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© 2016 Adriana Corrales Osorio
ECTOMYCORRHIZAL ASSOCIATIONS IN TROPICAL MONTANE FOREST: INSIGHTS INTO THEIR INFLUENCE ON NUTRIENT CYCLING AND FUNCTIONAL RESPONSES
TO SOIL FERTILITY
BY
ADRIANA CORRALES OSORIO
DISSERTATION
Submitted to fulfillment of the requirements for the degree of Doctor of Philosophy in Plant Biology
in the Graduate College of the University of Illinois at Urbana-Champaign, 2016
Urbana, Illinois
Doctoral Committee:
Professor James W. Dalling, Chair and Director of Research Professor Ken N. Paige Assistant Professor Katy Heath Associate Professor Anthony Yannarell Assistant Professor Scott Mangan
ii
Abstract
In Panamanian montane forest, the ectomycorrhizal tree Oreomunnea mexicana forms
monodominant stands where it accounts for up to 70% of individuals. Monodominance is
unexpected in tropical forest because the accumulation of host-specific pathogens is thought to
limit the local abundance of individual species. Although rare, monodominant forests have now
been recognized in all major tropical regions and include a wide diversity of tree taxa. Notably,
many monodominant species associate with a particular type of mutualist: ectomycorrhizal (EM)
fungi. Ectomycorrhizal associations are rare in tropical forests where most species associate with
arbuscular mycorrhizal fungi.
Oreomunnea associates with a diverse community of ectomycorrhizal fungi. In a survey
of Oreomunnea root tips using sanger sequencing, I identified 115 EM fungal taxa from 234 EM
Oreomunnea root tips collected from four sites across a soil fertility gradient. There was a high
compositional turnover in the EM fungal communities associated with Oreomunnea with a
significant effect of soil fertility on EM fungal compositional variation. In addition, analysis of
the phylogenetic beta diversity for Russula, the most abundant and diverse EM genus in the
community, revealed that Russula species show greater than expected phylogenetic dissimilarity
among taxa occupying sites with contrasting fertility.
Current theory on how monodominance is maintained has focused on alterations to plant-
microbial interactions. I tested three potential mechanisms by which EM fungi may potentially
allow a host tree species to achieve monodominance: (1) by conferring resistance to soil-borne
pathogens that are responsible for negative plant-soil feedback experienced by competing
species, (2) by connecting juveniles to adults through ectomycorrhizal networks that transfer
water, nutrients or carbon, and (3) by altering ecosystem nutrient economies, thereby reducing
the availability of limiting resources to competing species. After testing these three hypotheses I
found no evidence for positive feedback on Oreomunnea abundance caused by either pathogen
resistance or the formation of mycorrhizal networks. Instead, the presence of EM fungi was
associated with a reduction in inorganic nitrogen availability tightening the nitrogen cycle,
making it difficult for other, non-EM tree species to compete.
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Ectomycorrhizal fungi have been shown to respond differently to N addition depending
on their functional traits. I studied EM fungal communities associated with Oreomunnea in
control plots and plots that had received a nitrogen addition treatment for nine years, I found a
significant difference in the species composition of the EM fungal community between plot
treatments, and differences in the abundance of some genera. Members of the EM fungal genera
Laccaria and Lactarius, showed an increase in their relative abundance with N addition while
members of the genus Cortinarius showed a strong reduction in relative abundance. Increased N
availability in tropical ecosystems could result in a reduction in EM fungal taxa specialized in
organic N and P absorption (e.g., Cortinarius) along with a decrease in EM colonization of host
plants potentially having implications in soil C storage, and ecosystem N cycling ultimately
affecting forest productivity and diversity.
The isotopic composition of EM fruiting bodies has been shown to be a useful tool for
understanding the functional role of EM fungi in ecosystems. After analyzing the δ15N and δ13C
of Russula fruiting bodies, and its correlation with Oreomunnea host abundance and soil
inorganic N availability, I found that the isotopic composition of the Russula community reflects
increased host demand for ectomycorrhizal fungal nitrogen supply with a reduction in soil
inorganic nitrogen availability. These results are consistent with an increase in N sequestration
by EM fungi in sites with higher host abundance. Given the high correlation of host abundance,
N availability, and N transfer from EM fungi to the host (reflected in fruiting body δ15N) in our
system here I provide further evidence that the formation of Oreomunnea dominated forest is
facilitated by its associated EM fungi.
In conclusion I found that EM fungi are highly diverse in tropical montane forest and that
they can facilitate the formation of monodominant forest of EM associated tree species by
altering the N cycle. Also, I predicted increase in N availability due to atmospheric N deposition,
could potentially alter the interaction between EM fungi and their host plant potentially leading
to biotic and abiotic changes in the ecosystem.
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To my parents, Julia Osorio and Fernando Corrales,
Thank you for your great support and inconditional love.
and
To Dr. Esperanza Franco-Molano, the person that motivated me to explore the world of mycology
v
Acknowledgments
I would especially like to thank Dr. Jim Dalling for acting as my adviser, and for his
committed guidance and assistance during the research and preparation of my thesis. I would
also like to thank all the members of my committee for providing helpful suggestions and
comments during the development of this project. I would like to thank my collaborators Scott
Mangan, Ben Turner, Betsy Arnold, Astrid Ferrer, Clark Ovrebo, and Leho Tedersoo for all their
guidance in their topic of expertise and their useful comments to my thesis.
I would like to thank to the many people that have contributed their help to make this
project possible. I would like to thank my fellow lab mates Katie Heineman, Cecilia Prada,
Jennifer Jones, Camilo Zalamea, and Carolina Sarmiento for their help their help during the
development of the project. For their assistance with molecular lab work I would like to thank
Dr. Katy Heath and her lab personnel for hosting the DNA lab work, also Kayla Arendt, Jana
U’Ren, Pat Burke, and Sten Anslan. Also would like to thank Mike Masters, Dayana Agudo,
Aleksandra Bielnicka, and Iulianna Taritsa for help with resin bag and isotopic analysis and
Carmen Velasquez, Carlos Espinosa, Marggie Rodriguez, Pedro Caballero, Freddy Miranda, Jan
Miranda and Evidelio Garcia for their help with fieldwork and logistic support in Panama.
Finally I would like to thank Aaron DiMartino and Shawn Brown for their useful comments on
the early versions of several chapters.
There were also many institutions that made this project possible thanks to their generous
funding. That includes COLCIENCIAS (497-2009 Programa de Formación doctoral “Francisco
José de Caldas”), Smithsonian Tropical Research Institute Short-Term Fellowship and Pre-
doctoral fellowships, NSF Dissertation Improvement Grant (Award Id: 1501483), Tinker
Summer Research Fellowship, Francis M. and Harlie M. Clark Research support grant, Robert L.
Gilbertson Mycological Herbarium Grant (University of Arizona), and Dissertation Improvement
Grant from the Graduate College at University of Illinois.
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TABLE OF CONTENTS CHAPTER 1: INTRODUCTION .............................................................................................................. 1 CHAPTER 2: VARIATION IN ECTOMYCORRHIZAL FUNGAL COMMUNITIES ASSOCIATED WITH OREOMUNNEA MEXICANA (JUGLANDACEAE) IN A NEOTROPICAL MONTANE FOREST ................................................................................................................................. 5 CHAPTER 3: AN ECTOMYCORRHIZAL NITROGEN ECONOMY FACILITATES MONODOMINANCE IN A NEOTROPICAL FOREST ..................................................................... 53 CHAPTER 4: NITROGEN ADDITION ALTERS ECTOMYCORRHIZAL FUNGAL COMMUNITIES AND SOIL ENZYME ACTIVITIES IN A TROPICAL MONTANE FOREST . 85 CHAPTER 5: VARIATION IN STABLE ISOTOPES OF RUSSULA SPECIES ASSOCIATED WITH OREOMUNNEA MEXICANA IN A TROPICAL MONTANE FOREST ............................. 119 CHAPTER 6: CONCLUSIONS ............................................................................................................. 146
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Chapter 1: Introduction
Mycorrhizal associations – which are mutually beneficial associations between plants and
fungi-- play a critical role in nutrient acquisition and, therefore, the growth and survival of
plants. There are several kinds of mycorrhizas that occur across vascular plants, however two
types predominate: Arbuscular mycorrhizas (AM), that are most frequent associations in
neotropical ecosystems, and Ectomycorrhizas (EM), which dominate in boreal and many
temperate ecosystems (Smith & Read 2008). However, in contrast to this general pattern, some
tropical forests support EM tree species that reach high abundance and host a considerable
diversity of EM fungi (Peay et al. 2010, Smith et al. 2011).
A defining feature of tropical forests is the presence of a high biodiversity, where
hundreds of tree species occur in areas of a few hectares or less. While most attention from
ecologists has been directed toward understanding how these species coexist, less attention has
been given to the exception to this pattern, known as ‘monodominance’. Monodominant forests
arise when a single tree species accounts for the majority of individuals in a stand. Although an
infrequent occurrence, monodominant forests have now been recognized in all major tropical
regions and include a wide diversity of tree taxa. Monodominance is unexpected in tropical
forest because the accumulation of host-specific pathogens is thought to limit the local
abundance of individual species (Klironomos 2002, Bell et al. 2006, Mangan et al. 2010).
Current theory on how monodominance is maintained has therefore focused on alterations to
plant-microbial interactions. Notably, many monodominant species associate with
ectomycorrhizal fungi.
For my doctoral dissertation, I characterized the general structure of the ectomycorrhizal
(EM) fungal community in stands of the EM tree Oreomunnea mexicana (Juglandaceae) at
Fortuna Forest Reserve in western Panama. Oreomunnea is distributed from Mexico to Panama
at 900–2600 m.a.s.l., becoming dominant in some locations where it accounts for up to 70% of
individuals. Oreomunnea forms EM associations in contrast to almost all co-occurring species at
Fortuna. For my second chapter I described the EM fungal community associated with
Oreomunnea populations. Sequencing of fungal nrITS DNA revealed 115 EM fungi taxa
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(including 36 Russula) from 234 EM Oreomunnea root tips collected from four sites across a soil
fertility gradient. Soil fertility did not have an effect on overall EM fungal species richness,
however high compositional turnover was observed among sites located < 6 km apart, with a
significant effect of soil fertility on EM fungal compositional variation. Analysis of the
phylogenetic diversity of the Russula sequences also revealed greater than expected phylogenetic
dissimilarity among taxa occupying sites with contrasting fertility, suggesting that environmental
filtering is important in structuring EM fungal communities. This study was published in the
journal Mycorrhiza and is coauthored by Drs. Astrid Ferrer, Elizabeth Arnold, Benjamin Turner,
and James Dalling.
For my third chapter, I tested three hypotheses to explain how EM fungi allow a host tree
species to achieve local dominance: (1) by conferring resistance to soil-borne pathogens that are
responsible for negative plant-soil feedback, (2) by enabling transfer of water, nutrients or
carbon through ectomycorrhizal networks, and (3) by reducing the availability of nitrogen (N) to
competing species due to the capacity of EM fungi to access organic N sources via extracellular
enzymes. I tested for plant soil-feedback using a greenhouse experiment in which growth of
seedlings of five species was measured after addition of conspecific or heterospecific soil
inocula. I also tested for EM network effects in the field using nylon mesh to exclude hyphal
connections. I found no evidence for positive plant-soil feedback or that EM networks confer a
competitive advantage to Oreomunnea seedlings. However, I found ~3-fold higher nitrate and
ammonium concentrations outside than inside Oreomunnea-dominated forest. Additionally, I
found lower bulk soil stable δ15N in Oreomunnea-dominated forest compared with adjacent AM
mixed forest. Nitrification and denitrification processes strongly discriminate against 15N,
leaving behind 15N-enriched N in the soil. Therefore soils with low inorganic N and low
mineralization rates, show reduced δ15N of the soil N pool. This indicates a tighter N cycle in
Oreomunnea-dominated forest due to depletion of N from the soil organic matter pool by EM
fungi. This study was published in the journal Ecology Letters and was coauthored by Drs. Scott
Mangan, Benjamin Turner, and James Dalling.
For my fourth chapter I studied the changes in EM fungal community composition after
long-term N fertilization along with changes in the soil in enzymatic activity associated with
carbon (C) and nitrogen (N) mineralization. In N-limited temperate forests, long-term increases
3
in N availability can reduce the species richness and alter the community composition of EM
fungi. It has been proposed that differences in the sensitivity to N addition among EM fungi is
associated with fungal strategies of growth and colonization, and nutrient acquisition strategies
that can vary considerably among taxa. Consistent with this, my results revealed that nine years
of N fertilization are enough to significantly change the species composition of the EM fungal
community associated with Oreomunnea and affect the abundance of some genera. Members of
the EM fungal genera Laccaria and Lactarius, showed an increase in their relative abundance
with N fertilization while members of the EM fungal genus Cortinarius showed a strong
reduction in relative abundance. Consistent with this pattern, the genera Laccaria and Lactarius
have previously been reported to increase in abundance in response to high N availability and to
uptake labile N forms, while Cortinarius has been reported as showing a consistent negative
association with N availability and to be capable of absorbing organic N in temperate forest
(Lilleskov et al. 2011). I also found lower phosphatase activity in the soil in N addition plots
potentially linked with a reduction in the abundance of EM fungi, and reflected in lower EM
colonization of Oreomunnea roots. This study will be submitted to the journal Soil Biology and
Biogeochemistry.
For my fifth chapter I studied the variation in the isotopic composition of Russula species
found in a gradient of soil inorganic N availability and host abundance. The genus Russula is an
important component of the EM community at our study site and accounts for almost half of the
EM fungal species. I found that the Russula community average δ15N was positively correlated
with the abundance of the host species Oreomunnea mexicana while δ13C was negatively
associated with host abundance. I also found that the abundance of Oreomunnea was negatively
correlated with soil inorganic N availability as expected based on results from chapter two. This
δ15N enrichment and δ13C depletion of Russula fruiting bodies with increase in host abundance
and decrease in inorganic N availability is evidence of a higher transfer rate of N from Russula to
their host plant and higher transfer of C from the host plant to Russula due to a higher
Oreomunnea N demand. As proposed in chapter 2, low soil inorganic N availability conditions
under Oreomunnea dominated forest are created by competition between EM fungi and the
community of free living saprotrophs (Averill et al. 2014), suggesting that EM fungi create soil
conditions that increase Oreomunnea dependency on EM fungal N. These findings are in line
4
with the results from the second chapter of this dissertation supporting that EM fungi associated
with Oreomunnea can cause a reduction in soil inorganic N availability facilitating the
maintenance of Oreomunnea monodominant forest. Here I provide additional evidence that this
reduction in N availability could be caused by an increase in N sequestration by EM fungi in
sites with higher host abundance due to higher C transfers from Oreomunnea to its associated
EM fungi in sites with lower fertility.
In the final chapter I summarize the main results from each of the above-mentioned
chapters and give an overview of the mechanisms facilitating the monodominance of
Oreomunnea mexicana at Fortuna Forest Reserve. I also discuss the implications of high
abundance of EM fungi in tropical montane forest for soil carbon sequestration and N cycling.
5
Chapter 2: Variation in ectomycorrhizal fungal communities associated with Oreomunnea mexicana (Juglandaceae) in a Neotropical montane forest1
Introduction
Nutrient uptake and transfer via mycorrhizal associations strongly influences the growth
and survival of most plant species in nearly all of earth’s most species-rich and threatened
terrestrial biomes (Bonfante and Genre 2010; Smith and Read 2008). In tropical forests, trees
predominantly form associations with arbuscular mycorrhizal (AM) fungi (Glomeromycota)
(Béreau and Garbaye 1994, Janos 1983, McGuire 2008, Onguene and Kuyper 2001, St John
1980, St John and Uhl 1983). However, forests dominated by tree species that associate with
ectomycorrhizal (EM) fungi, especially Basidiomycota, have been recognized in all major
tropical regions (Becker 1983, Connell and Lowman 1989, Hart et al. 1989, Henkel 2003).
Ectomycorrhizal plants in lowland tropical forests belong mostly to the Dipterocarpaceae and
Fabaceae (primarily a narrow group of Caesalpinioideae), whereas Fagales (including members
of the Juglandaceae, Betulaceae and Fagaceae) frequently occur in montane sites (Itoh 1995,
Conway and Alexander 1992, Hart et al. 1989, Henkel 2003, Morris et al. 2008). In some cases
these EM species grow in ‘monodominant’ forests, wherein a single tree species accounts for
more than 50% of canopy trees in a stand (Connell and Lowman 1989). Why these
monodominant forests persist in otherwise diverse plant communities is not fully understood
(Peh et al. 2011).
Mast fruiting, low rates of disturbance, high tolerance of shade by seedlings, slow litter
decomposition, and escape from herbivory have been proposed as mechanisms to explain
tropical monodominance (reviewed by Peh et al. 2011). Strikingly, a common feature of many
monodominant tree species in tropical forests is the formation of EM associations (Connell and
Lowman 1989, Malloch et al. 1980, Henkel 2003). In temperate forests, natural isotope
1 This chapter appeared in its entirety in the Journal Mycorrhiza and is referred to later in this dissertation as “Corrales et al. 2016a”. Corrales, A.; Arnold A.E.; Ferrer, A; Turner, BL; and Dalling J.W. 2016. Variation in ectomycorrhizal fungal communities associated with Oreomunnea mexicana (Juglandaceae) in a Neotropical montane forest. Mycorrhiza 26:1–17. This article is reprinted with the permission of the publisher and is available from http://link.springer.com/article/10.1007/s00572-015-0641-8 and using DOI: 10.1007/s00572-015-0641-8
6
abundance and radio-isotopic labeling experiments have shown that some EM tree species can
develop EM networks, where hyphal connections transfer water, carbon, and nutrients from adult
to juvenile plants (Booth and Hoeksema 2010, Simard et al. 1997, Plamboeck et al. 2007, see
Simard et al. 2012 for review). In tropical forests, direct evidence of resource transfer among
individuals is currently lacking, but decreased survival and growth of seedlings when isolated
from neighboring plants is consistent with EM network effects (McGuire 2007, Onguene and
Kuyper 2002).
Ectomycorrhizal networks may increase survival of conspecific seedlings in a spatially
structured fashion, disproportionately increasing their abundance near adult trees (Henkel 2003,
McGuire 2007, Onguene and Kuyper 2002, Booth and Hoeksema 2010, Teste et al. 2009) in a
manner consistent with positive plant–soil feedbacks (reviewed by Bever et al. 2012). In turn, the
presence and strength of plant–soil feedback depends on the functional traits and taxonomic
composition of the EM fungal community (e.g., Dickie et al. 2002, O’Brien et al. 2010, Kennedy
et al. 2012). Determinants of EM fungal community composition remain poorly understood in
tropical forests. For example, there is conflicting evidence regarding host specificity in tropical
EM fungal communities (e.g., for evidence of host preference, see Morris et al. 2009, Tedersoo
et al. 2008, and Tedersoo et al. 2010a, for evidence of low host specificity, see Diédhiou et al.
2010, Smith et al. 2011, 2013, and Tedersoo et al. 2011). Similarly the influence of soil type on
EM fungal community composition remains unresolved, in part because EM fungal communities
associated with the same host species have not been studied across a range of soil conditions.
Here we examine EM fungal communities associated with Oreomunnea mexicana
(Standl.) J.-F. Leroy, a widely distributed neotropical tree in the walnut family (Juglandaceae),
and one of the few examples of a monodominant EM species in the Neotropics. In montane
forests in western Panama, Oreomunnea forms locally monodominant stands within otherwise
highly species-rich forest comprised mostly of taxa that form AM associations (Andersen et al.
2010). In this region, Oreomunnea forms dominant stands on several distinct soil types that are
distributed over a scale of only a few kilometers. These soils are derived from contrasting parent
materials and occur in areas that differ in the seasonality and quantity of annual rainfall
(Andersen et al. 2010), making this system unique for the study of EM fungal ecology.
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Preliminary field surveys of fungal fruiting bodies indicated that diverse communities of EM
fungi associate with Oreomunnea in these stands (A. Corrales et al. unpublished data).
In this first characterization of the EM fungal community associated with Oreomunnea,
we used data generated from root tips of seedlings, saplings, and adult trees across this landscape
to test four predictions. First, we predicted that infection frequency of EM fungi would be lower
in more fertile soils, consistent with the general view that benefits of EM fungi depend on soil
conditions (Treseder 2004). Second, we predicted that the diversity, composition, and
phylogenetic diversity of EM fungi would vary with soil fertility. Third, we expected to see (a)
commonalities in EM fungal communities shared across seedling, sapling, and adult life stages
of Oreomunnea, and that (b) community similarity among developmental stages would be
particularly strong in the lowest fertility soils, where selection for EM networks or particularly
beneficial symbionts would likely be strongest. Fourth, we expected lower phylogenetic diversity
of EM fungi in high-fertility sites, reflecting lower colonization rates and consequently lower
community diversity.
Methods
The study focused on stands of Oreomunnea mexicana (Juglandaceae; hereafter,
Oreomunnea) in three watersheds in a primary lower montane forest (1000–1400 m.a.s.l.) in the
Fortuna Forest Reserve in western Panama (Figure 2.1; hereafter, Fortuna; 8°45 N, 82°15 W).
Oreomunnea is a mid-elevational canopy tree distributed from southern Mexico to western
Panama at 900–2600 m.a.s.l. (Stone 1972). It produces ca. 100 mg, wind-dispersed fruits, which
can generate high-density seedling patches in the understory (Table 2.1). Oreomunnea is locally
dominant at some of our study sites, accounting for up to 70% of individuals and stand basal area
at the Honda watershed (A. Corrales unpublished data). Dominance by Oreomunnea is not
directly related to particular functional traits such as leaf chemistry (i.e., nitrogen (N) and
phosphorus (P)) and wood density, which are close to community averages for the area (K.
Heineman unpublished data). However, Oreomunnea forms EM associations (as previously
reported from Mexican populations; Quist et al. 1999), in contrast to almost all co-occurring tree
species at Fortuna. Other EM tree species that are present in the study area (i.e., Quercus
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insignis, Q. cf lancifolia, and Coccoloba spp.) occur at low densities (typically <10 individuals
>10 cm DBH per ha) and are present within and outside of Oreomunnea-dominated stands.
Climate records indicate that the mean annual temperature for Fortuna ranges from 19 to
22˚C (Cavelier 1996). Annual rainfall averages ca. 5800 mm at our sites in Hornito and Alto
Frio, and 9000 mm at our sites in Honda A and Honda B, although all were drier during our
study (Table 2.1; Figure 2.1). Hornito and Alto Frio typically have 1–2 months per year with
<100 mm of precipitation; in contrast, no months with <100 mm of rainfall have been recorded
over the seven-year period for which records are available at Honda A and Honda B (Andersen
et al. 2012, J. Dalling unpublished data).
In addition to differences in rainfall, these sites differ markedly in soil characteristics,
with contrasting pH, N, P and base cation availability (Table 2.1). These distinctive soil traits are
related to underlying geology: low-fertility Ultisols at Honda A and Honda B are derived from
rhyolite, whereas high fertility soils at Hornito (Ultisol) and Alto Frio (Inceptisol) are derived
from dacite and andesite (Andersen et al. 2012, B. Turner unpublished data). Sites differing in
soil fertility are ca. 6 km apart, with 200 m separating the two low fertility sites (Honda A and B)
and 1 km separating the two high fertility sites (Hornito and Alto Frio). A third soil type of
intermediate fertility derived from andesite separates the high and low fertility sites in this study,
and does not support populations of Oreomunnea (Andersen et al. 2010). Characteristics of our
study sites are shown in Table 2.1, and methods for distinguishing potential effects of spatial
proximity, fertility, and rainfall on community structure of EM fungi are described below.
Sampling of ectomycorrhizas
Root tips of Oreomunnea were collected at all sites between January and July 2012
(Table 2.2). At Honda A, Honda B, and Hornito, samples were collected from a total of 44
individuals per site: four adults per site (mean DBH = 50 cm) located > 50 m apart; five
seedlings (5 – 20 cm height) within 20 m of each adult; and five saplings (40 – 100 cm height)
within 20 m of each adult. The area where the trees were sampled was approximately 4500 ±
3500 m2 per site. Adults and juveniles of Oreomunnea were less common at Alto Frio, such that
17 individuals were sampled there (four adults, nine seedlings, and four saplings).
9
Five lateral roots were excavated 2–3 m from the trunk of each adult tree until fine roots that
were clearly connected to the tree were found. From each adult we collected up to 50 cm of total
root length representing multiple root branches. All roots obtained from adult trees were included
in field collections even if EM fungal infection was not visible macroscopically. The entire root
system of each focal seedling and sapling was collected.
Roots were stored in plastic bags and refrigerated within 2 h of collection. Each sample
was carefully cleaned with tap water, cut into 1 cm pieces, and observed with a dissecting
stereoscope. Three 1 cm pieces with EM structures were collected haphazardly from each sample
and preserved in 95% alcohol at 4˚C for DNA extraction. Infection frequency was calculated for
each sample as the number of root tips among 10 haphazardly chosen 1 cm fragments with
presence of an EM mantle observed under a dissecting microscope.
Molecular analysis
Molecular analyses followed Peay et al. (2011) with slight modifications. Genomic DNA
was extracted from EM root tips and the internal transcribed spacers and 5.8S rDNA of fungal
associates was amplified directly using the REDExtract–N–Amp plant PCR kit (following the
manufacturer’s instructions; Sigma-Aldrich) with primers ITS1F and ITS4 or ITS4B. PCR
conditions consisted of 95˚C for 1 min, and then 35 cycles of 95˚C for 30 sec, 52˚C for 30 sec,
and 72˚C for 45 sec, with a final extension time at 72˚C for 10 min. PCR amplicons were
visualized on 1.5% agarose gel stained with ethidium bromide or SYBR Green. Positive products
were cleaned using ExoSap–IT (Affymetrix; Santa Clara, CA, USA: 1.5 uL Exosap, 7.5 uL PCR
product) and sequenced bidirectionally using the Applied Biosystems BigDye Terminator v3.1
cycle sequencing kit and the original PCR primers on an Applied Biosystems 3730xl DNA
Analyzer (Foster City, CA, USA) at the University of Arizona Genetics Core (UAGC).
Sequences were assembled and quality scores were assigned using phred and phrap (Ewing and
Green 1998; Ewing et al. 1998) with orchestration by Mesquite v. 1.06
(http://mesquiteproject.org), and then manually verified and edited in Sequencher 5.1 (Gene
Codes Corporation, MI, USA) following U’Ren et al. (2012). Sequences were assigned to
operational taxonomic units (OTUs) using a 97% sequence similarity cutoff (see Smith et al.
2007a, Hughes et al. 2009) with Sequencher 5.1 (see Arnold et al. 2007, U’Ren et al. 2009).
10
Statistical analyses
Two-way ANOVA was used to assess the effect of developmental stage (seedling,
sapling and adult) and site (Honda A, Honda B, Hornito and Alto Frio) on infection frequency.
An alternative model was also assessed where sites were grouped into high fertility/low rainfall
sites (Hornito and Alto Frio) and low fertility/high rainfall sites (Honda A and B). Species
accumulation curves were used to compare OTU richness among developmental stages and sites.
Total species richness was estimated using the bootstrap estimator (Smith and van Belle 1984;
U’Ren et al. 2012). Singleton OTUs were excluded to provide a robust data set to compare the
diversity of EM fungal communities among sites and host developmental stages.
To explore broader patterns of EM fungal diversity we compiled records from EM fungal
inventories of temperate and tropical forests, including studies reviewed by Tedersoo et al.
(2012) and three more recent studies Diédhiou et al. (2014), Smith et al. (2013), and Kennedy et
al. (2012). We calculated diversity for data presented in each study using Fisher’s alpha, which is
robust to differences in sample size and compared temperate vs. tropical forests using a one-way
ANOVA.
Differences in EM fungal community composition among sites and developmental stages
were visualized by Nonmetric Multidimensional Scaling (NMDS). Only nonsingleton OTUs
were used in these analyses, allowing us to evaluate the distributions of the more common
species while reducing the potential for rare species, whose occurrence in the dataset may be
influenced by undersampling, to influence inferences about composition. Nonetheless, results
with and without singletons were very similar (results not shown). NMDS analyses were based
on Bray-Curtis dissimilarity matrices using abundance and presence-absence data. Significance
of visualized differences was determined using permutational analyses of dissimilarity
(ADONIS) using 200 permutations and a Euclidean distance matrix (Oksanen et al. 2008).
Because location, soil fertility, and rainfall patterns were correlated in this study, we used
a statistical approach to explore the interplay of these factors with regard to observed community
structure. A Mantel test based on 999 permutations first was used to examine the relationship of
species community composition to geographic distance among sites. However, geographic
11
proximity also reflected environmental similarity (Figure 2.1, Table 2.1). Therefore, a principal
component analysis (PCA) was used to reduce 14 environmental variables (see Table 2.1) to two
axes (describing 62.56% and 15.30% respectively of the total variance). Environmental variables
included in the PCA were site-specific annual rainfall from May 2012–April 2013, and soil
characteristics: bulk density (g cm-3), total C, inorganic N, and P (mg cm-3), NaOH–EDTA
inorganic P (µg cm-3), NaOH–EDTA organic P (µg cm-3), resin P (µg cm-3), NH4 (µg cm-3), NO3
(µg cm-3), K2SO4 extractable organic C (µg cm-3), and base cations Al, Ca, and K (cmol L-1). The
first two of the resulting PCA axes were used in a NMDS analysis, with correlation coefficients
between the PCA axes and the NMDS axes identifying differences between sites with
contrasting fertility and rainfall patterns (Ter Braak 1995). All statistical analyses were carried
out using the package vegan 2.0-6 in R 2.15.1 (R Development Core Team 2011).
Taxonomic placement
Taxonomic placement of OTUs was estimated by comparisons via BLAST with
GenBank (blastn; Altschul et al., 1990) and the UNITE database (Kõljalg et al. 2013). The
databases gave matching results with high confidence at the genus level for 92% of sequences
(Table 2.5). The remaining 8% of sequences showed <50% query length and were left as
undetermined (19 sequences representing 12 OTUs). Sequences that matched named sequences
at 91%–97% identity in GenBank and UNITE were identified only to genus. Genus names were
only assigned to OTUs when all sequences within the OTU returned the same genus name after
BLAST/UNITE searches. For Russula, species-level taxonomic placement of OTUs also was
informed by phylogenetic inference including GenBank sequence data from vouchered and
identified specimens (see below).
Phylogenetic analysis of Russula species
Preliminary collections of fruiting bodies in the study area indicated that Russula was
common in the EM fungal community associated with Oreomunnea. Given that this genus is an
important component of many EM fungal communities in the tropics (Peay et al. 2010; Smith et
al. 2011; Tedersoo et al. 2011) and appears to shift in community composition with changes in
soil N availability (Avis 2003, Avis et al. 2008, Lilleskov et al. 2002), it was chosen to test
12
hypotheses concerning phylogenetic diversity. To augment data on Russula distribution obtained
from root tips, Russula fruiting bodies were collected every two weeks throughout the study
period from January 2012 to July 2012 along four 50 m × 4 m transects in each site. Transects
were established in Oreomunnea stands at the same sites from which root tips were sampled,
averaging 150 m (± 110 m) linear distance from root tip sampling points. Macromorphology of
fresh Russula fruiting bodies was recorded in the field, and a sample of tissue was preserved for
DNA extraction. Sequences from fruiting bodies were obtained as described above. Vouchers of
fruiting bodies are deposited at the University of Arizona Robert L. Gilbertson Mycological
Herbarium (MYCO-ARIZ).
To evaluate the structure of Russula communities as a function of Oreomunnea
developmental stage and soil fertility/rainfall level, we inferred phylogenetic relationships of the
genus using 109 sequences representing root tips and fruiting bodies collected in this study
(Table 2.6), and 32 sequences downloaded from GenBank. Sequences were selected from
GenBank only if they were obtained from vouchered and identified specimens, and if there was a
>90% BLAST match with one or more sequences from the study site. This approach permitted
us to select high-quality, fully identified sequences from GenBank to estimate taxonomic
placement of Oreomunnea-associated species. Four sequences from voucher specimens of
Stereum hirsutum (Willd.) Pers. (AY854063), Amylostereum laevigatum (Fr.) Boidin
(AY781246), Gloeocystidiellum porosum (Berk. & M.A. Curtis) Donk (AY048881), and
Bondarzewia montana (Quél.) Singer (DQ200923) were used for the outgroup following Miller
and Buyck (2002) and Buyck et al. (2008).
Sequences were aligned using MUSCLE (Edgar 2004). The resulting alignment was
edited using Gblocks 0.91b (Castresana 2002) to exclude positions that were poorly or
ambiguously aligned. The final data set consisted of 639 characters and 144 terminal taxa. The
tree was inferred using maximum likelihood analysis using the GTR+I+Gamma model of
evolution in GARLI 2.0 (Zwickl 2006). Support was assessed using 1000 bootstrap replicates.
The resulting phylogenetic tree was used as input for two subsequent analyses. First, we
calculated Faith’s phylogenetic diversity index (PD) using the package Picante 1.5-2 in R
(Kembel et al. 2010) to compare the phylogenetic diversity of Russula within each
13
fertility/rainfall environment (i.e., alpha diversity). Faith’s PD, defined as the sum of the branch
lengths connecting all taxa within a local community (Faith 1992), was calculated based on
random subsets of 19 OTUs (the smallest number of Russula OTUs recovered per site).
Second, UniFrac permutation analysis (Lozupone et al. 2006, 2010) was used to
determine whether the phylogenetic structure of communities differed in a manner consistent
with environmental filtering. For comparisons among communities associated with different
sites, soil fertility/rainfall conditions, or developmental stages of Oreomunnea, the lengths of
branches in the phylogeny that were unique to each community were calculated and compared to
50 permutations in which assignment of taxa to communities was randomized. If the
environment or developmental stage selects for fungi that share phylogenetically-conserved
traits, then we would expect communities from similar environments or stages to share more
branch length (i.e., be more closely related) than the random expectation (Lozupone et al. 2006).
Results
Ectomycorrhizal fungi were common in roots of Oreomunnea at all developmental stages
and sites at Fortuna, Panama. Seedlings, saplings, and adults of Oreomunnea did not differ
significantly in infection frequency (i.e., the percentage of root tips with visible EM infection)
(F2,160 = 2.69, P = 0.07), although a trend suggested somewhat lower incidence in seedlings
(Figure 2.2).
Infection frequency by EM fungi differed significantly among sites (F3,159 = 2.88, P =
0.04), reflecting a significantly higher infection frequency at Alto Frio than Honda B (Tukey
HSD P = 0.043). Consistent with the differences among sites, we observed significant
differences in infection frequency when the sites were grouped by soil fertility/rainfall (P =
0.0195; Figure 2.2). There was no evidence that variation in infection frequency reflected a
meaningful interaction of site and developmental stage (F6,151 = 0.16, P = 0.98).
Richness and diversity of EM fungi
In total, 473 mycorrhizal root tips were collected from adult trees, saplings, and
seedlings. Sequences obtained from 234 root tips (49.3%; Table 2.2) yielded 115 OTUs (Figure
14
2.3). Overall EM fungal diversity was high (Shannon-Wiener Index: H’ = 4.56; Simpson’s
Index: 1-D = 0.99, Fisher’s alpha = 89.5; Table 2.4; see also Figure 2.3).
Diversity of EM fungi was similar among developmental stages of Oreomunnea (Table
2.3, Figure 2.4). In contrast to our prediction, EM fungal diversity was similar among sites, with
no apparent relationship to soil fertility/rainfall (Table 2.3, Figure 2.4).
Community composition of EM fungi
Although the community of EM fungi was highly diverse, accumulation curves were
asymptotic once singletons were removed (Figure 2.4). Consistent with our prediction, we found
that communities of EM fungi did not differ as a function of Oreomunnea developmental stage
(F2,30 = 1.09, P = 0.34). Only one species (Laccaria sp. 4) was found in all sites. Six OTUs
occurred in at least three sites (Table 2.5). Overall community composition was most similar
between Honda A and Honda B, which shared 16 OTUs.
ADONIS revealed significant differences in EM fungal community composition among
sites (F3,29 = 1.81, R2 = 0.17, P = 0.005). NMDS suggested differences in the EM fungal
community as a function of soil fertility/rainfall: the low fertility/high rainfall sites (Honda A and
B) grouped separately from the high fertility/low rainfall sites (Hornito and Alto Frio) (Figure
2.5). However, the Mantel test also revealed a significant, positive correlation between
geographic proximity and community similarity (R = 0.61, P = 0.001). Because geographic
proximity is positively associated with environmental similarity in our study, the relative
importance of spatial and environmental factors is difficult to interpret. We therefore examined
the importance of environmental factors alone by evaluating correlations of PCA axes with
NMDS. Only the first PCA axis (PC1), was significantly correlated with the first two axes of the
NMDS (R2 = 0.69, P = 0.001; Figure 2.5). PC1 accounted for 62.5% of the variation in
environmental variables, and was negatively associated with soil fertility and positively
associated with annual rainfall. Correlation of the PCA axis with the NMDS suggests
environmental filtering driven by rainfall and/or soil fertility influences EM fungal community
composition in addition to geographic proximity.
15
Taxonomic placement of EM fungi
Overall 99% of sequenced root tips were colonized by EM fungi belonging to 13 lineages
sensu Tedersoo et al. (2010): /amanita, /byssocorticium, /boletus, /clavulina, /laccaria,
/cortinarius, /elaphomyces, /cantharellus, /coltricia, /inocybe, /russula-lactarius, /sebacina,
/tomentella-thelephora, and /tricholoma. The most OTU-rich lineages were /russula-lactarius (36
OTUs from 97 root tips), /tomentella-thelephora (25 OTUs from 39 root tips), /cortinarius (14
OTUs from 27 root tips), /boletus (10 OTUs from 12 root tips), and /laccaria (5 OTUs from 17
root tips). Three root tips were colonized by members of the Strophariaceae, Marasmiaceae, and
a genus of Atheliaceae considered to be saprotrophic; these were excluded from further analysis.
Russula, Tomentella, and Laccaria were present in all sites. Laccaria was especially
abundant in the high fertility/low rainfall sites (Table 2.5). Cortinarius was not observed in Alto
Frio and was rarely observed in Hornito (high fertility/low rainfall sites), but was abundant in
Honda A and B, making up 16% and 26% respectively of the total number of sequenced root
tips. Boletus and Clavulina were found only in low fertility/high rainfall sites (Honda A, Honda
B), but were not common.
The four most abundant OTUs represented Russula (16% of sequences). These included
one unidentified species (Russula sp. 2), Russula cyanoxantha (Russula sp. 3), Russula puellaris,
and Russula cf. pectinata (see Table 2.5 for information about OTU and taxonomic distributions
across sites). Russula spp. were found in association with roots of adults, saplings, and seedlings
(Figure 2.6).
Phylogenetic diversity of Russula
The most commonly found clades in our surveys included representatives from roots as
well as fruiting bodies (Figure 2.7). In some clades, BLAST matches were consistent with
estimated taxonomy based on phylogenetic analysis, but in other cases taxonomic placement at
the species level differed between the two approaches. For example, one of the more common
morphotypes is related to R. cyanoxantha, since sequences from this study form a clade with
identified vouchers from Hibbet (GQ452059) and Smith et al. 2007b (DQ974758).
16
Phylogenetic diversity of Russula did not differ as a function of fertility/rainfall (PDhigh
fertility/low rainfall = 2.15, PDlow fertility/high rainfall = 3.53, t = -0.9521, df = 2, P = 0.506) (Table 2.3). We
found no significant structure in the Russula phylogeny as a function of Oreomunnea
developmental stage (Figure 2.7), either when comparing adults and seedlings (UniFrac analysis,
P = 0.29) or seedlings and saplings (P = 0.73). However, consistent with our prediction, the
phylogenetic composition of Russula communities differed significantly among sites (P < 0.001)
and when sites were grouped according to fertility/rainfall (P < 0.001). Significant differences
were observed between Alto Frio and Honda A (P < 0.001), Alto Frio and Honda B (P < 0.001),
and Honda B and Hornito (P = 0.01 – 0.05), but not between sites with similar fertility/rainfall.
Discussion
This is the first detailed inventory of EM fungi associated with the widely distributed
neotropical tree Oreomunnea mexicana, and one of the few analyses of EM fungal community
diversity in a tropical montane forest. Ectomycorrhizal tree species that have been studied in
detail in the neotropics include Dicymbe corymbosa (Fabaceae), Pakaraimaea dipterocarpacea
(Dipterocarpaceae), Quercus crassifolia and Q. laurina (Fagaceae), and Coccoloba spp. (Henkel
2003, Miller et al. 2000, Morris et al. 2009, Smith et al. 2011, Smith et al. 2013, Tedersoo et al.
2010a). Our study adds new data for a representative of the Juglandaceae in an area of high plant
species richness, and dramatic local-scale differences in soil types and rainfall patterns.
Our results reveal that species diversity of EM fungi associated with Oreomunnea is high
despite the small spatial scale at which this study was carried out. In conjunction with other
inventories, our data suggest that tropical forest EM fungal communities can be as species-rich
as those found in temperate forests (i.e., Diédhiou et al. 2014, Henkel et al. 2012, Smith et al.
2011). A comparison of EM fungal diversity in root tips using Fisher's alpha showed no
significant difference between temperate forests (mean Fisher’s alpha = 46.87, SD = 55.23) and
tropical forests studied thus far (mean Fisher’s alpha = 47.06, SD = 52.65; F1,46 = 0, P = 0.992).
When comparisons were restricted only to angiosperms, the results still did not differ
significantly (temperate forests, mean Fisher’s alpha = 62.02, SD = 80.97; P = 0.618).
17
All of the major EM fungal clades sensu Tedersoo et al. (2010b) encountered in this
study have been reported previously in both temperate and tropical EM fungi inventories
(Tedersoo et al. 2011, Phosri et al. 2012, Smith et al. 2011, Peay et al. 2010, Diédhiou et al.
2014). Members of the /russula-lactarius, /cortinarius, and /tomentella-thelephora lineages were
particularly abundant, accounting for 72% of OTUs. An overall dominance by Russula has been
found in other EM fungal inventories in tropical forests (Tedersoo et al. 2011, Henkel et al. 2012,
Phosri et al. 2012, Smith et al. 2011, Peay et al. 2010, Diédhiou et al. 2014), suggesting that this
is an important taxonomic group to focus on in future biogeographic and systematics-based
studies.
Consistent with the few previous studies of EM fungal communities in tropical forests,
we observed strong community dissimilarity of EM fungi across sites. For example, in
Fabaceae-dominated forest in Guyana, Smith et al. (2011) found significant differences in EM
fungal species composition among 19 sites with an average inter–site distance of 689 m. Peay et
al. (2010) found significant clustering in community composition in sites with similar soil types
in dipterocarp-dominated forest in Lambir Hills National Park, Sarawak (Malaysia). Our results
suggest that variation in EM fungal communities at small spatial scales also may be a feature in
montane tropical forests, expanding the scope for local turnover to greatly enhance alpha and
beta diversity at small spatial scales.
Geographic distance and environmental similarity are confounded in our study area, as
the distance between sites within the same soil/rainfall characteristics was small (0.2–1 km)
compared to the distance between sites with different characteristics (6 km). However, our
analyses suggest an important role of environmental factors, here defined as soil fertility and
rainfall patterns, in potentially filtering community structure.
At present, our sampling is not sufficient to define the relative importance of soil fertility
vs. rainfall patterns in shaping EM fungal communities in Oreomunnea. In general, high rainfall
sites tend to have low nutrient availability due to increased leaching (Austin and Vitousek 1998);
however, variation in fertility in our sites is determined by differences in underlying geology.
Variation in fungal community composition may also in part be driven by dispersal limitation,
which also could underlie a significant Mantel correlation between geographic distance and
18
community dissimilarity. In spite of our inability to identify the exact environmental variables
that drive EM fungal beta diversity, our study suggests that abiotic factors can drive high levels
of species turnover in EM fungal communities that associate with the same host species.
Turnover in the EM fungal community as a whole was echoed by turnover in OTU
assemblages among sites in key fungal genera. For example, even when singletons were
removed, 14 of 21 Russula OTUs found at the high fertility/low rainfall sites were not found in
either of the low fertility/high rainfall sites. UniFrac results also showed significant differences
in the phylogenetic relatedness of Russula communities across sites consistent with
environmental filtering: Russula communities associated with the two high fertility/low rainfall
sites were less closely related with the ones found in low fertility/high rainfall sites than expected
by chance.
Temperate Russula, particularly from the subsection Foetentineae, tend to inhabit more
fertile habitats with relatively higher N availability (Avis 2003, Avis et al. 2012), and have been
reported to increase in abundance in response to N fertilization in permanent plots (N addition of
5.4 or 17 g N m−2 yr−1 in oak savanna, Avis et al. 2003). Several authors have also noted that EM
fungi associated with N-rich habitats may form less beneficial or parasitic relationships with
their hosts (Johnson et al. 1997, Egger and Hibbett 2004, Avis 2012). It is possible that less
beneficial EM fungi reduce the competitive advantage of Oreomunnea relative to co-occurring
tree species in the more fertile sites studied here (Table 2.1).
In contrast, all of the 14 species of Cortinarius observed here were found infecting root
tips at the low fertility/high rainfall sites. Cortinarius can extract N from organic sources under
conditions of low N availability (Taylor et al. 2000, Lilleskov et al. 2002, Avis et al. 2003). More
research on functional traits may help us understand the influence of fertility and rainfall on
fungal community composition, and its effect on associated plant communities.
We found that diversity and composition of EM fungal communities did not differ among
developmental stages of Oreomunnea. Strikingly, some of the most abundant Russula OTUs
were found in all stages, and UniFrac analyses revealed the similarity of Russula communities in
both adults and seedlings, and seedlings and saplings. A similar result was found in seedlings
19
and adults of EM tree species in Guinean tropical rain forest, where the OTUs infecting several
developmental stages were the most abundant in the EM fungal community (Diédhiou et al.
2010).
Russula as a candidate for ectomycorrhizal network effects in Oreomunnea
Indirect evidence consistent with the existence of EM networks in tropical forest comes
from experiments showing that hyphal exclusion increases the mortality and decreases the
growth rate of EM seedlings (McGuire 2007, Onguene and Kuyper 2002). However, no study
has yielded direct evidence of resource transfer (i.e., nutrients or water) to seedlings, nor
identified the EM fungal potentially involved in such transfer in tropical forest. Analyses of EM
fungal composition is important, as EM networks effects are unlikely to strongly impact
recruitment of host trees unless common EM fungal taxa infect both seedlings and adults. At
Fortuna, network effects on Oreomunnea might be more likely to occur at the low fertility/high
rainfall sites of Honda A and B, where seedling densities are exceptionally high, and seedling
mortality rates are low below crowns (Table 2.1). In our surveys, OTUs representing Russula
cyanoxantha, Russula sp3, Russula puellaris, and Russula cf. pectinata accounted for 24% of
sequences in the low fertility/high rainfall sites. Further studies should target whether resource
transfer to seedlings occurs via members of this group.
Although inventories of fruiting bodies and root tips of EM fungi have been made in oak-
dominated Central American montane forests (Halling and Mueller 2005, Mueller et al. 2006,
Morris et al. 2009), this is one of the first below-ground surveys of EM fungi in a tropical
montane forest and provides insight into the diversity of EM fungal species associated with
Oreomunnea across sites varying in fertility and in the amount and seasonality of rainfall. The
rationale for this project was to provide information for future experiments that will explicitly
test for the existence of mycorrhizal networks and their effects on Oreomunnea seedling
performance. This information will be useful to uncover the factors driving plant–soil feedback
and EM host tree dominance in tropical montane forests.
20
Figures and Tables
Figure 2.1. Upper panel, location of Fortuna Forest Reserve, Panama. Lower panel, sampling
sites at Fortuna: circles represent low fertility/high rainfall sites (Honda A and B) and triangles
represent high fertility/low rainfall sites (Hornito and Alto Frio). Reproduced with modifications
from Andersen et al. (2010)
!!
Honda&B Honda&A
Hornito
Alto&Frio
!!
!!
!!
!!
21
Figure 2.2. Infection frequency (i.e., percent of root tips visibly infected by ectomycorrhizal
fungi) in roots of Oreomunnea mexicana (A) adults, saplings and seedlings in (B) four study
sites in Fortuna, Panama. Error bars indicate the 95% confidence interval around the mean.
Honda A (HA) and Honda B (HB) are low fertility/high rainfall sites; Alto Frio (AF) and Hornito
(HO) are high fertility/low rainfall sites. Sample sizes (n) indicate the number of root tips
sequenced
0
10
20
30
40
50
60
Age
% in
fect
ion
Adults Saplings Seedlings
n=60 n=49 n=54
A
0
10
20
30
40
50
60
Site
% in
fect
ion
HA HB HO AF
n=44 n=45 n=44 n=30
B
22
Figure 2.3. Species (OTU) accumulation curves of EM fungi colonizing root tips of
Oreomunnea mexicana at Fortuna. OTUs were based on 97% sequence similarity. The analysis
includes all sequences obtained in the present study. The grey solid line indicates observed OTU
richness; dotted grey lines represent the 95% confidence interval around observed richness; the
black solid line indicates the bootstrap estimate of total species richness; the dashed black line
indicates the accumulation curve for nonsingleton OTUs.
0 50 100 150 200
050
100
150
Number of root tips
Num
ber o
f OTU
s
BootstrapObserved richness95 %CINonsingleton
23
Figure 2.4. Ectomycorrhizal species accumulation curves and 95% confidence intervals based
on nonsingleton OTUs derived from 97% sequence similarity. Upper panel: Accumulation
curves per site for Honda A, and Honda B, Hornito, Alto Frio. Lower panel: Accumulation
curves for three developmental stages: seedlings 5–20 cm in height, saplings 40–100 cm in
height, and adults.
0 10 20 30 40 50 60
010
2030
4050
Honda A
Number of root tips
Num
ber o
f OTU
S
0 10 20 30 40 50 60
010
2030
4050
Honda B
Number of root tips
Num
ber o
f OTU
S
0 10 20 30 40 50 60
010
2030
4050
Hornito
Number of root tips
Num
ber o
f OTU
S
0 10 20 30 40
010
2030
4050
Alto Frio
Number of root tips
Num
ber o
f OTU
S
0 20 40 60 80
010
2030
4050
60
Adults
Number of root tips
Num
ber o
f OTU
S
0 20 40 60 80
010
2030
4050
60
Saplings
Number of root tips
Num
ber o
f OTU
S
0 10 20 30 40 50
010
2030
4050
60
Seedlings
Number of root tips
Num
ber o
f OTU
S
24
Figure 2.5. Nonmetric multidimensional scaling (NMDS) plot showing differences in the EM
fungal community composition among sites that differ in fertility and rainfall (high fertility/low
rainfall: triangles, low fertility/high rainfall: circles). The first PCA axis of environmental
variables (PC1) was significantly correlated with compositional variation (R2 = 0.69, P = 0.00;
see text for further explanation). Stress = 0.116
-0.4 -0.2 0.0 0.2 0.4
-0.4
-0.2
0.0
0.2
0.4
NMDS1
NMDS2
HAHBHoAF
PC1H L
25
Figure 2.6. (A) Relative abundance of OTUs based on 97% sequence similarity. Columns in
black show OTUs infecting adults, saplings, and seedlings. (B) Relative abundance of fungal
genera
0
1
2
3
4
5
6 1 6 11
16
21
26
31
36
41
46
51
56
61
66
71
76
81
86
91
96
101
106
111
116
Abu
ndan
ce (%
)
EM fungal taxa (OTUs)
A
44.1
14.5 13.4 7.5 4.3 2.7 2.2 1.6 1.6 1.6 1.1 1.1 1.1 0.5 0.5 0.5 0.5 0.5 0.5
0
10
20
30
40
50
Abu
ndan
ce (%
)
EM genus
B
26
Fig. 2.7 Results of maximum likelihood analysis of Russula obtained from mycorrhizal root tips (by direct PCR) and fruiting bodies in Oreomunnea mexicana-dominated stands at Fortuna, Panama (bold font), and exemplar taxa chosen as described in the text. Thickened branches indicate ≥ 70% bootstrap support. Strains from root tips are annotated to indicate the life stage (seedling, sapling, adult), site (low fertility/high rainfall in blue font: HA, Honda A; HB, Honda B; high fertility/low rainfall in green font: HO, Hornito; AF, Alto Frio), name and accession number for the closest match to the sequence in GenBank, and sequencing code. Sequences from fruit bodies are annotated to indicate site (those listed previously; collections also were made at ZA, Zarceadero; see supplementary material), name and accession number for the closest match to the sequence in GenBank, and sequencing code.
Root•Adult•HB•KM594970-AC408/Russula cf. pectinata HQ604835Russula pectinatoides EU819493
Russula illota DQ422024 Root•Seedling•AF•KM594875-AC150/Russula illota HQ677769
Root•Seedling•AF•KM594873-AC148/Russula illota HQ677769Root•Seedling•AF•KM594874-AC149/Russula illota HQ677769
Fruiting body•ZA•KM594814-AC545/Russula cf. pectinata HQ604835
Russula pectinatoides EU598185Russula amoenolens GU222264
Fruiting body•HA•KM594828-AC572/Russula cf. pectinata HQ604835
Fruiting body•AF•KM594802-AC497/Russula illota HQ677769Fruiting body•AF•KM594804-AC507/Russula foetens FJ845427
Fruiting body•ZA•KM594794-AC334/Russula cf. foetens DQ422023Russula foetens FJ845427
Root•Seedling•HO•KM595042-AC262/Russula cf. pectinata HQ604835
Root•Adult•HO•KM594922-AC277/Russula cf. pectinata HQ604835
Root•Adult•HO•KM594926-AC282/Russula cf. pectinata HQ604835
Root•Seedling•HA•KM594896-AC210/Russula cf. pectinata HQ604835
Root•Adult•HA•KM594893-AC195/Russula cf. pectinata HQ604835
Root•Adult•HB•KM594907-AC237/Russula cf. pectinata HQ604835
Fruiting body•HO•KM594818-AC588/Russula eccentrica EU598163
Fruiting body•AF•KM594803-AC501/Russula eccentrica EU598163Fruiting body•AF•KM594807-AC518/Russula eccentrica EU598163
Root•Adult•AF•KM594890-AC190/Russula eccentrica EU598163Root•Adult•HO•KM594944-AC350/Russula eccentrica EU598163
Root•Adult•AF•KM594963-AC394/Russula eccentrica EU598163
Root•Sapling•HO•KM594933-AC302/Russula nigricans JQ711972Root•Seedling•AF•KM594871-AC145/Russula nigricans EU597075Fruiting body•AF•KM594808-AC520/Russula nigricans EU597075
Fruiting body•AF•KM594806-AC517/Russula subnigricans AB291748
Uncultured Russula JN168741
Uncultured Russula JN168752
Root•Adult•HB•KM594908-AC239/Russula compacta GU229820Fruiting body•ZA•KM594790-AC317/Russula sp. DQ778002
Russula cyanoxantha DQ974758
Fruiting body•HB•KM594786-AC270/Russula cf. cyanoxantha EU598166
Fruiting body•HA•KM594809-AC521/Russula sp. DQ777996
Fruiting body•HB•KM594815-AC578/Russula cf. cyanoxantha EU598166
Fruiting body•HA•KM594829-AC579/Russula sp. DQ777996
Fruiting body•HB•KM594787-AC271/Russula cf. cyanoxantha EU598166Fruiting body•HB•KM594799-AC431/Russula sp. DQ777996
Fruiting body•HA•KM594817-AC584/Russula cf. cyanoxantha EU598166
Fruiting body•HA•KM594798-AC416/Russula cf. cyanoxantha EU598166
Root•Seedling•HB•KM595010-AC573/Russula sp. DQ777996
Fruiting body•HB•KM594812-AC527/Russula cf. cyanoxantha EU598166
Fruiting body•HB•KM594801-AC448/Russula cf. cyanoxantha EU598166
Fruiting body•HB•KM594819-AC590/Russula cf. cyanoxantha EU598166
Fruiting body•HA•KM594805-AC516/Russula sp. DQ777996
Root•Sapling•HA•KM594986-AC47B/Russula cf. cyanoxantha EU598166
Root•Sapling•HO•KM594931-AC296/Russula sp. DQ777996
Russula cyanoxantha GQ452059
Fruiting body•HA•KM594784-AC198/Russula cf. cyanoxantha EU598166
Fruiting body•HA•KM594797-AC403/Russula cf. cyanoxantha EU598166
Fruiting body•HB•KM594811-AC523/Russula sp. DQ777996
Root•Adult•HB•KM594978-AC450/Russula sp. DQ777996Root•Sapling•HB•KM594977-AC445/Russula sp. DQ777996
Fruting body•HO•KM594820-AC591/Russula cf. cyanoxantha EU598166
Root•Sapling•HO•KM594957-AC381/Russula crustosa EU598153Russula nitida EU598164
Root•Sapling•HB•KM594914-AC249/Russula sp. AF350057Root•Seedling•HA•KM594949-AC36/Russula crassotunicata DQ384580Root•Sapling•HA•KM594903-AC223/Russula crassotunicata DQ384580
Fruiting body•HO•KM594830-C581/Russula sp. DQ777996Russula variata EU819436
Root•Seedling•HO•KM594948-AC359/Russula sp. DQ777989
Root•Adult•HO•KM594881-AC158/Russula crenulata HQ604846Root•Seedling•HA•KM594991-AC494/Russula crenulata HQ604846
Root•Adult•HA•KM595054-C562/Russula crenulata HQ604846
Russula viscida FJ627039
Russula bicolor FJ845435
Root•Adult•HA•KM594998-AC537/Russula puellaris HQ604852
Root•Seedling•HB•KM594935-AC310/Russula puellaris HQ604852Root•Sapling•HB•KM594915-AC250/Russula puellaris HQ604852
Root•Sapling•HB•KM594918-AC257/Russula puellaris HQ604852
Root•Adult•HB•KM595051-AC442/Russula puellaris HQ604852Root•Adult•HA•KM595034-AC205/Russula puellaris HQ604852
Root•Adult•HB•KM595036-AC229/Russula puellaris HM189938
Fruiting body•HB•KM595014-AC72/Russula puellaris HQ604852Root•Sapling•HA•KM594904-AC225/Russula puellaris HQ604852
Root•Adult•HA•KM594992-AC495/Russula puellaris HM189941
Russula puellaris HQ604852
Russula fragilis JF899569Russula gracilis FJ845431
Russula odorata JQ888198
Fruiting body•AF•KM594821-AC603/Russula gracilis FJ845431Fruiting body•AF•KM594810-AC522/Russula puellaris HQ604852
Fruiting body•HO•KM594824-AC430/Russula sp. AY456357Fruting body•HO•KM594827-AC531/Russula sp. AY456357
Root•Sapling•HA•KM594901-AC221/Russula odorata JQ711877Root•Seedling•HB•KM594910-AC241/Russula odorata JQ711900
Russula puellaris HM189935
Fruiting body•ZA•KM594792-AC329/Russula sp. JF834367
Russula risigallina DQ422022Root•Sapling•HA•KM594898-AC215/Russula lutea HQ604848
Root•Adult•HA•KM594990-AC491/Russula cf. flavisiccans EU598156Fruiting body•HO•KM594816-AC580/Russula cf. flavisiccans EU598156
Russula xerampelina JF899571Russula paludosa GU373486
Fruiting body•HB•KM594785-AC269/Russula cf. favrei EF530934Fruiting body•HB•KM594800-AC434/Russula cf. favrei EF530934
Fruiting body•HO•KM594831-AC587/Russula sp. EU569264
Root•Adult•AF•KM595032-AC189/Russula rubellipes EU598175Root•Adult•HO•KM595043-AC288/Russula rubellipes EU598175Root•Sapling•HO•KM594946-AC357/Russula sp. EU598161
Fruiting body•AF•KM594825-AC503/Russula rubellipes 598175
Root•Seedling•HO•KM594887-AC173/Russula sp. EU598161Root•Sapling•HB•KM594976-AC441/Russula sp. EU598161
Fruiting body•HB•KM594788-AC272/Russula sp. EU598159
Root•Adult•HA•KM594993-AC504/Russula sp. EU598159
Root•Seedling•HA•KM594956-AC38/Russula sp. EU598159
Fruiting body•HB•KM594795-AC336/Russula sp. EU598159
Fruiting body•HA•KM594796-AC369/Russula sp. EU598159
Root•Seedling•HB•KM594975-AC427/Russula sp. EU598159Root•Sapling•HA•KM594902-AC222/Russula sp. EU598159
Fruiting body•HB•KM594813-AC528/Russula sp. EU598159Russula compacta EU598157
Root•Sapling•HB• KM595037-AC233/Russula solaris JN944007Root•Sapling•HA•594983-AC457/Russula occidentalis AY228349
Root•Sapling•HO•AC612/Russula sp. GU220376Fruiting body•ZA•KM594791-AC318/Russula sp. HQ604851Fruiting body•ZA•KM594793-AC331/Russula sp. HQ604851
Russula lepidicolor AY061687
Fruiting body•ZA•KM594783-AC132/Russula sp. EU569264Fruiting body•ZA•KM594789-AC305/Russula sp. EU569264
Root•Adult•AF•KM595025-AC100/Russula xerampelina HM240542
Root•Adult•AF•KM594837-AC624/Russula decolorans FJ845432
Russula subtilis GQ166871Fruiting body•HB•KM594822-AC52/Russula corallina JN944006
Russula subtilis EU598178Root•Adult•HA•KM594895-AC204/Russula olivacea AF418634
Root•Adult•HO•KM594942-AC345/Russula decolorans DQ367913
Russula integra HM189840
Root•Adult•AF•KM594833-AC615/Russula tenuiceps DQ974756Russula velenovskyi HM189951
Russula decolorans FJ845432
Fruiting body•ZA•KM594823-AC335/Russula sp. GU234030Fruiting body•AF•KM594826-AC511/Russula sp. GU234030
Russula xerampelina HM240542Russula sphagnophila AY061719
Russula firmula DQ422017
Stereum hirsutum AY854063Amylostereum laevigatum AY781246
Bondarzewia montana DQ200923Gloeocystidiellum porosum AY048881
Article title: Variation in ectomycorrhizal fungal communities associated with Oreomunnea mexicana (Juglandaceae) in tropical montane forests. Journal Mycorrhiza.Adriana Corrales1,4, A. Elizabeth Arnold2, Astrid Ferrer1, Benjamin L. Turner3, James W. Dalling1,3. 1) Department of Plant Biology, University of Illinois at Urbana-Champaign, Urbana-Champaign, IL 61801 USA. 2) School of Plant Sciences, University of Arizona, Tucson, AZ 85721 USA. 3) Smithsonian Tropical Research Institute, Apartado Postal 0843–03092, Republic of Panama.4) Corresponding author: Adriana Corrales- corrlssr@illinois.edu
27
Table 2.1. Characteristics of the study sites at Fortuna Forest Reserve, Panama. Honda A and Honda B are low
fertility/high rainfall sites, and Hornito and Alto Frio are high fertility/low rainfall sites. Soil data are expressed in
volume basis due to large variation in bulk density among plots.
Site Honda A Honda B Hornito Alto Frio
Elevation (m) 1175 1266 1404 1176
Annual rainfall 2013 (mm) 6055 6440 1990 1895
Soil variables
Geology Rhyolite Rhyolite Dacite Andesite
NaOH–EDTA inorg. P (µg cm-3) 17.9 15.1 24.3 27.7
NaOH–EDTA org. P (µg cm-3) 73.4 60.8 122.7 248.8
NH4 (µg cm-3) 2.2 1.8 1.8 3.8
NO3 (µg cm-3) 1.2 0.4 1.2 2.6
K2SO4 extract. org. C (µg cm-3) 152.2 92.0 95.3 92.8
pH in water 4.63 3.63 5.76 5.62
Total N (mg cm-3) 2.92 2.39 2.87 4.72
Total C (mg cm-3) 43.9 40.9 35.0 51.1
Total P (µg cm-3) 180.6 127.7 280.2 503.0
Resin P (µg cm-3) 0.2 1.9 2.2 1.4
Bulk density (g cm-3) 0.11 0.13 0.39 1.00
Al (cmol L-1) 1.1 1.3 0.5 0.0
Ca (cmol L-1) 0.0493 0.1046 4.9381 8.4702
K (cmol L-1) 0.0241 0.0248 0.1834 0.1225
Light variables
Canopy openness (%) 6.66 7.90 8.70 9.32
Vegetation variables
Community basal area >10 cm
(m2/ha) 45.6 46.5 52.9 40.7
No. Oreomunnea seedlings/m2 9.9 7.8 0.7 0.2
28
Table 2.1 (continued)
Site Honda A Honda B Hornito Alto Frio
Annual seedling mortality rate
(%) 0.31 0.17 0.19 0.5
Oreomunnea adults /0.1 ha 31 79 71 42
Oreomunnea basal area >10 cm
(m2/0.1ha) 1.59 2.48 2.58 1.80
29
Table 2.2. Number of ectomycorrhizal root tips sequenced, root tips collected, individuals
sampled, and OTUs observed for each site in Fortuna, and each developmental stage of
Oreomunnea.
Site Honda A Honda B Hornito Alto Frio Total
Root tips sequenced / total root tips collected (individuals sampled)
Adults 22/48 (4) 17/33 (4) 30/57 (4) 25/49 (4) 94/187 (16)
Saplings 30/52 (20) 35/67 (20) 19/54 (19) 4/10 (5) 88/183 (64)
Seedlings 12/25 (20) 15/34 (20) 14/30 (20) 11/14 (9) 52/103 (69)
Number of OTUs
Adults 17 13 23 15 55
Saplings 26 25 18 4 59
Seedlings 11 13 12 7 39
30
Table 2.3. Diversity of the EM fungal community for each site and for all developmental stages of
Oreomunnea. Simpson and Shannon indexes are based on 99 randomizations and restricted to 24
samples per site and 42 samples per developmental stage (consistent with the minimum number of
samples recovered per site or stage; see Table 2.2). Faith’s phylogenetic diversity index (PD) was
calculated based on sampling 19 Russula OTUs per site.
Index Honda A Honda B Hornito Alto Frio Seedlings Saplings Adults
Simpson 0.94 0.94 0.95 0.94 0.97 0.97 0.97
Shannon 3.07 3.07 3.09 2.57 3.67 3.64 3.61
Fisher’s alpha 73.3 33.9 64.7 28.5 79.4 78.3 55.5
PD 0.87 0.99 1.33 0.98
31
Table 2.4 Comparison of the results of this study with other studies of ectomycorrhizal species in tropical and
temperate biomes (adapted from Tedersoo et al. 2012). Columns indicate latitude in degrees (Lat), longitude in
degrees (Lon), elevation in meters (Elev), dominant host lineage (Host), Mean Annual Precipitation in mm (MAP),
diversity (Fisher’s alpha), number of samples analyzed (N) and number of ectomycorrhizal species found in study
site (OTUs based on 97% sequence similarity).
Reference Country Lat Lon Elev Host MAP Fisher’s
alpha N
No.
Species
Corrales et al.
(this study) Panama 9˚02’91"N 82˚32’46” W 1000 Juglandaceae 4000 89.5 234 115
Diédhiou et al.
2014
Southern
Guinea
8˚51’ and
7˚60’ N
9˚31’ and
8˚49’ W
500–
1752 Fabaceae
2500-
3000 182.1 332 189
Diédhiou et al.
2010 Guinea 8°50' 60" N 9° 31' 01" W 600 Fabaceae 3000 16.9 370 53
Morris et al.
2009 Mexico 18˚36’ N 99˚36’ W
2450–
2550 Fagaceae
1200-
1500 27.1 8000 154
Phosri et al.
2012 Thailand
16° 51' 14”
N 100° 31' 4” E 160 Dipterocarpaceae 1250 38.3 194 69
Peay et al.
2010 Malaysia 4°19' 59" N
113°49' 59"
E 140 Dipterocarpaceae 3000 37.7 589 106
Smith et al.
2011 Guyana 5°16' 1.2"N
59° 50' 24"
W 710 Fabaceae 3866 34.5 1020 118
Tedersoo et al.
2007
The
Seychell
es
4° 41' 17" S 55° 29' 6" E 480 Dipterocarpaceae 3500 4.3 135 15
Tedersoo et al.
2010a Ecuador 0° 41' S 76° 24' 0" W 232 Caryophyllales 3081 20.4 105 37
Smith et al.
2013 Guyana 5˚26’21” N 60˚04’43” 800
Dipterocarpaceae
and Fabaceae
2000 –
>2400
mm
19.8 255 52
Temperate studies
Avis et al. 2003
USA 45°25′ N 93°10′ W 450 Quercus/ Corylus 790 20.7 648 72
Avis et al.
2008 USA
41˚ 37’51”
N 87˚05’13” W 206 Quercus 945 129.5 1333 314
32
Table 2.4 (continued)
Reference Country Lat Lon Elev Host MAP Fisher’s
alpha N
No.
Species
Bahram et al.
2011 Estonia 58˚17’ N 27˚19’ E 35 Populus 620 312.55 122 103
Bahram et al.
2012 Iran 36°27 N 51°06’ E
100–
2700 Several spp
237-
552 141.34 1755 367
Bergemann
and
Garbelotto
2006
USA 40˚00’30”N 123˚57’00”
W 580 Lithocarpus 1125 59.28 382 119
Courty et al.
2008 France 48˚75’N 6˚35’E 250 Quercus 744 48.3 180 75
Dickie et al.
2010
New
Zealand 43˚9’12”S 171˚43’48”E 1000
Pinus and
Nothofagus 1447 61.98 354 118
Douglas et al.
2005
(Lodgepole
pine site)
USA 44˚27’21”N 110˚29’34”
W 2430 Pinus contorta 510 13.43 5570 81
Douglas et al.
2005 (Mixed
conifer site)
USA 44˚27’21”N 110˚29’34”
W 2430 Several spp 510 4.93 5933 35
Gao and Yang
2010 China 27°50′N 99°24′ E 4300 Kobresia spp 2000 48.11 150 70
Ishida et al.
2007 Japan
35°56′−
35°57′ N
138°48′−138
°49′ E
1350–
1500 Several spp 1596 66.25 1396 205
Izzo et al.
2005 USA 36°58′N 119°2′W 2100 Several spp 1250 27.05 1105 101
Jones et al.
2008 Canada
50° 18' 43
N
125° 28' 29
W 37–160 Several spp 2240 31.48 138 53
Kennedy et al.
2003 USA 37°54′ Ν 122°37′ W 600 Several spp 1250 16.5 442 56
33
Table 2.4 (continued)
Reference Country Lat Lon Elev Host MAP Fisher’s
alpha N
No.
Species
Kennedy et al.
2012 USA 42°10′52N 122°43′85W 1082 Arbutus/Pinaceae 611 51.85 537 126
Kjøller and
Clemmensen
2009
Sweden 57° 56' 13° 47' 132–300 Pinaceae 582-
818 30.72 969 107
Krpata et al.
2008 Austria 46°33’ N 13°42’ E 578 Populus tremula 1297 7.28 12020 54
Lang et al.
2011
German
y
51°05’20”
N 10°31′24′′E 350 Several spp 670 14.83 94893 130
Lian et al.
2006 Japan 39°56′ N, 141°14′ E 360–380 Pinus densiflora 1145 5.67 5499 39
Mühlmann
and Peintner
2008a, 2008b;
Mühlmann et
al. 2008
Austria 46°50′ N 11°01′ E 2280–
2450 Several spp 6.86 10000 50
Nara 2006 Japan 35° 19'N 138° 11'E 1450–
1600 Several spp 4854
4.99 6698 36
Palmer et al.
2008 USA 43° 57'N 91° 2'W 275 Several spp 820
17.13 233 46
Parrent and
Vilgalys 2007 USA 35° 57'N 79° 7'W 130 Pinus taeda 1140 33.55 1787 134
Pena et al.
2010
German
y 47°59 ︎ N 8°45 ︎ E 800 Fagus sylvatica 776 31.03
At least
515 89
Richard et al.
2005 France 42°20′ N 8°49′ E Several spp 750 77.73 393 140
Richard et al.
2011 France
43° 44′ 29′′
N 3° 35′ 45′′ E 270 Quercus ilex 908 38.11 1147 131
Roy et al.
2013 France
41° 59'–
45° 46'N
0° 38'–9°
17'E Alnus spp 284 21.34 1178 86
34
Table 2.4 (continued)
Reference Country Lat Lon Elev Host MAP Fisher’s
alpha N
No.
Species
Ryberg et al.
2009 Sweden 68°21’N 18°30’E
1010–
1040
Dryas
octopetala, Salix
reticulata
850 27.08 389 74
Ryberg et al.
2010 Sweden 68°20’N 18°30’E 980 Several spp 847 13.50 154 34
Smith et al.
2004 USA 43.5°N 118.5°W
1600–
1700
Pinus ponderosa,
Cercocarpus
ledifolius
173 66.44 480 140
Smith et al.
2005 USA 45.4°N 117.3°W
1300–
1600 Several spp 642 92.25 543 178
Smith et al.
2007b, 2009;
Morris et al.
2008
USA 39°17′ N 121°17′ W 400–
600 Several spp 710 30.65 11590 182
Taniguchi et
al. 2007 Japan 35°32′N 134°13′E Several spp 1083 11.79 284 38
Tedersoo et al.
2003 Estonia 58°17′N 27°19′E 36 Several spp 620 43.22 85 47
Tedersoo et al.
2006 Estonia 58°27’ N 22°00’ N 8 Several spp 550 98.15 468 172
Toljander et
al. 2006 Sweden 64°39′ N 18°30′ E 235 Several spp 570 12.50 2442 66
Twieg et al.
2007 Canada
50°22'-
50°58'
118°32'–
119°23'
500–
1200
Pseudotsuga
menziesii, Betula
papyrifera
663 30.31 938 105
Walker et al.
2005 USA
35°02′29′′
N 83°27′16′′ W
Quercus rubra,
Q. prinus 1800 32.72 291 75
35
Table 2.5 Ectomycorrhizal fungi found in roots of Oreomunnea mexicana and their distribution among develpmental stages and sites in the Fortuna Forest Reserve, Panama. OTUs were assigned to species if all the sequences in the OTU had a percentage of match ≥97% with vouchered sequences in GenBank, to genus if >90%-97%, and to family if ≤90%. Identification of Russula was augmented by phylogenetic analysis. The most abundant OTUs are shown in bold. Developmental stage abbreviations: A: Adult, Sa: Sapling, Se: Seedling; site abbreviations: AF, Alto Frio; HA, Honda A; HB, Honda B; HO, Hornito.
OTU BLAST ID Family Developmental
stages Sites GenBank accession numbers Abundance A Sa Se
AF
HA
HB HO
Russula sp1 Russulaceae 1 x 1 KM594827 Russula puellaris Russulaceae 13 x x x 6 7 KM595056,
KM595014, KM595034, KM594904, KM595036, KM594915, KM594917, KM594918, KM594935, KM594998, KM595051, KM594992, KM595060
Laccaria sp1 Hydnangiaceae 5 x x 3 2 KM594869, KM594870, KM594962, KM594835, KM594856
Byssocorticium atrovirens
Atheliaceae 4 x 3 1 KM594883, KM594839, KM594841, KM594843
Cortinarius sp1 Cortinariaceae 4 x x 4 KM594909, KM595012, KM594834, KM594845
Cortinarius sp2 Cortinariaceae 1 x 1 KM594851 Thelephoraceae sp9 Thelephoraceae 4 x x x 1 3 KM594953,
KM595004, KM594853, KM594857
Russula sp2 (cyanoxantha)
Russulaceae 7 x x x 1 5 1 KM594986, KM594931, KM594977, KM594978, KM595010, KM595061, KM594854
36
Table 2.5 (continued)
OTU BLAST ID Family Developmental
stages Sites GenBank accession numbers Abundance A Sa Se
AF
HA
HB HO
Russula sp3 Russulaceae 12 x x x 8 1 3 KM594858, KM594867, KM594872, KM594891, KM594967, KM594969, KM595008, KM595058, KM595059, KM595063, KM595064, KM595065
Clavulina sp1 Clavulinaceae 4 x x x 1 3 KM594984, KM594987, KM594861, KM594862
Russula nigricans Russulaceae 1 x 1 KM594871 Hydnum sp1 Hydnaceae 3 x 3 KM594876,
KM594877, KM594880
Tomentella sp1 Thelephoraceae 4 x x x 3 1 KM594884, KM594900, KM595006, KM594988
Russula sp5 Russulaceae 5 x x x 1 1 3 KM594887, KM595032, KM595043, KM594946, KM594976
Laccaria sp2 Hydnangiaceae 2 x x 1 1 KM594892, KM595048
Russula cf. pectinata Russulaceae 8 x x 2 1 5 KM594893, KM594896, KM594907, KM595041, KM595042, KM594922, KM594926, KM594927
Boletaceae sp Boletaceae 4 x 3 1 KM594960, KM594899, KM594937, KM595001
Russula crassotunicata Russulaceae 2 x x 2 KM594949, KM594903
37
Table 2.5 (continued)
OTU BLAST ID Family Developmental
stages Sites GenBank accession numbers Abundance A Sa Se
AF
HA
HB HO
Boletus cf. innixus Boletaceae 3 x 3 KM594906, KM594913, KM594919
Russula sp6 Russulaceae 4 x x x 3 1 KM594956,
KM594902, KM594975, KM594993
Fungal sp. JK-02M 2 x x 2 KM594924, KM594932
Boletus sp Boletaceae 1 x 1 KM594940 Russula eccentrica Russulaceae 3 x 2 1 KM594890,
KM594944, KM594963
Tomentella bryophila Thelephoraceae 3 x 2 1 KM594952, KM594965, KM594885
Cortinarius sp3 Cortinariaceae 4 x x x 1 3 KM595015, KM595039, KM595053, KM594971
Cortinarius obtusus Cortinariaceae 6 x x 2 4 KM595013, KM595023, KM595024, KM594972, KM594996, KM594838
Cortinarius herpeticus Cortinariaceae 1 x 1 KM594989 Laccaria sp3 Hydnangiaceae 3 x 1 2 KM594912,
KM595040, KM594982
Laccaria sp4 Hydnangiaceae 6 x x 1 1 2 2 KM595030, KM594925, KM595045, KM594997, KM595049, KM594848
Russula sp8 Russulaceae 4 x x 1 3 KM595005, KM595057, KM594865, KM594995
Cortinarius sp4 Cortinariaceae 3 x x 1 2 KM595022, KM594938, KM595007
Russula sp9 Russulaceae 2 x x 2 KM595025, KM595029
Boletus sp1 Boletaceae 1 x 1 KM595033
38
Table 2.5 (continued)
OTU BLAST ID Family Developmental
stages Sites GenBank accession numbers Abundance A Sa Se
AF
HA
HB HO
Lactarius leonardii Russulaceae 2 x 2 KM594941, KM594847
Chalciporus aff. rubinellus
Boletaceae 1 x 1 KM594868
Phylloporus centroamericanus
Boletaceae 2 x x 1 1 KM594879, KM594911
Russula crenulata Russulaceae 3 x x 2 1 KM594881, KM595054, KM594991
Thelephoraceae sp. Thelephoraceae 3 x x x 1 2 KM594860, KM594936, KM594974
Tomentella sp. Thelephoraceae 2 x 1 1 KM595011, KM594939
Russula decolorans Russulaceae 2 x 2 KM594942, KM594943
Laccaria sp5 Hydnangiaceae 2 x 2 KM594950, KM594954
Octaviania asterosperma Boletaceae 2 x 2 KM594955, KM594958
Lactarius sp1 Russulaceae 4 x x 1 3 KM595028, KM595038, KM594934, KM595003
Elaphomyces sp. Elaphomycetaceae
2 x x 2 KM595018, KM595020
Amanita sp. Amanitaceae 1 x 1 KM595044 Cortinarius sp5 Cortinariaceae 1 x 1 KM594849 Leccinum sp. Boletaceae 1 x 1 KM594894 Russula illota Russulaceae 3 x 3 KM594873,
KM594874, KM594875
Russula odorata Russulaceae 2 x x 1 1 KM594901, KM594910
Russula sp10 Russulaceae 1 x 1 KM594908 Russula sp11 Russulaceae 1 x 1 KM594970 Russula cf. flavisiccans Russulaceae 1 x 1 KM594990 Cortinarius sp6 Cortinariaceae 1 x 1 KM594850 Tomentella sp2 Thelephoraceae 2 x x 2 KM594920,
KM594929 Russula sp13 Russulaceae 2 x 1 1 KM595037,
KM594983 Tomentella sp3 Thelephoraceae 2 x 2 KM594866,
KM594968 Thelephoraceae sp2 Thelephoraceae 2 x x 1 1 KM594928,
KM595009
39
Table 2.5 (continued)
OTU BLAST ID Family Developmental
stages Sites GenBank accession numbers Abundance A Sa Se
AF
HA
HB HO
Lactarius cf. gerardii Russulaceae 1 x 1 KM594852 Sebacinaceae sp. Sebacinaceae 1 x 1 KM594864 Thelephoraceae sp3 Thelephoraceae 1 x 1 KM594973 Leratiomyces ceres Strophariaceae 1 x 1 KM594985 Inocybe sp1 Inocybaceae 1 x 1 KM595016 Rhodocollybia turpis Marasmiaceae 1 x 1 KM595017 Elaphomyces sp1 Elaphomycetace
ae 1 x 1 KM595019
Sebacina sp1 Sebacinaceae 1 x 1 KM595021 Tylopilus pseudoscaber Boletaceae 1 x 1 KM594859 Leptosporomyces galzinii Atheliaceae 1 x 1 KM595026 Russula variata Russulaceae 1 x 1 KM595027 Coltricia sp Hymenochaetac
eae 1 x 1 KM594863
Tomentella sp4 Thelephoraceae 1 x 1 KM595031 Amanita virosa Amanitaceae 1 x 1 KM594878 Tomentella sp5 Thelephoraceae 1 x 1 KM594882 Boletaceae sp Boletaceae 1 x 1 KM594886 Hydnum repandum Hydnaceae 1 x 1 KM594888 Elaphomyces sp2 Elaphomycetace
ae 1 x 1 KM594889
Russula olivacea Russulaceae 1 x 1 KM594895 Coltriciella dependens Hymenochaetac
eae 1 x 1 KM594897
Tomentella sp6 Thelephoraceae 1 x 1 KM595035 Russula lutea Russulaceae 1 x 1 KM594898 Cortinarius junghuhnii Cortinariaceae 1 x 1 KM594905 Russula sp16 Russulaceae 1 x 1 KM594914 Thelephoraceae sp4 Thelephoraceae 1 x 1 KM594916 Thelephoraceae sp5 Thelephoraceae 1 x 1 KM594921 Coltriciella dependens Hymenochaetac
eae 1 x 1 KM594923
Inocybe cicatricata Inocybaceae 1 x 1 KM594930 Lactarius sp2 Russulaceae 1 x 1 KM595046 Russula sp17 Russulaceae 1 x 1 KM594933 Tricholoma sulphureum Tricholomatacea
e 1 x 1 KM594945
Thelephoraceae sp6 Thelephoraceae 1 x 1 KM595047 Sebacina sp2 Sebacinaceae 1 x 1 KM594947 Russula sp20 Russulaceae 1 x 1 KM594948 Thelephoraceae sp7 Thelephoraceae 1 x 1 KM594951 Russula crustosa Russulaceae 1 x 1 KM594957 Russula sp21 Russulaceae 1 x 1 KM594959 Tomentella sp7 Thelephoraceae 1 x 1 KM594961 Cortinarius junghuhnii Cortinariaceae 1 x 1 KM594964 Cortinarius laetus Cortinariaceae 1 x 1 KM594966 Tomentella sp8 Thelephoraceae 1 x 1 KM594999
40
Table 2.5 (continued)
OTU BLAST ID Family Developmental
stages Sites GenBank accession numbers Abundance A Sa Se
AF
HA
HB HO
Thelephoraceae sp8 Thelephoraceae 1 x 1 KM595000 Clavulina sp2 Clavulinaceae 1 x 1 KM595002 Cortinarius sp9 Cortinariaceae 1 x 1 KM595050 Cortinarius sp Cortinariaceae 1 x 1 KM595052 Byssocorticium sp Atheliaceae 1 x 1 KM594979 Tomentella sp9 Thelephoraceae 1 x 1 KM594980 Inocybe tubarioides Inocybaceae 1 x 1 KM594981 Cortinarius sp10 Cortinariaceae 1 x 1 KM594994 Russula sp26 Russulaceae 1 x 1 KM595055 Russula sp27 Russulaceae 1 x 1 KM594833 Tomentella sp10 Thelephoraceae 1 x 1 KM594836 Russula sp28 Russulaceae 1 x 1 KM594837 Tomentella sp11 Thelephoraceae 1 x 1 KM594840 Tomentella ramosissima Thelephoraceae 1 x 1 KM594842 Tomentella sp12 Thelephoraceae 1 x 1 KM594844 Tomentella sp13 Thelephoraceae 1 x 1 KM594846 Clavulina sp3 Clavulinaceae 1 x 1 KM594855
41
Table 2.6 Distribution of Russula from Oreomunnea root tips in Fortuna Forest Reserve, Panama. Columns indicate the GenBank number for sequences generated in this study; sequence code; tentative ID; sites, developmental stages, and fertility levels of origin; whether the sequence was obtained from a root tip (RT) or fruiting body (FB) in Oreomunnea stands; and the top BLAST match. Sequences referenced here were used in phylogenetic reconstruction for measures of phylogenetic diversity and UniFrac analyses. Site abreviations HA: Honda A, HB: Honda B, HO: Hornito, AF: Alto Frio, ZA: Zarceadero (not included for root tip collections). Developmental stage abbreviations: A: Adult, Sa: Sapling, Se: Seedling.
GenBank accession
#
Sequence Code OTU BLAST ID Site Fertility Developmental
stages Tissue Best match in BLAST
KM594949 AC36 Russula crassotunicata HA Low Se RT Russula crassotunicata KM594956 AC38 Russula sp6 HA Low Se RT Russula sp.
KM594986 AC47B Russula sp3 (cyanoxantha) HA Low Sa RT Russula cf. cyanoxantha
KM594822 AC52 Russula sp29 HB Low FB Russula corallina KM595014 AC72 Russula sp2 HA Low A RT Russula puellaris KM595025 AC100 Russula sp4 AF High A RT Russula xerampelina KM594783 AC132 Russula sp23 ZA FB Russula sp. KM594871 AC145 Russula nigricans AF High Se RT Russula nigricans KM594873 AC148 Russula illota AF High Se RT Russula illota KM594874 AC149 Russula illota AF High Se RT Russula illota KM594875 AC150 Russula illota AF High Se RT Russula illota KM594881 AC158 Russula crenulata HO High A RT Russula crenulata KM594887 AC173 Russula sp5 HO High Se RT Russula sp. KM595032 AC189 Russula sp5 AF High A RT Russula rubellipes KM594890 AC190 Russula sp7 AF High A RT Russula eccentrica KM594893 AC195 Russula cf. pectinata HA Low A RT Russula cf. pectinata
KM594784 AC198 Russula sp3 (cyanoxantha) HA Low FB Russula cf. cyanoxantha
KM594895 AC204 Russula olivacea HA Low A RT Russula olivacea KM595034 AC205 Russula sp2 HA Low A RT Russula puellaris KM594896 AC210 Russula cf. pectinata HA Low Se RT Russula cf. pectinata KM594898 AC215 Russula sp16 HA Low Sa RT Russula lutea KM594901 AC221 Russula sp11 HA Low Sa RT Russula odorata KM594902 AC222 Russula sp6 HA Low Sa RT Russula sp. KM594903 AC223 Russula crassotunicata HA Low Sa RT Russula crassotunicata KM594904 AC225 Russula sp2 HA Low Sa RT Russula puellaris KM595036 AC229 Russula sp2 HB Low A RT Russula puellaris KM595037 AC233 Russula sp14 HB Low Sa RT Russula solaris KM594907 AC237 Russula cf. pectinata HB Low A RT Russula cf. pectinata KM594908 AC239 Russula sp12 HB Low A RT Russula compacta KM594910 AC241 Russula sp11 HB Low Se RT Russula odorata KM594914 AC249 Russula sp17 HB Low Sa RT Russula sp. KM594915 AC250 Russula sp2 HB Low Sa RT Russula puellaris KM594918 AC257 Russula sp2 HB Low Sa RT Russula puellaris KM595042 AC262 Russula cf. pectinata HO High Se RT Russula cf. pectinata KM594785 AC269 Russula sp26 HB Low FB Russula cf. favrei
KM594786 AC270 Russula sp3 (cyanoxantha) HB Low FB Russula cf. cyanoxantha
42
Table 2.6 (continued)
GenBank accession
#
Sequence Code OTU BLAST ID Site Fertility Developmental
stages Tissue Best match in BLAST
KM594787 AC271 Russula sp3 HB Low FB Russula cf. cyanoxantha KM594788 AC272 Russula sp6 HB Low FB Russula sp. KM594922 AC277 Russula cf. pectinata HO High A RT Russula cf. pectinata KM594926 AC282 Russula cf. pectinata HO High A RT Russula cf. pectinata KM595043 AC288 Russula sp5 HO High A RT Russula rubellipes voucher
KM594931 AC296 Russula sp3 (cyanoxantha) HO High Sa RT Russula sp.
KM594933 AC302 Russula sp18 HO High Sa RT Russula nigricans isolate KM594789 AC305 Russula sp23 ZA FB Russula sp. KM594935 AC310 Russula sp2 HB Low Se RT Russula puellaris voucher KM594790 AC317 Russula sp12 ZA FB Russula sp. KM594791 AC318 Russula sp24 ZA FB Russula sp. KM594792 AC329 Russula sp30 ZA FB Russula sp. KM594793 AC331 Russula sp24 ZA FB Russula sp. KM594794 AC334 Russula sp31 ZA FB Russula cf. foetens KM594823 AC335 Russula sp28 ZA FB Russula sp. KM594795 AC336 Russula sp6 HB Low FB Russula sp. KM594942 AC345 Russula sp10 HO High A RT Russula decolorans isolate KM594944 AC350 Russula sp7 HO High A RT Russula eccentrica voucher KM594946 AC357 Russula sp5 HO High Sa RT Russula sp. KM594948 AC359 Russula sp19 HO High Se RT Russula sp. KM594796 AC369 Russula sp6 HA Low FB Russula sp. KM594957 AC381 Russula crustosa HO High Sa RT Russula crustosa voucher KM594963 AC394 Russula sp7 AF High A RT Russula eccentrica voucher
KM594797 AC403 Russula sp3 (cyanoxantha) HA Low FB Russula cf. cyanoxantha
KM594970 AC408 Russula sp13 HB Low A RT Russula cf. pectinata
KM594798 AC416 Russula sp3 (cyanoxantha) HA Low FB Russula cf. cyanoxantha
KM594975 AC427 Russula sp6 HB Low Se RT Russula sp. KM594824 AC430 Russula sp27 HO High FB Russula sp.
KM594799 AC431 Russula sp3 (cyanoxantha) HB Low FB Russula sp.
KM594800 AC434 Russula sp26 HB Low FB Russula cf. favrei KM594976 AC441 Russula sp5 HB Low Sa RT Russula sp.
KM594977 AC445 Russula sp3 (cyanoxantha) HB Low Sa RT Russula sp.
KM594801 AC448 Russula sp3 (cyanoxantha) HB Low FB Russula cf. cyanoxantha
KM594978 AC450 Russula sp3 (cyanoxantha) HB Low A RT Russula sp.
KM595051 AC442 Russula sp2 HB Low A RT Russula puellaris voucher KM594983 AC457 Russula sp14 HA Low Sa RT Russula occidentalis KM594990 AC491 Russula cf. flavisiccans HA Low A RT Russula cf. flavisiccans KM594991 AC494 Russula crenulata HA Low Se RT Russula crenulata KM594992 AC495 Russula sp2 HA Low A RT Russula puellaris voucher
43
Table 2.6 (continued)
GenBank accession
#
Sequence Code OTU BLAST ID Site Fertility Developmental
stages Tissue Best match in BLAST
KM594802 AC497 Russula sp25 AF High FB Russula illota KM594803 AC501 Russula sp7 AF High FB Russula eccentrica voucher KM594825 AC503 Russula sp5 AF High FB Russula rubellipes voucher KM594993 AC504 Russula sp6 HA Low A RT Russula sp. KM594804 AC507 Russula sp33 AF High FB Russula foetens voucher KM594826 AC511 Russula sp28 AF High FB Russula sp.
KM594805 AC516 Russula sp3 (cyanoxantha) HA Low FB Russula sp.
KM594806 AC517 Russula sp34 AF High FB Russula subnigricans KM594807 AC518 Russula sp7 AF High FB Russula eccentrica voucher KM594808 AC520 Russula nigricans AF High FB Russula nigricans
KM594809 AC521 Russula sp3 (cyanoxantha) HA Low FB Russula sp.
KM594810 AC522 Russula sp27 HO High FB Russula puellaris voucher
KM594811 AC523 Russula sp3 (cyanoxantha) HB Low FB Russula sp.
KM594812 AC527 Russula sp3 (cyanoxantha) HB Low FB Russula cf. cyanoxantha
KM594813 AC528 Russula sp6 HB Low FB Russula sp. KM594827 AC531 Russula sp1 HO High FB Russula sp. KM594998 AC537 Russula sp2 HA Low A RT Russula puellaris voucher KM594814 AC545 Russula sp13 ZA FB Russula cf. pectinata KM595054 AC562 Russula crenulata HA Low A RT Russula crenulata KM594828 AC572 Russula cf. pectinata HA Low FB Russula cf. pectinata
KM595010 AC573 Russula sp3 (cyanoxantha) HB Low Se RT Russula sp.
KM594815 AC578 Russula sp3 (cyanoxantha) HB Low FB Russula cf. cyanoxantha
KM594829 AC579 Russula sp3 (cyanoxantha) HA Low FB Russula sp.
KM594830 AC581 Russula sp35 HO High FB Russula sp. KM594816 AC580 Russula cf. flavisiccans HO High FB Russula cf. flavisiccans
KM594817 AC584 Russula sp3 (cyanoxantha) HA Low FB Russula cf. cyanoxantha
KM594818 AC588 Russula sp36 HO High FB Russula eccentrica voucher
KM594819 AC590 Russula sp3 (cyanoxantha) HB Low FB Russula cf. cyanoxantha
KM594820 AC591 Russula sp3 (cyanoxantha) HO High FB Russula cf. cyanoxantha
KM594831 AC587 Russula sp23 HO High FB Russula sp. KM594821 AC603 Russula sp37 AF High FB Russula gracilis voucher KM595055 AC612 Russula sp20 HO High Sa RT Russula sp. KM594833 AC615 Russula sp21 AF High A RT Russula tenuiceps voucher KM594837 AC624 Russula sp22 AF High A RT Russula decolorans voucher
44
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Chapter 3: An ectomycorrhizal nitrogen economy facilitates monodominance in a neotropical forest2
Introduction
Tropical forests are renowned for high local species richness, often exceeding 100 tree
species ha-1 (Wright 2002). Nonetheless, exceptions to the general pattern of high-diversity and
low relative abundance exist throughout the tropics. These 'monodominant' communities, in
which a single tree species accounts for > 60% of basal area (Hart et al. 1989), include Dicymbe
corymbosa forests in Guyana, Gilbertiodendron dewevrei forests in central Africa, and some
dipterocarp forests in Southeast Asia (e.g., Dryobalanops aromatica¸ Shorea curtisii). Several
potential mechanisms that promote monodominance in tropical forest have been proposed, but a
general explanation remains elusive (Peh et al. 2011a).
Mechanisms to explain monodominance have focused either on the disturbance regime of
the forest, with low disturbance rates favoring competitive exclusion (Connell & Lowman 1989;
Hart et al. 1989), or on intrinsic traits of the species that confer a competitive advantage,
including low palatability to herbivores, slow rates of leaf litter decomposition, high seedling
shade-tolerance, and large seed size (Hart et al. 1989, Torti et al. 2001). However, a feature of
many monodominant species, both temperate and tropical, is that they form associations with
ectomycorrhizal (EM) fungi (Connell and Lowman 1989). Only 6% of neotropical tree species
are estimated to form associations with EM fungi, while 94% associate with arbuscular
mycorrhizal (AM) fungi (Table 3.1). In contrast, of the 22 tree species reported as
monodominant by Peh et al. (2011a), ten (45%) are EM and twelve (55%) are AM. As a
consequence, mechanisms have been sought that could account for how EM-associated plants
achieve monodominance at sites with similar soil conditions and disturbance regimes as those
2 This chapter appeared in its entirety in the Journal Ecology Letters and is referred to later in this dissertation as “Corrales et al. 2016b”. Corrales, A.; Mangan, S.A.; Turner, B.L.; and Dalling, J.W. 2016. An ectomycorrhizal nitrogen economy explains monodominance in a neotropical forest. Ecology Letters 19: 383–392. This article is reprinted with the permission of the publisher and is available from http://onlinelibrary.wiley.com/doi/10.1111/ele.12570/abstract and using DOI: 10.1111/ele.12570.
54
that support diverse communities of AM-associated trees (Connell and Lowman 1989, Dickie et
al. 2014).
Two mechanisms have been proposed for the EM facilitation of local monodominance.
First, EM networks consisting of hyphal connections linking plants could facilitate the transfer of
water, carbon (C), and nutrients from adults to seedlings (Simard et al. 2012). Ectomycorrhizal
networks are predicted to increase juvenile survival, disproportionately increasing their
abundance near adult trees (Teste et al. 2009, Simard et al. 2012). Consistent with this
hypothesis, seedlings of the monodominant species Paraberlinia bifoliolata in Cameroon, and
Dicymbe corymbosa in Guyana grew more slowly and died more frequently when isolated from
potential EM networks using exclosures (Onguene and Kuyper 2002, McGuire 2007). However,
these studies provided no direct evidence that EM networks transfer nutrients or photosynthate.
Moreover, neither study included a non-mycorrhizal plant species to assess exclosure effects
unrelated to mycorrhizal associations.
Second, recent reviews hypothesize that monodominance may arise through microbially
mediated positive plant-soil feedbacks (McGuire 2014). Plant-soil feedbacks (PSF) mediated by
EM fungi could lead to monodominance in two ways. First, the build-up of beneficial EM fungi
around adult host trees might favor the growth and survival of conspecific seedlings relative to
those of neighboring non-ectomycorrhizal seedlings. This positive plant-soil feedback is
expected to promote the dominance of EM plant species (Bever et al. 1997). Second, EM fungi
could weaken the strength of negative PSF produced by species-specific pathogens and increase
the survivorship rates of EM seedlings around conspecific adult trees (Bever 2003). Plant species
that exhibit weak negative feedbacks often dominate the community (Klironomos 2002, Mangan
et al. 2010), yet no experimental study has examined the strength of PSF in tropical EM tree
species.
In addition to network effects and altered plant-soil feedback, EM fungi might also
facilitate monodominance by altering N cycling, with detrimental effects on competing AM plant
species. Recent research has highlighted mycorrhizal association as an important functional trait
influencing species interactions and ecosystem processes (Phillips et al. 2013, Averill et al.
2014). Here we propose that EM associations facilitate monodominance via the "Microbial
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competition for N hypothesis" (referred to as the “organic nutrient-use hypothesis” in Dickie et
al. 2014). Ectomycorrhizal fungi have the enzymatic capability to access organic N directly from
soil organic matter, including the litter layer (Hobbie and Högberg 2012), implying that they
compete directly with saprotrophs for N (Orwin et al. 2011, Hobbie and Högberg 2012, Averill
et al. 2014). This enhanced competition is expected to reduce litter decomposition and N
mineralization rates in the presence of EM fungi compared to AM fungi (Connell and Lowman
1989, McGuire et al. 2010, Phillips et al. 2013). Slower decomposition rate and reduced N
availability could, in turn, favor EM plants when competing with AM plants (Orwin et al. 2011,
Phillips et al. 2013). Consistent with this hypothesis, lower concentrations of total N, ammonium
and nitrate have been reported below stands of monodominant forest relative to nearby mixed
forest (Torti et al. 2001, Read 2006, Brookshire and Thomas 2013, Dickie et al. 2014).
Here we evaluated these three potential mechanisms mediating monodominance in a
lower-montane tropical forest in Panama. We used a combination of field and growing house
experiments to test for mycorrhizal networks, plant-soil feedback, and changes in the N cycle in
forest dominated by Oreomunnea mexicana (Juglandaceae), a widely distributed EM tree species
that forms monodominant forest. We predicted that if dominance is promoted by mycorrhizal
networks, then disruption of hyphal networks would negatively affect Oreomunnea seedling
growth and survival and alter leaf tissue and leaf sugar C isotope ratios. In contrast, if dominance
is mediated by plant-soil feedback then we predicted that Oreomunnea seedling growth would
either be increased in the presence of soil inoculum from beneath Oreomunnea trees relative to
inoculum from competing species (positive PSF), or that the strength of negative PSF in the
presence of conspecific inoculum would be weaker for Oreomunnea than for competitors.
Finally, if dominance is mediated via the “Microbial competition for N hypothesis”, then we
predicted that mineral N supply would be reduced inside Oreomunnea-dominated forest, and that
bulk soil stable δ15N isotope ratio –an integrated measure of the N cycle– would be smaller
inside than outside Oreomunnea-dominated forest, indicating a tighter N cycle due to depletion
of N from the soil organic matter pool by EM fungi.
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Methods
Study site
The study was located in a primary lower montane forest (1000–1400 m a.s.l.) in the
Fortuna Forest Reserve in western Panama (hereafter Fortuna; 8°45 N, 82°15 W). Mean annual
temperature ranges from 19 to 22˚C, and annual rainfall from 5800 to 9000 mm (Andersen et al.
2012). Tree communities at Fortuna are diverse, containing between 61 and 153 species ha-1 ( >
10 cm DBH), with high compositional turnover reflecting variation in both rainfall and soils.
Oreomunnea mexicana is a canopy tree distributed between Mexico and Panama at elevations
from 900 to 2600 m. The distribution of Oreomunnea at Fortuna is patchy, with populations
occurring on both high and low fertility soils (Corrales et al. 2016a). On low fertility rhyolite-
derived soils, mixed forest is interspersed within Oreomunnea-dominated forest, where it
accounts for up to 70% of individuals and basal area (Andersen et al. 2010, Corrales et al.
2016a). Dominance by Oreomunnea appears unrelated to particular plant functional traits
previously associated with monodominance: foliar N, phosphorus (P), and C:N ratios are close to
community averages for the site (Adamek 2009) and seeds are small (approximately 100 mg).
However, Oreomunnea forms EM associations (Corrales et al. 2016a) in contrast to almost all
co-occurring tree species at Fortuna. Other EM tree species found at low abundance at the study
area are Quercus insignis, Q. cf. lancifolia, and Coccoloba spp.
Experimental assessment of EM network effects
We established a hyphal exclusion experiment to test whether Oreomunnea seedlings
benefit from EM hyphal connections to neighboring plants. Roupala montana (Proteaceae), a
non-mycorrhizal tree species that co-occurs with Oreomunnea, was used to assess the non-
mycorrhizal treatment effects of exclosures on seedling growth. Oreomunnea and Roupala
seedlings were planted in mesh exclosures inside and outside a ~25 ha Oreomunnea-dominated
forest. Individual seedlings were transplanted 2 m from each of 20 randomly selected
Oreomunnea trees (blocks) inside the Oreomunnea forest and 10 similar-sized heterospecific
trees 30 m outside the edge of the forest. Nearest-neighbour focal trees inside the Oreomunnea-
dominated forest were on average 40 m apart; trees outside were on average 45 m apart.
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Treatments consisted of: 1) fungal hyphal exclosures with 0.5 µm mesh to prevent
seedlings from connecting to EM networks; 2) root exclosures with 35 µm mesh to exclude roots
of neighboring plants, but allow hyphae access to the transplanted seedling (this treatment was
used to assess the effect of reduced root competition in the analysis of EM network effects); 3)
transplants without an exclosure, to assess the effect of soil disturbance on seedling growth (the
seedlings and surrounding soil was removed and replaced in a similar way to treatments 1 and 2);
4) control: A naturally occurring Oreomunnea seedling of similar size growing next to the other
treatments, but left untouched for comparison (inside Oreomunnea forest only). A total of 202
seedlings were established, 112 Oreomunnea (30 replicates × 3 exclosure treatments, and 22
control seedlings) and 90 Roupala (30 replicates × 3 exclosure treatments, no control). Mesh
exclosures were assembled following Nottingham et al. (2010) from 16 cm diameter, 20 cm deep
PVC piping, perforated along the sides and covered on the bottom and sides with the nylon mesh
corresponding to each treatment (Plastok, Birkenhead, UK).
Seedlings were transplanted from a nearby forest into the exclosure treatments between
August and October of 2013 and grown for 297 to 345 days. Previous studies that have reported
effects of EM networks on seedling mortality and RGR grew plants for between 5 and 12 months
(Onguene and Kuyper 2002, McGuire 2007). We therefore expected that 10 to 11 months would
be sufficient time to detect differences in our experiment. Seedlings were harvested and the
relative growth rate (RGR, mg g-1day-1) was calculated as the natural log of final dry mass minus
the natural log of initial dry mass divided by the number of days in the experiment. To estimate
initial seedling biomass 20 and 15 seedlings of Oreomunnea and Roupala from outside of the
experiment were harvested, and height, leaf number, and leaf area measured to develop biomass
models (Table 3.2). In a subset of the surviving Oreomunnea seedlings growing inside (n = 32)
and outside (n = 8) a sample of root tissue was saved to evaluate EM colonization by clearing in
10% KOH and staining with trypan blue. Colonization of 10 randomly chosen 2.0-cm root
sections per seedling was assessed using the grid-line intersect method (McGonigle et al. 1990).
Fresh leaf tissue was frozen immediately in liquid N for isotopic analysis. In tropical trees with
long leaf life spans, foliar isotope ratios can reflect nutrient uptake over months or years, and
may mask any signal of fungal C dependency (Hynson et al. 2012). We therefore analyzed leaf
soluble sugars, which represent recently acquired C. Sugars were extracted using ion exchange
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resins for sugar purification following Hynson et al. (2012). In addition, leaf tissue from a subset
of the surviving Oreomunnea seedlings growing inside (n =16) and outside (n = 12)
Oreomunnea-dominated forest was analyzed for C and N concentrations and stable isotope ratios
(δ13C and δ15N) by continuous flow isotope ratio mass spectrometry using an elemental analyzer
(Costech 4010) coupled to a Delta-V Advantage isotope ratio mass spectrometer (Thermo Fisher
Scientific, Bremen, Germany). Run precision for δ13C and δ15N was typically < 0.2‰.
Plant-soil feedback experiment
To determine whether Oreomunnea shows positive or negative PSF compared with co-
occurring species, seedlings of five tree species were grown in a fully factorial greenhouse
experiment. The species included were two EM plant taxa, Oreomunnea mexicana
(Juglandaceae) and Quercus insignis (Fagaceae), and three AM species, Guarea pterorhachis
(Meliaceae), Cupania seemannii (Sapindaceae), and Nectandra purpurea (Lauraceae). Species
were chosen based on seed availability and contrasting abundance in permanent 1-ha forest plots.
Species relative abundance of individuals with a diameter at breast height (DBH) >10 cm ranged
from 0.3% for Quercus to 7.7% for Oreomunnea.
Seedlings were grown in different soil treatments: each species was grown either in their
own soil inoculum ("conspecific" soil treatment), where pots received an inoculum of live soil
from underneath conspecifics representing 6% of the total soil volume (120 cm3), or in pots
inoculated with live soil from one of each of the other four species separately ("heterospecific"
soil treatment). Seven seedlings of each species were grown in each of the "heterospecific"
treatments and 10 were grown in the "conspecific" treatment. To control for differences in
abiotic conditions associated with the live soil, two additional replicates of each treatment were
established in which the inoculum was sterilized (Figure 3.7). A total of 240 sampling units were
used for the full experiment ("heterospecific" treatments = 5 species × 4 inoculum types × 7
replicates = 140 seedlings; "conspecific" treatments = 5 species × 10 replicates = 50 seedlings;
sterilized inoculum controls = 10 replicates × 5 species = 50 seedlings).
Between June and October 2013, seeds of the five species were collected from the forest
floor, surface sterilized with 1% NaClO and grown in sterile sand for 2 months prior to the start
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of the experiment. Oreomunnea seeds were also soaked in 1% HCl for 1 hour prior to planting to
break dormancy. Each species was grown in a 2-L pot (14 cm × 11 cm × 13 cm depth) filled with
steam-pasteurized soil collected from three relatively nutrient-rich sites in the Fortuna Reserve.
Seedlings were planted during November and December 2013 and grown under 18% full sun in
a growing house at Fortuna Forest Reserve. Inoculum was collected from underneath five adult
individuals of each species (about 500 g of soil per tree) and inoculum from each tree used
separately to keep track of individual inoculum sources. Nectandra seedlings were grown for 3
months, while the remaining species were grown for 5 to 6 months depending on growth rate.
Plants were watered once or twice per week and did not receive additional nutrients.
At harvest a subsample of root tissue was saved to evaluate EM colonization using the
same method described above. The roots of all plants were washed and all plant parts were dried
at 70°C for 48 h and weighed to quantify total biomass. To calculate leaf area, photographs of all
fresh leaves of each plant were analyzed using ImageJ (Schneider et al. 2012). For individual
plants, the RGR was calculated as above. Leaf Area Ratio (LAR) was calculated by dividing
total leaf area by total biomass. To estimate the initial seedling biomass 14 to 20 seedlings per
species were harvested, and height, number of leaves, and leaf area measured to develop biomass
models (Table 3.2).
Nutrient availability and uptake inside and outside Oreomunnea-dominated forest
To determine whether Oreomunnea forest influences N cycling in a way that is consistent
with the “Microbial competition for N hypothesis”, we compared resin-extractable nitrate,
ammonium and phosphate inside and outside the same Oreomunnea-dominated forest used for
the EM exclosure experiment. Twenty resin bags containing 5 g of mixed-bed anion and cation
exchange resins (Dowex Marathon Mr-3) sealed inside 220 µm polyester mesh were buried 2 cm
beneath the soil surface 0.5-1 m from 10 trees inside and 10 trees outside the Oreomunnea forest.
Bags were buried during August 2014 at the same locations where mesh cores were installed.
After incubation in situ for 18 days the resin bags were collected, rinsed with deionized water to
remove adhering soil, extracted with 75 mL of 0.5 M HCl, and then nitrate (+nitrite),
ammonium, and phosphate were determined by automated colorimetry on a Lachat QuikChem
8500 (Hach Ltd., Loveland, CO, USA). In addition, soils collected inside (n =10) and outside (n
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= 8) Oreomunnea forest were analyzed for C and N isotope ratios and total concentrations as
described above using a Flash HT analyzer (Thermo Scientific, Waltham, MA, USA).
Finally, we examined whether there was a relationship between tree abundance and soil
properties. Abundance was calculated for the two EM tree species (Oreomunnea mexicana and
Quercus insignis) and the four most abundant AM tree species (Ardisia sp., Dendropanax
arboreus, Cassipourea guianensis, and Eschweilera panamensis) as the sum of individuals >5
cm DBH in 20 × 20-m sub-plots located in two permanent 1-ha forest plots at the study site (n
=18 sub-plots; plots = Honda A and Honda B in Andersen et al. 2010). Soils data specific to
each sub-plot were collected in 2008 to 10 cm depth, and included soil ammonium (µg N g-1 soil
dry mass), nitrate (µg N g-1), total N (%), total C (%), total P (µg P g-1), and litter mass (g m-2)
(Table 3.3).
Statistical analysis
For the mesh exclosure study, a two-way ANOVA was used to compare seedling RGR,
isotopic data, and EM colonization among treatments inside and outside Oreomunnea forest,
with location (inside versus outside) and mesh treatment (0.5 µm, 35 µm, transplant, and control)
as fixed effects and replicates (trees) as a random effect. The mortality rates of seedlings were
compared using contingency tables and a Chi-square test of independence. Resin extractable
nitrate, ammonium and phosphate were not normally distributed, and were compared between
locations (inside versus outside) using a Wilcoxon test.
We used ANCOVA to analyze the effects of inoculum source and plant species (and their
interaction) on plant growth using the SAS procedure PROC MIXED. Estimated initial biomass
was included as a covariate. Within this model, we used a priori contrasts to isolate the
inoculum source × species interaction of each possible pair of species (Bever et al. 1997) to
determine the strength and direction of plant-soil feedback. In the case where seedlings
performed better in conspecific inoculum relative to that of heterospecifics, the interaction
coefficient (i.e. pairwise feedback) would be positive. Average feedback strength of a given
species was then determined by averaging all pair-wise interaction terms that involved that
species (Bever et al. 1997). The relationship between tree species abundance and strength of
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average feedback was examined using linear regression. The relative abundance of each species
was calculated for trees >10 cm DBH in permanent plots established where the species inoculum
was collected (Oreomunnea mexicana = Honda A, Cupania seemannii and Nectandra purpurea
= Pinola, Quercus insignis and Guarea pterorhachis = Hornito) (J.W. Dalling personal
communication).
Soil extractable C, N and δ15N were compared inside and outside the Oreomunnea forest
using one-way ANOVA. The relationship between the number of trees or basal area in sub-plots
and soil variables in permanent plots was analyzed using a simple linear regression after (log+1)
transformation of the independent variable (individuals and basal area).
Results
EM network effects
From an initial total of 202 seedlings established in the experiment (112 Oreomunnea and
90 Roupala), 173 survived until the end of the experiment: 86 Oreomunnea and 87 Roupala. Of
the 86 surviving Oreomunnea seedlings, 70 seedlings grew inside and 16 outside Oreomunnea
forest. There were no significant effects of hyphal and root exclosure treatments on growth rate
of Oreomunnea seedlings, with the exception of faster growth in the root exclosure treatment
(0.35 µm mesh) compared to the control (undisturbed) treatment (F3,78 = 2.67, P = 0.05) when
including seedlings both inside and outside Oreomunnea forest (Figure 3.1A). Therefore,
excluding hyphae (0.5 µm mesh) did not reduce seedling growth as predicted. Roupala did not
show significant differences among treatments (F2,78 = 0.39, P = 0.68; Figure 3.1B).
Oreomunnea mortality was significantly lower inside (9%) versus outside Oreomunnea
forest (34%; n = 112, X2 = 19.8, df = 1, P < 0.001), but there was no significant difference in its
growth rate (F1,78 = 1.05, P = 0.31; Figure 3.1C). Roupala grew significantly faster outside
Oreomunnea forest (F1,78 = 6.02, P = 0.02; Figure 3.1D), but mortality rate did not differ (2%
inside and outside; n = 90, X2 = 0.13, df = 1, P = 0.71).
Overall, there was no significant difference in EM colonization of Oreomunnea seedlings
among treatments (F3,34 = 0.81, P = 0.50). However, the percentage of EM colonization of
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Oreomunnea seedlings was significantly higher inside than outside Oreomunnea forest (F1,38 =
13.71, P < 0.001; Figure 3.2), and there was a significant site × treatment interaction (F1,34 =
9.46, P = 0.004) driven by significantly lower EM colonization (F1,2 = 38.06, P = 0.03) among
the few surviving seedlings analyzed from the 0.5 µm mesh treatment outside Oreomunnea
forest.
There were no significant differences among treatments for seedlings growing inside the
Oreomunnea forest for δ13C of the extracted leaf sugars (F3,28 = 1.97, P = 0.14), or for δ13C (F3,12
= 1.02, P = 0.7, Figure 3.1E) or δ15N (F3,12 = 0.46, P = 0.98, Figure 3.1F) of leaf bulk tissue.
Oreomunnea seedlings growing inside Oreomunnea forest did not differ in foliar δ13C
(F1,23=1.15, P = 0.35, Table 3.4). However, seedlings growing inside Oreomunnea forest were
significantly depleted in foliar 15N compared with seedlings growing outside (F = 17.33, P <
0.001, Table 3.4) and had significantly greater foliar N concentration (F1,23 = 6.55, P = 0.02,
Table 3.4).
Plant-soil feedback experiment
Monodominance could arise if beneficial EM fungi are restricted to forest dominated by
Oreomunnea and provide a competitive growth advantage to Oreomunnea seedlings. If so, we
predicted that Oreomunnea would show positive PSF or weaker negative PSF effects relative to
co-occurring species. However, Oreomunnea showed the strongest negative PSF among species
in the experiment (Figure 3.3, Table 3.5), and the strength of negative PSF was significantly
positively correlated with species relative abundance (Figure 3.3).
Differences in seedling and soil nutrient status inside and outside Oreomunnea forest
There was significantly lower resin extractable ammonium (Wilcoxon test W = 19, P =
0.021) and nitrate (W = 9, P = 0.002) inside compared to outside the Oreomunnea forest (Table
3.4), but there was no difference in resin-extractable phosphate across the same sites (W = 38, P
= 0.31, Table 3.4). There were also significantly higher concentrations of total C (P < 0.001)
and total N (P = 0.001, Table 3.4), and a higher C:N ratio (P < 0.001; Table 3.4) in soils inside
compared to outside Oreomunnea forest. Soils inside Oreomunnea forest were significantly
depleted in 15N (P < 0.001; Table 3.4).
63
The number of Oreomunnea individuals > 5 cm DBH in 20 × 20-m sub-plots in two 1-ha
plots was significantly negatively associated with the concentration of nitrate (R2 = 0.48, P <
0.001), and P (R2 = 0.21, P < 0.001) measured in the center of each subplot, but was not
significantly correlated with ammonium (R2 = 0.06, P = 0.15), total N (P = 0.79), total C (P =
0.65), or litter mass (P = 0.44, Figure 3.4). Results were similar when the regression analysis was
repeated using Oreomunnea basal area (Figure 3.5). Quercus insignis abundance was not
significantly correlated with any soil nutrients (Figure 3.6). However, all the most abundant AM
species were negatively associated with either ammonium or nitrate concentrations in the soil
(Table 3.6). Ardisia sp., Cassipourea guianensis, and Eschweilera panamensis were also
significantly positively correlated with the number of individuals or basal area of Oreomunnea
mexicana (Table 3.6).
Discussion
Here we provide the first simultaneous test of two prominent hypotheses to account for
monodominance in tropical forests. Our results suggest that neither the absence of negative PSF
nor the formation of EM networks can account for monodominance in our study system. Instead,
we provide evidence that Oreomunnea dominated forest is associated with lower availability of
soil ammonium and nitrate than nearby forest where Oreomunnea is infrequent or absent. This
reduced nutrient availability, presumably driven by depletion of readily mineralizable N from
soil organic matter by EM fungi, could confer Oreomunnea seedlings a competitive advantage
over AM or non-mycorrhizal species.
Plant-soil feedback and EM associations
Several authors have proposed that positive PSF experienced by EM plants underlie
monodominance in tropical forests (McGuire 2007, 2014). However, all of these studies identify
EM networks as the mechanism generating positive PSF. Here we differentiate microbially-
mediated PSF sensu Bever et al. (1997) from positive distance-dependent effects of EM
networks. To our knowledge, only two studies of microbially-mediated PSF have included EM
tree species, and in general negative feedbacks dominate over positive feedbacks (McCarthy-
64
Neumann and Ibañez 2012, Liu et al. 2012). In our study, Oreomunnea showed the strongest
negative PSF among the species tested, despite its high local abundance. This result contrasts
with findings from lowland tropical forest and temperate grasslands, where more abundant
species show weaker negative PSF (Klironomos 2002, Mangan et al. 2010). This is the first PSF
experiment ever done including montane tree species and more research in this topic is needed to
conclude if this is a widespread phenomenon or just a peculiarity of our system. The reduced
RGR of Oreomunnea when grown in its own inoculum indicates that the negative effect of
Oreomunnea species-specific pathogens is stronger than any potential positive effects that might
arise from more beneficial inoculum sources.
Mycorrhizal network effects
Previous experiments that have tested for the presence of EM networks in tropical
monodominant forests reported higher growth and survival for seedlings in contact with the roots
of conspecifics and EM mycelium relative to those isolated in mesh exclosures (Onguene and
Kuyper 2002, McGuire 2007). In contrast, our results show no evidence of C or N transfer from
adults to seedlings through EM networks based on either changes in seedling growth or survival
in response to hyphal exclosure treatments. The only difference in growth rate among treatments
was higher RGR in seedlings grown in 35-µm mesh exclosures compared with control seedlings,
which was probably due to release from belowground competition with roots of other plants.
However, the fact that we used already established (and EM-infected seedlings in the
experiment) could have reduced the likelihood Oreomunnea seedlings joined existing EM
networks.
The leaves of plants connected to EM networks have an isotopic composition that is
distinct from fully photosynthetic plants (Selosse and Roy 2009, Hynson et al. 2012). If plants
receive sugars via EM networks then we would expect that leaf tissue, and in particular leaf
soluble sugars, would be enriched in 13C (Selosse and Roy 2009, Hynson et al. 2012). Here we
found no differences in the isotopic composition of leaf tissue among exclosure treatments,
further suggesting that no EM networks were formed in this system.
65
Instead, this study shows that seedlings growing inside Oreomunnea-dominated forest
had higher EM colonization, lower foliar 15N, and higher foliar N concentration. Previous studies
have found a correlation between δ15N in plants and the degree of EM fungal colonization
(Hobbie and Colpaert 2003). The presence of EM colonization alters host plant δ15N by
transferring 15N depleted compounds to the plant while sequestering 15N-enriched compounds in
fungal tissue (Hobbie and Colpaert 2003). In the case of Oreomunnea seedlings growing inside
Oreomunnea forest, the lower 15N of their leaves could have been partially due to lower 15N in
the soil. However, a decrease in foliar 15N has been observed in EM plants experiencing
increased N limitation (McLauchlan et al. 2010, Craine et al. 2015), associated with a higher
dependency on EM fungi (Craine et al. 2015). These results suggest that the patchiness of
Oreomunnea populations in part reflects reduced mycorrhizal infection outside Oreomunnea
forest. The lack of appropriate or compatible EM inoculum outside Oreomunnea patches could
have strongly reduced survivorship rates due to a reduced capacity of seedlings to compete for N
with larger AM trees (Nara et al. 2006).
Microbial competition for N as a mechanism facilitating monodominance in tropical forest
A reduction in the decomposition rate of litter, and an increase in organic matter
accumulation has been frequently noted under EM dominated forest (Torti et al. 2001, Orwin et
al. 2011, Phillips et al. 2013, Averill et al. 2014). The mechanisms underlying this effect are not
fully understood, although it has been proposed that direct competition for N between EM fungi
and the community of free-living decomposers could reduce litter decomposition rates (Read and
Perez-Moreno 2003, Orwin et al. 2011). Competition for nutrients is proposed to result in the
accumulation of organic matter depleted in N under EM dominated forest, resulting in a
reduction in nutrient availability for AM or non-mycorrhizal plant species, as well as microbial
heterotrophs, that rely to a greater extent on inorganic N sources (Phillips et al. 2013).
Ectomycorrhizal plants growing in soils with low available N may have a competitive advantage
over AM plants because EM fungi are able to acquire N directly from organic matter when
supplied with sugar from their host plant (Phillips et al. 2013, Lindahl and Tunlid 2015).
Lower N availability could reduce plant diversity (Dickie et al. 2014) and ultimately
drive monodominance in tropical forests (Figure 3.8). The differences in soil 15N in our system
66
along with the higher total C, reflects N limitation and an accumulation of soil organic matter
with a higher C : N ratio. We propose that N depletion from the soil organic matter by EM fungi
increases N limitation. This results in a tightened N cycle, and is consistent with low rates of
nitrification, denitrification and gaseous N losses from the soil organic layer of our study site
reported over two years of measurements (Koehler et al. 2009, Corre et al. 2010). Nitrification
and denitrification strongly discriminate against 15N, enriching the soil in 15N (Houlton et al.
2006). Therefore, soils with low resin extractable inorganic N, and presumably therefore low
mineralization rates, show reduced δ15N of the soil available N pool (Martinelli et al. 1999). This
is supported by the finding that the abundance of Oreomunnea, as well as the most abundant AM
tree species, was negatively correlated with inorganic N concentration in the soil in permanent
plots.
Several studies have found results consistent with our findings of lower availability of
inorganic N inside than outside monodominant forest (Read et al. 1995, 2006, Torti et al. 2001,
Brookshire and Thomas 2013). Further, studies that failed to find differences in soil nutrients did
not measure plant available ammonium or nitrate in the soil (i.e. Hart et al. 1989, Conway and
Alexander 1992, Henkel 2003, Peh et al. 2011b).
Finally, differences in N mineralization between EM and AM dominated forests may be a
consequence of intrinsic differences in litter quality between EM and AM associated plants.
However, using a global database of leaf functional traits, Koele et al. (2012) found no
relationship between leaf traits and mycorrhizal type after correcting by phylogenetic placement.
At Fortuna, Oreomunnea is close to the community average for leaf traits associated with
decomposition (foliar C : N, Adamek 2009) and δ15N (Mayor et al. 2014).
We conclude that changes in N availability due to the ability of EM fungi to acquire N
directly from organic matter is the likely mechanism underlying Oreomunnea monodominance
in our study system. This positive density-dependent mechanism creates patterns consistent with
previous evidence in EM tree species. Furthermore, the reduction in N availability may explain
the occurrence of other monodominant forests, including Dicymbe corymbosa in Guyana,
Gilbertiodendron dewevrei in central Africa, and dipterocarp species in Southeast Asia.
67
Figures and Tables
Figure 3.1 Relative growth rate (RGR) of Oreomunnea (A) and Roupala (B) seedlings among treatments; RGR of
Oreomunnea (C) and Roupala (D) seedlings inside and outside Oreomunnea forest; Oreomunnea seedling δ13C (E)
and δ15N (F) of leaf bulk tissue among treatments inside Oreomunnea-dominated forest. Exclosure treatment
abbreviations: 0.5 µm (0.5), 35 µm (35), transplant (trans), and control. Error bars in the bar plots represent the
standard error of the mean. The boxplots were created using the median and the 25th and 75th percentiles and the
95% confidence interval of the median was used for the error bars.
0.0000
0.0025
0.0050
0.0075
0.0100
0.5
35control
trans
-35.0
-34.5
-34.0
-33.5
-33.0
0.5
35control
trans
p=0.70%
δ13C%%
-3-2-101
0.5
35control
trans
Treatment
δ15N%%
p=0.98%
0.000
0.002
0.004
0.006
inout
0.000
0.002
0.004
0.006
0.5
35control
trans
Treatment
0.000
0.002
0.004
0.006
0.008
inout
Inisd
e vs
Out
side
p=0.05*%
p=0.68%
p=0.31%
p=0.02*%
RGR%(mg%g61%d61)%%
A%C%
B%D%
E% F%
*%
*%
68
Figure 3.2 (a) Differences in the percentage of EM colonization of Oreomunnea seedlings
between sites inside Oreomunnea-dominated forest and 30 m outside in mixed AM dominated
forest and, (b) across exclosure treatments including seedling from both inside and outside
Oreomunnea forest.
P=0.00714 **
0
25
50
75
100
Inside OutsideInside vs Outside
EM
% c
olon
izat
ion
P=0.729
0
25
50
75
100
0.5 35 control transTreatment
EM
% c
olon
izat
ion
P=#0.007#**# P=0.729#A# B#
69
Figure 3.3 A. Strength of negative plant-soil feedback effects on relative growth rate in 240
seedlings of five tree species differing in abundance in permanent forest plots. AM species are
Guarea pterorhachis (GUAPTE), Cupania seemannii (CUPSEE), and Nectandra purpurea
(NECPUR). EM species are Oreomunnea mexicana (OREMEX) and Quercus aff insignis
(QUEINS). Bars indicate standard errors of the mean. (* P < 0.05, ** P < 0.01). B. Regression
model for the strength of plant-soil feedback vs species abundance individuals > 10 cm DBH in
five 1 ha permanent plots.
-0.004 -0.003 -0.002 -0.001 0.000
0.000
0.005
0.010
0.015
0.020
0.025
Strength of feedbackR
elat
ive
abun
danc
e of
tree
s >1
0 cm
DBH
OREMEX
GUAPTE CUPSEE
QUEINS
NECPUR
Str
en
gth
of fe
ed
ba
ck
-0.005
-0.003
-0.001
CUPSEE GUAPTE NECPUR OREMEX QUEINS
**
*A" B"
R2= 0.79, p= 0.029
70
Figure 3.4 Regression models for Oreomunnea basal area in m2 in 20 × 20 sub-plots and (a)
nitrate (mg N kg-1), (b) ammonium, (c) total N (%), and (d) total P (mg P kg-1) measurements in
the same sub-plots.
0.0 0.2 0.4 0.6 0.8 1.0
02
46
810
1214
log(BA_ore + 1)
Nitrate
0.0 0.2 0.4 0.6 0.8 1.0
05
1015
2025
3035
log(BA_ore + 1)
Ammonium
0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Log(Oreomunnea BA +1)
N
0.0 0.2 0.4 0.6 0.8 1.0
0500
1000
1500
Log(Oreomunnea BA +1)
P
Nitrate'(m
g'N'kg,1 )'
Ammon
ium'(m
g'N'kg,1 )'
Total'N
'(%)'
Total'P'(m
g'P'kg
,1)'
A" B"
C" D"
R2'='0.43,'P"<0.001***' R2'=',0.05,'P"='0.66'
R2'='0.04,'P"='0.22' R2'='0.002,'P"='0.32'
71
Figure 3.5 Regression models for the number of Oreomunnea individuals > 5 cm DBH in 20 ×
20 sub-plots of two 1 ha permanent plots and ammonium (mg N kg-1), nitrate (mg N kg-1), total
N (%), and total P (mg P kg-1) measured in the same sub-plots.
0 1 2 3 4
02
46
810
12
log(indiv_ore + 1)
Nitrate
0 1 2 3 4
05
1015
2025
3035
log(indiv_ore + 1)Ammonium
A" B"
0 1 2 3 4
0.0
1.0
2.0
3.0
Log(Oreomunnea abundance + 1)
N
0 1 2 3 4
0500
1000
1500
Log(Oreomunnea abundance + 1)
P
D"C"
R2=0.48,)p=<0.001***) R2=0.06,)p=0.16)
R2=.0.06,)p=0.98) R2=0.30,)p=0.01)*)
Nitrate)(m
g)N)Kg.1 ))
Total)N
)(%))
Ammon
ium)(m
g)N)Kg.1 ))
Total)P)(m
g)N)Kg.1 ))
72
Figure 3.6 Regression models for Quercus aff. insignis basal area in 20 × 20 sub-plots and (a)
nitrate (mg N kg-1), (b) ammonium, (c) total N (%), and (d) total P (mg P kg-1) measurements in
the same sub-plots.
0.0 0.2 0.4 0.6 0.8 1.0
02
46
810
1214
log(BA_Q + 1)
Nitrate
0.0 0.2 0.4 0.6 0.8 1.0
05
1015
2025
3035
log(BA_Q + 1)Ammonium
0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Log(Quercus BA + 1)
N
0.0 0.2 0.4 0.6 0.8 1.0
0500
1000
1500
Log(Quercus BA + 1)
P
Nitrate'(m
g'N'kg,1 )'
Ammon
ium'(m
g'N'kg,1 )'
Total'N
'(%)'
Total'P'(m
g'P'kg
,1)'
A" B"
C" D"
R2'=',0.06,'P"='0.79' R2'='0.04,'P"='0.21'
R2'='0.03,'P"='0.23' R2'='0.004,'P"='0.32'
73
Figure 3.7 A) Comparison of the Relative growth rate (RGR), and B) Mycorrhizal colonization of seedlings grown on sterile vs live inoculum. We grew 10 replicates of each species in sterile substrate using the same bulk soil and sterile inoculum of all the species (2 replicates of each species inoculum, to give a total of 10 replicates per species x 5 species = 50 pots with sterile inoculum). Seedlings grown in sterile inoculum had a significantly lower RGR compared with seedlings grown with a live inoculum independent of the inoculum sources (Figure 3.4A). This evidence suggests that the benefits of mycorrhizal associations on seedlings’ RGR were stronger than the negative effects caused by pathogens present in the soil. However, as shown in the paper, the effect of the species-specific pathogens found in the conspecific inocula had a stronger negative effect on Oreomunnea seedlings than pathogens found in heterospecific inocula.
We started the experiment with sterile seedlings grown in sterile sand from sterile seeds. At the end of the experiment roots of a subset of fourteen seedlings per species, four grown in sterile control and ten grown in live inoculum (two plants per species per inoculum source), was preserved in 96 % ethanol for quantification of mycorrhizal colonization. Seedlings grown in sterile control had very low or no colonization while seedlings grown in live inoculum had high colonization rates (Figure 3.4B). Notably, none of the Oreomunnea seedlings in sterile inoculum showed signs EM colonization at the end of the experiment, while seedlings in the live inoculum were highly colonized.
0
10
20
30
40
CUPSEE GUAPTE OREMEX QUINSSpecies
Myc
orrh
izal
col
oniz
atio
n (%
)
Inoculum
Live
Sterile
0.004
0.008
0.012
0.016
CUPSEE GUAPTE NECPUR OREMEX QUERCUSSpecies
Mea
n R
GR Inoculum
Live
Sterile
A" B"
74
Figure 3.8 Feedback loop to explain monodominance of Oreomunnea mexicana based on the
‘microbial competition for nitrogen hypothesis’. Abbreviations: Ectomycorrhizal (EM).
Hypothesis*for*EM*mediated*monodominance*
High%abundance%of%Oreomunnea)
High%abundance%of%EM%fungi%
EM%fungi%compete%with%saprophy8c%fungi%for%nitrogen%slowing%down%li:er%decomposi8on%
rates%
Low%decomposi8on%rates%produce%a%reduc8on%in%the%availability%of%inorganic%nitrogen%in%the%soil%
Increase%survivorship%and%growth%rate%of%
Oreomunnea%seedlings%
75
Table 3.1 List of mycorrhizal status inventories in neotropical areas used to calculate the average
percentage of EM vs AM trees in neotropical forest.
Reference Site Total spp %EM %AM
McGuire et al. 2008 Guyana 142 2.1 96.4 Béreau et al. 1997 French Guyana 75 5.3 94.7 Kottke et al. 2008 Ecuador 115 2.6 97.4 Thomazini 1974 Brasil 60 7 93 St. John 1980 Brasil 83 3 97 St. John and Uhl 1983 Venezuela 22 14 86 Moyersoen 1993 Venezuela 42 12 89 Lodge 1987 Puerto Rico 48 6 96 SD
39.61 4.4 4.3
Mean 73.38 6.4 93.6 Béreau M., Gazel M., & Garbaye, J. 1997. Les symbioses mycorhizienne des arbres de la foret tropicale humide de Guyane francaise. Canadian Journal of Botany 75:711–716. Kottke, I., Beck, A., Haug, I., Setaro, S., Jeske, V., Suárez, J.P. et al. 2008. Mycorrhizal state and new and special features of mycorrhizae of trees, ericads, orchids, ferns, and liverworts. In: Gradients in a Tropical Mountain Ecosystem of Ecuador (eds. Beck, E., Bendix, J., Kottke, I. & Makeschin, F.). Ecological Studies 198. Springer-Verlag Berlin Heidelberg. Lodge D.J. Resurvey of mycorrhizal associations in the El Verde rainforest, Puerto Rico. In: Sylvia D.M., Hung L.L., Graham J.H. (eds.) Mycorrhiza in the next decade practical applications and research priorities. Ins. Food Agric Sci, Univ of Florida Gainsville, p 127. McGuire KL, Henkel TW, Granzow de la Cerda I., Villa G., Edmund F., Andrew C. 2008. Dual mycorrhizal colonization of forest-dominating tropical trees and the mycorrhizal status of non-dominant tree and liana species. Mycorrhiza DOI 10.1007/s00572-008-0170-9. Moyersoen B. 1993. Ectomicorrizas y micorrizas vesiculo-arbusculares en Caatinga Amazonica del Sur de Venezuela. Scientia Guianae 3:1–82. St. John TV. 1980 A survey of mycorrhizal infection in an Amazonian rain forest. Acta Amazonica 10:527–533.; St. John T.V. & Uhl C. 1983. Mycorrhizae in the rain forest at San Carlos de Rio Negro, Venezuela. Acta Científica Venezolana 34: 233:237 Thomazini L.I. 1974. Mycorrhiza in plants of the ‘Cerrado’. Plant and Soil 41(3): 707-711.
76
Table 3.2 Biomass models used for the estimation of initial seedling biomass in the calculation
of RGR. LA = leaf area, D = diameter, H = height.
Species Intercept Total
LA
D H No.
leaves
F P R2
CUPSEE 69.709 9.729 17.98 < 0.001 0.4592
GUAPTE -63.67 10.69 47.32 43.25 < 0.001 0.816
NECPUR 71.29 67.79 -146.82 16.48 0.0004 0.7043
OREMEX 8.052 12.845 -15.367 8.547 0.003 0.4561
QUEINS 0.1645 0.2023 6.009 0.036 0.3337
ROUMON -1232 394.4 149.6 11.67 0.002 0.6213
77
Table 3.3 Soil variables values by sub-plot from two 1-ha permanent plots (Honda A = HA, and
Honda B = HB).
Plot Quadrant NO3
mg N kg-1
NH4
mg N kg-1
N
%
C
%
P
mg P kg-1
Litter
g m-2
Microbial N
mg N kg-1
Microbial C
mg C kg-1
HA 1 11.46 1.48 1.02 13.95 920.07 4.86 223.07 973.67
HA 2 0.07 1.55 0.99 19.80 542.51 3.71 158.60 816.37
HA 3 6.34 18.35 1.68 25.86 1056.23 2.50 151.41 821.22
HA 4 1.95 13.23 0.94 13.99 566.36 0.74 83.13 466.16
HA 5 10.44 3.49 1.55 23.41 781.59 1.57 161.69 1065.02
HA 6 0.92 13.87 0.61 11.54 358.77 2.59 111.63 631.15
HA 7 3.51 13.02 0.94 15.01 605.50 1.36 154.60 800.48
HA 8 4.12 3.84 0.65 9.79 410.94 4.47 137.04 725.38
HA 9 1.27 4.99 1.63 16.88 639.37 2.94 231.97 1081.67
HB 1 7.24 28.13 1.57 21.81 683.77 9.08 188.13 932.95
HB 2 8.44 20.02 1.28 16.34 1462.32 5.73 240.35 1010.29
HB 3 0.10 29.21 2.27 41.21 878.10 3.19 305.52 1605.51
HB 4 0.24 5.14 1.81 36.31 727.97 3.60 264.01 1407.24
HB 5 0.37 4.49 1.00 15.48 668.22 3.83 107.67 685.00
HB 6 -‐0.15 7.05 2.12 49.62 681.61 5.26 170.45 903.47
HB 7 2.66 2.78 0.79 13.72 596.03 9.57 72.60 232.18
HB 8 0.97 8.97 2.60 42.23 855.45 3.83 323.13 1490.16
HB 9 0.13 4.10 0.75 12.56 443.99 8.98 133.44 720.59
78
Table 3.4 Mean C : N, total C, total N, δ13C and δ15N of soils and seedlings, and soil ammonium,
nitrate, and phosphate (lg bag-1) measured using an 18- day resin bag incubation at sites either
inside Oreomunnea-dominated forest, or 30 m outside in mixed AM dominated forest.
Variable Locatio
n Soil n F p Seedlings n F p
C:N Inside 19.30 23.68
Outside 15.75 17 26.77 < 0.001 24.94 23 0.45 0.51
%C Inside 28.19 46.22
Outside 12.63 17 21.37 < 0.001 41.44 23 23.10 < 0.001
% N Inside 1.46 1.98
Outside 0.80 17 15.9 0.001 1.70 23 17.33 < 0.001
δ13C Inside -28.01 -33.84
Outside -28.55 17 8.65 < 0.01 -33.38 23 1.15 0.35
δ15N Inside 0.57 -1.18
Outside 2.84 17 26.31 < 0.001 0.87 23 6.55 0.02
Ammonium Inside 4.46
Outside 10.67 19 W=19 0.02
Nitrate Inside 4.33
Outside 12.4 19 W=9 <0.01
Phosphate Inside 0.28
Outside 0.33 19 W=38 0.31
79
Table 3.5 Average plant-soil feedback per species and pairwise feedback between species
included in the experiment. Species abbreviations are Guarea pterorhachis (Guapte), Cupania
seemannii (Cupsee), and Nectandra purpurea (Necpur). EM species are Oreomunnea mexicana
(Oremex) and Quercus aff. insignis (Queins).
Parameter Estimate
feedback
Standard
Error t-value P
Cupsee average -0.00138 0.00140 -0.99 0.3244
Guapte average -0.00284 0.00140 -2.03 0.0444
Necpur average -0.00089 0.00140 -0.64 0.5233
Oremex average -0.00381 0.00145 -2.62 0.0095
Queins average -0.00135 0.00141 -0.96 0.3402
Cupsee & Guapte -0.00111 0.00110 -1.01 0.3129
Cupsee & Necpur -0.00037 0.00110 -0.34 0.7367
Cupsee & Oremex -0.00174 0.00111 -1.57 0.1178
Cupsee & Queins 0.00046 0.00110 0.42 0.6746
Guapte & Necpur -0.00177 0.00110 -1.61 0.1089
Guapte & Oremex -0.00232 0.00114 -2.04 0.0429
Guapte & Queins -0.00050 0.00110 -0.44 0.657
Necpur & Oremex -0.00027 0.00111 -0.24 0.8109
Necpur & Queins 0.00062 0.00110 0.56 0.5755
Oremex & Queins -0.00329 0.00117 -2.81 0.0056
80
Table 3.6 Coefficient of correlation of two EM tree species (Oreomunnea mexicana and
Quercus aff. insignis) and the four most abundant AM tree species with the ammonium and
nitrate concentrations in the soil and with the number of individuals and basal area of
Oreomunnea in 20 × 20 sub-plots. The stars represent the significance of the correlation based on
its p-value = *** < 0.0001, ** < 0.001, * < 0.05.
Species NO3 (R2) NH4 (R2) Individuals
Oreomunnea
Basal area
Oreomunnea
Oreomunnea mexicana
No. of individuals (indv/ 20×20 sub-plot) 0.48 *** 0.06 - -
Basal area (m2/ sub-plot) 0.43 *** -0.05 - -
Quercus aff. insignis
No. of individuals (indv/ 20×20 sub-plot) 0.09 -0.06 -0.06 -0.06
Basal area (m2/ sub-plot) -0.06 0.04 -0.06 0.021
Ardisia sp.
No. of individuals (indv/ 20×20 sub-plot) 0.33** 0.21 * 0.58 *** 0.32 *
Basal area (m2/ sub-plot) 0.18 * 0.07 0.56 *** 0.31 **
Dendropanax arboreus
No. of individuals (indv/ 20×20 sub-plot) -0.06 0.09 0.03 -0.03
Basal area (m2/ sub-plot) -0.05 0.23 * -0.01 0.008
Cassipourea guianensis
No. of individuals (indv/ 20×20 sub-plot) -0.06 0.22 * 0.18 * 0.011
Basal area (m2/ sub-plot) -0.05 0.26 * 0.11 -0.01
Eschweilera panamensis
No. of individuals (indv/ 20×20 sub-plot) -0.02 0.20 * 0.22 * 0.18 *
Basal area (m2/ sub-plot) 0.04 0.08 0.24 * 0.11
81
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Chapter 4: Nitrogen addition alters ectomycorrhizal fungal communities and soil enzyme
activities in a tropical montane forest
Introduction
Anthropogenic nitrogen (N) deposition has increased over the last century (Matson et al.,
1999), extending across unmanaged ecosystems, with measureable impacts on tissue N
concentration in tropical forest (Hietz et al. 2011). Nitrogen deposition causes soil acidification,
reduces plant productivity, and changes plant species composition, soil C storage, and N cycling
(Hasselquist and Högberg 2014, BassiriRad, 2015). Nitrogen deposition is predicted to continue
to rise globally in the next decade with impacts expected on tropical and sub-tropical biodiversity
hotspots (Allen et al. 2011, Phoenix et al. 2012, Liu et al. 2013, Vet et al. 2014, BassiriRad
2015).
Communities dominated by ectomycorrhizal (EM) associated tree species are expected to
be particularly sensitive to increases in N availability. This is because EM fungi specialize in N
absorption from soil (Read 1991) and are particularly sensitive to increases in N availability
since host trees strongly reduce C allocation to their root symbionts under excess N conditions
(Treseder 2004, Högberg et al. 2010 Hasselquist and Högberg 2014). In N-limited temperate
forest it has been shown that long-term increases in inorganic N availability can also reduce the
species richness of EM fungi and alter EM fungal community structure and composition
(Arnebrant and Sördeström 1992, Peter et al. 2001, Lilleskov et al. 2002, Avis 2003, Pardo et al.
2011). However, effects of N addition on EM species composition may depend on the functional
traits of component EM fungal taxa (see Lilleskov et al. 2011 for review). It has been proposed
that these differences in sensitivity to N addition are associated with fungal exploration type and
enzymatic activity. Fungal exploration types are strategies of growth and colonization of the soil
environment that can vary considerably among taxa (Hobbie and Agerer 2010). Sensitive or
"nitrophobic" species usually have high enzymatic capabilities for organic N uptake, a process
that incurs a higher carbon cost to their hosts (Lilleskov et al. 2002). In contrast, "nitrophilic"
species are usually limited to the uptake of labile nitrogen forms, and incur a lower carbon cost
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to their hosts (Lilleskov et al. 2002). With increased N availability, nitrophobic EM species may
respond by reduced production of sporocarps (Arnolds 1991, Lilleskov et al. 2011), mycelia
growth rate (Wallander and Nylund 1992), and colonization of root tips (Treseder and Allen
2000, Peter et al. 2001), while nitrophilic species tend to increase in abundance.
Changes in the composition and functional trait composition of EM fungi associated with
increases in inorganic N availability are expected to have an impact on ecosystem processes
including soil C storage, N cycling, and plant community productivity (Treseder et al. 2012,
Treseder and Lennon 2015). Ectomycorrhizal associations play an important role in soil nutrient
dynamics due to their capacity to compete for organic nitrogen with the decomposer microbial
community (Phillips et al. 2013, Averill et al. 2014). Further, because different ectomycorrhizal
lineages vary in their capacity to uptake different N or P sources they differentially affect
ecosystem level processes (Koide et al. 2014). Changes in EM fungal community composition
could therefore result in changes in enzyme activity, with long-term consequences for N, P and C
cycling in the soil. In addition, changes in the EM community composition may influence the
competitive ability of EM associated plants, since nitrophilic EM fungal species may be less
beneficial or even parasitic on their host plants (Avis 2003, Avis 2012).
Montane forests in Central America are often dominated by tree species that form EM
associations. For example, montane cloud forests in Mexico and Costa Rica are often dominated
by several species of Quercus spp. (Halling 2001, Morris et al. 2008). Similarly, montane forest
in western Panama support EM fungal communities associated with Oreomunnea mexicana that
differ according to soil fertility (Corrales et al. 2016a). Thus, there is a critical need to
understand the response of EM associations to N addition in this region, if we are to predict the
response of this ecosystem to global change (Dalling et al. 2015). Here we take advantage of a
nine-year N addition experiment at a site with low background N availability to explore the
effect of N on soil enzyme activity and on the composition of the EM fungal community
associated with the canopy tree Oreomunnea mexicana. We predicted that an increase in N
availability would have strong impact on EM fungal community composition and infection by (i)
shifting the EM fungal community from one characterized by taxa with previously described
associations with low inorganic N environments (i.e., nitrophobic taxa) to one increasingly
characterized by abundant nitrophilic taxa, (ii) reducing the activity of enzymes associated with
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organic N acquisition, (iii) reducing the EM colonization of root tips consistent with a reduction
of C transfer from the host tree.
Methods
Study site
The study was located in a primary lower montane forest (1000–1400 m.a.s.l.) in the
Fortuna Forest Reserve in western Panama (hereafter, Fortuna; 8°45 N, 82°15 W). Climate
records indicate that the mean annual temperature for Fortuna ranges from 19 to 22˚C (Cavelier
1996) and annual rainfall averages ca. 5800–9000 mm (Andersen et al., 2012). Ambient N
deposition from rainfall in the area is 5 kg N ha−1 y−1 (Adamek et al. 2009, Koehler et al. 2009).
Soils at this site are infertile Ultisols derived from rhyolite (Table 4.1). The focal species,
Oreomunnea mexicana, is an EM canopy tree distributed from Mexico to Panama at 900–2600
m.a.s.l. (Stone, 1972) and forms monodominant stands at the study site (Corrales et al. 2016 a,b).
Nitrogen addition experiment
The N addition experiment consists of a paired-plot design with eight 40 × 40 m plots.
Four plots have been fertilized with urea (CO(NH2)2) at an annual rate of 125 kg N ha-1 divided
into four applications per year. The four remaining plots are untreated controls (Adamek et al.
2009). The experiment was started in February 2006 and it had been fertilized for 9 years at the
time of the study. Seven of the eight plots (four control and three fertilized) contain individuals
of the focal species Oreomunnea mexicana, including seedlings, saplings and adults.
Oreomunnea individuals > 10 cm DBH accounted for 15.5 ± 10.8 % of the total number of
individuals and 28.2 ± 13.4 % of the total basal area of the plots (Dalling et al. unpublished
data).
Soil data collection
Samples of the surface 0-10 cm of mineral soil were collected in nine points separated by
10 m inside each plot (nine internal interception points of 10 × 10 subplots) using a soil corer
and were pooled to generate one sample per plot prior to analysis. Ammonium, nitrate, and
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phosphate were measured using resin bags. Four resin bags containing 5 g of mixed-bed anion
and cation exchange resins (Dowex Marathon Mr-3) sealed inside 220 µm polyester mesh were
buried 2 cm beneath the soil surface in the central point of four 20 × 20 subplots and in the
central point of the plot. Bags were buried during October 2013. After incubation in situ for 21
days the resin bags were collected, rinsed with deionized water to remove adhering soil,
extracted with 75 mL of 0.5 M HCl, and then nitrate (+nitrite), ammonium, and phosphate were
determined by automated colorimetry on a Lachat QuikChem 8500 (Hach Ltd., Loveland, CO,
USA). Readily exchangeable phosphate (Resin P) was determined by extraction with anion-
exchange membranes (Turner and Romero 2009).
Microbial N was determined by chloroform fumigation and extraction in K2SO4 by
standard procedures (Brookes et al. 1985, Vance et al. 1987) following Turner and Wright
(2014). Microbial P was determined by hexanol fumigation and extraction with anion-exchange
membranes following Kouno et al. (1995) as modified in Turner and Romero (2010). Dissolved
organic carbon (DOC) and total dissolved N were determined simultaneously in K2SO4 extracts
on a TOC-VCHN analyzer (Shimadzu, Columbia, MD) after five-fold dilution of the extracts.
The activities of six hydrolytic enzymes were determined using fluorogenic substrates as
described previously (Turner 2010, Turner and Romero 2010). The enzymes and substrates were:
(i) acid phosphomonoesterase (Enzyme Commission (EC) number 3.1.3.2) assayed with 4-
methylumbelliferyl phosphate; (ii) phosphodiesterase (EC 3.1.4.1) assayed with bis-(4-
methylumbelliferyl) phosphate; (iii) β-glucosidase (EC 3.2.1.21) assayed with 4-
methylumbelliferyl β-D-glucopyranoside; and (iv) N-acetyl β-D-glucosaminidase (EC 3.2.1.52)
assayed with 4-methylumbelliferyl N-acetyl β-D-glucosaminide; (v) β-xylanase (EC 3.2.1.37)
assayed with 4-methylumbelliferyl β-D- xylopyranoside; (vi) cellulose 1,4- β-cellobiosidase (EC
3.2.1.91) assayed with 4-methylumbelliferyl β- D-cellobiopyranoside.
Substrates were purchased from Glycosynth Ltd (Warrington, UK) and dissolved in 0.4 %
methylcellosolve (2-methoxyethanol; 0.1 % final concentration in the assay) and then processed
following Turner and Wright (2014). Plates were incubated for 30 min at 26 °C to approximate
the daytime temperature in the upper 10 cm of soil in montane forests in western Panama (Ben
Turner Pers obs). The reaction was terminated by adding 50 µL of 0.5 M NaOH (final solution
89
pH > 11) and the fluorescence determined immediately on a FLUOstar Optima multi-detection
plate reader (BMG Labtech, Offenburg, Germany), with excitation at 360 nm and emission at
460 nm. Control wells were prepared for each substrate and contained substrate, buffer, and 1
mM NaN3 (no soil suspension). Blank wells contained soil suspension and buffer only (no
substrate). Standard wells contained buffer, 1 nmol methylumbelliferone (MU), and either soil
suspension or 1 mM NaN3 to account for the reduction of fluorescence in the presence of soil
(quenching). All enzyme activities are expressed as nmol MU g-1 soil (dry weight) min-1.
Sampling of ectomycorrhizas
To assess whether N addition had an impact on the EM fungal community, fine roots
from Oreomunnea were collected from N fertilized and control plots. Fine roots were collected
from three Oreomunnea adult trees, five saplings (40-100 cm height), and five seedlings (5-20
cm height) per plot for a total of 91 individuals. Two lateral roots were excavated 2-3 m from the
trunk of each adult tree until fine roots that were clearly connected to the tree were found. The
entire root system of each focal seedling and sapling was collected.
Fine roots were stored in plastic bags and refrigerated within 2 h of collection. Each
sample was carefully cleaned with tap water and cut into 2 cm pieces. Eighty centimeters of total
fine root length representing multiple root branches were collected from adults, 40 cm from each
sapling, and the entire root system of each focal seedling for a total of 192 samples (Table 4.2).
All roots obtained were included in field collections even if EM fungal infection was not visible
macroscopically. Samples were preserved in 2% CTAB in -20˚C until ready for DNA extraction.
In addition to the roots collected for DNA extraction, 20 cm of fine root of three adults, five
samplings and the entire root system of five seedlings was preserved in 96% ethanol for
quantification of EM fungal colonization.
DNA extraction and sequencing of the EM fungi
To identify EM fungi DNA was extracted from about 0.1 mg of ground root tissue from
each sample using the MOBIO PowerSoil DNA isolation kit (MoBio, Carlsbad, CA USA)
following the manufacturer instructions. PCR was performed using a mixture of six forward
primers (in equimolar concentration) analogous to ITS3 and a degenerate reverse primer
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analogous to ITS4 (hereafter referred to as ITS4ngs) following Tedersoo et al. (2014;
Supplemental material).
Amplicons were subjected to ligation of Illumina adaptors using two variants of the
TruSeq DNA PCR-free HT Sample Prep kit (Illumina Inc., San Diego, CA, USA). All samples
were sequenced in a 150 single Illumina MiSeq 2x300 paired-end run at the Estonian Genome
Center at University of Tartu.
Bioinformatics
Paired-end sequencing (2 × 300 bp) in the Illumina MiSeq sequencer resulted in 722 649
reads. Quality filtering and selection of representative sequences were done using methods
explained in supplementary material. Representative sequences for each OTU were
taxonomically assigned using BLASTn searches (options: word size = 7, gap penalty = -1, gap
extension penalty = -2 and match score = 1) retrieving the 10 best BLASTn matches. The
UNITE 7.0 beta data set (https://unite.ut.ee/repository.php) and Sanger sequences from fruiting
bodies and root tips collected in previous studies at the study area (including sequences from
Corrales et al. 2016 and Corrales et al. unpublished data) were used as a reference for BLASTn
taxonomic assignment of ITS.
Only OTUs with a match of > 80% with families previously reported as ectomycorrhizal
by Tedersoo et al. (2010) in the Blastn taxonomic assignment were included in the final database
for analysis. For the results and discussion we will refer to OTUs as “species” since 97% is a
broadly accepted cut off that usually represents species concept in EM fungi (Kõljalg et al.
2013).
Root staining and frequency of infection
A subsample of root tissue was saved to evaluate EM colonization. EM colonization was
evaluated in roots cleared in 10% KOH and stained with trypan blue. Infection of four randomly
chosen 2.0-cm root sections per seedling was assessed using the grid-line intersect method as
modified by McGonigle et al. (1990).
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Statistical analysis
Soil nutrients and enzyme activity was compared between treatments using ANOVA.
Phosphate, Microbial N : P, phosphodiesterease, and enzyme C : P were log transformed to
improve normality; other variables were normally distributed. Enzyme activity was compared
between treatments using raw data and also after standardization dividing enzyme activity by soil
microbial C.
OTUs with fewer than 10 sequences (n = 655) were excluded from alpha and beta
diversity analyses to provide a robust data set to compare EM fungal communities among N
treatments and host developmental stages (Smith and Peay 2014; Brown et al. 2015). Species
accumulation curves with the remaining 387 OTUs were used to compare richness among
developmental stages, and between the nitrogen addition treatment and control. Curves were
rarefied based on the smallest number of samples per developmental stage and per treatment
(Table 4.2). Alpha diversity indexes (Pielou evenness index and Fisher alpha) were calculated
per plot based of an equal number of samples and compared using ANOVA including treatment
(N addition and control) and developmental stage as fixed effects and plot as a random effect
after checking for equality of variance and residuals using Levene’s and Shapiro-Wilk test. The
Pielou index was calculated following Pielou (1966).
Nonmetric Multidimensional Scaling (NMDS) was used to compare EM fungal
community composition among N treatments and developmental stages. NMDS analyses were
based on Bray-Curtis dissimilarity matrices using abundance data for individual roots, and for
samples pooled per plot. The significance of differences between N treatments and
developmental stages was determined using permutational analyses of dissimilarity (ADONIS)
using 1000 permutations and a Euclidean distance matrix (Oksanen et al. 2008).
Principal component analysis (PCA) was applied separately on two soil datasets
(nutrients and enzymes), resulting in two axes of variation for soil nutrients (describing 78.77%
and 17.31% respectively of the total variance) and one axis for soil enzymes (describing 98.63%
of the total variance). Due to the high correlation between some variables, the nutrient and
enzyme dataset included a subsample of the measured soil variables that showed less than 50%
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correlation with any other variable in the data set. The soil variables included were: resin P (mg
P kg-1), NH4 (µg bag-1 day-1), NO3 (µg bag-1 day-1), PO4 (µg bag-1 day-1), DOC (mg C kg-1),
microbial N (mg N Kg-1), and microbial C : N (Table 4.1). The enzyme dataset included
phosphomonoesterase (MUP, nmol MU g-1 min-1), β-D-glucosaminidase (BG, nmol MU g-1 min-
1), xylanase (XYL, nmol MU g-1 min-1), cellobiohydrolase (CELLO, nmol MU g-1 min-1), and
enzyme N:P (Table 4.1). Enzyme N:P (Enz N:P) ratio represents the ratio between N-acetyl β-D-
glucosaminidase (an enzyme targeting organic N) and phosphomonoesterease (an enzyme
targeting organic P). Correspondingly, enzyme C:N (Enz C:N) refers to the ratio between β-
glucosidase (an enzyme targeting C) to N-acetyl β-D-glucosaminidase. The first axes of the
resulting PCA for the soil nutrient dataset (PC1.nut) and the first axis of the soil enzyme dataset
(PC1.enz) were used in the NMDS analysis of samples pooled per plot to find correlations
between soil variables and changes in the EM community composition with N treatments (Ter
Braak, 1995). To further explore the relationship between changes in EM community
composition and enzyme activity, a permutational analyses of dissimilarity (ADONIS, Oksanen
et al. 2008) was used with 200 permutations using the species abundance matrix per plot and a
subset of independent enzymes variables. All statistical analyses were carried out using the
package vegan 2.0-6 in R 2.15.1 (R Development Core Team 2011).
A GLM with Poisson errors was used to compare number of OTUs between treatments,
while negative binomial errors were used to test the effect of N addition on the relative
abundance of the eight most abundant individual genera, including treatment as a fixed effect. A
GLM with gamma errors was used to test whether enzyme activity was associated to the
abundance of the five most abundant genera. The goodness of fit of the models was determined
using a chi-squared test. Only the five most abundant genera were included in the multiple
regressions based on the degrees of freedom available.
To test for the effect of nitrogen addition, developmental stage and its interaction on the
abundance of the 100 most abundant species, a multivariate GLM analysis was performed using
manyglm in mvabund 3.10.4 package in R using a negative binomial model and sequence count
data following Peay et al. (2015). This analysis runs a univariate analysis to check for responses
of individual species to treatment effects (Wang et al. 2012). The univariate ANOVA analysis
was run using the “adjust” option that corrects probability values for multiple testing using a
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step-down resampling procedure (Wang et al., 2012). Species with probability values lower than
0.07 were considered significant due to the lower power of the univariate ANOVA compared to
multivariate ANOVA (Wang et al. 2012).
Finally, two-way ANOVA including treatment (N addition and control) and
developmental stage (seedling, sapling, and adult) as fixed effects and plot as a random effect
was used to assess the effect of developmental stage and N addition treatment on EM
colonization frequency.
Results
Changes in soil fertility and enzyme activity with N addition
Among soil chemical variables, only nitrate differed significantly between treatments,
with two-fold higher concentrations in N addition plots (Table 4.1). The activity of
phosphomonoesterase was significantly higher in control plots, while the β-glucosidase and N-
acetyl-glucosaminidase ratio (enzymatic C : N ratio) was significantly lower in control than N
addition plots (Table 4.1). However, none of the treatment effects on enzyme activity were
significant after standardization by microbial biomass (phosphomonoesterase F1,5 = 0.6, P = 0.5;
enzymatic C:N ratio F1,5 = 0.07, P = 0.8). Phosphomonoesterase activity was positively
correlated with activities of phosphodiesterase and N-acetyl-glucosaminidase, and negatively
correlated with the enzymatic C : N ratio (Table 4.3).
Diversity patterns
Illumina sequencing of Oreomunnea roots revealed high EM species richness for both the
four control plots and three N fertilized plots with 318 and 344 species respectively. The
community was dominated by Basidiomycota with 352 species and 19 genera; Ascomycota was
represented by 35 species and 3 genera. Russula was the most species-rich genus with 128
species, followed by Lactarius (47 spp), Tomentella (44 spp) and Cortinarius (36 spp). Overall,
215 of the species obtained by Illumina sequencing (56%) had as their closest match Sanger
sequences from fruiting body voucher specimens or root tips from the study area. Thirty-two
OTUs belonging to Cantharellaceae, Elaphomycetaceae, Russulaceae, and Thelephoraceae with
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a percentage of match > 80% were left as “unidentified” species. In terms of abundance, Russula,
Tomentella, Cortinarius, Cenococcum, Lactarius, Clavulina, Elaphomyces, and Laccaria were
the most abundant genera and accounted for 87% of the total number of reads.
Species diversity and evenness were not significantly different between control and N
fertilized plots (Figure 4.1, Table 4.4). In contrast, there were significant differences when
comparing across developmental stages, driven by significantly higher species diversity and
evenness in saplings compared to seedlings and adults based on Tukey HSD, P < 0.05 (Figure
4.1, Table 4.4).
EM fungal community composition differed between control and N fertilized plots when
pooling samples per plot (Adonis R2 = 0.22, F1,6 = 1.43, P = 0.005) or using individual samples
(Adonis R2 = 0.01, F1,191= 2.65, P = 0.005, Figure 4.2). This difference between treatments was
also reflected in the NMDS ordination where control plots grouped separately from N fertilized
plots (Figure 4.3a). In contrast, species composition did not differ among developmental stages
based on ADONIS analysis (Adonis R2 = 0.01, F2, 189 = 1.03, P = 0.34; Figure 4.3b).
None of the soil nutrient PCA axes showed a significant correlation with axes of EM
fungal community composition derived from the NMDS ordination. However, the first PCA axis
of the soil enzyme dataset (PC1.enz) was significantly correlated with the first two axes of the
NMDS (R2 = 0.84, P = 0.012; Figure 4.3a). PC1.enz accounted for 98.6% of the variation in soil
enzyme variables. The ADONIS analysis including individual soil variables and the species
abundance matrix showed that changes in phosphomonoesterase activity (MUP) were
significantly correlated with changes in EM species composition (Table 4.5).
Changes in abundance and species richness with N addition
Nitrogen addition did not affect species richness for any of the EM genera based on the
GLM results (Table 4.6). When the same analysis was done using abundance data, Cortinarius
showed a significant reduction from 15% of sequence reads to 6% with N addition (z = -3.57, P
<0.001). In contrast, Laccaria and Lactarius responded positively to N addition, increasing from
1% to 10% (z = 3.43, P < 0.001) and 5% to 9% (z = 2.27, P = 0.02), respectively (Figure 4.4,
Table 4.6).
95
At the species level, the multivariate GLM showed a significant effect of N addition (P =
0.001), developmental stage (P = 0.001), and their interaction (P = 0.001) on the abundance
distribution of the 100 most abundant species. The univariate ANOVA to test for differences in
abundance of individual species showed a significant effect of treatment on the abundance of
eight species. Laccaria sp. (OTU 20), Cortinarius sp. (OTU 87), Tomentella sp. (OTU 126), and
an unidentified Cantharellaceae (OTU 45) showed higher abundance in N addition plots; while
Cortinarius obtusus (OTU 137), two Cortinarius (species both identified as Cortinarius
junghuhnii OTUs 18 and 85), and Tomentella sp. (OTU 22) showed lower abundance in N
addition plots (Table 4.7). None of the genera showed a significant correlation with any of the
soil nutrients measured except for Lactarius that showed a positive association with nitrate (data
not shown).
The univariate ANOVA to test for changes in abundance of individual species also found
changes in abundance of eight species associated to different plant developmental stages. Three
species of Cortinarius (OTUs 18, 28, and 85), and an unidentified Cantharellaceae (OTU 45)
showed significantly higher abundance in seedlings compared with saplings and adult
individuals. Cenococcum sp. (OTU16) and Tomentella sp. (OTU22) showed a significantly
higher abundance in saplings and just one species of Tomentella (OTU 61) showed a higher
abundance in adult trees.
Association between genera and enzyme activity
The activities of phosphomonoesterase and phosphodiesterase were positively associated
with Cortinarius and negatively associated with Russula abundance. Phosphomonoesterase
activity was also associated positively with Cenococcum abundance (Table 4.8). The remaining
enzymes were not associated with any of the genera included in the model.
Effect of N addition and developmental stage in EM colonization
EM colonization in Oreomunnea root tips was significantly lower in the N addition
treatment (F1,64 = 19.9, P < 0.0001), but did not differ across development stages (F4,64 = 2.3, P =
0.07; Figure 4.5). There was no interaction between N addition treatment and developmental
stage (F2,64 = 1.03, P = 0.36).
96
Discussion
To our knowledge, this is the first study to explore the effects of long-term N addition on
tropical EM fungal communities. Our results indicate that N addition can induce changes in the
EM fungal community, with alterations in the relative abundance of fungal genera known to
differ in their enzymatic activity. Over the long term, changes in enzyme activity resulting from
altered EM communities might be expected to influence key ecosystem processes, including soil
carbon storage and the cycling of N and P.
Effect of N addition on species richness, community composition and EM colonization
Nitrogen addition did not have an effect on the number of EM species associated with
Oreomunnea roots. This result is consistent with studies in temperate forest showing no effect of
N addition on species richness in short-term N addition experiments (less than 5 y, Lilleskov et
al. 2011). However, a significant reduction in species richness has been found along N
deposition gradients in Europe that have been monitored for more than 40 years (Lilleskov et al.
2011, Suz et al. 2014), and therefore a future reduction in richness at our site is possible.
The nine years of N addition, however, were sufficient to significantly change EM
community composition. Both N addition treatment and enzyme activity were significantly
correlated with the NMDS axes of the EM community suggesting an effect of N on community
composition and function. The significant association of EM fungal community composition and
phosphomonoesterase activity could be interpreted as a change in the activity of enzymes
associated with access to phosphate (phosphomonoesterase, phosphodiesterase) and nitrogen (N-
acetyl-glucosaminidase) given the high correlation among these enzymes.
Changes in community composition also reflected altered abundance of some EM genera.
Laccaria and Lactarius had significantly higher relative abundance with N addition plots while
Cortinarius had significantly lower abundance. Likewise, regression analysis showed that
Lactarius was positively correlated with soil nitrate. These patterns are congruent with broader
affinities for N reported for these genera. In studies from temperate forest in Europe and North
America Laccaria and Lactarius consistently exhibit a positive response to high N availability,
and therefore represent “nitrophilic” genera, (Lilleskov et al. 2011) while Cortinarius has been
97
reported as showing a consistent negative association with N availability, and has therefore been
considered to be "nitrophobic" (Lilleskov et al. 2002, Wright et al. 2009, Lilleskov et al. 2011,
Suz et al. 2014). In turn N affinity reflects hyphal exploration traits: Lactarius, Laccaria, and
Tomentella have hydrophilic short and medium distance exploration types and seem to use labile
forms of soil N (Lilleskov et al. 2011) while Cortinarius has hydrophobic medium-distance
fringe exploration and seems to use organic N sources and associates with less fertile soils.
Interestingly, Russula abundance did not correlate with any of the soil variables included in this
study. This likely reflects high variability in resource acquisition traits in this genus (see Chapter
5). Russula species have been reported as showing both negative and positive responses to N
availability (Avis 2003, Avis et al. 2008, Cox et al. 2010). A study of Russula communities
across forest sites varying in N availability at the landscape level at Fortuna also showed
significant differences in the phylogenetic relatedness of Russula between sites with high and
low fertility consistent with environmental filtering (Corrales et al. 2016a). Intrageneric variation
in N affinity was also observed in this study. Cortinarius sp. (OTU 87) showed a positive
association with N addition while Cortinarius obtusus (OTU 137) showed a negative response
(Table 4.7).
Ectomycorrhizal colonization was reduced by N addition as expected (Arnebrant and
Söderström 1992, Egli 1996, Treseder and Allen 2000, Peter et al. 2001). This could have been
caused by a reduction in the C supply from Oreomunnea to its associated ectomycorrhizas or a
reduction in mycelium abundance in the soil (Wallander and Nylund 1992, Wallenda and Kotke
1998, Peter et al. 2001, Frey et al. 2004, Nilsson and Wallander 2003, Nilsson et al. 2007).
Lower EM mycelium abundance in the soil could also be an explanation for the lower enzyme
activity found in soils in the N addition plots.
Changes in soil enzyme activity with N addition
Nitrogen addition was associated with significant changes in soil enzyme activity.
Notably phosphomonoesterase activity was lower in the N addition plots. Also, there was a
significantly higher enzyme C : N ratio in N addition plots representing lower N-acetyl-
glucosaminidase activity (that targets N compounds) compared with β-glucosidase (that targets
byproducts of cellulose). A lower N-acetyl-glucosaminidase activity was expect due to a higher
98
availability of soil inorganic N for plants and microorganisms (Midgley and Phillips 2014).
Lower phosphomonoesterase activity in N addition plots is opposite to the expectations since the
production of phosphatase requires a high investment of N (Olander and Vitousek, 2000).
However Marklein and Houlton (2012) in a meta-analysis of the response of phosphatase to N
and P addition found that even though the average response of soil phosphatase was positive, 26
of the 85 studies included in the analysis showed a negative response of phosphatase to N
addition. These authors hypothesize that soil fertility, duration of the fertilization, soil organic
matter content, C : N : P ratio, and microbial community composition could be possible causes
for the negative response (Marklein and Houlton, 2012).
Lower enzyme activity in N addition plots may indicate a down-regulation of
phosphatase production from plant roots, a reduction in EM fungal associated enzymes, or may
simply reflect the size of the microbial biomass pool in the N addition treatment.
Ectomycorrhizal fungi have been shown to release phosphatase in pure culture (Louche et al.
2010) and to actively deplete organic phosphorus sources (Read and Perez-Moreno 2003). In
addition, phosphatase activity has been shown to increase with the formation of ectomycorrhizal
associations (Ali et al. 2009, van Aarle and Plassard 2010) and to show a higher activity in the
rhizosphere soil around EM root tips (Buée et al. 2005, Courty et al. 2006). Consistent with
reduced EM phosphatase activity, we observed significantly lower EM colonization of
Oreomunnea roots in N addition plots (Figure 4.5). However, the fact that there were no
differences in enzyme activity between treatments after standardization by microbial biomass
could mean that a reduction in overall microbial biomass caused the reduction in enzyme activity
in the N addition plots, rather than a direct down-regulation of enzyme production.
Ectomycorrhizal functional response to N addition
Although the enzymatic activity of EM taxa was not measured directly, covariation in
soil enzyme activity and EM community composition suggests a functional response to N
addition. Overall, phosphomonosterase and phosphodiesterase activity was positively associated
with the abundance of Cortinarius and negatively associated with the abundance of Russula.
This supports previous findings of higher enzymatic capability to access organic N and P from
99
soil organic matter and litter reported for Cortinarius species (Agerer 2001, Nygren and Rosling
2009, Lilleskov et al. 2011).
Implications of anthropogenic N deposition for tropical montane forest
Tropical montane forests have been shown to have a strong short-term response to
moderate nutrient inputs including a reduction of investment in below-ground biomass and
changes in tree species composition (Homeier et al. 2012, Dalling et al. 2015). Consequently, the
predicted increase in N availability in tropical ecosystems resulting from urbanization and
agricultural intensification (Hietz et al. 2011) could cause significant changes in plant species
composition and soil C storage (Homeier et al. 2012, Dalling et al. 2015). With regard to
mycorrhizal associations, soils supporting EM dominated forest tend to have a higher soil
organic matter content compared to soils supporting arbuscular mycorrhizal dominated forest
probably due to stronger competition of EM fungi with free-living decomposer communities for
organic sources of N present in litter (Averill et al. 2014). Changes in the EM community
composition and soil enzyme activity with N addition could have important implications in soil
C storage, and ecosystem N cycling, ultimately affecting forest productivity and diversity. Our
results indicate that N addition could cause a decrease in the abundance of “nitrophobic” EM
fungal species and reduce soil enzyme activity associated with the absorption of organic N and P
in tropical montane forest. A reduction in EM fungal taxa specialized in organic N and P
absorption (e.g., Cortinarius) along with a decrease in EM colonization of host plants could
cause a decrease in the abundance of EM associated plants that could have feedback effects on
decomposition rates and soil C storage. More research focusing in the interaction of the EM
fungi and the decomposer communities under different environmental conditions, and on EM
fungal enzyme activity including peroxidase, chitinase, laccase, and leucine aminopeptidase
could improve our understanding of the implications of N addition on soil processes under EM
dominated tropical forest.
100
Figures and Tables
Figure 4.1 Species-accumulation curves of EM fungi for N treatments and developmental stages
including OTUs with > 10 reads.
Number'of'samples'
Num
ber'o
f'OTU
s'Num
ber'o
f'OTU
s'
Seedlings'Saplings'Adults'
101
Figure 4.2. NMDS using EM fungal species associated to Oreomunnea root tips (stress = 0.13)
for nitrogen treatment based on sequence abundance from individual samples including
seedlings, saplings, and adults. Adonis analysis R2 = 0.01, F1,191 = 2.65, P = 0.005.
+N#Control#
NMDS1#
NMDS
1#
102
Figure 4.3 NMDS using EM fungal species associated to Oreomunnea root tips. A) NMDS
(stress = 0.09) for nitrogen treatment based on sequence abundance pooling species from all
samples in each 40 × 40 plot and the first PCA axis for soil enzyme activity (PC1.enz), B)
NMDS for developmental stages (stress = 0.09) using individual samples.
NMDS
2&NMDS
2&
NMDS1&
B&
PC1.enz&
A&
&&&&&&&&Control&
Seedlings&Saplings&Adults&
&&&&&+&N&
103
Figure 4.4 Changes in relative abundance of the eight most abundant genera associated to
Oreomunnea mexicana roots in control and nitrogen fertilized plots. Error bars represent
standard error of the mean.
Abundance (mean no. of sequences)
0 10 20 30 40 50
+NControl
Cenococcum(
Clavulina!!
Cor$narius!! *!
*!
*!
Elaphomyces!!
Laccaria!!
Lactarius!!
Russula!!
Tomentella!!
Genus!rela+ve!abundance!(%)!
+N!Control!
104
Figure 4.5 Mean percent EM colonization (± 1 SE) of Oreomunnea mexicana roots of adults,
saplings, and seedlings in control and nitrogen addition plots.
P < 0.0001
0
25
50
75
100
A_C A_N SAP_C SAP_N SEE_C SEE_NTreatment
EM %
col
oniz
atio
n
Adults'
P'<'0.001' Control''+N'
Saplings' Seedlings'
EM'colon
iza<o
n'(%
)'
105
Table 4.1 Mean and standard error of soil characteristics control and nitrogen fertilized (+N)
plots at Fortuna Forest Reserve, Panama. The probability value (P) was calculated using Anova
analysis.
Soil variables Control + N P
Soil nutrients
pH 4.62 ± 0.09 4.51 ± 0.08 0.3
Resin P (mg P Kg-1) 1.60 ± 0.42 0.97 ± 0.22 0.6
NH4 (µg bag-1 day-1) 29.7 ± 6.9 40.3 ± 7.4 0.9
NO3 (µg bag-1 day-1) 16.7 ± 2.8 37.9 ± 6.1 0.02*
PO4 (µg bag-1 day-1) 1.06 ± 0.40 2.53 ± 0.82 0.7
DOC (mg C Kg-1) 417 ± 23 365 ± 34 0.5
Microbial C (mg C Kg-1) 1134 ± 170 797.8 ± 94.2 0.4
Microbial N (mg N Kg-1) 237.3 ± 28.8 180.8 ± 19.6 0.3
Microbial P (mg P Kg-1) 34.3 ± 4.9 34.5 ± 1.98 0.9
Microbial C:N 4.61 ± 0.31 4.46 ± 0.25 0.7
Microbial C:P 37.01 ± 8.06 24.28 ± 4.29 0.5
Microbial N:P 7.82 ± 1.53 5.38 ± 0.75 0.4
Soil enzymes
Phosphomonoesterase (MUP, nmol MU g-1 min-1) 169.30 ± 16.5 117.32 ± 14.9 0.05*
Phosphodiesterase (BIS, nmol MU g-1 min-1) 32.73 ± 7.25 20.99 ± 3.28 0.1
β-glucosidase (BG, nmol MU g-1 min-1) 6.89 ± 0.64 6.36 ± 0.75 0.5
N-acetyl-glucosaminidase (NA, nmol MU g-1 min-1) 7.54 ± 1.21 6.60 ± 1.80 0.2
β-xylanase (XYL, nmol MU g-1 min-1) 2.48 ± 0.35 2.79 ± 0.17 0.6
Cellobiohydrolase (CELLO, nmol MU g-1 min-1) 0.64 ± 0.04 0.85 ± 0.17 0.9
BG:NA (Enzymatic C:N) 0.98 ± 0.08 1.19 ± 0.16 0.04*
BG:MUP (Enzymatic C:P) 0.04 ± 0.001 0.06 ± 0.01 0.3
NA:MUP (Enzymatic N:P) 0.04 ± 0.005 0.05 ± 0.007 0.9
106
Table 4.2 Number of root samples sequenced per plot and per developmental stage of
Oreomunnea. Replicate samples were collected from three adult trees, five saplings, and five
seedlings per plot.
Plot Adult Saplings Seedlings Total
51 11 9 6 26
52 16 8 5 29
53 11 10 6 27
54 12 8 7 27
55 12 10 5 27
56 12 10 5 27
58 12 12 5 29
Total 86 67 39 192
107
Tables 4.3. Correlation analysis between soil enzymes. Values in bold represent significant
correlation.
Enzyme MUP BIS BG NA XYL CELLO EnzC.N EnzC.P EnzN.P MUP 1.00
BIS 0.93 1.00
BG 0.52 0.61 1.00
NA 0.83 0.90 0.81 1.00
XYL 0.31 0.57 0.59 0.54 1.00 CELLO 0.11 0.21 0.84 0.46 0.62 1.00
EnzC.N -0.76 -0.74 -0.24 -0.75 -0.15 0.13 1.00 EnzC.P -0.45 -0.34 0.50 -0.08 0.25 0.78 0.63 1.00
EnzN.P 0.06 0.22 0.71 0.58 0.32 0.61 -0.24 0.51 1.00
108
Table 4.4 Diversity indexes for EM fungi associated with the roots of Oreomunnea mexicana for
N addition treatments and Oreomunnea developmental stages. Values for N treatments were
computed based on plot averages pooling samples from all individuals in each plot.
Developmental stages means were computed using individual samples.
Diversity
index
Total
OTU
Fisher’s
alpha
Pielou
Evenness
+N 344 139.48 0.6
Control 318 116.77 0.57
df
6 6
P
0.117 0.103
Adults
4.96 0.51
Saplings
5.85 0.58
Seedlings
4.38 0.52
df
2 2
P
< 0.001 < 0.01
109
Table 4.5 ADONIS analysis including the species abundance matrix and an independent subset
of soil enzymes to explore relationship between EM species composition and soil enzyme
activity. Abbreviations as in Table 1.
R2 (%) F value NMDS
MUP 23.2 1.62 0.02*
BG 14.1 0.98 0.53
XYL 18.1 1.26 0.17
CELLO 14.6 1.02 0.44
EnzN:P 15.8 1.11 0.35
110
Table 4.6 The ten most abundant ectomycorrhizal fungal genera found in roots of Oreomunnea
mexicana and their distribution among nitrogen fertilized (N) and control plots (C) at the Fortuna
Forest Reserve, Panama. The number of OTUs in each genus (No. OTUs) was quantified based
on OTUs with more than 10 reads in the total database. The relative abundance of each genus
(RA) was calculated as the percentage of the total sum of reads of the OTUs in the genus divided
by the total number of read in the plot. The probability values for relative abundance are based
on a negative binomial generalized linear model analysis. The probability values of the number
of OTUs are based on a generalized linear model with Poisson errors. The genera with
significant differences between N fertilized and control plots are shown in bold.
P Plot 51 (C) Plot 52 (N) Plot 53 (N) Plot 54 (C) Plot 55 (N) Plot 56 (C) Plot 58 (C)
Genus
No.
OTUs RA
No.
OTUs RA
No.
OTUs RA
No.
OTUs RA
No.
OTUs RA
No.
OTUs RA
No.
OTUs RA
No.
OTUs RA
Cenococcum 0.48 0.06 14 10.68 11 2.43 12 7.87 11 9.70 9 6.45 15 10.42 10 5.06
Clavulina 0.28 0.15 6 4.41 9 6.82 4 1.45 3 1.86 8 4.11 6 10.77 5 13.57
Cortinarius 0.54 <0.001 25 13.50 25 9.74 21 3.78 21 10.73 10 5.04 18 21.09 19 14.49
Elaphomyces 0.24 0.87 13 8.27 6 4.33 8 4.84 10 8.40 8 10.30 11 4.12 6 1.14
Laccaria 0.68 <0.001 4 0.45 7 10.74 6 3.81 7 3.39 5 15.72 5 0.94 5 0.88
Lactarius 0.15 0.02 33 3.53 40 9.57 36 10.71 26 4.79 36 8.32 37 5.75 28 6.42
Russula 0.23 0.35 67 28.37 87 33.98 85 25.86 69 34.93 52 18.61 63 24.88 69 36.06
Tomentella 0.68 0.12 23 13.09 25 16.44 27 32.34 20 9.70 20 10.49 20 5.20 27 14.06
111
Table 4.7 Species showing significant changes in abundance across treatment (T),
developmental stage (DS), and their interaction, direction of the response, and their closest match
in the local sequence database or GenBank database. ID% refers to the percentage of sequence
match with the closest match in GenBank and the e-value refers to the probability of finding a
match by chance when searching the GenBank database. Data are for the 100 most abundant
taxa; P values are corrected for multiple comparisons using step-down resampling procedure (see
methods).
Species OTU Effect Response
to N P
Accession
number Family ID% e-value
Laccaria sp. 0020 T + 0.04 AC863-FB Hydnangiaceae 99% 4.29533e-157
Cortinarius sp. 0087 T + 0.04 DQ102685 Cortinariaceae 93% 5.37017e-127
Unknown 0045 T,
T*DS
+ 0.07,
0.004
JQ991671 Cantharellaceae 89% 1.26704e-132
Tomentella sp. 0126 T + 0.005 AB587786 Thelephoraceae 93% 1.10285e-140
Cortinarius obtusus 0137 T - 0.01 AC509-RT Cortinariaceae 98% 7.35007e-159
Tomentella sp. 0156 T - 0.006 EF411110 Thelephoraceae 96% 1.57522e-153
Cenococcum sp. 0016 T * DS ns 0.027 FJ440882 Gloniaceae 98% 7.65076e-125
Cortinarius junghuhnii 0018 T * DS - 0.02 AC412-RT Cortinariaceae 99% 5.98332e-162
Tomentella sp. 0022 T * DS ns 0.06 AC316-RT Thelephoraceae 99% 2.15756e-162
Cortinarius junghuhnii 0085 T * DS - 0.07 AC412-RT Cortinariaceae 98% 2.10525e-158
Tomentella sp. 0183 T * DS ns 0.07 AC316-RT Thelephoraceae 99% 1.65929e-161
Tomentella sp. 0061 DS 0.06 UDB004090 Thelephoraceae 92% 2.16885e-131
Cortinarius sp. 0028 DS 0.03 AC604-RT Cortinariaceae 97% 1.05797e-144
112
Table 4.8 Multiple regression models for enzyme activity and soil abiotic variables. GLM were
run using a gamma error distribution for a continuous positive response variable. Variables with
stars denote significance based on the > |z| value.
Enzyme Intercept Cenococcum Cortinarius Lactarius Russula Tomentella
MUP 4.58 0.0001* 0.00013***
- -0.00003**
-
BIS 2.09 - 0.0002 ** - -0.00007* - NA - - - - - - BG - - - - - - XYL - - - - - - CELLO - - - - - -
113
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Chapter 5: Variation in stable isotopes of Russula species associated with Oreomunnea mexicana in a tropical montane forest
Introduction
A knowledge of fungal functional traits is essential to predict fungal responses to
environmental factors (McGill 2006, Petchey and Gaston 2006) and to understand the role of
fungi in ecosystem processes (Crowther et al. 2014, Koide et al. 2014, Aguilar-Trigueron et al.
2015, Treseder and Lennon 2015). Fungal functional traits are features of mycelial morphology
and physiology that result in differences in the capacity for carbon (C) storage and nutrient
uptake (Hobbie and Agerer 2010). For ectomycorrhizal (EM) fungi these traits include
differences in the capacity to absorb organic vs inorganic nitrogen (N), in the amount of C
allocated to biomass for nutrient transport (rhizomorphs), and in water stress tolerance (Koide et
al. 2014). Recent work has shown that these functional traits underlie variation in the
composition of EM communities and is reflected in the isotopic composition of fungal tissue
(Courty et al. 2010, Hobbie and Agerer 2010, Hasselquist and Högberg 2014).
The isotopic composition of fruiting bodies can be a useful tool to differentiate
saprotrophic (SAP) and EM fungi (Mayor 2009). During the transfer of nitrogen from fungal
tissue to plant roots a fractionation occurs where 14N is preferentially transferred to the host
plant. This results in an average 6.7‰ enrichment of fruiting bodies of EM fungi relative to those
of saprophytic fungi (Hobbie and Hogbert 2012). The amount of N transferred from EM fungi to
the host plant is also correlated with the δ15N composition of EM fruiting bodies (Hogbert et al.
1999a), and has been proposed as a proxy for the EM fungal sink strength for N in boreal forest
(Hasselquist and Högberg 2014). In addition, higher δ15N in fruiting bodies has also been
associated with the presence of a hydrophobic mantle, long distance hyphal exploration types,
foraging for N at deeper soil horizons, and greater enzymatic capabilities to acquire N from
organic sources (Hobbie and Agerer 2010, Lilleskov et al. 2011, Hobbie et al. 2012, Hobbie et al.
2014). In contrast, species that show lower δ15N generally have a hydrophilic mantle, short
distance exploration types, forage for N in the soil organic layer, and have lower enzymatic
capabilities (Hobbie and Agerer 2010, Lilleskov et al. 2011, Hobbie et al. 2012, Hobbie et al.
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2014). Finally fruiting bodies with higher δ15N are also associated with sites with lower nitrogen
availability (Hobbie and Agerer 2010; Figure 5.1).
In addition to N, the δ13C of fruiting bodies can also be used to distinguish between
ectomycorrhizal and saprophytic fungi since their carbon sources (plant sugars for EM fungi, and
cellulose and lignin for saprophytic fungi), have contrasting δ13C signatures (Högberg et al.
1999b). As with N, variation in δ13C reflects additional sources of variation, depending on the
δ13C composition of their host plant (Högberg et al. 1999b). First, EM fungi that receive C from
plants from the forest overstorey tend to have higher δ13C than EM fungi receiving C from plants
growing in the forest understory (Högberg et al. 1999b). Second, plants exposed to drought stress
have been shown to have a higher δ13C signature than plants growing in more humid conditions,
which could also affect EM fungi δ13C composition (Högberg et al. 1999b, Figure 5.1).
To date, the diversity patterns and functional ecology of EM fungi in tropical forest and
their influence in ecosystem processes remain, for the most part, poorly understood. The aim of
this study was to explore community and intra-generic variation in the isotopic composition of
Russula species found along a gradient of soil inorganic N availability in a Panamanian montane
forest. Russula is an important component of many EM fungal communities in the tropics, often
having the highest species richness in fruiting body and root tip inventories (e.g., Peay et al.
2010; Smith et al. 2011; Tedersoo et al. 2011, Corrales et al. 2016). This genus is also highly
variable in response to N fertilization with some species increasing in their abundance in high N
environments, while others decrease (Lilleskov et al. 2002; Avis et al. 2003; Avis et al. 2008,
Lilleskov et al. 2011). These differences in response among Russula species may reflect
interspecific niche differentiation due to differences in species functional traits. Consistent with
resource based niche partitioning, Corrales et al. (2016) reported that Russula species found in
soils with low fertility had a greater than expected phylogenetic distance from Russula species
found in high fertility soils in Oreomunnea forest in Panama. It may be possible therefore that
Russula species with different resource acquisition capabilities colonize habitats with contrasting
fertility.
Oreomunnea mexicana (Juglandaceae) is an EM tree distributed through Central America
and locally accounting for up to 70% of individuals and basal area in the forests where it occurs.
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At the Fortuna Forest Reserve in western Panama it has been shown that Oreomunnea inhabits
soils with both high and low soil fertility and associates with diverse EM fungal communities, of
which Russula is the dominant genus. At Fortuna, the presence of monodominant Oreomunnea
forest is associated with reduced inorganic N availability, which has been hypothesized to result
from competition between EM fungi and free-living saprotrophs for N. Here we analyze the δ15N
and δ13C of Russula fruiting bodies and its correlation with EM (principally Oreomunnea) host
abundance and soil inorganic N availability (Corrales et al. 2016b). We hypothesized that: (1)
Russula found in low N sites will possess greater enzymatic capabilities to uptake N from
organic sources, resulting in higher δ15N in fruiting bodies than sites with high N availability. (2)
High N availability will reduce the amount of C that the host plant supplies to EM fungi,
resulting in Russula fruiting bodies from N addition plots and sites with natural high inorganic N
availability that are more enriched in δ13C than Russula from sites with low inorganic N. (3) If
the enzymatic capabilities of Russula species are a conserved trait, then more closely related
species will have a more similar isotopic signature than more distantly related species no matter
which environment they inhabit. (4) Based on the proposed competition between EM and
saprophytes we predicted that Oreomunnea abundance will be highly correlated with soil
inorganic N availability across a wider gradient of N availability than described by Corrales et al.
(2016b).
Methods
Study area
The study focused on stands of Oreomunnea mexicana (Juglandaceae) in four watersheds
(Alto Frio, Honda, Hornito, and Zarceadero) in a primary lower montane forest (1000–1400
m.a.s.l.; Figure 5.2 and Table 2.1 in Corrales et al. 2016a) in the Fortuna Forest Reserve in
western Panama (8˚45’ N, 82˚15’W, Corrales et al. 2016a). Oreomunnea is a mid-elevational
canopy tree distributed from southern Mexico to western Panama at 900–2600 m.a.s.l. (Stone
1972). It produces ca. 100 mg, wind-dispersed fruits, which can generate high-density seedling
patches in the understory. Oreomunnea is locally dominant at some of our study sites, accounting
for up to 70% of individuals and stand basal area (Corrales et al. 2016a). Other EM tree species
that are present in the study area are Quercus insignis, Q. cf lancifolia, and Coccoloba spp.
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These species do not form monodominant forests and are found in low abundance in some of the
study sites. Climate records indicate that the mean annual temperature for Fortuna ranges from
19 to 22 °C (Cavelier 1996). Annual rainfall averages ca. 5800 mm at our sites in Hornito, Alto
Frio, and Zarceadero, and 9000 mm at our sites in Honda, although all were drier during our
study period (Table 5.1). Hornito and Alto Frio typically have 1–2 months per year with <100
mm of precipitation; in contrast, no months with <100 mm of rainfall have been recorded at
Honda, or Zarceadero (Andersen et al. 2012; J. Dalling unpublished data).
The selected sites were located under Oreomunnea dominated forest and differ markedly
in soil characteristics (Corrales et al. 2016a). The sites were classified as high, medium, and low
soil inorganic nitrogen (N) availability based on the sum of resin-extractable ammonium and
nitrate (Table 5.1; Andersen et al. 2010, Corrales et al. 2016a). A total of 148 resin bags
containing 5 g of mixed-bed anion and cation exchange resins (Dowex Marathon Mr-3 Supelco,
Bellefonte, PA, USA) sealed inside 220 µm polyester mesh were buried 2 cm beneath the soil
surface at each site in Oreomunnea forest. Bags were buried during August 2014. After
incubation in situ for 18 days, the resin bags were collected, rinsed with deionised water to
remove adhering soil, extracted with 75 mL of 0.5 M HCl, and then nitrate (+ nitrite) and
ammonium were determined by automated colorimetry on a Lachat QuikChem 8500 (Hach Ltd.,
Loveland, CO, USA). Oreomunnea basal area in these sites was measured in one 50 × 20 m plot
at each site where all Oreomunnea trees were measured and recorded in July 2011 and July
2014.
In addition, seven 40 × 40 plots belonging to a N addition experiment in the Honda
watershed were included as collection sites (four control plots and three N addition plots), where
125 kg N ha-1 have been applied annually over nine years (for description of the N addition
experiment see Chapter 3 and Adamek et al. 2009). Resin bags were buried in these plots during
October 2013 and August 2015 and were incubated for 18 days. Oreomunnea basal area was
measured in all the plots in 2015 (Dalling et al. unpublished data).
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Collection of the Russula fruiting bodies
Russula fruiting bodies were collected over a four year period between January 2012 and
June 2015. Macromorphology of fresh fruiting bodies was recorded in the field, and a sample of
tissue was preserved in 96% ethanol for DNA extraction from a subsample of the collections
(Table 5.1). Vouchers of fruiting bodies are deposited at the University of Arizona Robert L.
Gilbertson Mycological Herbarium (MYCO-ARIZ) and at the herbarium of University of
Central Oklahoma (UCO). Russula voucher collections were identified based on their
morphology and names assigned when possible. Because of the high diversity and challenging
taxonomy of Russula, and the possibility that collections represent new species, most species
names were assigned based on sequence similarity with a 97% similarity threshold used to
classify OTUs (explained below).
DNA extraction and amplification
Genomic DNA was extracted using the REDExtract-N-Amp plant PCR kit (following the
manufacturer’s instructions; Sigma-Aldrich). Primers ITS1F and ITS4B were used for species
identification following Corrales et al. (2016a).
Phylogenetic analysis
Phylogenetic relationships were inferred using 113 sequences from Russula fruiting
bodies collected at the study site and 31 sequences downloaded from GenBank as in Corrales et
al. (2016a). Four sequences from voucher specimens of Stereum hirsutum (Willd.) Pers.
(AY854063), Amylostereum laevigatum (Fr.) Boidin (AY781246), Gloeocystidiellum porosum
(Berk. & M.A. Curtis) Donk (AY048881), and Bondarzewia montana (Quél.) Singer
(DQ200923) were used for the outgroup following Miller and Buyck (2002) and Buyck et al.
(2008). Sequences were aligned using MUSCLE (Edgar 2004). The resulting alignment was
edited using Gblocks 0.91b (Castresana 2002) to exclude positions that were poorly or
ambiguously aligned. The final data set consisted of 580 characters and 144 terminal taxa. The
tree was inferred using maximum likelihood analysis using the GTR+I+Gamma model of
evolution and the bootstrap support was assessed using 1000 bootstrap replicates using the Ape
package in R (Paradis et al. 2004).
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Pagel’s λ was used to measure phylogenetic dependence of the isotopic composition and was
calculated using the function phylosig in the package phytools 0.1.2 in R (Revell 2012)
Stable isotopic composition analysis
A small piece of dry fruiting body (dried at 60˚C) was sampled from the stipe and pileus
of 93 Russula collections, and from nine saprophytic fungi collected at the same locations (Table
5.1). Samples were finely ground using glass beads in a TissueLyser II machine (QIAGEN, Inc.)
and then analyzed for C and N concentrations and stable isotope ratios (δ13C and δ15N) by
continuous flow isotope ratio mass spectrometry using an elemental analyser (Costech
Analytical, Valencia, California 4010) coupled to a Delta-V Advantage isotope ratio mass
spectrometer (Thermo Fisher Scientific, Bremen, Germany). Run imprecision for δ13C and δ15N
was typically < 0.2 ‰. To augment the database on saprophytic fungi, eight data points from
samples collected from Fortuna, published in Mayor et al. (2014) were included in the SAP
average.
Statistical analysis
To test for the effect of soil N availability on fruiting body isotopic composition a nested
ANOVA was done using isotopic composition (δ13C and δ15N) as dependent variables and sites
nested within N fertility level (LN: Low N, MN: Medium N, HN: High N, C: Control plots from
N addition experiment, N: N addition plots in N experiment) as independent variables.
Differences among soil N levels were calculated using a post-hoc Tukey’s test in R. We used
type II Major Axis (MA) regression to fit N content (N%) of fruiting bodies with δ15N using the
lmodel2 package in R (Legendre, 2011) following Heineman et al. (2016). δ15N was transformed
using the multiplicative inverse function (1/x) to improve linearity. In addition, a MA regression
between δ13C and δ15N was fitted to check for coupling between N and C transfer between EM
plant and EM fungi.
To test the effect of inorganic N availability on Russula fruiting body δ15N, a linear
regression was run using δ15N as a dependent variable and soil inorganic N availability
(measured with resin bags) as the independent variable. To test the effect of host abundance on
Russula δ15N, a linear regression was run using δ15N as a dependent variable and Oreomunnea
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basal area as independent variable after logarithmic transformation of both variables. To test the
effect of inorganic N availability and host abundance on Russula fruiting body δ13C, two linear
regressions with no variable transformations were run using δ13C as the dependent variable,
Oreomunnea basal area and soil inorganic N availability as independent variable, and mean
annual dry season rainfall (recorded from January to April during 2013 and 2014) as a covariate
to account for changes in water availability in the plant δ13C signature.
Results
Russula community
During the four years of the study, 161 Russula fruiting bodies were collected and 108
(68%) were sequenced (Table 5.1). The 108 sequenced collections were grouped into 52 OTUs
based on 97% similarity. The average number of Russula OTUs per site was 12, with the lowest
richness in the N addition plots (5 OTUs; Table 5.1). The Russula phylogenetic tree was well
supported based on the bootstrap values (Figure 5.3).
Effect of soil inorganic nitrogen availability on C and N isotope ratios of the Russula
community
There was high variation in the isotopic signature of Russula OTUs, ranging for δ13C
from -22.05 (in AC422 OTU S25) to -27.92 (in CO 5478 what OTU S7) and for δ15N from 1.06
(in AC648 what OTU S37) to 10.00 (in AC422 OTU S25, Figure 5.4).
The isotopic composition of δ13C (F4,79 = 2.70, P = 0.03) and δ15N (F4,79 = 3.63, P =
0.009) differed significantly among soils with different inorganic N availability. The δ13C of
Russula fruiting bodies collected from soil with naturally high inorganic N availability showed
significantly higher δ13C than Russula collected in sites with lower inorganic N availability or in
the N addition plots (Figure 5.5a). The δ15N of Russula fruiting bodies collected from the site
with the lowest soil inorganic N availability showed a significantly higher δ15N than Russula
collected in sites with higher N availability (Figures 5.5b).
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The community average δ13C of Russula fruiting bodies from the sites with the highest natural
soil inorganic N were closer to δ13C values of tropical saprophytic fungi (SAPF) while the
community average δ15N of Russula from the N fertilization plots were closer to the SAPF δ15N
values (Figure 5.6).
The community average δ15N was not significantly correlated with soil inorganic N
availability (R2 = 0.60, P = 0.08,) while δ13C was correlated with soil inorganic N availability
when using mean annual dry season precipitation as a covariate (R2 = 0.96, P = 0.02).
Community average δ15N was significantly negatively correlated with community average %N in
fruiting bodies (R2 = 0.99, P < 0.001; Figure 5.7A) and positively correlated with the abundance
of the host species Oreomunnea (R2 = 0.95, P = 0.002; Figure 5.7B). The abundance of
Oreomunnea was also negatively correlated with soil inorganic N availability (R2 = 0.70, P =
0.048; Figure 5.7C). The community average δ13C correlated with host tree abundance when
using mean annual dry season precipitation as a covariate (R2 = 0.92, P = 0.041). Finally,
Russula community δ15N was significantly correlated with δ13C when using precipitation during
the dry season as a covariate (R2 = 0.84, P = 0.048)
Based on Pagel’s λ, there was a significant phylogenetic dependence of δ15N (λδ15N =
0.66, P = 0.03), but not of δ13C (λδ13C = 6.61e-5, P > 0.99) in Russula fruiting bodies (Figure 5.8).
Discussion
Determinants of variation in Russula isotopic composition
Here we provide evidence that the δ15N of Russula fruiting bodies increases in proportion
to the abundance of Oreomunnea mexicana. In turn, Oreomunnea abundance increased with
decreasing soil inorganic N availability. Due to fractioning of N isotopes, EM fungi become
more enriched in 15N when they transfer more N to their host (Hogberg et al. 1999a). The greater
enrichment of δ15N in Russula fruiting bodies with an increase in host abundance and a decrease
in inorganic N availability is therefore consistent with higher N transfer rate from Russula to
their host plant under conditions of decreasing inorganic N availability. Changes in δ15N could
also be associated with differences in the soil depth at which EM access N (Hobbie et al. 2014).
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However as this study was limited to a single EM genus interspecific differences in EM foraging
patterns may be smaller than would be observed for the whole EM community.
The δ13C of the Russula communities was also correlated with Oreomunnea abundance,
suggesting a higher transfer of C from Oreomunnea to Russula in sites with higher Oreomunnea
abundance. However, the correlation with Oreomunnea abundance was only significant when
dry season precipitation was used as a covariate. Drought stress has been shown to increase the
δ13C signature in plants (Farquhar et al. 1989, Ehleringer et al. 1993). High nitrogen (HN) sites at
Fortuna (Hornito and Alto Frio, Table 5.1) typically have 1–2 months per year with < 100 mm of
precipitation; in contrast no months with < 100 mm of rainfall have been recorded in the other
sites (Corrales et al. 2016a). The higher δ13C of Russula fruiting bodies in HN sites compared to
other sites is therefore likely to be associated with seasonal drought stress.
Isotopic evidence for EM effects on ecosystem N cycling
The significant correlation between δ15N and δ13C along with increase of δ15N and
decrease in δ13C with host abundance indicates a coupling of tree C allocation to EM fungi with
EM fungi N transfer to Oreomunnea. Hasselquist and Hogberg (2014) proposed that EM
community δ15N could potentially be used as a relative index of EM fungal sink strength. This is
because in sites with low N availability, trees transfer more C to EM fungi in exchange for N,
and that C can then be invested in the production of more fungal biomass including fruiting
bodies. Sites with higher fungal biomass will in turn have a higher demand for soil N
immobilizing a higher proportion of soil N in their biomass. As a consequence, higher N transfer
from EM fungi to the host plant will result in higher 15N fractionation making EM fruiting bodies
more δ15N enriched and foliar δ15N more depleted.
Conditions of low soil inorganic N availability under EM dominated forest are proposed
to result from competition between EM fungi and the community of free living saprotrophs
(Averill et al. 2014, Corrales et al. 2016). Collectively, these results suggest that EM fungi
creating soil conditions that increase Oreomunnea dependency on EM fungal N via a feedback
loop (Figure 5.9). We would predict that sites with lower inorganic N availability and higher
δ15N in Russula fruiting bodies (i.e., the low N site) will have a higher fungal biomass and
128
therefore EM fungi will retain a larger amount of ecosystem N. This would imply that higher C
allocation from the tree to EM fungal species could decrease N availability for free-living
saprotrophs and non-EM plant species creating a positive feedback loop contributing to increased
abundance of Oreomunnea mexicana in sites with lower inorganic N availability (Figure 5.9).
Evidence for EM mediated positive feedback has been found in boreal forest, where EM fungi
may sustain rather than alleviate N limitation making their host plant more dependent of EM
fungal N (Näsholm et al. 2013, Franklin et al. 2014).
Effect of N addition on isotopic composition
There were no significant differences in the mean isotopic composition of fruiting bodies
from the control and N addition plots. These results contrast with those found from the sites
differing in natural soil inorganic N availability and suggest that an increase in N availability
over 9 years did not reduce C allocation from the host plant to Russula species. However, N
addition did seem to affect the number of Russula species producing fruiting bodies. Although
the transects located in the control and N addition plots were visited the same number of times,
only 17 collections belonging to 5 OTUs were found from N addition plots while 36 collections
belonging to 17 OTUs were found in control plots (Table 5.1). This change in Russula OTU
richness with N addition however was not reflected in the belowground community characterized
using Illumina sequencing in Oreomunnea root samples from the same plots (Corrales et al.
Chapter 4).
Interspecific variation in Russula isotopic composition
At Fortuna, interspecific variation of Russula δ15N showed significant phylogenetic
signal (Figure 5.8), with more closely related species showing more similar δ15N than more
distantly related species. These results are consistent with findings from Tedersoo et al. (2012),
who reported a strong effect of genetic distance on enzymatic activity, root tip mantle
morphology, and exploration type in Russula collected from an African lowland tropical forest.
However there was no correlation between isotopic composition and enzyme activity in this
study, probably due to differences in the isotopic composition of EM fungi in root tips compared
with fruiting bodies. We would expect that changes in the δ15N composition of Russula at our
129
study site could be explained at least partially by differences in species enzymatic capabilities
since species with greater access to organic N are expected to have higher δ15N content in their
fruiting bodies (Lilleskov et al. 2002).
It has also been shown that exploration type and mycelium hydrophobicity are associated
with differences in the N sources that EM species can access (Hobbie and Agerer 2010, Hobbie
et al. 2014). In general, hydrophobic species usually belong to long distance exploration types,
forage N at deeper soil horizons, and have higher enzymatic capabilities and δ15N, while
hydrophilic species show the opposite pattern (Hobbie and Agerer 2010, Lilleskov et al. 2011,
Hobbie et al. 2012, Hobbie et al. 2014). Hobbie et al. (2014), in a labeling experiment at the
Duke Forest FACE experiment, found that Russula and Lactarius that are usually classified as
hydrophilic genera showed lower δ15N compared with hydrophilic taxa as expected. However,
some hydrophilic genera (Laccaria and Amanita) showed a high δ15N similar to hydrophobic
species presumably due to exploration of deeper soil horizons. In addition, it has been shown that
organic N uptake is enzymatically costly for EM fungi and requires a high glucose supply from
the host plant (Lindahl and Tunlid 2014). We would therefore expect hydrophobic taxa to
receive more C from the host plant and to be more depleted in δ13C than hydrophilic taxa.
Russula has previously been classified as a hydrophilic genus with an average δ15N of
3.2±0.3 ‰ (Hobbie and Högbert 2012). However, the large range of variation in δ13C (-27.9 to -
22.05) and δ15N (1.06‰ to 10‰) found in this study suggests that Russula may be considerably
more functionally diverse than is generally recognized. Russula species have been reported to
have a variety of exploration types ranging from contact and short distance to medium distance
smooth types (Table 5.2, Agerer and Rambold 2004). Examination of mantle hydrophobicity
data reported on DEEMY database (www.deemy.de) also supports this; 29 Russula specimens
are reported as hydrophobic and 24 as hydrophilic (Table 5.2). In addition, Russula species have
been shown to exhibit contrasting abundance changes in response to N addition and N deposition
(Lilleskov et al. 2011). Collectively, this evidence indicates that Russula species vary widely in
their functional traits. This may be particularly true in tropical ecosystems where collections
records indicate that Russula is the most diverse EM genus.
130
Conclusions and future directions
The δ15N and δ13C isotopic composition of EM fruiting bodies is a useful tool with
potential to connect EM fungal species composition to ecosystem function. Here we demonstrate
that the isotopic composition of the Russula community reflects changes in host abundance and
inorganic N availability, presumably reflecting increased host demand for EM fungal N supply
with a reduction in soil inorganic N availability. Corrales et al. (2016b) demonstrated that the
presence of Oreomunnea mexicana and therefore its associated EM fungi is associated with a
reduction in soil inorganic N availability. Here we provide evidence that this is consistent with
an increase in N sequestration by EM fungi in sites with higher host abundance. Since host
abundance, N availability, and N transfer from EM fungi to the host (reflected in fruiting body
δ15N) all change in the same direction isotopes provide further evidence that the formation of
Oreomunnea dominated forest is facilitated by its associated EM fungi (Figure 5.9).
The high interspecific variation in Russula isotopic composition and it association with
other functional traits may explain why Russula abundance and diversity did not change with N
addition in Corrales et al. (Chapter 3). N addition may have increased the abundance of
nitrophilic species and reduced the abundance of nitrophobic species while richness at the genus
level was unchanged.
Future studies focusing on tropical Russula exploration types coupled with an N15
labeling experiment should be done to corroborate the soil depth at which Russula species forage
for nutrients and their actual exploration types and mantle properties. Functional trait databases
should also be enriched with more information on tropical species to improve our ability to
associate species composition with ecosystem function.
131
Figures and Tables
Figure 5.1. Direction of reported changes in δ15N and δ13C of EM fruiting bodies.
Arrows show only direction of the expected change but are not proportional to the
magnitude of those changes.
2
3
4
5
6
-26.0 -25.5 -25.0 -24.5 -24.0d13C
d15N
HF#
MF#
+N#
LF#
C#
SAP#
Forages#N
#at#d
eepe
r#soil#horizo
n#
Transfers#m
ore#N#to
#plant#
Higher#access#to#organic#N#so
urces##
Saprop
hy@c#A##Hydroph
ilic##A##Hydroph
obic##
Ectomycorrhizal##################################Saprophy@c#(plant#sugars)########################################(cellulose)##
δ13#C##
δ15 N##
##A#######Plant#drought#stress######+#Understorey##################Overstorey#
Högberg#et#al.#(1999)#
Hobbie#and#Högberg#(2012)#
More#δ13C#enriched#
More#δ1
5 N#dep
leted#
More#δ13C#depleted#
More#δ1
5 N#enriche
d#
132
Figure 5.2. Upper panel, location of Fortuna Forest Reserve, Panama. Lower panel, sampling
sites at Fortuna: LN, site with low inorganic noitrogen availability (Zarceadero); MN, sites with
medium N availability (Honda A and B); HN, sites with high N availability (Hornito and Alto
Frio); C and +N nitrogen addition and control plots, Upper panel reproduced with modifications
from Andersen et al. (2010)
LN#MN#
C#and#+N#
HN#
133
Figure 5.3 Results of maximum likelihood analysis of Russula obtained from fruiting bodies in Oreomunnea mexicana-dominated stands at Fortuna, Panama, and representative taxa chosen from GenBank as described in the text. Values in the branches represent bootstrap support. Sequences are annotated sequencing code.
AC52
AC132
AC198
AC269
AC270
AC272
AC305
AC317
AC318
AC329
AC331
AC334
AC336
AC430
AC434
AC457
AC497
AC501
AC507
AC516
AC517
AC518
AC520
AC521
AC522
AC528
AC531
AC545
AC572
AC578
AC579
AC581
AC580
AC588
AC591
AC587
AC603
RUSX71
RUSD32
RUSL87
RUSP86
RUSC57
RUSI40RUSV51
RUSS78
RUSX42
RUSF27
RUSA64
RUSI24
RUSP93
RUSP52
RUSG31
RUSF69
RUSP35
RUSO98
RUSC59
RUSC58
RUSV36RUSN64
RUSP85
RUSF17
RUSS19
RUSR22
RUSB35
RUSV39
RUSS71
RUSU41
RUSU52
STEH63AMYL46
GLOP81BONM23
AC335
AC503
AC511
AC691
AC706
AC707
AC716
AC719
AC720
AC723
AC724
AC769
AC783
AC792
AC801
AC803
AC805
AC815
AC819
AC822
AC823
AC830
AC834
AC856
AC858
AC864
AC865
AC872
AC874
AC878
AC881
AC893
AC896
AC907
AC911
AC912
AC917
AC927
AC1030
AC1031
AC1035
AC1044
AC1046
AC1047
AC1048
AC1049
AC1053
AC1054
AC1056
AC1058
AC1063
AC1064
AC1079
AC1083
AC1084
C AC688
C AC1040
AC271AC431AC448AC523AC527AC584
AC369
AC403AC416AC590
AC827AC859
AC837AC850
AC895
Root
100
78
69
32
10043
60
55
80
68
100
46
100
69
80
55
85
88
10090
100
57
50
50
8572
10088
97
99100
99
100
67
10060
94
10091
58
57
54
66 9966
89
100
93
72100
100100
74
97 10094
54100
82
94
76
10094
100
89
100
82
10051
100
49
52
82100
99
91
86
9578
89
53
69100
84
55
100
52
100
89
66
99
93
50
5990
92
50
96
58
68
47
63
93
93
64
70100
94
30
79
75
73
93
95
100
64
85
100
99
90
10091
34
78
74
95
86
60
39
100
100
100
90
134
Figure 5.4 OTU mean values for (A) δ13C and (B) δ15N. Dot colors represent sites with
contrasting soil inorganic N availability.
-22
-26
-30
-34
OTUS 97%
d13C
S7 C006 S36 S40 S5 S6 S1 C0019 S35 S3 S41 C0035 S31 S37 S33 S42 C003 S28 S29 C0020 S39 S26 S4 S27 S30 S38 S32 S25
C+NLNHNC_N
02468
OTUS 97%
d15N
S37 S32 S30 S35 S4 S29 S33 C0020 C003 S39 S26 S3 S41 C0035 C0019 S1 S36 S38 C006 S40 S31 S6 S42 S7 S27 S28 S5 S25
C+NLNHNC_N
-22
-26
-30
-34
OTUS 97%
d13C
S7 C006 S36 S40 S5 S6 S1 C0019 S35 S3 S41 C0035 S31 S37 S33 S42 C003 S28 S29 C0020 S39 S26 S4 S27 S30 S38 S32 S25
C+NLNHNC_N
02468
OTUS 97%
d15N
S37 S32 S30 S35 S4 S29 S33 C0020 C003 S39 S26 S3 S41 C0035 C0019 S1 S36 S38 C006 S40 S31 S6 S42 S7 S27 S28 S5 S25
C+NLNHNC_N
A"
B"
δ13$C$$
δ15N
$$
135
Figure 5.5 Russula fruiting bodies community average for (A) δ13C and (B) δ15N in soil
low (LN), medium (MN), and high (HN) inorganic N availability levels and for the
control and N addition plots (C and +N). Error bars represent ± 1SE of the mean.
-26.0
-25.5
-25.0
-24.5
-24.0
LN MN C HN +NTreatment
d13C
2
4
6
8
LN MN C HN +NTreatment
d15N
*"*"
LN"""""""""""""MN""""""""""""""C""""""""""""""HN"""""""""""""+N"
δ13 C"(‰
)""
δ15 N"(‰
)"
A" B"
a"
ab"
b"
ab"ab"
ab"ab"
b"
a"a"
Site"(ordered"by"N"availability)"LN"""""""""""""MN""""""""""""""C""""""""""""""HN"""""""""""""+N"
Site"(ordered"by"N"availability)"
136
Figure 5.6 Russula community average isotopic values for sites with low (LN), medium
(MN), and high (HN) inorganic N levels, and for control (C) and N addition (+N) plots
and for a comparison group of saprophytic fungi (SAP). Vertical and horizontal error bars
represent ± SE.
HF#
LN#
+N#
MN#
C#
SAP#
δ15 N#(‰
)#
δ13C#(‰)#
137
Figure 5.7 Linear regressions between (A) 1/ Russula community average of δ15N vs average percentage of N of fruiting bodies per site, (B) Natural logarithm Oreomunnea basal area vs Natural logarithm of Russula community average of δ13C, (C) Soil inorganic N availability vs Oreomunnea basal area.
2.0 2.5 3.0 3.5 4.0 4.5
1.2
1.4
1.6
1.8
2.0
LN Oreomunnea BA (m2/ha)
LN R
ussu
la c
omm
unity
d15
N
0.18 0.20 0.22 0.24 0.26
3.5
4.0
4.5
5.0
5.5
1 / Community d15 N
Com
mun
ity N
% in
frui
ting
bodi
es R2 = 0.99, P < 0.001
1"/"Russula"community"δ15N"
A"
B"
Russula"commun
ity"δ
15N"
0 10 20 30 40 50 60 70
010
2030
4050
60
Soil inorganic N (µg N/bag)
Ore
omun
nea
BA (m
2/ha
) R2= 0.70, P= 0.048C"
Oreomunnea"BA"(m2/ha)"
Oreom
unnea"BA
"(m2 /ha)"
R2"="0.95,"P"="0.002"
R2"="0.99,"P"<"0.001"
R2"="0.70,"P"="0.048"
138
Figure 5.8 Maximum likelihood phylogeny of Russula obtained from fruiting bodies and
trimmed to include only samples with isotope data. Bubbles at the tree tips represent
δ13C, δ15N. The size of the bubble is at scale with the δ13C, δ15N average values per OTU.
Annotations at tree tips correspond to the closest matching sequence in GenBank and the
sample collection number.
AC691AC864AC917AC865AC896AC1056AC1046AC803AC927AC707AC719AC1053AC1079AC706AC858AC724AC783AC1048AC872AC893AC878AC716AC801AC823AC827AC859AC805AC830AC881AC723AC769AC856AC1031
d13C
d15N
AC691AC864AC917AC865AC896AC1056AC1046AC803AC927AC707AC719AC1053AC1079AC706AC858AC724AC783AC1048AC872AC893AC878AC716AC801AC823AC827AC859AC805AC830AC881AC723AC769AC856AC1031
d13C
d15N
Russula nigricans (CO5211) Russula nigricans (AC626) Russula sp. (AC508) Russula atropurpurea (AC424) Russula aff. betularum (AC648) Russula sp. (AC574) Russula sp. (AC336) Russula sp. (AC629) Russula sp. (AC494) Russula sp. (AC518) Russula sp. (AC561) Russula sp. (AC449) Russula sp. (AC645) Russula sp. (AC614) Russula lutea (AC640) Russula corallina (CO5430) Russula sp. (AC558) Russula cf. favrei (AC381) Russula xerampelina (AC616) Russula cf. flavisiccans (AC454) Russula lepida (CO5478) Russula lepida (CO5435) Russula sp. (AC342) Russula sp. (AC462) Russula sp. (AC552) Russula cyanoxantha (AC554) Russula cf. cyanoxantha (CO5429) Russula variata (CO5440) Russula cerolens (AC601) Russula cerolens (AC632) Russula crassotunicata (AC607) Russula sp. (AC591) Russula sp. (AC422)!!
δ13C
!
δ15N
!
λδ13C!=!6.61e-5,!P!=!1!λδ15N!=!0.66,!P!=!0.03!*!!
139
Figure 5.9 Feedback loop to explain monodominance of Oreomunnea mexicana based on
the ‘microbial competition for nitrogen hypothesis’ (Corrales et al. 2016b) and EM
fruiting bodies δ15N. Abbreviations: Ectomycorrhizal (EM).
Hypothesis*for*EM*mediated*N*sequestra5on*
High%abundance%of%Oreomunnea)
High%abundance%of%EM%fungi%
EM%fungi%compete%with%saprophy8c%fungi%slowing%down%decomposi8on%rates%
and%reducing%the%availability%of%inorganic%nitrogen%in%the%soil%
Lower%soil%inorganic%N%availability%and%higher%
Oreomunnea%abundance%increase%demand%for%N%transfer%from%EM%fungi%
to%Oreomunnea%%
Increased%N%transfer%from%EM%fungi%is%coupled%with%increased%C%transfer%from%Oreomunnea%to%EM%fungi%suppor8ng%more%fungal%biomass%and%increasing%
N%sequestra8on%
Increase%survivorship%and%growth%rate%of%
Oreomunnea%seedlings%N*
C*
140
Table 5.1 Total soil inorganic N, dry season rainfall (Jan-April), and number of Russula
fruiting body collections, ITS sequences, OTUs, samples with isotope data and samples
with both isotope and sequence data per site. F and P values from ANOVA analysis
including all sites and a post-hoc Tukey’s test (as super indexes) are given for δ13C and
δ15N.
Fertility Sites Total soil N
(µg N/ bag)
Total rain dry season
(mm)
No. Collections
ITS sequences
No. OTUS Isotopes Both
High N Alto Frio, Hornito
40.48 97.75 43 20 17 25 2
Medium N Honda A, B
25.42 209.70 34 28 8 7 6
Low N Zarceadero 2.74 189.42 25 15 12 16 1
Control1 Plots 51, 54, 56, 58
33.65 209.70 36 29 17 31 24
N addition1 Plots 52, 53, 55
55.71 209.70 17 16 5 11 10
Total 158 108 52 93 43 1Control and N addition plots are from an N fertilization experiment established at the medium N site
141
Table 5.2 Summary of exploration type and presence of mantle hydrophobicity of all the
Russula species included in the DEEMY database (Agerer and Rambold 2004-2016).
Exploration type Hydrophobic Hydrophilic Both No information Total
Contact 1 18 1 11 31 Contact/Short distance
2
2
Medium distance smooth 22 2
2 26 Short distance 6 2 1
9
Total 29 24 2 13 68
142
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Tedersoo L, Bahram M, Jairus T, Bechem E, Chinoya S, Mpumba R, Leal M, Randrianjohany E, Razafimandimbison S, Sadam A, Naadel T, Koljalg U (2011) Spatial structure and the effects of
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host and soil environments on communities of ectomycorrhizal fungi in wooded savannas and rain forests of Continental Africa and Madagascar. Mol Ecol 20: 3071–3080.
Tedersoo L, Naadel T, Bahram M, Pritsch K, Buegger F, Leal M, Kõljalg U, Põldmaa K (2012) Enzymatic activities and stable isotope patterns of ectomycorrhizal fungi in relation to phylogeny and exploration types in an afrotropical rain forest. New Phytologist 195: 832–843.
Treseder KK, Lennon JT (2015) Fungal traits that drive ecosystem dynamics on land. Microbiology and Molecular Biology Reviews 79: 243–262.
Weber CF, Zak DR, Hungate BA et al. (2011) Responses of soil cellulolytic fungal communities to elevated atmospheric CO2 are complex and variable across five ecosystems. Environmental Microbiology 13: 2778–2793.
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Chapter 6: Conclusions
Main results and mechanisms facilitating monodominance of Oreomunnea mexicana
Results from Chapter two reveal that species diversity of EM fungi associated with
Oreomunnea is high and suggests that tropical EM fungal communities can be as species-rich as
those found in temperate forests (Diédhiou et al. 2014; Henkel et al. 2012; Smith et al. 2011).
Consistent with the few previous studies of EM fungal communities in tropical forests, we
observed strong community dissimilarity of EM fungi across sites.
Results from Chapter three shows that ectomycorrhizal communities play an important
role in soil nutrient dynamics due to their ability to acquire organic nitrogen via extracellular
enzymes. After running experiments to test for presence of EM networks and positive plant-soil
feedback our results suggest that neither of those mechanisms account for monodominance in
our study system. Instead, I found that Oreomunnea dominated forest is associated with lower
availability of soil ammonium and nitrate than nearby arbuscular mycorrhizal dominated forest.
This reduction in inorganic N availability is most likely associated with a tightened N cycle, with
low rates of nitrification, denitrification and gaseous N losses from the soil organic layer. I
propose that reduced N availability, driven by the depletion of readily mineralizable N from soil
organic matter by EM fungi, could confer Oreomunnea seedlings a competitive advantage over
AM or non-mycorrhizal species accounting for the dominance of this species.
Results from Chapter four suggest that nine years of N addition can cause a decrease in
the abundance of EM species adapted to low N availability and reduce soil enzyme activity
associated with the mineralization of organic N and P in tropical montane forest. A reduction in
the abundance of EM fungal taxa specialized in organic N and P absorption (e.g., Cortinarius)
along with a decrease in EM colonization of host plants could cause a decrease in the abundance
of EM associated plants with potential feedback effects on decomposition rates and soil C
storage.
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Results from Chapter five demonstrate that the δ13C and δ15N of the Russula community
reflect changes in Oreomunnea mexicana abundance and inorganic N availability, presumably as
a consequence of increased host demand for EM fungal N in response to a reduction in soil
inorganic N availability. I also demonstrated that the presence of Oreomunnea mexicana and its
associated EM fungi is correlated with a reduction in soil inorganic N availability consistent with
an increase in N sequestration by EM fungi in sites with higher host abundance. Given that sites
with lower inorganic N availability also show evidence of higher C transfer from the host plant,
EM fungi will have a higher fungal biomass and therefore will retain a larger amount of
ecosystem N. This would imply that higher C allocation from the tree to EM fungal species
could decrease N availability for free-living saprotrophs and non-EM plant species creating a
positive feedback loop contributing to increased abundance of Oreomunnea mexicana in sites
with lower inorganic N availability. This finding provides further evidence that the formation of
Oreomunnea dominated forest is facilitated by its associated EM fungi.
Implications of EM dominated tropical montane forest for N cycling and soil C storage
Tropical forests play a critical role in the global C cycle. They account for almost 40
% of global net primary productivity, store approximately 25 % of global biomass C, and
contribute 10 % of global soil C (Post et al. 1982, Townsend et al. 2011). Consequently, the
impact of global environmental change on tropical forest ecosystem function and feedbacks on
biogeochemical cycling is are important aspects of climate regulation at regional and global
scales this century (Malhi et al. 2008).
In general, the presence of EM fungi has been associated with reduced litter
decomposition rates and increased accumulation of soil organic matter due to strong competition
between EM fungi and the community of free-living saprotrophs for N (Averill et al. 2014). This
has been amply demonstrated in temperate forest, and has been termed the “mycorrhizal-
associated nutrient economy” where AM and EM dominated forest differ significantly in their
nitrogen, phosphorus, and carbon cycling (Phillips et al. 2013, Brzostek et al. 2015, Rosling et al.
2016). Here we provide evidence that indicates that the “mycorrhizal-associated nutrient
economy” model can also be applied to tropical montane forest. Here we demonstrate that soils
under Oreomunnea mexicana monodominant forest have lower inorganic N than those under
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adjacent AM-dominated forest. At a pan-tropical scale, Oreomunnea mexicana, and other
tropical monodominant EM trees such as Quercus spp, are predicted to have a significant impact
on global C storage.
Future directions on tropical ectomycorrhizal research
Understanding the ecology of complex inter-kingdom interactions like mycorrhizas
will contribute to advancing the frontiers of knowledge in biological science. EM associations
are the living interface between plants and soils and therefore play an important role in
regulating nutrient cycles in many forests (Alexander and Lee 2005). It is becoming increasingly
clear that EM fungal species exhibit inter- and intrageneric variation in their morphology and
physiology (Courty et al. 2010). Understanding fungal functional traits, including response and
effect traits, is essential to predicting the influence of fungi on ecosystem processes (Koide et al.
2014). In particular, EM fungal functional traits may have a strong influence on terrestrial C
cycling (Averill et al. 2014, Tresseder et al. 2012). Studies of how EM fungal community
composition and functional traits are coupled with soil C storage in tropical forest may therefore
improve predictions for C emissions in tropical forest and provide insights into how to manage
tropical forests for C sequestration.
The influence of Oreomunnea mexicana on nutrient cycling is likely context-dependent
and factors such as soil fertility, precipitation, temperature, and fungal community composition
may all be important. Because montane tropical forests are under increasing anthropogenic
pressure and are forecast to be strongly impacted by climate change, it is critical that we study
how these changes will impact the EM fungal communities associated with Oreomunnea and
other Juglandaceae and understand the role of both plants and fungi in nutrient cycling. Future
research should build on this work in tropical forest to further integrate fungal community
dynamics with ecosystem properties at larger spatial and temporal scales. Research should also
focus on understanding how shifts in fungal community composition caused by natural
environmental variation, climate change, or N deposition could differentially affect fungal taxa
with different functional traits, and as a consequence influence soil biogeochemical cycling.
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Analysis of the isotopic composition of Russula fruiting bodies was shown here to be a
useful tool for understanding the role of EM fungal communities on ecosystem processes. Future
studies focusing on tropical EM fungal species exploration types coupled with 15N labeling
experiments should be done to corroborate the soil depth at which EM fungal species forage for
nutrients and it associations with their exploration types and mantle properties. Functional trait
databases should be enriched with more information on tropical species to improve our ability to
associate species composition with ecosystem function.
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