university of hawaii · information to users this manuscript has been reproduced from the microfilm...
Post on 09-Aug-2020
4 Views
Preview:
TRANSCRIPT
INFORMATION TO USERS
This manuscript has been reproduced from the microfilm master. UMI
films the text directly from the original or copy submitted. Thus, some
thesis and dissertation copies are in typewriter face, while others may
be from any type of computer printer.
The quality of this reproduction is dependent upon the quality of thecopy submitted. Broken or indistinct print, colored or poor qualityillustrations and photographs, print bleedthrough, substandard margins,
and improper alignment can adversely affect reproduction.
In the unlikely. event that the author did not send UMI a complete
manuscript and there are missing pages, these will be noted. Also, ifunauthorized copyright material had to be removed, a note will indicate
the deletion.
Oversize materials (e.g., maps, drawings, charts) are reproduced by
sectioning the original, beginning at the upper left-hand comer and
continuing from left to right in equal sections with small overlaps. Each
original is also photographed in one exposure and is included in
reduced form at the back of the book.
Photographs included in the original manuscript have been reproducedxerographically in this copy. Higher quality 6" x 9" black and white
photographic prints are available for any photographs or illustrations
appearing in this copy for an additional charge. Contact UMI directly
to order.
University Microfilms InternationalA Bell & Howell Information Company
300 North Zeeb Road. Ann Arbor. Mr48106·1346 USA313,761·4700 800:521-0600
Order Number 9506224
Speciation, species boundaries, and the population biology ofIndo-west Pacific butterflyfishes (Chaetodontidae)
McMillan, William Owen, Ph.D.
University of Hawaii, 1994
V-M·I300 N. Zeeb Rd.Ann Arbor, MI 48106
SPECIATION, SPECIES BOUNDARIES, AND THE POPULATION BIOLOGY
OF INDO-WEST PACIFIC BUTTERFLYFISHES (CHAETODONTIDAE)
A DISSERTATION SUBMITTED TO THE GRADUATE DNISION OF THEUNIVERSITY OF HAWAIl IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
IN
ZOOLOGY
AUGUST 1994
By
W. Owen McMillan
Dissertation Committee:
Stephen R. Palumbi, ChairpersonE. Alison Kay
Leonard A. FreedChris Simon
Rebecca L. Cann
This dissertation is dedicated to Valerie W. McMillan - my wife and travel
companion. Off we go. Cheers .
iv
ACKNOWLEDGMENTS
This work would not have been possible without the help of many
people. I would especially like to acknowledge the support of Valerie
McMillan, without whom this research would not have been nearly as
enjoyable. Members of my dissertation committee, Becky Cann, Lenny Freed,
Alison Kay, Chris Simon, and especially my chairman and mentor, Steve
Palumbi, provided enlightened direction and discussions throughout the
evolution of this work. My lab mates, Bailey Kessing, Andrew Martin, Ed
Metz, Sandra Romano, Paul Armstrong, Frank Cipriano, Gail Grabowski, and
Tom Duda, always kept things fun. Added thanks go to Frank Cipriano, Ed
Metz, Carol Reeb, George Roderick and Sandra Romano for somehow
managing to read through my many spelling and grammatical errors to
provide thoughtful comments on earlier drafts of some of these chapters.
Richard Pyle proved an invaluable resource both as collector and
photographer and as someone who was always willing to talk about fishes,
IWP biogeography, and speciation. Thanks for collecting specimens go to C.
V. Dinar, Jane Culp, John Godwin, Randy Kosaki, Friedhelm Krupp, Jeff
Mahon, Tony Nahacky, and Larry Sharron. Lastly, Lee Weigt helped greatly
with gathering and analyzing the allozyme data. Financial support from the
Research Corporation of the University of Hawaii, Achievement Rewards for
College Scientists, Sigma Xi, The American Museum of Natural History, and
NSF (#DEB-9106870 and #DEB-92-24071) is greatly appreciated.
v
ABSTRACT
This study uses the pattern of nucleotide substitutions within two regions
of the mitochondrial genome to construct a phylog~netic and demographic
framework to explore species formation in allopatric groups of Indo-west Pacific
(IWP) butterflyfishes (Chaetodontidae). Strong parallels in the evolutionary
history of these groups, as inferred from substitutions within a 495 base pair (bp)
section of the mitochondrial cytochrome b gene (cyt b), suggest a link between
their formation and Pleistocene climatic fluctuations, and highlight a recent
period of significant demographic and evolutionary change across the IWP.
Variation within the hypervariable proline tRNA (tRNApro) end of
the mitochondrial control-region within the three Pacific species of one of
these groups reveals a rich history of demographic change marked by the
rapid coalesced of mtDNA variation into three major lineages followed by a
wave of demographic expansion. However, only one of these lineages shows
any species specificity. This lineage is confined to the Hawaiian
archipelago/Johnston Island endemic Chaetodon multicinctus. The
remaining two lineages are geographically widespread and are composed of
similar numbers of C. pelewensis and C. punctatofasciatus. This patterning of
mtDNA variation supports the genetic distinctiveness of the Hawaiian
species but argues for extraordinarily high levels of hybridization between C.
pelewensis and C. punctatofasciatus. Similar conclusions are suggested by the
distribution of allozyme variation among species.
The greater genetic differentiation of C. multicinctus is paralleled by
marked behavioral isolation. By contrast, in controlled pairing experiments,
C. pelewensis and C. punctatofasciatus do not pair assortatively. Field
VI
observations suggest that a broad hybrid zone joins the two species. In
localities across this zone, intermediately colored individuals are common
and pairing is random with respect to color pattern.
Despite being tightly coupled reproductively and evolutionary, C.
pelewensis and C. punctatofasciatus remain uncoupled phenotypically. Color
pattern differences change over a much narrower range than expected given the
complete homogenization of mtDNA and allozyme variation. Selection directly
on color pattern must be maintaining phenotypic integrity of these two color
variants while most other genes move freely across "species" boundaries.
However the nature of this selections remains enigmatic.
vii
TABLE OF CONTENTS
Acknowledgments v
Abstract vi
List of Tables x
List of Figures .. xi
Introduction 1
Chapter 1: Concordant Evolutionary Paterns Among Indo-west PacificButterflyfishes............................................................................................ 9
1.1 Abstract 9
1.2 Introduction 11
1.3 Materials and Methods 15
1.4 Results 20
1.5 Discussion......... 25
Chapter 2: Contrasting Patterns of Phenotypic and MtDNA VariationAmong Recently Differentiated Butterflyfishes (FamilyChaetodontidae).. 48
2.1 Abstract 48
2.2 Introduction 50
2.3 Materials and Methods 54
2.4 Results : 60
2.5 Discussion.. 69
Chapter 3: Random Mating and Species Boundaries in AllopatricCoral Reef Fishes................................................................................... 108
3.1 Abstract 108
3.2 Introduction 110
3.3 Materials and Methods 114
3.4 Results 120
3.5 Discussion 126
viii
Table1.1
1.2
1.3
1.4
2.1
2.2
2.3
2.4
2.5
3.1
3.2
3.3
3.4
LIST OF TABLES
Number of transitions and transversions in themitochondrial cyt b gene 34
Polymorphic nucleotide positions in the cyt b gene 35
Average within- and between-species levels ofmtDNA variation in species of butterflyfishes 37
Hierarchical structure within cyt b trees 39
Pattern of nucleotide substitution along thetRNAPro end of the control-region butterflyfishes 81
Pattern of variation in C. multicinctus, C.pelewensis and C. punctatofasciatus using a 500 bppiece of the cyt b gene and a 195 bp piece of thetRNAPro end of the control-region 82
Observed and expected number of basesubstitutions under a Poisson and negativebinomial model of substitution in the tRNAProend of the control-region of butterflyfishes 83
Average percent difference within and among thethree control-region lineages in C. multicinctus,C. pelewensis and C. punctatofasciatus 84
Number of individuals of C. multicinctus, C.pelewensis and C. punctatofasciatus with each ofthe three major mtDNA lineages 85
List of museum specimens 139
Number of different pairing experiments 140
Number of hetero- and conspecific pairs 141
Summary of controlled pairing experiments 142
x
Figure.
1.1
1.2
1.3
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
UST OF FIGURES
Geographic distribution of members of the a)"punctatofasciatus" and b) "rhombochaetodon"species-groups ~ 40
Cytochrome b phylogeny of some subgenera withinChaetodon 43
Cytochrome b phylogeny for individuals within a)the "punctatofasciatus" and b)"rhombochaetodon" species-groups 45
Plate of Chaetodon punctatofasciatus, C. pelewensisand C. multicinctus 87
PCR primers used to amplify the control-region ofbutterflyfishes 89
Neighbor-joining tree for 38 individuals of C.punctatofasciatus, C. pelewensis and C.multicinctus using a) a 195 bp portion of thecontrol-region and b) a 500 bp portion of the cyt Bgene 91
Rate of change in the tRNAPro end of the controlregion relative to the rate of change within the cytb gene in butterflyfishes 94
Average number of changes along a 195 bp segmentof the tRNAPro end of the control-region 96
Neighboring-joining cartoon of the three majorcontrol-region lineages within C.punctatofasciatus, C. ~lewensis and C.multicinctus 98
Pairwise differences among individuals in clades Aand B 103
Pairwise differences among individuals in each ofthe three major control-region lineages 105
xi
2.9 The proportion of the three major control-regionlineages across the Pacific 106
2.10 Hetero-specific and conspecific branch lengthcomparison 107
3.1 The three hybrid phenotypic classes 143
3.2 Color plates of the fishes collected in the SolomonIslands and Papua New Guinea 145
3.3 The geographic distribution of the five phenotypicclasses 156
3.4 Results of conspecific pairing experiments 158
3.5 Proportion of the five phenotype classes that werepaired and unpaired in the Solomon Islands 159
3.6 The observed and expected distribution pairs in theSolomon Islands 160
3.7 Pairing of "pure" C. punctatofasciatus typeindividuals in Papua New Guinea 161
3.8 Change in the proportion of "pure" C. pelewensisphenotypes across the Pacific relative to thechange in the proportion of mtDNA variation 162
xii
INTRODUCTION
Background
Understanding the process by which new species arise remains a
central question in biology. A key to untangling the origins of biodiversity
lies in gaining a solid understanding of the evolutionary history of groups of
closely related taxa. Only when viewed within a strong historical context can
factors associated with speciation become clear.
In this respect, there is a growing consensus that genetic data,
particularly DNA sequence data, may provide some of the keenest insights
into the history and mechanisms of speciation (Avise et. al., 1989). DNA
variation can be decomposed into neutral and non-neutral sites and
probabilistic models of nucleotide substitution can be used to determine the
phylogenetic relationships among organisms (Hillis and Moritz, 1990). This
approach can be applied at several different levels. Above the species level,
differences and similarities along a portion of DNA can be used to reconstruct
the pattern of branching events that gave rise to extant taxa. A similar
approach can be applied below the species-level. In this case, genetic changes
can be used to reconstruct the history of individuals that make up a species or
group of closely related species (Avise et aI., 1987). Examination of the
patterning of molecular variation at both levels, particularly when focusing
on newly formed groups, can yield rich insights into the history of species
formation.
Molecular analysis of genetic variation focusing at these two levels has
already greatly contributed to our current understanding of the process of
speciation (see Otte and Endler, 1989). The historical perspective gained from
these studies, coupled with an understanding of behavior, ecology, and
1
biogeography, has helped solidify many or our current paradigms of
speciation. However, most of the paradigms have been generated from
research on terrestrial organisms possessing restricted dispersal capabilities
(OUe and Endler, 1989). Relatively little critical focus has been directed at
speciation in organisms with high dispersal potential. This is especially true
in the marine realm, where taxa often possess vast ranges, huge population
sizes and extended dispersal capabilities due to long-lived planktonic larval
stages (Palumbi, 1992; Knowlton and Jackson, 1993). Classical allopatric
models predict that organisms with these types of population dynamics
should to be buffered from speciation, yet recent paleontological and genetic
research has exposed the speed at which evolutionary change can occur in
these groups (Kohn, 1990; Palumbi and Metz, 1991; Allmon et al., 1993;
Jackson et al., 1993; Kay, 1994). As a result, it is important to begin to augment
studies of speciation in low dispersal species with similar studies on high
dispersal animals (Palumbi, 1992).
Examination of speciation in the Indo-west Pacific Butterflyfishes
The above rational provides the intellectual foundation upon which
this dissertation research is built. By using the pattern of DNA sequence
variation within regions of mitochondrial DNA (mtDNA), I construct an
evolutionary and demographic framework within which to explore
speciation in Indo-west Pacific (IWP) marine taxa. I have taken a hierarchical
approach to this subject, beginning first with phylogenetic analysis above the
species level. I use the conclusions drawn from this initial phylogenetic work
to frame further finer-scale genetic analysis of newly formed species.
The IWP is an extraordinary area in which to blend information on
biogeography, ecology, and behavior with analysis of genetic variation to
2
explore the process of marine speciation. This region, spanning from the East
coast of Africa across the Indian ocean and into the Pacific to the archipelagoes
of Oceania, is the most species-rich of the marine biogeographic provinces.
Despite long standing interest in this diversity, there is little consensus as to
the evolutionary mechanisms that have given rise to the extraordinary
number of species across this region (Connell, 1978; Rosen, 1988). At the heart
of this problem is the difficulty in understanding how populations with long
lived planktonic larval stages phases can become separated long enough to
diverge into new species. In this respect, the challenges facing biogeographers
working in the IWP can be generalized to all biologist attempting to
understand diversification in taxa, such as wind pollinated plants, migratory
birds, or even ballooning spiders, that have the potential for the wide
dispersal of gametes or offspring.
I have focused on speciation in IWP butterflyfishes (Chaetodontidae).
This family of vividly colored fishes is common on coral reefs around the
world and has been the subject of a wealth of ecological, systematic,
biogeographic, and phylogenetic research (Reese, 1975; 1991; Burgess, 1978;
Anderson et al. 1981; Hourigan, 1987;Tricus, 1987; Blum, 1988, 1989; Leis,
1989). Thus, there exist a solid biological background upon which to frame
genetic results. Larvae of these fishes spend between 40 and 60 days feeding in
the plankton before settling and metamorphosing (Hourigan and Reese, 1987;
Leis, 1989). The ecology of juveniles and adults is intricately tied to coral
reefs, with many butterflyfishes feeding exclusively on corals or coral
associate invertebrates.
My research primarily targets two "species-groups" of the more than 90
IWP butterflyfishes. Both groups are composed of morphologically similar
3
species that differ primarily by color pattern. Each partitions the IWP among
four largely allopatric species. Similar allopatric distributions are common
among many apparently closely-related IWP marine taxa.
Whether or not the generalized biogeographic pattern of closely
related, yet allopatric, species reflects chance or has some underlying
deterministic explanation is the subject of the first part of my dissertation
research. Chapter 1 begins with an overview of the challenges facing
researchers attempting to understand the origins of high IWP diversity and
suggests that genetic analysis of newly formed groups may provide additional
insights into evolution in this region. To support this claim, I use sequence
variation within a 500 base pair (bp) portion of the mitochondrial cytochrome
B gene (cyt B) to reconstruct the evolutionary history of these two
butterflyfish groups. The two groups show remarkable parallels in the
pattern and the timing of differentiation. Because it is unlikely that such a
strong similarity would arise by chance in independently evolving lineages,
this concordance argues that speciation in these groups was influenced by
common environmental factors. In this case, the very low levels of genetic
differences suggest a link between differentiation and Pleistocene sea level
fluctuations.
Clearly, much additional work on the tempo of evolution is necessary
to gauge the effects of Pleistocene changes on biodiversity in this region.
However, the congruent pattern of rapid and recent diversification in these
butterflyfish groups joins a growing body of paleontological and genetic work
highlighting a recent period of significant demographic and evolutionary
change in this region (Crarne, 1987; Pauley, 1990; Kahn, 1990; Palumbi and
Metz, 1991; Kay, 1994). These results challenge earlier notions linking the
4
extraordinary diversity of this region to long-term stable environmental and
physical conditions (Ekman, 1953; Briggs, 1974). Pleistocene climatic changes
are thought to have profoundly affected patterns of biodiversity in other
marine faunal provinces (Allmon et al., 1993; Jackson et al., 1993). Future
ecological and biogeographic work within the IWP must be tempered with
the understanding that the very recent history of this region has, likewise,
been extremely turbulent.
Chapter 2 details the finer scale distribution of mtDNA variation
within the three Pacific species of one of these buttertlyfish species-groups.
The previous chapter ended with the suggestion that the recent history of the
Pacific has been notably dynamic. The goal of further research was to provide
a high resolution demographic framework in which to explore speciation in
this region in much greater detail. Indeed, the genealogical pattern of
mtDNA variation within Chaetodon multicinctus, C. punctatofasciatus, and
C. pelewensis traces a rich history of demographic change. MtDNA variation
coalesced into three distinct lineages, or groups of individuals that share a
common ancestor, early in the history of this group. This period of loss of
mtDNA variation was followed by a period of population expansion in which
mtDNA variation within each of the three lineages was buffered from
extinction.
Yet, only one of the three lineages shows any species-specificity with
the other two composed of individuals from different species. For this group,
the failure of the mlDNA gene tree to reflect species boundaries argues for
hybridization and introgression of mtDNA. For the Hawaiian endemic, C.
multicinctus, levels of hybridization suggested by the mtDNA data are low.
By contrast, between the south Pacific, C. pelewensis and the western Pacific,
5
C. punctatofasciatus, the mtDNA genealogy suggests extraordinary high levels
of hybridization associated with the complete homogenization of mtDNA
variation.
The conclusions about the evolutionary and genetic cohesion of these
species based on patterning of variation in the cytoplasmically inherited
mitochondrial genome extend to nuclear encoded loci. The distribution of
allozyme variation among these butterflyfishes mirrors the pattern of .
mtDNA variation. Chaetodon multicinctus is again genetically the most
distinct. Populations of C. pelewensis and C. punctatofasciatus separated by
7500 kilometers show no significant differences in the distribution of
allozyme variation.
My dissertation ends by more thoroughly dissecting the nature of
species boundaries and species-defining color pattern differences in this
group. Controlled pairing experiments indicate that the degree of genetic
differentiation parallels the degree of behavioral, and presumably
reproductive, isolation among species. In these experiments C. multicinctus
shows both a higher degree of assortative pairing and a significant reduction
of intra-sexual aggression relative to conspecific trials. In contrast, pairing
between C. pelewensis and C. punctatofasciatus appears to be random and
behavioral interactions among males of the two species are identical to the
interaction among conspecific males. There is no compelling evidence to
suggest that these two species have achieved the evolutionary cohesion to
merit their species-level distinction.
Observations in zones of sympatry between these two species reaffirm
this conclusion. A broad, approximately 3000 kilometer, hybrid zone joins
the range of these two species. In this zone, a large proportion of the
6
population is composed of individuals with color patterns intermediate
between the two parental forms. Moreover, in Papua New Guinea and in the
Solomon Islands pairing among pure parental and hybrid phenotypes is
random with respect to color pattern.
Despite being tightly coupled reproductively and genetically, C.
pelewensis and C. punctatofasciatus, none-the-less, remain uncoupled
phenotypically. Color pattern changes much more abruptly than expected
given the random distribution mtDNA and allozyme variation between the
two species. The obvious implication of the strong geographic structure to
phenotypic variation in the absence mtDNA and allozyme differentiation is
that selection is preventing the erosion of color pattern in the presence of
potentially homogenizing levels of gene flow.
However, the nature of selection on color pattern in these fishes
remains enigmatic. The evolutionary and ecological significance of the vivid,
non-cryptic coloration in butterflyfishes, as in most reef fishes, is poorly
understood (Lorenz, 1967; Peterman, 1971; Brockman, 1973; Potts, 1973;
Ehrlich et al., 1977; Allen, 1980; Kelly and Hourigan, 1983; Nuedecker, 1989).
The higher degree of behavioral and genetic isolation in C. multicinctus
suggests that divergence in color pattern can be paralleled by the evolution of
assortative pairing either through species recognition or mate selection. Yet,
the random pairing among C. punctatofasciatus and C. pelewensis argues that
color pattern differences are not providing similar cues in these two species.
Though neither the field observations nor controlled pairing experiments
reveal all the forces underlying pair-formation in these species, they clearly
suggest that mate choice or species recognition based on color pattern alone
are not playing a strong role. It therefore seems unlikely that the original
7
divergence of color pattern in this group was coupled with strong inter-sexual
selection or pressure for species recognition.
It is possible that color pattern in these butterflyfishes evolved under
intra-sexual social selection (sensu, West-Eberhard, 1983) or as an aposomatic
warning. Whatever the reason, selection preventing the breakdown of color
pattern between these two species must be strong to explain the discrepancies
between the distribution of genetic and phenotypic variation. Future
ecological work should help to distinguish between these alternatives.
8
CHAPTER 1
Concordant Evolutionary Patterns Among Indo-west Pacific Butterflyfishes
1.1. ABSTRACT
The tropical Indo-West Pacific (IWP) is the most diverse of the marine
biogeographic provinces. Despite considerable interest in the mechanisms
generating this extraordinary diversity, the lack of a good fossil record and little
information on the phylogenetic relationships among closely related species
have made it difficult to distinguish among competing scenarios for the origins
of new species in this region. A molecular phylogenetic approach to
biogeography may help illuminate the evolutionary process in the IWP by
providing a more detailed picture of the evolutionary relationships among
closely related species, induding an approximate timing of divergence, than
provided by earlier studies. In this study, we report strong parallels in the
evolutionary history of two monophyletic species groups of IWP butterflyfishes
(Family Chaetodontidae) as inferred from a 495 base pair (bp) section of the
mitochondrial cytochrome b gene (cyt b). An approximately 2.0% genetic break
within both groups clearly partitions individuals between the Indian Ocean and
Pacific Ocean species. The Pacific Ocean individuals of both complexes fall
within well-defined monophyletic clades. Genetic differences within the Pacific
clade are low, on average less than 1.0%, and fail to cluster by species boundaries.
Individuals from different species, separated by thousands of kilometers, often
9
possess identical cyt b sequences, whereas conspecifics from the same reefs can
show up to 1.5% difference. Failure of the mtDNA gene tree and species tree to
match could be the result of recent hybridization or may simply reflect shared
ancestral variation.
The striking concordance in phylogenetic patterns of two independent
species groups suggests that genetic differentiation was influenced by common
environmental factors. The very low levels of within- and between-species
genetic differences imply a recent divergence time and suggest a link between the
differentiation of these groups and Pleistocene climatic fluctuations. These
results paint a turbulent picture of the recent evolutionary history of the IWP.
Keywords: Indo-West Pacific, marine fishes, historical biogeography
Chaetodontidae, cytochrome b, mtDNA, PCR, speciation.
10
1.2. INTRODUCTION
The explorer-naturalists of the 19th century were awed by the
extraordinary richness of the tropical Indo-West Pacific (IWP) marine
environment. This region, extending from East Africa across the Indian ocean to
the archipelagoes of the central Pacific, is home to over 500 species of reef
building corals, 4000 species of gastropods, and 4000 species of fishes (Abbott, 1982;
Springer, 1982; Veron, 1986). The diversity of coral reef communities across this
region is rivaled only by the diversity of tropical rain forests (Connell, 1978).
Despite over a century of description and maps of the distribution of
species, the evolutionary mechanisms underlying the origins of high IWP
diversity remain unclear (Rosen, 1988). The long-lived planktonic larval stage of
many IWP marine organisms injects a potential for gene flow and dispersal that
makes it difficult to conceptualize the process by which marine populations
become isolated and ultimately diverge into new species (Valentine and
Jablonski, 1983). Indeed, the two patterns that most characterize IWP
biogeography highlight the difficulties facing researchers attempting to
understand species formation in this region. On one hand, there is a striking
attenuation of species diversity as one moves from the shallow seas surrounding
Indonesia, New Guinea, and the Philippines outward across the Pacific (and to a
lesser extent the Indian ocean), suggesting that long-lived planktonic larval
stages cannot completely overcome strong barriers to dispersal (Verrneij, 1987).
On the other hand, many IWP species' are distributed continuously from East
Africa to Polynesia and Hawaii, over a quarter of the earth's circumference,
underscoring the capacity of larval dispersal to achieve vast geographic ranges
(Ekman, 1953). Overlaid on these broad biogeographic patterns is the observation
that for angelfishes (Pisces: Pomacanthidae), damselfishes (Pisces:
11
Pomacentridae) and cone shells (Mollusca: Gastropoda), the geographic range of a
species is not clearly correlated with the length of larval life (Thresher and
Brothers, 1985; Perron and Kohn, 1985; Thresher et al., 1989; however see Reaka,
1980).
Attempts to understand the origins of IWP biodive~sity are further
hampered by the scarcity of information on the evolutionary history of closely
related species in this region (Rosen, 1988; however see Kohn, 1990; Kay, 1994).
The fossil record is at best patchy, and for most taxa is non-existent. Those
groups that do fossilize well often lack good taxonomic characters making
phylogenetic inference difficult (Rosen, 1988). A molecular-phylogenetic
approach to biogeography should help illuminate evolutionary processes in this
region by providing homologous characters that can be used in reconstructing
phylogenetic relationships among closely related species and, given assumptions
about the rate of change of these characters over time, provide a rough
approximation of the timing of speciation.
Here we examine genetic distances and phylogenetic relationships within
species groups of IWP butterflyfishes (family Chaetodontidae). Butterflyfishes
are common and conspicuous members of coral reefs around the world (Burgess,
1978). After a planktonic larval stage of 40-60 days, larvae settle on coral reefs
where they remain for the duration of their lives (Hourigan and Reese, 1987;
Leis, 1989). Their adult ecology is intricately tied to coral reef ecosystems, with
many species feeding exclusively on coral tissue or coral-associated invertebrates.
They have a long history of association with coral reefs stretching at least as far
back as the Eocene (Choat and Bellwood; 1991). Presently the family consists of
120 species, over 90% of which are restricted to the IWP (Blum, 1989).
12
We have specifically targeted two, the "punctatofasciatus" and
"rhombochaetodon", species groups within the subgenus Exornator. Although
Blum's (1988) morphological analysis failed to resolve the relationships among
22 species that comprise this subgenus, the monophyly of each group was
supported in osteological and color pattern features. These species groups have a
remarkably similar biogeographic pattern. Each is composed of four species,
differing primarily by color pattern, that carve the IWP into large non
overlapping allopatric ranges (Burgess, 1978; Allen, 1980; Blum, 1989) (figures
1.1a and 1.1b). This biogeographic pattern, in which related species or species
pairs partition the IWP, is evident in many IWP taxa (see Springer, 1982;
Donaldson, 1986; McManus, 1985; Blum, 1989). For example, Blum (1989)
identified 31 monophyletic species groups of IWP Butterflyfishes, of which 18
were allopatric or marginally sympatric,
In terrestrial and freshwater habitats, concordance in biogeographic
patterns among independent taxonomic groups is thought to reflect the action of
past vicariant events (Croizat et al., 1974; Platnick and Nelson, 1978; Cracraft and
Prum, 1988). However, within marine environments, the broad dispersal
capabilities of many organisms can obscure areas of endemism and make past
biogeographic barriers less obvious. As a result, the relationship between
biogeographic patterns and historical processes are much less clear (Reid, 1990).
For example, although species in both the "rhombochaetodon" and
"punctatofasciatus" species groups have a similar patterns of large non
overlapping geographic ranges, only the species-break at the Indonesian
archipelago is concordant between the two groups.
The two species groups partition the Pacific and Indian Oceans differently.
The "punctatofasciatus" group divides the Pacific among three species: a
13
Hawaiian/Johnson Island endemic, a western-Pacific species whose range
extends across Micronesia as far east as the Marshall Islands, and a south-Pacific
species whose range extends from the Solomon Islands to the Tuamotu and
Marquesas island groups of the eastern South Pacific. In contrast, the
"rhombochaetodon" complex partitions the tropical Pacific among two species.
Again there is a western-Pacific representative; however, the range of this species
does not extend beyond the Philippines. In this group, the range of the "South
Pacific" representative extends over much of the tropical Pacific north of the
equator (see figures l.la and 1.1b).
It is unclear if the differences in biogeographic patterns reflect underlying
differences in the history of speciation or indicate differences in the history of
dispersal following speciation in these groups. This study uses genetic
differences in a large section of the mitochondrial cytochrome b (cyt b) gene to
answer two questions about the biogeographic and phylogenetic patterns between
these groups. First, did both species groups arise contemporaneously or does the
pattern of divergence among species in each group suggest that they arose over
different time scales? Secondly, if they did arise contemporaneously, did they
form in parallel or are the phylogenetic patterns random with respect to
biogeographic patterns?
14
1.3. MATERIALS AND METHODS
To gauge both within- and between-species levels of genetic differences,
we determined the DNA sequence of a 495 base pair (bp) portion of the
mitochondrial cyt b gene from 6-15 individuals of each of the four species of the
"rhombochaetodon" and "punctatofasciatus" species groups. When possible, we
chose individuals to reflect a broad sample of a species across its geographic
range. Species and collection localities were as follows: for the
"rhombochaetodon" species group, C. paucifasciatus (Eilat, Israel, n=7), C.
madagascariensis (Mauritius Islands, n=7), C. xanthurus (Bali, Indonesia, n=3;
Philippines, n=4) and C. mertensii (Guam, n=4: American Samoa, n=4; Tahiti,
n=I), and for the "punctatofasciatus" species group, C. muIticinctus (Hawaii,
n=15); C. punctatofasciatus (Bali, Indonesia, n=7; Philippines, n=3; Guam, n=6), C.
pelewensis (Moorea, Tahiti, n=9; Paga Pago, American Samoa, n=2) and C.
guttatissimus (Mauritius Islands, n=6). In addition, this cyt b region was
sequenced for a single C. auriga, C. miliaris, C. tinkeri, C. burgessi. C. declivis, and
C. flavocoronatus, and four C. argentatus.
All fishes were collected between 1990 and 1992. Individuals were either
frozen at _700
C or preserved in 95% ethanol. With the exception of the
Philippine samples, all collections were made by WOM or by a biologist working
in the area. Individuals from the Philippines were acquired through the tropical
fish trade directly from a distributor.
Genomic preparations, peR amplification, and sequencing
For each fish, approximately 0.5 grams of gill or muscle tissue was
homogenized in 2 ml of cold grinding buffer (O.2M NaCl/0.05M EDTA, pH 8.0).
Tissue stored in 95% ethanol was soaked in 5 ml of grinding buffer on ice for
approximately 1 hour before grinding. Approximately 0.5 ml of this homogenate
15
was transferred into a 1.5 ml microcentrifuge tube. Sodium dodecyl sulfate (SDS)
and proteinase K (Sigma Chemicals) were added to the homogenate to a final
concentration of 1%(v iv) and 20 ug/rnl, respectively. Following an overnight
incubation at 50° C, this solution was extracted twice with equal volumes of
buffered phenol, once with an equal volume of phenol/chloroform/isoamyl
alcohol (25:24:1) solution, and once with an equal volume of
chloroform/isoamyl alcohol (24:1). DNA was recovered by cold-ethanol
precipitation (Sambrook et al., 1989). The resulting pellet was dissolved in 100
200 III of IX TE, and stored at _20° C until further use.
Approximately 100 ng of total cellular DNA was symmetrically amplified
during 40 cycles in a Perkin Elmer Cetus DNA Thermal Cycler in a 100111 reaction
volume with 1.5 units of Taq DNA polymerase (Perkin Elmer Cetus). DNA
primers used to initiate amplification and their position relative to the human
mtDNA sequence were (5'-ATTCTAACTGGACTATTCCTTGCC-3') (14881) and
(5'-ATTATCTGGGTCTCCGAA(C/T)AGGTT-3') (15490) (Anderson et al., 1981).
The standard thermal cycle profile was: 1) 45 seconds at 94° C, 2) 45 seconds at 55°
C and 3) 45 seconds at 72° C. Following amplification, the double stranded
product was collected by ethanol precipitation, resuspended in 10111 sterile water,
and electrophoresed through a 1% agarose/0.5X TAE gel. The resulting 609 bp
band was cut from the 1.0% agarose/O.5X TAE gel, Gene Cleaned(© biolab 101),
and resuspended in 7-30 III TE. Seven microliters of this product was used as
template for double-stranded sequencing (Palumbi et al., 1991). Both PCR
primers and an internal primer, (5'-GTAGTACTTCTCCT(C/T)CTAGTTAT-3'),
were used to initiate sequencing reactions. These reactions were carried out
using reagents and DNA polymerase enzyme provided in the Sequenase® 2.0
DNA sequencing kit (USB). Sequencing reactions were electrophoresed through
16
6-8% polyacrylamide/7M urea gels with a buffer density gradient, dried under
vacuum at 80° C, and exposed to autoradiographic film for 24-72 hours. Refer to
Palumbi et al. (1991) for the finer details of template preparation, sequencing, and
electrophoresis.
In general, only a single strand was sequenced per individual. However,
in the process of filling in sequence gaps, different PCR reactions were used.
Although this strategy does not eliminate sequencing artifacts produced by errors
in replication during peR amplification, it provided some internal check on the
DNA sequences obtained.
Sequence analysis
Sequences were aligned by eye to the published human cyt b sequence.
Phylogenetic analysis was performed with both parsimony and neighbor-joining
methods using the phylogenetic package PAUP and MEGA (3.03, Swofford, 1993;
1.01, Kumar et. al, 1993). The large number of taxa made parsimony analysis of
all possible topologies impossible. As a result, a subset of possible trees was
evaluated using the heuristic search setting on PAUP (Swofford, 1993). The tree
bisection and reconnection (TBR) branch swapping algorithm was chosen to
search for optimal trees within this framework. For parsimony analyses,
transversions were weighted 10 times more than transitions. This weighting
scheme was consistent with previous conclusions about the pattern of molecular
evolution in the cyt b gene (Irwin et al., 1991). The pairwise genetic distance
matrix used in neighbor-joining analyses were generated using a Kimura two
parameter model (Kimura, 1980). Trees constructed from distance matrices
estimated by maximum-likelihood, Tamura's distance method and Tamura and
Nei's distance method were identical (Felsenstein, 1993; Tamura, 1992; Tamura
and Nei, 1993).
17
Phylogenetic trees were rooted using aligned sequences from several
additional IWP chaetodontids from the subgenus Exornator~ miliaris), as well
as subgenera Roaops~ tinkeri, C. burgessi, C. declivis, C. flavocoronatus) and
Rabdophorus~ auriga). Of these three subgenera, Blum's (1988) cladistic
analysis of Chaetodon placed Rabdophorus at the most basal position. As a
result, the cyt b sequence for C. auriga was used to root phylogenetic trees
examining the relationships among subgenera. For these trees, a single cyt b
sequence was chosen as the representative for each species. For those species in
which more than one individual was examined, the most common cyt b
sequence was chosen to represent that species. This convention did not alter our
interpretation of the phylogenetic patterns but simplified graphic representation.
The relative strengths of major nodes within phylogenetic trees were
examined by bootstrap re-sampling the original data matrix 500 times
(Felsenstein,1985). Bootstrapped data sets were examined by distance methods
using the phylogenetic packages MEGA, and where possible by parsimony
analysis using PAUP (Swofford, 1993; Kumar et al., 1993).
We used the randomization procedure described by Faith (1991) and log
likelihood difference tests to evaluate alternative phylogenetic hypotheses
(Kishino and Hasegawa, 1989; PHYLIP 3.5, Felsenstein, 1993). The log-likelihood
differences test examines the mean and variance of log-likelihood differences
between alternative trees across all sites. If the observed difference in log
likelihoods was greater than 1.96 standard deviations then two trees were
considered significantly different. In Faith's (1991) procedure, the observed
18
differences in length between alternative phylogenies were compared to
differences obtained by randomly permuting the original data matrix 500 times
(program courtesy of G. Roderick, University of Hawaii, Honolulu, HI). In these
comparisons, the sequence of the outgroup taxa was not randomized.
19
1.4. RESULTS
Between-group variation
Over 90% of the 126 variable sites among the 15 Chaetodon species
examined occurred at the third position of a codon (appendix A). Corrected
genetic differences between species ranged from below 1.0% among members of
the "punctatofasciatus", "rhombochaetodon" and "tinkeri" species groups to
slightly greater than 17% for differences between C. auriga (subgenus
Rabdophorus) and members of both subgenera Roaops and Exornator (table 1.1).
Nearly all of the DNA substitutions occurring between species were transitions at
either 2- or 4-fold degenerate sites (appendix A). Transition/ transversion ratios
ranged from greater than 38:1 to less than 4:1 (table 1.1). The smaller ratios
occurred between the most divergent individuals and can undoubtedly be
attributed to multiple hits at the same nucleotide position (Desalle et al., 1987).
The actual transition/transversion ratio for the cyt b gene of these fishes probably
lies closer to the higher value, and similar to the 10:1 to 20:1 ratio reported in
mammals (Irwin et al., 1991). A total of only 7 replacement substitutions were
observed among the sample of Chaetodon species depicted in figure 1.2.
Phylogenetic analysis of these species clearly supports the monophyly of
both the "punctatofasciatus" and "rhombochaetodon" species groups (figure 1.2).
Branches leading to both complexes were well supported in bootstrap
replications and were defined by at least 11 substitutions using a 10:1 transition to
transversion weighting. This analysis, however, did not support the inclusion of
C. argentatus within the "rhombochaetodon" species group as previously
suggested by Blum (1989). Instead, this species fell between members of the
subgenus Roaops and the members of the "rhombochaetodon" species group.
Parsimony analysis grouped C. argentatus with members of the subgenus Roaops
20
(figure 1.2). In contrast, the neighbor-joining distance tree grouped C. argentatus
with members of the "rhombochaetodon" complex. Neither of these groupings
well supported by bootstrap analysis. This phylogenetic pattern suggests that the
subgenus Exornator, which includes both "rhombochaetodon" and
"punctatofasciatus" species complexes, is not a monophyletic group.
Within-group variation
Genetic differences among individuals were low and similar in both
"punctatofasciatus" and "rhombochaetodon" species groups. A total of 42
polymorphic nucleotide positions were observed within the "punctatofasciatus"
group, and 27 within the "rhombochaetodon" group, yielding a total of 32 and 18
unique cyt b sequences in each, respectively (table 1.2a and 1.2b). All save five of
these substitutions were silent transitions at the .third position of the codon. Of
these five, four were guanine to adenine transitions occurring at the first
position of the codon resulting in conservative valine to isoleucine amino acid
substitutions (French and Robson, 1983). The only transversion occurred at the
third position of a four-fold degenerate codon for a single C. pelewensis. The
maximum genetic difference observed in the 48 individuals of the
"punctatofasciatus" group was 3.2%, slightly lower than the 3.9% among the 31
individuals of the "rhombochaetodon" group.
Within-group variation was partitioned similarly in the two species
groups. In both, the majority of the within-group variation was partitioned
between the Pacific and Indian ocean basins. Both groups showed an
approximately 2.0% (calculated over all sites and corrected for within-species
variation) genetic break between species from each basin (tables l.3a and 1.3b).
For example, C. punctatofasciatus, C. pelewensis and C. multicinctus differ from
the Indian Ocean C. guttatissirnus by 1.7-2.0%. Similarly, within the
21
"rhombochaetodon" species group, the Pacific species, C. mertensii and C.
xanthurus, differed from the Indian Ocean C. madagascariensis by 2.3-2.6% and
from the Red Sea endemic, C. paucifasciatus, by 1.7-1.9% corrected sequence
divergence (table 1.3b).
The Indian/Pacific Ocean genetic break is clearly evident from the
phylogenetic relationships among individuals within each complex (figures 1.3a
and 1.3b). In these trees, the Pacific individuals of both groups cluster together
into a monophyletic group supported at the 0.005 level by topological dependent
tests (table 1.4) (Faith, 1991). Within the "punctatofasciatus" complex, there was a
distinct Indian Ocean/Pacific bifurcation. Individuals in each basin formed
monophyletic clusters supported in 70% and 81% of bootstrap replicates (figure
1.3a). The phylogenetic pattern within the "rhombochaetodon" species group
was slightly different. Within this group, the monophyletic Pacific clade was
rooted within the Indian Ocean group. Thus, the Indian Ocean species, C.
madagascariensis, possessed the most ancestral mitochondrial genotypes,
followed by the Red Sea endemic, C. paucifasciatus, and lastly the Pacific clade
containing C. mertensii and C. xanthurus (figure 1.3b). It was unclear if this
topology reflects the true evolutionary history of this group, the limited number
of phylogenetically informative sites, or stochastic variation caused by the
random assortment of mtDNA variation since differentiation. However, the
branching order of these groups was poorly supported in bootstrapped
replications. Neither log-likelihood nor randomization tests could reject the
hypothesis that, as in the "punctatofasciatus" species group, an initial bifurcation
divided this group into Indian and Pacific forms with subsequent differentiation
further partitioning each ocean (Kishino and Hasegawa, 1989; Faith, 1991;
Felsenstein, 1993) .
22
Genetic variation within ocean basins was much smaller than the 2%
difference observed between ocean basins. Chaetodon madagascariensis and C.
paucifasciatus , the Indian Ocean and Red Sea members of the
"rhombochaetodon" group differed by only 0.85% (corrected sequence
divergence) (table 1.3b). Despite the small genetic difference, these two species
were clearly genetically distinct. Levels of genetic variation within each species
were less than half of the corrected genetic differences between species (table
1.3b). In addition, with the exception of one C. madagascariensis possessing what
appears to be an ancestral haplotype, individuals of the two species grouped
together within the same region of the phylogenetic tree (figure 1.3b).
Similarly slight levels of genetic differences define the Pacific species of
both complexes. In these cases, however, the relationships among species were
more complicated. Average pairwise genetic diversity among all Pacific
individuals was only 0.89% in the "punctatofasciatus" species group and only
0.61 % among Pacific individuals of the "rhombochaetodon" group. Levels of
within-species genetic variation were similar to the corrected genetic differences
between species (tables 1.3a and 1.3b). Indeed, some individuals from different
species, collected thousands of kilometers apart, had identical cyt b sequences.
For example, the most common sequence, found in 6 of the 17 Pacific
individuals of the "rhombochaetodon" complex, occurred in individuals
sampled from Indonesia to American Samoa. Within the "punctatofasciatus"
complex, individuals possessing identical cyt b sequences were found in Hawaii,
Tahiti, Guam, and Indonesia, effectively across the entire West-Pacific Ocean.
Furthermore, the phylogenetic relationships among the Pacific individuals
examined were distinctly polyphyletic (figures 1.3a and 1.3b). This phylogenetic
pattern was not a function of the limited resolution of the cyt b data. Distinct
23
lineages are defined, albeit by few substitutions, within the Pacific. However,
with the exception of a single C. multicinctus specific clustering, these lineages
contain individuals from different species (figures l.3a and 1.3b). Trees
constrained to be monophyletic were significantly longer than the shortest
length non-constrained trees (p<O.OOl)(table 1.4).
24
1.5. DISCUSSION
The long-lived planktonic larval stage of many marine organisms allows
for the wide geographic dispersal of gametes that make it difficult to
conceptualize the processes by which populations become isolated and diverge
into new species (Mayr, 1954; Valentine and Jablonski, 1983). A central question
in the evolution of high IWP diversity, and marine diversity in general, is the
role that chance versus deterministic forces has played in species formation (see
Valentine and Jablonski, 1983). The nature of the IWP, with its many
archipelagoes separated by large expanses of open ocean, has led to the suggestion
that species form most often when dispersal events isolate small founding
populations (Mayr, 1954; Ladd, 1960; Valentine and Jablonski, 1983; [okiel, 1990).
Other biogeographers have argued that species formation is much less random.
Populations become isolated when geological or environmental changes erect
barriers that interrupt the pattern of gene flow across the range of a previously
well mixed ancestral population (Springer, 1982, 1988; Woodland, 1983, 1986;
McManus, 1985; Donaldson, 1986; Hocutt, 1987). The rise of the Isthmus of
Panama 3.5 million years ago is the most striking example of such an extrinsic
barrier in the marine environment (Bermingham and Lessios, 1993). However,
biogeographic barriers need not be so obvious. Changes in the spatial
distribution of habitat or alterations in current patterns can erect more subtle, yet
equally profound, divisions within marine populations (Reeb et al., 1990; Avise,
1993).
Similar levels of within-group cyt b sequence differences suggest that both
the "punctatofasciatus" and the "rhombochaetodon" species-complexes arose
contemporaneously. Moreover, the two groups show a remarkable degree of
phylogenetic concordance which includes a 2.0% genetic break between the
25
Indian and Pacific ocean and evidence for more recent differentiation within
ocean basins (figures 1.3a and 1.3b). This is especially true within the Pacific,
where very small differences among individuals highlight a recent period of
rapid species formation. In-so-much as it is unlikely that such a strong temporal
and phylogenetic concordance between independent species groups would arise
by chance, this result argues that similar historical events may underlie the
formation of both groups. This concordance provides the most compelling
support to date for the role that vicariant events have played in the evolutionary
history of this region (Croizat et al., 1974; Platnick and Nelson, 1978;
Bermingham and Avise, 1986; Cracraft and Prum, 1988; however see also, Endler,
1982).
The Indo-West Pacific has had a complicated geological and climatic
history marked by tectonic plate movement and collisions, volcanism, and
periodic sea level changes (Audley-Charles, 1981; Charlton, 1986; Chesner, 1991;
Acharyya and Basu, 1993). The central Indo-Malay region of highest species
diversity is formed by the confluence of 4 of the earth's tectonic plates (Audley
Charles, 1981). Several vicariant hypotheses have linked the generation of new
species within this region to the movement of these plates over time (Rotondo
et al., 1981; Springer, 1982, 1988;Woodland, 1986;Hocutt, 1987). Others, by
contrast, suggest that speciation has been much more recent, and argue that
changes in sea level associated with Pleistocene periods of global cooling isolated
regions across the IWP sparking a recent wave of species formation (Woodland,
1983, 1986; McManus, 1985; Donaldson, 1986).
The timing of speciation
Because the actual rate of cyt b evolution in butterflyfishes is unclear, we
can only suggest a rough temporal framework for speciation based on rate
26
estimates in taxa with good fossil records. These estimates have varied from
approximately 1.0% per million years (averaged over all sites) in sharks to
approximately 2.5% per million years in primates and ungulates (Brown et. aI.,
198~; Irwin et aI., 1991; Martin et al., 1992). The observed correlation between
metabolic rate and rates of molecular evolution suggests that the rate of cyt b
evolution in these fishes falls somewhere between the two (Martin and Palumbi,
1993). The oxygen consumption of 10-gram coral-feeding damselfish
Plectotroglyphidodon johnstonianus at rest is less than that an adult human at
rest (0.35 mg02/g/hr versus 0.31 mg02/g/hr), but still greater than the oxygen
consumption of a swimming lemon shark (0.28 mg02/g/hr) (Altmann and
Dittmer, 1968; Bushnell et al., 1989; Gochfeld, 1991).
Using this range for the rate of cyt b evolution suggests that butterflyfish
species groups diverged from each other 5-12 mya. Evolution within each group
has been much more recent, beginning with an initial differentiation between
the Indian and Pacific Oceans basins that occurred between 0.8-2.0 mya.
However, placing the divergence of these species within a clear temporal
framework is complicated. Random genetic drift of ancestral mtDNA variation
may be obscuring the timing of species formation, or the true branching patterns
in these species groups. For example individuals of the Red Sea endemic, c.
paucifasciatus, are on average 1.20% different from individuals of C.
madagascariensis (table 1.3). Correcting for within-species levels of genetic
differences suggest that these two species may have diverged between 340,000 and
850,000 thousand years ago (Wilson et al., 1985). However, this time scale may be
an over-estimation. This is because a mitochondrial gene genealogy does not
necessarily trace a species' history, which begins with the origin of barriers to
reproduction (Neigel and Avise, 1986; Palmilo and Nei, 1988). It is possible that
27
emerging species may have been fixed for very different ancestral mitochondrial
lineages. Because these lineages predate the isolation event our estimate of the
timing of divergence will be inflated. In addition, because lineages may not
reflect the actual pattern of species branching, the assortment of ancestral
variation may obscure the phylogenetic re.lationships among species. This is
especially true when a number of species diverge from each other over a short
period of time. Indeed, the odd phylogenetic position of the Red Sea endemic, C.
paucifasciatus, intermediate between Pacific and Indian Ocean species, argues
that random assortment of ancestral lineages may be obscuring the "true"
evolutionary history of these newly formed species groups.
Ancestral mitochondrial variation may be similarly obscuring the timing
of speciation and evolutionary relationships among the Pacific species of both
complexes. Within the Pacific, identical cyt b sequences are distributed broadly
and shared between recognized species (figures 1.3a and l.3b). Presently, it is
unclear if this phylogenetic pattern reflects the history of speciation or a history
of hybridization among species that have not evolved strong reproductive
barriers. The discrepancy between the mtDNA gene tree and the species
designations may reflect the incomplete sorting of ancestral variation in very
recently formed species (Neigel and Avise, 1986; Palmilo and Nei, 1988; Morin
and Kornberg, 1993). For neutral genes, polyphyly is predicted when the time
since speciation is short relative to the long-term effective population size
(Neigel and Avise, 1986). Assuming a generation time of 2 years in these
butterflyfishes and strict maternal inheritance of the mitochondrial genome, the
observed levels of within-ocean genetic variation suggests that the three species
of the "punctatofasciatus" complex shared a common maternal ancestor between
263,000 and 868,000 years (Tajima, 1983; Tricas , 1986). A similar time to common
28
ancestry is suggested for the two Pacific species of the "rhombochaetodon"
complex. Thus, populations maintaining a historical effective female
population size of only 132,000 to 434,000 individuals would be expected to share
mitochondrial lineages over this time period (see Neigel and Avise, 1986).
Given the demographic properties of these fishes, including enormous ranges,
high fecundities, and large present-day population sizes, even greater historical
effective population sizes would seem reasonable (however see Avise et aI.,
1988). For example, C. multicinctus is one of the most common butterflyfishes
on reefs around Hawaii and on Johnston Atoll. Females spawn year round, with
a peak from mid-May through mid-October, producing an estimated 200,000
300,000eggs annually (Reese, 1991).
If this scenario is correct, then the timing of speciation would be much
more recent than the 263,000-868,000 years suggested by the coalescence of
mtDNA variation. Traits, such as color pattern in butterflyfishes, that are
important in the social ecology of a species can coalesce rapidly under strong
social or sexual selection and are more likely to reflect the actual species
genealogy (Reese, 1975;West-Eberhard, 1983).
Alternatively, hybridization and the introgression of mtDNA genes could
potentially generate the observed discrepancy between the mtDNA gene tree and
species designations in the Pacific species of both groups (Takahata and Slatkin,
1984). The presence of a distinct C. guttatissimus sequence in a C.
punctatofasciatus collected on the Indian Ocean side of Indonesia suggests that
hybridization between species may take place in areas where they overlap (also
see Randall et al., 1977). Distinguishing between these alternatives, especially
when species have formed recently, is notoriously difficult (Slatkin, 1989).
None-the-less, the conclusion that the Pacific species of both groups formed
29
recently is supported by the low intra-ocean levels of mtDNA variation and by
slight nuclear genetic differences (Nei's D values ranging between 0.001 and 0.04)
among these species at allozyme loci (Appendix C).
Despite the difficult in reconstructing the evolutionary history of these
species groups from the history of mtDNA variation, the low levels of mtDNA
variation clearly place the origins of both species groups within the Pleistocene.
This temporal framework is an order of magnitude more recent than expected
from models linking IWP diversification to the movement of tectonic plates.
However, this timing is predicted by vicariant models focusing on recent
physical changes in this region associated wi~h Pleistocene sea level fluctuations.
Pleistocene changes and the origins of these species groups
As many as seven times during the past two million years, drops in sea
level of 100-150 meters stranded much of the shallow water shelf environment
of the IWP, restricting connections among the numerous ocean basins that
comprise this region (Porter, 1989). Although only the Red Sea was completely
isolated during lowered sea stands, concomitant changes in coral reef habitat and
ocean circulation patterns are speculated to have generally disrupted gene flow
patterns across the IWP. This is particularly true between the Indian and Pacific
ocean basins, where increased temperature and decreased salinity in the Banda
Sea are envisioned to have aided the separation of these two basins (Fleminger,
1986). Today, the ranges of a number of species and subspecies pairs from a broad
array of marine taxa abut at or near the Indonesian archipelago (Allen, 1980;
Woodland, 1983; McManus, 1985; Blum, 1989). For example, within
butterflyfishes,9 of the 31 species complexes show species or sub-species
distinctions in this area (Blum, 1989). A similar biogeographic pattern is evident
within rabbitfishes (Family Siganidae; Woodland.. 1983), copepods (Labidocers:
30
Undinula, Fleminger, 1986), anemonefishes (Amphiprion; Allen, 1972), and
hermit crabs (Uca; Crane, 1975). These patterns led Woodland (1983) to suggest
two focal centers of diversification: an Indian Ocean focus and another in the
Pacific Ocean. Unlike previous biogeographers (see Ekman, 1953; Briggs, 1974),
Woodland attributed the extraordinary high species diversity within the shallow
waters surrounding Indonesia, the Philippines and New Guinea to the
confluence of these separate biotas. The phylogenetic patterns within these two
butterflyfish complexes are consistent with this explanation. A similar 2.0% total
mtDNA genetic break between Indian and Pacific ocean populations of the
coconut crab, Birgus latro, underscores the broad role that this barrier played in
the historical biogeography of this region (Lavery, 1993).
It is much less clear what role sea level fluctuations may have played in
the rapid diversification within the Pacific. Unlike the physical barriers created
between the Red Sea, and the Indian Ocean and the Indian and Pacific Oceans,
drops in sea level do not obviously partition this region. It is possible that
Pleistocene sea level regressions may have acted subtly to subdivide the Pacific.
Today, the predominately westward-flowing surface currents of the tropical
Pacific are frequently interrupted by El Nino/Southern Oscillation (ENSO)
events. These current anomalies profoundly alter the direction and speed of
surface currents across this region, providing a potent mechanism for the
eastward transport of planktonic larvae (Richmond, 1990; Vermeij, 1990).
However, paleoclimate data suggest that wind patterns were more intense and
equatorial productivity higher during glacial maximums than during interglacial
periods (Molina-Cruz, 1977; Pedersen, 1983; Janecek and Rea, 1985; Chuey et al.,
1987; Rea, 1991; but see Rea, 1990). These data have led to the suggestion that the
westward flowing surface currents were stronger and ENSO events less frequent
31
during glacial maximums (Quinn, 1971). Therefore during glacial maximums, it
is possible that genetic exchange between the western Pacific and archipelagoes of
the eastern Pacific may have been interrupted long enough for new species to
form. Pollock (1992) also focused on possible alterations and intensification of
surface current patterns during glacial maximums to explain the apparent post
Pliocene speciation in the spiny lobster genus Panuliris.
Similar patterns of recent and rapid diversification within the Pacific is
emerging from genetic examination of other groups. The four Pacific members
of a third species group of Butterflyfishes, the "tinkeri" complex, likewise, show
slight differences «1.0%) at the 495 bp portion of the mitochondrial cyt b (figure
1.2). Similarly, the sea urchin Echinometra mathaei, previously thought to be a
single IWP species (see Mayr, 1954), has recently been shown to be composed of at
least four species which have all apparently arisen within the last 2.0 million
years (Uehara et al., 1986; Palumbi and Metz, 1991; Palumbi, 1992). These
emerging patterns suggest that whatever events led to the rapid differentiation of
these two butterflyfish groups in the Pacific, may have acted more broadly
affecting many taxa across this region. Additional work on paleo-oceanographic
and paleoclimatic conditions can only help clarify the relationship between
physical and oceanographic changes across the Pacific and diversification.
Conclusions
The evolutionary mechanisms underlying the extraordinary diversity of
the IWP are complex, reflecting an interplay between life history, adult ecology,
and historical events. Much additional work on the tempo and patterns of
evolution across the IWP is necessary to gauge the effects of recent historical
changes on biodiversity in this region. At the very least, the congruent patterns
of rapid and recent diversification in these groups highlights a turbulent period
32
in the history of this region. These results challenge the notion that the
extraordinary diversity of this region reflects long-term stable environmental
and physical conditions across this region (Ekman, 1953; Briggs, 1974). Profound
evolutionary changes associated with Pleistocene climate fluctuations have
recently been proposed for the temperate Atlantic and the Caribbean marine
faunas (Stanley, 1981; Jackson et al., 1993; Allmon et al., 1993). However, unlike
these other regions, extinction has apparently not played a significant role in the
recent history of the IWP (Vermeij, 1987). Indeed, the phylogenetic pattern
within these butterflyfishes, inferred from mtDNA sequences, suggests that this
region may have been characterized by extraordinary bursts of species formation.
A similar conclusion is emerging from the fossil record, which shows a rapid
increase in diversity of a number of IWP mollusks since the Miocene and
Pliocene, much of which may well have occurred within the Pleistocene (Kohn,
1990; Kay, 1994).
Significant demographic changes in IWP taxa, including both local
extinction and speciation, were probably associated with the most recent low
sea level stand which ended less than 10,000 years ago (Crame, 1987; Porter,
1989; Pauley, 1990; Springer, 1992). Future ecological and biogeographic work
in this region must be tempered with the understanding that the recent
history of this region has been tempestuous.
33
Table 1.1: Number of transitions/transversion (above diagonal) and genetic distances (below diagonal) DNA
substitutions in a 495 pb segment of the mitochondrial cytochrome b gene. Genetic distances were corrected for
multiple hits at the same nucleotide position using the Kimura 2-parameter model (Kimura, 1980) in which
tranversions were weighted 10 times transitions. See Appendix A for sequences.
2 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
~
1. C.tinklll
2. TYPE1
3. TYPE2
4. TYPE3
5. TYPE4
6. C.mer/C.xan
7. C.mer/C.xan
8. C.mertensii
9. C.madaagas.
10. C.paucifas.
11. C.argentatus
12. C.miliaris
13. C.multi.
14. cmu,Cpu,Cpe
15. C.punctato.
16. C.pelewensis
17. C.guttat.
18. C.auriga
•1.2
0.8
1.2
0.9
8.5
8 0
8 0
8.3
8 7
8.4
12.5
11.2
12.0
11.5
12.0
12.2
17 3
5/0
•0.2
0.5
0.7
8.7
8.4
7.8
8.1
8.7
7.2
11.7
10.7
11.5
10.7
11.2
11.2
16.9
4/0
1/0
0.2
0.5
8.1
7.6
7.5
7.8
8.3
8.0
11.5
10.8
11.5
11.0
11.5
11.8
16.2
5/0
2/0
I/O
•0.7
8.7
8.4
7.8
8.1
8.7
7.2
11.7
10.7
11.5
10.7
11.2
11.2
16.2
4/0
3/0
2/0
3/0
•9.0
8.7
8.0
8.4
8.9
7.5
12.0
10.4
11.2
10.4
11.0
11. 0
17.1
37/2
33/2
35/2
33/2
34/2
•0.4
1.7
2.7
1.9
8.7
14.1
12.2
13 .0
12.5
13.0
13.7
15.6
35/2
32/2
33/2
32/2
33/2
2/0
•1.7
2.7
1.9
8.7
13.6
12.2
13.0
12.5
13 .0
13.7
15.6
34/2
29/2
32/2
29/2
30/2
8/0
8/0
•1.9
1.5
8.1
14.5
12.5
13.3
12.8
13.3
13.5
16.2
35/2
30/2
33/2
30/2
31/2
12/0
12/0
8/0
1.2
7.5
13.9
11.9
13 .5
12.7
13.3
13 .5
15.9
38/2
33/2
36/2
33/2
34/2
9/0
9/0
7/0
5/0
7.5
13 .9
12.5
13.7
13.2
13.7
14.0
16.1
37/0
28/0
35/0
28/0
29/0
36/2
36/2
33/2
30/2
31/2
•12.4
13 .4
14.5
14.0
14.5
13.7
16.5
45/7
39/7
41/7
39/6
40/6
51/7
49/7
51/7
49/7
50/7
42/7
•10.9
10.9
10.9
10.4
10.4
17 .4
43/7
35/7
41/7
35/7
34/7
47/7
47/7
47/7
45/7
48/7
49/7
40/6
•1.4
0.6
1.0
2.5
17.6
46/7 44/7
38/7 35/7
44/7 42/7
38/7 35/7
37/7 34/7
50/7 48/7
50/7 48/7
50/7 48/7
50/7 48/7
53/7 51/7
53/7 51/7
40/6 40/6
7/0 3/0
• 4/0
0.8 •
0.4 0.4
2.3' 1.9
17.9 17.3
46/7
37/7
44/7
37/7
36/7
50/7
50/7
50/7
50/7
53/7
53/7
38/6
5/0
2/0
2/0
•1.9
17.9
47/7
37/7
45/7
37/7
36/7
53/7
53/7
51/7
51/7
54/7
50/7
38/6
12/0
11/0
9/0
9/0
•17.6
57/15
49/13
53/15
47113
50/13
53/13
53/13
54/13
53/13
55/13
51/15
53/15
57/16
58/16
56/16
58/16
57/16
•
Table 1.2: Species designation, collection site, and polymorphic nucleotide positions within a 495 bp portion of the mtDNA
cytochrome b gene region from a) the 33 individuals representing the four species that comprise the "punctatofasciatus" complex and
b) the 48 individuals representing the four species that comprise the "rhombochaetodon" species complex. In this table, dots indicate
positions that are identical to the most common sequence within each complex. Polymorphic sites highlighted in bold-face occur at
the first position of the codon. Missing nucleotide information is denoted by a"?".
A. The "punctatofasciatus" complex:1 1 1 1 111 122 2 2 222 2 ~ 2 3 3 3 3 3 3 3 3 3 444 4 4 4 4 4
2 4 6 8 9 9 2 2 3 567 8 901 3 4 677 8 B 9 0 0 122 4 7 8 9 0 1 1 3 4 5596 7 8 9 1 0 3 0 9 5 625 3 3 164 3 709 2 3 103 8 1 422 1 6 2 1 7 8 7 092
Species LocationC. rnul, C. pel, C. pun HI, Ta., Sa., Guam, Indo.(4) C C T Tee A CAT A A a T 0 A G A A T T Teo eTC C A A T T eGA eGA T C G CC. rnulticinctus Hawaii · · · · · · • A • • G • · · · · · · · · · · • G • • T • · · · • CC. mullicinetus Hawaii(2) · · · · · · · · · · · · · • C • · • T • · · · · · · · · · · · · • TC. multicinetus Hawaii(5) · · · · · · · · · · · • G • · · · · · · · · • G • · • T • · · CC. multieinelus Hawaii · · · · · · · · · · · • G • · · · · · · • G • · • T • · · • G CC. multicinelus Hawaii · · · · · · · · · ·· · · ? · · · · · · · · • G • · • T • · · · • C •
~ c. multicinctus Hawaii · · · · · · · · · · • A G • · · · · · · · · · • G • • T • · · · • CC. multicinctus Hawaii · · · · · · · · · · • G • • G • · · · · · · ? G • · • T • · · · • CC. rnult'cinctus Hawaii · · · · · · · · · · · • G • · · · · · · · · • G • · • T • · • A • CC. multicinctus Hawaii · · · · · · • G • · · · · · · · · • G • · • TC. punctatofasciatus Guam · · · · · · · · · · · · · · · · · · · · · · · · · • C • · · · · · · · ?C. punctatofasciatus Guam · · · · · · · · · · · · • G • · · · · • T • · · · · · · • T •C r\;netalofascialus Guam · • C • • T G • G • G • · · • G • · · · · · • T • · · · • A •C. punelalofascialus Guam · · · • T • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ?C. punctatofasciatus Guam · · · • T · · · · · · · · · · · · • C • • T • · · · · ·C. punelatofasciatus Philippines · · · · · · · • GA· · · · · · · · · · · · • T • · · · · · · · ?C. punctatofasciatus Philippines · · · · · · · · · · · • C • · • T • · · · · · • TC. punctatofasciatus Philippines · · · · · · · · · · · · · · · · · · · • GC. punctatofasciatus Indonesia T • · · · · • C • G • · • A • • C • · · · · · · · · · · • T A • T ?C. punelalofasciatus Indonesia • T • · · · · · · · · • G •C. punelalofascialus Indonesia · · · · · • T • · · · · · · · · · · · · · · · · · · · · · · · ? ?C. pelewensis Samoa · · · · · · · · · · · • C • · • T • · · · • CC. pelewensis Tahiti · ? · · · · · · · · · · · · · · · · · · · · • T • · · · · · · · · · · · ?C. pelewensis Tahili · · · · · · · · · · · · · · · · ··· · · · · · · · ·· · · · · • AC. pelewensis Tahiti(2) · · · · · · · · · • G • · · · · · · · · • TC. pelewensis Tahiti · · · · · · · · · • G • · · · · · · · · • T • GC. pelewensis Tahiti · · · · · · · • C • G • · · · · · · · · · · · · · • TC. pelewensis Tahiti · · · · · · · · • GA· · · · • A • · · · · • TC. pelewensis Tahiti · · · · · · · • G • · · · · · · · · · • T • · • AC. guttatissimus Mauritius T • · · · · · • C • G • · • A • • C • · · · • C · · · · • T A • T · · • AC. guttatissimus Maurilius(3), Indonesia T • · · · · • C • G • · · • A • • c • · · · · · · · · · • T A • T • · · • AC. gutlalissimus Indonesia T • • C • T • · • C • G • · · • A • , C • · · • C · · · · • T A • T • · • A
Table 1.2: Species designation, collection site, and polymorphic nucleotide positions within a 495 bp portion of the mtDNA cyt b gene
(continued).
B. The "rhombochaeiodon" complex:
Species
e. xan, e. mer.e. mer, e. xan,e. mertensiie. rnertensiiC. xanthuruse. xanthuruse. xanthurusC. madagascariensisC. madagascariensisC. madagascariensisC. madagascariensisC. paucifasciatus
~ C. paucifasciatusC. paucifascialus
Location
Indo., PhiL(3), Guam(2)Guam(2), Samoa(3), Indo.
TahitiSamoa
PhilippinesIndonesiaIndonesiaMauritiusMauritius
Mauritius(4)MauritiusRed SeatS)
Red SeaRed Sea
1 1 1 1 2 2 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 42 7 9 3 5 8 8 1 5 7 900 1 123 5 6 8 0 0 1 1 2 6 7:l 5 9 5 3 0 3 9 504 0 6 5 840 1 6 4 2 8 1 4 020
aCCTTATCCTCCAATGTGTCAACTATTT • . · · · . · · · · · . . . · . • CT • . · · • T • · · · · · · · • T G G T CT • C
A • · · · · · • GT • · . • G? · · · · · • C
T C • G C • T CT. · · · · · • CT. G • C G • CT C • ·C?TCT· · • CA' ACT • G • C G • CT C • • C • T CT' · · · • CT. G • C G • CT C • • C • T C • · · · · · · • CT. G • C G • CT C • · · • T CT' G • · · · · • T • • C • • C
• T T C • · · ? T CT. · · · · • CT. . • C • C• T T C • · · • T C T T • · · · • CT· • C • • C
Table 1.3: Average percent genetic variation within species (boldface, along
diagonal), percent genetic differences between species (d~) uncorrected (below
diagonal) and percent genetic differences between species corrected for within
species genetic variation (above diagonal) of butterflyfishes in the a)
"punctatofasciatus" species complex and b) "rhombochaetodon" species complex.
Uncorrected genetic difference values were calculated from by averaging all
pairwise differences, corrected for multiple substitutions at the same nucleotide
position, among individuals of each species. For corrected values, the average
level of within-species genetic variation was subtracted from the uncorrected
genetic difference values (Wilson et al., 1985). Numbers in parenthesis represent
the number of individuals sampled from each of the eight species.
A. The "punctatofasciatus" species complex(n=48):
PACIFIC INDIANC.multi. C. pmletata. C.pelewensis C. guttatissimus
C. multicinctus (n=15) 0.0060 0.0036 0.0024 0.0201
C. punctatofas. (n=16) 0.0110 0.0089 0.0004 0.0167
C.~Iewensis (ne l l ) 0.0084 0.0078 0.0060 0.0171
C. guttatissimus (n=6) 0.0250 0.0230 0.0220 0.0038
37
Table 1.3: Average percent genetic variation (continued).
B. The "rhombochaetodon" complex (n=31):
PACIFICc. mertensii C xanthurus
INDIANC. rnadagas. C. paucifas.
k mertensii (n=9) 0.0062
C. xanthurus (n=8) 0.0066
C. madagascar. (n=7) 0.0280
C. paucifasciatus (n=7) 0.0220
0.00165
0.0039
0.0300
0.0230
38
0.0234
0.0265
0.0031
.00120
0.0170
0.0191
0.0085
0.0039
Table 1.4: Hierarchical structure within the phylogenetic tree of members of the
"rhombochaetodon" and "punctatofasciatus" complexes. *,.,. represents
significant differences at the 0.005 level between the length of the observed
phylogeny and the length of the shortest length constrained phylogeny.
Significance values were obtained by comparing the observed differences
between two topologies to the difference obtained by randomly permuting the
original data matrix 500 times. In these comparisons the sequence of the
outgroup taxa, indicated by parenthesis, was not randomized.
A. "punctatofasciatus" complex:Indian Ocean species as outgroup to Pacific species <h:.miliaris ): .
Shortest length tree (figure 1.3a):Shortest length non-Indian Ocean outgroup:
Monophyly within Pacific <h:. guttatissimus)Shortest length monophyletic grouping:Shortest length non-monophyletic grouping (figure 1.3a):
B. "rhombochaetodon" complex:Indian Ocean/Red Sea species as outgroup to Pacificspecies <k tinkeri):
Shortest length tree (figure 1.3b):Shortest length non-Indian Ocean/Red Sea outgroup:
Monophyly within Pacific <h:. paucifasciatus ):Shortest length monophyletic grouping:Shortest length non-monophyletic grouping(figure 1.3b):
39
Length
8992***
55***46
6466***
24***18
+N
JohnstonAtoll
~waiianIS.IIdUl1IMarshall Is.
IlitH!I
:••. M~[S'~•...g~~~~~ "~'''~ Pitcairn
Scale (Kilometers) ~l~~~-~~~~=. . ----,
o 2001
"'"o
Figure 1.1a. The geographic distribution of the species within the" punctatofasciatus " species group (Q
; Chaetodon guttatissimus;§ I C. punctatofasciatus £2J ,C. pelewensis; andlITIl , C. multicinctus ).
Shading should be interpreted only as approximate distributions. For site specific distribution see
Blum (989). The @'s represent sampling localities.
+N... ,jiawaiian Is.
"".Johnston Atoll
~~~M~'qeusas Is.
~~~~.F;j;~. sooe~~~Pk<.;,n
Scale (Kilometers)I I Io 200
*'">-'
Figure Llb. The geographic distribution of the species within the" rhoinbochaetodon "species group (Illll, Chaetodon paucifasciatus fSJ ,..k madagascariensis § ,C. xanthurus ; andE1, c. mertensii ).
Shading should be interpreted only as approximate distributions. For site specific distribution see
Blum (1989). The @'s represent sampling localities.
Figure 1.2: Phylogenetic relationships among 15 species from three subgenera
within the large genus Chaetodon. This tree was constructed using parsimony
with C. auriga defined as the outgroup (see Blum, 1988). The neighbor-joining
tree differed only in the placement of C. argentatus, which grouped with the
members of the "rhombochaetodon" species group. The relative strength of
major nodes within this phylogeny is given by the percentage agreement in a
consensus of trees produced from a 500 bootstrap simulations of the data set
(above) and the number informative nucleotide changes (below). The
number of informative nucleotide substitutions is given in both transitional
(listed first) and transversional (listed second) changes .
42
C.declivis wilderi
C.dec1ivis wilderi Roaops100
18C. burgessi
693/2 C. fIavocoronatus
1
C.argentatus I Exornator17
C. mertensii85 1 C.xanthurus
17/2 C.mertensii1 C.xanthurus
C.mertensii Exornator99 C.paucifasciatus11 1
C. madagascar.1
C.miliaris17/3
92
17/2
100
18/3 C. pelewensisa
Exornator
4
~ Rabdophorus29/11L. ~ c. auriga
43
Figure 1.3a: Major phylogenetic relationships within the "punctatofasciatus"
species complex. The tree was constructed using the neighbor-joining
method from pairwise distance matrices estimated by the Kimura two
parameter model (Kimura, 1980). Support for major nodes within these trees
is shown as the percentage agreement in a consensus of trees produced from
500 bootstrap simulations of the data set (above) and the number informative
nucleotide changes (below). Shaded boxes represent clusters of individuals of
the same species with one exception. A C. punctatofasciatus collected from
the Indian Ocean side of Indonesia had the distinct C. guttatissimus genotype
(see table 1.1). Species symbols are as follows: C. punctatofasciatus (@), c.pelewensis (~ ) and C. multicinctus(tI). The tree was rooted with sequences
from C. miliaris and C. tinkeri. Phylogenetic trees constructed using
parsimony were nearly identical. Consistency and retention indexes for these
trees were 0.78 and 0.86. Since all phylogenetically informative substitutions
within groups were transitions, different transition/ transversion weighting
schemes did not alter the branching pattern.
44
(/)
(/) fU ~
::s .... =.... (/) ....- ~ ~ fU....
A. The" punctatofasciatus "complex.... r:: ~ 6 .... ....S 0 - fU0 .... ....- lU e ~fU "'0 .... ::s ..s::~ r:: ..s:: fU lU fU- c.. o 00 f- :t
81C. guttatissimus 5 2
3
Indian
100Pacific
f @ 1Ii () 1
18~ 1
78 @ 15 <t 1
ct 1
o 2
12C. multicinctus
@ 12
11
11
4 1 1 1 11
11
1 ..J.
I I1
0.0 1.0% sequence divergence
45
Figure 1.3b: Major phylogenetic relationships within the
"rhombochaetodon" species complex. This tree was constructed using the
neighbor-joining method from pairwise distance matrices estimated by the
Kimura two-parameter model (Kimura, 1980). Support for major nodes
within these trees is shown as the percentage agreement in a consensus of
trees produced from 500 bootstrap simulations of the data set (above) and the
number informative nucleotide changes (below). Shaded boxes represent
clusters of individuals of the same species. Species symbols are as follows: C.
xanthurus Q), and C. mertensii «(1]). This tree was rooted with sequences
from C. miliaris and C. tinkeri. Trees constructed using parsimony were
identical. Consistency and retention indexes for parsimony trees were 0.76
and 0.84. As in the "punctatofasciatus" species-complex, all phylogenetically
informative substitutions within groups were transitions.
46
lUeo
CIJ ~
lU ~ 0CIJ .... ~
~lU .E CIJ ....QJ ~ Q.,
C/'}....
~ Q., S 0 ....... -::J 0 ....S ....
"'t:S - lUlU "0 .... ::J ..c:
QJ
:E ~ ..c: lU lU
B. The" rhombochaetodon " complex ~ - c.. Co' Cf} E-o
C. madagascariensis 1
99 C. madagascariensis 6~
11
63
3 C. paucifasciatus 7
47
CHAPTER 2
Contrasting Patterns of Phenotypic and MtDNA Variation Among Recently
Differentiated Butterflyfishes (Family Chaetodontidae)
2.1. ABSTRACT
Three very closely related butterflyfishes (Family Chaetodontidae),
differing principally in color pattern, partition the tropical west-Pacific into large
allopatric ranges. Partial sequences from the hypervariable proline tRNA
(tRNAPro) end of the mitochondrial control-region in 138 individuals of these
three species reveal a rich history of demographic change. From a common
ancestor, MtDNA variation coalesced into three major lineages. This period of
rapid coalescence appears to have been followed by a wave of demographic
expansion in which newly arising variation accumulated in all three lineages.
Only one of the three major mtDNA lineages that formed during the early
history of this group shows any species or geographic specificity. This lineage is
confined to the Hawaiian archipelago/Johnston Island endemic Chaetodon
multicinctus. The remaining two lineages are geographically widespread and are
composed of similar numbers of C. pelewensis and C. punctatofasciatus with
occasional individuals of C. multicinctus. However, significant differences in
the distribution of these lineages in populations across the south and western
Pacific defines a broad cline with one lineage dominating populations in the
48
western Pacific and the other lineage more likely to be found in the eastern south
Pacific.
The present genealogical and geographic patterning of mtDNA variation
suggests that color pattern differences that define species likely emerged at the
same time as the three major mtDNA lineages and that hybridization accounts
for the present discrepancy between the three major mtDNA lineages and
taxonomic boundaries. Within this framework, the genetic distinctiveness of
the Hawaiian species indicates very low levels of hybridization with either C.
pelewensis or C. punctatofasciatus, possibly due to greater reproductive or
geographical isolation. Between C. pelewensis and C. punctatofasciatus, the
genealogy of mitochondrial variation argues for extraordinarily high levels of
hybridization (Nm values> 100 individuals per generation) and associated
introgression. Yet, characteristics which define these species, primarily color
pattern, change abruptly at borders of allopatric species ranges. These contrasting
patterns suggest that strong selection is maintaining the cohesion of species
specific color pattern in the presence of potentially homogenizing levels of gene
flow.
Keywords: marine fishes, mitochondrial DNA, control-region, gene tree, species
tree, hybridization, lineage sorting, Tropical Pacific.
49
2.2. INTRODUCTION
Because of its rapid rate of evolution, lack of recombination, and apparent
neutrality, the mitochondrial genome has become an important marker for
tracing the history of closely related species (Wilson et al., 1985; Whittam et al.,
1986; Avise et al., 1987; Harrison, 1989), However, in a number of instances,
discrepancies between the mtDNA gene genealogy and species boundaries
defined by phenotypic characters have been reported (Powell, 1983; Ferris et al.,
1983; Spolsky and Uzzell, 1984, 1986; Lamb and Avise, 1986; Carr et al., 1986;
Ovendon et al., 1987; Baker et al., 1989; Dowling, 1989; Moran and Kornfield,
1993). In some cases, these discrepancies can be attributed to the plasticity of the
characters used to infer taxonomic distinctions. For example, Ball et al., (1988)
attribute much of the observed morphological variation within Red-winged
blackbirds to environmental rather than genetically based differences in plumage
and size characteristics. In other instances, however, discrepancies between
mtDNA genes and "species-defining" phenotypic traits reflect underlying
differences in selective forces operating on different loci. For example,
discrepancies between mtDNA gene trees and species boundaries can arise
through hybridization even when there is strong selection against hybrid
offspring (see Takahata and Slatkin, 1984). Alternatively, such discrepancies can
occur when species have formed very recently. In this case, differences between a
mtDNA gene tree and species-defining traits may reflect differences in the
selective pressures acting during species formation (see Neigel and Avise, 1986;
Palmilo and Nei, 1988). For example, Moran and Kornfield (1993) attributed the
polyphyly of species in the rock-dwelling cichlid fishes of Lake Malawi to the
retention of ancestral mtDNA variation in very recently formed species. In these
fishes, strong selection on morphological and color variation during
50
differentiation apparently caused these traits to coalesce more rapidly than
mtDNA genes.
Rather than being viewed as a limitation of the phylogenetic resolving
power of mtDNA, discrepancies between gene trees and species boundaries can
yield rich insights into the evolutionary process. This is because the distribution
of neutral variants within and between closely related species provides a
powerful demographic framework for interpreting the nature and importance of
inter-specific differences (Neigel and Avise, 1986).
Here we focus on the discrepancy in the pattern of phenotypic and
mtDNA variation in a group of three closely related Pacific Butterflyfishes.
Members of this group of tropical marine fishes are common and conspicuous
inhabitants of coral reef communities around the world (Burgess, 1978; Allen,
1980). The three species, Chaetodon punctatofasciatus, C. pelewensis, and C.
multicinctus, partition the tropical west Pacific into large, nearly allopatric ranges
(see Blum, 1989). Chaetodon punctatofasciatus is found on coral reefs from the
Indonesian archipelago eastwards to the Marshall Islands and New Guinea
where it is replaced by C. pelewensis, whose range continues to the Society
Islands some 5000 kilometers to the east. Chaetodon multicinctus has the most
limited distribution of the three, and is restricted to reefs along the 1500
kilometer Hawaiian archipelago and at Johnson Island (see figure 2.9).
Previous phylogenetic and taxonomic evaluation of this group failed to
find any morphological differences (Burgess, 1978; Blum, 1988). However, these
species differ distinctly from one another in color patterning, Chaetodon
F!!nctatofasciatus and C. pelewensis are the most similar, differing primarily in
the striations along the body, which run vertically in C. punctatofasciatus but
diagonally in C. pelewensis (figure 2.1). Chaetodon multicinctus is more distinct.
51
Its underlying body stripes are similar to C. punctatofasciatus, but it lacks the
striking yellow wash, bright orange splash on the caudal fin, and the yellow eye
band that characterize it's congeners. These color pattern differences are
preserved across the broad geographic range of each species with little overlap
and no evidence for a broad gradient between species patterns (Burgess, 1978;
Allen, 1980; Blum, 1989;). For example, individuals of C. punctatofasciatus
collected from coral reefs around Guam strongly resembled the individuals
collected from reefs in Indonesia some 4000 kilometers to the west. The same
pattern is true for C. pelewensis where our Tahitian, Cook Island and Fijian
collections showed only minor variation around the phenotype depicted in
figure 2.1.
In contrast, mtDNA variation is not partitioned nearly so neatly among
the three species (Chapter 1). The morphological similarity of this group is
paralleled by only slight differences at the mitochondrial cytochrome b gene (cyt
b) which suggest that these species have diverged recently. Levels of genetic
differences in this gene region suggest that all three species diverged from their
Indian ocean sibling, C. guttatissimus, between 1-2 million years ago and from
each other within the last 300-800,000 years. However, within our sample of 41
individuals, cyt b sequences failed to cluster into species-specific lineages and it
was common to find individuals of different species from widely scattered
locations across the Pacific with identical cyt b sequences.
The small sample size and slight genetic differences in the cyt b sequences
limited insights into the reasons for the observed discordance between mtDNA
and phenotypic variation. As a result, we have expanded our original data set to
include sequences from a hypervariable portion of the mitochondrial control
region in 138 individuals from 3-4 populations across the range of each of the
52
three species. The high rate of evolution in the control-region allows us to
distinguish very closely related mtDNA haplotypes and makes it possible to
refine our picture of the genealogical and geographic patterning of mtDNA
variation among these species (Meyer et al., 1990; Vigilant et al., 1991; Wenink et
al, 1993). This high resolution mtDNA gene genealogy provides insights into
the evolutionary and demographic history of this group that are important for
evaluating the selective framework shaping the striking geographical and
phenotypic patterns that define these species.
53
2.3. MATERIALS AND METHODS
Fishes were collected from eight widely scattered localities across the
Pacific between 1990 and 1993. The species and populations sampled were as
follows: for Chaetodon multicinctus, Oahu, Hawaii (n=15), Midway Island
(n=ll), and Johnston Island (n=6); for C. pelewensis, Moorea, Society Islands
(n=9), Rarotonga, Cook Islands (n=18), Pago Pago, American Samoa (n=2), Viti
Levu, Fiji (n=21); and for C. punctatofasciatus, Guam (n=8), Palau (n=20),
Philippines (n=21) and Bali, Indonesia (n=7). In addition, 3 C. guttatissimus, the
Indian Ocean member of this species-group, were also included as an outgroup
(see Blum, 1989). Individuals were either frozen at _700
C or preserved in 95%
ethanol. With the exception of the Philippine and Fiji samples, all collections
were made by WOM or by a biologist working in the area. Individuals from the
Philippines and Fiji were acquired through the tropical fish trade directly from
collectors working in these areas. The collection is currently stored in the _700
C
freezer at the Pacific Biomedical Research Center, Kewalo Marine Lab, Honolulu,
Hawaii and is available upon request. Voucher specimens for each population
are deposited at the Bernice P. Bishop Museum, Honolulu, Hawaii.
Genomic preparations and PCR
For each fish, approximately 0.5 grams of gill or muscle tissue was
homogenized in 2 ml of cold grinding buffer (0.2M NaCl/0.05M EDTA, pH 8.0).
Tissue stored in 95% ethanol was soaked in 5 ml of grinding buffer on ice for
approximately 1 hour before grinding. Approximately 0.5 ml of this homogenate
was transferred into a 1.5 ml micro centrifuge tube. Sodium dodecyl sulfate (SDS)
and proteinase K (Sigma Chemicals) were added to the homogenate to a final
concentration of 1% (v /v) and 20 ~g/ml, respectively. Following an overnight
incubation at 50° C, this solution was extracted twice with equal volumes of
54
buffered phenol, once with an equal volume of phenol/chloroform/isoamyl
alcohol (25:24:1) solution, and lastly with an equal volume of a
chloroform/isoamyl alcohol solution (24:1). DNA was recovered by cold-ethanol
precipitation in the presence of sodium acetate (see Sambrook et aI., 1989). The
resulting pellet was dissolved in 100-200 JlI of sterile IX TE solution.
Approximately 100 ng of total cellular DNA was subjected to 40 cycles in a
Perkin Elmer Cetus DNA Thermal Cycler in a 100JlI reaction volume with 1.5
units of Taq DNA polymerase (Perkin Elmer Cetus). We used a 12s rRNA
primer (12sar) (5'-CATATTAAACCCGAATGATAITf-3') and a Chaetodon
specific control-region (C.R.-I) (5'-ACCATATTATGTACTAGGCAC-3') to
amplify most of the control-region of these fishes (figure 2.2). This primer was
designed from initial sequences obtained from PCR amplifications using the 12s
rRNA primer and a proline tRNA (tRNAPro) primer, (5'
CTACCTCCAACTCCCAAAGC-3') (see Palumbi et al., 1991). The standard
thermal cycle profile was: 1) 45 seconds at 94° C, 2) 45 seconds at 55° C and 3) 45
seconds at 72° C. Following amplification, the double stranded product was
collected by ethanol precipitation, resuspended in 10Jll sterile water, and
electrophoresed through a 1% agarose/O.5X TAE gel. The resulting
approximately 1200 base pair (bp) band was cut from the 1.0% agarose/D.Sx TAE
gel, Gene cleaned (© biolab 101), and resuspended in 7-30 JlI TE. Seven
microliters of this product were used as the template for double-stranded
sequencing reactions (Palumbi et aI., 1991). These reactions were carried out
using the reagents and DNA polymerase enzyme provided in the Sequenase®
2.0 DNA sequencing kit (USB). Sequencing reactions were electrophoresed
through 6-8% polyacrylamide/7M urea gels with a buffer density gradient, dried
under vacuum at 80° C and exposed to autoradiographic film for 24-72 hours.
55
Refer to Palumbi et al. (1991) for the details of template preparation, sequencing,
and electrophoresis.
Phylogenetic analysis
Sequences were aligned by eye and phylogenetic analysis was performed
using both maximum parsimony and Neighbor-joining distance methods
(Saitou and Nei, 1987; Swofford, 1993). For parsimony analysis the large number
of taxa made the evaluation of all possible topologies impractical. As a result, a
subset of possible trees was evaluated using the phylogenetic package PAUP (3.0s,
Swofford, 1993) under the heuristic search setting. The tree bisection and
reconnection (TBR) branch swapping algorithm was chosen to search for optimal
trees within this framework. To determine the effect that
transition/transversion weighting had on parsimony trees, we weighted
transversions 0, 10, or 20 times more than transitions. The genetic distances
between individuals used in the construction of Neighbor-joining trees were
corrected for multiple hits at the same nucleotide position in two ways. For the
cyt b data, we used Kimura's two parameter model (Kimura, 1980). For the
control-region data, we used the gamma correction on Tamura and Nei's (1993)
model to estimate ilij (MEGA, Kumar et al., 1993). This model was developed
specifically for the analysis of sequence data within the control-region where
strong transitional biases, differences in the types of transitional substitutions,
and site to site variation in the rate of substitution have all been observed
(Kocher and Wilson, 1991; Tamura and Nei, 1993; Kumar et al., 1993). For these
distance calculations we used k=0.6. We choose this value based on the fit of
initial control-region sequences to a negative binomial distribution with a
k=O.60. This parameter was estimated by iteration using equation 2.15 of
Southwood (1978):
56
klog(1+x/k)=log(NIno)
where ~ is the mean number of substitutions per site, N is the number of sites,
and llQ is the number of positions where no substitutions occurred. Our
estimated value of Is. was similar to values estimated from the tRNApro end of
the human control-regionfwakeley, 1993). All phylogenetic trees were rooted
using aligned sequences from C. guttatissimus. This species differed by
approximately 2.0% in a 500 bp portion of the mitochondrial cyt b and previous
phylogenetic analysis of a number of Indo-west Pacific butterflyfishes within the
subgenus Exornator identified C. guttatissimus as the most closely related
outgroup (Chapter 1).
The relative strengths of major nodes within our Neighbor-joining tree
were examined by bootstrap resampling of the original data matrix 100 times
using the computer program MEGA (Felsenstein, 1985; Kumar et al., 1993; Hillis
and Bull, 1993). In addition, we used the randomization procedure described by
Faith (1991) to evaluate alternative phylogenetic hypotheses. In this procedure,
the observed differences in length between alternative phylogenies were
compared to differences obtained by randomly permuting the original data
matrix 500 times (program courtesy of G. Roderick, University of Hawaii,
Honolulu, HI). In these comparisons, the sequence of the outgroup taxa was not
randomized.
Population analysis
We explored the spatial distribution of mitochondrial variation using
an analysis of molecular variance (AMOVA) (Excoffier et. aI., 1992) and FST
analyses (Hudson et al., 1992). In our AMOVA analysis we used the absolute
number of sequence differences between individuals as our Euclidean
57
distance. Three components of genetic variation were calculated: among
species, among populations within species, and within populations. These
components were considered to be significantly different from random if the
observed value was less than 95% of the values produced by 500 random
permutations of the squared Euclidean distance matrix (program courtesy of
L. Excoffier, University of Geneva, Carouge, Switzerland). FST values were
calculated as (1-Hw/Hb), where Hw is the average of all pairwise difference
among individuals from the same subpopulation and HQ. is the average of all
pairwise differences among individuals from different subpopulations.
Pairwise differences used in this analysis were corrected for multiple hits at
the same nucleotide position using a Jukes-Cantor model weighted for
differences in transition and transversion rates (Lynch and Crease, 1990). To
gauge the significance of the observed FST value it was compared to 500
randomly generated FST values (program available from S. R. Palumbi).
When the observed value was greater than 95% of the randomly generated
FST values, we concluded that there was significant genetic heterogeneity
between regions. The FST approach is based on an explicit island model of
population genetics and thus permits an estimate of migration between
geographic regions based on the relationship, FST =1/(1 + 2Nm), where N is
the effective number of breeding females within each locality and m is the
migration rate per generation.
We also utilized the phylogeographic method outlined in Slatkin and
Maddison (1989) to estimate the minimum number of migration events, §.,
consistent with a given phylogeny. In our data set, there were many equally
most parsimonious trees and §. was determined by averaging values obtained
from a subset of 500 to 1000 of these trees (see Slatkin and Maddison, 1989).
58
The effective migration rate consistent with our estimated value of §. and the
number of individuals examined was estimated using a computer program
provided by M. Slatkin. To determine if this value represented significant
population differentiation, it was compared to values of §. obtained from 500
randomly generated trees using MacClade (Maddison and Maddison, 1992). If
the observed value of §. was greater than 95% of the values in random trees,
then significant population differentiation between sampling locations was
assumed.
59
2.4. RESULTS
The DNA primers used to initiate PCR reactions routinely amplified an
approximately 1200 bp portion of the mitochondrial genome spanning most of
the control-region (figure 2.2). The 3' end of this piece aligned well with other
published vertebrate 115 rRNA sequences. However, of the 195 bp portion of the
5' end of this piece that we sequenced, we could only confidently align two small
segments to the tRNAPro end of control-region of Cichlids (figure 2.2). The
failure of most of our control-region segment to align was not surprising, the
tRNAPro end has been shown to be the most variable section of the control
region of a variety of vertebrates (Saccone et al., 1991; Meyer et al., 1991; Vigilant
et aI., 1991; Pesole et aI., 1992; Brown et aI., 1993; Wenink et aI., 1993). Cichlids,
which are in the same suborder as butterflyfishes (Percoidei), are more closely
related to Chaetodontids than any other taxa for which the mitochondrial
control-region has been sequenced. Never-the-less, the lineage that gave rise to
the Cichlids and the lineage that gave rise to the Chaetodontids likely diverged
over 80 million years ago (Choat and Bellwood, 1991).
The tRNApro end of the control-region of C. multicinctus, C. pelewensis
and C. punctatofasciatus proved to be hypervariable. One hundred twenty-six of
the 195 nucleotide positions of this A-T rich region were variable, yielding a total
of 130 unique sequences or mitochondrial haplotypes from our sample of 141
individuals. Four of these changes were single base-pair additions and the rest
were single nucleotide substitutions (table 1.1). Genetic differences between
individuals chosen at random, without replacement ranged from 1.6 to over 50%
corrected sequence difference. In these 70 pairwise comparisons, transitions
always out-numbered transversions. However, as many as five transversions
occurred between individuals. To reduce the effects of multiple substitution at
60
the same nucleotide position, we calculated the transition/transversion ratio
using only those randomly chosen pairs in which a single transversion was
observed. This ratio varied from 5:1 to 37:1 in our randomly chosen subsample
and we used the average of these comparisons, 20:1, as our estimation of the
transition/ transversion ratio. Similar high transitional bias has been reported in
the control-region of other vertebrates, and it appears to be a characteristic of
mitochondrial DNA evolution (Aquadro and Greenberg, 1983; Desalle et. al.
1987; Vigilant et al., 1991; Brown et al., 1992).
Cytochrome b/control-region comparison
For 38 of these 141 individuals we had previously sampled a 500 bp
segment of the mitochondrial cyt b gene (Chapter 1). The absolute number of
unique DNA sequences among this subset of individuals was similar in the
two gene regions, with nearly every individual sampled possessing a unique
cyt b and a unique control-region sequence. As a result, diversity measures,
such as the genotype diversity index (Ball et al., 1988), that estimate the
probability that two individuals sampled from a population have different
mitochondrial sequences, were high (~ 0.90) and similar for the two gene
regions (table 1.2). By contrast, absolute levels of sequence differences among
individuals were over an order of magnitude higher in the control-region
than in the cyt b region (table 1.2). For example, average pairwise difference
among these 38 individuals was only 0.85% in the cyt b portion compared to
over 25% within the much smaller control-region segment.
The phylogenetic relationships among individuals inferred from these
two portions of the mitochondrial genome were nearly identical. Both
regions identified three major mitochondrial lineages or clades (figures 2.3a
and 2.3b). In both regions, only clade C was species-specific, containing
61
individuals of the Hawaiian endemic, C. multicinctus. Clade A and B was
composed of individuals from two or more recognized species. For example,
clade B contained multiple C. multicinctus, C. pelewensis and C.
punctatofasciatus. Although these patterns were clear in both the cyt band
control-region data, the higher level of genetic variation within the control
region greatly extends the confidence that can be placed in these phylogenetic
groupings. In the tree constructed with the control-region sequences, lineages
were identified by at least 3, and as many as 13, substitutions and were
supported in over 70% of bootstrap replications (see Hillis and Bull, 1993).
Trees in which species were constrained into monophyletic groups were
nearly 50 steps longer. In contrast, these same groupings were distinguished
by at most a single substitution in the cyt b segment and were supported in
fewer than 40% of bootstrapped data sets. Topological dependent tests of trees
constructed using the cyt b data supported the polyphyly of these species
(p<O.005)(Faith, 1991); however monophyletic alternatives were only three
steps longer.
Although lineages A, B, and C appeared distinct, the phylogenetic
relationships among them was unclear. The particular branching pattern
depicted in figure 2.3a was not well supported in bootstrapped data sets and
was sensitive to the order in which sequences were added and the program
used to construct trees. As a result, these lineages are best regarded as equally
divergent clusters.
Relative rate of control-region evolution
The rate of evolution within this small segment of the control-region
. was extraordinarily high relative to evolution within the larger cyt b segment.
Plotting the difference among pairs of individuals in the control-region
62
against the differences among the same individuals within the cyt b gene
reveals the extraordinary rate at which change accumulates within this small
segment of the control-region (figure 2.4). Genetic differences in the control
region segment rose rapidly relative to changes in the cyt b gene region but
leveled off with increasing divergence suggesting that, despite distance
corrections, multiple substitutions at the same nucleotide position obscured
the true number of substitutions between distantly related pairs. Because
multiple hits were less likely to affect more closely related pairs we used these
pairs to estimate the relationship between change in the control-region and
change in the cyt b gene. For example, those individuals that differed by only
0.2% of their cyt b sequence (1 change out of 500 sampled) differed by, on
average, 8.1% of their control-region sequence. Similarly, those individuals
that differed by 0.4% of their cyt b sequence differed by 9.8% in the control
region, suggesting a relative control-region/cyt b rate of between 25:1 and 40:1.
None-the-less, we used the average of these values, 33:1, as a very crude
approximation of the relative rate of change between the two regions.
Assuming that the cyt b gene in butterflyfishes evolves between the 1.0% per
million years estimated in sharks and 2.5% per million years estimated in
mammals (Martin et al., 1992; Irwin et al., 1991), we estimate a rate of
evolution in this small segment of the control-region of between 33% and
82.5% per million years. This range was considerably higher than the 11.5
20% reported in other vertebrates (Vigilant et al., 1991; Brown et al., 1993).
However, given the heterogeneity in rates across the different domains of the
control-region, a difference in the rate of evolution of this magnitude was not
completely unexpected (Saccone et al., 1987; Saccone et al., 1991). For example,
Ward et al., (1991) suggested that the tRNAPro end of the human control-
63
region (hypervariable domain I) evolves at nearly 30% per million years,
whereas the tRNAphe hypervariable region (domain II) evolves at a rate
closer to 15% per million years. Rate heterogeneity over smaller spatial scales
was clearly evident within the control-region of Cichlids where much of the
variation in a 442 bp segment of the tRNAPro end of the control-region was
clustered in a small (150 bp) central domain (see figure 2.3, Sturmbauer and
Meyer, 1992). The 3' end of the 195 bp portion of the control-region that we
sequenced aligned well with a conservative area just 3' from this
hypervariable domain.
Phylogenetic relationships among the total sample of individuals
Extracting the phylogenetic relationships from the larger sample of
individuals was complicated by multiple substitutions at the same nucleotide
position. Among a subset of the many thousands of most parsimonious
trees, there were on average 3.1 changes per position along this short control
region segment. Indeed, it was not uncommon to have positions change in
excess of 10 times along shortest length phylogenetic trees (figure 2.5). As in
the human control-region, the observed number of base substitutions fit a
negative binomial much better than a Poisson model of evolution suggesting
significant constraints on base substitutions within this region (table 1.3)
(Kocher and Wilson, 1991; Wakeley, 1993). Many of the invariant positions
occurred singularly or in small blocks of 2-4 nucleic acids; however, there was
a region of approximately 20 nucleic acids beginning at position 150 that was
highly conserved. This region aligned well with the Cichlid control-region
sequence (figure 2.2). The other small region that we could confidently
aligned with the cichlid control-region sequence (l04-127) also showed a high
degree of base conservation. The distribution of transitional substitutions
64
and deletions were cluster in 3-5 bp units along this 195 bp region (figure 2.5),
Nearly all transversions occurred at positions inferred to have had more than
5 substitutions, possibly reflecting mutational hot-spots along this sequence.
Despite this extensive level of superimposed change, the three major
phylogenetic divisions identified earlier were reasonably well preserved in
the larger sample of 137 individuals (figures 2.6a-d. Both Clade A and C
continued to be strongly supported in bootstrapped replications of Neighbor
joining trees. Clade B, which contained over 57% of the individuals sampled,
however, was supported in fewer than 30% of these replications. In this case,
multiple changes at the same nucleotide position probably obscured the
phylogenetic signal. Average pairwise difference among individuals within
this group was over 11 %, and the two most divergent individuals differed by
over 38% (table 1.4). In addition, after eliminating the 73 invariant sites
identified from the examination of the entire data set, a nucleotide position
changed, on average, 2.8 times along most parsimonious arrangements of
individuals within this clade. Moreover, the distribution of positions with 0,
1,2,3, ., ., " n substitutions continued to depart from a random Poisson model
of change (not pictured). This pattern suggested more complicated constraints
on the nucleotide substitution in this region than expected from a simple
two-rate Poisson mixture model (Wakeley, 1993).
Support for the evolutionary distinctiveness of clade B, however, was
evident from the pattern of pairwise differences among individuals in this
lineage and individuals of the other two lineages. The frequency distribution
of these comparisons were notably bimodal suggesting the presence of two
distinct clades (Hudson and Slatkin, 1991). Comparisons of the absolute
number substitutions within and between individuals of clade A and B
65
showed two distinct peaks: a larger one at 15 substitutions and a smaller one
at 27 substitutions (figure 2.7). The larger peak at 15 substitutions represented
comparison among individuals within each clade which were distinctly
unimodal (figure 2.8). The peak at 27 substitutions represented between clade
comparisons. This pattern is also reflected in the much smaller average
within-clade genetic differences, which ranged from 1.8% to 11.1%, relative to
between-clade differences, which ranged from approximately 30% to over 45%
(table 1.4).
Relationships among individuals within lineages
A remarkable difference in the phylogenetic patterning among these 3
lineages was the five-fold reduction of variation within lineage C relative to
lineages A and B (table 1.4). As a result, the branch leading to this clade was
over three times longer than the branch leading to either clade A or B (figures
2.6a-c). However, the phylogenetic patterns within each of the three mtDNA
lineages showed a similar lack of phylogenetic structure. The more basal
nodes were short relative to the more terminal branches giving the
phylogeny of these lineages a distinct star-like pattern with little internal
clade structure (figures 2.6a-c). This lack of phylogenetic structure was best
visualized by the unimodal and Poisson-like distribution of pairwise
comparisons among individuals within each lineage (figures 2.8) (Ball et al.,
1990; Slatkin and Hudson, 1991).
Partitioning of genetic variation
Clade C was the only one of the three major lineages that showed any
striking geographic or species specificity. This clade was only found within
the geographic range of C. multicinctus (figure 2.6c). Nearly all (85%) of the 32
C. multicinctus examined fell within this shallow lineage, making this
66
species the most genetically distinct of the three. The remaining 5 C.
multicinctus fell along three terminal branches within clade B (figures 2.6b-c).
In contrast, individuals in clade A and B were scattered throughout the
Pacific and showed no clear species specificity (table 1.5). It was common for
individuals of different species from widely scattered locations across the
Pacific to possess very similar control-region sequences. For example, within
clade A, our Neighbor-joining tree contained 7 terminal branch pairs
composed of a C. punctatofasciatus and a C. pelewensis (the "*" in figure 2.6a).
In 2 of these 7 pairs, individuals were collected from Tahiti and the
Philippines, roughly 10,000 kilometers apart. A similar pattern is evident
within the large, highly diverse lineage B (figure 2.6b). In this case,
individuals collected as far apart as Hawaii and the Philippines had identical
control-region sequences. .
At the species level, mitochondrial variation was distributed randomly
between C. pelewensis and C. punctatofasciatus. Neither MANOVA
(variance among species =8.98%, p=0.1782), FST (FST=0.124, p=0.28), chi
squared [(chi-squared=1.514, p=0.212)(see Roff and Bentzen, 1989)], nor
phylogenetic approaches (s=34; p>0.05 ) could detect differences in distribution
of mtDNA variation between these two species. Despite this observation, .
mtDNA variation was not distributed randomly across the geographic range
of each species. A significant amount of the genetic variation between these
two species, nearly 24%, was partitioned among populations (MANOVA;
p=O.Ol). In particular, the proportion of lineages A and B within the seven
western and south Pacific populations examined differed significantly from
random (chi-square=13.8; p=0.035). The distribution of clade A and Bin
populations across the south and western Pacific suggests a broad cline, with
67
clade B dominating populations of the Western Pacific and clade A found in
higher frequencies within populations of the eastern South Pacific (figure 2.9).
The significant differences in the distribution of mtDNA variation among
populations of these two species could be attributed largely to the higher than
expected portion of clade B in the Tahitian samples and clade A in the
Indonesian samples. When these two peripheral populations were
eliminated there was little evidence for genetic structuring of populations
across the range of C. pelewensis and C. punctatofasciatus (chi square=4.72;
p=O.325).
68
2.5. DISCUSSION
Assuming that most variation in the mitochondrial genome is neutral,
then the pattern of branching events provides a powerful glimpse into the
demographic history of a species or group of closely related species (Tajima, 1983;
Hudson, 1990; Slatkin and Hudson, 1991; Rogers and Harpending, 1992;
Harpending et al., 1993). For these butterflyfishes, the mtDNA genealogy traces a
rich history of demographic change. The early history of this group was
apparently marked by the coalescence of mtDNA diversity into three major
mtDNA lineages. However, following the coalescence of mtDNA variation into
these lineages there appears to have been a shift in the demographic landscape of
these species. The Poisson-like pattern of pairwise differences among
individuals within each of the three major lineages argues for a recent period in
which mtDNA variation was effectively buffered from extinction (figure 2.7)
(Slatkin and Hudson, 1991; Rogers and Harpending, 1992; Harpending et al.,
1993). Although factors such as a purifying selection or extreme rate
heterogeneity [(k<O.l, if the underlying distribution of mutations in the control
region fits a negative binomial (Rogers, 1992)] can generate a similar unimodal
peak in the pairwise differences among extant individuals, this genealogical
pattern is most consistent with a wave of population expansion following a
population bottleneck (Di Rienzo and Wilson, 1991; Rogers and Harpending,
1992; Marjoram and Donnelly, 1994). In clade A and B, the high and similar
intra-clade levels of variation suggest that a period of rapid population growth
followed soon after the initial formation of these lineages. In this case,
population expansion resulted in the widespread geographic distribution of both
lineages. Clade C, likewise, shows a Poisson-like peak in the pairwise
comparisons among extant lineages (figure 2.7). However, the levels of within-
69
lineage variation are notably smaller (figure 2.6c). This lineage is the most
geographically restricted of the three and it is possible that this pattern reflects a
recovery from a recent genetic bottleneck that was not linked to the initial
formation of this lineage. Alternatively, the shallowness of this clade may
indicate that a more significant population bottleneck was associated with the
formation of this lineage.
Given the demographic history suggested by the control-region
sequences, the lack of a strong concordance between the three major mtDNA
clades and species boundaries is intriguing. Either the formation of the
species was completely decoupled from these demographic changes or
speciation occurred within this demographic backdrop and hybridization and
introgression has since eroded the initial concordance between mtDNA
lineages and species designations. These alternative scenarios paint
contrasting pictures for the recent evolutionary history of these species,
differing both with respect to the timing and demographic parameters
associated with differentiation.
Two scenarios and their evolutionary and demographic implications
Shared evolutionary history
Under certain demographic conditions, species will share ancestral
genetic variation for many generations after the origin of reproductive
isolation (Neigel and Avise, 1986; Pal milo and Nei, 1988). If the levels of
mtDNA variation are any indication, these three species have arisen recently.
Slight genetic differences among individuals in the cyt b gene region suggest a
maximum time-frame of divergence of between 300,000 and 800,000 years
(Chapter 1). Species maintaining an effective population size of only 150,000
400,000 females through speciation would be expected to share ancestral
70
lineages over this time period (Neigel and Avise, 1986). Given the
demographic properties of these species, which include large population sizes,
high fecundity, and vast dispersal potential due to planktonic larval stages
(Reese, 1991), it is possible that the observed patterning of mtDNA variation
among these species reflects ancestral patterns of gene flow among groups
that are no longer exchanging individuals.
If this scenario is correct, then the widespread distribution of similar
control-region sequences implies that speciation has occurred very recently,
well after the formation of clades A, B, and C. In this case genetic differences
between individuals of different species falling together at the terminal
branch tips of the mtDNA genealogy, represented by the >I- in figure 2.6a, are
the most likely to reveal the timing of speciation. In our Neighbor-joining
tree, these differences range from 0% to 10.34% with an average of 3.1%,
placing the origin of these species, based on our crude estimation of the rate of
molecular evolution within the last 37,000-94,000 years.
This scenario makes similar strong predictions about the demographic
process associated with species formation. The haploid, maternally inherited
mitochondrial genome is four times more sensitive to changes in population
size than are neutral nuclear loci (Wilson et al., 1985). Thus, the presence of
shared mitochondrial variation suggests that the differentiation of this group
proceeded without a severe, protracted population bottleneck. Presumably,
the color pattern differences that presently define these species consolidated
within this demographic framework. This scenario would be more plausible
if there were strong selective pressure on the loci coding for color pattern
during speciation. Although the evolutionary or ecological significance of
vivid coloration in butterflyfishes is unknown, color pattern may play an
71
important social or sexual role (Reese, 1975; Fisher, 1980; however see Ehrlich
et al., 1977). Because socially and sexually important traits can be subject to
strong directional selection during speciation they can change very rapidly
and coalesce quickly within emerging species (Lande, 1982; West-Eberhard,
1983). In contrast, neutral markers will be shared between newly formed
species until the time since the onset of reproductive isolation approaches 2-4
times the historical effective population size, or historical female population
size for mtDNA genes (Neigel and Avise, 1986; Palmilo and Nei, 1988).
Hybridization
Alternatively, the lack of clustering of species within the three mtDNA
lineages may be caused by recent mitochondrial gene flow. Under this
scenario, species defining characteristics (i.e., color pattern) may have formed
within the same demographic framework that caused the coalescence of
mtDNA variation into three lineages. In this case, the timing of speciation
can best be approximated from the average corrected genetic differences
between the three mtDNA lineages. This average, 26.7%, places speciation
between 325,000 and 800,0000 years ago or nearly an order of magnitude more
distance than the temporal framework suggested by the shared evolutionary
history scenario.
Subsequently, mitochondrial gene flow between species may have
eroded the initial mtDNA gene and species concordance. The species and
geographic specificity of clade C argues that this lineage was the original
lineage of C. multicinctus. Thus, under this scenario, the number of
individuals with mtDNA haplotypes that do not fall within this clade reflect
the levels of hybridization. Within our sample of 32 C. multicinctus, there are
5 individuals that posses haplotypes outside of clade C. Two pairs have
72
identical control-region sequences and probably descended from a single
hybridization event, yielding support for a minimum of three hybridization
events in the history of the individuals sampled. Given our sample size, this
phylogenetic pattern suggests an effective migration rate, Nm, between C.
multicinctus and either C. pelewensis or C. punctatofasciatus of
approximately 0.4 individuals per generation (see Slatkin and Maddison,
1989). A similar value, not dependent on a phylogenetic tree, was suggested
from the patterning of variation within and between species (FST=0.5227,
Nm=0.41) (Hudson et al., 1993).
The widespread mixing of similar control-region sequences between C.
punctatofasciatus and C. pelewensis suggests extraordinary high levels of
hybridization and associated introgression of mtDNA genes. Within our
Neighbor-joining tree there were 21 instances of a C. pelewensis and a C.
punctatofasciatus falling together at the terminal branch tips, see " in figure
2.6a. Across this entire phylogeny, there was phylogenetic evidence for a
minimum of 34 separate hybridization events. Identical estimates were
obtained from maximum parsimony trees. This genealogy suggests an
effective migration rate between these two species of nearly 300 individuals
per generation. A slightly greater estimate was obtained from the observed
FST value between species (FST=0.0007; Nm=713). Although both estimates
are likely biased upward, if hybridization accounts for the present distribution
of mtDNA variation between these two species, then the present pattern of
mtDNA variation highlights a history of substantial movement of mtDNA
genes between species boundaries (Hudson et al., 1993).
Because the mitochondrial genome does not code for genes likely to be
directly involved in reproductive isolation, this cytoplasmic marker has
73
frequently been observed to introgress across species boundaries at a rate
higher than nuclear encoded loci (Powell, 1983; Ferris et al., 1983; Spolsky and
Uzzell, 1984, 1986; Lamb and Avise, 1985; Baker et aI., 1989; Dowling, 1989;
however see Szymura et al., 1985; Szymura and Barton, 1986; Nelson et al.,
1987). Theoretical considerations suggest that even with very strong selection
against hybrids, the mitochondrial genome can introgress across species
boundaries at a rate determined principally by the frequency of hybridization
and the dispersal potential of the two parental species (Takahata and Slatkin,
1984). Under this scenario, the present patterning of mtDNA variation
suggests that the frequency of hybridization among these species can be quite
high. In addition, these fishes have a planktonic larval stage of over 40 days
(Tricus, 1987; Hourigan and Reese, 1987; Leis, 1989), indicating that
widespread dispersal and gene flow in these species may be qui te common. In
marine populations, a number of population genetic studies have
documented genetic homogeneity over vast areas presumably due to long
distance dispersal of planktonic larval stages (reviewed in Palumbi, 1992).
Distinguishing ancestry from hybridization
When the time since speciation is short, discriminating between the
retention of ancestral polymorphism and hybridization as alternative
explanations for the presence of shared gene lineages is notoriously difficult
(Slatkin, 1989). Despite this conclusion several features about the genealogical
and geographic patterning of mtDNA variation among these three
butterflyfishes lead us to support hybridization as the most likely explanation
for the observed discrepancy between the mtDN A tree and species
boundaries. However, it is important to note that none of these features by
themselves provide compelling support for either scenario. In all cases, a
74
similar pattern could, in theory, be attributed to the retention of ancestral
variation in very recently evolved species. Yet, in each case, the observed
genetic and geographical patterning of mtDNA variation can be more easily
explained by recent hybridization than by the retention of ancestral
polymorphism.
Branch tips and divergence
If shared history accounts for the widespread distribution of similar
mtDNA haplotypes among species, one would expect that any evolution
occurring after the onset of reproductive isolation would accumulate at the
branch tips first. For example, a panmictic ancestral population divided into
two emerging species will initially share many gene lineages and closely
related haplotypes. However, over time, new mutations will cause these
closely related haplotypes and gene lineages to diverge. At the same time,
mutation will create a new generation of very similar, but not identical
haplotypes. This new generation of similar haplotypes will only be found in
individuals of the same species. Thus, assuming that any time at all has
elapsed since speciation, divergence among species will be most evident at the
terminal branches of a gene genealogy. Specifically, one might expect that
branch tips containing individuals of the same species would be significantly
shorter than branch tips containing individuals of different species. In our
Neighbor-joining tree there are 19 cases in which individuals of different
species fell together at the terminal branches, represented by the * in figure
2.6a, and 14 instances in which conspecific individuals fell together, represent
by the ** in this figure. Figure 2.10 plots the frequency distribution of genetic
differences between individuals in these two types of terminal nodes.
Although the mean genetic difference is slightly higher in the heterospecific
75
versus conspecific comparisons, 3.1% versus 2.4%, we can detect no branch tip
divergence among these species (t=0.922; p=0.36). The shared ancestry model
is compatible with this observation only if speciation has occurred so recently
that no control-region divergence has occurred. By contrast, recent and high
levels of hybridization easily explains the lack of divergence between
individuals of the different species that fall together at the terminal tips of
our mtDNA genealogy.
Geographic patterning of mtDNA variation:
Additionally, the random distribution of mtDNA variation between C.
pelewensis and C. punctatofasciatus at the species level but not at the
population levels seems curious under the shared history scenario. The
shared history scenario requires that the observed cline in mtDNA haplotypes
predated speciation and that species boundaries consolidated around it. This
might be reasonable if there is strong differential selection on these mtDNA
lineages in different parts of the Pacific (see Endler, 1977). However, there
does not appear to be any strong association between these lineages and
obvious environmental or physical characteristics. In fact, differences in the
distributions of these lineages among populations are so minor, with the
proportion of lineage B never rising above 67%, as to almost preclude any
selective explanation. By contrast, if one assumes that mtDNA variation is
neutral, then any underlying structuring of mtDNA haplotypes would likely
be accentuated during speciation (Neigel and Avise, 1986). Thus, one would
expect genetic differences between species to build faster than genetic
differences between populations.
The present geographical distribution of mtDNA variation between
species, however, is easily incorporated into the hybridization scenario.
76
Under this scenario, lineage B, the dominant lineage in the Western Pacific,
was the original lineage of C. punctatofasciatus and lineage A, the dominant
lineage within the eastern South Pacific, was the original mtDNA lineage of
C. pelewensis. It is possible that lineages coalesced at speciation and were
initially restricted geographically in the central Malay region and in the
archipelagoes of the South Pacific, respectively. However, in the 325,000
800,0000 years since speciation, the high fecundity and wide dispersal
capabilities of these species have permitted each lineage to expand its
geographic range. To date, lineage B has been able to spread across the entire
range of C. pelewensis. Lineage A has done nearly as well, spreading as far
east as the Philippines (figure 2.9). Under the hybridization scenario, the non
random distribution of mtDNA among populations of these two species
merely suggests that expansion has not completely eroded the historical
pattern. Indeed, it is populations at the endpoints of each species range, the
Tahitian and Indonesian populations, that generate the observed genetic
differences among populations.
Genealogical patterning of mtDNA variation
Lastly, the presence of three distinct species and three distinct mtDNA
lineages is difficult to reconcile under the shared history scenario. This
concordance may have arisen by chance; however, the strong geographic and
species specificity of clade C, as well as, the non-random distribution of clade
A and B detailed above argue against this possibility. It seems more probable
that phenotypic and mtDNA evolution were contemporary and that the
species-specific color patterns formed within a demographic framework in
which populations were sufficiently small to cause the coalescence of
mtDNA variation.
77
Demographic changes, speciation, and hybridization
Interpreted as a whole, the patterning of mtDNA variation begins to
build a substantial case for hybridization as the most plausible explanation for
the widespread distribution of similar mtDNA sequences among these
species. This framework suggests that following phenotypic and mtDNA
differentiation, the enormous fecundity and the high dispersal potential
linked to long-lived planktonic larval stages allowed newly formed species to
expand population size and extend their geographic range. In the case of C.
pelewensis and C. punctatofasciatus, the newly emergent mtDNA lineages
and phenotypic lineages met. Reproductive barriers were apparently
insufficient to prevent these species from hybridizing and there appears to
have been only slight impediments to the geographic expansions of the
mitochondrial lineages of these two species.
In the presence of such pervasive mixing of mtDNA variation between
C. pelewensis and C. punctatofasciatus the geographic and species-specificity
of lineage C within C. multicinctus. is notable. On the one hand, this pattern
may merely reflect the extreme geographic isolation of this species. The
Hawaiian Islands (and Johnson Atoll approximately 1300 kilometers to the
southwest) are the most isolated group of islands within the Pacific. The
nearest known source population for C. punctatofasciatus lies in the Marshall
Islands nearly 2300 kilometers to the southwest and the source population of
C. pelewensis is nearly twice as far to the south. Thus, it is possible that few
individuals of C. punctatofasciatus or C. pelewensis ever reach Hawaii,
thereby limiting the possibility for hybridization. Previous allozyme stu.dies
of marine populations across this region have revealed slight but significant
genetic differences, generally associated with lower levels of heterozygozity,
78
between Hawaiian and other Pacific populations (Winans, 1980; Nishida and
Lucas, 1988). In addition, mitochondrial sequences of populations of
Hawaiian sea urchins show a similar pattern of reduced genetic diversity
relative to populations nearer the western Pacific (Palumbi and Metz, 1991;
Palumbi, 1994).
On the other hand, the genetic isolation of the Hawaiian endemic, C.
multicinctus, might be attributed to a greater degree of reproductive isolation.
Chaetodon multicinctus is the largest (Appendix C) and most phenotypically
distinct of the three species (see figure 2.1). Although the underlying body stripes
are similar to C. punctatofasciatus, C. multicinctus is almost totally white, lacking
the striking yellow and orange coloration that characterizes C. punctatofasciatus
and C. pelewensis. Chaetodon punctatofasciatus and C. pelewensis are more
similar to one another in size, differing primarily in the striations along the body
which run vertically in C. punctatofasciatus but diagonally in C. pelewensis
(figure 2.1). These color patterns change abruptly at the suture zone between
species (Appendix C). Thus, levels of hybridization and gene flow between
species capable of generating a nearly random pattern of mtDNA variation have
failed to erode species-defining color patterns. The contrasting patterns of
mtDNA and phenotypic variation suggest that strong selection is acting to
maintain color pattern differences. Positive assortative mating among species,
similar to that observed among different colored morphospecies of Western
Atlantic hamlets (Hypoplectrus: Serranidae) (Fisher, 1980), may account for the
sharp demarcation of phenotypic differences among these species. Future work
on the degree of reproductive isolation among species, as well as on the
patterning of variation at other genetic markers, will help interpret the cohesion
79
of phenotypic variation in these species within a background of potentially
homogenizing levels of gene flow.
80
Table 2.1: Pattern of nucleotide substitution in a 195 base portion of the
tRNAPro end of the mitochondrial control-region of Butterflyfishes.
NucleotideNucleotide Proportion Introduced'[Replaced of Sequencet G A T C Del
G 0.138(0.103-0.179) 82 3 2 0
A 0.396(0.349-0.426) 157 - 6 4 0
T 0.258(0.103-0.179) 0 2 116 0
C 0.180(0.221-0.282) 6 1 106 0
Insertion 0.021(0.153-0.021) 1 2 1 0t Averaged over a11141 sequences. Numbers in parenthesizes are the
minimum and maximum percentages observed.t Average number of unambiguous changes from a suite of 500 equally most
parsimonious trees generated from a heuristic search using the phylogenticprogram PAUP (Swofford, 1993). For this search transversions wereweighted 10 times transitions.
81
Table 2.2: Comparison of within and between species mtDNA variation
using a 500 bp segment of the cyt b gene and a 195 bp segment of the tRNAPro
end of the mitochondrial control-region.
GenotypeNumber of types diversity indext % seq. difference'[
CYfB CONTROl... CYfB CONfROL CYfB CONTROL
All individuals (n=38) 30 34 0.97 0.98 0.85 25.40
C. multicinctus (n=15) 10 12 0.90 0.97 0.67 13.9
C. punctatofasciatus (n=12) 11 12 0.98 1.00 0.99 17.4
C. pelewensis (n=l1) 9 11 0.97 1.00 0.71 18.5
t Genotype diversity is give by h = (1-~xi2)n/(n-1), whereXi is the frequency of the ith type and n is the sample size.:j: percentage sequence difference is the average pairwise divergence amongindividuals. For the cyt b data set, genetic differences between individualswere calculated using the Kimura 2-parameter model (Kimura, 1980). For thecontrol-region, distances among individuals were corrected using a TamuraNei model with a gamma correction (a=0.6) (Kumar et al., 1993).
82
Table 2.3: Observed and expected number of base substitutions under both a
Poisson and negative binomial model of nucleotide substitution in the
tRNAPro end of the mitochondrial control-region of butterflyfishes.
Estimated NUMBER OF SITESnumber of changes
per sitet Observed Poisson Negative binomialo 69 8.17 63.271 28 25.70 31.892 16 40.44 21.433 16 42.42 15.604 4 33.37 11.795 14 21.01 9.116 11 11.02 7.15
>7 32 7.88 29.57t The number of changes at each of the 195 sites was inferred from arandomly chosen most parsimonious tree generated during a heuristic searchusing the phylogenetic program PAUP (Swofford, 1992). The mean numberof changes per site along this tree (m=3.15) was used to compute the expectednumber under both the Poisson and negative binomial distribution (k=O.6).The negative binomial fit the observed distribution of changes per site farbetter than a Poisson model (chi-squared=12.426, p=O.09 and chisquared=586.616, p<O.0001, respectively).
83
Table 2.4: Average percent difference of all pairwise comparisons of genetic
difference between individuals within (along diagonal), between uncorrected
(below diagonal) and between corrected (above diagonal) for the three
mitochondrial lineages identified in this study. Corrected differences were
calculated by subtracting the average within-clade variation from the average
between clade variation (Wilson et al., 1985). Genetic distances between
individuals were corrected for multiple hits at the same nucleotide position
using the Tamura and Nei distance method with a gamma correction (a=0.6)
(Kumar et al., 1993).
A B C
Clade A 9.3 17.9 21.7
Clade B 28.1 11.1 40.5
CladeC 46.1 34.5 1.8
84
Table 2.5: Distribution of individuals within each of the three major mtDNA
lineages from populations across the Pacific.
Numberof Individuals Clade A Clade B CladeC
C. multicinctusMidway Island 11 0 2 9
Oahu Island 15 0 3 12Johnson Island 6 0 0 6
C. punctatofasciatusGuam 8 2 6 0Palau 20 3 17 0
Philippines 21 8 18 0Indonesia 7 0 7 0
C. pelewensisTahiti 9 6 3 0
Am. Samoa 2 0 2 0Cook Islands 18 7 11 0
Fiji 21 1 17 QTotals 138 81 30 27
85
Figure 2.1. Color plate of a) Chaetodon punctatofasciatus, b) C. pelewensis,
and c) C. multicinctus.
86
&l
Figure 2.2: peR primers used to amplify an approximately 1200 bp portion of the mitochondrial
genome spanning the control-region of butterflyfishes. The 3' end of this 1200 bp piece aligned well
with the 12s rRNA gene of other vertebrates. However, we were only able to align two small regions of
this piece with the tRNApro end of the control-region of Cichlids. Alignment of the Chaetodon
multicinctus sequence to both Cyathopharynx furcifer (Pisces: Cichlidae) and Crossostoma lacustre
(Pisces: Homalopteridae) sequence was done by eye. Numbers at the beginning of each sequence are the
position of the aligned regions relative to the 5' end of the 12s rRNA gene of C.lacustre and the
tRNApro end of the control-region of C. furcifer (see Tzeng, et al, 1992;Sturmbauer and Meyer, 1993).
Chaetodon mtDNA
tRNA-Pro[S, ~~C.R.-l iIRNA.Phe :;lIRNA-lhr 1 ~ 72J12sar
00\0
Control region:
ryathopl1drynx t urc i fer
Cbeet.cdon multicinctus
Cyat!lcpharynx turc i t or
Ctiee t ccton mv t c i c i nct us:
125 rRNA:crcsac sc cr-s Lacus r r-e
Ctiaet cdcn multicinctus
cros.scs r cr-e lacustre
Chaet cdcn mul r f c I nc r us
5' (111) 21 -CATA.AAGC-TAAGGGGTACAT- - - -AAACCATAGCTGTAATATTCAACTAACTATTTACTGAAAGCTAAACGATAGTTT-AAGACCGATCACACCTCTCACATAGTT
II 1111 I II I I11111 II III I I I II III III I II II I III II I I I I II I I5 ' - TGT ATTAGAT AAGAAG - ACTT f.CT AAAACC AAAGTAAT AAGAAGACCTTGA - TACTTATTGAGTAATGM - GAAAC1"TCTAACTeAGCT AAA TTTCATCCCGTCA
OJ' -AAGATATACCAAGTACCCACCATCCT-AT7MTTAA-TJ..ATA-TTTAATGTAGTAAGAGCCCACCATCAGTTGATTTC7CAATGATAACGGTTCTTGA
111111111111111111111 I I I III I II 11111 II 111111 111111 III 1111111111111 11115' - AAGACATACCAAGTACC-ACCATTTCTGTATAGGAAATAACl\ACCCM- - - - -TAAGAACCTACCATCGGTTGATATCTGAATGATAACGGTTATTGA
5' II 221) -AGGGTCGGTAAAACTCGTGCCAGCCACCGCGGTTATACGAr.AGACTCAAGTTGACAGTCACTCGGCGTAAAGT- -GGTTAAGATGAACMAAACTAAAGCCAAAT- r.CCTTCAAA
111111111111111111111111111111111111111111 I I 111111 II 1111111111 1111111 1111I1 II 111111111 11115 ' - AGGGCCGGTAAAACTCGTGCCAGCCACCGCGGTT1\T ACGAGAGGCCCT AGTTGATAGGTG - - CGGCGTAAAGGGTGGTTAAGGAGAGCAAGAAITAAAGCT AAGGGM"( "reTT· G
S' -ACTGTTATACGTTCTCGMGGTGAGAAGCCCAA
I 11 III I I I II I 11II1 IIS· -GCCGTCATACGCTTCTGAGTATC~AAAGCCMA
Figure 2.3a: Neighbor-joining tree for 38 individuals from three species of
butterflyfishes [Chaetodon multicinctus (n=15), c. pelewensis (nel l) and C.
punctatofasciatus (n=16)] inferred from substitutions within a 195 base
portion of the 5'-end of the mitochondrial control region. This tree was
constructed from pairwise distance matrices corrected for multiple
substitutions using a Tarnura-Nei distance with a gamma correction (a = 0.6)
(Kumar et aI., 1993). The relative strength of major nodes within this
phylogeny is given by the percentage agreement in a consensus of trees
produced from 100 bootstrap simulations of the data set (above each branch)
and the number of informative nucleotide changes (above each branch).
Chaetodon guttatissimus was used as an outgroup to root changes along this
tree. Trees constructed using parsimony with a 20:1, 10:1, and 1:1
transition/ transversion bias were nearly identical, differing only in the
position of individuals within each group. Consistency and retention indexes
for these trees generated under a 10:1 weighting scheme were 0.49 and 0.79 .
Collection localities are as follows: Ha- Oahu, Hawaii; Phil- Philippines;
Bali- Bali, Indonesia; AS- Pago Pago, American Samoa; TA- Moorea, Society
Islands.
90
B
c
13100
..------- c. pun Guam 4r---- C. multi Ha 2,Ha 9, C. pun Phil. 31..-_ C. pele AS 2
C. multi Ha 11C. pun Guam 3a
C. pun Phil. 4C. peleTH8
C. pun Guam 2C. pun Bali 1
1"---- C. pun Guam 4a'---- C. pele AS 1
IIL--- C. pele TH 2'-tL.----- C. pele TH 1
C. pun Bali 3C. pun Bali 5
C. pun Bali 4
C. multi Ha 1, Ha 6C. muitiHa5
C. multi HasC. multi Ha 7
C. multi Ha 10
C. multi Ha 12
C. multi Ha 4, Ha 14C. multi Ha 15
C. multi Ha 3C. multi Ha 13
70
5
4
62
a)
84
3
iii iii
C. pele TH5C. peleTH3
C. peleTH9..------ C. pele TH 4
C. peleTH7C. pun Guam 3
C. peleTH6C. pun Phil. 2
A
a 5% sequence difference
91
Figure 2.3b: Neighbor-joining tree for 38 individuals from three species of
butterflyfishes [Chaetodon multicinctus (n=15), c. pelewensis (n=l1) and C.
punctatofasciatus (n=16)] inferred from substitutions a 500 base portion of the
cyt B gene region (Note: this tree is not drawn to the same scale as 3.3a). This
phylogeny was constructed from pairwise distance matrices corrected for
multiple substitutions at the same nucleotide position using a Kimura two
parameter model (Kimura, 1980). The relative strength of major nodes
within this phylogeny is given by the percentage agreement in a consensus of.trees produced from 100 bootstrap simulations of the data set (above each
branch) and the number of informative nucleotide changes (above each
branch). Chaetodon guttatissimus was used as an outgroup to root changes
along this trees. Trees constructed using parsimony were nearly identical,
differing only in the position of individuals within each group. Consistency
and retention indexes for these trees were 0.85 and 0.85. Collection localities
are as follows: Ha- Oahu, Hawaii; Phil- Philippines; Bali- Bali, Indonesia;
AS- Pago Pago, American Samoa; TA- Moorea, Society Islands.
92
B
5
c
A
r-------- C.pun Guam 4
C. multi Ha 2,Ha 9
C. peleAS 2
C. pun Phil 3
C. pun Guam 3a
C. pun Guam 4a
C. multi Ha 11; C. pun Bali 3, Bali 1; C. pele TH 8, AS 1
C. peleTH2
c.punGuam2
C. pun Bali5
C. pun Phil 4
C. pun Bali 4
C. peleTH 1
C. multi Ha 1C. multi Ha 3, Ha 5,Ha lO,Ha 14
C. multi Ha4
C. multi Ha 7
C. multi Ha 8
% sequence difference
40 C. multi Ha 121 C. multi Ha 6
C. multi Ha 13
1..---C. pun Guam 3
C. pele TH 3, TH 6
C. peleTH4
C. peleTH5C. peleTH9
C. punPhi12 ~Ac. peleTH7 ~
I I
21
51
o
b)
93
......."0(l) 20.....u(l)
'"' 18'"'0u
"-"
l:: 16 .~ ..0 •.-~ 14'"'- •8 12
f t.....l:: •8 10 0C1J 0u 8l::(l)
'"' 6 4'~.-"0 4 .(l)
:g ......
20(l)
U::s 0l::~ 0 .2 .4 .6 .8 1.0 1.2 1.4 1.6 1.8 2.0 2.2
% nucleotide difference, cytochrome b (uncorrected)
Figure 2.4: Mean percentage nucleotide difference with one standard
deviation error bars in a 195 base-pair portion of the tRNAPro end of the
mtDNA control region relative to changes in a 500 bp segment of the
mitochondrial cyt B gene for all pairwise comparisons of individuals within
clade B (see figure 2.2a). Percent differences within the control region were
corrected for multiple hits at the same nucleotide position using the Tamura
Nei distance method with a gamma correction (a=0.6).
94
Figure 2.5: Average number of substitutions per position along a 195 bp portion of the tRNApro end of
the control-region estimated from 500 equally most parsimonious trees generated from a single
heuristic search on the computer program PAUP (Swofford, 1991). These trees were constructed
assuming that transitions were 10 times more likely to occur than transversions. The two large shaded
bars represent the two regions that aligned well with the control-region of the Cichlid, Cyathopharynx
\0(Jl
furcifer (Pisces: Cichlidae) (see figure 1). White bars represent those positions where trans versions
occur. A "d" represents a position where a sequence addition was observed..
suouruusqns JO # a~huaAV
Q)us::Q)
~Q)ens::o'SoQ)
""'.-4e....s::oubes::o
.-4!tl
s::o..............enoc,
o-\J"')......oC"l
\J"')N
96
Figure 2.6: Neighbor-joining cartoon of the three major mtDNA lineages.
The size of each shaded box indicates the average pairwise genetic distances
(width) and the number of individuals (height) within each. Support for
each clade is given by the percentage agreement in a consensus of trees
produced from 100 bootstrap simulations of the data set. Chaetodon
guttatissimus was used as an outgroup to root this tree. The telescoped
portion represents the relationships among a) the 30 individuals of c.
punctatofasciatus (blacken boxes) and C. pelewensis (shaded boxes) that fell
within clade A, b) the 81 individuals of C. punctatofasciatus (blacken boxes),
C. pelewensis (shaded boxes), and C. multicinctus (shaded circle) within clade
B and c) the 27 individuals of C. multicinctus (shaded circle) within clade C.
Trees constructed using parsimony with a 10:1 transition/transversion
bias were similar, but not identical. In these trees, members of clade Band
clade C fell within distinct lineages. However, individuals in clade A
clustered at the base of these trees, as in the neighbor-joining tree, but failed to
group within a defined lineage. Forcing these individuals into a
monophyletic lineage produced trees that were only 7 steps longer.
Consistency and retention indexes for parsimony trees were extremely low at
0.25 and 0.77, respectively.
97
100
a)
72
36
1~111o 5
% sequence difference
11IIII, Tahi~!.Cook 1.1**L.-_ 1IiII, FIJI
iii, Tahiti I., Philippines *
iii , Cook Islands
II, Fiji I.,Palau *
L..-__II , Fiji
III, Tahiti., Philippines It.L
• , Philippines r-*., Philippines kl.*
.,Palau ~
11m, Tahiti ~_
• , Philippines~11II, Cook Islands I
• , Philippines *m,Cook Islands
Ill, Tahiti
11m, Fiji I.,Guam *
11III, Tahiti
1Dl, Cook Islands I*.,Guam
IL-_ 16,Cook Islands
m, Cook Islands III, Philippines *
• , PalauL.-__ ., Philippines
I I I I "o 5% sequence difference
98
Figure 2.6: Collection location and phylogenetic relationships and among
individuals within clade B (continued),
99
I " " Ia 5% sequence difference
Indonesiac::J Fiji
c::J Fiji_ Palau
Fiji_ Guam\----;;;;;;;; _ Indonesia
'------I _ Palau_ PalauI::::J Cook Islands_ Guam
_ Palau'------..:; r:::J Cook Islands
---- 0 Hawaii (2)c::J Cook Islands
.J ..--- r:::J Cook Islands.--. ~F~ ..
I::J Fiji Jl
L~==:;;-~ - Philippines_ Guam- Philip.pines_ Parau
_ Philippinesr.::::J Tahiti
I::::::J Fiji_ Guam
_ Palau_ Indonesia
c::::J Fijic::J Fiji
CO -Hawaii (2), Philippines_ Palau
l'::J '& Hawaii_ Palau
L... Philippines
_ Philippines.. Philippines
II ,...---- t::J Am. Samoa_ Palau~---r--_ Philippines
.. Palau
I ....--c:. F~hiliPPines.....--- c:::::J Cook Islands
111 ~Fi~alaua::::J £iii
- Iiiiii Philippines_ Palau
.. Palau
t::J Cook Islands_ Philippines
r:::::2 Fiji_ Palauc::::l Am. Samoa
L- Philippines--z-- c::J Tahiti
c::::::I Cook IslandsE::::] Cook Islands~Fiji,,--- c::II Cook Islands
_ Palaut::J Fiji
_-_..:- _ Philippines_ Indonesia
r---- c::J Fiji_ Indonesia
t::::::J Cook Islandsr::J Cook Islands
r---L=~-~ PalauG - Guam'---- .. uam
1.- Indonesia..------- _ Tahiti
I I II I Ia 5
% sequence ditference
c\
36
100
72
b)
100
Figure 2.6: Collection location and phylogenetic relationships and among
individuals within clade C (continued).
101
c)72
36
100
I I I I I Ia 5
% sequence difference
102
\
@ , Hawaii (2)@,Hawaii@,Hawaii® ' Ha., Johnston (2)@,Midway@,Hawaii
® ' Johnston Atoll@,Midway@,Hawaii
®,Hawaii
@,Hawaii@ , Ha (2), Johnston~ ,Hawaii
@ , Johnston Atoll@,Midway@,Midway
@,Midway~, Midway@,Midway@ , Johnston Atoll
I I I I I Ia 5
% sequence difference
350
300(/)
~0
250(/).-1-0resS- 2000u..... 15001-0QJ
..0 100E;j
Z 50
00 5 10 15 20 25 30 35 40
Number of pairwise differences
Figure 2.7: Frequency distribution of pairwise differences among individuals
in clades A and B (see figure 2.6).
103
Figure 2.8: Frequency distribution of pairwise differences among individuals
in a) clade A, b) clade B, and c) clade C (see figure 2.6).
104
a) 70
en 60~ Clade A0
50en'crt:l0.. 40E0u
30......01-0
~ 20
E;j 10Z
00 5 10 15 20 25 30 35 40
Number of pairwise differences
b) 450
en 400~0 350 Clade Ben.-1-0rt:l 3000..
§ 250u
...... 2000
aJ 150..0E 100;j
Z 50
00 5 10 15 20 25 30 35 40
c)Number of pairwise differences
90
80en~ 70 Clade C0en
'C 60rt:l0..E 500u 40......01-0 30Q)
..020E
;:l
Z 10
5 10 15 20 25 30 35 40Number of pairwise differences
105
I I IKilometers
tN
2000o
Oahu, n=15
' .. C. pelewensis
~ .{~tl:n~,
Cook I I . ~ ..s anus, 1l=~8 . " ,
' ..
C. multicinctus
'itFiji, n=21
..- \
......,-~ ..., ..
C. punctatofasciatus
~
aQ\
Figure 2.9: Proportion of the three major mtDNA lineages in the populations sampled: 0, clade A; _,
clade B; ~, Clade C. Larger squares represent the approximate geographic range of each species. See Blum
(1989) fo site specific distributions of each species
......o""-J
4
(/)
~ 3.9(/).....I-<ro
~8 2
......o!n
"S::l 1Z
o<0.5 0.5-1.0' '1.0-1.5' '1.5-2.0
• heterospecific pairs
II conspecific pairs
5.0-5.5 5.5~.0
% sequence difference between terminal pairs
Figure 2.10: Frequency distribution of the branch length of terminal nodes containing individuals of the
same species (shaded bars) versus those containing individuals of different species (blacken bars).
CHAPTER 3
Random Mating and Species Boundaries in Allopatric Coral Reef Fishes
3.1. ABSTRACT
Three closely related butterflyfishes (Chaetodontidae) that differ in
color pattern, partition the tropical west Pacific into large allopatric ranges.
Previous mtDNA and allozyrne analysis supported the evolutionary
distinctiveness of only the Hawaiian endemic, Chaetodon rnulticinctus.
MtDNA and allozyme variation was distributed randomly between C.
pelewensis and C. punctatofasciatus. Pairing experiments with C. pelewensis
and C. punctatofasciatus suggest that the greater genetic differentiation in C.
multicinctus is paralleled by greater behavioral and presumably reproductive
isolation. Chaetodon multicinctus pairs assortatively and heterospecific trials
show significantly lower levels of intra-sexual aggression relative to that
observed in conspecific trials. By contrast, pairing between C. pelewensis and
C. punctatofasciatus appears to be random with respect to color pattern.
Additionally, levels of intra-sexual aggression between these two species are
similar to that observed in conspecific trials. Observations of C. pelewensis
and C. punctatofasciatus in the zones of sympatry yield similar conclusions.
A broad, approximately 3000 kilometer, hybrid zone joins the geographic
ranges of the two species. In localities across this zone, individuals with color
patterns intermediate between the two parental forms are common.
108
Moreover, in both Papua New Guinea and the Solomon Islands, pairing
among "pure" parental and "hybrid" phenotypes is random with respect to
color pattern.
Despite being tightly coupled reproductively and evolutionary, C.
pelewensis and C. punctatofasciatus remain uncoupled phenotypically. Color
pattern difference between the two species change over a much narrower
range than expected given the complete homogenization of variation at
presumably neutral loci. These results suggest that selection directly on color
pattern, and probably little else, is maintaining phenotypic integrity of these
two color variants while most other genes move freely across "species"
boundaries.
109
3.2. INfRODUcnON
Phenotypically similar groups that have allopatric or parapatric
distributions have always posed interesting challenges in evolutionary
biology. Because many of the traits that differ in these groups are similar to
characteristics used to define species, these forms (e.g., species, subspecies,
races) are thought to represent the first stage of diversification and thus
provide a context within which to explore the nature and timing of barriers to
gene flow (Mayr, 1942). Moreover, in many instances, allopatric forms meet
and form hybrid zones which reveal the extent to which reproductive
isolation and ecological coexistance can occur (Harrison, 1990). Information
on the levels and patterning of cytological, biochemical, morphological, and
genetic variation across hybrid zones coupled with behavioral and ecological
studies have provided insightful views into the nature of species distinctions.
When multiple data sets are available, it is the concordance or discrepancies
between different types of data that have provided the best clues into the
origins of reproductive isolation, the maintenance of genetic cohesion, and
the ecological and evolutionary significance of inter-specific differences
(Avise and Saunders, 1984; Littlejohn and Watson, 1985; Barton and Hewitt,
1985; Harrison, 1990; Hewitt, 1989; Moore and Price, 1993; Mallet, 1993; Parsons
et al., 1993).
Nearly all studies examining the genetic, reproductive and
evolutionary cohesion of parapatric or allopatric taxa have focused on
terrestrial or freshwater groups with limited dispersal potential (Barton and
Hewitt, 1981; Avise and Saunders, 1984; Littlejohn and Watson, 1985;
Szymura and Barton, 1986; Rand and Harrison, 1989; Moore and Price, 1993;
Mallet, 1993; Parsons et al., 1993). There have been few examinations of
110
hybrid zone complexes in the marine environment, where organisms often
posses extended dispersal capabilities due to a long-lived planktonic larval
stage (however see Schopf and Murphy, 1973; Bert and Harrison, 1988). Yet,
the stability of a hybrid zone and the flow of genes and gene complexes across
it is thought to rely on a balance between selection and gene flow (Barton and
Hewitt, 1985; Moore and Price, 1993). In terrestrial systems, where dispersal
distance per generation is often measured in meters or kilometers, tension
zones between taxa can become trapped and stabilized in areas of low
population density (Barton and Hewitt, 1985; Hewitt, 1989). However, in
marine organisms with long-lived planktonic larval stages, dispersal is a
much more potent force and hybrid zones between taxa are likely to be much
more dynamic.
Here we focus on the genetic and reproductive cohesion in a group of
three closely related allopatric West Pacific butterflyfishes, Chaetodon
multicinctus, C. pelewensis and£ punctatofasciatus. Species differ from one
another primarily in color pattern (figure 2.1). In these bright non-cryptically
colored reef fishes, color pattern distinctions are often the only diagnostic trait
between closely related Butterflyfishes, the differences among these species
are greater than among some broadly sympatric species (Burgess, 1978; Blum,
1988; Blum, 1989). As a result, early taxonomist gave each color variant a
species level distinct. Subsequent collections showed little phenotypic
variation and recent revisions of this family have not challenged the original
distinctions (Burgess, 1978; Allen, 1980; Blum, 1988).
However, mtDNA and allozyme variation is partitioned far less neatly
among the three species. Sequences of the mtDNA control-region suggest
that species diverged in allopatry 300,000-800,000 years ago. Yet, these data
111
confirm the evolutionary distinctiveness of only C. multicinctus. MtDNA
variation is distributed randomly between C. pelewensis and C.
punctatofasciatus and argues for extraordinary high levels of hybridization
and introgression of mtDNA variation (Chapter 2). Allozyme variation is
partitioned similarly. Genetic differences between C. multicinctus and either
C. pelewensis or C. punctatofasciatus suggest very low levels of gene flow
(Nm<l). By contrast, allozyme variation is distributed randomly between
populations of C. pelewensis and C. punctatofasciatus separated by over 7500
kilometers consistent with high levels of hybridization and introgression of
many nuclear-encoded genes.
Because color pattern may be important in the reproductive and social
ecology of these brightly colored reef fishes, it may be under strong selection
(Lorenz, 1966; Peterman, 1971; Reese, 1975; Ehrlich, 1977; Kelly and Hourigan,
1983). Adults of all three species form long-lasting male/female pairs that
defend contiguous, non-overlapping, feeding territories and color pattern
may be an important component of mate choice or species recognition in this
group.
We examine the degree of assortative pairing among these species
using both controlled pairing experiments and field observations in zones of
sympatry. In the controlled pairing experiments, pair formation and
behavior of adults was examined using individuals of each species collected
from phenotypically homogeneous populations. In these areas, social
interactions are restricted to phenotypically similar individuals and the
pairing behaviors under controlled experimental conditions probably reflect
innate rather than conditioned discrimination of phenotypic differences. For
C. pelewensis and C. punctatofasciatus we also examined pairs in a narrow
112
zone bounded by Papua New Guinea (PNG), the Solomon Islands (SI) and the
northern portion of the Great Barrier Reef (GBR) where both species co-occur
(Blum, 1989). Several authors have reported seeing fishes with intermediate
color patterns in this area (Randall et al., 1977; Allen, 1980; Blum, 1989; Allen
and Swainston, 1992). Yet the abundance and fate of these reputed hybrids, as
well as behavioral interactions among the two siblings remained unclear.
113
3.3. MATERIALS AND METHODS
Characterization of phenotypic variation
Of the three species, C. multicinctus, the Hawaiian archipelago and
Johnston Island endemic, is phenotypically the most distinct. Although the
underlying vertical body stripes are similar to C. punctatofasciatus, it lacks the
striking yellow wash, the bright orange coloration on the caudal fin, and a
yellow eye band. Chaetodon pelewensis and C. punctatofasciatus are more
similar, possessing nearly identical yellow and orange patterning but differing
in the orientation of the underlying black body stripes which run diagonally
in C. pelewensis and vertically in C. punctatofasciatus.
We have assembled collections of all three species from 13 widely
distributed locations across the western Pacific between 1990 and 1993. Our C.
multicinctus individuals were collected from Midway Island (n=14), Oahu
(n=20) and Molokai (n=70) in the high Hawaiian Islands and from Johnston
Atoll (n=25). Chaetodon pelewensis were collected from Moorea, Society
Islands (n=9), Rarotonga, Cook Islands (n=21), Viti-Levu, Fiji (n=87).
Chaetodon punctatofasciatus were collected from Bali, Indonesia (n=12), the
Philippines (n=28), Palau (n=82), Guam (n=8), and the Marshall Islands (n=l).
In addition, we visually sampled three locations, two in the 51 and one
within PNG, in the zone of overlap between C. pelewensis and C.o
punctatofasciatus. The collection is currently stored in the -70 C freezer at
the Pacific Biomedical Research Center, Kewalo Marine Lab, Honolulu,
Hawaii and is available upon request. Voucher specimens for each
population were deposited at the Bernice P. Bishop Museum, Honolulu,
Hawaii.
114
We augmented our collections with previous collections housed in the
Bernice P. Bishop Museum in Honolulu, Hawaii and the California Academy
of Sciences in San Francisco, California. Most specimens in these museums
were collected within the last forty years and included individuals from
Pitcairn Island (n=8), the Marquesas (n=3), the Society Islands and Tuamotus
archipelago (n=35), the Philippines (n=7), the Caroline Islands (n=5), Guam
(n=3), Palau (n=6), and the Marshall Islands (n=20) (see table 3.1).
We did not observe any striking variation in the phenotype of the C.
multicinctus across its geographic range. However, C. pelewensis and C.
punctatofasciatus showed considerable phenotypic variation. In these two
species, individuals were given a phenotype score between 1 and 5 based on
deviations from the "pure" parental phenotype. Type 1 individuals possessed
distinct and sharp diagonal bands characteristic of C. pelewensis. whereas the
black bars of type 5 individuals ran vertically, typical of C. punctatofasciatus.
Type 2 and 4 individuals were clearly allied with one of the "pure"
phenotypes yet showed slight variations. For example, in a type 4 individual,
some vertical bands bifurcate, muddling the otherwise sharp pattern of the
"pure" C. punctatofasciatus phenotype. Type 2 individuals are similarly
muddled but none-the-less could be clearly classed with C. pelewensis. Type 3
individuals, however, were strikingly intermediate and could not be reliably
classed into either C. punctatofasciatus or C. pelewensis (see figure 3.1). It is
important to note that these phenotype classes did not reflect the underlying
genetics of color pattern, which are unknown in these fishes, but only our
interpretation of pattern differences.
Because these designations reflected differences in the underlying dark
banding pattern they did not fade with preservations and we were able to
115
clearly phenotype fishes fixed in formalin and preserved in alcohol for over
ninety years.
Random mating and pair-bonding experiments
Behavioral experiments
An initial pilot study indicated that strong pairs formed between adult
C. multicinctus collected from different reefs around Oahu within hours after
being released into a large, 2500 gallon, donut-shaped aquarium at the Hawaii
Institute of Marine Biology. The dimensions of this tank were approximately:
6 meters outer diameter, 4.3 inner diameter and a height of 1 meter. This
tank, which lacks corners, allows for the continual forward movement of
individuals. To further approximate natural conditions the bottom of this
tank was lined with small coral heads of Porites compressaJ P. lobada,
Montipora verrucossa, and Pocillopora meanadrina collected from patch reefs
in Kaneohe Bay, Oahu, Hawaii. All four species of coral are common within
the range of C. multicinctus and closely related species, if not the same
species, are common within the range of C. punctatofasciatus and C.
pelewensis (Hourigan, 1987; Hourigan, 1989; O. McMillan, personal
observation). The behavior of pairs in tank experiments was similar to the
behavior of pairs in nature with individuals spending the majority of their
time feeding and swimming in very close proximity to each other.
In the tank experiments, two males of different species were placed in
the tank together with a female that was the same species as one of the males.
The adults used in these experiments were collected from five widely
scattered locations across the Pacific and shipped to Oahu, Hawaii. Chaetodon
punctatofasciatus were collected from Palau or the Philippines, C. pelewensis
were collected from either Fiji or the Cook Islands and C. multicinctus were
116
collected from reefs around Molokai, Hawaii. Each of these regions were well
entrenched within the geographic range of a given species and, with the
possible exception of the Fijian sample, all individuals shared a consistent
phenotype pattern. Within Fijian populations, a small proportion of the
population (less than 10%) showed patterning of stripes along the body wall
that were slightly muddled (type 2) relative to the "pure" C. pelewensis type
(figure 2.1).
Fishes were received via overnight air cargo from July 1992-April 1993.
After clearing U'S. customs, fishes were unpacked and 15-25 conspecifics were
housed together in large, 600 gallon, saltwater holding tanks at Kewalo
Marine Lab, Honolulu, Hawaii. In order to prevent the outbreak of disease,
all fishes were subjected to an eleven day incubation and treatment period.
During this period, individuals were bathed three times for 30 minutes in
1ml of formalin per gallon of sea water. This concentration of formalin was
fatal to the egg, juvenile, and adult stages of many of the common
ectoparasites that threaten these fishes in captivity but did no apparent harm
to the fishes (Stoskopf, 1993). Each of the three formalin treatments was
followed by three non-treatment days. During all time in captivity, with the
exception of the actual pairing experiments, fishes were fed a specialized diet
designed by the Waikiki aquarium.
Following the treatment and quarantine period, fishes were sexed by
catheterization (Ross, 1984). The soft portion of the dorsal fin of females was
marked with a small hole to facilitate identification in pairing experiments.
Fishes were placed into individual 5 gallon holding tanks where they were
allowed to recover for 2-3 days. After this recovery period, two males and a
female of approximately the same size were transported to Hawaii Institute of
117
Marine Biology, released into the donut tank and allowed to acclimate over
night.
Behavioral observations were made by WOM from a blind within the
center of the tank. One-way glass allowed WOM to monitor the behavior of
the group without the fish being aware of his presence. Fishes were observed
during 4 to 5, 10-minute observational periods in which 1) the amount of
time the female spent with each male 2) the amount of time the two males
were together and 3) the aggressive interactions among individuals were
recorded. For these observations, "together" was defined as within 0.25
meters of each other. Aggressive interactions included attacks, chases, and
dorsal fin flexing displays. Each of these observational periods were separated
by one hour. All data were recorded directly using a computer-based events
recorder (BEAST version 3.0, Losey, 1988). Following the experiment, fishes
were collected, weighted, and the standard length ~as measured. Individuals
were only used in one experiment.
Between 5 and 9 trials of all possible combinations of two males and a
female among the three species were examined (table 3.2). These
heterospecific trials were compared to 12 conspecific trials (four per species) in
which two males and a female of the same species were placed in the tank
together. For statistical analysis, the observational periods were grouped and
the mean time for each event was used to characterize a trial. A pair was
considered to have formed if a male and female spent more than 50% of the
observational period within close proximity to one another. The paired-male
was designated as the focal male. In the cases (n=5) when a female associated
with both males more than 50% of the time, the male that she spent the most
time with was declared the focal male.
118
Field observations
In addition to the controlled pair-forming experiments, we examined
natural pairs of C. pelewensis and C. punctatofasciatus in areas where the
ranges of the two species overlap. We established three sites in a transect
across this zone. Two of these sites were from adjacent island groups within
the 51 separated by roughly 150 kilometers. The third was along the north
coast of PNG near Madang, some 1600 kilometers to the west.
The phenotypes of both paired and unpaired individuals were
examined during transect dives. In these dives, the observer (WOM) swam
in one direction maintaining a depth of between 10 and 20 meters below sea
level. He recorded the phenotype of every individual and whether or not
that individual was paired. As noted previously, paired individuals often
swim in very close proximity to each other. In addition, non-overlapping
territories were generally small, 50-100 square meters in areas of high coral
cover, making it possible to positively identify the members of a pair (Reese,
1975; Tricas, 1989). In instances when only a single individual was initially
observed, we continued to monitor that individual for one minute. If no pair
was located then that individual was scored as a solitary individual. During
these dives, we also scored the phenotype of any juvenile individuals that
were encountered. A transect lasted roughly 70 minutes and covered a
distance of approximately 300 meters.
119
3.4. RESULTS
Patterning of phenotypic variation
With the exception of PNG and the SI, our collections were composed
predominately of individuals exhibiting one or the other "pure" phenotype
(figure 3.3). The highest frequency of "non-pure" phenotypic classes occurred
in Fiji, where approximately 10% of individuals showed slight variation from
the "pure" C. pelewensis patterning. Museum specimens from the
Philippines, Palau, and Tahiti collected over 40 years ago showed little
deviation from those collected between 1990 and 1993.
By contrast, within the two locations in the 51 [the New Georgia Islands
(n=64) and the Russell Islands (n=250)] individuals showed almost every
conceivable phenotypic pattern (figure 3.2). Nearly 70% of the individuals
examined had phenotypes that were intermediate between parental types.
Roughly a third of these intermediate phenotypes could not easily be allied
with either parental phenotype (i.e. type 3) (figure 3.1).
Within PNG, individuals showed a distinct sift towards C.
punctatofasciatus-like phenotypes (types 4 and 5). Over half of the 86 adults
observed along the barrier reef off Madang lagoon had a "pure" C.
punctatofasciatus pattern. Of the remaining half, 22 individuals possessed the
similar type 4 phenotype. There were only four individuals with a "pure" C.
pelewensis phenotype.
In both PNG and the 51 deviations within any of the "hybrid"
phenotype classes were extreme and it was clear that our five phenotypic
classes did not adequately categorize color pattern variation. Nor did these
classes take into consideration striking asymmetry in color pattern on either
side of an individual. For example, one individual (not collected) had the
120
pattern of a C. pelewensis on one side and the pattern of C. punctatofasciatus
on the other. We collected and photographed 37 individuals from the 51 and
PNG. From this collection, 8 of the 10 individuals given a type 3 designation
showed very different patterning on either side. The remaining type 3
individuals showed a moderate degree of asymmetry (figure 3.2). Color
pattern asymmetry was less pronounced in type 2 and type 4 individuals. For
these two classes, most (8 of 13) individuals showed a moderate degree of
color pattern asymmetry, 1 showed a high degree of asymmetry, and 4 were
nearly symmetrical. By contrast all"pure" phenotypes had identical, or
nearly identical, color patterns on either side.
Behavioral experiments
Conspecific trials
The interactions among individuals in the trials in which two males
and a female of the same species were placed into the donut tank together
were identical in all three species. In these trials, a single male-female pair
always formed and this pair fed and swam together for on average 82% of the
observational period. In contrast, the female spent less than 1% of the
observational period with the remaining unpaired male (figure 3.4a). This
was because whenever the unpaired male came within visual range, it was
attacked by the paired male. Aggression between males could be quite violent
involving both chases, bites, and spears with extended dorsal spines. The
unpaired male was clearly subordinate. He actively avoided contact with the
pair and it was not uncommon for him to spend most of the observational
period hiding beneath a coral head as far away as possible from the pair. In all
controls, females exhibited very little aggression towards either male (figure
3.4b).
121
In controls there was no indication that size, measured in either
standard length or weight, was a good predictor of which male paired with
the female. In half of the controls the larger male formed a pair with the
female and in the other half it was the smaller male (p>O.25, binomial test).
Heterospecific trials
The results of the 18 trials between individuals of C. punctatofasciatus
and C. pelewensis were qualitatively and quantitatively similar to the
conspecific controls. In these trials, a single male-female pair always formed
and this pair spent nearly 85% of the observational period in close proximity
to each other and away from the remaining unpaired male. There was no
evidence for positive assortative pairing among males and females of C.
punctatofasciatus and C. pelewensis under these experimental conditions
(table 3.3). Nearly as many heterospecific as conspecific pairs formed in these
trials suggesting few pre-mating barriers to reproduction in these two species
(chi-square=O.277, p=0.27 based on 500 randomizations, Roff and Benson,
1989). The behavioral interactions among males were identical to the
interactions among conspecific males (chi-square=1.42, p=0.464; z=-0.934
Mann-Whitney U-test, p=0.3498). In all but one trial, when the two males
were observed to come in contact, the paired male was extremely aggressive
towards the unpaired male (table 3.4). This high level of intra-male
aggression was particularly telling because it suggested that the two species
failed to distinguish between hetero- and conspecific color patterning.
Six 'out of 8 heterospecific pairs occurred between a male C.
punctatofasciatus and a female C. pelewensis (Fisher's exact test, p=O.OOl).
However, it is unclear if this result was a function of chance, was attributable
to a better physical condition of C. punctatofasciatus males or reflected some
122
innate dominance of C. punctatofasciatus over C. pelewensis. Neither size
nor a simple measure of condition (weight/length ratio) played an obvious
role (p>O.25, binomial test).
By contrast, the Hawaiian endemic, C. multicinctus, exhibited a much
higher degree of behavioral isolation. In trials between C. multicinctus and C.
pelewensis only a single heterospecific pair formed, indicating a high degree
of assortative pairing (table 3.3) (chi-square=7.543, p=O.001 based on 500
randomizations). This pair was between a male C. pelewensis and a female C.
multicinctus. In addition, males were often less aggressive towards
heterospecific males than they were to conspecific males in the control trials
(chi-square = 8.896, p=0.004) (table 3.4). This effect was variable, however, and
was only seen in 6 of the 10 trials where contact between males was observed.
In these trials, less than 2.0% of the time that males were in contact was spent
in aggressive posturing. In the remaining 4 trials, levels of aggression were
high and identical to what was observed in conspecific trials. The two types of
reactions suggest that, although there was some degree of
behavioral/reproductive differentiation between these two species,
differentiation was not absolute.
A similar, but slightly more complex, pattern was evident in the
behavioral trials between C. multicinctus and C. punctatofasciatus. In 4 of the
13 trials, a heterospecific pair formed and assortative pairing was not clear
(chi-square=3.343; p=0.06 based on 500 randomizations). In each case, the
heterospecific pair was between a female C. punctatofasciatus and a much
larger male C. multicinctus. As in the C. pelewensis and C. multicinctus trials
there were significant differences in the levels of male/male aggression
compared to conspecific controls (table 3.4) (chi squares=9.976, p=0.03), though
123
again, there was a clear bimodality to the responses of males towards one
another. Either they were highly aggressive or they very nearly completely
ignored each another.
Behavior of pairs in zones of overlap
Theories for the reinforcement of pre-mating isolation predict a higher
degree of species discrimination in sympatric relative to allopatric
populations (see Butlin, 1989). Thus it becomes important to verify the
pattern of random pairing among C. pelewensis and C. punctatofasciatus
observed among allopatric individuals with observations of the pattern of
pair formation in areas where both species co-occur.
Nearly eighty-five percent of the 306 adults scored in the 51 were
observed as pairs. There was no significant difference in the phenotypes of
individuals that were paired versus individuals that were not paired (figure
3.5) (chi-squared=3.038 and 5.04, p=0.55). In addition, in the 130 pairs
examined there was no compelling evidence for positive assortative pairing
among similarly patterned fishes (figure 3.6) (chi-squared=5.040, p=0.98). In
other words, an individual's phenotype was not correlated with whether or
not it was paired and, if paired, with the phenotype of it's partner.
We might expect any pattern of assortative mating to be strongest for
"pure" phenotypes. This is because, assuming color pattern is polygenetic,
individuals with "pure" phenotypes are more likely to be recruits from
"pure" parental populations. Thus, any innate preferences for assortative
mating may not have broken down by repeated hybridization and
backcrossing. There were a total of fifty-one pairs containing a "pure" C.
pelewensis phenotype. Within this group there were 10 instances in which
two "pure" individuals were paired, 13 instances in which a "pure" type 1
124
individual paired with a phenotypically similar type 2 individual, and 29
cases in which a "pure" C. pelewensis phenotype paired with types 3-5. This
distribution was not significantly different from that expected under a model
of random pairing (chi-square=1.4, p=0.49) suggesting few barriers to
reproductive isolation. There were too few individuals of "pure" C.
punctatofasciatus phenotypes (type 5) in these two populations to do a parallel
type of analysis.
Within PNG, although the majority of individuals possessed C.
punctatofasciatus-like phenotypes (type 4 and 5), there was no compelling
evidence for positive assortative mating. Individuals with "pure" C.
punctatofasciatus phenotypes were just as likely to be paired with a similar
phenotype (type 4 or 5) as with an individual with a hybrid or C. pelewensis
like pattern (chi square=0.42, p=O.81) (figure 3.7).
125
3.5. DISCUSSION
Among the three similar species studied, only C. multicinctus appears
to have achieved the evolutionary, genetic, and reproductive cohesiveness
consistent with its species-level designation. Chaetodon pelewensis and C.
punctatofasciatus, show only superficial differences at both allozyme and
mtDNA loci (Chapter 2 and Appendix C). Coupled with the lack of
differentiation at presumably neutral genetic markers is a lack of pre-mating
barriers to reproduction. Pairing, the basis for mating in these fishes (Reese,
1991; but see Lobel, 1989) appears to be random among individuals collected
in allopatry and those observed in sympatry. These genetic and behavioral
results suggest that there is little inhibiting the movement of genes between
these two species, and provide no compelling evidence to suggest that either
has achieved the evolutionary cohesion to merit their species-level
distinction.
Despite this conclusion, color pattern distinctions remain true across
the vast majority of each species' range (figure 3.3). Levels of gene flow
between Philippine populations of C. punctatofasciatus and Fijian
populations of C. pelewensis are far higher than levels expected to prevent
the accumulation of neutral differences by genetic drift (Appendix C). Yet,
these populations show completely different distributions of phenotypic
variation, with all 30 individuals collected from reefs around the Philippines
possessing the "pure" C. punctatofasciatus phenotype and all individuals
(n=88) from Fiji possessing a C. pelewensis-like phenotype. Plotting the
proportion of "pure" C. pelewensis-like color pattern in localities along a
roughly linear transect across the tropical Pacific yields a "sharp" change in
the distribution of phenotypes between Fiji and the 51 (figure 3.8). By
126
contrast, the proportion of one of the two main mitochondrial lineages in
these same populations changes only slightly over 10,000 kilometers. These
incongruent patterns suggest two different possibilities. Either color pattern is
not genetically determined in these fishes and thus does not reflect
evolutionary history, or selection directly on color pattern, and probably little
else, is maintaining phenotypic integrity while other markers move freely
between "species."
Environmental cline
In many fishes, color pattern is known to be highly heritable and can be
controlled by a single gene or by a suite of many autosomal and sex-linked
genes (McPhail, 1969; Endler, 1980; McAndrew et al., 1988; Teve et al., 1989).
As a result, it has always been assumed that color pattern in butterflyfishes is,
likewise, under genetic control. However, little is known about the genetics
of color pattern in this family, raising the possibility that color pattern is at
least partially determined by environmental conditions. It is difficult to
imagine an environmental gradient that would maintain tight control over
color pattern from the Pitcairn to Fiji (some 5000 kilometers), change so
abruptly, and then be constant again from PNG to the Marshall Islands and
Indonesia. Temperature, which has been shown to have an effect on pattern
formation of butterfly wings, shows little systematic change across the tropical
Indo-west Pacific (Reynolds, 1988).
The only obvious environmental difference among reefs across this
area is a strong west-east attenuation of species diversity (Stehli and Wells,
1971; Rosen, 1988; Belasky and Runnegar, 1993). Coral diversity drops from
over 68 genera on reefs within the shallow seas surrounding the Philippines
to less than 34 genera on reefs in Tahiti (Rosen, 1988). The "species" are
127
obligate corallivores, and diet may shift in different parts of the Pacific. Diet
has been shown to effect color pattern in many aquarium kept fishes, albeit
subtlety, and it is possible that some food item, or lack thereof, is contributing
to the patterning of phenotypic variation (B. Carlson, pers. comm.).
Several observations argue against diet shifts as a major factor
contributing to the strong color pattern gradient. First, a number of closely
related coral feeding butterflyfishes show no variation in color pattern across
this region. Second, these "species" are coral feeding generalists, feeding on
most corals within their territory in roughly the proportion of their
abundance (Hourigan, 1987). Many of the most abundant corals in outer reef
habitats where these species are found are distributed broadly from Indonesia
through French Polynesia (Veron, 1986). Lastly, individuals with very
different phenotypes often occupy the same territory and presumably feed on
the same suite of coral species. Thus, it seems unlikely that adult diet
influences color pattern in these fishes. However, color pattern crystallizes
quickly upon settlement and newly recruited juveniles possess adult
coloration, suggesting that color patterns may be fixed at or prior to settlement
(McMillan, personal observation). As a result, differences in larval and very
early juvenile diet may be contributing to the observed phenotypic gradient.
Secondary contact and hybrid zone dynamics
On the other hand, color pattern in these butterflyfishes may indeed be
under genetic control. The genealogical pattern of extant mtDNA variation
in this group is most consistent with a scenario in which the color pattern
differences that define species arose in allopatry between 300,000-800,000 years
ago (Chapter 2). Thus, the extreme phenotypic variation in the SI and PNG
may represent hybridization following secondary contact. In this case, the
128
obvious implication of the strong geographic structure to phenotypic
variation in the absence of mtDNA and allozyme differentiation is that
selection is preventing the erosion of color pattern outside the zone of
secondary contact and in the presence of potentially homogenizing levels of
gene flow.
Although it is unclear how color pattern is inherited in these fishes,
the extreme phenotypic variation, including the observation of asymmetrical
color patterns on either side of many intermediate classed individuals, argues
that color pattern is probably under complicated genetic control involving
multiple gene loci and epigenetic developmental pathways. The asymmetry
in color pattern is particularly interesting because it is often associated with
developmental instability resulting from both environmental and genetic
stress (Palmer and Strobeck, 1986; Leary and Allendorf, 1989). Hybrids often
show increased morphological asymmetry relative to parental populations,
presumably due to the break-up of intrinsic co-adaptive gene complexes
(Leary et al., 1985; Zakharov, 1981, Graham and Felley, 1985; Ross and
Robertson, 1990; Markow and Ricker, 1991; but see Lamb et al., 1990).
Asymmetry is more prevalent in hybrids from genetically distinct parental
taxa. In these cases, the asymmetry is evident in a many morphological traits
and is often associated with reduced viability (Leary and Allendorf, 1989).
Presently, it is unclear if the pattern of asymmetry extends to other
morphological characters as well, or is restricted to color pattern in these
butterflyfishes. However, the shear number of hybrids, coupled with the
observation that individuals with hybrid phenotypes are paired and the high
levels of "interspecific" gene flow suggested by the pattern of mtDNA and
allozyme markers argue that hybrids between the two color forms are viable.
129
In many respects, the behavioral, genetic, and phenotypic patterns in
these fishes parallel the patterns reported in well studied hybrid zones in
neotropical butterflies and North American birds (Moore, 1987; Moore and
Buchanan, 1985; Mallet and Barton, 1989a and 1989b; Moore et al., 1991;
Mallet, 1993; Moore and Price, 1993). In zones of sympatry, mating between
races of Heliconius erato butterflies and the red and yellow-shafted flickers,
Colaptes auratus, is random and hybrids are viable (Mallet, 1993; Moore and
Price, 1993). In addition, with the exception of wing coloration in races of
Heloconius and plumage coloration in northern flickers, parental
populations show only slight differences at presumably neutral genetic
markers (Grudzien and Moore, 1986; Grudzien et al., 1987; Mallet, 1993).
In both Heloconius butterflies and northern flickers, strong frequency
dependent selection is envisioned to be contributing to the discrepancy
between phenotypic and genetic variation. For Heliconius, differential
predation on oddly patterned butterflies is thought to be maintaining the
distinction between races. This selection has been measured and shown to be
quite high, nearly 52% against foreign rnorphs (Mallet and Barton, 1989b). For
northern flickers the mechanism of frequency dependent selection is less
clear. Although red- and yellow-shafted flickers differ primarily by plumage
characteristics, the hybrid zone very neatly follows several ecological
gradients (Moore and Price, 1993). Moore and Price (1993) proposed that both
ecological selection against hybrids and frequency dependent social selection
on plumage traits contribute to the narrowness of the cline between
subspecies. In either case, average selection against loci involved in or closely
linked to plumage traits must be high to explain the width of the hybrid zone
(Moore and Price, 1993).
130
Similar frequency-dependent selection regimes may operate in these
butterflyfishes. Under this model, the ability of a fish to survive and
reproduce must depend on its color pattern relative to the color pattern of
other conspecifics on the reef where it settles. In this case, two obvious
questions need to be explored. First, how strong is selection on color pattern,
and second, how is it acting? For the first question, previous theoretical
models can provide a reasonable estimate of selection preventing the erosion
of color pattern given the width of the hybrid zone and some idea of dispersal
distance in these fishes. As for as the second question, these results only
begin to dissect the nature of selection operating on color patterns in these
fishes.
How strong is selection on color pattern?
For the dynamics of a wide variety of hybrid zones, the width, defined
as the inverse of the maximum slope of change between alleles or polygenetic
traits, is a function of dispersal potential and the strength of selection (Barton
and Hewitt, 1981; Szyrnura and Barton, 1986; Barton and Gale, 1993; Moore
and Price, 1993). For a hybrid zone maintained by frequency dependent
selection, simple mathematical models suggest that effective selection, ~
acting on a typical gene involved in the determination of a polygenetic trait is
roughly equivalent to (2.8cr/w)2, where q is the average dispersal, defined as
the standard deviation in parent to offspring distance, and w is the width of
the cline (Barton, 1983; Mallet and Barton, 1989a; Moore and Price, 1993).
Although our collections are widely scattered, the change in proportion of
"pure" C. pelewensis-like phenotypes across this transect suggests a hybrid
zone width of approximately 3000 kilometers (figure 3.8).
131
This spatial scale is at least an order of magnitude broader than most
terrestrial hybrid zones (Harrison, 1990). For example, the width of hybrid
zones in races of Heliconius butterflies are 10-100 kilometers (Mallet, 1993).
Similarly, in flickers, the hybrid zone between the red-and yellow-shafted
subspecies seldom exceeds 300 kilometers. However, the dispersal potential
of these fishes is likely orders of magnitude greater than these terrestrial
examples. For example, in Heliconius butterflies, estimates of dispersal
distance range between 0.7 and 2.3 kilometers per generation (Mallet, 1993).
In flickers, although some juveniles are known to disperse up to two
hundred kilometers, most fledglings nest near the parents and adults show
strong breeding site fidelity. These birds, likewise, show marked population
structuring at both mitochondrial genes and allozymes (Moore and
Buchanan, 1985; Grudzien and Moore, 1986; Grudzien et al., 1987; Moore et
al., 1991). In contrast, butterflyfishes have a larval stage that drifts in the
plankton 40-60 days before settling (Tricas, 1987; Hourigan and Reese, 1987;
Leis, 1989). Although the details of larval dispersal in these fishes are
unknown, this stage is known to facilitate transport of larvae of other marine
species with similar long-lived planktonic larval stages thousands of
kilometers. For example, Caribbean reef fishes are commonly transported as
far north as New York, some 2000 kilometers from the nearest reproductive
populations (Allen, 1980). Similarly, during el Nino years, waves of Indo
west Pacific marine organisms settle in the Galapagos nearly 5000 kilometers
from the nearest source population (Richmond, 1990). Strong directional
currents certainly aid dispersal in these cases; however, these examples serve
to underscore the scale of dispersal in marine organisms with planktonic
larval stages.
132
Assuming a moderate dispersal distance of 200-500 kilometers per
generation for these fishes, we estimate a per locus selection coefficient acting
against homogenization of color pattern genes of between 0.04 and 0.22. If
only three loci are involved in the determination of color pattern in these
fishes then total selection acting against the introgression of color pattern may
be as high as 66%. Clearly our estimation of selection on color pattern genes
is sensitive to our assumption about dispersal in these fishes. However,
given complete homogenization of allozyme and mtDNA variation and high
estimated levels of migration between locales separated by 7500 kilometers,
we believe that this estimation is conservative.
The nature of selection on color pattern in butterflyfishes
If our estimation of dispersal distance is correct then selection
maintaining the observed gradient in color pattern is quite strong. The
evolutionary and ecological significance of the vivid, non-cryptic coloration
in butterflyfishes, as in most reef fishes, is poorly understood (Lorenz, 1967;
Peterman, 1971; Brockman, 1973; Potts, 1973; Ehrlich et al., 1977; Allen, 1980;
Kelly and Hourigan, 1983; Nuedecker, 1989). Butterflyfishes have a well
developed visual cortex and visual cues are thought to be important in both
inter- and intra-specific recognition (Peterman, 1971; Reese, 1975, 1989; Snow
and Rylander, 1982; Bauchot et al., 1989). Many teleosts can neurally or
hormonally alter color pattern and these changes have been demonstrated to
be important in intra-specific communication (Barlow, 1963; Lanzing and
Bower, 1974; Nelissen, 1976; Hailman, 1979; Rowland, 1979). Although
butterflyfishes are sexually monomorphic and capable of only slight
modifications in color, bright coloration may similarly be an important intra
specific social signal for either species-recognition, mate attraction (epigamic
133
sexual selection), or intra-sexual selection (Zumpe, 1965; Lorenz, 1966;
Peterman, 1971; Reese, 1975). Alternatively, bright conspicuous coloration in
butterflyfishes may have evolved as aposomatic warning, or disruptive
signals directed towards potential predators (Gosline, 1965; Neudecker, 1989).
What do the behavioral and genetic patterns reveal about the nature of
color pattern in this group? First, the higher degree of behavioral and genetic
isolation in C. multicinctus suggests that divergence in color pattern can be
paralleled by the evolution of assortative pairing either through species
recognition or mate selection. However, the random pairing among C.
punctatofasciatus and C. pelewensis argues that color pattern differences are
not providing similar cues in these two species. Our behavioral experiments
extend this conclusion to phenotypically homogenous allopatric populations.
Though neither the field observations nor our controlled pairing
experiments reveal all the forces underlying pair-formation in these species,
they clearly suggest that mate choice or species recognition cues based on color
pattern are not playing a strong role. It therefore seems unlikely that the
original divergence of color pattern in this group was coupled with strong
inter-sexual selection or pressure for species recognition.
It is possible that intra-sexual social selection, similar to that proposed
to maintain differences between red and yellow-shafted flickers, may be
maintaining the color pattern differences in these butterflyfishes. In this case,
strong selection among individuals of the same sex for territorial space
provides a likely mechanism. For C. multicinctus, the establishment of a
territory is thought to be a prerequisite for successful reproduction (Tricas,
1987; Hourigan, 1987). Removal experiments suggest that competition for
open territorial space in C. multicinctus is fierce. When one individual of a
134
pair of C. multicinctus is removed, the remaining individual quickly
establishes a new mate (Hourigan, 1987). Similarly, after a pair is removed, a
new pair quickly establishes itself within the bounds of the original territory
(Hourigan, 1987). These results indicate a substantial population of "floater"
individuals waiting for space to become available and suggest that intra
sexual competition for space may be quite strong (Hourigan, 1987; 1989).
Such strong intra-sexual competition was evident in our pairing
experiments among all three species. The outcome of these experiments was
determined essentially by male-male encounters. One male is clearly
dominant and the female always associates with him. In a number of
experiments (n=5) when two heterospecific males were used in a second
experiment, this time with a female from a different species, the original
dominance hierarchy was maintained and the new female paired with the
dominate male. Likewise, in experiments in which two females and a male
were added to the tank, there was strong female/ female aggression and the
pair formed between the male and dominate female (n=3) (McMillan,
unpublished data). Similar evidence for strong intra-sexual competition
comes from field observations of C. multicinctus. In this species, territorial
defense is directed primarily towards members of the same sex (Hourigan,
1989). Because both males and females compete for open space in these
species, intra-sexual social pressures on color pattern would extend to both
sexes.
Thus, it is possible that color pattern in these territorial reef fishes
evolved under strong intra-sexual social pressure (sensu, West-Eberhard,
1983) and has failed to erode because individuals that settle outside the range
of their conspecifics fail to establish a territory. In the one-on-one social
135
situation set up in the pairing experiments, one male was able to dominate
the space independent of color pattern. In the more complex social network
of a coral reef, many individuals are likely competing for a limited number of
open territories. Under these situations, oddly marked individuals may not
provide the necessary social signals to keep other fishes competing for
territorial space from continually attacking and eventually displacing them
from territories. Color pattern is thought to be an important social signal in
many avian species and changes in social status often follow experimental
manipulation of plumage characters (Butcher and Rower, 1987). For example,
the red epaulet of the male red wing blackbird is important in territorial
defense. In this species, males set up and defend breeding territories from
other males. If the epaulet is blackened, intrusion into a male's territory
increases and he is frequently displaced (Smith, 1972).
Observations in the 51, where phenotypic variation is extreme and
unrelated to an individual's ability to hold a territory, clearly indicate that the
frequency-dependent selection on color pattern can break down. But, it is the
rapidity at which a break down occurs that is the critical dynamic of this
theory. In short-lived species, where adult turn-over is high, the social
dynamics of a community may change quickly. However, in a long-lived
species, like these butterflyfishes, where pairs are stable and can maintain the
same territory for more than four years, the social dynamics of a community
may change much slower (Hourigan, 1989; Reese, 1991). Under these social
dynamics, frequency-dependent selection against the odd phenotype may
erode more slowly relative to the flow of neutral genes.
In PNG, C. punctatofasciatus-like phenotypes (type 4 and 5) dominate
the population but adults with odd phenotypes hold territories and mating
136
appears to be random. It is possible that the proportion of odd-colored
individuals in this population, roughly 27%, has exceeded some threshold
beyond which frequency-dependent selection is no long acting. Alternatively,
it is possible that recruitment of the juvenile fish and their growth into the
adult population is what is color-type dependent. Predation, which has
shown to be a powerful force in maintaining a narrow hybrid zone among
races of Heliconius butterflies, may be playing a role in the pattern of
phenotypic variation in these fishes, especially among juveniles.
Butterflyfishes are underrepresented in the gut content of piscivorous fishes
(Hiatt and Strasburg, 1960; Hobson, 1974; Hourigan, 1987). Their laterally
compressed bodies and sharp dorsal spines are thought to make them difficult
to eat and has lead to the suggestion that the bright, vivid colors serve as an
aposomatic advertisement to potential predators (Gosline, 1965; Neudecker,
1989). Indeed, chaetodontids do not flee when large predators are nearby.
Instead, they turn sideways and display their lateral body markings
(Neudecker, 1989; McMillan, personal observation). Thus, it is possible that
oddly patterned juveniles are more heavily preyed upon, inhibiting the
erosion of color pattern differences.
Conclusions
It is clear that despite being tightly coupled reproductively and
genetically, C. pelewensis and C. punctatofasciatus, none-the-less. remain
uncoupled phenotypically. Is the hybrid zone between the two forms
stationary, as the hybrid zone between red- and yellow-shafted flickers (Moore
and Buchanan, 1993), or are the color forms in the process of homogenizing?
The patterning of phenotypic and genetic variation provided by this study
represents but a single point in the history these forms that likely emerged
137
hundreds of thousands of years ago (Chapter 2). Given that the dynamics of
hybrid zones are tightly coupled to dispersal, this hybrid zone between these
high dispersal forms is likely to be quite turbulent. Further study can only
help elucidate the forces shaping morphological and genetic variation in
these butterflyfish.
138
Table 3.1: Specimens of Chaetodon punctatofasciatus and C. pelewensis
examined from collections at the California Academy of Science, San
Francisco, California and the Bernice Pauley Bishop Museum, Honolulu,
Hawaii.
Location no. Collection Dates Catalogue numbersMarshall Is. 20 1967-1970,1986,1972 BPBM 1776, 6245, 6298, 8230, 8244,
10782-10783; CAS 58959, 42230
Palau 6 1966-1970;1955-1956 BPBM 6841, 8294,10192; CAS 37896-97
Guam 3 1968;1922 BPMB 4189, 8451
Caroline Is. 5 1953, 1959 CAS 37898
Philippines 7 1901, 1931-33, 1949 SU 9609, 25791, 29899;CAS 52775
Society Is. / 35 1956-58;1967-1971 BPBM 6053, 6070, 6120,8357, 13569,
Tuamotus 17264; CAS 37899-902
Pitcairn I 8 1970-1971 BPBM 13245, 16555, 16937
Marquesas 3 1971 BPBM 11670, 11769
139
Table 3.2: Number of pair-formation experiments matching a single female
with a conspecific and a heterospecific male.
,C. multicinctus C. peJewensis C. punctatQfasciahls
6C. multi./C. pele.
fII' C. multi.ZC. punct,
C. pele. / c. punct.
5
6
140
9
7
9
Table 3.3: Results of controlled pairing experiments matching a single female
with a conspecific and a heterospecific male for each of the three possible
hetero-specific comparisons. ,a)
C. pelewensis
~ C. punetatofasciatus
C. pelewensis C. punctato.
3 2
6 7
,b)
C. pelewensis
ti'c I"-=. mu ticmctus
C. pelewensis C. multicinctus
5 1
0 5
,c)
C. multicinctus
ti' c. punetatofasciatus
C. multicinctus C. punctato.
6 3
0 Aq
141
Table 3.4: Summary of the results of pair-bonding experiment among the
three species. For this table, levels ~f male/male aggression within
heterospecific and control trials was categorized into strong (>50% of the time
males were within visual contact they were involved in aggressive displays)
and slight «30% of the time that males were in visual contact were they
involved in aggressive displays). "nc" signifies that no male/male contact
was recorded.
# hetero. MALE/MALE AGGRESSION# trials pairs Strong nc slight
Con trols 12 9 0 3e. punctato.ZC, pele. 18 8 12 1 5e. pele./ e. multi. 11 1 . 4 1 6**e. punctato'/c. multi. 13 4 3 3 7**** denotes a significant difference in levels of male/male aggression at the0.01 level between heterospecific and conspecific trials based on a chi-squaredtest. For this test, the probability density curve of possible chi-squared valuesfor a given sample size was estimated by 500 randomizations (see Rolf andBenson, 1989). If the observed chi-square value was greater than 495 of therandomly generated values then a significant difference was assumed. MannWhitney U-tests in which the actual proportion of time males were involvedin aggressive displays yielded identical results.
142
Figure 3.1: Plate of the three hybrid phenotypic classes. Type 2 individuals (a)
resemble the phenotype of C. pelewensis but with definite deviations from
the typical diagonal banding pattern. Similarly, type 4 (b) individuals
resemble C. punctatofasciatus but with slight muddling relative to normally
distinct vertical bands. Type 3 (c) individuals are striking in their variation
and can not be confidently allied with either of the two parental types.
143
Figure 3.2: Color plate of individuals collected in a) the Russell Islands, SI b) New
Georgia Islands, SI and c) Madang, PNG.
145
Figure 3.2: Color plate of individuals collected in a) the Russell Islands, 51 b) New
Georgia Islands, 51 and c) Madang, PNG (continued).
147
Figure 3.2: Color plate of individuals collected in a) the Russell Islands, 51 b) New
Georgia Islands, 51 and c) Madang, PNG (continued).
149
Figure 3.2: Color plate of individuals collected in a) the Russell Islands, 51 b) New
Georgia Islands, 51 and c) Madang, PNG (continued).
151
Figure 3.2: Color plate of individuals collected in a) the Russell Islands, 51b) New
Georgia Islands, 51 and c) Madang, PNG (continued).
153
......UlUl
Figure 3.3: The geographic distribution of the five phenotype classes of C. punctatofasciatus and C.
pelewensis. The wheel represents the proportion of each of the five classes observed within a given
location: ., "pure" C. pelewensis (type 1);~ , type 2; 1m, type 3; II , type 4 and; D, "pure" c.
punctatofasciatus (type 5) (see figures 2.1 and 3.1). Locations with an asterisk (*) beside the sample size were
augmented with museum specimens (see table 3.1). The smaller circles represent regions where 5 or fewer
individuals were examined.
2
3
4
C. pelewensis
.~
C. multicinctus
Marshall Is.(n::21")
Kilometers
·f'!' ".(n=87) . ...•. I . ta.e .• Pitcairn (n=11 )'~I., .
sOlom~n':i~n"31"1' , , Society. ,.(n~4~'i N"': Cook Is.(n=21) .
r-----. •
o 2000
c. punctatofasciatus
.....~
Figure 3.4: Results of pairing experiments among conspecifics. Figure 3.4a
depicts the amount of time female spent with each male during conspecific
pair-forming trials. In 3.4b, levels of aggression, defined as the proportion of
time two individuals were in visual contact and involved in aggressive
displays, are plotted for each of three possible pairwise interactions.
157
a) 1.0'"00.-I-<OJ 0.8P..-res~
.90.6.....
res:>I-<OJen. ..00 0.4.......0~0.- 0.2.....I-<0P..0I-<~ 0.0
Female with Female withfocal male non-focal male
b) 1.0-r----------------------,.....ures'E 0.88s::.-OJ 0.6.9............os:: 0.4.9.....I-<oP..o 0.2t
o.o~--Aggression
towardsnon-focal maleby focal male
Aggressiontowards
non-focal maleby female
158
Intra-pairazzrcssionbb... - .............. v ..
30-.-----------------r-----,paired
unpaired
25
20s::0..........C'a
.-<;j0.. 1500......0
~10
5
o1 2 3
Phenotype class
4 5
Figure 3.5: Histogram of the proportion of the 5 different phenotype classes
among individuals that were paired (blacken bars) versus the proportion of
the 5 different phenotype classes among individuals that were not paired
(shaded bars).
159
• observed-~., II expected-
:ll~lmn
0;:;::
~I~ lI!'..WI ~r ::;:;:
~H: ;..
;:::;:: :~:::: ;::::;: :\~- ~?~ {=:= :.::; :;::::::j::;: .:. :;::::: ::::. ::;:: ~~r
'.' ;:;::: .;::;)(if] :;:;:: :;:{ ;jf :::::;
- t=: .:::. ....:: .::;:;,-.:: ::::::. "::::: f\]~:t w~:: :;::::: :::;:;:
;:::;:; :::. :::::::~~r:;::;: :::::: .::::> :~f : ;
- ::::;: ::::.:. ;:;:;:: : ; ; ::t!1!1!l:
:;:;:!: ;:;:: '.:.:
h ::.::'~ j:~: \W
:~~~i~:f:· :;:::::
I:.::::: f".. \!j ;~i~ii~
~.- ::::;::;/
::;:::;t1~ t:~ ~~t "'.jf= ::::::; )j:
'.' [~j~~:~ ::;:;:; :;:;: .:.::: :;:;:; {j] ;:;::j:!.f:~.
.:.'::r
i::~::r }j~
::::;: r:j~ .:. ::::.- :':..- :::.,: ::::::: ".'::: :tj: fj: :::::: "'.
I
:::::: -x. j:j;::; :f(j :ff .:.; :.::=- I",
lilll:i,:'
:f: .:;::.: :::;:: :;:;:: :j:::; ::::~: ;::;: ~:~::
i'.'::
f:~: :!!t!:lll
if!!;:::::j;:
~:t (:: :::~~~ i~j~~- !!!i]t::::::: :::.:. ~t~::::::: .:.:.:;
~:f'.' :::;::. ::::::: :;:;~ I:
.;.:.;
:~irr:::::;.
I.;. :;:::::
2 r~: uma ?r a .:::::: a 2 II,Jj ::::::- - ~ .... l- I- l- I- - ::::: l- I-I
1/1 1/2 1/3 1/4 1/5 2/2 2/3 2/4 2/5 3/3 3/4 3/5 4/4 4/5 5/5
Phenotype of individuals in pairs
18
16
14trJ1-4"; 12c,'010
1-4Q) 8
164
2
o
Figure 3.6: The observed (blacken bars) and expected (shaded bars)
distribution under a model of random mating of the 15 possible types of pairs
between individuals of C. pelewensis, C. punctatofasciatus, and hybrids in the
Solomon Islands. Expected distributions for each of the 15 possible were
calculated assuming that the observed proportion of each phenotypic class
within the Solomon Islands represented the underlying pool of adults. For
this estimation, we grouped adults from both locations within the Solomon
Islands. We felt justified in pooling these samples because there were no
significant differences in the distribution of phenotypes between the two
Island groups (chi-square=6.359, p=0.17).
160
• Observed
[ill] Expected
24,-----------;=:============::1,22
20
rJ) 18J-<.....cU 16
0...'0 14
aJ 12
S 10
Z 8
6
4
2
o
Phenotype of individuals in pairs
Figure 3.7: The observed (blacken bars) and expected (shaded bars)
distribution of combinations of "pure" C punctatofasciatus phenotypes paired
with other "pure" C punctatofasciatus phenotypes, type 4 phenotypes, or
hybrid/C pelewensis -like phenotypes in New Guinea. The expected
distribution under random mating was calculated as explained in figure 3.6.
161
1
.9Q)
~g .8Q)
...t:~ .7....tilt::~ .6Q).....Q)
P!.
rJl .5
e~ .4P-.
.......o .3t::o
1:o .2P-.e
P-. .1
o
Palau
2000 4000 6000
Cook Islands Pitcairn Island
1Tahiti
.9
.8
-e.7 ~
t"ClClJs::
.6 ~Zc
.5 S......0
.4 s::.9.....I-<0
.3 P..0I-<c,
.2
.1
08000 10000 12000
Distance from Bali,Indonesia(kilometers)
Figure 3.8: Change in the proportion of "pure" C. pelewensis phenotypes
(blacken circles) (type 1) plotted against the distance (in kilometers) from Bali,
Indonesia compared to change in the proportion of one of the two distinct
mtDNA lineages (shaded circles) revealed in our recent analysis of mtDNA
variation in these two species (see Chapter 1).
162
APPENDIX AA 495 bp portion of the mitochondrial cytochrome b gene for 18 Chaetodonspecies. C. tinkeri was arbitrarily chosen as the representative sequence.Sequences are from 5'-3' and begin at the first codon position. Numbering isaccording to the human mitochondrial sequence (Anderson et al., 1981). A"." represents sequence identity. Missing nucleotide information is denotedby a "?".
Species Sequence ...
•• T
.. T
• .T· .T
· .T.. T
• .A• .A• .A· .A· .A
• .A• .A• .C
.. C• .C
• .C• .C
??
• .C
• .C
• .C
· .C.. C
• .C
??? ??? ??? t .,
•• C
•• T•• T
[149661GTT GCC ACA GCA TTT TCT TCT GTC GCC CAT ATC TGC CGA GAC GTC[45]
.??
.??
.??
.??
C.tinkeriC.declivis wilderiC.flavocoronatusC.burgessiC.declivis wilderiC.mer/C.xanC.mer/C.xanC.mertensiC.madagascariensisC.paucifasciatusC.ar:gentatusC.miliarisC.multicinctusC.mult/C.pun/C.peleC.punctatofasciatusC.pelewensisC.gutatissimusC.auriga
AAC TAC GGC TGA TTA ATC CGC AATC.tinkeriC.declivis wilderiC.flavocoronatusC.burgessiC.declivis wilderiC.mer/C.xanC.mer/C.xanC.mertensiC.madagascariensisC.paucifasciatusC.argentatusC.miliarisC.multicinctusC.mult/C.pun/C.peleC.punctatofasciatusC.pele~lensis
C.gutatissimusC.auriga
.. T
.. T
.. T
.. T
.. T
.. T
.. A• .A.. A• .A• .A
• .T.. T .. A .. C
.. T .. A
.. T .. A
.. T .. A
.. T .. A
.. T .. AC.. ..A .. C
[15011]CTT CAC GCC AAC GGT GCC TCC[90]· .C
• .C.. C• .C.. C
• .C
• .C
· .C
· .C.. C.. c ??? ???.. C.. C
· .C
• .C.. C• .C
163
APPENDIX A (continued)A 495 bp portion of the mitochondrial cytochrome b gene for 18 Chaetodonspecies.
[15056JC.tinkeri ATA TTC TTT ATC TGT ATT TAC ATG CAC ATC GGC CGA GGA CTT TAT[135]C.declivis wilderi ., TC.flavocoronatus ., TC.burgessi .. TC.declivis wilderi .. TC.mer/C.xan · .C · .T · .TC.mer/C.xan .. T ooTC.mertensi · .T ooTC.madagascariensis · .T · .T · .CC.paucifasciatus · .T · .T · .CC.argentatus .. C · . T · .CC.miliaris · .T · .A · .T · .? · .CC.multicinctus Goo .. T ooA · .TC.mult/C.pun/C.pele G.. .. T · .A · .TC.punctatofasciatus G.. ooT · .A · .TC.pelewensis G.• ooT · .A ooTC.gutatissimus G.. ., T · .A · .T · .CC.auriga · .C · .C ooC · .T C.T · .T · .T · .C · .C
[15101)C.tinkeri TAC GGC TCA TAC CTT TAT AAA GAA ACA TGA AAC GTC GGC GTA GTA[180]C.declivis wilderi · .TC.flavocoronatus · .TC.burgessi · .TC.declivis wilderiC.mer/C.xan · .G ooT · .G .. TC.mer/C.xan · .G · .T · .G .. TC.mertensi · .G · .T ooG " TC.madagascariensis · .G · .T ooG .. TC.paucifasciatus · .G .. T · .G .. TC.argentatus · .T · .G · .GC.miliaris ooT · .C · .G .. T Aoo .. TC.multicinctus · .T · .C · .C Aoo · .T .. TC.mult/C.pun/C.pele · .T · .C · .C A.. · .T · .TC.punctatofasciatus ooT · .C · .C Aoo .. T .. TC.pelewensis .. T · .C ooC Aoo .. T .. TC.gutatissimus ooT · .C · .C · .G Aoo .. T .. TC.auriga ooT .. G ooT · .C · .G A.. .. T · .C
[15146]C.tinkeri CTC CTC CTT CTA GTC ATG ATA ACC GCC TTC GTA GGG TAT GTC CTC[225jC.declivis wilderiC.flavocoronatusC.burgessiC.declivis wilderiC.mer!C.xan ooT · .T ooA • .1\ ooC .. TC.mer/C.xan · .T · .T ooA • .1\ .. C · . TC.mertensi ooT ooT ooA • .1\ · ,C · . TC.madagascariensis · .T · .A · .A .. C · .TC.paucifasciatus ooT · .T · .A , ,1\ , ,C .. TC.argentatus · .C · .T • .1\ · .A · .CC.miliaris · .T ooT ooA • .G · .A · .TC. mu It icinct us .. T ooC .. T · .A GC.C.mult/C.pun!C.pele · .T · .C · .T • .1\ GCGC.punctatofasciatus · .T · .C · .T ooA GC.C.pelewensis · .T · .C ooT ooA GCGC.gutatissimus · .T · .C · .T · .A GC. · .AC.auriga " T ooA GC. ooT .. T .. T · ./\
164
APPENDIX A (continued)A 495 bp portion of the mitochondrial cytochrome b gene for 18 Chaetodonspecies.
[15191JC.tinkeri CCC TGA GGA CAA ATG TCA 1'1'1' TGA GGG GCT ACT GTA ATT Ace AAT[270JC.declivis wilderi .. GC.flavocoronatuse.burgessi · .GC.declivis wilderie.mer/e.xan · .G · .e •• TC.mer/C.xan .. G · .C •• Te.mertensi · .G · .1'e.madagascariensis · .G · .1' · .ee.paucifasciatus · .G •. T · .ee.argentatus .. C .. C .• 1' · .ee.miliaris • .1' · .e .. C •. T · .Ce.multicinctus · .1' · .A · .1' · .Ce.mult/e.pun/e.pele · .1' .. A · .1'e.punctatofasciatus • .1' .. A · .1'e.pelewensis .. 1' · .A •. Te.gutatissimus · .1' · .Ae.auriga · .A · .G · .A · .e · .A · .e •. T •• T
[ 15236Je.tinkeri CTe CTC TeT Gee GTe eCT TAT ATT GGA AAC Ace TTA GTA CAA TGA1315)C.declivis wilderie.flavocoronatuse.burgessiC.declivis wilderie.mer/C.xan · .e · .C C..e.mer/C.xan · .e · .C e ..e.mertensi · .C · .e c ..e.madagascariensis · .e e ..e.paucifasciatus · .e C.GC.argentatus ??? ??? ??? ??? ??? ??? ???
e.miliaris • .1' .. 1' · .e · .C · .e .. Ge.multicinctus · .C • .1' •. T
e.mult/C.pun/e.pele · .e · .e •• T •• T
e.punctatofasciatus · .C .. C •• T .• TC.pelewensis · .C · .C •• 'I' .• Te.gutatissimus · .C · .C •• T .• T
e.auriga · .1' .. 1' · .? · .C · .C · .1' C.?
[15281]e.tinkeri ATe TGA GGA GGC 1'1'1' Tce GTA GAC AAC GCC ACC TTA ACT eGA TTe[360Je.declivis wilderiC.flavocoronatuse.burgessie.declivis wilderie.mer/C.xan .. 1' · .G .. 1' · .T e.Ge.mer/e.xan · .1' · .G · .1' •• T e.Ge.mertensi · .1' .. G •• T .• T e.GC.madagascariensis · .1' · .G •• T .• T e.Ge.paucifasciatus •• T · .G · .1' •• T e.Ge.argentatus · .G · .1' C .. · .ee. miliaris .. G · .e .. 1' · .eC.multicinctus · .G .. 1' •• T .. 1' .. 1'e.mult/e.pun/C.pele .. G · .G • .1' .. 1' • .1' •. 1'C.punctatofasciatus · .G •• T .. 1' · .1' .• 1'e.pelewensis · .G •• T •. 1' .. 1' .• 1'C.gutatissimus .. G •• 'I' • .1' • .1' .• 1'C.auriga · .G · .G · .e · .A .. 1' e .. · .e
165
APPENDIX A (continued)A 495 bp portion of the mitochondrial cytochrome b gene for 18 Chaetodonspecies.
[15326]C.tinkeri TTC GCT TTC CAC TTC CTC CTC CCT TTC ATC ATT GCA GCC GTA ACC[405]C.declivis wilderiC.flavocoronatusC.burgessiC.declivis wilderiC.mer/C.xan · .C · .T · .CC.mer/C.xan · .C · .T · .CC.mertensi · .T · .C · .GC.madagascariensis · .C · .T · .CC. paucifasciat us .. T · .CC.argentatus · .C .. T · .TC.miliaris · .T · .C · .T .. T .. TC.multicinctus · .C · .T · .T · .GC.mult/C.pun/C.pele · .C .. T · .T · .T · .GC.punctatofasciatus · .C · .T · .T · .Gc.pelewensis · .C .. T " T · .T · .GC.gutatissimus · .C · .T · .T · .TC.auriga · .C · .C · .T · .G · .T .. T
[15371]C.tinkeri ATG GTT CAT CTA ATG TTC CTC CAC CM ACG GGC TCT AAC AAC CCC[450]C.declivis wilderi · .AC.flavocoronatus · .AC.burgessi · .AC.declivis wilderi · .AC.mer/C.xan · .A · .C · .A · .AC.mer/C.xan · .A · .C · .A · .AC.mertensi · .C · .A · .AC.madagascariensis · .C · .C · .AC.paucifasciatus · .A · .C · .C · .A · .AC.argentatus · .C · .C · .A · .T · .AC. miliaris · .A · .T .. T · .A · .G .. TC.multicinctus · .C · .G .. T · .A · .A .. T .. TC.mult/C.pun/C.pele · .C · .G .. T · .A · .A .. TC.punctatofasciatus · .e .. G .. T · .A .. A .. T .. Te.pelewensis · .e · .G .. T · .A · .A .. T .. Te.gutatissimus · .G .. T · .A · .A .. T '".. ,e. auriga · .A A.. · .e · .A · .e
[154161e.tinkeri eTA GGC eTG AAe TeG GAT ATA GAC AAG ATe TCA TTe CAT eCT TAC!495je.declivis wilderiC.flavocoronatusC.burgessiC.declivis wilderiC.mer/C.xan .. T · .A · .CC.mer/C.xan · .A · .CC.mertensi .. T · .C · .A · .CC.madagascariensis .. T · .C · .A · .'-
C.paucifasciatus .. T · .C · .A · .CC.argentatus T .. · .A .. T .. A · .C · .A .. T · .CC.miliaris .. T · .A C.. ??? ??? ??? ??? ??? ??? ???C.multicinctus .. T · .A · .G .. T · .CC.mult/C.pun/C.pele .. T · .A .. G .. T · .C · .CC.punctatofasciatus .. T · .A · .G .. T · .C · .CC.pelewensis .. T · .A · .G .. T · .C · .CC.gutatissimus · .A .. T · .A · .G " T · .C · .CC.auriga .. T · .C · .T · .A · .C · .C
166
APPENDIX B
A 195 bp portion of the tRNAPro end of the mitochondrial control-region for141 individuals of C. multicinctus (n=32),c. pelewensis (n=501 C.punctatofasciatus (n=561 and C. guttatissimus (n=3). Sequnces are from 5' to3' with a ''." and "_" represents sequence identity and a gap, respectively.Missing nucleotide information is denoted by a"?".
Species Location Seguence
• •• TT ••••••••••• -C • C •••••••••••• A ••••••••••••
•.•. T. ..••••.•.. - ...• '" ••......•.•..••....•.•••••••••••••••• - ••••••••••••••••••• C ••••••••
••••••••• G•••••• - ••••••••••••••••••••••••••••
•••• T••••••••••• - ••••••••••••••••••••••••••• ,
................ - .
................ - .
•••• T••••••••••• - ••••••••••••••••••••••••••••
•••• T••••••••••• - ••••••••••••••••••••••••••••
................ - AC .
45GTACCAGATAAGAAGA-ATTACTAAAACCAAAGTAATAAGAAGAC... TT -C .•••• T••••••••••• - ••••••••••••••••••••••••••••
•••• T•.••••••••• - ••••••••••••..••••.•••••.•••••••••••• G•••••• - ••••••••••••••••••••••••••••
••• TT•••••.•.••• -C •••..•••..•••••.•••••.••.•••••• T••••••••••• - ••••••••••••••••••••••••••••
... T? G•••••• -G •••••••••.•••...•••••..•••.
· .. TT C -C A ••• C .· .. TTG -C A G.• •• TT ••••••••••• -C •••••••••••••• A •••••••••• G•· .. TT C ••••••• -G •••••••••••••• A ••••••••••••
Is TT - AC. GC G .???TT -G.C AC.. C .· .. TT C A. -G AC.. C G .· .. TT C -G C.. C G T????????????????-G AC.. C G o
?. TT ••••••••••• -C •••••••••••••. A ••.••••.•••.• •• TT ••••••••••• -G • C •••••••••••• AC.. C ••• G ••••
· .. TT. A A. -G A .••• TT ••••••••••• -T ••••••.••••.•• A •••.•..•..•.
· .. TT... C ..•..•• -G •.••.•••••••.• AC •• C .•• G ••••
• •• TT ••••••••••• -C •••••••••••••• A ••••••••••••
••• TT •••••••••• G-G •••••••••••••• A ••••••••••••
· .. TT G-G •••••••••••••• A ••••••••••••
• •• TT • A ••.•••••• -C •..••••••••••• A ••••••••••••
• •• TT ••• C ••••••• -G •••••••••••••• A ••••••• G••••
•••• T••••••••••• -T •••••••••••••• A ••• C ••••••••
· .. TT -G . C AC.. C G ••• T• •• TT ••• C ••••••• - ••••••••••••••• A ••••••••••• T
· .. TT -G AC.. C G ••••
• •• TT ••••••••••• -G •••••••••••••• A ••••••••••••
· .. TT -G ••••.•.••..••. AC G•••.A T -G AC.. C GG .
Hawaii(2)Hawaii(2l,PhilHawaiiHa(2),JI,MI(2)HawaiiHa(l), JI(2)HawaiiHawaiiHa, MI (2)
HawaiiHawaiiHawaiiJohnson IslandJohnson Island,Johnson IslandMidway IslandMidway IslandMidway IslandMidway IslandMidway IslandMidway IslandMidway IslandMidway IslandAmerican SamoaAmerican SamoaTahitiTahitiTahiti, CookTahitiTahitiTahitiTahitiTahitiTahitiCook IslandsCook IslandsCook IslandsCook .IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook Islands
C. multicinctusC. multi,C. puncta.C. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisc. pelewensisC. pelewensis
167
C. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatcfasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatus
Cook IslandsCook IslandsFijiFijiFi jiFijiFijiFijiFijiFijiFijiFi jiFijiFijiFijiFijiFijiFijiFijiFijiFijiFijiFijiGuamGuamGuamGuamGuamGuamGuamGuamPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesIndonesiaIndonesiaIndonesiaIndonesiaIndonesiaIndonesiaIndonesiaPalauPalauPalauPalauPalau
... TT C -C .• • • . • • • . . . • • • A ,.· .. TT 0 • ; ••••• GC A•••••• A•••••... TT o' '" -C •. . • • 0 •••• 0" .AC 0 •••• ,.
'" TT.......• 0" -C •• • • • • • • • • • • • • A••••••••••••
· .. TT .. 0 •••••••• - ••••••••••••••• AC .. C... G....... TT o' •• -G •••••••••••••• A ••••••••••.•
... TT 0" •••• -C •• o ••••••••••• A ••••••••••••· .. TT . A ••••••••• -C •••••••••••••• A ••••••••• 0 ••
... TT -C •• • • • • • o' 0 •••• A ••••••••••••
... TT -C .•..•.•••••••.•••••••......
'" TT '" -C •••••••••••••• A •••••••••• ,.
A •• TT -G •••••••••••••• A ••••••••••••
· .. TT . A -G A T· .GTT 0 • - ••••••••••••••• AC . . C • • • G .· T -T AC .. C .· .. TT -C T A .... TT o' -C •• • • • • • • • • • • . • A ••••••••••••••• T ••••• 0 ••••• • -C •••••••.•.••. . A •••••••.•••.
• ••• T ••••••• 0 ••• -G ••••••••..•••• AC G ••.•
••• TT ••• 0 ••••••• -G ••••.•••••.••• A •••.••••.•••
• •• TT ••••••••• A. -G •••••••••••••. A ••••••.•.•. T
· .. TT -G •• 0 ••••••••••• A •••••••• G •••
... TT -G.C A G T
.... T C •••••• • - •••••••••••• o' .A ••••••••••..
... TT.. o' 0" ••• G-G 0 T AC .. C G .· •• TT •••••••• 0 •• -C 0 ••••••••••••• A ••••• G •• G •••
• •• TT • A ••••••••• -C •••••••••••••• A ••••••••••••
... TT -Co •• • • • • . • • • • • • A ••••••••••••
... TT C ••••••• -G •••••••••••••• AC .. C ••• G ••••
• .•• T •• • C ••••• • • - ••••• 0 •••••• 0 •• A ••••••••••• T
????????????????-? A.....•......? ? ? ? ? ? •• C -G AC .. C G .••• TT •• o' •••••• • -C •• 0 •••••••••• GA " ..• ••• T ••••••••••• -T G • A ••• C ••••••••
· .. TT -C • • • • • • • • . . T A G .••• TT •••••.••••• -C •••• 0 ••••••••• A ••••••••••••... TT -C ••• . . . . . • • . . • • A ••••••••••••
• .• TT .••••.••••• -G •.••••••.••••. AC G.••.••• TT .••••••••• • -C ••• • . . . . • • . . . . A ••••••••••••••• TT •• o ••••••• • -C ..• • • • . . • . . • • • A ••••••••••••
••• TT •. • C •••••• • -G •••••••••••••• AC . . C •• •G ••••
... TT -G •••••••••••••• AC G ••••
· .. TT -G . C AC .. C G .· .. TT C -G AC.. C G .... TT -G •••.•...••••.• A •••••• " .• "
• .• TT ••• C ••.•••• -G •••••••••••••• A ••• C ••••••••
· .. TT C -G AC .. C G T
••• TT •.•••.•••• . -C ••.. • • • G•••••• A ••••••••••••· .. TT C - .. A ACG . C G .••• TT •••••••••• • -C •.•••.•••••...• C•..•••..•..
•.. TT ••••••••••. -C ••••••• G•••••• A ••••••••••••
••• TT •••• , .••• o. -C .••••.••.•.•.• A •••••...•••.
· .. T G-G AC .. C ••• G .... TT C ••••••• - ••••••••••••••• A ••••••••••••... TT G-C A ••••••••••••••• TT •••• , •••••• -G •••••.•••.•..• A •.••••...• "
• •• TT ••••••••••• - ••••••••••••••• A ••••••••• AGT
•.• TT .•••••••••• -G •.•.••.••••••• A .•....•.•.••
••• TT •••• , •••••• -C •••••••• , •••••••••••••••• G.
••. TT •••••••..•. -G •.••..••.•••.. A .....•••.••.
· •. TT .•••.•.•••• -G ••••••.••••.•• AC G.•.•
... TT -C T· .. TT . A -C A T
168
C. punctatofasciatus PalauC. punctatofasciatus PalauC. punctatofasciatus PalauC. punctatofasciatus PalauC. punctatofasciatus PalauC. punctatofasciatus PalauC. punctatofasciatus PalauC. punc~atofasciatus PalauC. punctatofasciatus PalauC. punctatofasciatus PalauC. punctatofasciatus Palau'-. punctatofasciatus PalauC. punctatofasciatus PalauC. punctatofasciatus PalauC. punctatofasciatus PalauC. guttatissimus Mauritius Is.C. guttatissimus Mauritius Is.C. guttatissimus Mauritius Is.
••• TT •• G•••••••• -C •••••• G •.••••• A •••.••••••••••• TT??? •• , .??G-C •.•••••••••••• A .•••••••••••
••• TT •• • C •••••• • - ••••••••••••••• A••••••••••••••• TT •••••••••• • -C .• . . • • • . . • • . , .A ••...•.••. , .••• TT ••••••••••• -G.C •••.•••••••. A ••••••• G •• GT• •• TT ••••.•••••• -C •••••••••••••• A •••••••••• G •••• TT •• • C •••••• • - ••••••••••••••• AC • • C •• •G ••••
••• TT •.••••••• A.-C ••••••.••••••• A .•••••••••••••• TT ••••••••••• -C •• • • . • • • • . . • . . A ••••••••••••• ••• T ••••••••••• -T •••••••••••••• A •• • C •••• • • • •
• •• TT • A••••••••• -G •••••••••••••• A ••••••••••• T• •• TT • A •••••••• G-G •••••••••.•••• A .••••.••••• T• •• TT ••••••••••• -C •••••••••••••• A •••••••••• G •
••• TT •••••••••• • -C •••..•...••.. •A •••••..•••••••• TT •••••••••• • -C ••..••....•.• . A ••••••••••••• •• T •••••••••••• - •••••••• G •••••• AC •• C ••••••••• •• T ••••• ; •••••• - ••••.•••••••••. AC •• C ••• G•••." .TT.A ••• G••••• - •••••••••••.••• AC•••••. G ••.•
169
APPENDIX B (continued)Control region sequences.
Species Location Seguence
•..••.. G.- ••• - ••..•••••.••...••••...•••......
......... - - .
....•.... - - .•. G G.. G .••••••••• - ••• - •••••••••••.••.•••••••.• C •.••••
· .... - .. C- .. A- .... G.. A.... G... C.... G.C. C.. C..
90CTTAAATAT-TTG-TTGAATAGTGAAAAAATTTCTAGGTTAGTTA· .. G. - .. C- .. A- ...• G.. A.... G... C....• AC. C.. C..
••••••••• - ••• - ••••••••••• G•••••••••••••••.•••· .. GT- .•. - .. A- .. A. G.• A G GAC.C.. C..
· .. G.- - .. A? . A. G.. A G C AC.C........G.- - .. A- .... G.. A G C AT.C .. C..· .. G. - -C. A- .. AGG .. A GG C T .· .. G.-G .. - .. A- G.. A G C ATCC .. C..
Is .. CCG .-C .C- .. A- G.. A.. G.G C.. T. GACC C..· CC.. -C .. - .. A- G.. A. AG . G C.. T... CC C..TCC .. - .•. - .. A- G.. A.. G.G C.. T.. ACC C..TCC .. -C .C- .. A- G.. A.. G.G C.. T.. ATC C..TCC .. -C .C- •. A- G.. A.. G.G C.. T.. ACC C..· .. G.- - .. A- .. A. G.. A G AC.C.. C..· CC.. -C. C- .. A- .... G.. A.. G. G C.. T. GAC .... C..... G.-C •. - .. A- .. A.G .. A G C AT .C .. C....... - ... - .. A- .. A.G .. A G , GAC.C.. C..TCC .. -C .C- .. A- ... GG .. A.. G.G C.. T.. ATC ... C.... C.. - ... - .. A- .. A.G .. A G.. GC AC .C .. C..· .. G. -C .. - .. A- GGC . A G C AT . C.. C..· .. G. -C .. - .. A- GG .. A G AT. C.. C..· .. G. -C .. - .. A- G.. A G C GACCC .. C..TCC .. -C . C- .. A- G.. A.. G. G.. GC .. T.. ATC C..· .. G. -C .. - .. AA G.. A.. G. G AC.C ..CC .. -C.C- .. A- G AG .G C.. T.. ATC C..· .CG. - .•. - .. A- GG .. A G C AT.C .. C...CC .. -C .C- .. A- .. AGG .. A.. G.G T.. ACCC .. C..... G.-C .. - .. A- .... G.. A G C AT.C .. C...CC .. -C.C- .. A-C .AGG.. A G ,. T.. ACC C...CC.. -C. C- .. A- G.. A.. G.G C GACC C..· - - .. A- G.. A.. G. G C ACC C..· - - .. A- G.. A G C AC.C .· .. G. - - .. A- .. A. G.• A G AC. C.. C..... G.- T.. A- G.. A G C AC .C .. C..TCC .. -C .C- .. A- G.. A.. G.G C.. TC.ACC... C..· .. G. -C .. - .. A- GGC.A G C AT. C.. C....... - ... - .. A- .. A.G .. A G C GAC ....C..
Hawaii (2)Hawaii (2) ,PhilHawaiiHa(2),JI,MI(2)HawaiiHa(1),JI(2)HawaiiHawaiiHa,MI(2)HawaiiHawaiiHawaiiJohnson IslandJohnson IslandJohnson Island~lidway IslandMidway IslandMidway IslandMidway IslandMidway IslandMidway IslandMidway IslandMidway IslandAmerican SamoaAmerican SamoaTahitiTahitiTahiti, CookTahitiTahitiTahitiTahitiTahitiTahitiCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsFi jiFijiFi jiFijiFiji
C. multicinctusC. mUlti,C. puncta.C. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensis
170
C. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusc. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatus
FijiFijiFijiFijiFijiFijiFijiFijiFijiFijiFijiFijiFijiFijiFijiFijiGuamGuamGuamGuamGuamGuamGuamGuamPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesIndonesiaIndonesiaIndonesiaIndonesiaIndonesiaIndonesiaIndonesiaPalauPalauPalauPalauPalauPalauPalauPalauPalauPalauPalauPalau
· .. G. - - .. A- .. A.G.. A G AC. C.. C..... G.-C .. - .. A- .. A.G.. A•... G.....•... AC.C .. C..... G.- .. C- .. A- G.• A.. G.G C AC.C .. C..... G.-C .. - .. A- G.. A.... G C AC .C .. C..... G. -C .. - .. A- GG .. A•... G C AT. C.. C..· .... - ... - .. A-C .AGG .. A•. G.G. G.C GAC.C.. C...CCG.-C .C- .. A- .• A.G.. A.. G.G C.. T.. ACC C..· .. G.-C .• - .. AA G.• A.. GGG AC. C ...... - ..• - •• A- .. A.G .. A•. G. G AC.C .. C.." . G. - ... - .. A- .• A. G.. A•. G. G•........ AC.C .. C..· .• G.- ..• - .. A- .• A. G.• A.... G C • • • • • AT. C.. C...CCT .-C .C- .CA- ••.. G.• A.AG. G C.. T.. ACC ... C..... G. - A.• A- .. AGGC. A G C AC.C .. C....... - - .. A-C.AGG.. A G C AC.C .. C..... G.-C .. - .. A- ... GG .. A G.G.C AT.C .. C...CC.. -G.C- .. A- .. A.G .. A.AG. G C.. T.. ACC ... C..· .. G.-C .. - .. A- .• AGG .. A.. G.G C AT.C .. C..TCC .. -C.C- .. A- .•.. G.. A•. G.G C ACC ... C..... G.-G .. - .. A- .. A.G .. A•.. GG AC.C .. C..... G.- ..• - •. A- .•.. G.. A G C GACCC .. C..... G.- - .. A- .. A.G .. A G AC.C .. C..TCC .. -C . C- .. A- .•.. G.. A.. G. G C.. TC. ACC ... C..· .CG. -.G. - .. A- ... GG .. A.. G.G C AT.C .. C..... G.- ... - .. A-..• AGGC. A•... G C AC. C.. C..TCC .. -C.C- .. A- .... G.. A.. G.G C.. T.. ATC ... C..· .. G. - ..• - .. A- .. A.G .. A•..• G AC.C .. C..... G.-C .• - •. AA .•.. G.. A•. G AC. C .... G.- - .. A- .. A.GC.A .. G.G AC.C .. C..... G.- .. C- .. A- G.. A.. G. G C AC. C.. C..... G.- ... - .. A- G.. A G C •• • • • AC.C .. C...CC.. -C .C- •. A-C .. GG .. A G C • • T.. ACC ... C..· .. G. -C .. - .. A- .... G.. A G C AC . C.. C..... G.-C .. - .. A- .. A.G .. A G C AC.C .. C..TCC .. - .. C- .. A- .... G.. A.. G. G C.. T.. ACC C..· .CG.-C. C- .. A-C. A. G.. A.. G. G C.. T.. ACC C..TCC .. - .. C- .. A- G.. A. AG.G C.. T... CC C..TCC •. - .. C- .. A- G.. A..G.G C.. T.. ACC C..... GG-C.. - .. A- GG .. A G C •• • • • AT.C .. C..... G.- ... - .. A- .. A.G .. A G C • • • • • ATC ,..CC.. -CGC- .. A- T.. A.. G.G C.. T.. ATC C..· .. G. - ... - .. A- G.. A... GG c ..... AC . C.. C..TCC •. - .. C- .. A- G.. A..G.G C • • T.. ACC ... C..... G.- .. C- .. A- .. A.G .. A G C AC.C .. C..· .... - .. C- .. A- .•..... A G C AC. C.. C..· .. G. - - .. A- .. A. G.. A G AC. C.. C...CC.. -C .. -.CA- ..... C.T G CC.. T.C.C .... G.-C .. - .. A-.CAGG.. A G C AC C..T.. G. - ... - .. A- GG .. A.. G. G C AC. C.. C..... G.-C .. - .. A- GG .. A G C •• • • • AT.C .. C..· C... -C .. - .. A- G.. A G C AT. C.. C..· .. GG-C.. - .. A- GG .. A G C GAT.C.. C..· ... G... C- .. A- G.. A.•.. G C AC. C.. C..... G.-C .. - .. A- GG .. A G T.. AT .C .. C...CC •• -C .C- .. A-C •. GG .. A G C • • T.. ACC ... C..· .. G. - .. C- .. A- G.. A G C • • • • • AC.C .. C..· .. G. - - .. A- G.. A G C GACCC .. C..· .. G. - - .. A- G.. A G C AC. C.. C..... G.- - .. A- .. A.G .. A G C AC.C .. C..· .CG.- - .. A- ... GG G. G C AT.C .. C..••• G.- .•• - •• A- •••• G.. A •••• G ••. C •.••• AC .C •. C ..
.CC.. -C.C- .. A- G.. A.AG.G C.. T.. ACC ... C..· .ATG-C.. - .. A- G.. A.... G C..... AC.C .. C..TCC .. - .. C- .. A- G.. A.. G. G C.. T.. ACC ... C..
171
C. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. guttatissimusC. guttatissimusC. guttatissimus
PalauPalauPalauPalauPalauPalauPalauPalauMauritius Is.Mauritius Is.Mauritius Is.
• •••• -C •• - •. A- •• A. G •• A •••. G••••••••• AC • C •• C .•
••• G.- ••• - •• A- •••• G •• A •••• G••• C ••••• AC.C .• C ••
• •• G .-C •• - •• AA•••• G •• A •• G .G ••••••••• AC. C •••••• •••• - ••• - •• A-C •• GG •• A •••• G••• C ••••• AC • C •• C ••
• •••• - ••• - •• A-C. AGG •• A ••• GG.G.C •• T .GAT. C •• C ••T.ATG-C •• - •• A- •••• G •• A •••• G.•. C •.••• AC.C •. C ••
• •• G.- ••• - •• A- .• A .GC .A •••• G••••••••. AC.C •• C ••
••• G.- ••• - •• A- •••• G •• A •••• T ••• C ••••• AC ••.• C ••.CC.G-G .• -.CA- ••••• C.C.AG.G •.•. C ••• CAC.C •••••.CC •• -A.C- .CA- ••••• C. T ••••••••...•• CAC .C •••••
..C •• -C •• - .CA- ••••• C.C ••••••••. C ••• CAC .C •••••
172
APPENDIX B (continued)Control region sequences.
Species Location Sequence
•••••• A•••••••••••••••••••••••••••••••••••••••••••••••.•.•••••••••• T ••.••••.••••••.• T •••....
••••••••••••••••••••••••••••••••• C ••••••••••••
.AT .. CA T C.. C TT A•••••.••.•.••••••••••••••••••.•••.•.•• T ••••...
.A .• .G •••••••••••••••••••••••••••••••••••••••••••
.AT A T C.. C TT A
136AGCTTTGTCCCGTCAAAGATACACCAAGTATCATCATCCCTGTATG.AT ..CA C. T C .. C TT A
.AT A T C.. C TT A
.A CA T C.. C TT" A· TTC.. A T C.. C T A.AT A T C .. C TT A
Is .. A •••• A•••••••••••••••••••••••••• C••• T ••••••••· AT A C T CGC..AT A•••••••••••••••••••••••••••••• T ••••••• A.A •••• A.T C .•• T .•.•...•
.A •••• A•••••••••••••••••••••.•••• C••• T ••••••••
.AT A T C.. C T .
.A •••• A•••••••••••••••••••••••••• C••• T ••••••• A
.AT A T C.. C T A
.AT A T C.. C TT A
.A A.T C T .
.AT A.T T C.. C TT .
.AT A T C.. C TT A
.. T A T C.. C TT , .CAGAT .. CA T C.. C •.•••.•.... A.A A.T C T .· .T A T T C.. C T A· AT A C T GC.· .TC .. A.T •..•.••..••••••...••. C •• C •.• TT ••••.. A.AT A C T ..AT A T C.. C TT A.AT A•••••••••••••••••••••••••• C••••••••••••
.p.,••• • A••••••.••••••••••••••••••• C••• T •.••••••
.AT A T C .. C T A
.ATC.CA T TT A
.ATC .. A T C.. C T •••.•.•
.AT .. CA T C TT A
.AT A C C.. C T .· . T A T C.. C TT A.AT A T C.. C T A
Hawaii (2)Hawaii (2), PhilHawaiiHa(2) ,JI,MI (2)HawaiiHa(1),JI(2)HawaiiHawaiiHa,MI(2)HawaiiHawaiiHawaiiJohnson IslandJohnson IslandJohnson IslandMidway IslandMidway IslandMidway IslandMidway IslandMidway IslandMidway IslandMidway Islar.dMidway IslandAmerican SamoaAmerican SamoaTahitiTahitiTahiti, CookTahitiTahitiTahitiTahitiTahitiTahitiCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsFijiFijiFi jiFijiFiji
C. multicinctusC. mUlti,C. puncta.C. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. mul ticinct usC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pe Lcwen s i sC. pelewensisC. pelewensisC. pelewensisC. pe.lewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisc. pelewensisC. pelewensisC. pelewensis
173
C. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusc. punctatofasciatusc. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatus
FijiFijiFijiFijiFijiFijiFijiFijiFijiFijiFijiFijiFijiFijiFijiFijiGuamGuamGuamGuamGuamGuamGuamGuamPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesPhilippinesIndonesiaIndonesiaIndonesiaIndonesiaIndonesiaIndonesiaIndonesiaPalauPalauPalauPalauPalauPalauPalauPalauPalauPalauPalauPalau
.AT ••• A ••••••.••••••• T •••.•••• C •• C ••• TT •••.•••
.AT ••• A •••••••••••••• T •••••••• C •• C ••• T ••••••••
.AT •• CA •••••••••••• C. T •••••••• C •• C ••• 'IT •••••• A
• AT •• CA •••••••••••••• 'I ••••••••••• C ••• 'IT •••••. A
•• T ••• A•••••••••••••• T •••••••• C •• C ••• TT •••••• A
• A •••• A•••••••••••••• 'I ••••••••••• C ••• 'IT ••.••• A
.A •••• A •••••• G ••••••••••••••••••• C ••• 'I ••••• G •.
•• T ••• A••• T •••••••••• T •.•••••• C •. C .•• T ••••••. A
.ATC .• A•••••••••••••• 'I •••••••• C •• C ••• 'IT •••••••
.ATC •• A•••••••••••••• T •••••••• C •• C ••• T ••.•••• A
.AT •• CA ••• T •..••••••• T •.••••••••• C .•• TT •••••• A
•••••• A. 'I ••••••••••••••.•.••••••• C ••• T ••••••••
· .TC •• A•••••••••••••• T •.•••••• C •• C ••• TT •••••. A
.A •••• A •••••••••••• C.T ••••.•••••• C .••• T .••••••
• • 'I ••• A••••••••••••••••••••.•• C •• C ••• 'IT •••.•• A
.AT ••• A••••.••••••••••..•••••• " .C ••• 'I •..•• GC.
.AT ••• A•••••••••••••• T •••••••••.• C ••• TT •••••• A
.A •••• A •••••••••••.•••.•••••.•••• C ••• T •..•••••• ATC •• A. T •••.•••••••• 'I •••••••• C •• C ••• 'IT •.•..•.
GAT ••• A•••••••••••••• 'I •••••.•. C •• C ••••••••••• A
.AT ••• A••••.•••••••.• T ...••••• C •• C ••• TT ••.••.•
.AT ••• A •••••••••••••••••••••••••••••• 'I •..•••••
· • 'I ••• A. T •••••••.••••••••••••• C •• C .•• 'IT ••.••• A
• • TC •• A•••••••.•••.•• 'I ..••••.• C •• C ••• 'IT ..•••• A
.A •••• A. 'I ••••••••••• G •••.•••••••• C .•• 'I ••.•.•••
.AT ••• A •••••.•••••••• T •••••••• C .• C ••• TT ••.••••
• .T ••• A ••. T •••••••••• T •••••.•• C •• C ••• T •..••.• A
.ATC •• A.••••••••••. C. TG .•.•••. C •• C .•. 'IT •..••.•
• ATC • CA •••••••.•••••• 'I •.•.•••. C •• C ••• 'IT •..••.•
.AT •. CA ••.•.•••..•••• T ••..••.•.•• C ••• TT.C ...• A
.AT ••• A.•••••••••••••••.••••••••• C .•.••.••• G ..
.ATC .CA ••••.••••••••• 'I ••.•.•••.•• C ••• 'IT ••.••. A
.ATC •• A•.••••••.••••• T .••••••• C .• C ••• 'IT ..•••••
.A •••• A•••••••••••••••••••••••••• C •••••••• C •• A
.AT •.• A••••••..••••••••....•...•.••.• 'I ••••..•.
.AT ••• A•.••..•••.•.•••..•••.••••• C ••• T •... CGC.
.AT •.• A.••••.•.•••.••••....••.... C •.• T ••••••. A
.AT ••• A.•••.•.••••.•••••..••.• C •• C •.• TTTC •.•• A
••••.• A••• T •.•••.•••• T ••••.••• C .• C ••• TT .•••.. A
.A •.•• A.T .••••••••. C •••....••.••• C .•• T •.•••••.
.AT •• CA ••••••.•.••••• T •.••...• C •• C ••• TT .••.•• A
.A •••• A •••• A ••••••••••••••••••••• C ••• T •••• C •• A
.AT •• CA •••• A.•••••. C. T •....••. C •. C •.• TT •••.•. A
.AT .• CA .•.••••••••. C •••..••••. C •• C ..• TT •••.•• A
.AT ••• A.•.•••••.••..• T ••..••.• C •• C .•• TT .•••.• A
.A •. C.A •••••.•••••.••••••••.••••. C •.• T .••.•• C.
· . T. C. A••..••.••••.•• T •..•.••. C •• C ••• 'IT ••••.. A
• • TC •• A••.••••••••..• 'I ••...•.• C .• C ••• 'IT .••.•• A
• .T •.• A•••••.•••..••• T •••••••• C •• C •.• TT •••••• A
.AT •.• A ••• 'I ••.•••••.• T ••...••• C •• A ••• TT ••••.• A
.AT ••• A.•.•..••.••••• T •..•.••• C .• C ••• TT •••••. A
.AT .. CA .•••••••••.. C. T •..•.••. C .• C •.• TT ••••.. A
•. 'I •.• A••••..•••••.•. T •••...•. C •• C •.• TT •••... A
.A •••• A•••••••••••••••••••••••••• C ••• ? ..•. G ••
.AT •. CA. 'I C. 'I •..•...• C .• C ••• TT .••••• A
GAT •.. A •••••.•••..•.• T •...•••• C •• C •••••.••.•• A
.AT .• CA.G ••...•••••.. 'I ••••••.••.•••.• TT •••••. A
• ATC . CA ••••••••.••••• 'I ••••.•• GC •• C ••• 'IT .•••.. A
• • TC •• A. 'I .••...•.••.•••....••• C •. C ••• 'IT .•••.• A
.AT •• CA •••••••..••.•. T ••••.••..•• C •.. TT ....•• A
.ATC .. A•••••.••••••.••••••••.••.• C •.• T •.••.• C.
•• T .•• A••••.••••••••• 'I •••••••• C •• C ••• TT •••••• A
.AT •.• A•.•••...••....•.....•••••• C •.• T .••••. AT
174
C. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. guttatissimusC. guttatissimusC. guttatissimus
PalauPalauPalauPalauPalauPalauPalauPalauMauritius Is.Mauritius Is.Mauritius Is.
.AT ••• A•••••••••••••• T •••••••• C •• C .•• TT .•••..•
.AT •• CA•••••••••••••• T ••••••••••• C ••• TT •...•• A•• T ••• A ••• T •••••••••• T •••••••• C •• C ••• T ••••••• A
• A•.•• A••• T •••••••• C • T •••••••.••• C•.• TT •••••• A.A.C •• A•••••••••••• C. T ••••••••••• C ••• T •• C •••• A•• T••• A•••••••••••••• T •••••••• C •. C ••• TT •••••. A.AT ••• A•••••••••••••••.••••••• C •• C ••• TT ••.••••.AT •• CA•••••••••••••• T •••••••• C •• C ••• TT •••..• A.A •• C.A. T •••••••••••••••••••••••• C ••• TT •••.•• A.A .• C .A ••••. C ••••••••••••..•••••• C ••• T •••... CA.A •• C .A •••••••••• , •••••••..•••••• C•.• T .••... CA
175
APPENDIX B (continued)Control region sequences.
Species Location Sequence
• .- ••••••• C •••••.•.•••••••••••.••.•••.•.••••••
• .- •••••••••••••••••••••••••••••••••••••••• G .•
.G- •.•••.•••••.•.••••.••••.•••••..••••••.•••..• .- •••••••••••••••••.•••••••••••••••••• G••••••• .- ..•••••••••••.••••••••.•.•.•••..••. T ••••...
· .- •••••••••••••••.••.••.••••••.•..••.• G••.•..
GG-.A.... TM .....•.............. G..... T.... GA.
GG-.A M T A..G- .A M ........................•. T GA.G. -. A AA T .C.. GA.G.-.A G.M •......................... T.C A.
Is .. G-.A •... TM T T.GG-.A TAA T.C .. GT.CG- AA T.C.. GT ..G-.A TM .....•.................... T.C ... T..G-GA AA T.C .. GT.GG-. A A A T GG.GG-.A TM T.C T.GG-. A••• G • M •••••••••••••••••••••••••• T •••.• A.GG-. A. C A A T G..G-.A.C .. TM T.C .. GT.GG-.A.C A A T A.GG-. A G. AA T A.GG-. A G. AA T .C AGGG- .A •..•. M •••••••••..•.•..•.• , ....•• T ..•.. A..G-.A.C .. TM T. ??????
GG-.A TAA T A..G-.A TM T .CT .GT.GG-. A CA TG GA.GG-.A TAA T.C .. GT.GG- • A••• G •M ••••.••••••••••••••••••••• T •C •.• A.GG-.A TA T.C .GG-.A TA T.C T.GG-.A GTM T TA.G.-.A A T A.GG-.A.C A A T AGG. - . A A GA T A..G- TAA G T .C • . CT.GG-.A G.AA T G.GG-. A. C ••• A •• , •••••••..••••.••..•.•••• T A.
182Hawaii(2) AA-AGATAACGGCCCAATAAGAACCTACCATCAGTTGACATCTACAHawaii (2), Phil GG-.A...•. M G T GA.Hawaii .. - A .Ha(2) ,JI (1) ,MI (2 .. - .Hawaii .. - .Ha (1), J. I • (2), •• • •••••••••..••••..•••••.•••..•••••••......•
Hawaii .. - ..............•............................Hawaii .. - .....•..........•..........................Ha , M.I. (2), GG-.A.C A A.. ooT GG.Hawaii .. - A .Hawaii .. - G.GHawaii .. - .....•.....................................Johnson Island .. - T.Johnson IslandJohnson IslandMidway IslandMidway IslandMidway IslandMidway IslandMidway IslandMidway IslandMidway IslandMidway IslandAmerican SamoaAmerican SamoaTahitiTahitiTahiti, CookTahitiTahitiTahitiTahitiTahitiTahitiCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsFijiFijiFijiF'ijiFiji
C. multicinctusC. multi,C. puncta.C. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. m'ulticinct usC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewens.1.sC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisc. pelewensisC. pelewensisC. pelewensisc. pelewensisc. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensis
176
C. pelewensis FijiC. pelewensis FijiC. pelewensis FijiC. pelewensis FijiC. pelewensis FijiC. pelewensis FijiC. pelewensis FijiC. pelewensis FijiC. pelewensis FijiC. pelewensis FijiC. pelewensis FijiC. pelewensis FijiC. pelewensis FijiC. pelewensis FijiC. pelewensis FijiC. pelewensis FijiC. punctatofasciatus GuamC. punctatofasciatus GuamC. punctatofasciatus GuamC. punctatofasciatus GuamC. punctatofasciatus GuamC. punctatofasciatus GuamC. punctatofasciatus GuamC. punctatofasciatus GuamC. punctatofasciatus PhilippinesC. punctatofasciatus PhilippinesC. punctatofasciatus PhilippinesC. punctatofasciatus PhilippinesC. punctatofasciatus PhilippinesC. punctatofasciatus PhilippinesC. punctatofasciatus PhilippinesC. punctatofasciatus PhilippinesC. punctatofasciatus PhilippinesC. punctatofasciatus PhilippinesC. punctato fasciat lIS PhilippinesC. punctatofasciatus PhilippinesC. punctatofasciatus PhilippinesC. punctatofasciatus PhilippinesC. punctatofasciatus PhilippinesC. punctatofasciatus PhilippinesC. punctatofasciatus PhilippinesC. punctatofasciatus PhilippinesC. punctatofasciatus PhilippinesC. punctatofasciatus PhilippinesC. punctatofasciatus IndonesiaC. punctatofasciatus IndonesiaC. punctatofasciatus IndonesiaC. punctatofasciatus IndonesiaC. punctatofasciatus IndonesiaC. punctatofasciatus IndonesiaC. punctatofasciatus IndonesiaC. punctatofasciatus PalauC. punctatofasciatus PalauC. punctatofasciatus PalauC. punctatofasciatus PalauC. punctatofasciatus PalauC. punctatofasciatus PalauC. punctatofasciatus PalauC. punctatofasciatus PalauC. punctatofasciatus PalauC. punctatofasciatus PalauC. punctatofasciatus PalauC. punctatofasciatus Palau
GG-GA A A T GG.GG-GA.C A A T AGGG-.A AA T GA.GG-.A A T A.GG- .A G. M T.C A.GG-.A A T.C .. GA..G-.A TM T.C .. CT.GG-.A M T A.GG- .A.C A A T GA.GG-.A A A T, A.GG- . A•••.• A ••••••••••••••••••••••••••• T •••.. A..G-.A TM T.C .. GG..G- .A.C M T A.GG- .A A T.C .. GA..G-.A G.A G T GA.GG-.A TM T,C .. GT.GG- .A G.M T A.GG-.A A T.CT .. T.GG- .A.C A A T GA.GG-.A M T A..G-.A A A T ??
GG- T Po.A • • • • • • • • • • • • • • • • • • • • • • • • • • T • C •• CT.
G .-.A .•••• CA T A..G-.A.C M T A...G- .A.C .. TM T.C T.G. - .A.C A A T.C .. GA.GG-.A TM T A.GG- .A. C A A T GG.GG-.A A , T A.GG-.A CAG T A.GG-.A TA T.C .GG- .A A T GAGGG- .A. C A A T GA.GG- •••.••. M ...••.•••••••....••••••.•. T •C ..• T .GG- T.A T.C .. G..GG-.A TM T.C .. GT.GG- M T.C .. GT.G. - .A G.M G T.C AGG.-.A M T A..G-.A.C .. TAA T.C T.GG- .A M G T A.GG- TM T.C T.GG-.A M G T A.GG-.A M T A..G- .A A A T GA.G.-.A TM T TT.GG-.A G.A T G.GG-.A TA T.. T .. A..G-.A G.A T A.G. - . A M T . C.. GA ..G-.A G.AA T .C A.GG-.A TM T GA.GG- .A G.M T.C A.GG-.A TM T.C A.GG-·.A M T GA.
GG-.A AA G T A.GG-.A A T A.GG-.A A T GA.GG- . A CA T GA..G-.A A T A.GG-.A TM T .C .. GT.GG-.A ••.• TM ••.•..•••..•.•...••....•.. T .•••. G.GG- M T.C .. GT.
177
C. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. punctatofasciatusC. guttatissimusC. guttatissimusC. guttatissimus
PalauPalauPalauPalauPalauPalauPalauPalauMauritius Is.Mauritius Is.Mauritius Is.
GG- .A.C •• TA •••••••••••••••••••••• A •••• T •••. GA.
GG- .A.C ••• A•••••••••••••••••.••••••••• T •..•• A.
GG-.A ••••• M •••••••••••••••••••••••••. T .•••. A.GGG• A•••• TA•••••••••.•••••.••.•••••••• T •C •. GA•
•• G.A ••••• M •••••••••••.•••••••••••.•• T.C •• GA.
GG-.A •••• TM •••••••••••••••••.•••••••• T •••.• G..G- .A •••• TA ••••••.••••••••••.•••• A•••• T •••. GA.
.G-.A ••••• M ••••••••••••••.••••••••••• T ••••• A.GG- . A•••• TTA •••••••••••••••••••••••••• T .••. TT .G.-.A ••••• TA••••••••.••••••••••••••••• T ••.. TT.G.-.A •••• TM ••••••••.•••••.••.•••.•••. T •.•• TT.
178
APPENDIX B <Continued)Control region sequences.
Species
C. multicinct usC. mUlti,C. puncta.C. multicinctusC. multicinctusC. multicinct usC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinct usC. mu Lt icinct usC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. multicinctusC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC.' pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensisC. pelewensis
Location
Hawaii (2)Hawaii (2), PhilHawaiiHa(2), JI (1) ,MI (2)HawaiiHa (1), J. I • (2) ,
HawaiiHawaiiHa, M.I.(2),HawaiiHawaiiHawaiiJohnson IslandJohnson IslandJohnson IslandMidway IslandMidway IslandMidway IslandMidway IslandMidway IslandMidway IslandMidway IslandMidway IslandAmerican SamoaAmerican SamoaTahitiTahitiTahiti, Cook Is.TahitiTahitiTahitiTahitiTahitiTahitiCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook IslandsCook I s landsCook IslandsCook IslandsCook IslandsCook IslandsCook Ls LandsCook IslandsFi jiFi jiFijiFijiFiji
Sequence
195TGATAACGGTTAT
... C .
... C .
... C .
... C ..
••• C •••••••••
... C ..
... C ..••• C ..
••• C •••••••••
••• C .
••• C ..
••• C ..••• C ..
... C ..
••• C .
... C ..
....... A.....
?????????????
?????????????
..... .. .. C ...
..... G.......
C .
179
C. pelewensis Fiji .. .......... 0 •••
C. pelewensis Fiji . .... G •...•..
C. pelewensis Fiji . ............C. pelewensis Fiji . ........ c ...C. pelewensis Fiji . ............C. pelewensis Fiji ..0 ..........C. pelewensis Fiji . ............C. pelewensis Fiji . ............C. pelewensis Fiji . ............C. pelewensis Fiji ..G ..........
C. pelewensis Fiji . ............C. pelewensis Fiji • •••••••• 0 •••
C. pelewensis Fiji . .....................C. pelewensis Fiji .. ........................C. pelewensis Fiji .. .......................C. pelewensis Fiji .. ......................C. punctatofasciatus Guam .....................C. punctatofasciatus Guam .. ......................C. punctatofasciatus Guam .. .......................C. punctatofasciatus Guam . ...................C. punctatofasciatus Guam ......................C. punctatofasciatus Guam ........................C. punctatofasciatus Guam .......................C. punctatofasciatus Guam ...................C. punctatofasciatus Philippines .. .....................C. punctatofasciatus Philippines .. ......................C. punctatofasciatus Philippines .. .....................C. punctatofasciatus Philippines ....................C. punctatofasciatus Philippines .. ....................C. punctatofasciatus Philippines .. .......................C. punctatofasciatus Philippines .. .......................C. punctatofasciatus Philippines ?????????????C. punctatofasciatus Philippines .. ...... A ....C. punctatofasciatus Philippines .. ...................C. punctatofasciatus Philippines .. ....................C. punctatofasciatus Philippines . ....................C. punctatofasciatus Philippines .. ...................C. punctatofasciatus Philippines ..?? .................C. punctatofasciatus Philippines . ..................C. punctatofasciatus Philippines .. .....................C. punctatofasciatus Philippines .. .....................C. punctatofasciatus Philippines .. .....................C. punctatofasciatus Philippines .. ..................C. punctatofasciatus Philippines .. ....................C. punctatofasciatus Indonesia .. .....................C. punctatofasciatus Indonesia .. ............C. punctatofasciatus Indonesia . ............C. punctatofasciatus Indonesia . ............C. punctatofasciatus Indonesia .............C. punctatofasciatus Indonesia . ............C. punctatofasciatus Indonesia .............C. punctatofasciatus Palau . ............C. punctatofasciatus Palau . ............C. punctatofasciatus Palau .............C. punctatofasciatus Palau . ............C. punctatofasciatus Palau .............C. punctatofasciatus Palau .............C. punctatofasciatus Palau . ............\.,. punctatofasciatus Palau . ............C. punctatofasciatus Palau .............C. punctatofasciatus Palau . ............C. punctatofasciatus Palau .............C. punctatofasciatus Palau . ............
180
C. punctatofasciatus Palau .. ........................C. punctatofasciatus Palau •.••• Co 0 •••••
C. punctatofasciatus Palau .. ........................C. punctatofasciatus Palau .. ........................C. punctatofasciatus Palau ••••• 0 oA .••••C. punctatofasciatus Palau .. ........................C. punctatofasciatus Palau .. ........................C. punctatofasciatus Palau .. ........................C. guttatissimus Mauritius Is. 0 ........................
C. guttatissimus Mauritius Is. .. ........................C. guttatissimus Mauritius Is. .. ........................
181
APPENDIXC
Draft 9 May 1994
Allozyme variation among recently formed Pacific Butterflyfishes
(Chaetodontidae)
W. Owen McMillant, Lee Weigt+§, and Stephen R. Palumbi"
tDepartment of Zoology and
Kewalo Marine Laboratory
University of Hawaii
Honolulu, Hawaii 96822
(808)539-7311
(808)599-4817(fax)
owenm@uhunix.uhcc.hawaiLedu
palumbi@uhunix.uhcc.hawaiLedu
tSmithsonian Tropical Research Institute
Apartado 2072
Balboa, Republic of Panama
(507)28-0980
(507)28-0516(fax)
§Alternate Mailing Address: Smithsonian Tropical Research Institute
Unit 0948, APO, AA 34002-0948 USA
182
ABSTRACT
Three closely related butterflyfishes (Chaetodontidae), that differ in
color pattern, partition the tropical west Pacific into large allopatric ranges.
Variation among species at 31 allozyme loci mirrors the pattern of variation
observed in mitochondrial DNA. Genetic differences among the three species
are slight, consistent with their recent differentiation. Similar to the pattern
in mtDNA only the Hawaiian and Johnston Island endemic, Chaetodon
multicinctus, shows significant of genetic differentiation at allozyme loci
(Nei's D=0.04). Genetic differences between C. pelewensis and C.
punctatofasciatus are slight (Nei's D=0.002), consistent with very high
migration rates (Nm>14) between these two species at locales separated by
over 7500 kilometers.
183
INTRODUCTION
Three allopatric butterflyfishes, Chaetodon multicinctus, C. pelewensis,
and C. punctatofasciatus differ primarily with respect to color pattern.
Previous mitochondrial DNA (mtDNA) analysis suggested that all three
species diverged from the Indian Ocean, C. guttatissimus, between 800,000 and
1,600,000 years ago and from each other between 300,000 and 800,000 years ago
(McMillan and Palumbi, submitted). Yet, these data confirm the evolutionary
distinctiveness of only C. multicinctus. MtDNA variation is distributed
randomly between C. pelewensis and C. punctatofasciatus with little
indication of significant subdivision across the combined range of both
species.
Although much of the observed mtDNA variation could have
predated speciation, the genealogical and geographic distribution of mtDNA
variation argues that hybridization and inter-specific introgression is a more
probable explanation for the failure of the mtDNA gene tree to reflect species
boundaries (McMillan and Palumbi, submitted). The mtDNA has been
observed to move across species boundaries even when there are strong
reproductive barriers (Powell, 1983; Ferris et al., 1983;Spolsky and Uzzell,
1984; Lamb and Avise, 1986; Carr et al., 1986; Baker et al., 1989; Dowling et al.,
1989). Theoretical work suggests that the rate of introgression is determined
primarily by the level of gene flow (Takahata and Slatkin, 1984). These
butterfishes have a 40-60 day planktonic larval stage are probably capable of
dispersing gametes over hundreds, if not thousands, of kilometers (Tricas,
1987; Hourigan and Reese, 1987; Leis 1989).
To augment our previous mtDNA work, we provide a description of
the variation in this group at allozyme loci. Allozymes provide a rough
184
meter with which to gauge the extent of gene flow among species at
presumably neutral nuclear markers (however see Karl and Avise, 1992).
Because the markers are more likely to be linked to loci and co-adaptive gene
complexes under strong selective pressure they may show a very different
pattern of introgress relative to mtDNA (Barton, 1983; Takahata and Slatkin,
1984). The extent to which these two different genetic markers show similar
or different patterns of introgression sheds light on the evolutionary and
genetic cohesion of these species, and provides a better foundation upon
which to examine the significance of interspecific color pattern differences in
this group.
185
MATERIALS AND METHODS
Approximately 30 individuals from each of the three species were
examined for genetic differences at allozyme loci using standard starch gel
electrophoresis (Murphy, et al., 1990). All thirty individuals were taken from
phenotypically homogeneous populations at the approximate center of each
species' geographic distribution. The sampling locations were as follows: for
C. multicinctus - Molokai, Hawaii (n=32); for C. punctatofasciatus - the
Philippines (n=30); for C. pelewensis - Viti-Levu, Fiji (n=27).
The heart, liver, eyes, brain and a small portion of skeletal muscle were
removed from each individual and homogenized in two volumes of
grinding buffer (0.25M sucrose, 2% phenoxyethanol) and centrifuged at 14,000
rpm for 10 min at 4° C. The resulting supernatant was removed, absorbed
onto filter wicks, and electrophoresed through slabs of 11% polymerized
starch using four different running buffers (table 1). Thin slices of starch gels
were stained for 19 different enzyme systems that yielded a total of 31 distinct
loci (table 1). Histological staining methods followed Aebersold et al. (1987).
Following staining, gels were photographed and allelic variation was scored
alphabetically in order of decreasing anodal mobility of the protein produced.
Allele frequencies at polymorphic loci were determined and genotype
frequencies at polymorphic loci were tested for conformity to Hardy
Weinberg expectations. Nei's (1978) unbiased genetic distances (D) .were
calculated between all species. The significance of the observed differences
between jackknifed average genetic differences was determined as outlined by
Muller and Ayala (1982), using a program provided by Lessios (1990).
In addition, F-statistics were computed between the three species.
Because the FST approach is based on an explicit island model of population
186
genetics, it permits an estimate of migration between geographic regions
based on the relationship, Nm=(1-FST)/4FST where Nm is the effective
number of individuals within each locality and m is the migration rate per
generation (Wright, 1951).
187
RESULTS
Of the thirty-one loci examined, 12 were monomorphic across all three
species and 9 showed only slight variation. Significant polymorphism (major
allele at less than 0.95 frequency) was evident at the remaining ten loci (table
2). Significant deviation (p<0.05) of genotype frequencies from Hardy
Weinberg equilibrium within each of the three localities was evident at only
the PEP A locus in Fijian populations of C. pelewensis. However, because at
least one deviation at the 0.05% level was expected to occur by chance in our
39 chi-squared tests, we attributed this observation to sampling artifact rather
than a biologically meaningful pattern.
Overall genetic differences among species at allozyme loci were very
small and similar to levels previously reported among conspecific
populations of marine fishes across the Pacific (Winans, 1980; Rosenblatt and
Waples, 1986; Planes, 1993). There were no fixed allelic differences among the
three species and only slight shifts in the frequencies of alternate alleles at
different loci (table 2). Of the three, C. multicinctus was genetically the most
distinct with Nei's D of 0.040 and 0.046 from C. punctatofasciatus and C.
pelewensis, respectively (table 3). At two loci, GPI 2 and MDH 2, the
dominant allele in Hawaiian populations of C. multicinctus was present in
very low frequency within C. punctatofasciatus and C. pelewensis. At other
polymorphic loci, C. multicinctus showed much smaller levels of variation
and had significantly lower levels of heterozygozity relative to it's two
siblings (table 4). Estimates of effective migration rates between C.
multicinctus and its two siblings were less than an Nm value of 1.0, a level
thought to prevent the accumulation of genetic differences by genetic drift
(Slatkin, 1987) (table 5).
188
By contrast, C. punctatofasciatus and C. pelewensis were nearly
identical at allozyme loci. Nei's D between these two species was very slight
(ca., 0.002) and not significantly different from zero. The largest difference in
the allele frequency occurred at the MDH2 loci. Within C. pelewensis, the A
allele occurred at a frequency of nearly 34% within Fijian populations. In
contrast, in Philippine populations of C. punctatofasciatus that same allele
reached a frequency of less than 10% (table 3). At other polymorphic loci,
allele frequencies were nearly identical despite the nearly 7500 kilometers
separating the two species. As a result, FST values between these two
locations were very small, 0.019 (0.023,0.009: 95% confidence interval),
suggesting a very high effective per generation migration rate (Nm=13)
between the eastern and western Pacific (table 5).
189
DISCUSSION
The patterning of variation at allozyme loci among these similar
butterflyfishes mirrors the pattern of mtDNA variation. Genetic
differentiation is slight among species consistent with a recent formation of
the group. None-the-less, similar to the pattern of mtDNA variation, the
Hawaiian/Johnston Island endemic, C. multicinctus, exhibits clear genetic
differences at allozyme loci suggesting low levels of intra-specific gene flow
(table 5) (McMillan and Palumbi, submitted). In addition, the lower levels of
heterozygozity in this species at allozyme loci are paralleled by much lower
levels of mtDNA variation in C. multicinctus relative to C. pelewensis or C.
punctatofasciatus (McMillan and Palumbi, submitted).
By contrast, both allozyme and mtDNA variation is distributed
randomly between Fijian populations of C. pelewensis and Philippine
populations of C. punctatofasciatus. The genealogy of mtDNA variation
suggests that these two species diverged in allopatry between 300,000 and
800,000 years ago and the present homogenization of mtDNA variation is the
result of high levels of high levels of hybridization and associated
introgression of mtDNA. Because allozyme differences accumulate much
slower than mtDNA differences, some of the biochemical similarity between
these two species may reflect historical association (Wilson et al., 1985; Reeb
and Avise, 1990). However, the strong concordance between estimates of
migration between Fiji and Philippine populations based on allozymes and
those based on mtDNA argues for contemporary movement of nuclear, as
well as mtDNA, variation across species boundaries (table 5). Long distance
dispersal during the 40-60 planktonic feeding larval stage probably accounts
for the widespread homogenization of genetic variation in these two species.
190
This genetic pattern indicates that, despite their species-level distinction, C.
punctatofa.sciatus and C. pelewensis are tightly coupled evolutionary.
Despite this conclusion, color pattern distinctions remain true across
the vast majority of each species' range (Blum, 1989). Levels of gene flow
between Philippine populations of C. punctatofasciatus and Fijian
populations of C. pelewensis are far higher than levels expected to prevent
the accumulation of neutral differences by genetic drift (Slatkin, 1987). Yet,
these populations show completely different distributions of phenotypic
variation, with all 30 individuals collected from reefs around the Philippines
possessing a typical C. punctatofasciatus color pattern and all individuals
examined from the Fiji possessing a typical C. pelewensis color pattern. The
obvious implication of this discrepancy is that strong selection on color
pattern is preventing the breakdown of species-specific color patterns in the
background of potentially homogenizing levels of gene flow.
191
LITERATURE CITED
Baker, R J., S. K. Davis, R. D. Bradley, M. J. Hamilton, and R. A. Van der
Bussche. 1989. Ribosomal-DNA, mitochondrial DNA, chromosomal,
and allozymic studies on a contact zone in the pocket gopher, Geomys.
Evolution 43:63-75.
Barton, N. H. 1983. Multilocus clines. Evolution 37:454-471.
Blum, S. D. 1989. Biogeography of the Chaetodontidae: an analysis of
allopatry among closely related species. Envir. Biol. Fish. 25: 9-31.
Carr, S. M., S. W. Ballinger, J. N. Deer, L. H. Blankenship, and J. W. Bickham.
1986. Mitochondrial DNA analysis of hybridization between sympatric
white-tailed deer and mule deer in west Texas. Proc. Natl. Acad. Sci. USA
83:9576-9580.
Dowling, T. E., G. R. Smith, and W. M. Brown. 1989. Reproductive isolation
and introgression between Notropis cornutus and Notropis
chrysocephalus (family Cyprinidae): comparison of morphology,
allozymes, and mitochondrial DNA. Evolution 43:620-643.
Ferris, S. D., R. D. Sage, C.-H. Huang, J. T. Nielsen, U. Ritte, and A. C. Wilson.
1983. Flow of mitochondrial DNA across a species boundary. Proc. Natl.
Acad. Sci. USA 80:2290-2294.
Hourigan, T. F. and E. S. Reese. 1987. Mid-ocean isolation and the evolution
of Hawaiian reef fishes. Trends Ecol. Evol. 2:187-191.
192
Lamb and Avise, 1986. Directional introgression of mitochondrial DNA in a
hybrid population of tree frogs: the inference of mating behavior. Proc.
Nat!. Acad. Sci. USA 83:2526-2530.
Leis, J. M. 1989. Larval biology of butterflyfishes (Pisces, Chaetodontidae):
what do we really know? Envir. BioI. Fish. 25:87-100.
Lessios, H. A. 1990. A program for calculating Neils genetic distances and
their jackknifed confidence intervals. J. Hered. 81:490.
Mueller, L. D., and F. J. Ayala. 1982. Estimation and interpretation of genetic
distance in empirical studies. Genet. Res. Camb. 40:127-137.
Nei, M. 1978. Estimation of average heterozygosity and genetic distance from
a small number of individuals. Genetics 89:583-590.
Nishida, M. and S. J. Lucas. 1988. Genetic differences between geographic
populations of the crown-of-thorn starfish throughout the Pacific region.
Marine Biology 98: 359-368.
Karl, S. A. and J. C. Avise. 1992. Balancing selection at allozyme loci in
oysters: implications from nuclear RFLPSs. Science 256:100-102.
Planes, S. 1993. Genetic differentiation and restricted larval dispersal of the
convect surgeonfish Acanthurus triostegus in French Polynesia. Mar.
Ecol. Prog. Ser. 98:2237-246.
Powell, J. R. 1983. Interspecific cytoplasmic gene flow in the absence of
nuclear gene flow: evidence from Drosophila. Proc. Natl. Acad. Sci. USA
80:492-495.
193
Reeb, c. A. and J. C. Avise. 1990. A genetic discontinuity in a continuously
distributed species: mitochondiral DNA in the American Oyster,
Crassostrea virginica. Genetics 124(2):397-406.
Rosenblatt, R. H. and R. S. Waples. 1986. A genetic comparison of allopatric
populaitons of shore fish species from the eastern and central Pacific
Ocean: dispersal of vicariance? Copeia 1986:275-284.
Spolsky, C. and T. Uzzell. 1984. Natural interspecies transfer of
mitochondrial DNA in amphibians. Proc. Natl. Acad. Sci. USA 81:5802
5805.
Slatkin, M. 1987. Gene flow and the geographic structure of natural
populations. Science 236:787-792.
Takahata, N. and M. Slatkin. 1984. Mitochondrial gene flow. Proc. Natl.
Acad, Sci. USA 81:1764-1767.
Tricas, T. C. 1987. Life history and behavioral ecology of Chaetodon
multicinctus. Ph. D. dissertation, University of Hawaii, Honolulu.
Wilson, A. c, R. L. Cann, S. M. Carr, M. George, U. B. Gyllensten, K. M.
Helm-Bychowski, R. G. Higuchi, S. R. Palumbi, E. M. Pranger, R. D. Sage,
and M. Stoneking. 1985. Mitochondrial DNA and two perspectives on
evolutionary genetics. Biol. J. Linn. Sci. 26:375-400.
'Alright, S. 1951. The genetical structure of populations. Ann. Eugen. 15:322
354.
194
Winans, G. A. 1980. Geographic variation in the milkfish, Chanos chanos. 1.
Biochemical evidence. Evolution 34:558-574.
195
Table 1: Enzymes, enzyme abbreviations, Enzyme Commission numbers,
number of loci, and gel buffers used in our electrophoretic analysis of
Chaetodon punctatofasciatus, C. pelewensis, and C. multicinctus.
Enzyme Abbr. EC No Loci BuffertAspartate aminotransferase AAT 2.6.1.1 2 TC,CTCreatine kinase CK 2.7.3.2 4 R WDiaphorase DIA 1.6.2.2 1 R WEsterase EST 3.1.1.1 3 R WGlucose-6-phosphate dehydrogenase G6PD 1.1.1.49 1 TVDGlucose phosphate isomerase GPI 5.3.1.9 2 CTlsocitrate dehydrogenase ICD 1.1.1.42 2 TCLactate dehydrogenase LDH 1.1.1.27 2 TVDMalate dehydrogenase MDH 1.1.1.37 2 CTMalate dehydrogenase (NADP+) MDPH 1.1.1.40 2 TCMannose phosphateisomerase MPI 5.3.1.8 1 TVDpeptidase B PEPB 3.4.13.,. 2 R Wpeptidase 5 PEPS 3.4.13." 1 R Wpeptidase A PEPA 3.4.13." 1 R WPhosphoglucomutase PGM 2.7.5.1 1 TC, CTPhosphogluconate dehydrogenase PGDH 1.1.1.44 1 CTSuperoxide dismutase SOD 1.15.1.1 1 TVDTriose phosphate isomerase TPI 5.3.1.1 2 R Wt CT: amine-citrate, pH 6.0 (Clayton and Tretiak, 1972); TC: tris-citrate, pH 8.0(Selander, et al., 1971); RW: tris-citrate, pH 8.5 (Ridgway, et al., 1970); TVB: trisversene-borate.. pH 8.0 (Selander, et al., 1971).
196
Table 2: Allele frequencies for the 19 variable loci in Chaetodon multicinctus.
C. punctatofasciatus, and C. pelewensis.
SpeciesLocus allele C. multi. C. puncta. C. pele.AAT1 A 0.043
B 0.020C 0.980 1.000 0.957
AAT2 A 0.018B 1.000 0.982 1.000
DIA A 0.909 1.000 0.978B 0.091 0.022
EST1 A 0.045 0.161 0.177B 0.955 0.732 0.694C 0.089 0.097D 0.018 0.016E 0.016
EST2 A 0.076B 0.924 0.982 1.000C 0.018
EST3 A 1.000 0.963 0.968B 0.037 0.032
G6PD A 0.280 0.278 0.261B 0.700 0.667 0.696C 0.020 0.056 0.043
GPIl A 0.985 0.946 0.935B 0.015 0.032C 0.016D 0.054 0.016
GPI2 A 0.036B 0.607 0.758C 0.048D 1.000 . 0.357 0.194
197
Table 2 (continued)
ICD2 A 0.879 1.000 1.000B 0.121
MEl A 0.020B 0.980 0.875 0.957C 0.100 0.043D 0.025
ME2 A 0.980 0.975 0.978B 0.020 0.025 0.022
MDHI A 0.016B 1.000 1.000 0.984
MDH2 A 0.985 0.089 0.339B 0.015 0.911 0.661
PEPB2 A 1.000 0.964 1.000B 0.018C 0.018
PEPS A 0.030 0.036 0.016B 0.970 0.964 0.984
PEPA A 0.271 0.273B 1.000 0.708 0.727C 0.021
PGM3 A 0.016B 1.000 0.982 0.984C 0.018
SOD A 0.018B 1.000 0.982 1.000
198
Table 3: Matrix of Nei (1978) unbiased genetic distance coefficients (D) based
on allelic variation at 31 presumptive loci.
C. punctatofasciatusPhilippines
C. punctatofasciatus
C. pelewensis 0.046
C. pelewensisFiii
C. multicinctusHawaii
C. multicinctus 0.040 0.002
199
Table 4: Summary of genetic variability at 31 loci in C. multicinctus, C.
pelewensis, and C. punctatofasciatus (standard errors in parentheses).
C. multicinctus(Hawaii)
0.042(0.016)
0.041(0.015)
Directcount
Mean heterozygosityHardy
Weinbergexpectedt
35.5
Percentageof loci
polymorphict1.4
(0.1)
Mean no.of allelesper locus
31.2(0.7)
Mean samplesize perlocusSpecies
C. punctato. 26.1 1.7(Philippines) (0.6) (0.2)
C. pelewensis 29.4 1.7(Fiji) (0.6) (0.2)
48.4
45.2
0.094(0.30)
0.080(0.026)
0.090(0.028)
0.088(0.029)
t A locus was considered polymorphic if more than one allele was detected.:I: Unbiased estimate (see Nei, 1978)
200
Table 5: FST values and values of Nm (in parentheses) among species
estimated from both allozyme variation (below diagonal) and mitochondrial
control-region sequences (above diagonal). For mtDNA data see McMillan
and Palumbi, submitted.
C. punctatofasciatusPhilippines
C. punctatofasciatus
C. pelewensisFiji
0.018 (27)
C. rnulticinctusHawaii
0.479 «1)
C. pelewensis
C. multicinctus
0.019 (13)
0.253 «1) 0.230 (<1)
201
0.480 «1)
REFERENCES
Abbott, R T. 1982. Kingdom of the seashell. CrownPress, New York.
Acharyya, K. S. and P. K. Basu. 1993. Toba ash on the Indian subcontinent
and its implications for correlation of late Pleistocene alluvium.
Quaternary Research 40:10-19.
Allen, G. R 1972. The anemonefishes, their classification and biology. T.F.H.
publications, Neptune City.
Allen, G. R 1980. Butterfly and angelfishes of the world, vol 2. Wiley
Interscience, New York.
Allen, G. R, and R Swainston. 1992. The fishes of New Guinea. Kristen
Press, Inc., Madang.
Allmon, W. D., G. Rosenberg, R. W. Portell, K. S. Schindler. 1993. Diversity
of Atlantic coastal plain mollusks since the Pliocene. Science 260:1626
1229.
Altman, P. L. and D. S. Dittmer. 1972. Biology data book. Fed. Am. Soc. Exp.
Biol., 2nd Ed., Bethesda.
Anderson, G. R V., A. H. Ehrlich, P. R. Ehrlich, J. D. Roughgarden, B. C.
Russel, and F. H. Talbot. 1981. The community structure of coral reef
fishes. Am. Nat. 117:476-495.
Audley-Charles, M. G. 1981. Geological history of the region of Wallace's
line. Pp. 24-35 in T. C. Whitmore, ed. Wallace's line and Plate tectonics.
Clarendon Press, Oxford.
202
Avise, J. C. 1992. Molecular population structure and the biogeographic
history of a regional fauna: a case history with lessons for conservation
biology. Oikos 63:62-76.
Avise, J. C. and N. C. Saunders. 1984. Hybridization and introgression among
species of sunfish (Lepomis): analysis by mitochondrial DNA and
allozyme markers. Genetics 108:237-255.
Avise, J. c.,J. Arnold, R. M. Ball, E. Bermingham, T. Lamb, J. E. Neigel, C. A.
Reeb, and N. C. Saunders. 1987. Intraspecific phylogeography: The
mitochondrial DNA bridge between population genetics and systematics.
Ann. Rev. Ecol. Syst. 18: 489-522.
Avise, J. c., R. M. Ball, and J. Arnold. 1988. Current versus historical
population sizes in vertebrate species with high gene flow: a comparison
based on mitochondrial DNA lineages and inbreeding theory for neutral
mutations. Mol. BioI. Evol. 5:331-344.
Baker, R. J., S. K. Davis, R. D. Bradley, M. J. Hamilton, and R. A. Van der
Bussche. 1989. Ribosomal-DNA, mitochondrial DNA, chromosomal,
and allozymic studies on a contact zone in the pocket gopher, Geomys.
Evolution 43:63-75.
Ball, R. M., J. E. Neigel, and J. C. Avise. 1990. Gene genealogies within the
organismal pedigrees of random-mating populations. Evolution 44:360
370.
203
Ball, R. M., S. Freeman, F. C. James, E. Bermingham, and J. C. Avise. 1988.
Phylogeographic population structure of Red-winged Blackbirds assessed
by mitochondrial DNA. Proc. Nat1. Acad. Sci. USA 85:1558-1562.
Barlow, G. 1963. Ethology of the Asian teleost Badis badis. II. Motivation
and signal value of the color patterns. Anim. Behav. 11:97-105.
Barton N. H, and K. Gale. 1993. Genetic analysis of hybrid zones. Pp 13-45 in
R. Harrison, ed. Hybrid zones and the evolutionary process. Oxford
University Press, New York.
Barton, N. H. 1983. Multilocus clines. Evolution 37:454-471.
Barton, N. H. and G. M. Hewitt. 1981. The genetic basis of hybrid inviability
between two chromosomal races of the grasshopper Podisma pedestris.
Heredity 47:367-383.
Barton, N. H. and G. M. Hewitt. 1985. Analysis of hybrid zones. Ann. Rev.
Eco1. Syst. 16:113-148.
Bauchot, R., J. Ridet, and M. Bauchot. 1989. The brain organization of
butterflyfishes. Environ. Bio. Fishes. 25:205-219.
Belasky, P. and B. Runnegar. 1993. Biogeographic constraints for tectonic
reconstructions of the Pacific region. Geology 21:979-982.
Bermingham E. and H. Lessios. 1993. Rate variation of protein and
mitochondrial DNA evolution as revealed by sea urchins separated by the
Isthmus of Panama. Proc. Nat1. Acad. Sci. USA 90:2734-2738.
204
Bermingham, E., and J. C. Avise. 1986. Molecular zoogeography of
freshwater fishes in the southeastern United States. Genetics 113:939-965.
Bert, T. M. and R. G. Harrison. 1988. Hybridization in western Atlantic stone
crabs (genus Menippe): Evolutionary history and ecological context
influence species interactions. Evolution 42:528-544.
Blum, S. D. 1988. Osteology and phylogeny of the Chaetodontidae (Pisces:
Perciformes). Ph. D. Dissertation, University of Hawaii, Honolulu.
Blum, S. D. 1989. Biogeography of the Chaetodontidae: an analysis of
allopatry among closely related species. Envir. Biol. Fish. 25: 9-31.
Brockman, H. J. 1973. The function of poster coloration in the beaugregory,
Eupomacenrus leucostictus (Pomacentridae, Pisces). Z. Tierpsychol. 33:13
34.
Brown, J. R., A. T. Beckenbach, and M. J. Smith. 1993. Intra-specific DNA
sequence variation of the mitochondrial control-region of the white
sturgeon (Acipenser transmontanus). Mol. BioI. Evol, 10:326-341.
Brown, W. M., E. M. Pranger, A. Wang, and A. C. Wilson. 1982.
Mitochondrial DNA sequences of primates: tempo and mode of
evolution. J. Mol. Evol. 18:225-239.
Burgess, W. E. 1978. Butterflyfishes of the world. T. F. H. Publications,
Neptune City.
205
Bushnell, P. G., P. L. Lutz, and S. H. Gruber. 1989. The metabolic rate of an
active, tropical elasmobranch, the lemon shark (Negaprion brevirostris).
Exp. BioI. 48:279-283.
Butcher, G. S. and S. Rohwer. 1987. The evolution of conspicuous and
distinctive coloration for communication in birds.
Butlin, R. 1989. Reinforcement of premating isolation. Pp 158-179 in D. Otte
and J. A. Endler, eds. Speciation and its consequences. Sinauer Press,
Massachusetts.
Carr, S. M., S. W. Ballinger, J. N. Deer, L. H. Blankenship, and J. W. Bickham.
1986. Mitochondrial DNA analysis of hybridization between sympatric
white-tailed deer and mule deer in west Texas. Proc. Natl. Acad. Sci. USA
83:9576-9580.
Charlton, T. R. 1986. A plate tectonic model of the eastern Indonesia
collision zone. Nature 319:394-396.
Chesner, C. A., W. 1. Rose, A. Deino, R. Drake and J. A. Westgate. 1991.
Eruptive history of earth's largest quaternary caldera (Toba, Indonesia)
clarified. Geology 19:200-302.
Cheuy, J. M., D. K. Rea, and N. G. Pisias. 1987. Late Pleistocene
paleoclimatology of the central equatorial Pacific: a quantitative record of
eolian and carbonate deposition: 28:323-339.
Choat, J. H. and Bellwood 1991. Reef fishes: their history and evolution. Pp.
39-66 in P. Sale, ed. The ecology of fishes on coral reefs. Academic Press,
Inc. San Diego.
206
Connell, J. H. 1978. Diversity in tropical rain forests and coral reefs. Science
199:1302-1310.
Cracraft, J. and R. O. Prum. 1988. Patterns and processes of diversification:
speciation and historical congruence in some neotropical birds.
Evolution 42:603-620.
Crame, J. A. 1986. Late Pleistocene molluscan assemblages from the coral
reefs of the Kenya coast. Coral Reefs 4:183-196.
Crane, J. 1975. Fiddler crabs of the world (Oeypodidaeg: genus Uca).
Princeton University Press, Princeton.
Croizat, L. G., G. Nelson and D. E. Rosen, 1974. Centers of origins and related
concepts. Syst. Zool. 23: 265-287.
Desalle, R. T. Freedman, E. M. Prager, and A. C. Wilson. 1987. Tempo and
mode of sequence evolution in mitochondrial DNA of Hawaiian
Drosophila. J. Mol. Evol. 26:157-164.
Di Rienzo, A. and A. C. Wilson. 1991. The pattern of mitochondrial
variation is consistent with an early expansion of the human population.
Proc. Natl. Acad. Sci. USA 88:1597-1601.
Donaldson, T. J. 1986. Distribution and species richness patterns of Indo
West Pacific Cirrhitidae: support for Woodland's hypothesis. Pp 623-628
in T. Uyeno, R. Arai, T. Taniuchi and K. Matsuura, eds. Indo-Pacific fish
biology, proceedings of the second international conference on Indo
Pacific fishes. Ichthyological Society of Japan, Tokyo.
207
Dowling, T. E., G. R. Smith, and W. M. Brown. 1989. Reproductive isolation
and introgression between Notropis comutus and Notropis
chrysocephalus (family Cyprinidae): comparison of morphology,
allozymes, and mitochondrial DNA. Evolution 43:620-643.
Ehrlich, P. R., F. H. Talbot, B. C. Russell, and G. R. V. Anderson. 1977. The
behavior of chaetodontid fishes with special reference to Lorenz's "poster
colouration" hypothesis. J. Zoo1. London 183:213-228.
Ekman, S. 1953.. Zoogeography of the sea. Sidgwick and Jackson, London.
Endler, J. A. 1977. Geographic variation, speciation, and clines. Princeton
University Press, Princeton, New Jersey.
Endler, J. A. 1980. Natural selection on color pattern in Poecilia reticulata.
Evolution 34:76-91.
Endler, J. A. 1982. Problems in distinguishing historical from ecological
factors in Biogeography. Amer. Zoo1. 22:441-452.
Excoffier, L., P. E. Smouse, and J. M. Quattro. 1992. Analysis of molecular
variance inferred from metric distances among DNA haplotypes:
application to human mitochondrial DNA restriction data. Genetics
131:479-491.
Faith, D. P. 1991. Cladistic permutation tests for monophyly and
nonmonophyly. Syst. Zoo1. 40:366-375.
Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the
bootstrap. Evolution 39:783-791.
208
Felsenstein, J. 1993. PHYLIP: phylogenetic inference package, version 3.5.
University of Washington, Seattle.
Ferris, S. D., R. D. Sage, C.-H. Huang, J. T. Nielsen, U. Ritte, and A. C. Wilson.
1983. Flow of mitochondrial DNA across a species boundary. Proc. Natl.
Acad. Sci. USA 80:2290-2294.
Fisher, E. A. 1980. Speciation in the Hamlets (Hypoplectrus: Serranidae)- a
continuing enigma. Copeia 1980:649-659.
Fleminger, A. 1986. The Pleistocene equatorial barrier between the Indian
and Pacific oceans and a likely cause for Wallace's line. Pp. 84-97 in A. C.
Pierrot-Bults, S. van der Spoel, B. J. Aahuraner, and R. K. Johnson, eds.
Pelagic biogeography. UNesco technical papers in marine science. No. 49.
French, S. and B. Robson. 1983. What is a conservative substitution? J. Mol.
Evo1. 19:171-175.
Gochfeld, D. J. 1991. Energetics of a predator-prey interaction: corals and
coral-feeding fishes. Pacific Science 46:246-256.
Gosline, W. A. 1965. Thoughts on systematic work in outlying areas. Syst.
Zoo1. 14:59-61.
Graham, J. H. and J. D. Felley. 1985. Genomic co-adaptation and
developmental stability within introgressed populations of Enneacanthus
glorioslls and E. obesus (Pisces, Centrarchidae). Evolution 39: 104-114.
209
Grudzien, T. A. and W. S. Moore. 1986. Genetic differentiation between the
yellow-shafted and Red-shafted subspecies of the Northern Flicker.
Biochemical Systematics and Ecology 14:451-453.
Grudzien, T. A., W. S. Moore, J. R. Cook, D. Tagle. 1987. Genetic population
structure and gene flow in the northern flicker (Colaptes auratus) hybrid
zone. Auk 104:654-664.
Hailman, J. P. 1979. Environrnentallight and conspicuous colors. Pp.289-354
in E. H. Burtt, [r., ed. The behavioral significance of color. Garland STPM
Press, New York.
Harpending, H. c., S. T. Sherry, A. R. Rogers, and Mark Stoneking. 1993. The
genetic structure of ancient human populations. Current Anthropology
4:483-496.
Harrison, R. G. 1989. Animal mitochondrial DNA as a genetic marker in
population and evolutionary biology. Trends Ecol. Evol, 4:6-11.
Harrison, R. G. 1990. Hybrid zones: windows on evolutionary process.
Oxford Surveys in Evolutionary Biology 7:69-139.
Hewitt, G. M. 1989. The subdivision of species by hybrid zones. Pp 85-110 in
D. Otte and J. A. Endler, eds. Speciation and its consequences. Sinauer
Press, Massachusetts.
Hiatt, R. W. and D. W. Strasburg. 1960. Ecological relationships of the fish
fauna on coral reefs on the Marshall Island. Ecol. Monogr. 30:65-127.
210
Hillis, D. M. and J. J. Bull. An empirical test of bootstrapping as a method for
assessing confidence in phylogenetic trees. Syst. Biol. 42:182-192.
Hillis, D. M., and C. Moritz. 1990. Molecular systematics. Sinauer Press.,
Sunderland, MA.
Hobson, E. S. 1974. Feeding 'relationships of teleostean fishes on coral reefs in
Kona, Hawaii. U. S. Fish. Bull. 77:915-1031.
Hocutt, C. H. 1987. Evolution of the Indian Ocean and the drift of India: a
vicariant event. Hydrobiol. 159:203-223.
Hourigan, T. F. 1987. The behavioral ecology of three species of
butterflyfishes (Family Chaetodontidae). Ph. D. Dissertation, University
of Hawaii, Honolulu.
Hourigan, T. F. 1989. Environmental determents of butterflyfish social
systems. Envir. Biol. Fishes 25:61-78.
Hourigan, T. F. and E. S. Reese. 1987. Mid-ocean isolation and the evolution
of Hawaiian reef fishes. Trends Ecol. Evol. 2:187-191.
Hudson, R. R. 1990. Gene genealogies and the coalescent process. Oxfor.
Surv. Evol. BioI. 7:1-44.
Hudson, R. R., M. Slatkin, and W. P. Maddison. 1992. Estimation of levels of
gene flow from DNA sequence data. Genetics 132:583-589.
Irwin, D. M., T. D. Kocher, and A. C. Wilson. 1991. Evolution of the
cytochrome b gene of mammals. J. Mol. Evol. 32: 128-144.
211
Jackson, J. B., P. Iung, A. G. Coates, L. S. Collins. 1993. Diversity and
Extinction of tropical American mollusks and emergence of the Isthmus
of Panama. Science 260:1624-1626.
Janecek, T. R. and D. K. Rea. 1985. Quaternary fluctuations in the northern
hemisphere trade winds and westerlies. Quater. Res. 24:150-163.
[okiel, P. L. 1990. Transport of reef corals into the great barrier reef. Nature
347:665-367.
Kay, E. A. 1994. Evolutionary radiations in the Cypraeidae. Journal of
Molluscan Studies (in press).
Kelly, C. D. and T. Hourigan. 1983. The function of conspicuous coloration
in chaetodontid fishes: a new hypothesis. Am. Behavior 31:
Kimura, M. 1980. A simple method for estimating evolutionary rate of base
substitution through comparative studies of nucleotide sequences. J. Mol.
Evo!. 16:111-120.
Kishino, H. and M. Hasegawa. 1989. Evaluation of the maximum likelihood
estimate of the evolutionary tress topologies form DNA sequence data,
and the branching order in Hominoidea. J. Mol. Evol. 29:170-179.
Knowlton, N. and J. B. C. Jackson. 1993. Jack-of-all-trades or master of some?
New taxonomy and niche partitioning on coral reefs. Trends Ecol. Evol.
(in press).
Kocher, T. D. and A. C. Wilson. 1991. Sequence evolution of mitochondrial
DNA in humans and chimpanzees: Control-region and a protein-coding
212
region. Pp. 391-413 in S. Osawa and T. Honjo, eds., Evolution of life.
Spring-Verlag, New York.
Kohn, A. J. 1990. Tempo and mode of evolution in Conidae. Malacologia
32:55-67.
Kumar, S., K. Tamura, and M. Nei. 1993. MEGA: molecular evolutionary
genetics analysis, version 1.01. Pennsylvania State University, University
Park.
Ladd, H. S. 1960. Origins of the Pacific island molluscan fauna. Amer. J.
Science. 258:137-150.
Lamb and Avise, 1986. Directional introgression of mitochondrial DNA in a
hybrid population of tree frogs: the inference of mating behavior. Proc.
Nat!. Acad. Sci. USA 83:2526-2530.
Lamb, T., J. M. Novak, and D. L. Mahoney. 1990. Morphological asymmetry
and interspecific hybridization: a case study using hylid frogs. J. Evol,
BioI. 3:295-309.
Lande, R. 1982. Models of speciation by sexual selection of polygenic traits.
Proc. NatI. Acad. Sci. USA 78:3721-3725.
Lansing, W. R. J. and C. C. Brower. 1974. Development of colour patterns in
relation to behavior in Tilapia mossarnbica (Peters). J. Fish BioI. 6:29-41.
Lavery, S. 1993. Ph. D. dissertation. Dept of Zoology, University of
Queensland, Brisbane, Australia
213
Leary, R. F. and F. W. Allendorf. 1989. Fluctuating asymmetry as an indicator
of stress: implications for conservation biology. Trends Ecol. Evol. 4:214
217.
Leary, R. F., F. W. Allendorf, and K. L. Knudsen. 1985. Developmental
instability and high meristic counts in interspecific hybrids of salmonid
fishes. Evolution 39:1318-1326.
Leis, J. M. 1989. Larval biology of butterflyfishes (Pisces, Chaetodontidae):
what do we really know? Envir. BioI. Fish. 25:87-100.
Littlejohn, M. J. and G. F. Watson. 1985. Hybrid zones and homogamy in
Australian frogs. Ann. Rev. Ecol. Syst. 16:85-112.
Lobel, P. S. 1989. Spawning behavior of Chaetodon multicinctus
(Chaetodontidae); pairs and intruders. Envir. BioI. Fish. 25:125-130.
Lorenz, K. 1967. On aggression. Bantam, New York.
Losey, G. 1988. Behavior events acquisition and analysis system, "BEAST".
Windward Technology, Kaneohe, Hawaii.
Lynch, M. and T. J. Crease. 1990. The analysis of population survey data on
DNA sequence variation. Mol. BioI. Evol. 7:377-394.
Maddison, W. P., and D. R. Maddison. 1992. MacClade: analysis of phylogeny
and character evolution. Sinauer Press, Sunderland.
Mallet, J. L. B. 1993. Speciation, raciation, and color pattern evolution in
Heliconius butterflies: Evidence from hybrid zones. Pp. 226-260 in R.
214
Harrison, ed. Hybrid zones and the evolutionary process. Oxford
University Press, New York.
Mallet, J. L. B. and N. H. Barton. 1989a. Inference from clines stabilized by
frequency-dependent selection. Genetics 122:967-976.
Mallet, J. L. B. and N. H. Barton. 1989b. Strong natural selection in a
warning-color hybrid zone. Evolution 43:421-431.
Marjoram, P. and P. Donnelly. 1994. Pairwise comparisons of mitochondrial
DNA sequences in subdivided populations and implications for early
human evolution. Genetics 136:673-683.
Markow, T. A. and J. P. Ricker. 1991. Developmental stability in hybrids
between the sibling species pair, Drosophila melanogaster and Drosophila
simulans. Genetica 84:115-121.
Martin, A. P and S. R. Palumbi. 1993. Body size, metabolic rate, generation
time and the molecular clock. Proc. Natl. Acad. Sci. USA 90:4087-4091.
Martin, A. P., G. J. P. Naylor, and S. R. Palumbi. 1992. Rates of mitochondrial
DNA evolution in sharks are slow compared with mammals. Nature
357:153-155.
Mayr, 1942. Systematics and the origins of species. Columbia University
Press, New York.
Mayr, E. 1954. Geographic speciation in tropical echinoids. Evolution 8:1-18.
215
McAndrew, B. J., F. R. Roubal, R. J. Roberts, A. M. Bullock, and I. M. McEwen.
1988. The genetics and histology of red, blond, and associated colour
variants in Oreochrornis niloticus. Genetica 76:127-137.
McManus, J. W. 1985. Marine speciation, tectonics and sea-level changes in
southeast Asia. Proc. 5th Int. Coral Reef Congr. 4:133-138.
McPhail, J. D. 1969. Predation and the evolution of a stickleback
(Gasterosteus). J. Fish. Res. Bd. Canada 26:3183-3208.
Meyer, T. D. Kocher, P. Basasibwaki, and A. C. Wilson. 1990. Monophyletic
origin of Lake Victoria cichlid fishes suggested by mitochondrial DNA
sequences. Nature 347:550-553.
Molina-Cruz, A. 1977. The relation of the southern trade winds to upwelling
process during the last 75,000 years. Quater. Res. 8:324-338.
Moore, W. S. 1987. Random mating in the northern flicker hybrid zone:
implications for the evolution of bright and contrasting plumage patterns
in birds. Evolution 41:539-546.
Moore, W. S. and D. B. Buchanan. 1985. Stability of the northern flicker
hybrid zone. Evolution 39:135-151.
Moore, W. S. and J. T. Price. 1993. Nature of selection in the northern flicker
hybrid zone and its implication for speciation theory. Pp. 196-225 in R.
Harrison, ed. Hybrid zones and the evolutionary process. Oxford
University Press, New York.
216
Moore, W. S., J. H. Graham, and J. T. Price. 1991. Mitochondrial DNA
variation in the Northern Flicker (Colaptes auratus, Aves). Mol. Bio!.
Evol. 8:327-344.
Moran, P. and I. Kornfield. 1993. Retention of an ancestral polymorphism in
the Mbuna species Flock (Teleostei: Cichlidae) of Lake Malawi. Mol. Biol.
Evol. 10:1015-1029.
Neigel, J. E. and J. C. Avise. 1986. Phylogenetic relationships of
mitochondrial DNA under various demographic modes of speciation.
Pp. 515-534 in S. Karlin and E. Nevo, eds. Evolution process and theory.
Academic Press, New York.
Nelissen, M. 1976. Contribution to the ethology of Tropheus moorii
Boulenger (Pisces, Cichlidae) and a discussion of the significance of its
colour patterns. Rev. Zool. Afr. 90:17-29.
Neudecker, S. 1989. Eye camouflage and false eyespots: chaetodontid
responses to predators. Envir. BioI. Fishes 25: 143-147.
Nishida, M. and S. J. Lucas. 1988. Genetic differences between geographic
populations of the crown-of-thorn starfish throughout the Pacific region.
Marine Biology 98: 359-368.
Otte, D. and J. A. Endler. 1989. Speciation and its consequences. Sinauer
Press, Sunderland, Massachusetts.
Ovenden, J. R., A. G. MacKinlay and R. H. Crozier. 1987. Systematics and
mitochondrial genome organization of Australian rosellas (Aves:
Platycercidae). Mol. BioI. Evoi. 4:52-543.
217
Palmer, R. A. and C. Strobeck. 1986. Fluctuating asymmetry: measurement,
analysis, and patterns. Ann. Rev. Ecol. Syst. 17:391-421.
Palmilo, P. and M. Nei. 1988. Relationships between gene trees and species
trees. Mol. BioI. Evoi. 5:568-583.
Palumbi, S. R. 1992. Marine speciation on a small planet. TREE 7:114-117.
Palumbi, S. R. and E. C. Metz. 1991. Strong reproductive isolation between
closely related tropical sea urchins (genus Echinometra). Mol. Biol, and
Evol. 8:227-239.
Palumbi, S. R., A. Martin, S. Romano, W. O. McMillan, L. Stice, and G.
Grabowski. 1991. The simple fool's guide to PCR. Dept. of Zoology,
University of Hawaii, Honolulu.
Parsons, T. J., S. L. Olson, M. J. Braun. 1993. Unidirectional spread of
secondary sexual plumage traits across an avian hybrid zone. Science
260:1643-1646.
Pauley, G. 1990. Effects of late Cenozoic sea-level fluctuations on the bivalve
faunas of tropical oceanic islands. Paleobiology 16:415-434.
Pedersen, T. F. 1983. Increased productivity in the eastern equatorial Pacific
during the last glacial maximum (19,000 to 14,000 yrs B. P.). Geology 11:16
19.
Perron, F. E. and A. J. Kohn. 1985. Larval dispersal and geographic
distribution in coral reef gastropods of the genus Conus. Proc. Fifth
Intern. Coral Reef Congr. 4:95-100.
218
Pesole, G, E. Sbisa, G Preparata, and C. Saccone. 1992. The evolution of the
mitochondrial d-loop region and the origin of modern man. Mol. Biol,
Evol. 9:587-598.
Peterman, R. M. 1971. A possible function of colour in coral reef fishes.
Copeia 2:330-331.
Platnick, N. 1. and G. Nelson. 1978. A model of analysis for historical
biogeography. Syst. Zoo1. 27:1-16.
Pollock, D. E. 1992. Paieoceanography and speciation in the spiny lobster
genus Panulirus in the Indo-Pacific. Bull. Mar. Sci. 51:135-146.
Porter, S. C. 1989. Some geological implications of average quaternary glacial
conditions. Quaternary Research 32:245-261.
Potts, G. 1973. The ethology of Labroides dimidiatus on Aldabra. Anim.
Behav. 21:250-291.
Powell, J. R. 1983. Interspecific cytoplasmic gene flow in the absence of
nuclear gene flow: evidence from Drosophila. Proc. N atl. Acad. Sci. USA
80:492-495.
Quinn, W. H. 1971. Late quaternary meteorological and oceanographic
developments in the equatorial Pacific. Nature 229:330-331.
Rand, D. M. and R. C. Harrison. 1989. Ecological genetics or a mosaic hybrid
zone: mitochondrial, nuclear, and reproductive differentiation of crickets
by soil type. Evolution 43:432-449.
219
Randall, J. E., G. R. Allen, and R. C. Steene. 1977. Five probable hybrid
butterflyfishes of the genus Chaetodon from the central and western
Pacific. Rec. West. Aust. Mus. 6:3-26.
Rea, D. K. 1990. Aspects of atmospheric circulation: the late Pleistocene (0
950,000 yr) record of eolian deposition in the Pacific Ocean. Palaeogeo.,
Palaeodim., Palaeoecol 78:217-227.
Rea, D. K. 1991. Late Pleistocene paleoclimatology of the central equatorial
Pacific: flux patterns of biogeneic sediments. Paleoceanography 6:227-244.
Reaka, M. L. 1980. Geographic range, life history patterns, and body size in a
guild of coral-dwelling mantis shrimps, Evolution 34:1019-1030.
Reeb, C. A. and J. C. Avise. 1990. A genetic discontinuity in a continuously
distributed species: mitochondrial DNA in the American Oyster,
Crassostrea virginica. Genetics 124(2):397-406.
Reese, E. 1975. A comparative field study of the social behavior and related
ecology of reef fishes of the family Chaetodontidae. Z. Tierpsychol. 37:37
61.
Reese, E. S. 1989. Orientation behavior of butterflyfishes (family
Chaetodontidae) on coral reefs: spatial learning of route specific
landmarks and cognitive maps. Environ. Biol. Fish. 25:79-86.
Reese, E. S. 1991. How behavior influences community structure of
butterflyfishes (Family Chaetodontidae) on Pacific coral reefs. Ecology
international Bulletin 19:29-41.
220
Reid, D. G. 1990. Trans-arctic migration and speciation induced by climate
change: the biogeography of Littorina (Mollusca: Gastropoda). Bull. Mar.
Sci. 47:35-49.
Reynolds, R. W. 1988. A real-time global sea-surface temperature analysis.
Journal of Climate 1:75-86.
Richmond, R. H. 1990. The effects of the El Nino/Southern Oscillation on
the dispersal of corals and other marine organisms. Pp. 127-140 in P. W.
Glynn, ed. Global ecological consequences of the 1982-1983 El Nino
Southern Oscillation. Elsevier Press, Amsterdam.
Roff, D. A. and P. Bentzen. 1989. The statistical analysis of mitochondrial
DNA polymorphisms: chi-square and the problem of small samples.
Mol. BioI. Evol. 6:539-545.
Rogers, A. R. 1992. Error introduced by infinite-sites model. Mol. BioI. Evol.
9: 1181-1184.
Rogers, A. R. and H. Harpending. 1992. Population growth makes waves in
the distribution of pairwise genetic differences. Mol. BioI. Evol. 9:552-569.
Rosen, B. R. 1988. Progress, problems and patterns in biogeography of reef
corals and other tropical marine organisms. Helgolander Meeresunters
42:269-301.
Ross, K. G. and J. L. Robertson. 1990. Developmental stability, heterozygosity,
and fitness in two introduced fire ants (Solenopsis invicta and S. richteri)
and their hybrid. Heredity 64:93-103.
221
Ross, R. M. 1984. Catheterization: a non-harmful method of sex
identification for sexually monomorphic fishes. Prog. Fish. Cult. 46:151
152.
Rotondo, G. M., V. G. Springer, G. A. J. Scott, and S. O. Schlanger. 1981. Plate
movement and island integration-a possible mechanism in the
formation of endemic biotas, with special reference to the Hawaiian
Islands. Syst. Zoo1. 30:12-21.
Rowland, W. J. 1979. The use of color in intraspecific communication. Pp.
379-421 in E. H. Burtt, [r., ed. The behavioral significance of color.
Garland STPM Press, New York.
Saccone, c., G. Pesole and E. Sbisa. 1991. The main regulatory region of
mammalian mitochondrial ONA: structure-function model and
evolutionary pattern. J. Mol. Evo1. 1991 33:83-91.
Saccone, c., M. Attimonelli and E. Sbisa. 1987. Structural elements highly
preserved during the evolution of the d-loop-containing region in
vertebrate mitochondrial DNA. J. Mol. Evo1. 25:205-211.
Saitou, N. and M. Nei. 1987. The Neighbor-joining method: a new method
for reconstructing phylogenetic trees. Mol. BioI. Evo1. 4:406-425.
Sambrook, J., E. F. Fritsch and T. Maniatis. 1989. Molecular cloning: a
laboratory manual, 2nd edition. Cold Spring Harbor Laboratory, Cold
Spring Harbor, New York.
222
Schopf, T. M. and 1. S. Murphy. 1973. Protein polymorphism of the
hybridization seas tars Asterias forbesi and Asterias vulgaris and
implications for their evolution. Biol. Bull. 145:589-597.
Slatkin, M. 1989. Detecting small amounts of gene flow from phylogenies of
alleles. Genetics 121:609-612.
Slatkin, M. and R. R. Hudson. 1991. Pairwise comparison of mitochondrial
DNA sequences in stable and exponentially growing populations.
Genetics 129:555-562.
Slatkin, M. and W. P. Maddison. 1989. A cladistic measure of gene flow
inferred from the phylogenies of alleles. Genetics 123:603-613.
Smith, D. G. 1972. The role of the epaulets in the Red-winged Blackbird,
Agelaius phoeniceus social system. Behavior 41:251-268.
Snow, J. 1. and M. K. Rylander. 1982. A quantitative study of the optic system
of butterflyfishes (Family Chaetodontidae). J. Hirf. 23:121-125.
Southwood, T. R. E. 1978. Ecological methods. Chapman and Hall, London.
Spolsky, C. and T. Uzzell. 1984. Natural interspecies transfer of
mitochondrial DNA in amphibians. Proc. Natl. Acad. Sci. USA 81:5802
5805.
Springer, V. G. 1982. Pacific plate biogeography, with special reference to
shore fishes. Smithsonian Contributions to Zoology, Number 367.
Smithsonian Institution Press, Washington. D.C.
223
Springer, V. G. 1988. The Indo-Pacific fish genus Ecsenius. Smithsonian
Contributions to Zoology, Number 465. Smithsonian Institution Press,
Washington, D.C.
Springer, V. G. and J. T. Williams. 1990. Widely distributed pacific plate
endemics and lowered sea-level. Bull. Mar. Sci. 47: 631-640.
Stanley, S. M. 1981. Neogene mass extinction of western Atlantic mollusks.
Nature 293:457-459.
Stehli, F. G. and J. W. Wells. 1971. Diversity and age patterns in hermatypic
corals. Systematic Zoology 20:115-126.
Stoskopf, M. K. 1993. Fish Medicine. W. B. Saunders Co., Philadelphia.
Sturmbauer, C. and A. Meyer. 1992. Genetic divergence, speciation and
morphological stasis in a lineage of African cichlid fishes. Nature
358:578:581.
Sturmbauer, C. and A. Meyer. 1993. Mitochondrial phylogeny of the endemic
mouthbrooding lineages of cichlid fishes from Lake Tanganyika in
eastern Africa. Mol. BioI. Evol. 10:751-768.
Swofford, D. 1993. Phylogenetic analysis using parsimony (PAUP), Version
3.1.1. University of Illinois, Champaign.
Szymura, J. M. and N. H. Barton. 1986. Genetic analysis of a hybrid zone
between the fire-bellied toads Bombina bombina and B. variegata, near
Cracow in southern Poland. Evolution 40:1141-1459.
224
Szyrnura, J. M., C. Spolsky and T. Uzzell. 1985. Concordant change in
mitochondrial and nuclear genes in a hybrid zone between two frog
species (genus Bombina). Experentia 41:1469-1470.
Tajima, F. 1983. Evolutionary relationships of DNA sequences in finite
populations. Genetics 105:437-460.
Takahata, N. and M. Slatkin. 1984. Mitochondrial gene flow. Proc. Natl.
Acad. Sci. USA 81:1764-1767.
Tamura, K. 1992. Estimation of the number of nucleotide substitutions when
there are strong transition-transversion and G+C-content biases. Mol.
BioI. Evol. 9:678-687.
Tamura, K. and M. Nei. 1993. Estimation of the number of nucleotide
substitutions in the control region of mitochondrial DNA in humans and
chimpanzees. Mol. BioI Evol. 10:512-526.
Teve, D., M. Rezk, R. O. Smitherman. 1989. Genetics of body color in Tilapia
mossambica. Journal World Aquaculture Society 20:214-222.
Thresher, R. E. and E. B. Brothers. 1985. Reproductive ecology and
biogeography of Indo-West Pacific Angelfishes (Pisces: Pomacanthidae).
Evolution. 39:878-887.
Thresher, R. E., P. L. Colin, and L. J. Bell. 1989. Planktonic duration,
distribution, and population structure of western and central pacific
damselfishes (Pomacentridae), Copeia 1989:420-434.
225
Tricas, T. C. 1987. Life history and behavioral ecology of Chaetodon
multicinctus. Ph. D. dissertation, University of Hawaii, Honolulu.
Tricas, T. C. 1989. Determents of feeding territory size in the corallivorious
butterflyfish, Chaetodon multicinctus. Animal Behavior 37:830-841.
Tzeng, C-S, C-F Hui, S-C Shen and P. C. Huang. 1992. The complete
nucleotide sequence of the Crossostoma lacustre mitochondrial genome:
conservation and variations among vertebrates. Nucleic Acids Research
20:4853-4858.
Uehara, T., M. Shingaki and K. Taira. 1986. Taxonomic studies in the sea
urchin, genus Echinometra from Okinawa and Hawaii. Zoo1. Sci. 3:1114.
Valentine J. W. and D. Jablonski. 1983. Speciation in the shallow sea: general
patterns and biogeographic controls. Pp. 201-2.26 in R. W. Sims, J. H. Price
and P. E. S. Whalley, eds. Evolution, time and space: the emergence of
the biosphere. Academic Press, London.
Vermeij, G. J. 1987. The dispersal barrier in the tropical Pacific: implications
for molluscan speciation and extinction. Evo1. 41: 1046-1058.
Vermeij, G. J. 1990. An ecological crisis in an evolutionary context: el nino in
the eastern Pacific. Pp. 505-518 in P. W. Glynn, ed. Global ecological
consequences of the 1982-1983 EI Nino-Southern Oscillation. Elsevier
Press, Amsterdam.
Veron, J. E: N. 1986. Corals of Australia and the Indo-Pacific. Angus and
Robertson, Sydney.
226
Vigilant, L. M., M. Stoneking, H. Harpending, K. Hawkes, and A. C. Wilson.
1991. African populations and the evolution of human mitochondrial
DNA. Science 236:1503-1507.
Wakeley, J. 1993. Substitution rate variation among sites in hypervariable
region I of human mitochondrial DNA. J. Mol. Evol. 37:613-623.
Ward, F. H., B. L. Frazier, K. Dew-Iaer, and S. Paabo. 1991. Extensive
mitochondrial diversity within a single Amer-indian tribe. Proc. Natl.
Acad. Sci. USA 88:8270-8274.
Wenink, P. W. A. J. Baker, and M. G. J. Tilanus. 1993. Hypervariable-control
region sequences reveal global population structuring in a long-distance
migrant shorebird, the Dunlin (Calidris alpina). Proc. Natl. Acad. Sci.
USA 90:94-98.
West-Eberhard, M. J. 1983. Sexual selection, social competition and
speciation. The Quarterly Review of Biology 58:155-183.
Whittam, T. S., A. G. Clark, M. Stoneking, R. L. Cann, and A. C. Wilson. 1986.
Allelic variation in human mitochondrial genes based on patterns of
restriction site polymorphisms. Proc. Natl. Acad. Sci. USA 83:9611-9615.
Wilson, A. c., R. L. Cann, S. M. Carr, M. George, U. B. Gyllensten, K. M.
Helm-Bychowski, R. G. Higuchi, S. R. Palumbi, E. M. Pranger, R. D. Sage,
and M. Stoneking. 1985. Mitochondrial DNA and two perspectives on
evolutionary genetics. BioI. J. Linn. Sci. 26:375-400.
Winans, G. A. 1980. Geographic variation in the milkfish, Chanos chanos. I.
Biochemical evidence. Evolution 34:558-574.
227
Woodland, D. J. 1983. Zoogeography of the Siganidae (Pisces): an
interpretation of distribution and richness patterns. Bull. Mar. Sci. 33:713
717.
Woodland, D. J. 1986. Wallace's line and the distribution of marine inshore
fishes. Pp. 453-469 in T. Uyeno, R. Arai, T. Taniuchi, and K. Matsuura,
eds. Indo-Pacific fish biology, proceedings of the second international
conference on Indo-Pacific fishes. Ichthyological Society of Japan, Tokyo.
Zakharov, V. M. 1981. Fluctuating asymmetry as an index of development
homeostasis. Genetica 13:241-256.
Zumpe, D. 1965. Laboratory observations on the aggressive behavior of some
butterfly fishes (Chaetodontidae). Z. Tierpsychol. 22:226-236.
228
top related