmecophytes are most likely to coevolve with ants because all plant
reproduction occurs in the presence of ants Their floral defenses would thus be expected to be
both stronger and more specific to their resident ants than those other plants visited by an array
of more opportunistic ant species Some evidence supports this Willmer et al (2009) found that
resident ants of acacias were repelled by flowers with more aggressive ant species showing
stronger repellence than less aggressive ants Additionally ants not normally associated with
acacias did not respond to acacia floral volatiles More studies are needed in other systems and
should take advantage of phylogenetic approaches examining floral traits in closely related
myrmecophytic and non-myrmecophytic plant species to determine whether ants commonly
select for floral defenses against ants in myrmecophytes
Acknowledgements
For comments and discussion we thank the Frederickson lab particularly Eric Youngerman
Lina Arcila Hernandez and Kyle Turner as well as Alison Parker and Jordan Pleet MEF
acknowledges financial support from an NSERC Discovery Grant a Connaught New Researcher
23
Award an Ontario Ministry of Economic Development and Innovation Early Researcher Award
and the University of Toronto ARC was supported by an Ontario Graduate Scholarship and
Sigma Xi
24
References
Adler L S 2001 The ecological significance of toxic nectar Oikos 91409ndash420
Agarwal V M and N Rastogi 2007 Role of floral repellents in the regulation of flower visits
of extrafloral nectary-visiting ants in an Indian crop plant Ecological Entomology 3359ndash
65
Altshuler D L 1999 Novel interactions of non-pollinating ants with pollinators and fruit
consumers in a tropical forest Oecologia 119600ndash606
Ashman TL 2000 Pollinator selectivity and its implications for the evolution of dioecy and
sexual dimorphism Ecology 812577-2591
Ashman TL and E A King 2005 Are flower-visiting ants mutualists or antagonists A study
in a gynodioecious wild strawberry American Journal of Botany 92891ndash5
Ballantyne G and P Willmer 2012 Floral visitors and ant scent marks noticed but not used
Ecological Entomology 37402-409
Ballantyne G and P Willmer 2012 Nectar theft and floral ant-repellence a link between
nectar volume and ant-repellent traits PloS One 7e43869
Baker H G and I Baker 1978 Ants and flowers Biotropica 1080
Barrett S C H 2002 Sexual interference of the floral kind Heredity 88154ndash159
Baroni Urbani C and M L de Andrade 1997 Pollen eating storing and spitting by ants
Naturwissenschaften 84256ndash258
25
Beattie A J C Turnbull R B Knox and E G Williams 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany
71421ndash426
Beattie A J C Turnbull T Hough S Jobson and R B Knox 1985 The vulnerability of
pollen and fungal spores to ant secretions evidence and some evolutionary implication
American Journal of Botany 72606ndash614
Blancafort X and C Goacutemez 2005 Consequences of the Argentine ant Linepithema humile
(Mayr) invasion on pollination of Euphorbia characias (L) (Euphorbiaceae) Acta
Oecologica 2849ndash55
Bleil R N Bluumlthgen and R R Junker 2011 Ant-plant mutualism in Hawailsquoi Invasive ants
reduce flower parasitism but also exploit floral nectar of the endemic shrub Vaccinium
reticulatum (Ericaceae) Pacific Science 65291ndash300
Bluumlthgen N G Gottsberger and K Fiedler 2004 Sugar and amino acid composition of ant-
attended nectar and honeydew sources from an Australian rainforest Austral Ecology
29418ndash429
Burkle L A R E Irwin and D A Newman 2007 Predicting the effects of nectar robbing on
plant reproduction implications of pollen limitation and plant mating system American
Journal of Botany 941935ndash1943
Byk J and K Del-Claro 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333ndash38
26
Caballero P C G Ossa W L Gonzaacuteles C Gonzaacutelez-Browne G Astorga M M Muruacutea and
R Medel 2013 Testing non-additive effects of nectar-robbing ants and hummingbird
pollination on the reproductive success of a parasitic plant Plant Ecology 214633ndash640
Carvalheiro L G E R M Barbosa and J Memmott 2008 Pollinator networks alien species
and the conservation of rare plants Trinia glauca as a case study Journal of Applied
Ecology 451419ndash1427
Chamberlain S A and J N Holland 2008 Density-mediated context-dependent consumer-
resource interactions between ants and extrafloral nectar plants Ecology 891364ndash1374
Creighton W S 1967 Studies on free colonies of Cryptocerus texanus Satschi (Hymenoptera
Formicidae) Psyche 7434ndash42
Cursach J and J Rita 2011 Implications of the reproductive biology of the narrow endemic
Naufraga balearica (Apiaceae) for its conservation status Plant Systematics and Evolution
298581ndash596
Cushman JH and Addicott JF In Huxley CR and DF Cutler (eds) 1st edition Ant-plant
interactions Oxford University Press Oxford
de Jong T J N M Waser and P G Klinkhamer 1993 Geitonogamy the neglected side of
selfing Trends in Ecology and Evolution 8321ndash325
de Vega C M Arista P L Oritz C M Herrera and S Talavera 2009 The ant-pollination
system of Cytinus hypocistis (Cytinaceae) a Mediterranean root holoparasite Annals of
Botany 4517ndash8
27
Dukas R 2001 Effects of perceived danger on flower choice by bees Ecology Letters 4327ndash
333
Dutton E M and M E Frederickson 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561ndash569
Edwards D P and D W Yu 2008 Tolerating castration by hiding flowers in plain sight
Behavioral Ecology and Sociobiology 6395ndash102
Frederickson M E 2009 Conflict over reproduction in an ant-plant symbiosis why Allomerus
octoarticulatus ants sterilize Cordia nodosa trees The American Naturalist 173675ndash81
Fritz R S and D H Morse 1981 Nectar parasitism of Asclepias syriaca by ants effect on
nectar levels pollinia insertion pollinaria removal and pod production Oecologia 50316ndash
319
Galen C 1983 The effects of nectar thieving ants on seedset in floral scent morphs of
Polemonium viscosum Oikos 41245ndash249
Galen C 2005 Catching ants with honey an experimental test of distraction and satiation as
alternative modes of escape from flower-damaging ants Oecologia 14480ndash87
Galen C and B Butchart 2003 Ants in your plants effects of nectar-thieves on pollen fertility
and seed-siring capacity in the alpine wildflower Polemonium viscosum Oikos 101521ndash
528
Galen C and J Cuba 2001 Down the tube pollinators predators and the evolution of flower
shape in the alpine skypilot Polemonium viscosum Evolution 551963-1971
28
Galen C and J C Geib 2007 Density-dependent effects of ants on selection for bumble bee
pollination in Polemonium viscosum Ecology 881202ndash1209
Galen C R Kaczorowski S L Todd J Geib and R A Raguso 2011 Dosage-dependent
impacts of a floral volatile compound on pollinators larcenists and the potential for floral
evolution in the alpine skypilot Polemonium viscosum The American Naturalist 177258ndash
272
Garciacutea M B R J Antor and X Espadaler 1995 Ant pollination of the palaeoendemic
dioecious Borderea pyrenaica (Dioscoreaceae) Plant Systematics and Evolution 19817ndash
27
Gaume L M Zacharias and R M Borges 2005 Ant ndash plant conflicts and a novel case of
castration parasitism in a myrmecophyte Evolutionary Ecology Research 7435ndash452
Graystock P and W O H Hughes 2011 Disease resistance in a weaver ant Polyrhachis
dives and the role of antibiotic-producing glands Behavioral Ecology and Sociobiology
652319ndash2327
Gόmez J M 2000 Effectiveness of ants as pollinators of Lobularia maritima effects on main
sequential fitness components of the host plant Oecologia 12290ndash97
Gόmez J M and R Zamora 1992 Pollination by ants consequences of the quantitative effects
on a mutualistic system Oecologia 91410ndash418
Gόmez J M R Zamora J A Hόdar and D Garciacutea 1996 Experimental study of pollination
by ants in Mediterranean high mountain and arid habitats Oecologia 105236ndash242
29
Gonccedilalves-Souza T P M Omena J C Souza and G Q Romero 2008 Trait-mediated effects
on flowers artificial spiders deceive pollinators and decrease plant fitness Ecology
892407ndash2413
Gonzaacutelvez F G L Santamariacutea R T Corlett and M A Rodriacuteguez-Gironeacutes 2012 Flowers
attract weaver ants that deter less effective pollinators Journal of Ecology 10178ndash85
Guerrant E O and P L Fiedler 1981 Flower defenses against nectar-pilferage by ants
Biotropica 1325ndash33
Harley R 1991 The greasy pole syndrome In Huxley CR and DF Cutler (eds) 1st edition
Ant-plant interactions Oxford University Press Oxford
Hansen D M and C B Muumlller 2009 Invasive ants disrupt gecko pollination and seed
dispersal of the endangered plant Roussea simplex in Mauritius Biotropica 41202ndash208
Hickman J C 1974 Pollination by ants a low-energy system Science 1841290ndash1292
Holland J N S a Chamberlain and T E X Miller 2011 Consequences of ants and
extrafloral nectar for a pollinating seed-consuming mutualism ant satiation floral
distraction or plant defense Oikos 120381ndash388
Houmllldobler B and Wilson E O 1990 The Ants 1st Edition Harvard University Press
Cambridge
Holway DA L Lach AV Suarez ND Tsutsui and TJ Case 2002 The causes and
consequences of ant invasions Annual Review of Ecology and Systematics 33181-233
30
Horskins K and Turner VB 1999 Resource use and foraging patterns of honeybees Apis
mellifera and native insects on flowers of Eucalyptus costata Australian Journal of
Ecology 24221-227
Horvitz C C and D W Schemske 1984 Effects of ants and an ant-tended herbivore on seed
production of a neotropical herb Ecology 651369ndash1378
Inouye DW 1980 The terminology of floral larceny Ecology 611251-1253
Inouye DW and O R J Taylor 1979 A temperate region plant-ant-seed predator system
consequences of extra floral nectar secretion by Helianthella quinquenervis Ecology 601ndash
7
Irwin RE 2003 Impact of nectar robbing on estimates of pollen flow conceptual predictions
and empirical outcomes Ecology 84485-495
Irwin RE and Maloof JE 2002 Variation in nectar robbing over time space and species
Oecologia 133525-533
Iwanami Y and T Iwadare 1978 Inhibiting effects of myrmicacin on pollen growth and pollen
tube mitosis Botanical Gazette 13942ndash45
Izzo TJ and Vasconcelos HL 2002 Cheating the cheater domatia loss minimizes the effects
of ant castration in an Amazonian ant plant Oecologia 133200-205
Janzen D H 1977 Why donrsquot ants visit flowers Biotropica 9252
Johnson SD Hargreaves AL and Brown M 2006 Dark bitter-tasting nectar functions as a
filter of flower visitors in a bird-pollinated plant Ecology 872709-2716
31
Junker R A Y C Chung and N Bluumlthgen 2007 Interaction between flowers ants and
pollinators additional evidence for floral repellence against ants Ecological Research
22665ndash670
Junker R R R Bleil C C Daehler and N Bluumlthgen 2010a Intra-floral resource partitioning
between endemic and invasive flower visitors consequences for pollinator effectiveness
Ecological Entomology 35760ndash767
Junker R R and N Bluumlthgen 2010b Floral scents repel facultative flower visitors but attract
obligate ones Annals of botany 105777ndash82
Junker R R C C Daehler S Doetteri A Keller and N Bluumlthgen 2011a Hawaiian ant ndash
flower networks nectar-thieving ants prefer undefended native over introduced plants with
floral defenses Ecological Monographs 81295ndash311
Junker R R J Gershenzon and S B Unsicker 2011b Floral odor bouquet loses its ant
repellent properties after inhibition of terpene biosynthesis Journal of Chemical Ecology
371323ndash31
Kawakita A and M Kato 2002 Floral biology and unique pollination system of root
holoparasites Balanophora kuroiwai and B tobiracola (Balanophraceae) American
Journal of Botany 891164ndash1170
Koopowitz H and TA Marchant 1998 Postpollination nectar reabsorption in the African
epiphyte Aerangis verdickii (Orchidaceae) American Journal of Botany 85508-512
Kirk W D J 1984 Ecological studies on Thrips imaginis Bagnall (Thysanoptera) in flowers of
Echium plantagineum L in Australia Australian Journal of Ecology 99ndash18
32
Kerner A 1878 Flowers and their unbidden guests C Kegan Paul London
Kessler D and I T Baldwin 2007 Making sense of nectar scents the effects of nectar
secondary metabolites on floral visitors of Nicotiana attenuata The Plant Journal 49840ndash
54
Koptur S and N Truong 1998 Facultative ant-plant interactions nectar sugar preferences of
introduced pest species in South Florida Biotropica 30179-189
Lach L 2005 Interference and exploitation competition of three nectar-thieving invasive ant
species Insectes Sociaux 52257ndash262
Lach L 2007 A mutualism with a native membracid facilitates pollinator displacement by
Argentine ants Ecology 881994ndash2004
Lach L 2008a Argentine ants displace floral arthropods in a biodiversity hotspot Diversity and
Distributions 14281ndash290
Lach L 2008b Floral visitation patterns of two invasive ant species and their effects on other
hymenopteran visitors Ecological Entomology 33155ndash160
Lach L 2013 A comparison of floral resource exploitation by native and invasive Argentine
ants Arthropod-Plant Interactions 7177-190
Luo C W K Li X M Chen Z Y Huang and K U N Li 2012 Ants contribute significantly
to the pollination of a biodiesel plant Jatropha curcas Environmental Entomology
411163ndash1168
33
Maleacute PJ G C Leroy A Dejean A Quilichini and J Orivel 2011 An ant symbiont directly
and indirectly limits its host plantrsquos reproductive success Evolutionary Ecology 2655ndash63
Maloof J E 2001 The effects of a bumble bee nectar robber on plant reproductive success and
pollinator behavior American Journal of Botany 881960ndash1965
Maloof J E and D W Inouye 2000 Are nectar robbers cheaters or mutualists Ecology
812651ndash2661
Moreau CS CD Bell R Vila SB Archibald and NE Pierce 2006 Phylogeny of the ants
diversification in the age of the angiosperms Science 312101-104
Nahas L M O Gonzaga and K Del-Claro 2012 Emergent impacts of ant and spider
interactions herbivory reduction in a tropical savanna tree Biotropica 44498ndash505
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506ndash514
Newman D A and J D Thomson 2005 Interactions among nectar robbing floral herbivory
and ant protection in Linaria vulgaris Oikos 110497ndash506
Nicklen E F and D Wagner 2006 Conflict resolution in an ant-plant interaction Acacia
constricta traits reduce ant costs to reproduction Oecologia 14881ndash7
Norment C J 1988 The effect of nectar-thieving ants on the reproductive success of Frasera
speciosa (Gentianaceae) American Midland Naturalist 120331ndash336
34
Orivel J L Lambs P-J G Maleacute C Leroy J Grangier T Otto A Quilichini et al 2011
Dynamics of the association between a long-lived understory myrmecophyte and its
specific associated ants Oecologia 165369ndash76
Palmer T M D F Doak M L Stanton J L Bronstein E T Kiers T P Young J R
Goheen et al 2010 Synergy of multiple partners including freeloaders increases host
fitness in a multispecies mutualism Proceedings of the National Academy of Sciences
10717234ndash9
Peakall R A J Beattie and S H James 1987 Pseudocopulation of an orchid by male ants a
test of two hypotheses accounting for the rarity of ant pollination Oecologia 73522ndash524
Peakall R and A J Beattie 1989 Pollination of the orchid Microtis parviflora R Br by
flightless worker ants Functional Ecology 3515ndash522
Peakall R C J Angus A J Beattie 1990 The significance of ant and plant traits for ant
pollination in Leporella fimbriata pollination Oecologia 84457ndash460
Puterbaugh M N 1998 The roles of ants as flower visitors experimental analysis in three
alpine plant species Oikos 8336ndash46
Prŷs-Jones OE and Willmer PG 1992 The biology of alkaline nectar in purple toothwort
(Lathraea clandenstina) ground level defenses Biological Journal of the Linnean Society
45373-388
Raine NE Willmer P and Stone GN 2002 Spatial structuring and floral avoidance
behaviour present in ant-pollinator conflict in a Mexican ant-acacia Ecology 833086-
3096
35
Ramsey M 1995 Ant pollination of the perennial herb Blandfordia grandiflora (Liliaceae)
Oikos 74265ndash272
Renault CK LM Buffa and Delfino MA 2005 An aphid-ant interaction effects on
different trophic levels Ecological Research 2071-74
Rico-Gray V and L B Thien 1989 Effect of different ant species on reproductive fitness of
Schomburgkia tibicinis (Orchidaceae) Oecologia 81487ndash489
Romero G Q P A P Antiqueira and J Koricheva 2011 A meta-analysis of predation risk
effects on pollinator behaviour PLoS One 6e20689
Rosumek FB FAO Silveira F de S Neves NP de U Barbosa L Diniz Y Oki F Pezzini
et al 2009 Ants on plants a meta-analysis of the role of ants as plant biotic defenses
Oecologia 160537ndash549
Roubik D W 1982 The ecological impact of nectar-robbing bees and pollinating
hummingbirds on a tropical shrub Ecology 63354ndash360
Rudgers J A 2004 Enemies of herbivores can shape plant traits selection in a facultative ant-
plant mutualism Ecology 85192ndash205
Schatz B and WT Wcislo 1999 Ambush predation by the ponerine ant Ectatomma ruidum
Roger (Formicidae) on a sweat bee Lasioglossum umbripenne (Halictidae) in Panama
Journal of Insect Behavior 12641-663
36
Schaffer W M D W Zeh S L Buchmann S Kleinhaus M V Schaffer and J Antrim
1983 Competition for nectar between introduced honey bees and native North American
bees and ants Ecology 64564ndash577
Schemske D W 1980 The evolutionary significance of extrafloral nectar production by Costus
woodsonii (Zingiberaceae) an experimental analysis of ant protection Journal of Ecology
68959ndash967
Schiestl F P and F Glaser 2011 Specific ant-pollination in an alpine orchid and the role of
floral scent in attracting pollinating ants Alpine Botany 1221ndash9
Schubart HOR AB Anderson 1978 Why donrsquot ants visit flowers A response to D H
Janzen Biotropica 10310-311
Schuumlrch S M Pfunder and B A Roy 2000 Effects of ants on the reproductive success of
Euphorbia cyparissias and associated pathogenic rust fungi Oikos 16ndash12
Stanton ML Palmer TM Young TP Evans A and Turner ML 1999 Sterilization and
canopy modification of swollen thorn acacia tree by a plant-ant Nature 401 578-581
Stephenson AG 1981 Toxic nectar deters nectar thieves of Catalpa speciosa American
Midland Naturalist 105381-383
Stout J C and D Goulson 2001 The use of conspecific and interspecific scent marks by
foraging bumblebees and honeybees Animal Behaviour 62183ndash189
Sugiura S T Abe and S Makino 2006 Loss of extrafloral nectary on an oceanic island plant
and its consequences for herbivory American Journal of Botany 93491ndash495
37
Svensson L 1985 An estimate of pollen carryover by ants in a natural population of
Scleranthus perennis L (Caryophyllaceae) Oecologia 66373ndash377
Tsuji K A Hasyim and K Nakamura 2004 Asian weaver ants Oecophylla smaragdina and
their repelling of pollinators Ecological Research 19669ndash673
Turner R C J J Midgley P Barnard R E Simmons and S D Johnson 2012 Experimental
evidence for bird pollination and corolla damage by ants in the short-tubed flowers of
Erica halicacaba (Ericaceae) South African Journal of Botany 7925ndash31
Visser D M G Wright and J H Giliomee 1996 The effect of the Argentine ant
Linepithema humile (Mayr) (Hymenoptera Formicidae) on flower-visiting insects of
Protea nitida Mill (Proteaceae) African Entomology 4285ndash287
Wagner D 2000 Pollen viability reduction as a potential cost of ant association for Acacia
constricta (Fabaceae) American Journal of Botany 87711ndash715
Wagner D and A Kay 2002 Do extrafloral nectaries distract ants from visiting flowers An
experimental test of an overlooked hypothesis Evolutionary Ecology Research 4293ndash305
Wang C Y Luo Y Tai D An and Y Kou 2008 Ants pollinate Neottia listeroides
(Orchidaceae) in Sichuan China 46836ndash846
Wyatt R 1980 The impact of nectar-robbing ants on the pollination system of Asclepias
curassavica Bulletin of the Torrey Botanical Club 10724ndash28
Wheeler WM and IW Bailey 1920 The feeding habits of Pseudomymine and other ants
Transactions of the American Philosophical Society 22235-279
38
Willmer P G and S A Corbet 1981 Temporal and microclimatic partitioning of the floral
resources of Justicia aurea amongst a concourse of pollen vectors and nectar robbers
Oecologia 5167ndash78
Willmer P G and G N Stone 1997 How aggressive ant-guards assist seed-set in Acacia
flowers Nature 388165ndash167
Willmer P G C V Nuttman N E Raine G N Stone J G Pattrick K Henson P Stillman
et al 2009 Floral volatiles controlling ant behaviour Functional Ecology 23888ndash900
Young T P C H Stubblefield and L a Isbell 1997 Ants on swollen-thorn acacias species
coexistence in a simple system Oecologia 10998ndash107
Yu D W and N E Pierce 1998 A castration parasite of an ant-plant mutualism Proceedings
of the Royal Society B Biological Sciences 265375ndash382
39
Tables
Table 11 Studies of ant-flower or ant-pollinator interactions that included a measure of plant fitness Each study system is listed only
once except when results varied between years or when different studies examined male and female fitness Significant effects of ants on
plant fitness are either lsquopositiversquo or lsquonegativersquo non-significant effects are lsquoneutralrsquo When ants significantly affected only one fitness
component but several were measured (eg fruit set and seed production) we still consider the result significant
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Acacia
drepanolobium
Crematogaster
nigriceps
Proportion
fruiting
Negative Destruction of floral buds Young et al 1997
Asclepias exaltata Several Pollinia inserted
removed
Negative for pollinia
insertion neutral for
pollinia removal
Exploitative competition Wyatt 1980 T
40
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Asclepias syriaca Several
Fruit set pollinia
removed
Negative for fruit
set neutral for
pollinia removal
Exploitative competition Fritz and Morse
1981 T
Cordia nodosa Allomerus cf
demerarae
Fruit set Negative Destruction of floral buds Yu and Pierce
1998
Costus woodsonii Wasmannia
auropunctata
Seed set Positive Unknown ant pollination
suggested (but unlikely)
Schemske 1980
Eritrichum aretioides F neorufibarbis
gelida
Seed set Negative Ants damaged flowers Puterbaugh 1998
41
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Euphorbia characias Several Seed set Neutral Plants in Argentine ant-
invaded area set less seed
than those in un-invaded
sites but an ant-exclusion
experiment did not produce
significant results
Blancafort and
Gόmez 2005
Ferocactus wislizeni Solenopsis xyloni
and others
Seed set Unknown negative Interference and exploitative
competition S xyloni
reduced seed set relative to
other ants but the study did
not include a no-ant control
Ness 2006
42
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Frasera speciosa F neorufibarbis
and Formica sp
Seed set Neutral Nocturnal pollination
occurring when ants were
less active may have
compensated for reduced
diurnal pollinator visitation
Norment 1988
Gypsophila struthium Several Seeds per
inflorescence
Neutral No effect despite large
numbers of ants on flowers
Gόmez et al
1996
Hirtella physophora Allomerus
decemarticulatus
Fruit set Negative Destruction of flowers Orivel et al
2011
Humboldtia brunonis Crematogaster
dohrni
Fruit set Negative Destruction of flowers Gaume et al
2005
43
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Lepidium subulatum Several Percentage of
ovules setting
seed
Positive Unknown Gόmez et al
1996
Leucospermum
conocarpodendron
Several Seed set Neutral Site not pollen limited Lach 2008
Linaria vulgaris Several ldquoFemale fitness
indexrdquo
(combination of
number of seeds
fruits flowers
and seed weight)
Positive Reduction in florivory and
seed predation nectar
robbing holes increased ant
visitation
Newman and
Thomson 2005
44
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Melastoma
malabathricum
Oecophylla
smaragdina
Seed set Positive Ants deterred less efficient
pollinators increasing
visitation by the more
efficient pollinator
Gonzaacutelvez et al
2012
Metrosideros
polymorpha
Several Fruit set Neutral Ants reduced nectar
collection rates by foraging
bees but had no effect on
pollen collection rates or
fruit set
Junker et al
2010a
Orexis alpine F neorufibarbis
gelida
Seed set Neutral Autogamy or not pollen
limited
Puterbaugh 1988
45
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Ouratea spectabilis Cephalotes
pusillus and others
Seeds per fruit Neutral positive C pusillus had no effect on
seed number but other ants
collectively increased seed
set
Byk and Del
Claro 2010
Pachycereus schottii Several Seed set Neutral positive In one year ants showed a
positive effect (seed
number 2008) but no effect
with increased sample sizes
Chamberlain and
Holland 2008
Polemonium
viscosum
F neorufibarbis
gelida
Seed set Negative Ants damaged styles Galen 1983
46
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Polemonium
viscosum
F neorufibarbis
gelida
Pollen removal Neutral Ants did not affect pollen
removal even at high ant
densities
Galen and Geib
2007 T
Protea nitida Linepithema
humile and others
Seed set Neutral positive L humile had no effect on
P nitida seed set but native
ants increased it in one year
ants may cause pollinators
to relocate
Lach 2007
Psychotria
limonensis
Ectatomma spp Fruit set Positive Suggested increased
pollinator relocation
Altshuler 1999
47
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Roussea simplex Technomyrmex
albipes
Seed set Negative Interference and exploitative
competition
Hansen and
Muumlller 2009
Tristerix aphyllus Several Fruit set Neutral Nectar robbing ants
minimally reduced nectar
levels and had no effect on
hummingbird visitation
Caballero et al
2013
Vaccinium
reticulatum
Several Fruit set Neutral Ants reduced florivory but
did not affect fruit set
Bleil et al 2011
T Study measured male fitness
Study of sterilizing ants showed short-term decrease in female fitness long-term effects are unclear (see text)
48
Table 12 Studies demonstrating either i) ant pollination through an exclusion experiment or ii) that ants transfer pollen (or pollen
analogue) between flowers strongly suggesting ant pollination
Plant species Ant species Ant pollination Evidence Reference
Alyssum purpureum Several Yes Exclusion experiment Gόmez et al 1996
Arenaria tetraquetra Several Yes Exclusion experiment Gόmez et al 1996
Balanophora kuroiwai Leptothorax sp Suggested Exclusion experiment and
observed pollen transfer
and pollen tube formation
but did not see if plants set
seed
Kawakita and Kato 2002
Blandfordia grandiflora Iridomyrmex sp Yes Exclusion experiment Ramsey 1995
Borderea pyrenaica Several Yes Exclusion experiment Garciacutea et al 1995
49
Plant species Ant species Ant pollination Evidence Reference
Cytinus hypocistis Several Yes Exclusion experiment de Vega et al 2009
Epipactis thunbergii Camponotus
japonicas
Suggested Observed transfer of
pollinia from ants to
orchids
Sugiura et al 2006
Euphorbia cyparissias Several Yes Exclusion experiment Schuumlrch et al 2000
Fragaria virginiana Several Very likely Ant-only treatments set
comparable seed to flying
pollinator-only treatment
seed set increased with
increasing ant visitation
Ashman and King 2005
Frasera speciosa Several Yes Exclusion experiment Norment 1988
Frankenia thymifolia Several Yes Exclusion experiment Gόmez et al 1996
50
Plant species Ant species Ant pollination Evidence Reference
Hormathophylla spinosa Proformica
longiseta
Yes Exclusion experiment Gόmez and Zamora 1992
Jatropha curcas
Several
Tapinoma
melanocephalum
most common
Yes Exclusion experiment Luo et al 2012
Leporella fimbriata Myrmecia urens Yes Observed pollinia transfer
and subsequently
confirmed as ant
pollination
Peakall et al 1987
51
Plant species Ant species Ant pollination Evidence Reference
Lobularia maritima Several
Camponotus
micans most
common
Yes Exclusion experiment Gόmez 2000
Microtis parviflora
Iridomyrmex
gracilis
Yes Observation used
laboratory colonies to
replicate observations
Peakall and Beattie 1989
Naufraga balearica
Several
especially
Plagiolepis
pygmaea and
Lasius grandis
Suggested Observation over three
years of study only ants
were observed visiting
ants carried pollen
Cursach and Rita 2011
52
Plant species Ant species Ant pollination Evidence Reference
Neottia listeroides Leptothoras sp
Paratrechina sp
Suggested Observed transfer of
pollinia from ants to
orchids
Wang et al 2008
Paronychia pulvinata Formica
neorufibarbis
gelida
Yes Exclusion experiment Puterbaugh 1998
Polygonum cascadense Formica argentea
Very likely Grew plants in greenhouse
open to flying visitors with
minimal seed set only
plants with access to ants
set significant seed
Hickman 1974
53
Plant species Ant species Ant pollination Evidence Reference
Retama sphaerocarpa Several Yes Exclusion experiment Gόmez et al 1996
Scleranthus perennis
Several
especially
Formica
rufibarbis
Suggested Ants transferred pollen
analogue to stigmas and
carried pollen on bodies
Svensson 1985
Sedum anglicum Proformica
longiseta
Yes Exclusion experiment Gόmez et al 1996
Trinia glauca Several Yes Exclusion experiment Carvalheiro et al 2008
54
Chapter 2 Ants and ant scent reduce bumblebee pollination of artificial flowers
Cembrowski AR Tan MG Thomson JD and Frederickson ME
In press The American Naturalist
All authors designed the experiment ARC and MGT performed the experiment ARC and MEF
performed statistical analyse
s and ARC wrote the manuscript with input from all authors
Abstract
Ants on flowers can disrupt pollination by consuming rewards or harassing pollinators but it is
difficult to disentangle the effects of these exploitative and interference forms of competition on
pollinator behavior Using highly rewarding and quickly replenishing artificial flowers that
simulate male or female function we allowed bumblebees (Bombus impatiens) to forage (1) on
flowers with or without ants (Myrmica rubra) and (2) on flowers with or without ant scent cues
Bumblebees transferred significantly more pollen analogue both to and from ant free flowers
demonstrating that interference competition with ants is sufficient to modify pollinator foraging
behavior Bees also removed significantly less pollen analogue from ant scented flowers than
from controls making this the first study to show that bees can use ant scent to avoid harassment
at flowers Ant effects on pollinator behavior possibly in addition to their effects on pollen
viability may contribute to the evolution of floral traits minimizing ant visitation
55
Introduction
Trait-mediated indirect interactions arise when a focal species causes phenotypic changes
including behavioral modifications in a second species and these effects cascade to still other
species (Werner and Peacor 2003) Although trait-mediated indirect interactions are often studied
in food webs (eg Werner and Peacor 2003 Preisser et al 2005) they are not limited to trophic
interactions For example the threat of predation can change the behavior of a mutualist thus
affecting its partners (Suttle 2003) In animal-pollinated plants predators can disrupt pollination
directly through density-mediated indirect interactions (Dukas 2005) or via trait-mediated
indirect interactions by changing pollinator behavior When predators are present pollinators
may switch to visiting less rewarding flowers or they may avoid flowers altogether potentially
reducing the amount of pollen donated or received by a flower and thus decreasing plant fitness
(Gonccedilalves-Souza 2008 Ings and Chittka 2009) Similarly competition for floral rewards with
other flower visitors may cause behavioral changes in pollinators (Maloof and Inouye 2000
Ohashi et al 2008) Previous research has shown that bees may spend less time visiting flowers
depleted of rewards (Thomson 1986) and may avoid flowers bearing scent marks that indicate
recent visits by other individuals of the same or different bee species as these flowers are likely
to be depleted (Stout and Goulson 2002)
Whereas competition between pollinators may not be particularly costly for the plant
because both competitors are likely to provide pollination services competition between
pollinators and organisms that do not usually provide a pollination service such as ants has a
greater potential to reduce plant fitness Ants are common floral visitors they are attracted to
flowers for nectar (Lach 2007) and in some cases pollen (Byk and Del Claro 2010) and will
often defend these resources against other flower visitors (Altshuler 1999) However ants rarely
56
contribute much useful pollination antibiotic secretions present on their cuticles kill pollen
grains (Beattie et al 1985 Dutton and Frederickson 2012) and when foraging in flowers ants
sometimes impede female function by damaging stigmas (Galen and Cuba 2001) We will
henceforth consider ants to be non-pollinators and will contrast them to animals like bees that
are pollinators Ants are capable of competing with pollinators in two ways (1) by consuming
floral rewards and reducing their availability to pollinators (ie exploitative competition) and (2)
by directly antagonizing and excluding pollinators from flowers (ie interference competition)
Many plants benefit from the presence of ants numerous plant species recruit ants using
extrafloral nectar food bodies or domatia because ants provide protection against folivores (Heil
and McKey 2003) Similarly some plants have extrafloral nectaries on or near reproductive
tissues that recruit ants that deter florivores (eg Inouye and Taylor 1979) However plants
may incur reproductive costs as a result of their association with ants Some ant species that nest
in myrmecophytes sterilize flowers to shunt plant resources from reproduction to vegetative
growth allowing for greater ant colony growth (Frederickson 2009) Also ants visiting
extrafloral nectaries may be attracted to floral nectaries where they can harass pollinators (Ness
2006) There is growing evidence that many angiosperms have evolved floral traits that prevent
ants from accessing flowers during anthesis (Willmer and Stone 1997 Ballantyne and Willmer
2012) The effects of flower-visiting ants can be substantial Ants can decrease the frequency
duration or species diversity of pollinator visits to flowers (Lach 2008 Hansen and Muumlller 2009
Junker et al 2010 Gonzaacutelvez et al 2012) all of which potentially impact pollen donation and
receipt In some studies changes to pollinator behavior induced by ants have also been linked to
seed set
It is difficult however to disentangle the intertwined effects of exploitative and
interference competition in nature If ants disrupt pollination plants may often evolve traits to
57
deter ants from flowers (Willmer et al 2009) From a practical standpoint this would then limit
our ability to study the effects of ants on pollinator visitation because ants would not visit
flowers Here we used artificial flowers to explore ant-bee interactions in the absence of floral
defenses against ants We used highly rewarding quickly replenishing artificial flowers to
examine how direct harassment (ie interference competition expressed through behavior)
changes bee foraging behavior and how this affects donation and receipt of a pollen analogue
(powdered food dye) while minimizing the effects of exploitative competition We predicted
that flowers visited by ants would both donate and receive less pollen analogue than flowers
without ants because bees would avoid flowers with ants or leave them sooner Because scent
plays a large role in bumblebee communication and flower choice (Stout and Goulson 2002) we
also tested whether the presence or absence of ant scent on artificial flowers would affect pollen
analogue donation because of changes in the foraging behavior of bumblebees
Methods
i) Subjects
Myrmica rubra is an invasive ant in eastern North America with a range and habitat preferences
overlapping that of Bombus impatiens a common bumblebee Because M rubra visits flowers
(A Cembrowski pers obs) the two species likely interact in nature We collected 12 M rubra
colonies in the fall of 2011 and 2012 from Toronto Ontario Canada and the surrounding area
and maintained them in environmental chambers on artificial diet (Dussutour and Simpson 2009)
and a 1410 LD schedule (light 600-2200) We used these colonies as sources of M rubra
workers for experiments
58
Workers from commercially supplied colonies of Bombus impatiens (Biobest Canada
Ltd) foraged on artificial flowers in flight cages (either 24 x 24 x 21 m or 79 x 34 x 20m) at
the University of Toronto We tested a total of five bee colonies four colonies were used in the
ant presence trials and two of these and one additional colony were used in the ant scent trials
Flight cages had overhead fluorescent lights attached to timers In contrast to most previous
studies in which bees have been trained and tested individually the entire worker force of a bee
colony was free to forage at will in our experiments Colonies were trained to forage on artificial
flowers for at least four days before being used in trials After being used for a trial the colony
was not used for at least two days to reduce dye carryover between trials Between trials
bumblebees were fed pollen and given sugar water
ii) Artificial flowers
The flowers (Fig 1 Thomson et al 2012 see also Makino 2008) consisted of glass jars filled
with 30 WV sucrose solution Sugar water travelled by capillary action up a sewing-thread
wick to a hole in a blue-painted lid accumulating in a knot that acts as a nectary Flowers
depleted by visitors were quickly replenished via capillary action and were non-rewarding only
very briefly after visits taking less than a minute to accumulate 05 microl note that bumblebee
visits to a single flower are often separated by several minutes or more (A Cembrowski
unpublished data)
Unlike most previous artificial flower designs these flowers allow for estimation of male
and female fitness because we can measure both the amount and type of dye received by female
flowers (see (d) Dye quantification below) In order to access nectar on a ldquomalerdquo flower a bee
must crawl through a circular ldquoantherrdquo of brush-like weather stripping material dusted in a
59
consistent manner with our pollen analogue powdered food dye (FDampC 5 or 6) which is
transferred to the bee in the process On a ldquofemalerdquo flower a bee crawls through a sticky plastic
reinforcement that functions as a ldquostigmardquo receiving dye from the beersquos body Male flowers
dispensed dye particles over multiple bee visits and could still dispense dye at the end of trials
They resemble many real flowers or flower heads in that pollen is dispensed gradually over time
but less dye is available for transfer with each subsequent bee visit (Harder and Thomson 1989)
iii) Flight cage trials
We conducted 6 ant scent trials between 22 March and 4 June 2012 and the remaining trials (16
ant presence trials and 9 ant scent trials) between 9 November 2012 and 6 January 2013 Ant
presence trials lasted for eight hours and ant scent trials lasted for four hours Artificial flowers
were prepared and placed individually in small plastic containers treated with Fluon (Insect-a-
Slip BioQuip Products Inc) to prevent ants from escaping In ant presence trials we used 32
flowers (16 male and 16 female flowers) arranged in an eight-by-four array with flowers spaced
45 cm apart between rows and 30 cm within rows Ant scent trials used 20 flowers (10 male and
10 female flowers) in a four-by-five array with flowers spaced 30 cm apart between and within
rows In all of the ant presence trials and 9 of the ant scent trials we counted the number of bees
actively foraging after one hour to get a measure of colony activity
a) Ant presence trials
We examined the effects of ants on the amount of dye donated by male flowers and received by
female flowers in sixteen trials testing four bee colonies four times each The containers of eight
randomly chosen male flowers and eight randomly chosen female flowers received 15 M rubra
60
workers each the other 16 flowers remained free of ants Isolated from their colonies M rubra
workers have nowhere to deposit sugar water they collect and therefore may become sated
nonetheless a force of 15 ant workers was enough to maintain visitation to the nectaries where
bees foraged while still rarely having more than one or two ants visit the nectary at any time As
ants attacked bees bees would occasionally carry or throw attacking ants off flowers but despite
this some ants maintained their presence at the nectary throughout the trials Because ants the
size of M rubra consume liquids slowly (~017 ndash 024 microlmin Davidson et al 2004) flowers
replenished nectar more quickly than ants consumed it We used two colors of dye (FDampC 5 and
FDampC 6) to differentiate male flowers with and without ants In two trials for each bee colony
anthers of male flowers with ants were coated with FDampC 5 dye while brushes of male flowers
lacking ants were coated with FDampC 6 dye In the other two trials we reversed dye colors to
control for effects of dye type and color Dye color was assigned in a random order Artificial
flowers were placed in randomly assigned positions in the array before we opened the colony
and allowed the bees to begin foraging
b) Scent trials
We explored the effect of ant scent on dye donation in fifteen trials testing three bee colonies in
six five and four trials each Bees were first exposed to ants by allowing the bees to forage for
eight hours on twenty flowers (ten male and ten female) of which five male flowers had 15 ants
and all others had none No dye was used during this ldquoexposurerdquo day We then collected all but
the five ant-free male flowers in the flight cage leaving these flowers to keep bees foraging
Next we individually stored five new male lids in Fluon-treated containers with ten M rubra
workers Five control male lids were put in identical containers lacking ants The following
61
morning we removed the remaining flowers and set out ten new male and female flowers in a
random spatial arrangement using the lids having or lacking ant scent Thus the ant-scented
flowers were not in the same positions as the ant-visited flowers on which the bees were trained
Only five male flowers had ant scent and the other 15 flowers (five male and ten female) did not
We used the same two dye colors as in i) to differentiate male flowers with and without ant
scent and randomized which color was used for ant-scented flowers between trials We collected
and replaced stigmas from female flowers after one hour and collected the stigmas again after
four hours In the first trial performed we also collected stigmas after two hours but due to the
small amount of dye transferred this was not repeated
iv) Dye quantification
After each trial we quantified the amount of dye transferred to female flowers using a
spectrophotometer We removed the stigmas from female flowers placed them in test tubes
added 51 mL of distilled water to each tube and vortexed each tube thoroughly to ensure the
dye was evenly diluted These were diluted further as needed if the absorbance exceeded the
sensitivity range of the spectrophotometer In the first six scent trials each stigma was analyzed
separately and the dye amounts were summed to obtain a total amount of each dye color
transferred to female flowers in each trial In all the ant presence trials and the other nine scent
trials we opted to treat the experiment as the unit of replication Therefore we combined stigmas
from each treatment type (ant-visited or ant-free) in a test tube and measured the total amount of
each dye color transferred in each treatment in each trial
Because we put different dye colors on male flowers with and without ants we could use
the amount of each dye color donated to all female flowers to measure the reproductive success
62
of ant-visited and ant-free male flowers The total amount of dye (of both colors) received by
female flowers with and without ants was our measure of female reproductive success We
calculated the amount of each dye color in the sample by measuring absorbance at 428 or 486
nm and converting absorbance to micrograms following computational methods for overlapping
spectra (Blanco et al 1989)
v) Statistical analyses
In two trials (one ant presence and one ant scent) most female flowers received no dye because
of low bee activity so we excluded these trials from analyses Because dye reception and
donation values were non-normally distributed we square-root transformed the data before
examining the effects of ant presence or ant scent on dye transfer in ANCOVAs For ant
presence trials we included ant presence on male and female flowers and their interaction as
main effects and the total amount of dye transferred in each trial as a covariate to account for
the large variation in overall dye transfer among trials For ant scent trials we included ant scent
as the main effect time (one or four hours) as a repeated measure the interaction between scent
and time and the total amount of dye transferred as a covariate Covariate by treatment (ant
presence or ant scent) interactions were never significant and so were excluded in final analyses
All analyses were conducted in JMP (v 1000)
Results
Bees usually started foraging within minutes of the beginning of the trial and continued until
flowers were collected from the flight cage or the lights were extinguished An average of 34 plusmn
012 and 40 plusmn 015 (mean plusmn SE) bees were foraging after an hour in the ant presence and ant
63
scent trials respectively Male flowers with ants donated significantly less dye than male flowers
lacking ants (Fig 22a F155 = 4019 p lt 00001) Similarly female flowers with ants received
significantly less dye than female flowers lacking ants (Fig 22a F155 = 461 p = 0036) There
was no significant interaction between ant presence on male flowers and ant presence on female
flowers (F155 = 118 p = 028) Flowers with ant scent donated significantly less dye than
flowers without ant scent (Fig 22b F125 = 11216 p lt 00001) There was no significant effect
of time (F125 = 121 p = 028) or interaction between time and scent (F125 = 111 p = 030) in
the model
Discussion
In this study ants altered bumblebee pollination behavior and exhibited trait-mediated indirect
interactions with (artificial) flowers Through interference competition M rubra workers
significantly affected the pattern of dye transfer by B impatiens causing a preferential flow of
dye from male flowers lacking ants to female flowers lacking ants If manifested in nature such
effects would reduce the reproductive success of plants visited by ants through both male and
female function
Ants could have changed the attractiveness of artificial flowers in at least two ways First
as ants interrupted or altogether prevented bumblebees from foraging at nectaries bumblebees
may have learned to avoid flowers with ants due to the relative inefficiency of foraging at these
flowers something bumblebees take into account (Heinrich 2004) Second bees attempt to
minimize their risk of being attacked during foraging bouts which ants did to visiting bees Ants
often attacked or harassed bees by biting grasping and appearing to sting visiting bees
preventing them from accessing the nectary or reducing their time on flowers (see online video
64
1) and bees sometimes avoided ant-tended flowers entirely This harassment was sometimes
physically traumatic (online video 1) and bees often appeared to have trouble flying after being
attacked by ants Previous research has demonstrated that bumblebees leave or avoid flowers
where they have been harassed (Jones and Dornhaus 2011) and avoid foraging where there is
visual or olfactory evidence of a predator or predation event (Abbott 2006 Goodale and Nieh
2012) Though it is unclear if bumblebees viewed ants as competitors or predators they
responded similarly to flowers having ants as they do to flowers housing predators (Gonccedilalves-
Souza et al 2008)
To avoid artificial flowers with ants bees likely used a combination of visual and
olfactory cues Previous research has shown that bees respond to conspicuous predators or
predator ldquodummiesrdquo on flowers (Suttle 2003 Gonccedilalves-Souza et al 2008) decreasing the
frequency and duration of their visits to these flowers Thus bees may have been able to avoid
flowers harboring ants by sight alone However even in the absence of ants bees still
preferentially visited flowers lacking ant scents (Fig 22b) suggesting that they had learned to
associate ant scent with harassment Bees are adept at associative learning (Wright and Schiestl
2009) and can learn to recognize unique scents left behind by both conspecific and heterospecific
flower visitors (Stout et al 1998) Bees can use these various scents often arising from tarsal
gland deposits (Stout et al 1998) to recognize recently visited flowers that are less likely to be
profitable (Stout and Goulson 2002) Recently Ballantyne and Willmer (2012) demonstrated that
bees learn to associate ant scents with unrewarding artificial flowers and decrease their visitation
to these flowers Our results complement their findings by showing that bees can associate ant
scent with harassment at otherwise rewarding flowers
In our study ant scent caused bees to adjust their foraging strategy and decreased the
amount of pollen analogue that was donated by ant-scented flowers Thus the effects of
65
interference competition with ants on flowers can extend beyond immediate interactions and may
have fitness consequences for plants even when ants are absent Like other olfactory cues these
effects are likely transitory (Stout et al 1998) Although more dye was still donated by male
flowers lacking ant scent than those with ant scent the ratio of dye donated was on average
closer to equality in hours 2-4 than in the first hour (Fig 22b) This effect may be partially
driven by dye depletion of male flowers without ant scent but the lack of a strong corresponding
decrease in flowers with ant scent suggests that visitation patterns became more similar We did
not test whether B impatiensrsquo avoidance of ant scent was an innate or a learned behavior but
previous work has shown that Bombus terrestris does not innately avoid flowers with ant scent
(Ballantyne and Willmer 2012)
Ants may be necessary for plant survival and growth but can be costly for plant
reproduction The evolution of ant attractants such as extrafloral nectaries in some plant lineages
suggests that the costs of ants can be outweighed by their protective abilities However in this
study flowers visited by ants received and donated significantly less dye although they did
retain some sexual function The net benefit of having ants depends on whether ants increase
plant fitness by reducing herbivory more than they decrease plant fitness by disrupting
pollination Alternatively plants may actually benefit from the costs of ants to plant
reproduction ants that castrate flowers may be better defenders increasing plant survival or
vegetative growth when plants are young allowing for increased reproduction later in life when
the plant is colonized by less aggressive non-castrating ants (Frederickson 2009 Palmer et al
2010) In some plant species pollinator harassment by ants may even be beneficial Altshuler
(1999) reported that Ectatomma ants greatly increased fruit set of Psychotria limonensis despite
reductions in pollinator visitation rates presumably due to increases in the rate of pollen
outcrossing Similarly Gonzaacutelvez et al (2012) found that the presence of Oecophylla
66
smaragdina ants on Melastoma malabathricum flowers reduced visitation by less effective
pollinators and increased visitation by more effective Xylocopa bees increasing plant fitness
Cases of ants being beneficial to flowers appear to be the minority and many plant
species have traits that limit ant access to flowers Several studies have detailed floral volatiles
that are thought to mimic ant alarm pheromones and thus repel ants (Junker and Bluumlthgen 2008
Willmer et al 2009) Other plants use structural modifications such as narrow corollas or
slippery stems to limit ant access to flowers (reviewed in Willmer et al 2009) Exploitative
competition between ants and pollinators (Lach 2005 Ballantyne and Willmer 2012) as well as
the lethal effects of ant antibiotic secretions on pollen grains (Beattie et al 1985 Dutton and
Frederickson 2012) may have resulted in selection on plants to reduce ant visits to flowers Our
results suggest that trait-mediated indirect interactions resulting from interference competition
between ants and bees may favor plants that defend their flowers and their pollinators against
ants
Acknowledgements
We thank H Rusnock and S Meadley-Dunphy for assistance J Dix for help with flight cage
construction and Biobest for bumblebee colonies We are grateful to the Thomson and
Frederickson labs for guidance particularly T Makino funded by JSPS for helping to refine
artificial flowers J Ogilvie for help with bees K Prior for M rubra colony collections and K
Turner for study design suggestions The comments of the editor and two anonymous reviewers
prompted improvements in the article especially with regards to study design MEF and JDT
acknowledge NSERC Discovery Grant funding The University of Torontorsquos Faculty of Arts and
Science supported MGRTrsquos participation
67
68
References
Abbott K R 2006 Bumblebees avoid flowers containing evidence of past predation events
Canadian Journal of Zoology 841240-1247
Altshuler D L 1999 Novel interactions of non-pollinating ants with pollinators and fruit
consumers in a tropical forest Oecologia 119600-606
Ballantyne G and Willmer P 2012 Floral visitors and ant scent marks noticed but not used
Ecological Entomology 37402-409
Beattie A J Turnbull C Knox R B and Williams E G 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany
71421-426
Blanco M Runiaga H Maspoch S and Tarin P 1989 A simple method for
spectrophotometric determination of two-components with overlapped spectra Journal of
Chemical Education 66178-180
Byk J amp Del Claro K 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333-38
Davidson D W S C Cook and R R Snelling 2004 Liquid-feeding performances of ants
(Formicidae) ecological and evolutionary implications Oecologia 139255ndash66
Dukas R 2005 Bumble bee predators reduce pollinator density and plant fitness Ecology
861401-1406
Dussutour A and Simpson S J 2009 Communal nutrition in ants Current Biology 19740-
744
69
Dutton E M and Frederickson M E 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561-569
Frederickson ME 2009 Conflict over reproduction in an ant-plant symbiosis why Allomerus
octoarticulatus ants sterilize Cordia nodosa trees American Naturalist 173675-681
Galen C and Cuba J 2001 Down the tube pollinators predators and the evolution of flower
shape in the alpine skypilot Polemonium viscosum Evolution 551963-1971
Gonccedilalves-Souza T Omena P M Souza J C and Romero G Q 2008 Trait-mediated
effects on flowers artificial spiders deceive pollinators and decrease plant fitness Ecology
892407-2413
Gonzaacutelvez F G Santamariacutea L Corlett R T and Rodriacuteguez-Gironeacutes M A 2012 Flowers
attract weaver ants that deter less effective pollinators Journal of Ecology 10178ndash85
Goodale E and Nieh JC 2012 Public use of olfactory information associated with predation
in two species of social bees Animal Behaviour 84919-924
Harder LD and Thomson JD 1989 Evolutionary options for maximizing pollen dispersal of
animal-pollinated plants American Naturalist 133323-344
Heil M and McKey D 2003 Protective ant-plant interactions as model systems in ecological
and evolutionary research Annual Review of Ecology Evolution and Systematics 34425-
453
Heinrich B 2004 Bumblebee economics 2nd
edn Harvard University Press Cambridge
Ings T C and Chittka L 2009 Predator crypsis enhances behaviourally mediated indirect
effects on plants by altering bumblebee foraging preferences Proceedings of the Royal
Society B 2762031-2036
70
Inouye D W and Taylor O R Jr 1979 A temperate region plant-ant-seed predator system
consequences of extra floral nectar secretion by Helianthella quinquenervis Ecology 601-
7
Junker R R and Bluumlthgen N 2008 Floral scents repel potentially nectar-thieving ants
Evolutionary Ecology Research 10295ndash308
Junker R R Bleil R Daehler C C and Bluumlthgen N 2010 Intra-floral resource partitioning
between endemic and invasive flower visitors consequences for pollinator effectiveness
Ecological Entomology 35760ndash767
Jones E I and Dornhaus A 2011 Predation risk makes bees reject rewarding flowers and
reduce foraging activity Behavioral Ecology and Sociobiology 651505-1511
Lach L 2005 Interference and exploitative competition of three nectar-thieving invasive ant
species Insect Sociaux 52257-262
Lach L 2007 A mutualism with a native membracid facilitates pollinator displacement by
Argentine ants Ecology 881994-2004
Lach L 2008 Argentine ants displace floral arthropods in a biodiversity hotspot Diversity and
Distributions 14281-290
Makino T T and Sakai S 2007Experience changes pollinator responses to floral display size
from size-based to reward-based foraging Functional Ecology 21854863
Maloof J E and Inouye D W 2000 Are nectar robbers cheaters or mutualists Ecology
812651ndash2661
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506-514
Ohashi K Leslie A and Thomson JD 2008 Trapline foraging by bumble bees V Effects of
experience and priority on competitive performance Behavioral Ecology 19936-948
71
Palmer T M Doak D F Stanton M L Bronstein J L Kiers E T Young T P Goheen
J R and Pringle R M 2010 Synergy of multiple partners including freeloaders
increases host fitness in a multispecies mutualism Proceedings of the National Academy
of Sciences 10717234-17239
Preisser E L Bolnick D I and Benard M E 2005 Scared to death The effects of
intimidation and consumption in predator-prey interactions Ecology 86501-509
Stout J C Goulson D and Allen J A 1998 Repellent scent-marking of flowers by a guild of
foraging bumblebees (Bombus spp) Behavioral Ecology and Sociobiology 43317-326
Stout J and Goulson D 2002 The influence of nectar secretion rates on the responses of
bumblebees (Bombus spp) to previously visited flowers Behavioral Ecology and
Sociobiology 52239-246
Suttle K B 2003 Pollinators as mediators of top-down effects on plants Ecology Letters
6688-694
Thomson JD 1986 Pollen transport and deposition by bumble bees in Erythronium influences
of floral nectar and bee grooming Journal of Ecology 74329-341
Thomson JD Ogilvie J E Makino T T Arisz A Raju S Rojas-Luengas V and Tan M
G R 2012 Estimating pollination success with novel artificial flowers effects of nectar
concentration Journal of Pollination Ecology 9108-114
Tsuji K Hasyim A and Nakamura K 2004 Asian weaver ants Oecophylla smaragdina and
their repelling of pollinators Ecological Research 19669-673
Ulrich S and Bertsch A 1990 Do foraging bumblebees scent-mark food sources and does it
matter Oecologia 82137-144
Werner E E and Peacor S D 2003 A review of trait-mediated indirect interactions in
ecological communities Ecology 841083-1100
72
Willmer P G Nuttman C V Raine N E Stone G N Pattrick J G Henson K Stillman
P McIlroy L Potts S G and Knudsen J T 2009 Floral volatiles controlling ant
behaviour Functional Ecology 23888-900
Wright G A and Schiestl F P 2009 The evolution of floral scent the influence of olfactory
learning by insect pollinators on the honest signaling of floral rewards Functional Ecology
23841-851
73
Figures
Figure 21 Photograph of artificial flower lids A = anther L = lid N = nectary R = region
where anther or stigma is placed S = stigma T = thread W = weight
74
Figure 22 Dye transferred (mean plusmn 1SE) per trial by B impatiens workers A) from artificial
male and to artificial female flowers in the presence (unfilled circles) or absence (filled circles)
of Myrmica rubra workers and B) from artificial male flowers with (unfilled circles) or without
(filled circles) M rubra scent to ant-free female flowers in the 1st or the 2
nd to 4
th hours of the
trials
75
Chapter 3 Not just for the bees pollen consumption is common among tropical
ants
Cembrowski AR Reurink G and Frederickson ME
Planned submission to Biotropica
ARC and MEF designed the study ARC and GR did laboratory work ARC performed statistical
analyses and wrote the manuscript with input from other authors
Abstract
Although palynivory or pollen consumption is widespread among other hymenopterans there
are few accounts of palynivory in ants To quantify how often ants consume pollen we subjected
adult workers and larvae from 75 species of neo- and paleo-tropical ants to acetolysis a process
that destroys most organic material but leaves behind pollen grains In over half of the species
we examined ants contained a few pollen grains (lt 5) and ants of several Camponotus species
contained abundant pollen grains (gt50) We tested for an association between trophic level and
palynivory using stable nitrogen isotope ratios (δ15
N) but we did not find a significant
correlation we found pollen grains in lsquoherbivorousrsquo omnivorous and carnivorous ants We
suggest that our results indicate sporadic opportunistic pollen consumption by ants in tropical
forests
Introduction
For those organisms that can digest it efficiently pollen can be an excellent food source
Depending on the plant species pollen can contain up to 60 protein as well as lipids
76
carbohydrates and numerous trace nutrients (Roulston and Cane 2000) Pollen feeding
(palynivory) occurs in many taxa including birds mammals and arthropods (Roulston and Cane
2000) but it is best known among the Hymenoptera particularly bees Although adult bees feed
mainly on plant exudates (ie nectar) nearly all bee species rely on pollen as the chief protein
source for developing larvae (Willmer 2011) Closely related to bees ants have similar
nutritional requirements adult workers need carbohydrates whereas larvae are fed a diet rich in
protein (Dussutour and Simpson 2009) Ants also exploit nectar heavily and regularly visit
flowers (eg Herrera et al 1984 Bluumlthgen et al 2004) but unlike bees are not believed to
widely consume pollen Instead ants are thought to receive most of the protein in their diet from
predation or scavenging (eg Floren et al 2002)
However ants may not be as reliant on animal protein sources as commonly assumed In
tropical forests arboreal ants can have similar δ15
N ratios a measure of trophic level as
herbivores suggesting limited carnivory and substantial feeding on plant-derived protein sources
(Davidson et al 2003) One potential protein source is pollen This idea is supported by scattered
accounts of pollen consumption by ants from other ecosystems including deserts (Ness 2006)
boreal forests (Czechowski et al 2009) and tropical savannas (Byk and Del Claro 2010) One
ant genus Cephalotes is widely recognized to consume pollen (Creighton 1967 Baroni Urbani
and de Andrade 1997 Byk and Del Claro 2010) However beyond this genus our knowledge of
pollen consumption by ants has largely been limited to direct observations (but see Czechowski
et al 2011) and it is unknown if palynivory is truly rare among ants or simply under-reported
We borrowed a technique commonly used in palynology acetolysis to assess the frequency of
pollen consumption by ants in 75 neo- and paleo-tropical ant species This technique allowed us
to examine many more ant species than we could have observed directly
77
Methods
Adult worker ants were collected by hand at baits or from leaf litter using Winkler extractors
while larvae along with adults were collected by hand from nests Collections were made by L
Arcila Hernandez and J Sanders in the primary tropical rainforest surrounding the Centro de
Investigaciόn y Capacitaciόn Rio Los Amigos (CICRA 12deg34rsquoS 70deg05rsquoW) in Peru in 2010-
2011 and by E Youngerman from lowland primary and secondary rainforests in Madang
Province Papua New Guinea (PNG) in 2011-2012 We sorted ant samples to morphospecies and
then identified them to genus and when possible to species using existing keys which were
unavailable for PNG ants Ant samples used for acetolysis were whole adult workers guts from
adult workers and whole larvae
We used acetolysis to look for pollen grains inside ants In acetolysis organic material is
dissolved in acetic anhydride and sulfuric acid leaving behind stained pollen exines When
possible we used adult and larval ants of the same species from different colonies or bait
locations in an attempt to sample more representatively We prepared samples by first washing
them in 70 ethanol to remove any pollen present on the samplersquos outer surface before placing
them individually in micro-centrifuge tubes with ~05mL of a 91 mixture of acetic anhydride
sulfuric acid We then heated each sample at 95degC for 20min allowing tissues to dissolve Next
we centrifuged samples at 1000 RPM for 5min and decanted the liquid supernatant before
adding ~05mL of glacial acetic acid to the precipitate centrifuging again as above We decanted
the samples and added a drop of distilled water
78
We examined a 150μL sub-sample of this solution for pollen grains under a light
microscope Larvae gave less than 150μL of solution following acetolysis so we sampled the
entire amount We identified pollen grains by their staining and their surface architecture For
most species we examined at least three adult workers and three larvae but for some species
fewer than three adult workers or larvae were available (Tables 31 and 32)
We investigated the relationship between trophic level as measured by δ15
N ratios and
pollen consumption by ants Stable isotope (SI) data was available for some (n =23) of the
Peruvian species from a separate project in which collections overlapped Samples were stored in
EtOH and dried at 50degC overnight We used heads legs and thoraxes (but not gasters) of ants
for SI analysis Often single individuals did not provide enough material for analysis and we
combined multiple individuals to achieve necessary weights up to 10 for small ant species
Samples were analyzed at the Boston University Stable Isotope Laboratory using a Finnigan
Delta-S analyzer For other Peruvian species for which we had samples but not corresponding
stable isotope data (n =25) we took the median δ15
N ratio value of that speciesrsquo genus and
assigned it to the species in question Using pollen presenceabsence data for these 48 species
we used logistic regression to correlate pollen presence and δ15
N ratio using R (v 2151)
Results
We found pollen grains in 37 of the 197 adult workers 20 of the 102 larvae and 38 of the 75 ant
species we examined (Tables 31 and 32) The proportion of individuals containing pollen did
not differ significantly between larvae and adults (χ2
1 = 002 p = 088) The number of pollen
grains found was often low (median = 3) but highly variable (range 1-468) Also pollen
79
presence was inconsistent among conspecific ant samples of species that contained pollen 31
had pollen in only one of the larvae or adults examined In only three species (two Cephalotes
and one Myrmelachista) did all examined adults contain pollen
Highly lsquoherbivorousrsquo carnivorous and omnivorous ants contained pollen grains (Fig
31) There was no correlation between δ15
N ratio and pollen presence (χ2 = 150 df = 1 p =
022) In fact several carnivorous species contained pollen grains (eg Odontomachus sp 1
Pachycondola sp1)
Discussion
Pollen consumption by ants appears to be more common than suspected The majority of species
(51) had at least one individual that had consumed pollen and in 20 multiple individuals
contained pollen Numbers of grains found tended to be low however with most individuals
containing only a few grains Ants may use pollen as only a small portion of their diet exploiting
a host of other protein-containing foods more heavily including other low- δ15
N foods (eg
honeydew epiphylls Houmllldobler and Wilson 1990 Davidson et al 2003)
There are a few reasons why pollen may not be heavily exploited by ants Pollen requires
much post-consumption processing to extract its nutrients These nutrients are located in the
pollenrsquos cytoplasm which is protected from direct digestion by the exines and intines of the
grains Organisms must either break through these layers or cause the pollen grains to germinate
or pseudo-germinate to access and digest the cytoplasm (Roulston and Cane 2000) This can be
achieved by consuming a sugar-containing solution (such as nectar) along with pollen However
animals that are not adapted to feed on pollen may not be very efficient at this process only
80
extracting nutrients from some of the grains (Herrera and Martiacutenez del Rio 1998) Thus while
ants can likely get some nutrients from pollen other forms of more easily processed protein may
be more attractive
Pollen may also pose handling difficulties for ants Despite the large number of studies
that have recorded ants visiting flowers (Chapter 1) only a few papers detail ants harvesting
pollen directly from anthers Byk and Del Claro (2010) reported that Cephalotes pusillus ants
removed almost all of the available pollen from Ouratea spectabilis flowers and at least two
other studies describe ants consuming pollen along with nectar from flowers (Horskins and
Turner 1999 Ness 2006) Unlike bees which possess both morphological (eg specialized hairs
for holding pollen) and behavioral adaptations (eg stereotyped pollen-packing motions) ants
have no apparent mechanisms to increase the amount of pollen harvested and are likely limited
by the number of grains they can ingest or carry Rather than collect pollen directly from flowers
it appears more likely that ants collect it haphazardly in the environment leading to a smaller
number of pollen grains consumed at any one time It is known that at least Cephalotes ants
engage in such behavior Creighton (1967) observed Cephalotes texanus collecting pollen grains
trapped by Citrus x paradisi leaf hairs
There is a chance that the small numbers of pollen grains we observed came from
secondary pollen consumption (ie consuming a palynivorous organism) or consumption of
liquid containing pollen While we cannot rule this out we believe our results represent actual
targeted pollen consumption by ants Cephalotes ants known palynivores (Creighton 1967
Baroni Urbani and de Andrade 1997) contained similar numbers of pollen grains as many of the
pollen-containing ant species that we examined As well in at least once instance ants that we
found containing pollen grains were in the process of preying on non-pollen consuming
81
organisms (eg a Pachycondola species that was carrying a termite in its mandibles) or were
attracted by sugar baits
Contrary to our expectations there was no relationship between an ant speciesrsquo δ15
N ratio
and whether it had consumed pollen (Fig 32) We expected palynivory to be more common in
herbivorous ants because pollen presents a widespread source of protein a potentially limiting
resource The lack of pattern might reflect how available pollen grains are in the environment
While other forms of food may be preferable ants tend to be opportunistic foragers (Carroll and
Janzen 1973) and there is likely little cost associated with the consumption of scattered pollen
grains during foraging trips beyond the volume they occupy in the gut Some genera were found
to consistently consume pollen In support of previous observations (Baroni Urbani and de
Andrade 1997 Byk and Del Claro 2010) Cephalotes species were among the most common
pollen consumers Camponotus species also consistently contained pollen grains of the eight
species examined five contained pollen and pollen was found in species from both Peru and
PNG Camponotus species also contained the two largest numbers of pollen grains found (468
and 271) Taken together these results suggest that they may be another as yet unrecognized
commonly palynivorous genus
In general larvae did not contain large numbers of pollen grains (ranging from 1-38
grains) This is counter to what is seen in bees where larvae are the main recipients of pollen
(Willmer 2011) This difference may be because ant larvae are continually fed via trophallaxis
by adults throughout their development whereas bee larval cells are provisioned with pollen and
then sealed while larvae complete their development If both adults and larvae are able to extract
nutrients from pollen we would expect to see a preferential flow of nutrients from pollen to
larvae but not necessarily the grains themselves
82
Unlike palynivory by organisms that also provide pollination services pollen
consumption by ants is unlikely to benefit plants Ants rarely act as pollinators (Beattie et al
2004) They have pollen-killing antibiotic secretions on their cuticles (Dutton and Frederickson
2012) and they can compete with other flower visitors reducing flower visitation rates (Ness
2006) The negative impacts of flower-visiting ants are thought to have led to the evolution of
ant-repellent floral volatiles (reviewed in Willmer 2009) These volatiles can pre-empt
competition between ants and pollinators and may often be located within the pollen itself
(Willmer 2009) If floral volatiles truly are a defense against ant visitation our results suggest an
additional evolutionary pressure to not only prevent ant-pollinator interactions but to also
protect plant gametes from occasional consumption by a non-pollinating visitor
Acknowledgements
We would like to thank Jon Sanders Lina Arcila Hernandez and Eric Youngerman for providing
samples the Thomson lab for use of supplies the Pierce lab for funding support during JSrsquo and
EYrsquos collections and the staff of CICRA and the New Guinea Binatang Research Center for
logistical support The Peruvian Ministry of Agriculture provided permits (Nos 394-2009-AG-
DGFFS-DGEFFS and 299-2011-AG-DGFFS-DGEFFS) MEF acknowledges financial support
from an NSERC Discovery Grant a Connaught New Researcher Award an Ontario Ministry of
Economic Development and Innovation Early Researcher Award and the University of Toronto
ARC was supported by an Ontario Graduate Scholarship and Sigma Xi
83
References
Baroni Urbani C and M L de Andrade 1997 Pollen eating storing and spitting by ants
Naturwissenschaften 84256ndash258
Beattie A J C Turnbull R B Knox and E G Williams 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany 71421ndash
426
Bluumlthgen N G Gottsberger and K Fiedler 2004 Sugar and amino acid composition of ant-
attended nectar and honeydew sources from an Australian rainforest Austral Ecology
29418ndash429
Byk J and K Del-Claro 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333ndash38
Carroll C R and D H Janzen 1973 Ecology of foraging ants Annual Review of Ecology and
Systematics 4231ndash257
Creighton W S 1967 Studies on free colonies of Cryptocerus texanus Satschi (Hymenoptera
Formicidae) Psyche 7434ndash42
Czechowski W B Markό and A Radchenko 2009 Rubbish dumps reveal the diet of ant
colonies Myrmica schencki Em and Myrmica rubra (L) (Hymenoptera Formicidae) as
facultative pollen-eaters Polish Journal of Ecology 56737ndash741
Czechowski WB Markoacute K Erős and E Csata 2011 Pollenivory in ants (Hymenoptera
Formicidae) seems to be much more common than it was thought Annales Zoologici
61519ndash525
84
Dussutour A and S J Simpson 2009 Communal nutrition in ants Current Biology 19740ndash4
Dutton E M and M E Frederickson 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561ndash569
Floren A A Biun and K E Linsenmair 2002 Arboreal ants as key predators in tropical
lowland rainforest trees Oecologia 131137ndash144
Grant B R 1996 Pollen digestion by Darwinrsquos finches and its importance for early breeding
Ecology 77489-499
Herrera L G and C Martiacutenez del Rio 1998 Pollen digestion by new world bats effects of
processing time and feeding habits Ecology 792828ndash2838
Houmllldobler B and Wilson E O 1990 The Ants 1st Edition Harvard University Press
Cambridge
Horskins K and Turner V B 1999 Resource use and foraging patterns of honeybees Apis
mellifera and native insects on flowers of Eucalyptus costata Australian Journal of
Ecology 24221-227
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506ndash514
Roulston T H I and J H Cane 2000 Pollen nutritional content and digestibility for animals
Plant Systematics 222187ndash209
Van Tets I G 1997 Extraction of nutrients from Protea pollen by African rodents Belgian
Journal of Zoology 12759-65
Willmer P G 2011 Pollination and Floral Ecology 1st Edition Princeton University Press
New Jersey
85
Willmer P G C V Nuttman N E Raine G N Stone J G Pattrick K Henson P Stillman
et al 2009 Floral volatiles controlling ant behaviour Functional Ecology 23888ndash900
86
Tables
Table 31 Numbers of adult worker and larval ants examined for each species collected in Peru
Numbers of pollen grains found in individual samples are given in parentheses after the sample
size when there were no grains no numbers are given
Genus Species Number of adults analyzed
(numbers of grains found)
Number of larvae analyzed
(numbers of grains found)
Dolichoderinae
Azteca sp 1 3 0
sp 2 2 0
sp 3 0 2
sp 4 3 (1) 0
sp 5 3 (2) 0
Dolichoderus decollatus 3 (66) 0
sp 1 3 0
sp 2 3 1 (2)
sp 3 3 3
sp 4 0 2
Ecitoninae
Labidus sp 1 3 0
Formicinae
Camponotus sp 1 3 3
sp 2 3 (82) 1 (2)
sp 3 3 (3) 0
sp 4 0 2
87
sp 5 8 (11) 0
sp 6 4 (7 16 468) 0
sp 7 5 (12) 0
sp 8 2 (1) 0
Paratrechina sp 1 4 (11) 0
Myrmelachista sp 1 3 (5 11 18) 0
sp 2 0 3
Trachymyrmex sp 1 3 (21) 0
sp 2 3 0
Myrmicinae
Atta sp 1 3 0
Cephalotes atratus 3 (2 2 21) 0
placidus 3 (1 4 21) 0
sp 1 3 0
sp 2 1 0
sp 3 2 0
Crematogaster sp 1 3 1
sp 2 3 (35) 0
sp 3 9 (7) 0
sp 4 2 0
Megalomyrmex sp 1 3 0
sp 2 0 2
sp 3 4 (7) 0
sp 4 3 0
Monomorium sp 1 0 3 (2)
88
Pheidole sp 1 0 3
sp 2 3 0
sp 3 0 3
Procryptocerus sp 1 3 0
Solenopsis sp 1 0 3
sp 2 3 (3 5) 0
Wasmannia sp 1 3 0
Ponerinae
Pachycondola sp 1 3 (3) 0
Odontomachus sp 1 3 (34) 0
Pseudomyrmicinae
Pseudomyrmex sp 1 3 0
sp 2 3 0
sp 3 3 (9) 0
sp 4 0 3
sp 5 0 3
89
Table 32 Numbers of adult worker and larval ants examined for each species collected in Papua
New Guinea Numbers of pollen grains found in individual samples are given in parentheses
after the sample size when there were no grains no numbers are given
Genus Species Number adults analyzed
(grains found)
Number larvae analyzed
(grains found)
Aenictinae
Aenictus sp 1 3 3 (1 1)
Dolichoderinae
Philidris sp 1 3 1 (4)
Tapinoma sp 1 2 3 (38)
Technomyrmex sp 1 0 3 (12 12)
Ectatomminae
Rhytidoponera sp 1 1 3 (2)
Formicinae
Acropyga sp 1 3 3 (2)
sp 2 3 3
sp 3 3 3
Anoplolepsis sp 1 3 (1) 3 (1)
Calomyrmex sp 1 3 3 (1)
Camponotus sp 1 3 (1 271) 3
sp 2 1 3
sp 3 2 0
Nylanderia sp 1 3 3
Opisthopsis sp 1 3 3
Polyrachis sp 1 1 3 (4)
90
Myrmicinae
Crematogaster sp 1 3 3
sp 2 3 (32) 3
Monomorium sp 1 3 (1) 3 (1)
sp 2 3 3 (1 1 1)
Pheidole sp 1 3 3
Pristomyrmex sp 1 3 3 (11)
Pyramica sp 1 3 0
Ponerinae
Odontomachus sp 1 3 3 (4)
91
Figures
Figure 31 Photographs of pollen grains found in two ant species a) Dolichoderus sp 2 and b)
Camponotus sp 6 Scale bars = 50 microm
92
Figure 32 Relationship between trophic level as measured by δ15
N ratio and pollen presence
for 48 neotropical ant species 1 Atta sp 5 Azteca spp 8 Camponotus spp 5 Cephalotes spp 4
Crematogaster spp 5 Dolichoderus spp 1 Labidus sp 3 Megalomyrmex spp 1 Monomorium
sp 2 Myrmelachista spp 1 Odontomachus sp 1 Pachycondola sp 1 Paratrechina sp 3
Pheidole sp 4 Pseudomyrmex spp 2 Solenopsis spp and 1 Wasmannia sp
93
Concluding Remarks
Ants have traditionally been considered to be unwelcome flower visitors they consume floral
rewards ostensibly meant for pollinators while rarely providing pollination services themselves
In this thesis I investigate the validity of this assumption and use an experimental study and an
observational study to examine two ways that ants may impact plant reproduction
In the first chapter I review the literature on ants as flower visitors I show that the common
assumption of ants always being categorically negative for plant reproduction is incorrect while
ants may reduce floral attractiveness to pollinators the net effect of ants on plant fitness
measures is variable ranging from positive to negative to neutral I also discuss ants as
pollinators and ways that plants may minimize ant visitation to flowers before finishing with a
section highlighting questions that still need investigation
In the second chapter I explore how interference competition with ants can structure bumblebee
foraging behavior and how this may alter pollen movement patterns Using artificial flowers with
male or female function I allowed Bombus impatiens bumblebees to forage on flowers that
either did or did not have Myrmica rubra ants on them Ant presence significantly reduced the
amount of pollen analogue donated and received by flowers Building on these results I found
that the presence of ant scent alone elicited similar changes to bumblebee behavior reducing the
amount of pollen analogue donated by male flowers My results show that interference
competition is sufficient to alter bumblebee foraging behavior and that the effects of ants on
pollinators may extend after ant visitation to flowers Further study is needed to determine if bees
are able to differentiate between ant species by visual or olfactory cues Their ability to do so
94
could affect how generalized their response to ant presence is and may help to better predict how
ants will affect plant reproduction
In the final chapter I investigate how common palynivory is amongst tropical ant species I used
acetolysis and light microscopy to examine whole ants larvae and ant guts from Papua New
Guinea and Peru I found that tropical ants consistently contain pollen grains albeit often in
small numbers (lt5) However some genera notably Camponotus and Cephalotes consistently
contained higher numbers providing the first evidence that Camponotus may be a palynivorous
genus The small numbers of pollen grains present may indicate that ants preferentially exploit
other more easily digestible high-protein foods over pollen Future studies should investigate
exactly what role pollen plays in ant diets by supplementing the diet of captive ant colonies with
pollen This would allow us to examine how pollen consumption affects colony demography as
well as how efficiently ants digest pollen and whether ants exhibit pollen preferences
I bring up several pressing and unanswered questions within this thesis and highlight three here
First numerous plant species possess chemical or physical traits assumed to increase plant
fitness by reducing ant visitation However this has rarely been tested and instead research has
focused predominately on documenting ant repellence We should move away from this and
begin examining plant fitness in the presence or absence of these repellent traits to see how
beneficial these traits truly are Second how flower visiting ants impact male fitness has been
almost completely ignored in favor of female fitness Future studies should investigate how ant
visitation affects both pollen donation and pollen movement patterns Lastly research involving
antibiotics present on ant cuticles and their effect on pollen viability has been too small in scope
often limited to demonstrating the pollen killing effects of ant secretions Investigation into
95
whether plants have evolved any resistance to the secretions of the local ant community or why
some plants appear unaffected by their ant partners may yield exciting results
In conclusion ants have the capacity to strongly affect plant reproduction both positively and
negatively The fact that these diametrically opposing forces exist means we cannot approach
flower-visiting ants as either positive or negative a priori Instead we should strive to work
towards a better understanding of what is occurring in the system of interest and what role ants
play within it
96
Copyright Acknowledgments
Chapter 2 ldquoAnts and ant scent reduce bumblebee pollination of artificial flowersrdquo is current in
press in The American Naturalist Request for its inclusion here was sought and approved
7
digest pollen and whether ants like bees feed it to larvae as a protein source is unknown At
least two studies have found that ant larvae consume pollen providing some support for this idea
(Wheeler and Bailey 1920 and Chapter 3)
b) Interference competition
Ants are relatively unique among flower visitors in defending floral resources from competitors
(but see Willmer and Corbett 1981 Roubik 1982) This defense ranges from chance encounters
with ants attacking visitors when flower occupancy overlaps (Ashman and King 2005 Junker et
al 2007) to actively patrolling inflorescences or constructing protective galleries around
nectaries (Horvitz and Schemske 1984 Gaume et al 2005 Hansen and Muumlller 2009)
Interference competition even in the absence of exploitative competition is sufficient to alter
pollinator behavior (Chapter 2)
The strength and outcome of interference competition depends on the identities of the ant
and the pollinator Pollinators might not alter their visitation patterns in response to non-
aggressive ants (Junker et al 2007) but more antagonistic ants may elicit avoidance behavior
before or soon after the visitor lands (Willmer and Corbet 1982 Altshuler 1999 Schuumlrch et al
2000 Table 11) Pollinator size may help predict their reaction to ants because ants may deter
only small-bodied pollinators (Gonzaacutelvez et al 2012) It is not clear whether flower visitors view
ants as aggressive competitors or as potential predators rarely ants prey on pollinators (Schatz
and Wcislo 1999) but in most cases ants merely cause pollinators to relocate
How well pollinators recognize and avoid ant-visited flowers is unresolved Collectively
pollinators have lower visitation rates to flowers visited by ants (Romero et al 2011) This is
8
dependent on pollinator identity however dipterans do not appear to discriminate between ant-
visited and ant-free flowers while bees can identify and avoid ant-visited flowers Bees likely
use a combination of visual and olfactory cues to recognize ant-visited flowers They can
identify previous conspecific and some heterospecific insect visitors by the hydrocarbon
ldquofootprintsrdquo they leave during foraging (Stout and Goulson 2001) Bumblebees can detect ant
hydrocarbons although this has been shown only in the laboratory (Ballantyne and Willmer
2012 Chapter 2) No study has yet examined if bees can use sight alone to detect ants but
several suggest it (Junker et al 2007 Gonzaacutelvez et al 2012) Some bees may discriminate
between ant species when visiting flowers (eg Lach 2008b) which follows if not all ant-
pollinator interactions are equally costly The ability of bees to do so will affect how generalized
their avoidance behaviors will be If they can discriminate between species bees may avoid
specific flowers visited by more ldquocostlyrdquo ants However if they cannot bees might either avoid
all ant-visited flowers or none depending on how negative the average interaction with an ant is
iii) Interactions with flower and pollinator antagonists
Ants can defend the flowers they visit Numerous plant species have circumfloral or bracteal
nectaries (ie extrafloral nectaries around flowers) and ants attracted to these nectaries can
reduce bud ovule or flower predation increasing plant fitness (Inouye and Taylor 1979 Rico-
Gray and Thien 1989 Sugiura et al 2006) Although the presence of EFNs (extrafloral nectaries)
so close to floral nectaries (sometimes less than 1 cm Ness 2006) might increase discovery and
exploitation of floral nectar by ants only one study has investigated this hypothesis finding no
relationship between the proximity of EFNs to floral nectaries and antsrsquo use of floral nectar
(Galen 2005) Holes made by nectar robbers are functionally analogous to circumfloral nectaries
9
and can also attract ants that defend flowers (Newman and Thomson 2005) and nectar-thieving
ants can similarly prevent florivory in some systems (Lach 2007 Bleil et al 2011)
Ants can also reduce the abundance of flower-dwelling predators or parasites of
pollinators Ambush predators such as crab spiders often forage at flowers where they may
reduce pollinator visitation rates and plant fitness (Dukas 2001 Gonccedilalvez-Souza et al 2008)
Ants can decrease spider abundance and richness on plants (Rudgers 2004 Renault et al 2005
Nahas et al 2012) and some evidence suggests that they can do the same on flowers (Lach
2008) In select cases ants may also reduce the abundance of pollinator parasitoids (Holland et
al 2011) Whether ants increase plant reproductive success by removing pollinator antagonists
has not yet been examined but should be If ants create lsquoenemy-freersquo flowers pollinators might
prefer to land on flowers with ants
iv) Net effects
Table 11 compiles studies in which the authors recorded ants consuming floral rewards or
interacting with pollinators and measured some aspect of plant fitness (eg fruit or seed set
pollen deposition) We included only a single study per plant species unless subsequent studies
found conflicting results or measured the fitness of another plant sex Records of ant pollination
are included in a separate table (Table 12)
Table 11 reveals relatively few examples of flower-visiting ants negatively affecting
plant fitness given how costly ant visitation is assumed to be The net effect of ants on plant
reproductive success is actually quite variable with similar numbers of studies finding positive
negative or neutral effects of ant presence Surprisingly when ants do have negative effects it is
10
usually because they directly damage floral structures and not because they prevent pollinators
from visiting flowers and successfully transferring pollen Why are ants not more often negative
for plant reproduction when this is commonly assumed to be true
First ants may compensate for reducing pollinator visitation rates by improving
pollination quality As pollinators move between flowers on a plant they often deposit self-
pollen on stigmas (ie geitonogamy) In some plants with late-acting self-incompatibility ovules
fertilized by self-pollen are subsequently aborted reducing the number of seeds that can be
produced (Barrett 2002 and references therein) Geitonogamy can also reduce offspring viability
or vigor (de Jong et al 1993) and cause pollen discounting a reduction in the amount of pollen
available for outcrossing because of self-pollination (Barrett 2002) By competing with
pollinators ants might decrease rates of geitonogamous pollination if pollinators then visit fewer
sequential flowers within a plant Ants might also cause pollinators to fly further between plants
potentially increasing the genetic diversity of pollen arriving at stigmas Although such effects
have been reported for other floral larcenists (Maloof 2001 Irwin 2003) to our knowledge these
hypotheses have not been investigated in relation to ants
Second ants and pollinators do not always forage for the same resources possibly
allowing them to partition floral rewards If ants forage for nectar but not pollen and flowers can
be pollinated efficiently by visitors looking to gather mostly pollen and little or no nectar then
plant reproduction will not be affected by ants consuming floral nectar Junker et al (2010a)
found that honeybees foraged for both pollen and nectar in the absence of ants but reduced their
foraging on nectar but not pollen when ants could access flowers resulting in no difference in
fruit set between treatments Whether pollinators commonly adjust their foraging behaviors in
response to ants warrants further research
11
Third ants are somewhat unique among floral larcenists in that they often provide direct
benefits to the plants they visit In the studies listed in Table 11 ants sometimes decreased
herbivory on plants (Chamberlain and Holland 2008 Bleil et al 2011) or otherwise aided the
plant (eg ant presence repelling smaller less effective pollinators Gonzaacutelvez et al 2012) If the
positive effects of ants cancel or outweigh the negative effects of flower-visiting ants on
pollinators then ant visitation may be neutral or even positive for plant fitness
Fourth ants may impose fitness costs to plants not captured in the studies in Table 11
Nectar robbers have stronger negative effects when plants are pollen-limited (Burkle et al 2007)
but only one study in Table 11 assessed whether flowers would set more seed if they received
more pollen finding no evidence of pollen limitation (Lach 2008a Table 11) Even if ants
decrease pollinator visitation rates there is limited scope for ants to decrease plant reproductive
success when plants are not pollen-limited Furthermore nearly all the studies in Table 11
measured only female fitness Only three quantified aspects of male fitness (Wyatt 1980 Fritz
and Morse 1981 Galen and Geib 2007) and we know of only one other study in a system in
which ants occasionally pollinate that has also measured male fitness (Ashman 2000) All four
studies measured pollen or pollinia removal rates and none found any effect of flower-visiting
ants on male fitness However whether other measures of male fitness (eg seeds sired pollen
deposition patterns) would reveal similar or different effects needs investigation
Finally although floral defenses against ants (see 3 below) may prevent us from
observing the negative effects of ants on plant reproductive success we consider this unlikely
Several studies have examined interactions between ants and plants that share little or no
coevolutionary history usually in the context of ant invasions Although invasive ants often
recruit to flowers in larger numbers than native ants they do not appear to reduce plant
12
reproductive success (Blancafort and Goacutemez 2005 Lach 2007 Lach 2008a but see Hansen and
Muumlller 2009) And in Hawaii where angiosperms evolved for millions of years in the absence of
ants and are thought to have secondarily lost floral defenses against ants (Junker et al 2011a)
there is no evidence that the preponderance of recently introduced flower-visiting ants negatively
affects plant fitness (Junker et al 2010a Bleil et al 2011)
2 Ants as pollinators
Ants may directly benefit plant reproduction by acting as pollinators but this is a relatively rare
occurrence Ants have long been considered poor pollinators (Kerner 1878) Initially this was
based on morphological and behavioral differences between ants and recognized pollinators
Worker ants are wingless and often relatively hairless making them poor pollen dispersers As
well because many ants exhibit Ortstreue (literally ldquoplace fidelityrdquo) ants were thought to return
to the same branch or flower repeatedly effecting no cross pollination (Houmllldobler and Wilson
1990) Though largely true these traits do not necessarily preclude ants from acting as
pollinators (Table 12) Although they commonly have restricted foraging ranges (Houmllldobler and
Wilson 1990) some ants can effect outcrossing (eg Ashman and King 2005 Carvalheiro 2008)
and many ants are as hairy if not hairier than their pollinating counterparts
In the late 1970s the ldquoantibiotic hypothesisrdquo was put forward to explain the rarity of ant
pollination Ants use antibiotic secretions to protect themselves against the fungal and bacterial
pathogens common in their nests and environs Iwanami and Iwadare (1978) found that one of
these compounds myrmicacin also greatly reduces pollen germination rates Subsequently
Beattie et al (1984 1985) found that short periods of contact (le 30 minutes) with an antrsquos
integument caused pollen grains to fail to germinate Some of these pollen-killing compounds
13
were traced back to the ant metapleural gland which produces hygienic secretions (Houmllldobler
and Wilson 1990) although contact with ants lacking metapleural glands can also reduce pollen
viability (Beattie et al 1985 Dutton and Frederickson 2012) Ant mandibular or anal gland
secretions and potentially ant venom may likewise defend ants against pathogens and have
similar effects on pollen germination (Beattie et al 1985 Graystock and Hughes 2011)
Nonetheless ant pollination has been documented repeatedly There are at least 18 cases
in which ant pollination has been shown experimentally and five others in which observations
strongly suggest it (Table 12) The majority of these plants fit the ldquoant pollination syndromerdquo
originally proposed by Hickman (1974) dense stands of short plants with small flowers and
easily accessible nectaries Typically ant-pollinated plants are associated with hot and dry
climates (principally in the Mediterranean) but dry alpine environments may also favor the
evolution of ant pollination (eg Svensson 1985 Puterbaugh 1998 Schiestl and Glaser 2011) In
most cases ants appear to be the sole or major pollinator of the plant in question (Hickman 1974
Peakall et al 1990 de Vega et al 2009) although ants sometimes make only supplementary
contributions to pollination (Schuumlrch et al 2000 Ashman and King 2005) This suggests that co-
evolution between ants and plants may not be necessary for ants to be useful pollinatorsmdashwhich
is surprising given the potential negative effects of ants on plant reproduction
Ant-pollinated plants cope with ant secretions in a variety of ways Several plant species
may simply tolerate pollen damage (Gόmez and Zamora 1992 de Vega et al 2009) apparently
possessing no overt adaptations to prevent it These plants might receive enough pollen to
overcome reduced pollen viability Other plants may place pollen on ant body parts thought to
have fewer secretions thereby potentially escaping damage (eg on ant legs Ramsey 1995) or
attach pollen to ants with specialized structures avoiding direct contact with the ant integument
14
(Peakall et al 1990) It is not known whether pollen evolves resistance to ant secretions or
whether plants deposit pollen on ants which have less disruptive secretions although these
possibilities have been suggested elsewhere (eg Garciacutea et al 1995 Ramsey 1995)
One key question regarding the effects of ant secretions on pollen grains urgently needs
to be resolved Several studies have suggested that the pollen-killing secretions of ants may have
contributed to the evolution of floral defenses against ants (eg Wagner 2000 Galen and Geib
2007) For this to be true ants must decrease male fitness by damaging pollen grains before they
leave flowers and are deposited on receptive stigmas Only two studies have investigated the
effects of ant secretions on pollen still in anthers (Wagner 2000 Galen and Butchard 2003) and
both constrained ants unnaturally to flowers for significantly longer than ants would normally
stay on flowers Although these studies found strong reductions in pollen vigor it is unclear if
ant secretions decrease male fitness under natural ant visitation conditions
3 Floral defenses against ants
Plants are not passive recipients of flower visitors and have some ability to ldquochooserdquo which
organisms can access floral rewards Flowers resist ants using both structural and chemical traits
More thorough investigation is needed to determine whether these traits evolved to prevent or
minimize the negative effects of ants on plant reproductive success (ie whether these traits can
truly be considered defenses against ants see 4ia below) It is unlikely however that ants
experience reciprocal selection to circumvent floral resistance traits because the fitness benefits
to ants of accessing flowers are probably small Thus there may be little scope for
coevolutionary arms races between floral defenses against ants and ant counter-adaptations
15
i) Structural resistance traits
a) Restricting ant access
Numerous plant species have physical structures preventing ant access to flowers Thin pedicels
can limit antsrsquo gripping abilities and make ants easier to dislodge when flowers are moved by
wind (Gaume et al 2005) and waxy secretions can render stems too slippery for ants to climb
(Harley 1991) Structures surrounding flowers such as water moats or dense hairs may similarly
keep ants from accessing floral rewards (Willmer et al 2009) Many such traits function as broad
filters and prevent not only ants but most crawling insects from reaching flowers Thus it is
difficult to determine whether ants florivores or both acted as selective agents favoring the
evolution of physical barriers to flowers
Flower shape may also reduce ant-pollinator conflict Long narrow corollas or nectar
spurs can prevent all but the smallest ants from accessing floral nectaries (Bluumlthgen et al 2004
Agarwal and Rastogi 2007) while still allowing long-tongued pollinators access to rewards
Selection to exclude nectar-thieving ants in such a manner however may be counteracted by
pollinator preferences for wider corollas that allow easier flower handling (Galen and Cuba
2001) Additionally these types of flowers may be more prone to robbery (Irwin and Maloof
2002) limiting their usefulness in preventing nectar larceny
b) Separation in space or time
Plants may separate floral rewards and ants temporally or spatially to minimize ant impacts on
plant reproduction Several authors have proposed that myrmecophytes have adapted to the
16
sterilization or otherwise detrimental activities of ants by locating domatia or food rewards on
different branches than flowers (Izzo and Vasconcelos 2002 Raine et al 2002 Edwards and Yu
2008) Temporal separation of ant and pollinator activities could likewise reduce conflict though
evidence is limited Some acacia flowers dehisce during periods of low ant activity (Willmer and
Stone 1997 Nicklen and Wagner 2006) but it is unclear if this is in response to ants
temperature or pollinator activity Timing of nectar secretion may similarly reduce ant-pollinator
competition If ants are active prior to pollinators they may deplete floral nectar and reduce
pollinator visitation rates (Schaffer et al 1984) However if nectar is secreted when ants are
inactive pollination may occur unimpeded (Norment 1988) This temporal separation could
favor pollinators that are active when ants are not (eg Wyatt and Morse 1981) but this is
poorly documented Further studies particularly focusing on flowers with the potential for
nocturnal and diurnal pollination may provide more examples
c) Extrafloral nectaries (EFNs)
One hypothesis put forward to explain the evolution of EFNs proposes that EFNs ldquodistractrdquo ants
from consuming floral nectar (Kerner 1878 Wagner and Kay 2002) Wagner and Kay (2002)
tested this hypothesis by constructing model plants using artificial nectaries assigned at random
to represent either floral or extrafloral nectaries They found that the presence of EFNs could
reduce ant exploitation of floral nectar However as highlighted by Galen (2005) this design is
closer to a plant making a surplus of flowers some of which will avoid ant visitation if visitation
rates increase sublinearly with increasing flower number Producing extra flowers to escape ant
visitation appears to be a viable strategy in some systems (Ashman and King 2005 Galen 2005)
However Galen (2005) found that applying honey (simulating extrafloral nectar) to P viscosum
17
leaves had the opposite effect and increased ant visits to flowers Only two studies both in
Pachycereus schottii have found evidence for the distraction hypothesis under field conditions
(Chamberlain and Holland 2008 Holland et al 2011)
There is limited empirical support for the distraction hypothesis but abundant evidence
that EFNs attract ants that defend plants against herbivores (reviewed in Rosumek et al 2009)
To function as attractants the presence of EFNs must increase ant abundance on plants
However because of the increased numbers attracted EFNs would not be expected to reduce the
abundance of ants on nectar rewards or be able to satiate the ants recruited Only if ant
abundance stops increasing at a reward density much lower than that available on the plant
would EFNs serve to distract ants In this case the amount of rewards may overwhelm ants
leaving some flowers underexploited However even in P schottii ants still visited flowers
(Chamberlain and Holland 2008) In cases where ant abundance would continue to increase with
additional rewards it is unlikely that EFNs function as distractions
ii) Chemical resistance traits
a) Toxic nectar
One of the earliest proposed floral defenses against ants was toxic nectar (Janzen 1977)
However studies testing Janzenrsquos (1977) hypothesis uncovered only a very few isolated
examples (eg Catalpa speciosa Stephenson 1981 Crinum erubescens Guerrant and Fiedler
1981) in short most floral nectar is not toxic or repellent to ants (Baker and Baker 1978
Schubart and Anderson 1978) In fact ants sometimes prefer floral nectar over solutions with
equivalent sugar concentrations (Koptur and Truong 1998)
18
In contrast floral nectar can repel other flower visitors (eg birds Johnson et al 2006
lepidopterans Kessler and Baldwin 2007) Repellency is often caused by plant secondary
metabolites which may occur in nectar as a by-product of their production elsewhere in the
plant where they function as defenses against herbivory (Adler 2001) Alternatively toxic or
repellent nectar may be adaptive repellent nectar may promote effective pollination by reducing
the amount of reward any one pollinator takes spreading nectar over several pollinator visits
(Kessler and Baldwin 2007) or it may function as a filter selecting for specific pollinators
(Johnson et al 2006) The latter may explain why repellent nectar is not more common
legitimate pollinators in addition to floral larcenists may be deterred from visiting flowers with
repellent nectar This may be particularly true of nectars that would repel ants which are closely
related to bees nectar distasteful to ants may often also be distasteful to bees and those nectars
acting as a filter for bees may still be palatable to ants For example Prŷs-Jones and Willmer
(1992) found that the ammonia-containing nectar of Lathraea clandestina is readily consumed
by both bees and ants even though it deters birds that act as nectar thieves Although floral
nectar itself rarely repels ants Guerrant and Fielder (1981) found that the presence of macerated
petals within nectar can often keep ants away Alkaloids in petals may be responsible and might
explain why ants seldom act as primary nectar robbers these secondary compounds may prevent
ants from chewing through corollas
b) Floral volatiles
Repellent floral volatiles appear to be a more widespread form of chemical resistance against
ants These were reviewed recently by Willmer et al (2009) and will be discussed here only
briefly Numerous plant taxa produce floral scents repellent to ants including myrmecophilous
19
myrmecophytic and non-ant associated plants in both temperate and tropical ecosystems
(Willmer et al 2009 Junker et al 2011a Ballantyne and Willmer 2012) In three large studies
floral volatiles that repel ants were detected in about a third of all plants tested (Willmer et al
2009 Junker et al 2011a Ballantyne and Willmer 2012) Pollen is often the source of ant-
repellent volatile compounds (Willmer et al 2009 Ballantyne and Willmer 2012) but they can
be emitted from other floral parts including petals That pollen is often the source of repellence
could suggest that preventing ant visitation may be more important in protecting male than
female function Additionally because repellence would decrease as pollen is removed from
anthers ants may return and defend flowers more quickly than if repellent chemicals were in
longer-lasting structures (eg petals) In some cases floral volatiles are thought to mimic ant
alarm pheromones (Willmer et al 2009) However floral scents elicit inconsistent reactions
across ant species ranging from repellence to attraction (Agarwal and Rastogi 2007 Ballantyne
and Willmer 2012)
Producing structural or chemical resistance traits may be costly for plants Native
Hawaiian plants which have little recent coevolutionary history with ants appear to have
secondarily lost floral ant repellents (Junker et al 2011a) Both Willmer et al (2009) and Junker
et al (2011a) found that highly chemical repellent flowers did not have strong physical defenses
and vice versa suggesting trade-offs between the two (but see Ballantyne and Willmer 2012)
Like toxic nectar floral ant repellents might sometimes deter both pollinators and ants For
example Galen et al (2011) found that 2-phenylethanol in P viscosum floral scent repels both
flower-damaging F neorufibarbis ants and the plantrsquos main pollinator Bombus kirbyellus
Interestingly four recent studies have found that floral scents can also attract ants
(Agarwal and Rastogi 2007 Edwards and Yu 2008 Schiestl and Glaser 2011 Gonzaacutelvez et al
20
2012) One study (Schiestl and Glaser 2011) has shown that floral scents attract ants to a
potentially ant-pollinated plant and such systems may provide many more examples
4 Future directions
We have highlighted areas in need of further investigation throughout the review Here we focus
on several questions we consider high priorities for future research
i) Floral defenses against ants
a) Do floral resistance traits increase plant fitness
The existence ant-repellent floral traits is often taken as evidence that ants negatively affect plant
reproductive success However despite the abundance and variety of mechanisms excluding ants
from flowers few studies show that ants reduce plant fitness when they visit flowers (Table 11)
This absence is particularly apparent with repellent floral volatiles which may be the most
widespread floral resistance trait (Willmer et al 2009 Junker et al 2011a Ballantyne and
Willmer 2012) To our knowledge only one study has shown that floral ant repellents benefit
plants with repellent P viscosum flowers avoiding damaging ant visits (Galen et al 2011)
Research is urgently needed to investigate if these traits increase plant fitness by excluding ants
or if ant visitation is inconsequential More studies taking advantage of standing variation in
traits (Galen et al 2011) or creating it artificially (eg Junker et al 2010b) will allow us to
distinguish between the two
21
b) If ant visitation is often not costly what caused the evolution of repellent traits and
what maintains them Similarly are these repellent traits truly defenses against ant
visitation or are they generalized defenses against a range of floral antagonists
Ant-repellent floral traits are widespread and their production may be costly (Willmer et al
2009 Junker et al 2011a) The studies in Table 11 however indicate that flower-visiting ants
often do not disrupt plant reproduction particularly outside of myrmecophytes What then
selected for these traits One possibility is that these repellents are not directed solely at ants
Instead a community of antagonistic flower visitors may exert diffuse selection on plants
thereby selecting for and maintaining these traits Structural modifications may repel crawling
insects while chemical repellents such as floral volatiles may repel both flying and non-flying
insects (Junker et al 2011b) How much overlap there is in floral volatiles that repel both ants and
other antagonists needs investigation Until we examine how specific these defenses are to ants
we risk overstating the importance of ants in their evolution
ii) Do ants alter pollen movement patterns or reduce male fitness
We are aware of only four studies that have measured if ant visitation impacts male fitness
(Wyatt 1980 Fritz and Morse 1981 Ashman 2000 Galen and Geib 2007) While none of these
studies found an effect all measured only amounts of pollen or pollinia removed However this
measure does not adequately encompass male fitness alterations to pollen movement patterns
and eventual deposition location may be more important to male function than changes in
removal rates If as suggested by some studies (Altshuler 1999 Lach 2007) ants increase
pollinator relocation rates they may decrease pollen discounting increasing male fitness
However if this increased relocation reduces overall pollen export rates ant competition with
22
pollinators may depress male function Studies using pollen-tracking methods such as fluorescent
dyes will allow us to see how ants change pollen movement patterns and methods like paternity
analysis will reveal how competition between ants and pollinators alters male fitness
iii) Coevolution between ants and plants do myrmecophytes exhibit stronger more
specific defenses against ants
The floral traits of myrmecophytes are most likely to coevolve with ants because all plant
reproduction occurs in the presence of ants Their floral defenses would thus be expected to be
both stronger and more specific to their resident ants than those other plants visited by an array
of more opportunistic ant species Some evidence supports this Willmer et al (2009) found that
resident ants of acacias were repelled by flowers with more aggressive ant species showing
stronger repellence than less aggressive ants Additionally ants not normally associated with
acacias did not respond to acacia floral volatiles More studies are needed in other systems and
should take advantage of phylogenetic approaches examining floral traits in closely related
myrmecophytic and non-myrmecophytic plant species to determine whether ants commonly
select for floral defenses against ants in myrmecophytes
Acknowledgements
For comments and discussion we thank the Frederickson lab particularly Eric Youngerman
Lina Arcila Hernandez and Kyle Turner as well as Alison Parker and Jordan Pleet MEF
acknowledges financial support from an NSERC Discovery Grant a Connaught New Researcher
23
Award an Ontario Ministry of Economic Development and Innovation Early Researcher Award
and the University of Toronto ARC was supported by an Ontario Graduate Scholarship and
Sigma Xi
24
References
Adler L S 2001 The ecological significance of toxic nectar Oikos 91409ndash420
Agarwal V M and N Rastogi 2007 Role of floral repellents in the regulation of flower visits
of extrafloral nectary-visiting ants in an Indian crop plant Ecological Entomology 3359ndash
65
Altshuler D L 1999 Novel interactions of non-pollinating ants with pollinators and fruit
consumers in a tropical forest Oecologia 119600ndash606
Ashman TL 2000 Pollinator selectivity and its implications for the evolution of dioecy and
sexual dimorphism Ecology 812577-2591
Ashman TL and E A King 2005 Are flower-visiting ants mutualists or antagonists A study
in a gynodioecious wild strawberry American Journal of Botany 92891ndash5
Ballantyne G and P Willmer 2012 Floral visitors and ant scent marks noticed but not used
Ecological Entomology 37402-409
Ballantyne G and P Willmer 2012 Nectar theft and floral ant-repellence a link between
nectar volume and ant-repellent traits PloS One 7e43869
Baker H G and I Baker 1978 Ants and flowers Biotropica 1080
Barrett S C H 2002 Sexual interference of the floral kind Heredity 88154ndash159
Baroni Urbani C and M L de Andrade 1997 Pollen eating storing and spitting by ants
Naturwissenschaften 84256ndash258
25
Beattie A J C Turnbull R B Knox and E G Williams 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany
71421ndash426
Beattie A J C Turnbull T Hough S Jobson and R B Knox 1985 The vulnerability of
pollen and fungal spores to ant secretions evidence and some evolutionary implication
American Journal of Botany 72606ndash614
Blancafort X and C Goacutemez 2005 Consequences of the Argentine ant Linepithema humile
(Mayr) invasion on pollination of Euphorbia characias (L) (Euphorbiaceae) Acta
Oecologica 2849ndash55
Bleil R N Bluumlthgen and R R Junker 2011 Ant-plant mutualism in Hawailsquoi Invasive ants
reduce flower parasitism but also exploit floral nectar of the endemic shrub Vaccinium
reticulatum (Ericaceae) Pacific Science 65291ndash300
Bluumlthgen N G Gottsberger and K Fiedler 2004 Sugar and amino acid composition of ant-
attended nectar and honeydew sources from an Australian rainforest Austral Ecology
29418ndash429
Burkle L A R E Irwin and D A Newman 2007 Predicting the effects of nectar robbing on
plant reproduction implications of pollen limitation and plant mating system American
Journal of Botany 941935ndash1943
Byk J and K Del-Claro 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333ndash38
26
Caballero P C G Ossa W L Gonzaacuteles C Gonzaacutelez-Browne G Astorga M M Muruacutea and
R Medel 2013 Testing non-additive effects of nectar-robbing ants and hummingbird
pollination on the reproductive success of a parasitic plant Plant Ecology 214633ndash640
Carvalheiro L G E R M Barbosa and J Memmott 2008 Pollinator networks alien species
and the conservation of rare plants Trinia glauca as a case study Journal of Applied
Ecology 451419ndash1427
Chamberlain S A and J N Holland 2008 Density-mediated context-dependent consumer-
resource interactions between ants and extrafloral nectar plants Ecology 891364ndash1374
Creighton W S 1967 Studies on free colonies of Cryptocerus texanus Satschi (Hymenoptera
Formicidae) Psyche 7434ndash42
Cursach J and J Rita 2011 Implications of the reproductive biology of the narrow endemic
Naufraga balearica (Apiaceae) for its conservation status Plant Systematics and Evolution
298581ndash596
Cushman JH and Addicott JF In Huxley CR and DF Cutler (eds) 1st edition Ant-plant
interactions Oxford University Press Oxford
de Jong T J N M Waser and P G Klinkhamer 1993 Geitonogamy the neglected side of
selfing Trends in Ecology and Evolution 8321ndash325
de Vega C M Arista P L Oritz C M Herrera and S Talavera 2009 The ant-pollination
system of Cytinus hypocistis (Cytinaceae) a Mediterranean root holoparasite Annals of
Botany 4517ndash8
27
Dukas R 2001 Effects of perceived danger on flower choice by bees Ecology Letters 4327ndash
333
Dutton E M and M E Frederickson 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561ndash569
Edwards D P and D W Yu 2008 Tolerating castration by hiding flowers in plain sight
Behavioral Ecology and Sociobiology 6395ndash102
Frederickson M E 2009 Conflict over reproduction in an ant-plant symbiosis why Allomerus
octoarticulatus ants sterilize Cordia nodosa trees The American Naturalist 173675ndash81
Fritz R S and D H Morse 1981 Nectar parasitism of Asclepias syriaca by ants effect on
nectar levels pollinia insertion pollinaria removal and pod production Oecologia 50316ndash
319
Galen C 1983 The effects of nectar thieving ants on seedset in floral scent morphs of
Polemonium viscosum Oikos 41245ndash249
Galen C 2005 Catching ants with honey an experimental test of distraction and satiation as
alternative modes of escape from flower-damaging ants Oecologia 14480ndash87
Galen C and B Butchart 2003 Ants in your plants effects of nectar-thieves on pollen fertility
and seed-siring capacity in the alpine wildflower Polemonium viscosum Oikos 101521ndash
528
Galen C and J Cuba 2001 Down the tube pollinators predators and the evolution of flower
shape in the alpine skypilot Polemonium viscosum Evolution 551963-1971
28
Galen C and J C Geib 2007 Density-dependent effects of ants on selection for bumble bee
pollination in Polemonium viscosum Ecology 881202ndash1209
Galen C R Kaczorowski S L Todd J Geib and R A Raguso 2011 Dosage-dependent
impacts of a floral volatile compound on pollinators larcenists and the potential for floral
evolution in the alpine skypilot Polemonium viscosum The American Naturalist 177258ndash
272
Garciacutea M B R J Antor and X Espadaler 1995 Ant pollination of the palaeoendemic
dioecious Borderea pyrenaica (Dioscoreaceae) Plant Systematics and Evolution 19817ndash
27
Gaume L M Zacharias and R M Borges 2005 Ant ndash plant conflicts and a novel case of
castration parasitism in a myrmecophyte Evolutionary Ecology Research 7435ndash452
Graystock P and W O H Hughes 2011 Disease resistance in a weaver ant Polyrhachis
dives and the role of antibiotic-producing glands Behavioral Ecology and Sociobiology
652319ndash2327
Gόmez J M 2000 Effectiveness of ants as pollinators of Lobularia maritima effects on main
sequential fitness components of the host plant Oecologia 12290ndash97
Gόmez J M and R Zamora 1992 Pollination by ants consequences of the quantitative effects
on a mutualistic system Oecologia 91410ndash418
Gόmez J M R Zamora J A Hόdar and D Garciacutea 1996 Experimental study of pollination
by ants in Mediterranean high mountain and arid habitats Oecologia 105236ndash242
29
Gonccedilalves-Souza T P M Omena J C Souza and G Q Romero 2008 Trait-mediated effects
on flowers artificial spiders deceive pollinators and decrease plant fitness Ecology
892407ndash2413
Gonzaacutelvez F G L Santamariacutea R T Corlett and M A Rodriacuteguez-Gironeacutes 2012 Flowers
attract weaver ants that deter less effective pollinators Journal of Ecology 10178ndash85
Guerrant E O and P L Fiedler 1981 Flower defenses against nectar-pilferage by ants
Biotropica 1325ndash33
Harley R 1991 The greasy pole syndrome In Huxley CR and DF Cutler (eds) 1st edition
Ant-plant interactions Oxford University Press Oxford
Hansen D M and C B Muumlller 2009 Invasive ants disrupt gecko pollination and seed
dispersal of the endangered plant Roussea simplex in Mauritius Biotropica 41202ndash208
Hickman J C 1974 Pollination by ants a low-energy system Science 1841290ndash1292
Holland J N S a Chamberlain and T E X Miller 2011 Consequences of ants and
extrafloral nectar for a pollinating seed-consuming mutualism ant satiation floral
distraction or plant defense Oikos 120381ndash388
Houmllldobler B and Wilson E O 1990 The Ants 1st Edition Harvard University Press
Cambridge
Holway DA L Lach AV Suarez ND Tsutsui and TJ Case 2002 The causes and
consequences of ant invasions Annual Review of Ecology and Systematics 33181-233
30
Horskins K and Turner VB 1999 Resource use and foraging patterns of honeybees Apis
mellifera and native insects on flowers of Eucalyptus costata Australian Journal of
Ecology 24221-227
Horvitz C C and D W Schemske 1984 Effects of ants and an ant-tended herbivore on seed
production of a neotropical herb Ecology 651369ndash1378
Inouye DW 1980 The terminology of floral larceny Ecology 611251-1253
Inouye DW and O R J Taylor 1979 A temperate region plant-ant-seed predator system
consequences of extra floral nectar secretion by Helianthella quinquenervis Ecology 601ndash
7
Irwin RE 2003 Impact of nectar robbing on estimates of pollen flow conceptual predictions
and empirical outcomes Ecology 84485-495
Irwin RE and Maloof JE 2002 Variation in nectar robbing over time space and species
Oecologia 133525-533
Iwanami Y and T Iwadare 1978 Inhibiting effects of myrmicacin on pollen growth and pollen
tube mitosis Botanical Gazette 13942ndash45
Izzo TJ and Vasconcelos HL 2002 Cheating the cheater domatia loss minimizes the effects
of ant castration in an Amazonian ant plant Oecologia 133200-205
Janzen D H 1977 Why donrsquot ants visit flowers Biotropica 9252
Johnson SD Hargreaves AL and Brown M 2006 Dark bitter-tasting nectar functions as a
filter of flower visitors in a bird-pollinated plant Ecology 872709-2716
31
Junker R A Y C Chung and N Bluumlthgen 2007 Interaction between flowers ants and
pollinators additional evidence for floral repellence against ants Ecological Research
22665ndash670
Junker R R R Bleil C C Daehler and N Bluumlthgen 2010a Intra-floral resource partitioning
between endemic and invasive flower visitors consequences for pollinator effectiveness
Ecological Entomology 35760ndash767
Junker R R and N Bluumlthgen 2010b Floral scents repel facultative flower visitors but attract
obligate ones Annals of botany 105777ndash82
Junker R R C C Daehler S Doetteri A Keller and N Bluumlthgen 2011a Hawaiian ant ndash
flower networks nectar-thieving ants prefer undefended native over introduced plants with
floral defenses Ecological Monographs 81295ndash311
Junker R R J Gershenzon and S B Unsicker 2011b Floral odor bouquet loses its ant
repellent properties after inhibition of terpene biosynthesis Journal of Chemical Ecology
371323ndash31
Kawakita A and M Kato 2002 Floral biology and unique pollination system of root
holoparasites Balanophora kuroiwai and B tobiracola (Balanophraceae) American
Journal of Botany 891164ndash1170
Koopowitz H and TA Marchant 1998 Postpollination nectar reabsorption in the African
epiphyte Aerangis verdickii (Orchidaceae) American Journal of Botany 85508-512
Kirk W D J 1984 Ecological studies on Thrips imaginis Bagnall (Thysanoptera) in flowers of
Echium plantagineum L in Australia Australian Journal of Ecology 99ndash18
32
Kerner A 1878 Flowers and their unbidden guests C Kegan Paul London
Kessler D and I T Baldwin 2007 Making sense of nectar scents the effects of nectar
secondary metabolites on floral visitors of Nicotiana attenuata The Plant Journal 49840ndash
54
Koptur S and N Truong 1998 Facultative ant-plant interactions nectar sugar preferences of
introduced pest species in South Florida Biotropica 30179-189
Lach L 2005 Interference and exploitation competition of three nectar-thieving invasive ant
species Insectes Sociaux 52257ndash262
Lach L 2007 A mutualism with a native membracid facilitates pollinator displacement by
Argentine ants Ecology 881994ndash2004
Lach L 2008a Argentine ants displace floral arthropods in a biodiversity hotspot Diversity and
Distributions 14281ndash290
Lach L 2008b Floral visitation patterns of two invasive ant species and their effects on other
hymenopteran visitors Ecological Entomology 33155ndash160
Lach L 2013 A comparison of floral resource exploitation by native and invasive Argentine
ants Arthropod-Plant Interactions 7177-190
Luo C W K Li X M Chen Z Y Huang and K U N Li 2012 Ants contribute significantly
to the pollination of a biodiesel plant Jatropha curcas Environmental Entomology
411163ndash1168
33
Maleacute PJ G C Leroy A Dejean A Quilichini and J Orivel 2011 An ant symbiont directly
and indirectly limits its host plantrsquos reproductive success Evolutionary Ecology 2655ndash63
Maloof J E 2001 The effects of a bumble bee nectar robber on plant reproductive success and
pollinator behavior American Journal of Botany 881960ndash1965
Maloof J E and D W Inouye 2000 Are nectar robbers cheaters or mutualists Ecology
812651ndash2661
Moreau CS CD Bell R Vila SB Archibald and NE Pierce 2006 Phylogeny of the ants
diversification in the age of the angiosperms Science 312101-104
Nahas L M O Gonzaga and K Del-Claro 2012 Emergent impacts of ant and spider
interactions herbivory reduction in a tropical savanna tree Biotropica 44498ndash505
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506ndash514
Newman D A and J D Thomson 2005 Interactions among nectar robbing floral herbivory
and ant protection in Linaria vulgaris Oikos 110497ndash506
Nicklen E F and D Wagner 2006 Conflict resolution in an ant-plant interaction Acacia
constricta traits reduce ant costs to reproduction Oecologia 14881ndash7
Norment C J 1988 The effect of nectar-thieving ants on the reproductive success of Frasera
speciosa (Gentianaceae) American Midland Naturalist 120331ndash336
34
Orivel J L Lambs P-J G Maleacute C Leroy J Grangier T Otto A Quilichini et al 2011
Dynamics of the association between a long-lived understory myrmecophyte and its
specific associated ants Oecologia 165369ndash76
Palmer T M D F Doak M L Stanton J L Bronstein E T Kiers T P Young J R
Goheen et al 2010 Synergy of multiple partners including freeloaders increases host
fitness in a multispecies mutualism Proceedings of the National Academy of Sciences
10717234ndash9
Peakall R A J Beattie and S H James 1987 Pseudocopulation of an orchid by male ants a
test of two hypotheses accounting for the rarity of ant pollination Oecologia 73522ndash524
Peakall R and A J Beattie 1989 Pollination of the orchid Microtis parviflora R Br by
flightless worker ants Functional Ecology 3515ndash522
Peakall R C J Angus A J Beattie 1990 The significance of ant and plant traits for ant
pollination in Leporella fimbriata pollination Oecologia 84457ndash460
Puterbaugh M N 1998 The roles of ants as flower visitors experimental analysis in three
alpine plant species Oikos 8336ndash46
Prŷs-Jones OE and Willmer PG 1992 The biology of alkaline nectar in purple toothwort
(Lathraea clandenstina) ground level defenses Biological Journal of the Linnean Society
45373-388
Raine NE Willmer P and Stone GN 2002 Spatial structuring and floral avoidance
behaviour present in ant-pollinator conflict in a Mexican ant-acacia Ecology 833086-
3096
35
Ramsey M 1995 Ant pollination of the perennial herb Blandfordia grandiflora (Liliaceae)
Oikos 74265ndash272
Renault CK LM Buffa and Delfino MA 2005 An aphid-ant interaction effects on
different trophic levels Ecological Research 2071-74
Rico-Gray V and L B Thien 1989 Effect of different ant species on reproductive fitness of
Schomburgkia tibicinis (Orchidaceae) Oecologia 81487ndash489
Romero G Q P A P Antiqueira and J Koricheva 2011 A meta-analysis of predation risk
effects on pollinator behaviour PLoS One 6e20689
Rosumek FB FAO Silveira F de S Neves NP de U Barbosa L Diniz Y Oki F Pezzini
et al 2009 Ants on plants a meta-analysis of the role of ants as plant biotic defenses
Oecologia 160537ndash549
Roubik D W 1982 The ecological impact of nectar-robbing bees and pollinating
hummingbirds on a tropical shrub Ecology 63354ndash360
Rudgers J A 2004 Enemies of herbivores can shape plant traits selection in a facultative ant-
plant mutualism Ecology 85192ndash205
Schatz B and WT Wcislo 1999 Ambush predation by the ponerine ant Ectatomma ruidum
Roger (Formicidae) on a sweat bee Lasioglossum umbripenne (Halictidae) in Panama
Journal of Insect Behavior 12641-663
36
Schaffer W M D W Zeh S L Buchmann S Kleinhaus M V Schaffer and J Antrim
1983 Competition for nectar between introduced honey bees and native North American
bees and ants Ecology 64564ndash577
Schemske D W 1980 The evolutionary significance of extrafloral nectar production by Costus
woodsonii (Zingiberaceae) an experimental analysis of ant protection Journal of Ecology
68959ndash967
Schiestl F P and F Glaser 2011 Specific ant-pollination in an alpine orchid and the role of
floral scent in attracting pollinating ants Alpine Botany 1221ndash9
Schubart HOR AB Anderson 1978 Why donrsquot ants visit flowers A response to D H
Janzen Biotropica 10310-311
Schuumlrch S M Pfunder and B A Roy 2000 Effects of ants on the reproductive success of
Euphorbia cyparissias and associated pathogenic rust fungi Oikos 16ndash12
Stanton ML Palmer TM Young TP Evans A and Turner ML 1999 Sterilization and
canopy modification of swollen thorn acacia tree by a plant-ant Nature 401 578-581
Stephenson AG 1981 Toxic nectar deters nectar thieves of Catalpa speciosa American
Midland Naturalist 105381-383
Stout J C and D Goulson 2001 The use of conspecific and interspecific scent marks by
foraging bumblebees and honeybees Animal Behaviour 62183ndash189
Sugiura S T Abe and S Makino 2006 Loss of extrafloral nectary on an oceanic island plant
and its consequences for herbivory American Journal of Botany 93491ndash495
37
Svensson L 1985 An estimate of pollen carryover by ants in a natural population of
Scleranthus perennis L (Caryophyllaceae) Oecologia 66373ndash377
Tsuji K A Hasyim and K Nakamura 2004 Asian weaver ants Oecophylla smaragdina and
their repelling of pollinators Ecological Research 19669ndash673
Turner R C J J Midgley P Barnard R E Simmons and S D Johnson 2012 Experimental
evidence for bird pollination and corolla damage by ants in the short-tubed flowers of
Erica halicacaba (Ericaceae) South African Journal of Botany 7925ndash31
Visser D M G Wright and J H Giliomee 1996 The effect of the Argentine ant
Linepithema humile (Mayr) (Hymenoptera Formicidae) on flower-visiting insects of
Protea nitida Mill (Proteaceae) African Entomology 4285ndash287
Wagner D 2000 Pollen viability reduction as a potential cost of ant association for Acacia
constricta (Fabaceae) American Journal of Botany 87711ndash715
Wagner D and A Kay 2002 Do extrafloral nectaries distract ants from visiting flowers An
experimental test of an overlooked hypothesis Evolutionary Ecology Research 4293ndash305
Wang C Y Luo Y Tai D An and Y Kou 2008 Ants pollinate Neottia listeroides
(Orchidaceae) in Sichuan China 46836ndash846
Wyatt R 1980 The impact of nectar-robbing ants on the pollination system of Asclepias
curassavica Bulletin of the Torrey Botanical Club 10724ndash28
Wheeler WM and IW Bailey 1920 The feeding habits of Pseudomymine and other ants
Transactions of the American Philosophical Society 22235-279
38
Willmer P G and S A Corbet 1981 Temporal and microclimatic partitioning of the floral
resources of Justicia aurea amongst a concourse of pollen vectors and nectar robbers
Oecologia 5167ndash78
Willmer P G and G N Stone 1997 How aggressive ant-guards assist seed-set in Acacia
flowers Nature 388165ndash167
Willmer P G C V Nuttman N E Raine G N Stone J G Pattrick K Henson P Stillman
et al 2009 Floral volatiles controlling ant behaviour Functional Ecology 23888ndash900
Young T P C H Stubblefield and L a Isbell 1997 Ants on swollen-thorn acacias species
coexistence in a simple system Oecologia 10998ndash107
Yu D W and N E Pierce 1998 A castration parasite of an ant-plant mutualism Proceedings
of the Royal Society B Biological Sciences 265375ndash382
39
Tables
Table 11 Studies of ant-flower or ant-pollinator interactions that included a measure of plant fitness Each study system is listed only
once except when results varied between years or when different studies examined male and female fitness Significant effects of ants on
plant fitness are either lsquopositiversquo or lsquonegativersquo non-significant effects are lsquoneutralrsquo When ants significantly affected only one fitness
component but several were measured (eg fruit set and seed production) we still consider the result significant
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Acacia
drepanolobium
Crematogaster
nigriceps
Proportion
fruiting
Negative Destruction of floral buds Young et al 1997
Asclepias exaltata Several Pollinia inserted
removed
Negative for pollinia
insertion neutral for
pollinia removal
Exploitative competition Wyatt 1980 T
40
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Asclepias syriaca Several
Fruit set pollinia
removed
Negative for fruit
set neutral for
pollinia removal
Exploitative competition Fritz and Morse
1981 T
Cordia nodosa Allomerus cf
demerarae
Fruit set Negative Destruction of floral buds Yu and Pierce
1998
Costus woodsonii Wasmannia
auropunctata
Seed set Positive Unknown ant pollination
suggested (but unlikely)
Schemske 1980
Eritrichum aretioides F neorufibarbis
gelida
Seed set Negative Ants damaged flowers Puterbaugh 1998
41
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Euphorbia characias Several Seed set Neutral Plants in Argentine ant-
invaded area set less seed
than those in un-invaded
sites but an ant-exclusion
experiment did not produce
significant results
Blancafort and
Gόmez 2005
Ferocactus wislizeni Solenopsis xyloni
and others
Seed set Unknown negative Interference and exploitative
competition S xyloni
reduced seed set relative to
other ants but the study did
not include a no-ant control
Ness 2006
42
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Frasera speciosa F neorufibarbis
and Formica sp
Seed set Neutral Nocturnal pollination
occurring when ants were
less active may have
compensated for reduced
diurnal pollinator visitation
Norment 1988
Gypsophila struthium Several Seeds per
inflorescence
Neutral No effect despite large
numbers of ants on flowers
Gόmez et al
1996
Hirtella physophora Allomerus
decemarticulatus
Fruit set Negative Destruction of flowers Orivel et al
2011
Humboldtia brunonis Crematogaster
dohrni
Fruit set Negative Destruction of flowers Gaume et al
2005
43
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Lepidium subulatum Several Percentage of
ovules setting
seed
Positive Unknown Gόmez et al
1996
Leucospermum
conocarpodendron
Several Seed set Neutral Site not pollen limited Lach 2008
Linaria vulgaris Several ldquoFemale fitness
indexrdquo
(combination of
number of seeds
fruits flowers
and seed weight)
Positive Reduction in florivory and
seed predation nectar
robbing holes increased ant
visitation
Newman and
Thomson 2005
44
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Melastoma
malabathricum
Oecophylla
smaragdina
Seed set Positive Ants deterred less efficient
pollinators increasing
visitation by the more
efficient pollinator
Gonzaacutelvez et al
2012
Metrosideros
polymorpha
Several Fruit set Neutral Ants reduced nectar
collection rates by foraging
bees but had no effect on
pollen collection rates or
fruit set
Junker et al
2010a
Orexis alpine F neorufibarbis
gelida
Seed set Neutral Autogamy or not pollen
limited
Puterbaugh 1988
45
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Ouratea spectabilis Cephalotes
pusillus and others
Seeds per fruit Neutral positive C pusillus had no effect on
seed number but other ants
collectively increased seed
set
Byk and Del
Claro 2010
Pachycereus schottii Several Seed set Neutral positive In one year ants showed a
positive effect (seed
number 2008) but no effect
with increased sample sizes
Chamberlain and
Holland 2008
Polemonium
viscosum
F neorufibarbis
gelida
Seed set Negative Ants damaged styles Galen 1983
46
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Polemonium
viscosum
F neorufibarbis
gelida
Pollen removal Neutral Ants did not affect pollen
removal even at high ant
densities
Galen and Geib
2007 T
Protea nitida Linepithema
humile and others
Seed set Neutral positive L humile had no effect on
P nitida seed set but native
ants increased it in one year
ants may cause pollinators
to relocate
Lach 2007
Psychotria
limonensis
Ectatomma spp Fruit set Positive Suggested increased
pollinator relocation
Altshuler 1999
47
Plant species Ant species Fitness measure Direction of effect Proposed mechanism Reference
Roussea simplex Technomyrmex
albipes
Seed set Negative Interference and exploitative
competition
Hansen and
Muumlller 2009
Tristerix aphyllus Several Fruit set Neutral Nectar robbing ants
minimally reduced nectar
levels and had no effect on
hummingbird visitation
Caballero et al
2013
Vaccinium
reticulatum
Several Fruit set Neutral Ants reduced florivory but
did not affect fruit set
Bleil et al 2011
T Study measured male fitness
Study of sterilizing ants showed short-term decrease in female fitness long-term effects are unclear (see text)
48
Table 12 Studies demonstrating either i) ant pollination through an exclusion experiment or ii) that ants transfer pollen (or pollen
analogue) between flowers strongly suggesting ant pollination
Plant species Ant species Ant pollination Evidence Reference
Alyssum purpureum Several Yes Exclusion experiment Gόmez et al 1996
Arenaria tetraquetra Several Yes Exclusion experiment Gόmez et al 1996
Balanophora kuroiwai Leptothorax sp Suggested Exclusion experiment and
observed pollen transfer
and pollen tube formation
but did not see if plants set
seed
Kawakita and Kato 2002
Blandfordia grandiflora Iridomyrmex sp Yes Exclusion experiment Ramsey 1995
Borderea pyrenaica Several Yes Exclusion experiment Garciacutea et al 1995
49
Plant species Ant species Ant pollination Evidence Reference
Cytinus hypocistis Several Yes Exclusion experiment de Vega et al 2009
Epipactis thunbergii Camponotus
japonicas
Suggested Observed transfer of
pollinia from ants to
orchids
Sugiura et al 2006
Euphorbia cyparissias Several Yes Exclusion experiment Schuumlrch et al 2000
Fragaria virginiana Several Very likely Ant-only treatments set
comparable seed to flying
pollinator-only treatment
seed set increased with
increasing ant visitation
Ashman and King 2005
Frasera speciosa Several Yes Exclusion experiment Norment 1988
Frankenia thymifolia Several Yes Exclusion experiment Gόmez et al 1996
50
Plant species Ant species Ant pollination Evidence Reference
Hormathophylla spinosa Proformica
longiseta
Yes Exclusion experiment Gόmez and Zamora 1992
Jatropha curcas
Several
Tapinoma
melanocephalum
most common
Yes Exclusion experiment Luo et al 2012
Leporella fimbriata Myrmecia urens Yes Observed pollinia transfer
and subsequently
confirmed as ant
pollination
Peakall et al 1987
51
Plant species Ant species Ant pollination Evidence Reference
Lobularia maritima Several
Camponotus
micans most
common
Yes Exclusion experiment Gόmez 2000
Microtis parviflora
Iridomyrmex
gracilis
Yes Observation used
laboratory colonies to
replicate observations
Peakall and Beattie 1989
Naufraga balearica
Several
especially
Plagiolepis
pygmaea and
Lasius grandis
Suggested Observation over three
years of study only ants
were observed visiting
ants carried pollen
Cursach and Rita 2011
52
Plant species Ant species Ant pollination Evidence Reference
Neottia listeroides Leptothoras sp
Paratrechina sp
Suggested Observed transfer of
pollinia from ants to
orchids
Wang et al 2008
Paronychia pulvinata Formica
neorufibarbis
gelida
Yes Exclusion experiment Puterbaugh 1998
Polygonum cascadense Formica argentea
Very likely Grew plants in greenhouse
open to flying visitors with
minimal seed set only
plants with access to ants
set significant seed
Hickman 1974
53
Plant species Ant species Ant pollination Evidence Reference
Retama sphaerocarpa Several Yes Exclusion experiment Gόmez et al 1996
Scleranthus perennis
Several
especially
Formica
rufibarbis
Suggested Ants transferred pollen
analogue to stigmas and
carried pollen on bodies
Svensson 1985
Sedum anglicum Proformica
longiseta
Yes Exclusion experiment Gόmez et al 1996
Trinia glauca Several Yes Exclusion experiment Carvalheiro et al 2008
54
Chapter 2 Ants and ant scent reduce bumblebee pollination of artificial flowers
Cembrowski AR Tan MG Thomson JD and Frederickson ME
In press The American Naturalist
All authors designed the experiment ARC and MGT performed the experiment ARC and MEF
performed statistical analyse
s and ARC wrote the manuscript with input from all authors
Abstract
Ants on flowers can disrupt pollination by consuming rewards or harassing pollinators but it is
difficult to disentangle the effects of these exploitative and interference forms of competition on
pollinator behavior Using highly rewarding and quickly replenishing artificial flowers that
simulate male or female function we allowed bumblebees (Bombus impatiens) to forage (1) on
flowers with or without ants (Myrmica rubra) and (2) on flowers with or without ant scent cues
Bumblebees transferred significantly more pollen analogue both to and from ant free flowers
demonstrating that interference competition with ants is sufficient to modify pollinator foraging
behavior Bees also removed significantly less pollen analogue from ant scented flowers than
from controls making this the first study to show that bees can use ant scent to avoid harassment
at flowers Ant effects on pollinator behavior possibly in addition to their effects on pollen
viability may contribute to the evolution of floral traits minimizing ant visitation
55
Introduction
Trait-mediated indirect interactions arise when a focal species causes phenotypic changes
including behavioral modifications in a second species and these effects cascade to still other
species (Werner and Peacor 2003) Although trait-mediated indirect interactions are often studied
in food webs (eg Werner and Peacor 2003 Preisser et al 2005) they are not limited to trophic
interactions For example the threat of predation can change the behavior of a mutualist thus
affecting its partners (Suttle 2003) In animal-pollinated plants predators can disrupt pollination
directly through density-mediated indirect interactions (Dukas 2005) or via trait-mediated
indirect interactions by changing pollinator behavior When predators are present pollinators
may switch to visiting less rewarding flowers or they may avoid flowers altogether potentially
reducing the amount of pollen donated or received by a flower and thus decreasing plant fitness
(Gonccedilalves-Souza 2008 Ings and Chittka 2009) Similarly competition for floral rewards with
other flower visitors may cause behavioral changes in pollinators (Maloof and Inouye 2000
Ohashi et al 2008) Previous research has shown that bees may spend less time visiting flowers
depleted of rewards (Thomson 1986) and may avoid flowers bearing scent marks that indicate
recent visits by other individuals of the same or different bee species as these flowers are likely
to be depleted (Stout and Goulson 2002)
Whereas competition between pollinators may not be particularly costly for the plant
because both competitors are likely to provide pollination services competition between
pollinators and organisms that do not usually provide a pollination service such as ants has a
greater potential to reduce plant fitness Ants are common floral visitors they are attracted to
flowers for nectar (Lach 2007) and in some cases pollen (Byk and Del Claro 2010) and will
often defend these resources against other flower visitors (Altshuler 1999) However ants rarely
56
contribute much useful pollination antibiotic secretions present on their cuticles kill pollen
grains (Beattie et al 1985 Dutton and Frederickson 2012) and when foraging in flowers ants
sometimes impede female function by damaging stigmas (Galen and Cuba 2001) We will
henceforth consider ants to be non-pollinators and will contrast them to animals like bees that
are pollinators Ants are capable of competing with pollinators in two ways (1) by consuming
floral rewards and reducing their availability to pollinators (ie exploitative competition) and (2)
by directly antagonizing and excluding pollinators from flowers (ie interference competition)
Many plants benefit from the presence of ants numerous plant species recruit ants using
extrafloral nectar food bodies or domatia because ants provide protection against folivores (Heil
and McKey 2003) Similarly some plants have extrafloral nectaries on or near reproductive
tissues that recruit ants that deter florivores (eg Inouye and Taylor 1979) However plants
may incur reproductive costs as a result of their association with ants Some ant species that nest
in myrmecophytes sterilize flowers to shunt plant resources from reproduction to vegetative
growth allowing for greater ant colony growth (Frederickson 2009) Also ants visiting
extrafloral nectaries may be attracted to floral nectaries where they can harass pollinators (Ness
2006) There is growing evidence that many angiosperms have evolved floral traits that prevent
ants from accessing flowers during anthesis (Willmer and Stone 1997 Ballantyne and Willmer
2012) The effects of flower-visiting ants can be substantial Ants can decrease the frequency
duration or species diversity of pollinator visits to flowers (Lach 2008 Hansen and Muumlller 2009
Junker et al 2010 Gonzaacutelvez et al 2012) all of which potentially impact pollen donation and
receipt In some studies changes to pollinator behavior induced by ants have also been linked to
seed set
It is difficult however to disentangle the intertwined effects of exploitative and
interference competition in nature If ants disrupt pollination plants may often evolve traits to
57
deter ants from flowers (Willmer et al 2009) From a practical standpoint this would then limit
our ability to study the effects of ants on pollinator visitation because ants would not visit
flowers Here we used artificial flowers to explore ant-bee interactions in the absence of floral
defenses against ants We used highly rewarding quickly replenishing artificial flowers to
examine how direct harassment (ie interference competition expressed through behavior)
changes bee foraging behavior and how this affects donation and receipt of a pollen analogue
(powdered food dye) while minimizing the effects of exploitative competition We predicted
that flowers visited by ants would both donate and receive less pollen analogue than flowers
without ants because bees would avoid flowers with ants or leave them sooner Because scent
plays a large role in bumblebee communication and flower choice (Stout and Goulson 2002) we
also tested whether the presence or absence of ant scent on artificial flowers would affect pollen
analogue donation because of changes in the foraging behavior of bumblebees
Methods
i) Subjects
Myrmica rubra is an invasive ant in eastern North America with a range and habitat preferences
overlapping that of Bombus impatiens a common bumblebee Because M rubra visits flowers
(A Cembrowski pers obs) the two species likely interact in nature We collected 12 M rubra
colonies in the fall of 2011 and 2012 from Toronto Ontario Canada and the surrounding area
and maintained them in environmental chambers on artificial diet (Dussutour and Simpson 2009)
and a 1410 LD schedule (light 600-2200) We used these colonies as sources of M rubra
workers for experiments
58
Workers from commercially supplied colonies of Bombus impatiens (Biobest Canada
Ltd) foraged on artificial flowers in flight cages (either 24 x 24 x 21 m or 79 x 34 x 20m) at
the University of Toronto We tested a total of five bee colonies four colonies were used in the
ant presence trials and two of these and one additional colony were used in the ant scent trials
Flight cages had overhead fluorescent lights attached to timers In contrast to most previous
studies in which bees have been trained and tested individually the entire worker force of a bee
colony was free to forage at will in our experiments Colonies were trained to forage on artificial
flowers for at least four days before being used in trials After being used for a trial the colony
was not used for at least two days to reduce dye carryover between trials Between trials
bumblebees were fed pollen and given sugar water
ii) Artificial flowers
The flowers (Fig 1 Thomson et al 2012 see also Makino 2008) consisted of glass jars filled
with 30 WV sucrose solution Sugar water travelled by capillary action up a sewing-thread
wick to a hole in a blue-painted lid accumulating in a knot that acts as a nectary Flowers
depleted by visitors were quickly replenished via capillary action and were non-rewarding only
very briefly after visits taking less than a minute to accumulate 05 microl note that bumblebee
visits to a single flower are often separated by several minutes or more (A Cembrowski
unpublished data)
Unlike most previous artificial flower designs these flowers allow for estimation of male
and female fitness because we can measure both the amount and type of dye received by female
flowers (see (d) Dye quantification below) In order to access nectar on a ldquomalerdquo flower a bee
must crawl through a circular ldquoantherrdquo of brush-like weather stripping material dusted in a
59
consistent manner with our pollen analogue powdered food dye (FDampC 5 or 6) which is
transferred to the bee in the process On a ldquofemalerdquo flower a bee crawls through a sticky plastic
reinforcement that functions as a ldquostigmardquo receiving dye from the beersquos body Male flowers
dispensed dye particles over multiple bee visits and could still dispense dye at the end of trials
They resemble many real flowers or flower heads in that pollen is dispensed gradually over time
but less dye is available for transfer with each subsequent bee visit (Harder and Thomson 1989)
iii) Flight cage trials
We conducted 6 ant scent trials between 22 March and 4 June 2012 and the remaining trials (16
ant presence trials and 9 ant scent trials) between 9 November 2012 and 6 January 2013 Ant
presence trials lasted for eight hours and ant scent trials lasted for four hours Artificial flowers
were prepared and placed individually in small plastic containers treated with Fluon (Insect-a-
Slip BioQuip Products Inc) to prevent ants from escaping In ant presence trials we used 32
flowers (16 male and 16 female flowers) arranged in an eight-by-four array with flowers spaced
45 cm apart between rows and 30 cm within rows Ant scent trials used 20 flowers (10 male and
10 female flowers) in a four-by-five array with flowers spaced 30 cm apart between and within
rows In all of the ant presence trials and 9 of the ant scent trials we counted the number of bees
actively foraging after one hour to get a measure of colony activity
a) Ant presence trials
We examined the effects of ants on the amount of dye donated by male flowers and received by
female flowers in sixteen trials testing four bee colonies four times each The containers of eight
randomly chosen male flowers and eight randomly chosen female flowers received 15 M rubra
60
workers each the other 16 flowers remained free of ants Isolated from their colonies M rubra
workers have nowhere to deposit sugar water they collect and therefore may become sated
nonetheless a force of 15 ant workers was enough to maintain visitation to the nectaries where
bees foraged while still rarely having more than one or two ants visit the nectary at any time As
ants attacked bees bees would occasionally carry or throw attacking ants off flowers but despite
this some ants maintained their presence at the nectary throughout the trials Because ants the
size of M rubra consume liquids slowly (~017 ndash 024 microlmin Davidson et al 2004) flowers
replenished nectar more quickly than ants consumed it We used two colors of dye (FDampC 5 and
FDampC 6) to differentiate male flowers with and without ants In two trials for each bee colony
anthers of male flowers with ants were coated with FDampC 5 dye while brushes of male flowers
lacking ants were coated with FDampC 6 dye In the other two trials we reversed dye colors to
control for effects of dye type and color Dye color was assigned in a random order Artificial
flowers were placed in randomly assigned positions in the array before we opened the colony
and allowed the bees to begin foraging
b) Scent trials
We explored the effect of ant scent on dye donation in fifteen trials testing three bee colonies in
six five and four trials each Bees were first exposed to ants by allowing the bees to forage for
eight hours on twenty flowers (ten male and ten female) of which five male flowers had 15 ants
and all others had none No dye was used during this ldquoexposurerdquo day We then collected all but
the five ant-free male flowers in the flight cage leaving these flowers to keep bees foraging
Next we individually stored five new male lids in Fluon-treated containers with ten M rubra
workers Five control male lids were put in identical containers lacking ants The following
61
morning we removed the remaining flowers and set out ten new male and female flowers in a
random spatial arrangement using the lids having or lacking ant scent Thus the ant-scented
flowers were not in the same positions as the ant-visited flowers on which the bees were trained
Only five male flowers had ant scent and the other 15 flowers (five male and ten female) did not
We used the same two dye colors as in i) to differentiate male flowers with and without ant
scent and randomized which color was used for ant-scented flowers between trials We collected
and replaced stigmas from female flowers after one hour and collected the stigmas again after
four hours In the first trial performed we also collected stigmas after two hours but due to the
small amount of dye transferred this was not repeated
iv) Dye quantification
After each trial we quantified the amount of dye transferred to female flowers using a
spectrophotometer We removed the stigmas from female flowers placed them in test tubes
added 51 mL of distilled water to each tube and vortexed each tube thoroughly to ensure the
dye was evenly diluted These were diluted further as needed if the absorbance exceeded the
sensitivity range of the spectrophotometer In the first six scent trials each stigma was analyzed
separately and the dye amounts were summed to obtain a total amount of each dye color
transferred to female flowers in each trial In all the ant presence trials and the other nine scent
trials we opted to treat the experiment as the unit of replication Therefore we combined stigmas
from each treatment type (ant-visited or ant-free) in a test tube and measured the total amount of
each dye color transferred in each treatment in each trial
Because we put different dye colors on male flowers with and without ants we could use
the amount of each dye color donated to all female flowers to measure the reproductive success
62
of ant-visited and ant-free male flowers The total amount of dye (of both colors) received by
female flowers with and without ants was our measure of female reproductive success We
calculated the amount of each dye color in the sample by measuring absorbance at 428 or 486
nm and converting absorbance to micrograms following computational methods for overlapping
spectra (Blanco et al 1989)
v) Statistical analyses
In two trials (one ant presence and one ant scent) most female flowers received no dye because
of low bee activity so we excluded these trials from analyses Because dye reception and
donation values were non-normally distributed we square-root transformed the data before
examining the effects of ant presence or ant scent on dye transfer in ANCOVAs For ant
presence trials we included ant presence on male and female flowers and their interaction as
main effects and the total amount of dye transferred in each trial as a covariate to account for
the large variation in overall dye transfer among trials For ant scent trials we included ant scent
as the main effect time (one or four hours) as a repeated measure the interaction between scent
and time and the total amount of dye transferred as a covariate Covariate by treatment (ant
presence or ant scent) interactions were never significant and so were excluded in final analyses
All analyses were conducted in JMP (v 1000)
Results
Bees usually started foraging within minutes of the beginning of the trial and continued until
flowers were collected from the flight cage or the lights were extinguished An average of 34 plusmn
012 and 40 plusmn 015 (mean plusmn SE) bees were foraging after an hour in the ant presence and ant
63
scent trials respectively Male flowers with ants donated significantly less dye than male flowers
lacking ants (Fig 22a F155 = 4019 p lt 00001) Similarly female flowers with ants received
significantly less dye than female flowers lacking ants (Fig 22a F155 = 461 p = 0036) There
was no significant interaction between ant presence on male flowers and ant presence on female
flowers (F155 = 118 p = 028) Flowers with ant scent donated significantly less dye than
flowers without ant scent (Fig 22b F125 = 11216 p lt 00001) There was no significant effect
of time (F125 = 121 p = 028) or interaction between time and scent (F125 = 111 p = 030) in
the model
Discussion
In this study ants altered bumblebee pollination behavior and exhibited trait-mediated indirect
interactions with (artificial) flowers Through interference competition M rubra workers
significantly affected the pattern of dye transfer by B impatiens causing a preferential flow of
dye from male flowers lacking ants to female flowers lacking ants If manifested in nature such
effects would reduce the reproductive success of plants visited by ants through both male and
female function
Ants could have changed the attractiveness of artificial flowers in at least two ways First
as ants interrupted or altogether prevented bumblebees from foraging at nectaries bumblebees
may have learned to avoid flowers with ants due to the relative inefficiency of foraging at these
flowers something bumblebees take into account (Heinrich 2004) Second bees attempt to
minimize their risk of being attacked during foraging bouts which ants did to visiting bees Ants
often attacked or harassed bees by biting grasping and appearing to sting visiting bees
preventing them from accessing the nectary or reducing their time on flowers (see online video
64
1) and bees sometimes avoided ant-tended flowers entirely This harassment was sometimes
physically traumatic (online video 1) and bees often appeared to have trouble flying after being
attacked by ants Previous research has demonstrated that bumblebees leave or avoid flowers
where they have been harassed (Jones and Dornhaus 2011) and avoid foraging where there is
visual or olfactory evidence of a predator or predation event (Abbott 2006 Goodale and Nieh
2012) Though it is unclear if bumblebees viewed ants as competitors or predators they
responded similarly to flowers having ants as they do to flowers housing predators (Gonccedilalves-
Souza et al 2008)
To avoid artificial flowers with ants bees likely used a combination of visual and
olfactory cues Previous research has shown that bees respond to conspicuous predators or
predator ldquodummiesrdquo on flowers (Suttle 2003 Gonccedilalves-Souza et al 2008) decreasing the
frequency and duration of their visits to these flowers Thus bees may have been able to avoid
flowers harboring ants by sight alone However even in the absence of ants bees still
preferentially visited flowers lacking ant scents (Fig 22b) suggesting that they had learned to
associate ant scent with harassment Bees are adept at associative learning (Wright and Schiestl
2009) and can learn to recognize unique scents left behind by both conspecific and heterospecific
flower visitors (Stout et al 1998) Bees can use these various scents often arising from tarsal
gland deposits (Stout et al 1998) to recognize recently visited flowers that are less likely to be
profitable (Stout and Goulson 2002) Recently Ballantyne and Willmer (2012) demonstrated that
bees learn to associate ant scents with unrewarding artificial flowers and decrease their visitation
to these flowers Our results complement their findings by showing that bees can associate ant
scent with harassment at otherwise rewarding flowers
In our study ant scent caused bees to adjust their foraging strategy and decreased the
amount of pollen analogue that was donated by ant-scented flowers Thus the effects of
65
interference competition with ants on flowers can extend beyond immediate interactions and may
have fitness consequences for plants even when ants are absent Like other olfactory cues these
effects are likely transitory (Stout et al 1998) Although more dye was still donated by male
flowers lacking ant scent than those with ant scent the ratio of dye donated was on average
closer to equality in hours 2-4 than in the first hour (Fig 22b) This effect may be partially
driven by dye depletion of male flowers without ant scent but the lack of a strong corresponding
decrease in flowers with ant scent suggests that visitation patterns became more similar We did
not test whether B impatiensrsquo avoidance of ant scent was an innate or a learned behavior but
previous work has shown that Bombus terrestris does not innately avoid flowers with ant scent
(Ballantyne and Willmer 2012)
Ants may be necessary for plant survival and growth but can be costly for plant
reproduction The evolution of ant attractants such as extrafloral nectaries in some plant lineages
suggests that the costs of ants can be outweighed by their protective abilities However in this
study flowers visited by ants received and donated significantly less dye although they did
retain some sexual function The net benefit of having ants depends on whether ants increase
plant fitness by reducing herbivory more than they decrease plant fitness by disrupting
pollination Alternatively plants may actually benefit from the costs of ants to plant
reproduction ants that castrate flowers may be better defenders increasing plant survival or
vegetative growth when plants are young allowing for increased reproduction later in life when
the plant is colonized by less aggressive non-castrating ants (Frederickson 2009 Palmer et al
2010) In some plant species pollinator harassment by ants may even be beneficial Altshuler
(1999) reported that Ectatomma ants greatly increased fruit set of Psychotria limonensis despite
reductions in pollinator visitation rates presumably due to increases in the rate of pollen
outcrossing Similarly Gonzaacutelvez et al (2012) found that the presence of Oecophylla
66
smaragdina ants on Melastoma malabathricum flowers reduced visitation by less effective
pollinators and increased visitation by more effective Xylocopa bees increasing plant fitness
Cases of ants being beneficial to flowers appear to be the minority and many plant
species have traits that limit ant access to flowers Several studies have detailed floral volatiles
that are thought to mimic ant alarm pheromones and thus repel ants (Junker and Bluumlthgen 2008
Willmer et al 2009) Other plants use structural modifications such as narrow corollas or
slippery stems to limit ant access to flowers (reviewed in Willmer et al 2009) Exploitative
competition between ants and pollinators (Lach 2005 Ballantyne and Willmer 2012) as well as
the lethal effects of ant antibiotic secretions on pollen grains (Beattie et al 1985 Dutton and
Frederickson 2012) may have resulted in selection on plants to reduce ant visits to flowers Our
results suggest that trait-mediated indirect interactions resulting from interference competition
between ants and bees may favor plants that defend their flowers and their pollinators against
ants
Acknowledgements
We thank H Rusnock and S Meadley-Dunphy for assistance J Dix for help with flight cage
construction and Biobest for bumblebee colonies We are grateful to the Thomson and
Frederickson labs for guidance particularly T Makino funded by JSPS for helping to refine
artificial flowers J Ogilvie for help with bees K Prior for M rubra colony collections and K
Turner for study design suggestions The comments of the editor and two anonymous reviewers
prompted improvements in the article especially with regards to study design MEF and JDT
acknowledge NSERC Discovery Grant funding The University of Torontorsquos Faculty of Arts and
Science supported MGRTrsquos participation
67
68
References
Abbott K R 2006 Bumblebees avoid flowers containing evidence of past predation events
Canadian Journal of Zoology 841240-1247
Altshuler D L 1999 Novel interactions of non-pollinating ants with pollinators and fruit
consumers in a tropical forest Oecologia 119600-606
Ballantyne G and Willmer P 2012 Floral visitors and ant scent marks noticed but not used
Ecological Entomology 37402-409
Beattie A J Turnbull C Knox R B and Williams E G 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany
71421-426
Blanco M Runiaga H Maspoch S and Tarin P 1989 A simple method for
spectrophotometric determination of two-components with overlapped spectra Journal of
Chemical Education 66178-180
Byk J amp Del Claro K 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333-38
Davidson D W S C Cook and R R Snelling 2004 Liquid-feeding performances of ants
(Formicidae) ecological and evolutionary implications Oecologia 139255ndash66
Dukas R 2005 Bumble bee predators reduce pollinator density and plant fitness Ecology
861401-1406
Dussutour A and Simpson S J 2009 Communal nutrition in ants Current Biology 19740-
744
69
Dutton E M and Frederickson M E 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561-569
Frederickson ME 2009 Conflict over reproduction in an ant-plant symbiosis why Allomerus
octoarticulatus ants sterilize Cordia nodosa trees American Naturalist 173675-681
Galen C and Cuba J 2001 Down the tube pollinators predators and the evolution of flower
shape in the alpine skypilot Polemonium viscosum Evolution 551963-1971
Gonccedilalves-Souza T Omena P M Souza J C and Romero G Q 2008 Trait-mediated
effects on flowers artificial spiders deceive pollinators and decrease plant fitness Ecology
892407-2413
Gonzaacutelvez F G Santamariacutea L Corlett R T and Rodriacuteguez-Gironeacutes M A 2012 Flowers
attract weaver ants that deter less effective pollinators Journal of Ecology 10178ndash85
Goodale E and Nieh JC 2012 Public use of olfactory information associated with predation
in two species of social bees Animal Behaviour 84919-924
Harder LD and Thomson JD 1989 Evolutionary options for maximizing pollen dispersal of
animal-pollinated plants American Naturalist 133323-344
Heil M and McKey D 2003 Protective ant-plant interactions as model systems in ecological
and evolutionary research Annual Review of Ecology Evolution and Systematics 34425-
453
Heinrich B 2004 Bumblebee economics 2nd
edn Harvard University Press Cambridge
Ings T C and Chittka L 2009 Predator crypsis enhances behaviourally mediated indirect
effects on plants by altering bumblebee foraging preferences Proceedings of the Royal
Society B 2762031-2036
70
Inouye D W and Taylor O R Jr 1979 A temperate region plant-ant-seed predator system
consequences of extra floral nectar secretion by Helianthella quinquenervis Ecology 601-
7
Junker R R and Bluumlthgen N 2008 Floral scents repel potentially nectar-thieving ants
Evolutionary Ecology Research 10295ndash308
Junker R R Bleil R Daehler C C and Bluumlthgen N 2010 Intra-floral resource partitioning
between endemic and invasive flower visitors consequences for pollinator effectiveness
Ecological Entomology 35760ndash767
Jones E I and Dornhaus A 2011 Predation risk makes bees reject rewarding flowers and
reduce foraging activity Behavioral Ecology and Sociobiology 651505-1511
Lach L 2005 Interference and exploitative competition of three nectar-thieving invasive ant
species Insect Sociaux 52257-262
Lach L 2007 A mutualism with a native membracid facilitates pollinator displacement by
Argentine ants Ecology 881994-2004
Lach L 2008 Argentine ants displace floral arthropods in a biodiversity hotspot Diversity and
Distributions 14281-290
Makino T T and Sakai S 2007Experience changes pollinator responses to floral display size
from size-based to reward-based foraging Functional Ecology 21854863
Maloof J E and Inouye D W 2000 Are nectar robbers cheaters or mutualists Ecology
812651ndash2661
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506-514
Ohashi K Leslie A and Thomson JD 2008 Trapline foraging by bumble bees V Effects of
experience and priority on competitive performance Behavioral Ecology 19936-948
71
Palmer T M Doak D F Stanton M L Bronstein J L Kiers E T Young T P Goheen
J R and Pringle R M 2010 Synergy of multiple partners including freeloaders
increases host fitness in a multispecies mutualism Proceedings of the National Academy
of Sciences 10717234-17239
Preisser E L Bolnick D I and Benard M E 2005 Scared to death The effects of
intimidation and consumption in predator-prey interactions Ecology 86501-509
Stout J C Goulson D and Allen J A 1998 Repellent scent-marking of flowers by a guild of
foraging bumblebees (Bombus spp) Behavioral Ecology and Sociobiology 43317-326
Stout J and Goulson D 2002 The influence of nectar secretion rates on the responses of
bumblebees (Bombus spp) to previously visited flowers Behavioral Ecology and
Sociobiology 52239-246
Suttle K B 2003 Pollinators as mediators of top-down effects on plants Ecology Letters
6688-694
Thomson JD 1986 Pollen transport and deposition by bumble bees in Erythronium influences
of floral nectar and bee grooming Journal of Ecology 74329-341
Thomson JD Ogilvie J E Makino T T Arisz A Raju S Rojas-Luengas V and Tan M
G R 2012 Estimating pollination success with novel artificial flowers effects of nectar
concentration Journal of Pollination Ecology 9108-114
Tsuji K Hasyim A and Nakamura K 2004 Asian weaver ants Oecophylla smaragdina and
their repelling of pollinators Ecological Research 19669-673
Ulrich S and Bertsch A 1990 Do foraging bumblebees scent-mark food sources and does it
matter Oecologia 82137-144
Werner E E and Peacor S D 2003 A review of trait-mediated indirect interactions in
ecological communities Ecology 841083-1100
72
Willmer P G Nuttman C V Raine N E Stone G N Pattrick J G Henson K Stillman
P McIlroy L Potts S G and Knudsen J T 2009 Floral volatiles controlling ant
behaviour Functional Ecology 23888-900
Wright G A and Schiestl F P 2009 The evolution of floral scent the influence of olfactory
learning by insect pollinators on the honest signaling of floral rewards Functional Ecology
23841-851
73
Figures
Figure 21 Photograph of artificial flower lids A = anther L = lid N = nectary R = region
where anther or stigma is placed S = stigma T = thread W = weight
74
Figure 22 Dye transferred (mean plusmn 1SE) per trial by B impatiens workers A) from artificial
male and to artificial female flowers in the presence (unfilled circles) or absence (filled circles)
of Myrmica rubra workers and B) from artificial male flowers with (unfilled circles) or without
(filled circles) M rubra scent to ant-free female flowers in the 1st or the 2
nd to 4
th hours of the
trials
75
Chapter 3 Not just for the bees pollen consumption is common among tropical
ants
Cembrowski AR Reurink G and Frederickson ME
Planned submission to Biotropica
ARC and MEF designed the study ARC and GR did laboratory work ARC performed statistical
analyses and wrote the manuscript with input from other authors
Abstract
Although palynivory or pollen consumption is widespread among other hymenopterans there
are few accounts of palynivory in ants To quantify how often ants consume pollen we subjected
adult workers and larvae from 75 species of neo- and paleo-tropical ants to acetolysis a process
that destroys most organic material but leaves behind pollen grains In over half of the species
we examined ants contained a few pollen grains (lt 5) and ants of several Camponotus species
contained abundant pollen grains (gt50) We tested for an association between trophic level and
palynivory using stable nitrogen isotope ratios (δ15
N) but we did not find a significant
correlation we found pollen grains in lsquoherbivorousrsquo omnivorous and carnivorous ants We
suggest that our results indicate sporadic opportunistic pollen consumption by ants in tropical
forests
Introduction
For those organisms that can digest it efficiently pollen can be an excellent food source
Depending on the plant species pollen can contain up to 60 protein as well as lipids
76
carbohydrates and numerous trace nutrients (Roulston and Cane 2000) Pollen feeding
(palynivory) occurs in many taxa including birds mammals and arthropods (Roulston and Cane
2000) but it is best known among the Hymenoptera particularly bees Although adult bees feed
mainly on plant exudates (ie nectar) nearly all bee species rely on pollen as the chief protein
source for developing larvae (Willmer 2011) Closely related to bees ants have similar
nutritional requirements adult workers need carbohydrates whereas larvae are fed a diet rich in
protein (Dussutour and Simpson 2009) Ants also exploit nectar heavily and regularly visit
flowers (eg Herrera et al 1984 Bluumlthgen et al 2004) but unlike bees are not believed to
widely consume pollen Instead ants are thought to receive most of the protein in their diet from
predation or scavenging (eg Floren et al 2002)
However ants may not be as reliant on animal protein sources as commonly assumed In
tropical forests arboreal ants can have similar δ15
N ratios a measure of trophic level as
herbivores suggesting limited carnivory and substantial feeding on plant-derived protein sources
(Davidson et al 2003) One potential protein source is pollen This idea is supported by scattered
accounts of pollen consumption by ants from other ecosystems including deserts (Ness 2006)
boreal forests (Czechowski et al 2009) and tropical savannas (Byk and Del Claro 2010) One
ant genus Cephalotes is widely recognized to consume pollen (Creighton 1967 Baroni Urbani
and de Andrade 1997 Byk and Del Claro 2010) However beyond this genus our knowledge of
pollen consumption by ants has largely been limited to direct observations (but see Czechowski
et al 2011) and it is unknown if palynivory is truly rare among ants or simply under-reported
We borrowed a technique commonly used in palynology acetolysis to assess the frequency of
pollen consumption by ants in 75 neo- and paleo-tropical ant species This technique allowed us
to examine many more ant species than we could have observed directly
77
Methods
Adult worker ants were collected by hand at baits or from leaf litter using Winkler extractors
while larvae along with adults were collected by hand from nests Collections were made by L
Arcila Hernandez and J Sanders in the primary tropical rainforest surrounding the Centro de
Investigaciόn y Capacitaciόn Rio Los Amigos (CICRA 12deg34rsquoS 70deg05rsquoW) in Peru in 2010-
2011 and by E Youngerman from lowland primary and secondary rainforests in Madang
Province Papua New Guinea (PNG) in 2011-2012 We sorted ant samples to morphospecies and
then identified them to genus and when possible to species using existing keys which were
unavailable for PNG ants Ant samples used for acetolysis were whole adult workers guts from
adult workers and whole larvae
We used acetolysis to look for pollen grains inside ants In acetolysis organic material is
dissolved in acetic anhydride and sulfuric acid leaving behind stained pollen exines When
possible we used adult and larval ants of the same species from different colonies or bait
locations in an attempt to sample more representatively We prepared samples by first washing
them in 70 ethanol to remove any pollen present on the samplersquos outer surface before placing
them individually in micro-centrifuge tubes with ~05mL of a 91 mixture of acetic anhydride
sulfuric acid We then heated each sample at 95degC for 20min allowing tissues to dissolve Next
we centrifuged samples at 1000 RPM for 5min and decanted the liquid supernatant before
adding ~05mL of glacial acetic acid to the precipitate centrifuging again as above We decanted
the samples and added a drop of distilled water
78
We examined a 150μL sub-sample of this solution for pollen grains under a light
microscope Larvae gave less than 150μL of solution following acetolysis so we sampled the
entire amount We identified pollen grains by their staining and their surface architecture For
most species we examined at least three adult workers and three larvae but for some species
fewer than three adult workers or larvae were available (Tables 31 and 32)
We investigated the relationship between trophic level as measured by δ15
N ratios and
pollen consumption by ants Stable isotope (SI) data was available for some (n =23) of the
Peruvian species from a separate project in which collections overlapped Samples were stored in
EtOH and dried at 50degC overnight We used heads legs and thoraxes (but not gasters) of ants
for SI analysis Often single individuals did not provide enough material for analysis and we
combined multiple individuals to achieve necessary weights up to 10 for small ant species
Samples were analyzed at the Boston University Stable Isotope Laboratory using a Finnigan
Delta-S analyzer For other Peruvian species for which we had samples but not corresponding
stable isotope data (n =25) we took the median δ15
N ratio value of that speciesrsquo genus and
assigned it to the species in question Using pollen presenceabsence data for these 48 species
we used logistic regression to correlate pollen presence and δ15
N ratio using R (v 2151)
Results
We found pollen grains in 37 of the 197 adult workers 20 of the 102 larvae and 38 of the 75 ant
species we examined (Tables 31 and 32) The proportion of individuals containing pollen did
not differ significantly between larvae and adults (χ2
1 = 002 p = 088) The number of pollen
grains found was often low (median = 3) but highly variable (range 1-468) Also pollen
79
presence was inconsistent among conspecific ant samples of species that contained pollen 31
had pollen in only one of the larvae or adults examined In only three species (two Cephalotes
and one Myrmelachista) did all examined adults contain pollen
Highly lsquoherbivorousrsquo carnivorous and omnivorous ants contained pollen grains (Fig
31) There was no correlation between δ15
N ratio and pollen presence (χ2 = 150 df = 1 p =
022) In fact several carnivorous species contained pollen grains (eg Odontomachus sp 1
Pachycondola sp1)
Discussion
Pollen consumption by ants appears to be more common than suspected The majority of species
(51) had at least one individual that had consumed pollen and in 20 multiple individuals
contained pollen Numbers of grains found tended to be low however with most individuals
containing only a few grains Ants may use pollen as only a small portion of their diet exploiting
a host of other protein-containing foods more heavily including other low- δ15
N foods (eg
honeydew epiphylls Houmllldobler and Wilson 1990 Davidson et al 2003)
There are a few reasons why pollen may not be heavily exploited by ants Pollen requires
much post-consumption processing to extract its nutrients These nutrients are located in the
pollenrsquos cytoplasm which is protected from direct digestion by the exines and intines of the
grains Organisms must either break through these layers or cause the pollen grains to germinate
or pseudo-germinate to access and digest the cytoplasm (Roulston and Cane 2000) This can be
achieved by consuming a sugar-containing solution (such as nectar) along with pollen However
animals that are not adapted to feed on pollen may not be very efficient at this process only
80
extracting nutrients from some of the grains (Herrera and Martiacutenez del Rio 1998) Thus while
ants can likely get some nutrients from pollen other forms of more easily processed protein may
be more attractive
Pollen may also pose handling difficulties for ants Despite the large number of studies
that have recorded ants visiting flowers (Chapter 1) only a few papers detail ants harvesting
pollen directly from anthers Byk and Del Claro (2010) reported that Cephalotes pusillus ants
removed almost all of the available pollen from Ouratea spectabilis flowers and at least two
other studies describe ants consuming pollen along with nectar from flowers (Horskins and
Turner 1999 Ness 2006) Unlike bees which possess both morphological (eg specialized hairs
for holding pollen) and behavioral adaptations (eg stereotyped pollen-packing motions) ants
have no apparent mechanisms to increase the amount of pollen harvested and are likely limited
by the number of grains they can ingest or carry Rather than collect pollen directly from flowers
it appears more likely that ants collect it haphazardly in the environment leading to a smaller
number of pollen grains consumed at any one time It is known that at least Cephalotes ants
engage in such behavior Creighton (1967) observed Cephalotes texanus collecting pollen grains
trapped by Citrus x paradisi leaf hairs
There is a chance that the small numbers of pollen grains we observed came from
secondary pollen consumption (ie consuming a palynivorous organism) or consumption of
liquid containing pollen While we cannot rule this out we believe our results represent actual
targeted pollen consumption by ants Cephalotes ants known palynivores (Creighton 1967
Baroni Urbani and de Andrade 1997) contained similar numbers of pollen grains as many of the
pollen-containing ant species that we examined As well in at least once instance ants that we
found containing pollen grains were in the process of preying on non-pollen consuming
81
organisms (eg a Pachycondola species that was carrying a termite in its mandibles) or were
attracted by sugar baits
Contrary to our expectations there was no relationship between an ant speciesrsquo δ15
N ratio
and whether it had consumed pollen (Fig 32) We expected palynivory to be more common in
herbivorous ants because pollen presents a widespread source of protein a potentially limiting
resource The lack of pattern might reflect how available pollen grains are in the environment
While other forms of food may be preferable ants tend to be opportunistic foragers (Carroll and
Janzen 1973) and there is likely little cost associated with the consumption of scattered pollen
grains during foraging trips beyond the volume they occupy in the gut Some genera were found
to consistently consume pollen In support of previous observations (Baroni Urbani and de
Andrade 1997 Byk and Del Claro 2010) Cephalotes species were among the most common
pollen consumers Camponotus species also consistently contained pollen grains of the eight
species examined five contained pollen and pollen was found in species from both Peru and
PNG Camponotus species also contained the two largest numbers of pollen grains found (468
and 271) Taken together these results suggest that they may be another as yet unrecognized
commonly palynivorous genus
In general larvae did not contain large numbers of pollen grains (ranging from 1-38
grains) This is counter to what is seen in bees where larvae are the main recipients of pollen
(Willmer 2011) This difference may be because ant larvae are continually fed via trophallaxis
by adults throughout their development whereas bee larval cells are provisioned with pollen and
then sealed while larvae complete their development If both adults and larvae are able to extract
nutrients from pollen we would expect to see a preferential flow of nutrients from pollen to
larvae but not necessarily the grains themselves
82
Unlike palynivory by organisms that also provide pollination services pollen
consumption by ants is unlikely to benefit plants Ants rarely act as pollinators (Beattie et al
2004) They have pollen-killing antibiotic secretions on their cuticles (Dutton and Frederickson
2012) and they can compete with other flower visitors reducing flower visitation rates (Ness
2006) The negative impacts of flower-visiting ants are thought to have led to the evolution of
ant-repellent floral volatiles (reviewed in Willmer 2009) These volatiles can pre-empt
competition between ants and pollinators and may often be located within the pollen itself
(Willmer 2009) If floral volatiles truly are a defense against ant visitation our results suggest an
additional evolutionary pressure to not only prevent ant-pollinator interactions but to also
protect plant gametes from occasional consumption by a non-pollinating visitor
Acknowledgements
We would like to thank Jon Sanders Lina Arcila Hernandez and Eric Youngerman for providing
samples the Thomson lab for use of supplies the Pierce lab for funding support during JSrsquo and
EYrsquos collections and the staff of CICRA and the New Guinea Binatang Research Center for
logistical support The Peruvian Ministry of Agriculture provided permits (Nos 394-2009-AG-
DGFFS-DGEFFS and 299-2011-AG-DGFFS-DGEFFS) MEF acknowledges financial support
from an NSERC Discovery Grant a Connaught New Researcher Award an Ontario Ministry of
Economic Development and Innovation Early Researcher Award and the University of Toronto
ARC was supported by an Ontario Graduate Scholarship and Sigma Xi
83
References
Baroni Urbani C and M L de Andrade 1997 Pollen eating storing and spitting by ants
Naturwissenschaften 84256ndash258
Beattie A J C Turnbull R B Knox and E G Williams 1984 Ant inhibition of pollen
function a possible reason why ant pollination is rare American Journal of Botany 71421ndash
426
Bluumlthgen N G Gottsberger and K Fiedler 2004 Sugar and amino acid composition of ant-
attended nectar and honeydew sources from an Australian rainforest Austral Ecology
29418ndash429
Byk J and K Del-Claro 2010 Nectar- and pollen-gathering Cephalotes ants provide no
protection against herbivory a new manipulative experiment to test ant protective
capabilities Acta Ethologica 1333ndash38
Carroll C R and D H Janzen 1973 Ecology of foraging ants Annual Review of Ecology and
Systematics 4231ndash257
Creighton W S 1967 Studies on free colonies of Cryptocerus texanus Satschi (Hymenoptera
Formicidae) Psyche 7434ndash42
Czechowski W B Markό and A Radchenko 2009 Rubbish dumps reveal the diet of ant
colonies Myrmica schencki Em and Myrmica rubra (L) (Hymenoptera Formicidae) as
facultative pollen-eaters Polish Journal of Ecology 56737ndash741
Czechowski WB Markoacute K Erős and E Csata 2011 Pollenivory in ants (Hymenoptera
Formicidae) seems to be much more common than it was thought Annales Zoologici
61519ndash525
84
Dussutour A and S J Simpson 2009 Communal nutrition in ants Current Biology 19740ndash4
Dutton E M and M E Frederickson 2012 Why ant pollination is rare new evidence and
implications of the antibiotic hypothesis Arthropod-Plant Interactions 6561ndash569
Floren A A Biun and K E Linsenmair 2002 Arboreal ants as key predators in tropical
lowland rainforest trees Oecologia 131137ndash144
Grant B R 1996 Pollen digestion by Darwinrsquos finches and its importance for early breeding
Ecology 77489-499
Herrera L G and C Martiacutenez del Rio 1998 Pollen digestion by new world bats effects of
processing time and feeding habits Ecology 792828ndash2838
Houmllldobler B and Wilson E O 1990 The Ants 1st Edition Harvard University Press
Cambridge
Horskins K and Turner V B 1999 Resource use and foraging patterns of honeybees Apis
mellifera and native insects on flowers of Eucalyptus costata Australian Journal of
Ecology 24221-227
Ness J H 2006 A mutualismrsquos indirect costs the most aggressive plant bodyguards also deter
pollinators Oikos 113506ndash514
Roulston T H I and J H Cane 2000 Pollen nutritional content and digestibility for animals
Plant Systematics 222187ndash209
Van Tets I G 1997 Extraction of nutrients from Protea pollen by African rodents Belgian
Journal of Zoology 12759-65
Willmer P G 2011 Pollination and Floral Ecology 1st Edition Princeton University Press
New Jersey
85
Willmer P G C V Nuttman N E Raine G N Stone J G Pattrick K Henson P Stillman
et al 2009 Floral volatiles controlling ant behaviour Functional Ecology 23888ndash900
86
Tables
Table 31 Numbers of adult worker and larval ants examined for each species collected in Peru
Numbers of pollen grains found in individual samples are given in parentheses after the sample
size when there were no grains no numbers are given
Genus Species Number of adults analyzed
(numbers of grains found)
Number of larvae analyzed
(numbers of grains found)
Dolichoderinae
Azteca sp 1 3 0
sp 2 2 0
sp 3 0 2
sp 4 3 (1) 0
sp 5 3 (2) 0
Dolichoderus decollatus 3 (66) 0
sp 1 3 0
sp 2 3 1 (2)
sp 3 3 3
sp 4 0 2
Ecitoninae
Labidus sp 1 3 0
Formicinae
Camponotus sp 1 3 3
sp 2 3 (82) 1 (2)
sp 3 3 (3) 0
sp 4 0 2
87
sp 5 8 (11) 0
sp 6 4 (7 16 468) 0
sp 7 5 (12) 0
sp 8 2 (1) 0
Paratrechina sp 1 4 (11) 0
Myrmelachista sp 1 3 (5 11 18) 0
sp 2 0 3
Trachymyrmex sp 1 3 (21) 0
sp 2 3 0
Myrmicinae
Atta sp 1 3 0
Cephalotes atratus 3 (2 2 21) 0
placidus 3 (1 4 21) 0
sp 1 3 0
sp 2 1 0
sp 3 2 0
Crematogaster sp 1 3 1
sp 2 3 (35) 0
sp 3 9 (7) 0
sp 4 2 0
Megalomyrmex sp 1 3 0
sp 2 0 2
sp 3 4 (7) 0
sp 4 3 0
Monomorium sp 1 0 3 (2)
88
Pheidole sp 1 0 3
sp 2 3 0
sp 3 0 3
Procryptocerus sp 1 3 0
Solenopsis sp 1 0 3
sp 2 3 (3 5) 0
Wasmannia sp 1 3 0
Ponerinae
Pachycondola sp 1 3 (3) 0
Odontomachus sp 1 3 (34) 0
Pseudomyrmicinae
Pseudomyrmex sp 1 3 0
sp 2 3 0
sp 3 3 (9) 0
sp 4 0 3
sp 5 0 3
89
Table 32 Numbers of adult worker and larval ants examined for each species collected in Papua
New Guinea Numbers of pollen grains found in individual samples are given in parentheses
after the sample size when there were no grains no numbers are given
Genus Species Number adults analyzed
(grains found)
Number larvae analyzed
(grains found)
Aenictinae
Aenictus sp 1 3 3 (1 1)
Dolichoderinae
Philidris sp 1 3 1 (4)
Tapinoma sp 1 2 3 (38)
Technomyrmex sp 1 0 3 (12 12)
Ectatomminae
Rhytidoponera sp 1 1 3 (2)
Formicinae
Acropyga sp 1 3 3 (2)
sp 2 3 3
sp 3 3 3
Anoplolepsis sp 1 3 (1) 3 (1)
Calomyrmex sp 1 3 3 (1)
Camponotus sp 1 3 (1 271) 3
sp 2 1 3
sp 3 2 0
Nylanderia sp 1 3 3
Opisthopsis sp 1 3 3
Polyrachis sp 1 1 3 (4)
90
Myrmicinae
Crematogaster sp 1 3 3
sp 2 3 (32) 3
Monomorium sp 1 3 (1) 3 (1)
sp 2 3 3 (1 1 1)
Pheidole sp 1 3 3
Pristomyrmex sp 1 3 3 (11)
Pyramica sp 1 3 0
Ponerinae
Odontomachus sp 1 3 3 (4)
91
Figures
Figure 31 Photographs of pollen grains found in two ant species a) Dolichoderus sp 2 and b)
Camponotus sp 6 Scale bars = 50 microm
92
Figure 32 Relationship between trophic level as measured by δ15
N ratio and pollen presence
for 48 neotropical ant species 1 Atta sp 5 Azteca spp 8 Camponotus spp 5 Cephalotes spp 4
Crematogaster spp 5 Dolichoderus spp 1 Labidus sp 3 Megalomyrmex spp 1 Monomorium
sp 2 Myrmelachista spp 1 Odontomachus sp 1 Pachycondola sp 1 Paratrechina sp 3
Pheidole sp 4 Pseudomyrmex spp 2 Solenopsis spp and 1 Wasmannia sp
93
Concluding Remarks
Ants have traditionally been considered to be unwelcome flower visitors they consume floral
rewards ostensibly meant for pollinators while rarely providing pollination services themselves
In this thesis I investigate the validity of this assumption and use an experimental study and an
observational study to examine two ways that ants may impact plant reproduction
In the first chapter I review the literature on ants as flower visitors I show that the common
assumption of ants always being categorically negative for plant reproduction is incorrect while
ants may reduce floral attractiveness to pollinators the net effect of ants on plant fitness
measures is variable ranging from positive to negative to neutral I also discuss ants as
pollinators and ways that plants may minimize ant visitation to flowers before finishing with a
section highlighting questions that still need investigation
In the second chapter I explore how interference competition with ants can structure bumblebee
foraging behavior and how this may alter pollen movement patterns Using artificial flowers with
male or female function I allowed Bombus impatiens bumblebees to forage on flowers that
either did or did not have Myrmica rubra ants on them Ant presence significantly reduced the
amount of pollen analogue donated and received by flowers Building on these results I found
that the presence of ant scent alone elicited similar changes to bumblebee behavior reducing the
amount of pollen analogue donated by male flowers My results show that interference
competition is sufficient to alter bumblebee foraging behavior and that the effects of ants on
pollinators may extend after ant visitation to flowers Further study is needed to determine if bees
are able to differentiate between ant species by visual or olfactory cues Their ability to do so
94
could affect how generalized their response to ant presence is and may help to better predict how
ants will affect plant reproduction
In the final chapter I investigate how common palynivory is amongst tropical ant species I used
acetolysis and light microscopy to examine whole ants larvae and ant guts from Papua New
Guinea and Peru I found that tropical ants consistently contain pollen grains albeit often in
small numbers (lt5) However some genera notably Camponotus and Cephalotes consistently
contained higher numbers providing the first evidence that Camponotus may be a palynivorous
genus The small numbers of pollen grains present may indicate that ants preferentially exploit
other more easily digestible high-protein foods over pollen Future studies should investigate
exactly what role pollen plays in ant diets by supplementing the diet of captive ant colonies with
pollen This would allow us to examine how pollen consumption affects colony demography as
well as how efficiently ants digest pollen and whether ants exhibit pollen preferences
I bring up several pressing and unanswered questions within this thesis and highlight three here
First numerous plant species possess chemical or physical traits assumed to increase plant
fitness by reducing ant visitation However this has rarely been tested and instead research has
focused predominately on documenting ant repellence We should move away from this and
begin examining plant fitness in the presence or absence of these repellent traits to see how
beneficial these traits truly are Second how flower visiting ants impact male fitness has been
almost completely ignored in favor of female fitness Future studies should investigate how ant
visitation affects both pollen donation and pollen movement patterns Lastly research involving
antibiotics present on ant cuticles and their effect on pollen viability has been too small in scope
often limited to demonstrating the pollen killing effects of ant secretions Investigation into
95
whether plants have evolved any resistance to the secretions of the local ant community or why
some plants appear unaffected by their ant partners may yield exciting results
In conclusion ants have the capacity to strongly affect plant reproduction both positively and
negatively The fact that these diametrically opposing forces exist means we cannot approach
flower-visiting ants as either positive or negative a priori Instead we should strive to work
towards a better understanding of what is occurring in the system of interest and what role ants
play within it
96
Copyright Acknowledgments
Chapter 2 ldquoAnts and ant scent reduce bumblebee pollination of artificial flowersrdquo is current in
press in The American Naturalist Request for its inclusion here was sought and approved