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GENETIC VARIATION AMONG
POPULATIONS AND SPECIES OF GROUPERS AND CORALGROUPERS
(OSTEICHTHYES: SERRANIDAE: EPINEPHELINAE)
By
JOEL L. CARLIN
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2003
Copyright 2003
by
Joel L. Carlin
Dedicated in loving memory of my parents, F. J. Carlin and K. M. Carlin.
ACKNOWLEDGMENTS
W. J. Lindberg and B. W. Bowen deserve recognition for mentorship and project
development. I thank M. Brenner, C. Cichra, M. Miyamoto, and D. Murie for excellent
graduate committee service. J. Acevedo, J. C. Avise, A. L. Bass, L. Bullock, G. Burgess,
R. W. Chapman, J. Colborn, D. DeMaria, A. M. Ecklund, A. Francisco, S. Gardieff, T.
Gomes, J. Horrocks, S. Johnson, S. A. Karl, T. Maggio, W. O. McMillan, A. Miles, K.
Moots, A. Muss, D. R. Robertson, R. H. Robins Jr., C. Rocha, L. Rocha, P. Santos, T.
Streelman, D. Weaver and D. Wyanski generously contributed time, expertise, and
samples. The Florida Museum of Natural History provided curatorial support; and the
Kansas University Natural History Museum loaned tissue samples.
Some samples were sequenced by the University of Florida DNA Sequencing Core
and/or the Nevada Genomics Center. A portion of this work was funded by a National
Science Foundation grant (NSF Award No. 9727048) jointly awarded by the Population
Biology and Biological Oceanography Divisions to B. W. Bowen and D. R. Robertson.
Field work was also supported in part by the Smithsonian Tropical Research Institute,
with logistic support from the US Air Force, the Direccão das Pescas do São Tomé, the
Administrator of Ascension Island, and the Governor of St. Helena Island. I was funded
by the International Women’s Fishing Association, the Florida Chapter of the American
Fisheries Society, the PADI Foundation, PADI Project AWARE, and the UF Department
iv
of Fisheries and Aquatic Sciences. All work complied with required animal care and use
guidelines.
v
TABLE OF CONTENTS
page
ACKNOWLEDGMENTS ................................................................................................. iv
LIST OF TABLES........................................................................................................... viii
LIST OF FIGURES ........................................................................................................... ix
ABSTRACT.........................................................................................................................x
CHAPTER
1
INTRODUCTION ........................................................................................................1
Statement of Purpose ................................................................................................... 1
Why Study Groupers?.................................................................................................. 2
How Should Genetic Variation Be Assessed?............................................................. 3
Molecular Tools.................................................................................................... 3
Optimality Criteria................................................................................................ 4
Species Concepts .................................................................................................. 6
Gene trees are not species trees......................................................................7
Trees show relationship; not status ................................................................7
Operational and theoretical species concepts may not be the same ...............7
Summary...................................................................................................................... 8
2
ANCIENT DIVERGENCES AND RECENT CONNECTIONS IN TWO
TROPICAL ATLANTIC REEF FISHES Epinephelus adscensionis AND Rypticus
saponaceous (PERCOIDEI: SERRANIDAE)...........................................................10
Introduction................................................................................................................ 10
Materials and Methods .............................................................................................. 13
Results........................................................................................................................ 17
Rock Hind........................................................................................................... 17
Greater Soapfish ................................................................................................. 18
Discussion.................................................................................................................. 20
Biogeographic Barriers: Caribbean versus Brazil ............................................. 28
Biogeographic Barriers: West versus East Atlantic .......................................... 30
vi
3
PHYLOGENETIC RELATIONSHIPS AMONG SOME GROUPER AND
CORALGROUPER SPECIES, WITH COMMENTS ON THE TRIBE
EPINEPHELINI (PERCOIDEI: SERRANIDAE: EPINEPHELINAE). ..................43
Introduction................................................................................................................ 43
Materials and Methods .............................................................................................. 47
Results........................................................................................................................ 52
Cytochrome b ..................................................................................................... 53
Nuclear Intron..................................................................................................... 56
Discussion.................................................................................................................. 57
Monophyly of the Epinephelini.......................................................................... 60
Conclusions and Recommendations................................................................... 62
4
CONCLUSIONS ........................................................................................................76
APPENDIX COMMENTS ON GROUPER SPECIES RELATIONSHIPS ....................78
Species Relationships within a Reduced Epinephelini.............................................. 78
The Hind Clade................................................................................................... 78
The True Grouper Clade..................................................................................... 79
The Haifa and Leather Bass Clades.................................................................... 80
The Mycteroperca Clade .................................................................................... 81
The American Clade........................................................................................... 82
The Pacific Grouper and Promicrops Group...................................................... 83
Final Thoughts ........................................................................................................... 84
LIST OF REFERENCES...................................................................................................86
BIOGRAPHICAL SKETCH ...........................................................................................101
vii
LIST OF TABLES
Table
page
2-1
Genetic variation in the rock hind Epinephelus adscensionis and
greater soapfish Rypticus saponaceous..................................................................33
2-2
Haplotype occurrences and sample sizes in Epinephelus adscensionis.................34
2-3
Population pairwise differentiation observed in Epinephelus adscensionis ..........36
2-4
Absolute haplotype frequencies and sample sizes in Rypticus saponaceous ........37
2-5
Population pairwise differentiation observed in Rypticus saponaceous................38
3-1
Four alternate classifications of grouper and coralgrouper genera........................66
3-2
Nucleotide composition of DNA sequence alignments from
percoid fishes sampled at cytochrome b (cyt b) and LDHA intron 6 ....................67
3-3
Results of a posteriori topology tests among 50% majority-rule
consensus trees generated from epinephelin cytochrome b...................................68
3-4
Results of a posteriori topology tests among 50% majority-rule
consensus trees generated from epinephelin LDHA6 introns................................69
3-5
Results of topology tests among unconstrained gene trees and
gene trees with a priori topological constraints .....................................................70
viii
LIST OF FIGURES
Figure
page
2-1
Biogeographic divisions of the tropical Atlantic with sample localities ...............39
2-2
Distances among Epinephelus adscensionis haplotypes (A–HH)
and selected congeners, as indicated by a neighbor-joining (NJ) tree...................40
2-3
Distances among Rypticus haplotypes (1–31) and selected
congeners, as indicated by a neighbor-joining (NJ) tree........................................41
2-4
Frequency distributions for pairwise haplotype distances for
cytochrome b from Epinephelus adscensionis and Rypticus saponaceous ...........42
3-1
Phylogenetic hypotheses of epinephelin evolution................................................71
3-2
The most likely gene tree at cytochrome b as selected by SH tests, showing
derived species only...............................................................................................72
3-3
The most likely gene tree at cytochrome b as selected by SH tests, showing
ancestral species only.............................................................................................73
3-4
The most likely epinephelin gene tree at LDHA6 as selected
by SH tests .............................................................................................................74
3-5
Hypothetical phylogeny of the groupers and their allies .......................................75
ix
Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
GENETIC VARIATION AMONG
POPULATIONS AND SPECIES OF GROUPERS AND CORALGROUPERS
(OSTEICHTHYES: SERRANIDAE: EPINEPHELINAE)
By
Joel L. Carlin
May 2003
Chair: William J. Lindberg
Cochair: Debra J. Murie
Major Department: Fisheries and Aquatic Sciences
I assessed variability at a portion of the cytochrome b mitochondrial (mtDNA) gene
at two taxonomic scales in the groupers and related sea basses (subfamily Epinephelinae,
tribe Epinephelini). Intraspecific variation in a grouper (the rock hind Epinephelus
adscensionis) and a related outgroup species (the greater soapfish Rypticus saponaceous)
clustered into distinct phylogeographic units with boundaries concordant to some
biogeographic provinces. The intraspecific study indicated that pelagic larval duration
(PLD) and faunal boundaries may only partially determine the limits of gene flow in
western Atlantic serranids, and that historical patterns may be important in determining
haplotype distribution. To elucidate these historical patterns, a comparison among
species of groupers was made at both the cytochrome b gene and a noncoding nuclear
intron. Although both mtDNA and nuclear data were concordant in some issues, the
LDHA6 intron was not useful in phylogenetic reconstruction of tribal relationships. The
x
grouper tribe Epinephelini was not a monophyletic group in the mtDNA gene genealogy,
because of the insertion of other families and subfamilies between the coralgroupers
(Plectropomus and Variola) and other grouper genera. These data are concordant with
other mtDNA loci and with previously described patterns of larval morphology.
Taxonomic revisions to the Epinephelini are suggested.
xi
CHAPTER 1
INTRODUCTION
Statement of Purpose
How well does the history of a single gene correlate with described taxonomy, and
what does this tell us about the evolutionary relationships of groupers in different parts of
the world? Here I describe the genetic variation within and among species of groupers
and their allies (fish in the serranid tribe Epinephelini) at a mitochondrial protein-coding
gene (cytochrome b) commonly used for elucidating fish population genetics and
phylogenetics. I examine this gene at two different scales. First, I describe the
distribution and similarity of intraspecific alleles within a single ocean among
(presumably) intraspecific demes. Second, I examine similarity and common features of
alleles across the entire tropics in order to reconstruct evolutionary relationships among
(presumably) noninterbreeding species. Both provide a gene genealogy that may or may
not concur with our knowledge of grouper variability as seen in other lines of evidence
(e.g., larval dispersal potential, species distribution, larval and adult morphology, other
genetic studies). To rigorously and thoroughly compare known taxonomy to the genetic
patterns, four supporting questions had to be answered:
•
Is there sufficient genetic variation to construct robust hypotheses of relationship?
•
Are the patterns of genetic variation concordant with previously described
morphological, geological, or distributional patterns?
•
Are genetic patterns in groupers concordant with patterns seen in other taxa, and
what are the historical and biogeographical implications of these patterns?
•
What are the implications of these data for management and conservation?
1
2
The answers to these questions depend greatly on the two different scales examined
(population demographic processes vs. speciation within a family), but both studies
involve related study organisms, laboratory techniques, and analytical tools.
Why Study Groupers?
The tribe Epinephelini (the groupers, coralgroupers, coneys, and lyretails)
comprises tropical and subtropical marine fishes that have considerable economic
importance and ecological importance as large demersal predators. Several smaller
species are popular as aquarium fish. The excellent food quality of the larger groupers
supports a variety of fisheries, from cyanide fishing in the Indo-Pacific (IMA Indonesia
2001) to extensive line and trap fisheries in Europe and the Americas (Coleman 2001).
The marine commercial catch of groupers for the United States alone has a mean annual
worth of nearly $3 million, not including the equal or greater recreational catch (NMFS
2002). Hermaphroditism, group spawning, relatively slow maturation, and low fecundity
are common to many grouper species (Domeier and Colin 1997). Therefore, this group is
particularly vulnerable to overfishing (Musick et al. 2000, Sadovy 2001).
Knowing the genetic variation within populations can lead to inferences about
variation in reproductive and recruitment success, some of the most elusive parameters to
measure in wild marine populations (Grant and Bowen 1998). One challenge in
identifying different genetic phylogroups is the possible ecological differences among
alternate forms found in sympatry (Gilles et al. 2000). Data from populations sampled
before and after fishing have shown loss of alleles and allozyme heterozygosity in the
slow-growing orange roughy (Smith et al. 1991) and successful founder populations of
introduced grouper have been documented (Planes and Lecaillon 1998). Estimates of
variation at different geographic and taxonomic scales could be applied to models for
3
currently exploited species whose past genetic diversity is not known. Finally, the
cryptic variation in some exploited species has hampered fishery data collection in the
Indo-West Pacific because of taxonomic confusion (K. Rhodes, U. of Hong Kong, pers.
comm.). The management of multispecies reef fish fisheries, the identification of
biodiversity ‘hotspots’ for conservation, and studies of comparative biology all require a
taxonomy that reflects biological history (Collette and Vecchione 1995, Sweijd et al.
2000, Vecchione et al. 2000), but currently no such phylogenetic resolution is available
for serranids (Gill and Kemp 2002). The identification of cryptic regional lineages would
help quantify the international trade in groupers. For instance, nondestructive surveys of
saltwater aquarium species within consumer countries such as the United States could
reveal the geographic distribution of unregulated fishing efforts.
How Should Genetic Variation Be Assessed?
Molecular Tools
A variety of tools are able to quantify genetic variation, from measures of protein
mobility variation (allozyme electrophoresis) to differential display of gene expression
(microarray analysis). Numerous reviews have listed the assumptions, capabilities, and
drawbacks of each method (Féral 2000, Haig 1998, Neigel 1997, Sweijd et al. 2000).
Hillis et al. (1996) arranged available techniques along spectra of cost and
discriminatory power (e.g., for population genetics, higher taxonomy). Discriminatory
power hinges on how much variation is present in the examined genetic material or
product. Genetic variation is generated by mutation, and retained mutations can be
observed among populations and species to generate average mutation rates, which can
vary widely among types of organism and types of marker (Hewitt 2001).
4
Thus experimental design in phylogeography and molecular systematics makes
assumptions about available variance in the taxa at a certain genetic locus and seeks to
balance these two factors:
•
•
The best laboratory method capable of measuring the variation.
The cost of analyzing the sample size necessary to address the question of interest.
Most of the molecular data generated for the study of grouper populations and
species were from DNA sequences of the mitochondrial cytochrome b gene. The
relationship between mutation rate and statistical power means that questions addressed
by this technique at this locus may not be answerable by other loci and techniques;
likewise other techniques can expand or refine aspects of this study.
Optimality Criteria
Discrimination and classification of compared units involves selecting
philosophical approaches to guide the data analysis. Many approaches operate under an
assumption (implicitly or explicitly) that mutation is rare; and therefore that complex
mutational patterns are less likely than simpler ones (Occam’s razor). Each philosophical
approach has algorithms that generate numerous possible phylogenetic trees and then
attempt to identify the best tree. The analytical approaches to phylogenetics differ in how
they measure a tree’s optimality.
Molecular phylogenies typically are constructed using one of four optimality
criteria (all of which were used to some extent here): distance, parsimony, likelihood,
and posterior probability methods. Distance methods optimize the shortest tree as
measured directly by some metric, most simply by fit of taxa to overall similarity in the
number of nucleotide substitutions. Distance measures therefore assume that similarity
among individuals is not due to chance, and that similar units are historically related and
5
not the result of evolutionary convergence. Parsimony methods minimize this
assumption by not considering all data. Instead, parsimony optimizes the shortest
number of changes, but only considers those features shared by two or more taxa.
Likelihood methods use the frequency of nucleotide differences (and lack of differences)
to calculate the probability that the observed data would generate a given phylogenetic
pattern. Likelihood methods can emphasize both differentiation and ‘sameness,’ and
produce results easily evaluated by statistical testing. Posterior probability (Bayesian)
methods also use likelihood calculations, but instead of evaluating a model’s ability to fit
observed data, Bayesian methods incorporate prior knowledge of mutation rate and
evolutionary history into the likelihood calculations, resulting in a tree with a probability
that a relationship could be the result of a model given any data, observed or not.
Optimality criteria also differ in sensitivity to an uneven distribution of variance.
The lack of close relatives to very divergent individuals can result in highly differentiated
taxa being lumped together in a false sister relationship. This long branch attraction
results when an algorithm, unable to cluster two extremely different taxa to any other
lineages, clusters the divergent pair together whether or not they truly share a common
history. The best way to overcome long branch attraction is to include the true relatives
(both extant and extinct) of the problem taxa, requiring foreknowledge of the true
phylogeny. Unable to sample all described species of groupers worldwide, I searched for
criteria that would be least affected by the long branch attraction problem. Studies of this
problem by Takahashi and Nei (2000) and my own preliminary analyses (Carlin 2002)
were inconclusive as to which criterion and algorithm performed best for the amount of
data available.
6
Chapter 2 addresses the null hypothesis that two serranid species each comprise a
single freely interbreeding population. If these species are regulated as a cohesive unit,
then it is doubtful that similarity between two individuals is due to chance convergence
between genetically independent lines. Therefore, I make the a priori choice of distance
methodology as the primary optimality criterion. In case the distance assumption of
minimal convergence is incorrect, I also conduct a parsimony analysis.
Chapter 3 addresses the genetic history of species and genera of groupers and their
relatives. I have the a priori expectation that these genetic patterns might be the result of
millions, if not tens of millions, of years of divergent evolution. Therefore, I consider
distance methods inappropriate for these data. My anticipation of a problem with long
branch attraction meant that I considered multiple optimality criteria. I compare results
from various parsimony methods to posterior probability analyses, under the assumption
that real relationships are more likely to be identified under all criteria.
Thus the phylogenetic trees presented are the simplest patterns as defined by
various criteria. I realize, however, that the simplest explanation is not necessarily the
most correct. Therefore I examined other lines of evidence, such as biogeographic
provinces (Chapter 2); or phylogenetic trees from other genetic loci (Chapter 3). In both
studies I endeavor to give alternate interpretations of the data, including the placement of
species boundaries.
Species Concepts
The optimality criteria and their associated algorithms are used to identify, but not
describe, historical relationships. I propose that delimiting evolutionary relationships is
the purview of phylogenetics, while the description of organismal sets is a question of
taxonomy. I have used three guidelines to discuss the results.
7
Gene trees are not species trees
A cytochrome b genealogy shows the apparent relationship among lineages
sampled from single individuals. The genealogy is therefore a reconstruction of observed
results, interpreting this strictly as a historical record for all individuals throughout some
period of geologic time changes the reconstruction into a phylogenetic hypothesis. The
investigator must optimize the sampling design to make this assumption believable.
Trees show relationship; not status
Single gene genealogies can provide important information even if the status of the
member species is in question. If two lineages do not seem to exchange alleles across a
proposed barrier, then there is support for the barrier’s existence. If several lineages
seem to have recently evolved in the Caribbean, then there is support for the Caribbean as
a promoter of genetic diversification.
Operational and theoretical species concepts may not be the same
Some species concepts describe the act of speciation, and some describe the
criterion for identifying species in the field or laboratory. Most attempt to do both, but
the criteria for identifying a species as distinct (an operational species concept) is based
upon a model of evolution (the theoretical species concept). Most morphological and
genetic surveys of tropical reef fishes (of which the groupers are a part) and large reef
invertebrates have revealed that the most diverse free-swimming families have all
experienced an explosion of speciation in the past few million years. The resulting
genetic variation within and among marine fauna tends to be a collection of very ancient
and very recent lineages in a patchy geographic mosaic.
Norris (2000) showed several mechanisms of speciation in the pelagic environment
that were invoked to explain the patchy mosaic of diversity in the sea (e.g., Lessios et al.
8
2001, Palumbi 1996) not explained by simple tectonic boundaries. Such a complex
pattern is a reasonable expectation in the groupers. Some groupers hybridize among
species (Glamuzina et al. 2001, van Herwerden et al. 2002) and genera (Bostrom et al.
2000), while there is an apparent lack of interbreeding between cryptic units within other
grouper species (Gilles et al. 2000). If grouper diversification is very recent, then the
ancestral units of some species may possibly be the same modern species that can be seen
today, meaning that the ancestral species should not be regarded as a separate
evolutionary unit. In contrast, ancient fish lineages are often found in remote areas or
with highly specialized life histories; and these may represent apparent anagenetic change
(i.e., evolutionary change without lineage splitting).
I view speciation in dispersive marine fauna (i.e., not gobies or blennies) to be best
described by an evolutionary species concept, in which cohesive lineages through time
may split off or coalesce. While the evolutionary species concept shows a view of
evolution that may (or may not) be accurate for the groupers, the operationality of the
evolutionary species concept is made difficult by its predictive nature and the lack of a
fossil record for groupers. An operational concept that accepts limited hybridization
among lineages, recognizes persistent ancestors, and has the ability to discriminate
recently evolved units is the PSC-1 variant (Brooks and McLennan 2002) of the
phylogenetic species concept (Cracraft 1983, Cracraft 1987). However, because gene
trees represent limited sampling of evolutionary history, I revised taxonomy only when
supported by other lines of evidence.
Summary
Within this dissertation, I elucidate evolutionary relationships in a large set of
widely distributed and highly variable warmwater marine fishes by comparing observed
9
cytochrome b gene genealogies to information from morphology, distribution, and other
loci. In Chapter 2, my dissertation examines variation of cytochrome b to describe gene
flow limits in the recent ecological past among populations of the rock hind (the grouper
Epinephelus adscensionis) and the greater soapfish (Rypticus saponaceous, in a sister
subfamily to groupers). The variation (or lack thereof) in cytochrome b at this scale is
compared to nongenetic evidence from the groupers (their pelagic larval dispersal ability)
and from many fish families (described zoogeographic boundaries). My sampling design
is optimized to address whether or not each of these species are monophyletic units (i.e.,
a single cohesive lineage distinct from other taxa) with free gene flow (i.e., panmixia)
across their geographic distributions. In addition to reconstructing relationships among
individuals and localities, I use an analysis of molecular variance test to determine if
different alleles are evenly distributed, as predicted by a panmictic model. Secondly, I
use the same locus to examine variation across multiple grouper species to build a
phylogenetic hypothesis (evolutionary tree) that describes evolution in the groupers. My
sampling is optimized to address whether the grouper tribe (Epinephelini) is a
monophyletic unit. As in the Chapter 2 phylogeographic study, I compare the
cytochrome b genealogy to other lines of evidence, including a nuclear noncoding region
and previous hypotheses derived from morphology and a different mitochondrial gene.
Finally, the data from both studies should expand our knowledge of gene flow in
groupers, contributing to identifying management units and delimiting areas of
biodiversity concentration.
CHAPTER 2
ANCIENT DIVERGENCES AND RECENT CONNECTIONS IN TWO TROPICAL
ATLANTIC REEF FISHES EPINEPHELUS ADSCENSIONIS AND RYPTICUS
SAPONACEOUS (PERCOIDEI: SERRANIDAE).
Introduction
Studies of population genetic structure in marine organisms often reveal low levels
of differentiation (Bucklin and Wiebe 1998, Graves 1998, Ward et al. 1994). In reef
fishes, which are typically nonmigratory as juveniles and adults (Leis 1991), little or no
genetic population structure has been observed among widely separated localities in the
Caribbean Sea. Shulman and Bermingham (1995) examined mitochondrial DNA
(mtDNA) from eight Caribbean species. Lacson (1992) surveyed 33 allozyme loci from
five Caribbean species. Collectively these studies included six taxonomic families
exhibiting a wide range of pelagic larval durations (from approximately 13 to 122 days).
Estimates of divergence among localities within the Caribbean Sea and Gulf of Mexico
were low (ΦST = 0.000 to 0.172, Shulman and Bermingham 1995; ΦST = 0.004 to 0.035,
Lacson 1992). On the same geographic scale, an allozyme survey of the damselfish
Stegastes partitus showed no significant population structure (Lacson 1992, Lacson et al.
1989). The lack of divergence between distant localities is usually attributed to egg and
larval dispersal over hundreds or several thousand kilometers (see Brothers and Thresher
1985, Johannes 1978), a mechanism invoked to explain the presence of reef fish species
on isolated islands (e.g. Allen and Robertson 1997, Robertson 2001). Shulman and
Bermingham (1995: 908) concluded that “understanding dispersal and consequent
10
11
genetic differentiation among populations will require that we direct our attentions to the
capabilities and behaviors of larvae that result in limitation or augmentation of dispersal
distances.”
Briggs (1995) described two distinct biogeographic provinces within the tropical
northwest Atlantic: a West Indian Province containing the islands from Cuba and the
Bahamas to Grenada and the isolated northerly island of Bermuda (Figure 2-1). The
West Indies fauna are distinct from those of the Caribbean biogeographic province,
which includes southern Florida, the southern Gulf of Mexico, and the coasts of Central
America and northern South America. Because these provinces are separated by as little
as 60 km, Briggs (1995) proposed that strong, fast currents could act as vicariant barriers
at the Florida Straits in the north, the Yucatán Channel in the west, and between Trinidad
and Grenada in the south. However, to date, genetic studies have not indicated a
restriction of gene flow between the Antilles and continental locations (Rocha et al. 2002,
Shulman and Bermingham 1995).
If a reef area as large as the Caribbean Sea and Gulf of Mexico can be connected by
contemporary gene flow, then at what scale can one expect to find genetic isolation of
reef populations? Many of the reef species surveyed in the Caribbean also occur in other
biogeographic provinces of the Atlantic. If multiple biogeographic provinces are
considered, evolutionary genetic partitions could be detectable despite a relatively
lengthy pelagic larval duration. Significant divergences have been observed between
biogeographic provinces in antitropical species (e.g., Ball et al. 2000, Bowen and Grant
1997); and tropical species may also show genetic structure between discontinuous
12
biogeographic zones (Banford et al. 1999, Colborn et al. 2001, Muss et al. 2001, Planes et
al. 1993, Rocha et al. 2002).
For Atlantic reef species, all of the major biogeographic provinces are
discontinuous (Briggs 1995). Coral reefs in the Gulf of Guinea (West Africa) are
separated from Brazilian counterparts by an oceanic gap of 4,000 km, a distance
postulated to be an effective barrier to dispersal (Rosen 1976). Caribbean reefs are
separated from Brazilian reefs by 2,300 km of coastline dominated by soft bottoms and
riverine outflows from the Amazon and Orinoco rivers. These outflows form coastally
trapped discharge zones of hyposaline water (Bonilla et al. 1993, Longhurst 1998),
potentially decreasing effective latitudinal dispersal in the tropical western Atlantic.
These barriers are invoked to explain the high endemism of the Brazilian and Caribbean
provinces, and this level of isolation may be higher than is currently recognized. Recent
research indicates a potentially large number of undescribed endemic reef fish species in
the Brazilian province (Bernardi et al. 2000, Floeter and Gasparini 2000, Floeter et al.
2001, Rocha and Rosa 1999), and this finding also may be applicable to the poorly
studied reefs in West Africa.
Vicariant barriers between Atlantic reef provinces may be sufficient to allow
allopatric speciation in some cases (Bernardi et al. 2000, Muss et al. 2001). In other
cases, there has clearly been some dispersal, gene flow, or colonization in recent
evolutionary time, as evidenced by tropical Atlantic reef fish species that occur on both
sides of the Atlantic. One possible mechanism for mediating gene flow between the
Brazilian and East Atlantic biogeographic provinces in particular is the presence of
oceanic islands (e.g., Ascension, Saint Helena, and Trindade Island) (Figure 2-1). While
13
small in size, the islands that interrupt the deep mid-Atlantic expanse may serve as
stepping stones for transoceanic colonization (Briggs 1995).
With these issues in mind, the genetic variation in two amphi-Atlantic reef fishes
was surveyed. The rock hind, Epinephelus adscensionis Osbeck (1971), is a serranid fish
that occurs in shallow water from Bermuda to Brazil, on the mid-Atlantic ridge islands,
and in the eastern Atlantic from the Gulf of Guinea to Morocco (Böhlke and Chaplin
1993). The greater soapfish, Rypticus saponaceous Bloch and Schneider (1801), is
another amphi-Atlantic serranid species (as defined by Guimarães 1999) that has a
distribution similar to the rock hind. Both species are demersal reef fishes (Thresher
1980), such that long-range dispersal is limited to the pelagic larval stage (Leis 1991).
The pelagic larval duration of rock hind and greater soapfish have not been reported, but
a 40-day average is proposed for serranids (Lindeman et al. 2000). If barriers to dispersal
by pelagic larvae exist in these amphi-Atlantic reef fishes, then sustained interruptions in
gene flow may be evident on a broad geographic scale. Molecular genetic analysis of
rock hind and greater soapfish from all major biogeographic zones within their ranges
should therefore elucidate the population structure in these reef fishes; and may
illuminate general biogeographic patterns for Atlantic reef species.
Materials and Methods
Samples of E. adscensionis, R. saponaceous, and selected congenerics were
collected from multiple locations within the Caribbean, Brazilian, and mid-Atlantic
biogeographic provinces, and eastern Atlantic region (Figure 2-1). Tropical northwest
Atlantic specimens of E. adscensionis were collected from South Carolina, the Florida
Keys, the Bahamas and Barbados. Brazilian rock hind specimens were collected from
Trindade Island and the mainland states of Vitoria and Bahia. Mid-Atlantic ridge
14
collections were made at Ascension Island and Saint Helena. Eastern Atlantic E.
adscensionis were obtained from São Tomé (Gulf of Guinea). Specimens of R.
saponaceous were obtained from Grenada and Barbados (tropical NW Atlantic); Vitoria
and Bahia (Brazilian province); Ascension and Saint Helena (mid-Atlantic ridge
province); and the eastern Atlantic localities of São Tomé and Cape Verde. Fish were
collected during snorkel and SCUBA diving (using polespears and microspears)
primarily by D. R. Robertson and B. W. Bowen. In most locations, gill and/or muscle
samples were taken and stored in a saturated-salt DMSO buffer (Amos and Hoelzel
1991).
Total genomic DNA was isolated with organic extraction (using phenol,
chloroform, and isoamyl alcohol) and precipitated with sodium acetate in a 95% ethanol
solution. Isolated DNA was resuspended in 100 µL TE (10 mM Tris and 1 mM EDTA,
pH 8.0). An approximately 750 base pair (bp) fragment of the mitochondrial cytochrome
b gene was amplified via the polymerase chain reaction (PCR; Saiki et al. 1985) with
primers Cyb-09H (5'-GTGACTTGAAAAACCACCGTTG-3', Song et al. 1994) and Cyb07L (5'-AATAGGAAGTATCATTCGGGTTTGATG-3', Taberlet et al. 1992). The
amplification reaction mix included 3 mM MgCl, 40 nM of each primer, 17.5 nM of each
dNTP, 0.80 µL of Taq DNA polymerase (Promega, Inc., Madison WI) and 3.5 µL of 10x
PCR buffer (Promega, Inc.) in 35 µL total volume. Amplification temperature regime
included an initial 3-minute denaturation at 94ºC and final extension of 72ºC for three
min, with 25 cycles of 30 sec at 94ºC, 30 sec at 53ºC, and 40 sec at 72ºC for E.
adscensionis. Amplifications of R. saponaceous DNA used an identical protocol, except
for an annealing temperature of 54ºC and a reaction buffer with 1 mM MgCl2.
15
Oligonucleotide primers were removed by Ultrafree-MC centrifugal filter units
(Millipore Corp., Billerica MA) and/or simultaneous digestion with exonuclease I and
shrimp alkaline phosphatase (USB Corp., Cleveland OH).
Amplification products were denatured and DNA sequencing reactions were
conducted with fluorescently labeled dideoxy terminators according to manufacturer's
recommendations (ABI Model 800, Applied Biosystems, Inc., Foster City, CA). Labeled
extension products were gel separated and analyzed with an automated sequencer (ABI
Models 373A and 377) in the University of Florida DNA Sequencing Core. Resulting
fragments were aligned and edited using Sequencher ver. 3.0 (Gene Codes Corp., Ann
Arbor, MI). All samples were sequenced in forward direction using Cyb-09H; or in the
reverse direction using Cyb-19H (5'-CTCACTGCTCGGACTCTG-3') for some samples
of E. adscensionis or using Cyb-22H (5'-GTGAARTTGTCTGGGTCTCCT-3') for some
samples of R. saponaceous. Both forward and reverse sequences were obtained for at
least one representative sample for each haplotype, to assure accuracy of haplotype
assignments. Sequences were aligned with representative species of Epinephelus (20
spp.) and Rypticus (2 spp.) to confirm identification and provide outgroup comparisons.
Haplotypes were also compared with the complete cytochrome b sequences from Homo
sapiens (GenBank Accession V00662, Anderson et al. 1981) and Epinephelus sp. A
(GenBank Accession AF143193, Allegrucci et al. 1999) as additional quality controls.
For clarity of discussion, rock hind haplotypes are designated by letter abbreviations,
while numerals are used for greater soapfish haplotypes.
Population genetic parameters were calculated with Arlequin ver. 2.0 (Schneider et
al. 2000). Nucleotide diversity (π; Equation 10.5 in Nei 1987) and haplotype diversity
16
(h; Equation 8.5 in Nei 1987) were calculated for each biogeographic province. Genetic
distances (d) were calculated using the substitution model of Tamura and Nei (1993)
corrected for unequal mutation rate (α = 0.50; Yang 1996) with a 3:1 transition to
transversion (ti:tv) ratio. The distribution of pairwise differences among haplotypes
generated a mismatch distribution (Rogers 1995, Rogers and Harpending 1992), with the
sum of squared differences in frequency at each mismatch class, or raggedness index (r;
Harpending 1994). A resampling simulation (100 bootstraps) generated an expected
distribution of pairwise differences using population-expansion parameters estimated by
a least squares nonlinear regression (Schneider and Excoffier 1999).
The proportion of genetic diversity within and among localities was estimated with
an analysis of molecular variance (AMOVA) in the Arlequin ver. 2.000 software package
(Excoffier et al. 1992). The total variance explained by population-level differences
(ΦST) was estimated to infer degree of population subdivision. A population pairwise
differentiation test was performed using a 10,000-step Markov chain simulation
(Schneider et al. 2000). Significance (overall α = 0.05) was determined after full
Bonferroni correction for multiple comparisons (individual P = 0.002 for rock hind and
P = 0.0034 for greater soapfish).
Both majority-rule Tamura-Nei neighbor-joining (NJ) and maximum parsimony
(MP) trees were constructed and evaluated using 500 bootstrap replicates. The NJ tree
includes two congeners: Epinephelus analogus (n = 3) and E. labriformis (Figure 2-2).
Rather than reporting only the most parsimonious phylogram(s), a consensus tree
considering slightly less parsimonious solutions was constructed. This was reported only
if there were few alternate solutions (i.e., < 25 trees < 5 steps longer than the most
17
parsimonious cladogram). All phylograms were constructed using Paup* β ver. 4.0b
(Swofford 2002).
Results
Rock Hind
A total of 109 E. adscensionis were surveyed, yielding 593 bp of resolved sequence
per individual. No extra termination codons were observed in comparison with the
Allegrucci et al. (1999) serranid sequence. Sequence comparisons revealed a transitionrich data set (ti:tv ratio = 23:5) that included a total of 34 haplotypes (d = 0.002 to 0.044;
Tables 2-1, 2-2). However, only seven haplotypes were observed in more than one
specimen. These sequences revealed low nucleotide diversity (π = 0.008) and moderate
haplotype diversity (h = 0.586), a common pattern in marine fishes (Grant and Bowen
1998). The AMOVA indicated very strong population structure for E. adscensionis (ΦST
= 0.867, P < 0.001). Differentiation tests indicated significant population structure, with
pairwise ΦST’s > 0.9 between Florida and all other localities except for comparisons with
South Carolina and Trindade Island, for which low sample sizes (n = 3 and 2,
respectively) precluded testing (Table 2-3). The population structure is characterized by
deep divergence between the southeastern United States haplotypes and those of all other
locations and the abundance of haplotype A (64.2% of all rock hind samples) outside
Florida/South Carolina. The mean sequence divergence between Florida/South Carolina
haplotypes and all other haplotypes was d = 0.040 (range: 0.034 to 0.044). Removing
the highly divergent Florida/South Carolina samples from AMOVA decreased diversity
measures of the remaining samples (h = 0.481, π = 0.001, ΦST = 0.056, P < 0.001).
Significant differentiation was also found in comparing the Bahamas sample with the
18
Ascension and St. Helena samples (Table 2-3), possibly reflecting the predominance of
haplotype A in the mid-Atlantic localities, in contrast to its absence in available Bahamas
specimens (n = 5) (Table 2-2).
The MP tree had the same structure as the NJ tree: two primary branches in
E. adscensionis with bootstrap values ≥ 0.90. All equally parsimonious trees had
unresolved polytomies among those haplotypes found together in only one population
(and usually differing by only one mutation). The close relationship among haplotypes
within each branch of the rock hind phylogeny also created a bimodal mismatch
distribution (r = 0.070, q0 = 1.169, q1 = 1.169). When a distribution was estimated after
removal of the Florida/South Carolina samples, no significant difference was found
between the observed haplotype distribution and an equilibrium distribution simulated
under a rapid population expansion model (τ = 1.126, P = 0.550). Aside from haplotype
A, only haplotype V (one specimen each from Barbados and St. Helena, 6,745 km apart),
was found in multiple biogeographic provinces. The Caribbean province, however, is
split by the presence of 21 to 29 nucleotide differences between haplotypes D to I from
the Florida Keys and haplotypes A and J to V in the Bahamas and Barbados.
Greater Soapfish
A total of 86 greater soapfish specimens were analyzed, including 27 specimens
from the East Atlantic Region (Cape Verde and São Tomé) that diverged notably from
samples in the remainder of the range (d = 0.044, range: 0.037 to 0.052). These fish
matched conventional descriptions of R. saponaceous (Fischer et al. 1981) and were not
consistent with the only other congeneric in the East Atlantic (Guimarães 1999).
Analyses of 682 bp of cytochrome b yielded a conventional pattern of mutations
19
(ti:tv = 27:6) and a total of 31 R. saponaceous haplotypes (Tables 2-1, 2-4) with
somewhat higher genetic diversity (π = 0.019, h = 0.929) than the rock hind (π = 0.008, h
= 0.586). As in rock hind, the dataset was influenced strongly by a bifurcation between
samples in one portion of the range (eastern Atlantic for this species) and a branch
containing all other collection sites (western and central Atlantic). The divergence
between the eastern Atlantic haplotypes and all others yielded strong population structure
(ΦST = 0.876, P < 0.001). Large pairwise ΦST’s were calculated in comparisons between
Cape Verde or São Tomé with all other localities (ΦST = 0.906 to 0.963, Table 2-5); all
but one of these comparisons were significant differentiation tests after Bonferroni
correction (α = 0.003).
Reanalysis of the R. saponaceous haplotypes, after excluding the divergent East
Atlantic samples, reveals significant overall population structure (ΦST = 0.372, P <
0.001). Nineteen specimens (22% of the total) possessed a haplotype unique within this
study; haplotypes 1 and 5 are the only ones present in more than one biogeographic
province. Within the West Atlantic samples, the low degree of haplotype sharing
resulted in significantly different pairwise differentiation in comparisons between Brazil
versus Ascension, Brazil versus St. Helena and Grenada versus St. Helena (ΦST = 0.327
to 0.695, P < 0.001; Table 2-5). To examine the influence of small sample size in the
Caribbean localities, an additional analysis was conducted considering the Barbados and
Grenada as a single population (h = 0.917; π = 0.003; overall ΦST = 0.886, P < 0.001;
West Atlantic overall ΦST = 0.354, P < 0.001). In this case only the comparison between
the combined Caribbean sample and São Tomé changed in significance (ΦST = 0.927, P
= 0.002). The distribution of observed differences between all pairs of haplotypes (r =
20
0.194, q0 = 0.000, q1 = 2.646) was significantly different from the equilibrium model of
rapid population expansion (τ = 4.430, P ≥ 0.49).
Analysis of three Rypticus species available in this study indicates that the two
lineages of putative R. saponaceous described here may not be monophyletic (67%
bootstrap support), and indeed form an unresolved polytomy with five specimens (three
haplotypes) of Rypticus bicolor, an eastern Pacific species, and R. subbifrenatus, another
amphi-Atlantic species. Mean interspecific (R. saponaceous vs. R. bicolor) pairwise
sequence divergence was d = 0.047 (range: 0.043 to 0.055). The only other soapfish
reported from the eastern Atlantic, R. subbifrenatus, was collected at the same locality in
São Tomé as the putative R. saponaceous specimens. The mean genetic distance
between R. saponaceous and R. subbifrenatus was d = 0.115 (range: 0.112 to 0.124),
greater than that found between the East Atlantic and other samples of R. saponaceous (d
= 0.044).
Discussion
Several patterns concerning the phylogeographic histories of these two species can
be recovered from these data. First, both rock hind and greater soapfish intraspecific
phylogenies are characterized by a single major bifurcation, with one branch restricted to
a single region while the other is widespread through multiple biogeographic provinces.
Haplotypes within the divergent branches exhibit a shallow history and rapid coalescence
to a common ancestor. For the rock hind, a single haplotype was shared over most of the
species’ distribution, and the large number of “twigs” is consistent with a population
bottleneck and subsequent expansion from two ancestral (and divergent) lineages (Avise
2000). In contrast, greater soapfish exhibited less haplotype sharing among
21
biogeographic provinces, and a null model of rapid population expansion was not
consistent with the structure of the western and central Atlantic lineages. A
reconstruction of the biogeographic and evolutionary history in greater soapfish and rock
hind is attempted by first considering the prominent, relatively ancient, separations
between phylogroups.
The greatest interlocality divergences found in rock hind and greater soapfish (d =
4.4% and 5.2%, respectively) are relatively deep for intraspecific comparisons of marine
fishes (see reviews by Avise 2000, Grant and Bowen 1998). However, descriptions of
relatively ancient cytochrome b divergences in marine fishes are accumulating. Bernardi
et al. (2000) observed a divergence of d = 2.3% in the parrotfish Sparisoma rubripinne
between two eastern Atlantic (São Tomé) and two Caribbean specimens. Divergences of
d = 0.052 to 0.127 were observed between five phylogroups in biogeographic provinces
for the amphi-Atlantic reef blenny Ophioblennius atlanticus (Muss et al. 2001). The
deep splits observed in rock hind, greater soapfish, parrotfish, and blenny are of an
equivalent magnitude to the divergences observed in cytochrome b between
Newfoundland and Norwegian samples of the capelin Mallotus vilosus (d = 5.7%; Birt et
al. 1995). These values approach divergences between sister species of other marine
fishes (Johns and Avise 1998), but are less than that described between cryptic sympatric
species in another grouper, Epinephelus marginatus (d = 0.158; Gilles et al. 2000).
The deep divergences observed in E. adscensionis and R. saponaceous also invoke
the possibility of cryptic species. This may be particularly likely for the greater soapfish,
based on a Pliocene divergence between eastern and western Atlantic specimens. The
molecular evolutionary rate of cytochrome b has been estimated to range from 1 to 3
22
%/MY (Canatore et al. 1994, Fleischer et al. 1998, Irwin et al. 1991). Although rates
may vary among taxa (Mindell and Thacker 1996), a molecular clock rate of 1 to 2
%/MY is applied. This would place the earliest divergence date between Atlantic R.
saponaceous and the East Pacific R. bicolor to 3.5 MYA (range = 2.1 to 5.5 MYA), a
timeframe concordant with the last contact between East Pacific and West Atlantic fishes
(about 3.1 to 3.5 MY ago; Coates and Obando 1996). This benchmark is used to estimate
other divergence dates in the evolutionary history of soapfishes, and provisionally apply
this rate to rock hind, with the recognition that divergence dates for these species are not
calibrated and must be interpreted with caution.
Based on the molecular clock rate of 1 to 2%/MY, the deepest mtDNA lineages in
E. adscensionis coalesce at approximately 2.7 MY, and the deepest lineages in R.
saponaceous coalesce at about 3.3 MY. Evidence from oxygen isotope ratios in fossil
foraminifera indicates the shoaling and separation of waters about the Isthmus of Panama
from 5 to 8 MYA, even before the complete emergence of present-day Panama (Collins
et al. 1996, Keigwin 1982). The resulting hydrostatic imbalance caused the major current
systems of the Atlantic to increase in velocity, then slowed to present levels by ~ 2.5 MY
BP to produce the general circulation patterns that persist today (Haug and Tiedemann
1998, Maier-Reimer et al. 1990). Thus, it is possible that divergence occurred among
Atlantic Rypticus populations following the closure of the central American isthmus (and
subsequent responses in Atlantic circulation) due to decrease in dispersal opportunities
across the tropical Atlantic.
An obvious alternative is that the specimens collected in Cape Verde and São Tomé
were misidentified members of another previously described Rypticus species. This
23
possibility is unlikely for three reasons: first, the collection protocol was designed to
distinguish R. saponaceous from similar congeners. Specimens of R. saponaceous in the
West Atlantic have been collected syntopically with R. bistrispinus (Guimarães 1999);
these species were differentiated according to coloration following the criteria of Smith
(1997). East Atlantic collections included specimens of the only other soapfish described
from this region (R. subbifrenatus; Courtenay 1970). Second, the phylogenetic analyses
include R. subbifrenatus collected at São Tomé and show that it is highly divergent from
both eastern and western Atlantic R. saponaceous as well as the East Pacific R. bicolor.
Third, Cape Verde and São Tomé specimens had no gross departures from prior
descriptions of R. saponaceous, the species with the greatest meristic variability in the
genus (Courtenay 1967, Guimarães 1999). The molecular evidence, in combination with
the field observation and geographic considerations, indicate the strong possibility of two
species within the currently recognized R. saponaceous.
Regardless of taxonomic considerations, the cytochrome b gene tree of greater
soapfishes indicates that eastern and western Atlantic lineages initially diverged at about
the time of the closure of the Isthmus of Panama. Subsequently, or at nearly the same
time (Figure 2-4), the ancestor of R. bicolor (in the east Pacific) diverged from R.
saponaceous (in the West Atlantic). This finding invokes the East Atlantic—East Pacific
generalized track proposed by Rosen (1976) to explain the distribution of shorefishes.
Under Rosen’s hypothesis, the greatest vicariant separation in this region should be
between East and West Atlantic, based on the opening of the Atlantic some 80 MY ago.
Subsequently, a more recent vicariant separation occurred between the West Atlantic and
East Pacific, based on the closure of a central American seaway. Some aspects of fish
24
phylogeography can support this theory (see Banford et al. 1999), and the
bicolor/saponaceous split does seem to fit the closure of the West Atlantic/East Pacific
connection. However, the East-West split in Atlantic greater soapfishes estimated at 3.3
MY is much too shallow to be based on sea floor spreading. For this aspect of greater
soapfish phylogeography, dispersal in an accelerated circulation system (see above) is a
more likely explanation.
A similar divergence was observed among rock hind, with one mtDNA lineage
restricted to the northwest Caribbean (Florida Keys) and the southeast United States. The
other lineages were observed throughout the tropical Atlantic, and most notably at two
Caribbean collecting sites (Barbados and the Bahamas). How can one explain this
curious finding of a major genetic separation of lineages separated by <100km of open
water? As above, the possibility of misidentification must be considered. This is
especially pertinent for rock hind, because there are several similar congeners in the
Caribbean. However, misidentification is unlikely for two reasons: First, the samples
were collected by personnel specifically trained to distinguish rock hind from other
epinephelines. Second, the Florida/South Carolina specimens were collected by several
expeditions and by different trained persons and it is therefore unlikely that all parties
collected the wrong species at 100% frequency.
What biogeographic process could explain the divergent mtDNA lineages within
the tropical West Atlantic rock hind? No obvious differences in size, coloration, or
external morphology among rock hind specimens were observed, so it is possible that
what is currently called one species consists of separate species: an insular species found
in the eastern part of the greater Caribbean and a continental species with a currently
25
unknown range. As in soapfish, the lineages appear to be allopatric, although Florida and
South Carolina sample sizes are small. Fast-flowing currents in the Florida Straits have
been proposed as vicariant barriers between the West Indian biogeographic province
(including the Bahamas) and a Caribbean province containing the Florida Keys (Figure 21; Briggs 1995). However, this strong divergence among rock hind is exceptional,
because all the prior surveys of Caribbean Sea reef fishes revealed low or no population
structure across such distances. In particular, Shulman and Bermingham (1995) showed
that, for each of eight reef fish species (not including rock hind or soapfish), populations
in Barbados and Florida shared haplotypes both with each other and with other
continental and island locations in the Caribbean.
Another alternative is a long-term divergence between the divergent Caribbean
lineages and those in the Brazilian province (as observed in other reef species; Bernardi
et al. 2000, Muss et al. 2001), followed by recent dispersal into the West Indies from
Brazil. Grenada, as one of the southernmost outposts of reef habitat in the Caribbean, is
suitably located to encounter larvae drifting north on the Brazilian Current. Based on
species distributions, geography, and ocean circulation patterns, Grenada is one of the
gateways for occasional dispersal between Brazil and the Caribbean (Rocha et al. 2002).
The genetic data support this hypothesis: while not found in abundance (n = 7), greater
soapfish haplotype 1 was found in specimens from Grenada, Barbados, and Brazil; and
rock hind haplotype A was observed in both Brazil and Barbados. Genetic data from
large population samples of both species at many sites scattered throughout the tropical
northwest Atlantic will be needed to (i) define the distributions of the two main lineages
of rock hind and show how they relate to insular/continental biogeographic subdivisions
26
of that region, (ii) show whether those rock hind lineages are allopatric, and (iii) show
how the phylogeography of the greater soapfish within the Caribbean relates to that of the
rock hind. As the latter species were sampled only in the southeastern Caribbean this
study cannot say whether there are lineage subdivisions in that species within the tropical
NW Atlantic.
Bowen et al. (2001) demonstrated that trumpetfish phylogroups, separated by
geography for 3 to 4 MY, hybridized in renewed contact in the South Atlantic. As noted
by Knowlton (2000), reproductive isolation in sympatry is an acid test for speciation, and
rock hind may be in the early stages of this test. The reproductive compatibility of the
two main lineages of both rock hind and greater soapfish may determine whether rock
hind have speciated in isolation, or whether this is a single species characterized by deep
divergence and subsequent admixture (see Veron 1995).
Against the backdrop of strong divergence, both species exhibit many slight
differentiations (often by a single transition) within the major lineages. The distribution
of these lineages among western and central Atlantic greater soapfish yields significant
population genetic structure, but this is not the case in rock hind. The relative abundance
of a widespread haplotype among biogeographic provinces and regions (as seen in rock
hind haplotype A), accompanied by many closely related haplotypes, yielded a low level
of population structure in rock hind samples from the Bahamas to São Tomé. The lack of
heterogeneity in rock hind may be due to extensive gene flow between biogeographic
provinces and regions. Alternately, the shallow gene genealogy with widespread
haplotype sharing could arise from selection or demographic processes, working either
singly or in combination (Grant and Bowen 1998). Such demographic processes include
27
the effects of temporal variation in recruitment success and large fluctuations in
population size.
A shallow but diverse gene genealogy can be generated by extremes in recruitment
success (Hedgecock 1994), where all recruits to a year-class were generated by a few
successful mating events. While the population may have a large number of spawners, in
sweepstakes reproduction successful cohorts of recruits are drawn from only the few
propagules that meet a specific and stringent combination of abiotic and biotic factors.
Typically, sweepstakes recruitment produces chaotic patchiness in allele distribution at
small temporal and spatial scales (Avise 2000). While collections from single localities
in multiple years were not attempted, the samples collected in this study span several
years effort and (for Brazil and the southeastern U.S.) involved multiple localities. In
contrast to chaotic patchiness, a consistency of pattern across years and samples were
observed (e.g., the widespread abundance of haplotype A and the presence of the same
haplotypes in South Carolina specimens collected in 1994 and Florida specimens
collected in 1999).
Population fluctuations also eliminate mtDNA lineages, such that the female
effective population size may be orders of magnitude lower than indicated by census size.
A survey of haplotypes in such a species may therefore reveal a large number of closely
related haplotypes. If rock hind represents a single species with significant population
structure (in mtDNA), then pairwise comparisons between haplotypes would create an Lcurve of similar shape to the expected Poisson distribution. The additional smaller peaks
in the mismatch distributions (an abundance of large interhaplotype distances; Figure 24) could be caused by rapid population expansion (Rogers et al. 1996). Overall, a
28
waveform result may indicate a rapid recovery from a recent genetic bottleneck (Rogers
and Harpending 1992). Similar patterns have been observed in other tropical fishes, such
as the mullet (Rocha-Olivares et al. 2000) and parrotfishes (Dudgeon et al. 2000). These
data support the growing body of evidence for large-scale instability of marine
populations over recent evolutionary time (e.g., Bowen and Grant 1997, Bucklin and
Wiebe 1998, Grant and Bowen 1998, Lavery et al. 1996, Zane et al. 1998).
The known species distribution of rock hind and greater soapfish implies a
connection (either past or present) among reef habitats in multiple tropical biogeographic
provinces and regions. There are considerable differences in the degree of genetic
heterogeneity between species and between localities. Therefore an examination of the
haplotype distribution in a geographic context may elucidate both potential differences in
life history between these reef fishes as well as the effectiveness of biogeographic
barriers.
Biogeographic Barriers: Caribbean versus Brazil
The reef-building corals of Brazil are highly distinct from those of the Caribbean
(see Figure 49; Veron 1995), providing longstanding evidence for recognition of those
areas as separate, large-scale biogeographic regions (Briggs 1974). Recent studies have
documented that Caribbean and Brazilian reef provinces are also highly distinct for fish
species, in terms of their distributions (Floeter and Gasparini 2000; Floeter et al. 2001;
Rocha and Rosa 2001), taxonomic distinctions (Rocha and Rosa 1999), and evolutionary
genetics (Bernardi et al. 2000, Bowen et al. 2001, Muss et al. 2001, Rocha et al. 2002).
In contrast, comparisons in both species from western Atlantic localities did not
reveal significant pairwise differentiations between the West Indian (i.e., not including
the Florida/South Carolina rock hind) and Brazilian biogeographic provinces. However,
29
this may be influenced by the smaller sample sizes in the West Indian samples (with the
important exception of 19 rock hind from Barbados). The sharing of haplotypes between
the two provinces in both species studied here (Tables 2-2, 2-3) indicates recent or
ongoing population connections. Are these species capable of routinely crossing the
Amazonian plume vicariant barrier (described above)? One mechanism to accomplish
this is that the extent of these species’ PLD (presently unknown for both species) may
allow recruitment of Brazilian haplotypes into the Caribbean (Figure 2-1). However, a
prolonged PLD alone does not explain the presence of population genetic structure
elsewhere in the range.
Perhaps larval transport is not the only method of dispersal between Brazilian and
Caribbean provinces. Benthic (juvenile and adult) stages could occupy sparse hardbottom habitat between the Caribbean and northeast Brazil, as they do in subtropical
waters of the eastern United States (Randall 1967). Alternately, they could occupy
certain soft-bottom habitats such as the sponge communities under the Amazon and
Orinoco plumes (Collette and Rützler 1977).
The possibility of dispersal (or population connections) through alternative habitats
is especially relevant for soapfishes. Effective dispersal between the Caribbean and
Brazilian reef systems requires survival across long distances without coral reef habitat,
but also the capability to withstand or avoid the immense halocline imposed by the
Amazon-Orinoco outflows (Longhurst 1998). The ability to exist in hyposaline waters is
not unknown in the soapfishes, and is in fact a unifying character of Rypticus nigripinnus
and R. bicolor in the Pacific (Guimarães 1999; D. R. Robertson, STRI Panama, pers.
obs.). Perhaps other soapfishes have wide salinity tolerances, as indicated by the
30
presence of Rypticus randalli in mangrove habitats in the Hawaiian archipelago (D.
Greenfield, U. Hawaii, pers. comm.). Courtenay (1967) suggested that R. saponaceous
might create shallow burrows in mud, although whether this actually occurs is not known
(Guimarães 1999). Nonetheless, the genetic findings for R. saponaceous is consistent
with a Caribbean-Brazil link via softbottom habitat.
Biogeographic Barriers: West versus East Atlantic
The reef fishes of the East Atlantic have been understudied, and it is certain that
additional species await discovery. In the comparisons of target species across the
tropical Atlantic Ocean, the rock hind samples were characterized by the widespread
distribution of haplotype A in Brazil, the mid-Atlantic Ridge, and East Atlantic. While
this may be due to demographic processes unrelated to vicariance (see Discussion above),
the nearly ubiquitous occurrence of haplotype A indicates that the rock hind has been
able to colonize across long distances in the recent past.
In contrast, the greater soapfish had moderate population structure (ΦST = 0.327 to
0.424, Table 2-5) between Brazil and the central Atlantic, and a strong (possibly specieslevel) distinction between the East Atlantic populations and elsewhere (pairwise ΦST’s >
0.90). The slight differentiations between haplotypes 1, 5, and 18 indicate a very rapid
coalescence to an ancestor capable of colonization across the western-central Atlantic
(Table 2-4). Collectively, these three haplotypes extend from Grenada and Barbados to
Brazil to Ascension and St. Helena, and may support the ‘stepping-stone’ colonization
pattern across the open ocean barrier of the central Atlantic. St. Helena exhibited a very
small haplotype diversity relative to other greater soapfish samples: 14 of 16 specimens
had haplotype 18, implying either highly differential survival of lineages, or too little
31
time for divergence to occur in St. Helena soapfish in the cytochrome b region.
Similarly, the lowest haplotype diversities in rock hind were in the Ascension and St.
Helena samples.
The low haplotype diversity in the Ascension/St. Helena province, coupled with
the restricted haplotype distributions of soapfish on São Tomé suggest that intermediate
localities may be used as ‘stepping-stones,’ but the lack of widespread haplotype in the
greater soapfish indicates that this reef fish is not as able to cross the Atlantic as readily
as the rock hind.
The reason for the difference in oceanic dispersal ability between rock hind and
greater soapfish is not readily apparent. Pelagic larval duration, while a tempting
potential predictor of population structure, is one of many factors influencing genetic
differentiation. The roles of oceanographic currents, freshwater plumes, recruitment
success, and niche competition surely play a part in the connections and divisions among
Atlantic coral reef fauna. Although each species undoubtedly meets unique challenges
the lack of concordance might be enhanced by stochastic processes such as rare long
distance dispersal, sweepstakes recruitment, local extirpation, and genetic drift in small
founder populations. This is especially relevant to the rock hind, in which there is
evidence for an evolutionary separation (approximately 3.2 MY) within the Caribbean
Sea, but evidence of common ancestry between Brazil and West Indies populations.
While this could be due to vicariance, other demographic and historical processes may
also explain a pattern that appears to be very unusual among Caribbean reef fishes.
The distribution of mtDNA diversity in both rock hind and greater soapfish offers
insight into how biogeography influences speciation in the marine realm. The geography
32
and oceanography of the Atlantic basin may promote intraspecific (or intrageneric)
partitions (Banford et al 1999, Graves 1995, Muss et al 2001, Tringali and Wilson 1993).
The distances among these biogeographic provinces and the durations of pelagic larvae
are not the only relevant factors in shaping patterns of genetic structure in panoceanic
species. Stochastic colonizations and utilization of isolated islands allow for contact
between Atlantic regions. The different responses to the mid-Atlantic and the AmazonOrinoco barriers by two confamilial and even congeneric species (e.g., Rocha et al. 2002)
illustrate how complex interactions may be involved in “simple” predictors of population
structure (e.g., dispersive larvae, adults restricted in habitat type, modern surface current
patterns, straight-line distances between habitats). Rock hind and greater soapfish thus
provide examples of the dynamic evolutionary processes of population isolation and
differentiation which may typify the superficially continuous distributions of marine
organisms.
Table 2-1. Genetic variation in the rock hind Epinephelus adscensionis and greater soapfish Rypticus saponaceous
Location
South Carolina
Florida
Bahamas
Grenada
Barbados
Brazil
Trindade
Ascension
Saint Helena
Cape Verde
São Tomé
E. adscensionis
n
3
9
5
—
19
16
2
21
17
—
17
h
0.667 ± 0.314
0.889 ± 0.091
1.000 ± 0.127
—
0.731 ± 0.109
0.625 ± 0.139
0.000 ± 0.000
0.186 ± 0.110
0.228 ± 0.130
—
0.331 ± 0.143
π
0.002 ± 0.002
0.003 ± 0.002
0.004 ± 0.003
—
0.001 ± 0.001
0.002 ± 0.002
0.000 ± 0.000
0.000 ± 0.000
0.001 ± 0.001
—
0.001 ± 0.001
R. saponaceous
n
—
—
—
5
4
23
—
11
16
18
9
h
—
—
—
0.900 ± 0.161
1.000 ± 0.177
0.913 ± 0.035
—
0.873 ± 0.089
0.242 ± 0.135
0.863 ± 0.042
0.722 ± 0.097
π
—
—
—
0.003 ± 0.002
0.003 ± 0.002
0.003 ± 0.002
—
0.003 ± 0.002
0.001 ± 0.001
0.004 ± 0.002
0.003 ± 0.002
33
Total:
109
0.586 ± 0.058
0.008 ± 0.004
86
0.929 ± 0.016
0.019 ± 0.009
Note: The number of specimens (n), the haplotype diversity (h) and nucleotide diversity (π) for each locality and in total are listed. Some species
were not collected (—) at some localities. Sample size at Trindade I. (n = 2) precludes statistical treatment.
Table 2-2. Haplotype occurrences and sample sizes observed in Epinephelus adscensionis
South Carolina
n=3
—
2
1
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
Florida
9
—
—
—
3
2
1
1
1
1
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
Bahamas
5
—
—
—
—
—
—
—
—
—
1
1
1
1
1
—
—
—
—
—
—
—
—
—
—
—
—
—
Barbados
19
10
—
—
—
—
—
—
—
—
—
—
—
—
—
2
1
1
1
1
1
1
1
—
—
—
—
—
Brazil
16
10
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
1
1
1
1
1
Trindade
2
2
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
Ascension
21
19
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
St. Helena
17
15
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
1
—
—
—
—
—
São Tomé
17
14
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
Total
109
70
2
1
3
2
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
2
1
1
1
1
1
34
Haplotype
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T
U
V
W
X
Y
Z
AA
Table 2-2. Continued
Haplotype
BB
CC
DD
EE
FF
GG
HH
South Carolina
n=3
—
—
—
—
—
—
—
Florida
9
—
—
—
—
—
—
—
Bahamas
5
—
—
—
—
—
—
—
Barbados
19
—
—
—
—
—
—
—
Brazil
16
1
—
—
—
—
—
—
Trindade
2
—
—
—
—
—
—
—
Ascension
21
—
1
1
—
—
—
—
St. Helena
17
—
—
—
1
—
—
—
São Tomé
17
—
—
—
—
1
1
1
Total
109
1
1
1
1
1
1
1
35
36
Table 2-3. Population pairwise differentiation observed in Epinephelus adscensionis
Florida
Bahamas
−
Barbados
+
−
Brazil
+
−
−
Ascension
+
+
−
−
St. Helena
+
+
−
−
−
São Tomé
+
−
−
−
−
−
Florida
Bahamas
0.911
Barbados
0.947
0.165
Brazil
0.937
0.125
0.039
Ascension 0.973
0.363
0.043
0.055
St. Helena 0.962
0.238
0.018
0.025
0.007
São Tomé 0.962
0.246
0.028
0.030
0.007
0.000
Note: The pairwise ΦST values are given below the diagonal, while above the diagonal are
significant (+) or not significant (−) pairwise differentiation as observed under Markov chain
simulation, based on an overall P < 0.05 after full Bonferroni correction. Trindade and South
Carolina samples were not included because of low sample sizes.
37
Table 2-4. Absolute haplotype frequencies and sample sizes in Rypticus saponaceous
Haplotype
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
Grenada
n=5
2
1
1
1
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
Barbados
4
1
—
—
—
1
1
1
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
Brazil
23
4
—
—
—
3
—
—
5
3
1
1
1
1
1
1
1
1
—
—
—
—
—
—
—
—
—
—
—
—
—
—
Ascension St. Helena Cape Verde São Tomé
11
16
18
9
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
1
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
4
14
—
—
2
—
—
—
1
—
—
—
1
—
—
—
1
—
—
—
1
—
—
—
1
—
—
—
—
1
—
—
—
—
5
2
—
—
3
4
—
—
3
3
—
—
3
—
—
—
2
—
—
—
2
—
Total
86
7
1
1
1
5
1
1
5
3
1
1
1
1
1
1
1
1
18
2
1
1
1
1
1
1
7
7
6
3
2
2
38
Table 2—5.
Population pairwise differentiation observed in Rypticus saponaceous
Grenada
Brazil
−
Ascension
−
+
St. Helena
+
+
−
Cape Verde
+
+
+
+
São Tomé
−
+
+
+
−
Grenada
Brazil
0.128
Ascension
0.456
0.327
St. Helena
0.695
0.424
0.005
Cape Verde 0.906
0.909
0.911
0.938
São Tomé
0.929
0.919
0.928
0.963
0.042
Note: The pairwise ΦST values are given below the diagonal, while above the diagonal are
significant (+) or not significant (−) pairwise differentiation as observed under Markov chain
simulation, based on an overall P < 0.05 after full Bonferroni correction. Barbados specimens
were not included because of low sample size.
39
Figure 2-1. Biogeographic divisions of the tropical Atlantic with sample localities.
Collection localities of greater soapfish Rypticus saponaceous (shaded
triangles), rock hind Epinephelus adscensionis (open triangles), or both
species (circles). The tropical biogeographic divisions discussed in the text
are indicated as follows: the West Indian Province including Bermuda (gray
stippled area); the Caribbean Province (western solid line); the Brazilian
Province (gray line); the Ascension-St. Helena Province (double circles); and
the East Atlantic Region including Cape Verde (eastern solid line and circle).
The dotted gray line indicates the area of intrusion by Amazonian discharge,
the Brazilian province north of this area is devoid of coral-type reefs.
40
Figure 2-2. Distances among Epinephelus adscensionis haplotypes (A–HH) and selected
congeners, as indicated by a neighbor-joining (NJ) tree. For those nodes with
> 70% retention after 500 bootstrap iterations, the bootstrap support is
indicated at the node.
41
Figure 2-3. Distances among Rypticus haplotypes (1–31) and selected congeners, as
indicated by a neighbor-joining (NJ) tree. For those nodes with > 70%
retention after 500 bootstrap iterations, the bootstrap support is indicated
above the node.
42
Figure 2-4. Frequency distributions for pairwise haplotype distances for cytochrome b
from Epinephelus adscensionis and Rypticus saponaceous.
CHAPTER 3
PHYLOGENETIC RELATIONSHIPS AMONG SOME GROUPER AND
CORALGROUPER SPECIES, WITH COMMENTS ON THE TRIBE EPINEPHELINI
(PERCOIDEI: SERRANIDAE: EPINEPHELINAE).
Introduction
The tribe Epinephelini (the groupers, coralgroupers, coneys, and lyretails) is a set
of tropical and subtropical marine fishes easily recognized by two confluent dorsal fins
and an oblong body adapted to demersal predation (Cavalcanti 1999). Hermaphroditism,
group spawning, relatively slow maturation, and low fecundity are common to many
grouper species (Domeier and Colin 1997), making these apex predators particularly
vulnerable to overfishing (Musick et al. 2000, Sadovy, 2001). Several smaller species are
popular as aquarium fish, while the excellent food quality of the larger groupers supports
a variety of fisheries, from cyanide fishing in the Indo-Pacific (IMA Indonesia 2001) to
extensive line and trap fisheries in Europe and the Americas (Coleman et al. 2001). The
marine commercial catch of groupers for the United States alone has a mean annual
worth of nearly $3 million, not including the equal or greater recreational catch (NMFS
2002). The management of multispecies reef fish fisheries, the identification of
biodiversity ‘hotspots’ for conservation, and studies of comparative biology all require a
taxonomy that reflects biological history (Collette and Vecchione 1995, Sweijd et al.
2000, Vecchione et al. 2000), but currently no such phylogenetic resolution is available.
Groupers and their allies are members of the percoid family Serranidae, possibly a
nonmonophyletic family and "classificatory waste-basket" (Johnson 1983). The limits of
this family were established by Gosline (1966), who diagnosed three serranid
43
44
subfamilies, the Anthiinae, Serraninae, and the grouper subfamily Epinephelinae.
Kendall’s (1976) examination of predorsal bones and associated pterygiophores
identified two evolutionary trajectories within Serranidae: an ancestral lineage including
subfamilies Serraninae and Anthiinae and a derived lineage including the Epinephelinae
with the Grammistinae, the latter uniting the soapfishes and their allies by combination of
the Liopropominae, Grammistidae, and Pseudogrammidae of Gosline (1960). The
subfamily Epinephelinae was presumed monophyletic because of the absence of an
autogenous distal radial on the first dorsal-fin pterygiophore (Johnson 1983). Further
modification was suggested by Johnson (1988), who included the soapfish tribes
Diploprionini, Liopropomini, and Grammistini as derived taxa within Epinephelinae, and
removed the percichthyid species Niphon spinosus into a fifth epinepheline subfamily,
the Niphonini. The Niphonini was proposed as the common ancestor to the soapfish
tribes and the monophyletic epinephelins. Please note that ‘epinephelines’ refers to the
grouper-soapfish subfamily Epinephelinae (following the usage of Johnson 1988), while
‘epinephelin’ denotes the grouper tribe Epinephelini. Support for a monophyletic
Epinephelini was proposed as the shared presence of elongate spinelets on the second
dorsal and caudal fins in postflexion larvae (Johnson and Keener 1984). Baldwin and
Johnson (1993) investigated soapfish tribes and genera, but did not investigate the
epinephelins because of a prohibitive lack of material. The literature review by Heemstra
and Randall (1993) elevated the tribe Epinephelini to a monophyletic subfamily (Table 31). No phylogenetic analysis was presented, but characters supporting a monophyletic
Epinephelinae included possession of: 10 precaudal and 14 caudal vertebrae, one
supernumeray spine on the first dorsal fin pterygiophore, well-developed supramaxilla,
45
depressible inner teeth, a membrane attaching the proximal third of inner pelvic fin ray to
the body, and larvae with the second dorsal fin spine and pelvic fin spine elongated and
serrate (Heemstra and Randall 1993). These features define the ingroup for consideration
in this study and are treated as members of the serranid tribe Epinephelini (sensu Johnson
1983).
Major difficulties in elucidating epinephelin relationships are the tribe’s
circumtropical distribution, impressive diversity (159 species in 15 genera sensu
Heemstra and Randall 1993) and, like most coral reef fishes, a near total lack of fossil
record (Bellwood and Wainwright 2002). The Epinephelini includes several complexes
of sympatric and parapatric species distinguished primarily by color pattern and
combinations of overlapping meristic characters (e.g., the reticulated groupers of the
Indo-West Pacific). Some species pairs differ only in distribution and color pattern (e.g.,
Epinephelus multinotatus, E. cyanopodus, E. trophis, and E. flavocaeruleus), while
marked color pattern or meristic differences exist between distant populations within
single widely distributed species (e.g., Epinephelus fasciatus, Mycteroperca acutirostris).
Recognized species may consist of separate noninterbreeding units (Carlin et al. in press,
Gilles et al. 2000, Heemstra and Randall 1993) and hybrids have been described between
some species and genera (Bostrom et al. 2000, Glamuzina et al. 2001). Resolution of
such species complexes by molecular techniques addresses not only evolutionary history
(e.g., McMillan and Palumbi, 1997) but allows for more informed management in
multispecies fisheries (Collette and Vecchione 1995, Sweijd et al. 2000).
The number of accepted genera differs between Atlantic and Indo-Pacific biologists
(e.g., Grove and Lavenberg 1997, Humann et al. 2002, Robins et al. 1986, Sadovy and
46
Cornish 2000). Smith (1971) used phenetic analysis of meristic and skeletal features to
arrange American grouper species into a phylogenetic hypothesis that proposed early
separate divergences of Paranthias and Mycteroperca from an Epinephelus lineage. The
Epinephelus lineage then diverged nearly simultaneously among Cephalopholis,
Alphestes, Dermatolepis, and a lineage that eventually gave rise to all American
Epinephelus and the genus Promicrops. Based upon this hypothesis, the four genera
derived from the Epinephelus lineage were demoted to subgenera. Examinations of
spinelet development in larvae upheld the subgeneric demotions of Smith (1971), but
with a few exceptions, especially within Cephalopholis and Alphestes (Johnson and
Keener 1984). Inclusion of the Pacific genus Plectropomus rooted a cladogram using 12
larval characters, contradicting an early divergence of Mycteroperca and Paranthias
(Leis 1986). Instead, Plectropomus was the most ancestral genus with available larvae,
and a trichotomy of Mycteroperca, Paranthias, and Epinephelus was the most derived,
with Gonioplectrus (based upon an incomplete series) and Cephalopholis as an
intermediate genus (Leis 1986). The binomial nomenclature of Heemstra and Randall
(1993) was chosen as a reference taxonomy for this study.
Systematic resolution of the epinephelines and related taxa would do more than
standardize a long-standing taxonomic debate. Comparative studies of fisheries ecology,
reproductive biology, behavior, or functional morphology with a phylogenetic component
require knowledge of sister taxon relationships to determine character polarity. The
epinepheline fishes usually exhibit large size, aggressive predation, a tendency to form
spawning aggregations, relatively slow growth, and low fecundity; all of which make
epinephelines both ecologically important as primary demersal predators and highly
47
vulnerable to overexploitation (Coleman et al. 2001, Musick et al., 2000). Fisheries
regulations are, in part, based on knowledge assuming that field identifications are
accurate and that recognized species are real biological entities. Such assumptions are
also made in attempts to delineate areas of endemicity, speciation, and biodiversity loss
(Gill and Kemp 2002, Roberts et al. 2002). Recently, the veracity of these assumptions
in large marine species complexes has been questioned, with the six races of Epinephelus
fasciatus (sensu Heemstra and Randall 1993) suggested as a prominent example of a
cryptic complex waiting to be discovered (Gill and Kemp 2002).
In this study, I tested the validity of tribe Epinephelini by comparing posterior
probability (Bayesian) and maximum parsimony (MP) gene trees from a partial sequence
of the mitochondrial cyt b gene and intron 6 of the nuclear lactose dehydrogenase gene.
The phylogenetic status of species was evaluated by replicate sampling within a priori
species whenever possible. Specifically, I generated gene trees to: evaluate the utility of
cyt b and LDHA6 gene trees as markers for the subfamily level in percoids; test the
accepted monophyly of the grouper and coralgrouper tribe Epinephelini; determine the
most likely phylogenetic position of the epinephelin genera Aethaloperca, Cromileptes,
Saloptia, and Variola (which have no genetic data in the literature); and test if the
molecular data observed here are as likely to be explained by other published
phylogenetic hypotheses (Figure 3-1; Craig et al. 2001, Leis 1986, Smith 1971). I then
suggest systematic revisions where appropriate and present a phylogenetic hypothesis for
further testing.
Materials and Methods
Tissue samples were obtained from multiple localities per species whenever
possible, collected from port samples or during snorkel and SCUBA diving using
48
polespears and microspears. The Ichthyology Division of the Kansas University Museum
of Natural History (KU) provided some tissue samples. In most locations, gill and/or
muscle samples were taken and stored in a saturated-salt DMSO buffer (Amos and
Hoelzel 1991). Several samples each were chosen to represent the range of both
geographic distance and genetic divergence observed across the species range of
Epinephelus adscensionis and Rypticus saponaceous (Carlin et al. in press).
Total genomic DNA was isolated either by organic extraction (phenol:chloroform:
isoamyl alcohol) and 95% ethanol precipitation, or by using the DNeasy Isolation Kit
(Promega, Inc., Madison WI). Isolated DNA was resuspended in 200 mL TE (10 mM
Tris and 1 mM EDTA, pH 8.0). An approximately 750-base pair (bp) fragment of the
mitochondrial cyt b gene was amplified via the polymerase chain reaction (PCR; Saiki et
al. 1985) with primers Cyb-09H (5'-GTGACTTGAAAAACCACCGTTG-3'; Song 1994)
and Cyb-07L (5'-AATAGGAAGTATCATTCGGGTTTGATG-3'; Taberlet et al. 1992).
The amplification usually included 3 mM MgCl, 40 nM of each primer, 17.5 nM of each
dNTP, 2.30 units of Taq DNA polymerase (Promega, Inc.), and 3.5 µL of 10x PCR
buffer (Promega, Inc.) in 35 µL total volume. Amplification temperature regime
included an initial 3-minute denaturation at 94ºC and final extension of 72ºC for 3minutes, with 25 cycles of 30-seconds at 94ºC, 40-seconds at 48ºC, and 1-minute at 72ºC.
The nuclear LDHA6 intron was directly amplified from genomic DNA isolations using
primers LDHA6F and LDHA6R, with amplification conditions varying only slightly
from published protocol (Quattro and Jones 1999).
Oligonucleotide primers were removed by Ultrafree-MC centrifugal filter units
(Millipore Corp., Billerica MA) and/or simultaneous digestion with exonuclease I and
49
shrimp alkaline phosphatase (USB Corp., Cleveland OH; Silva et al. 2001).
Amplification products were denatured and DNA sequencing reactions were conducted
with fluorescently labeled dideoxy terminators according to manufacturer's
recommendations (ABI Model 800, Applied Biosystems, Inc., Foster City, CA). Labeled
extension products were gel separated and analyzed with an automated sequencer (ABI
Models 310, 373A and 377) by the author, the University of Florida Sequencing Core,
and/or the Nevada Genomics Center. All samples were sequenced at least twice with
amplification primers; some cyt b samples were sequenced internal to the amplification
targets using primers Cyb-19H (5'-CTCACTGCTCGGACTCTG-3'), Cyb-21L (5’GTTTTGATGTGTGGTGGAG T-3’), or Cyb-22H (5'-GTGAARTTGTCTGGGTCT
CCT-3').
Resulting chromatograms were edited using Sequencher ver. 3.0 (Gene Codes
Corp., Ann Arbor, MI) and combined with cyt b sequences in GenBank from perciform
fishes. Sequences were aligned by sum-of-pairs progressive pairwise alignment using the
Needleman-Wunsch (1970) algorithm in Sequencher and manually inspected. Intron
chromatograms were visually examined for multiple peaks at a single nucleotide position.
Such samples were presumed as representing heterozygotes and removed from further
analyses. Presumed homozygote (single signal) samples were sequenced at least twice.
Sequences were compared to those in GenBank by BLAST alignment. GenBank
accessions of serranids, as well as any percoid accessions with ≥ 85% sequence match by
Sequencher included in the final alignment.
Nucleotide polymorphism, pairwise distances between sequences, and phylogenetic
estimations were performed using Paup*β5.10 (Swofford 2002). A Χ2 test of base
50
frequency homogeneity was performed considering all positions and third nucleotide
position only, with the acknowledgment that this test assumes phylogenetic
independence. Phylogenetic signal was examined by testing for signal among random
trees (g1 test; Hillis et al., 1996) and visually assessed by plotting transitions against
transversions and proportional distance (p-distance). Three partition homogeneity tests
with 100 replicates each (Farris et. al. 1994, 1995) were used to test for incongruence
between the nuclear intron and mitochondrial cytochrome b loci. The tests considered:
epinephelin taxa with sequence data at both loci; all taxa with data at both loci; and all
available taxa, including those lacking sequenced data for one locus. Single-locus
alignments include comparisons at multiple taxonomic levels, ensuring different amounts
of relevant signal for different questions. Therefore, signal assessment was repeated with
increasingly more inclusive sets of taxa, considering all data versus alternate codon
positions (for cyt b) and indel states (LDHA6). Additionally, numbers of variable,
parsimony-informative, and parsimony-uninformative sites were calculated for all taxa,
the subfamily Epinephelinae only, and tribe Epinephelini only. Neighbor joining (NJ)
trees were constructed using proportional distances (p-distances) and then resampled by
500 bootstraps. Sequences united at a p-distance NJ bootstrap frequency >90% were
assumed to be natural groupings and to maximize computational efficiency, nodes very
highly supported under a simple NJ model subsequently defined topological constraints
imposed on all parsimony analyses.
Two separate methods of phylogenetic estimation were used: maximum parsimony
and posterior probability analysis. A partial search of available treespace was conducted
under the maximum parsimony criterion, following the recommendations of Takahashi
51
and Nei (2000), for the given sample size and variance in internode length. Cladograms
were constructed by 50 iterations of heuristic searches by random taxon addition from a
random tree with the NJ constraints (see above), with each additional iteration followed
by the tree bisection-reconnection branch swapping protocol (Swofford and Begle 1993).
Strict and majority-rule consensus trees were constructed from equally parsimonious MP
trees. Three arbitrarily chosen weighting schemes were utilized in analyses of cyt b:
considering all sites with equal weights, weights reflecting the transition:transversion bias
previously reported in fish cyt b studies (Song 1994), and weighting incorporating both a
codon position bias (3:5:1 for first, second and third positions) and a 1:4 ti:tv bias. The
LDHA6 locus was analyzed both with indels (equal weights at all sites, 1:2 ti:tv, 1:2 ti:tv
and 1:5 nucleotide:indel) and without indels (equal weights at all sites, 1:2 ti:tv).
Posterior probability (Bayesian) analysis was conducted by the Monte Carlo
Markov chain algorithm in MrBayes 2.01, which approximates the results of a likelihood
analysis with bootstrap resampling (Huelsenbeck and Ronquist 2001). Two separate
Bayesian analyses evolved four chains (three heated, one cold) from an initial random
tree for 2 million generations, sampling a tree every 200 generations. The first Bayesian
analysis ran with three chains heated at power=0.2 with estimated priors. The second
analysis involved three chains heated at power=0.4 with unknown (flat) priors (e.g., equal
rates of substitution across sites). The first 10% of generated trees were discarded as
burn-in, and consensus phylograms (strict and majority-rule) were constructed from the
remaining trees.
The prior probabilities for some Bayesian analysis (power=0.2) were generated
from a model of DNA substitution for each locus. Model selection utilized Paup*β5.10
52
(Swofford 2002) and Modeltest 2.0 (Posada and Crandall 1998). A bottom-up
hierarchical likelihood ratio test (ηLRT; Cunningham et al. 1998, Posada 2001) selected
substitution models at each level of comparison. The ηLRT was conducted among
epinephelin sequences only with one randomly chosen sequence per a priori species to
minimize model estimation error from incomplete taxon sampling.
Topological comparisons were carried out in two steps. First, gene trees generated
under different weighting schemes were compared at each locus to best estimate
sequence evolution, using the Shimodaira-Hasegawa (SH) test (Shimodaira and
Hasegawa 1999). The SH test corrects for multiple comparisons among a priori trees
generated from the same dataset but may be overly conservative, with a probability of
correct rejection being less than α (Shimodaira 2002, Shimodaira and Hasegawa 1999).
The tree construction method indicated as optimal by the SH test was used to generate
new gene trees but with additional topological constraints as suggested by a priori
hypotheses of relationship (e.g., Craig et al. 2001, Johnson 1983). A comparison of the
tree with minimal constraints versus the a priori topology was performed using the
Kishino-Hasegawa (KH; Kishino and Hasegawa 1989) test. The SH and KH tests were
performed in Paup*v.5.10β; both indicate a topology as optimal if significant
improvement in likelihood score is found relative to an alternate topology.
Results
A total of 52 identified epinepheline species were available from 11 genera (out of
a possible 159 species in 15 genera listed by Heemstra and Randall (1993). Three
epinepheline genera (comprised of 7 described species) were not available for analysis.
Potential outgroup taxa included the subfamily Serraninae (9 species from 3 genera) and
53
the subfamily Grammistini (2 species from 2 genera). BLAST alignments of GenBank
sequences from 10 percoid families resulted in successful alignments among generated
sequences and accessions in the perciform families Moronidae, Percichthyidae and
Polyprionidae. The cyt b alignment also included six haplotypes identified only to genus:
Epinephelus GA-1999 (GenBank Accession AF143193; Allegrucci et al. 1999) and
Epinephelus sp. A (collected by the author from commercial markets in Hong Kong), 1
Mycteroperca sp. A (mid-Atlantic islands of the Azores, Portugal), 2 specimens of
Serranus sp. A (Gulf of Guinea, East Atlantic; from the tissue collection of D. R.
Robertson, STRI-Panama), and 1 Aporops spp. (KU 804; an unidentified specimen of 6
mm TL).
All sequence data were combined into a single alignment and tested for
incongruence by partition homogeneity testing. A significant incongruence (P = 0.010)
was detected between the nuclear and mitochondrial data when testing all taxa (n = 87),
all taxa sequenced at both loci (n = 71), and epinephelin taxa with sequence data at both
loci (n = 69). Subsequent results are from separate analyses of cytochrome b and
LDHA6.
Cytochrome b
A total of 118 individuals yielded 87 unique haplotypes for 665 bp from the cyt b
mitochondrial gene. The alignment was poor in guanines (Table 3-2), similar to studies
of cyt b in other percoid families (e.g., Near et al. 2000, Song et al. 1998). DNA
substitution frequencies varied by steric type (transition vs. transversion) and codon
position, indicating multiple substitutions per site (i.e., saturation), with all or nearly all
third position sites parsimony informative (Table 3-2). The number of transitions (ti) did
not increase linearly with the transversions (tv) in plots of absolute numbers of
54
substitutions for most pairwise comparisons. Instead, pairwise comparisons of all taxa
while considering all codon positions have a wide range of transversions (from 1 to 78) if
more than 45 transitions are present between compared taxa. All nucleotide sites in the
third codon position were variable. The g1 tests detected significant phylogenetic signal
for all comparisons (P = 0.010). The ηLRT resulted in selection of a general time
reversible (GTR) model (Hillis et al. 1996) incorporating a Γ correction (α = 0.584) and
some sites (47.05%) invariable.
Two or more specimens were sequenced for 22 species (42.30% of species
surveyed). Mean pairwise divergence between interspecific haplotypes within genera
was d = 13.90 % (range: 0.02 to 18.9%). The greatest differentiation within a genus
occurred between haplotypes of Epinephelus adscensionis and E. itajara (a difference of
35 ti and 90 tv). The least interhaplotype divergence estimates occurred between
Plectropomus leopardus vs. P. maculatus (9 ti, 0 tv), Epinephelus niveatus vs. E.
niphobles (10 ti, 0 tv), Mycteroperca bonaci vs. M. rosacea (8 ti, 3 tv), and E.
chlorostigma vs. E. cyanopodus (47 ti, 4 tv). Multiple haplotypes within a single species
were monophyletic for each species (>98% support after 500 bootstrap and 500 jackknife
iterations). However, strong support was also recorded for deep divergences within
monophyletic Epinephelus adscensionis (d = 4.3%), Rypticus saponaceous (d = 4.4%),
and Variola louti (d = 10.4%). Of the 24 species with >1 specimen, haplotypes were
shared across oceans in Cephalopholis cruentata (Bahamas and São Tomé), Epinephelus
adscensionis (Bahamas and São Tomé), Rypticus saponaceous (São Tomé and Cape
Verde; Ascension and Florida), and Variola louti (Australia and Seychelles; Australia and
55
Philippines). Two specimens of M. phenax from the Gulf of Mexico shared a haplotype
with the unidentified M. spp. A from the Azores.
Translation of the 87 haplotypes reduced the dataset into 66 unique amino acid
sequences with 40 parsimony informative characters out of a possible 221 amino acids.
A p-distance NJ tree resampled by 500 bootstrap iterations united two haplotypes of E.
itajara, all Plectropomus sequences, and unified serranines with anthiines. The
serranines, anthiines, and the outgroups Morone and Dicentrarchus were separated with
poor (62%) bootstrap support from all epinepheline and grammistine amino acid
sequences. Comparisons of cyt b amino acids may be able to distinguish percoid families
and subfamilies, but does not have sufficient variation for more exclusive phylogenetic
studies. Nodes supported in the amino acid alignment also had >97% support in the
nucleotide p-distance NJ tree after 500 bootstrap iterations; such nodes were used to
define topological constraints for parsimony analyses. MtDNA constraint nodes united
all haplotypes within Alphestes, Hypoplectrus, Lateolabrax, Morone, Mycteroperca,
Plectropomus, Variola, and all sequences within a priori species (as defined by Heemstra
and Randall 1993) except for .
Cyt b gene trees constructed under a variety of methods varied little in topology
from the p-distance tree. Because MP cladograms were constructed using a constraint
defined by NJ bootstrapping, some agreements between NJ and MP trees are biased.
However no such constraints were defined for Bayesian analyses. Posterior probability
explanations of the cyt b data provided the most likely explanations of the data (Table 33), although this tree was not significantly different by SH tests from either the p-distance
phylogram or a MP cladogram using extensive weighting. Because SH tests could detect
56
few significant topological differences among alternate methods for this dataset, the
Bayesian gene tree (power = 0.4) is presented (Figures 3-2 and 3-3), as this may provide
a better estimate of branch lengths among taxa (Braun and Kimball 2002).
Nuclear Intron
Direct sequencing of LDHA6 amplicons resulted in unambiguous sequences for
nearly a third of samples utilized in the cyt b analysis. The only nonepinepheline taxa to
amplify at the LDHA6 locus were Rypticus saponaceous (tribe Grammistini) and
Stereolepis gigas (family Polyprionidae). The lack of serranine, anthiine, and other
outgroup amplicons may be due to mispriming; no combination of published LDHA6
primers nor custom primers designed from epinephelines resulted in amplification. Two
amplification products, approximately 130 or 190 bp in length, were sequenced from 81
specimens from 52 species in the cyt b dataset. A total of 71 unique sequences were
aligned manually under a bias against novel gaps, resulting in 67 of 186 positions
occupied by indels for one or more sequence. The alignment had 70 indels and a nearly
equal number of transitions and transversions (Table 3-2). Phylogenetic signal was
assessed for the entire alignment (with indels as a fifth base in Paup*), the alignment
without indels, and with an alignment of indel positions alone in a nucleotide
presence/absence matrix. The greatest percentage of parsimony informative sites
(94.7%) occurred in the indels-only matrix (Table 3-2). The nucleotides-only alignment
was best explained in ηLRT by a GTR+I+Γ model with 39.3% invariant sites and a shape
parameter α = 0.870.
Thirteen of the 22 species sampled with more than one specimen possessed >1
genotype per species. No genotypes were found in >1 species. Only 2 species (M. fusca
57
and R. saponaceous) of the 13 species variable at this locus were united strongly in a
monophyletic group with strong support in a p-distance NJ tree after 500 bootstrap
iterations. An indels-only NJ tree clustered alleles into three phylogroups. One group of
21 alleles included the percichthyid Stereolepis gigas, the epinepheline Aethaloperca
rogaa, 4 Cephalopholis spp., and all Plectropomus specimens. The group of 40 alleles
included Anyperodon, Cromileptes, and all Mycteroperca specimens. The Mycteroperca
group has eight insertions 1 to 6-bp long and one 13-bp deletion relative to the
Cephalopholis-Plectropomus group. A third group containing 5 Epinephelus spp. had an
indel pattern intermediate to the Mycteroperca and Cephalopholis-Plectropomus groups,
but with a unique 5-bp deletion. Almost no significant differences could be detected
among gene trees for this locus (Table 3-4), and this is interpreted here as an indicator of
the poor quality of phylogenetic information – some nodes were supported at all trees and
others collapsed into polytomies at all trees. The simplest model that incorporates all
information available, a parsimony cladogram with equal weighting but considering both
sequence and indel position data, is presented in Figure 3-4.
Discussion
Presented here is the largest collection of serranid DNA sequences to date, and
substantial evidence for a nonmonophyletic Epinephelini (Figure 3-3). Robust gene trees
are best constructed using a large amount of sequence data from as wide a geographic
and taxonomic sampling as possible (Pollock et al. 2002, Wakeley 2000, Zwickl and
Hillis 2002), but the resultant gene tree may still not reflect the true phylogeny (Nichols
2001, Pamilo and Nei 1988). The nearly 200 species necessary to completely resolve
relationships among the groupers and their potential outgroups is prohibitive to most
single investigations. The large number of sequences analyzed, and the widely varying
58
branch lengths due to the inclusion of both family and intraspecific comparisons
dramatically lowered estimates of phylogenetic signal. However, the topologies of cyt b
consensus trees constructed under alternate optimality criteria and different character
weighting options varied only slightly, usually in the placement of the same highly
divergent sequences (e.g., the monotypic genus Aethaloperca). Moreover, many of the
relationships suggested by Craig et al. (2001) (e.g., polyphyly of the genus Epinephelus)
are confirmed by this study.
Significant incongruence exists between the intron alignment and the mitochondrial
locus. Combining alignments allows examination of all available evidence (Wiens and
Reeder 1995) and the ability of the partition homogeneity test to correctly identify
incongruence has been questioned (Dolphin et al. 2000; Yoder et al. 2001). However,
LDHA6 differs from cytochrome b by the having less than half the number of species
present in the mitochondrial alignment. More problematical is proper alignment of the
numerous gaps in the LDHA6 alignment. Two different size classes of LDHA6 allele
were amplified. The longer alleles tended to form unresolved polytomies including both
derived and ancestral mtDNA sequences. Some species shown as monophyletic in
mtDNA haplotype with high bootstrap support (>95%) were not monophyletic at
LDHA6. Variola louti specimens yielded either long or short LDHA6 alleles. The
monophyly of each of these species is therefore challenged at this intron, with numerous
undescribed polyphylys and paraphylys. Alternately, the length differences among
alleles precludes accurate alignment, especially in placement of small gaps. Therefore
separate analyses of cyt b and LDHA6 were conducted and subsequent phylogenies were
compared to both eachother and to prior hypotheses of relationship.
59
Significant length differences were detected among all trees (Table 3-4), indicating
a failure for either locus to support any hypothesis from alternate evidence. In contrast,
visual comparison confirms that some relationships suggested at mtDNA loci (either cyt
b or 16S) were not refuted by intron data, many relationships were supported by all three
datasets, and many of the clades identified may have support in morphological characters
as well. Concordance among alternate trees is illustrated in a majority-rule consensus
tree (Figure 3-5) constructed from taxa common to the cyt b, LDHA6, and 16S (Craig et
al. 2001) gene trees. Therefore the phylogeny in Figure 3-5 represents current
understanding of grouper molecular evolution and provides a framework for examining
intrafamily relationships.
Evolution among tribes in the Serranidae has been summarized as a split between
two evolutionary lineages, an ancestral anthiin-serranin lineage and a lineage from which
first the Niphonini, then a monophyletic Epinephelini, then a monophyletic Grammistini
were derived ( Johnson 1983, Johnson and Keener 1984, Kendall 1976). These data
present robust support for a common grammistin-epinephelin lineage within the
Serranidae (as suggested by Craig et al. 2001, Johnson 1983). Several species, sequenced
directly and accessed from GenBank, were used to sample the anthiin-serranin lineage
and basal percoid families. The Hypoplectrus, Paralabrax, and Serranus sequences
formed a well-supported monophyletic group, albeit including a polyphyletic Serranus
(the monophyly of Serranus may not be supported at other loci as well, M. McCartney,
University of North Carolina at Wilmington, pers. comm.). Both loci support the
ancestral position of Plectropomus and Saloptia relative to Epinephelus and its allies
(Heemstra and Randall 1993, Leis 1986, Smith 1964).
60
Monophyly of the Epinephelini
Viewed conservatively, the cyt b gene tree places the grammistins and the anthiinserranin lineage into an unresolved trichotomy with the monophyletic epinephelin genus
Variola. Together the three clades serve as a sister group to a lineage including both
percichthyids and the rest of the epinephelin genera (Figure 3-3). Thus the placement of
Plectropomus and Variola makes Epinephelini paraphyletic as defined by Johnson
(1983). Three of the four Variola specimens in the LDHA6 gene tree (Figure 3-5) were
sister taxa to alleles from the grammistin Rypticus and one allele from Plectropomus
maculatus, termed an ancestral genus on the basis of larval morphology (Leis 1986),
giving slight evidence for inclusion of Variola within the epinephelin-grammistin lineage
as opposed to the anthiin-serranin lineage.
The 16S gene tree by Craig et al. (2001) placed two species of Plectropomus as a
sister group to the grammistins, making the tribe Epinephelini paraphyletic if
Plectropomus was included. Plectropomus and Rypticus differ by approximately 12% at
16S (author’s estimate). Craig et al. (2001) upheld a monophyletic Epinephelini based
upon morphological evidence of Johnson (1983), invoking long branch attraction to
explain the Plectropomus-Rypticus affinity. The level of divergence separating the basal
genera Plectropomus and Variola (Epinephelini) from Rypticus and Aporops
(Grammistini) is at or close to saturation for both LDHA6 and cyt b (pairwise sequence
divergences ranging from d = 25.2 to 26.6% and 16.5 to 20.2%, respectively). Longbranch attraction is to be expected at such great divergences, yet the grammistin
sequences consistently clustered with Plectropomus (as opposed to Serranus or
Stereolepis or Lateolabrax) at cyt b under different weighting schemes, including all
bases, and not considering bases from the third codon position.
61
A similar clustering at a nuclear intron may be less coincidental. Johnson (1983)
chose to place the ancestral Niphon spinosus into a separate tribe rather than as a basal
taxon within Epinephelini. Such an arrangement correctly reflects the phylogenetic
relationships if the Epinephelini is monophyletic. If the taxonomic classification is to
continue to reflect genealogic patterns, then the genera Plectropomus, Saloptia, and
Variola would be removed to a separate tribe, named after its oldest generic name (ICZN
2000). Creation of a Plectropomini identifies the outgroup to the grammistin lineages
and restores monophyletic status to the rest of the Epinephelini. Additional taxa,
characters from a locus slower in mutation rate, and larval character states for Variola are
needed to resolve intertribe relationships within the Epinephelinae.
The genus Variola contains only two described species, Variola albomarginata and
V. louti, both easily recognized at the generic level by an oblong body form and lunate
caudal tail, hence lyretails as their common name. The species lack larval descriptions,
but differ in adult and juvenile coloration and relative pelvic-fin length, with V.
albomarginata often mistaken for the more common V. louti (Heemstra and Randall
1993). Only one V. albomarginata was available in this study, and comparisons with 6
specimens of V. louti (2 were presumptive LDHA6 heterozygotes) had surprising results.
The V. albomarginata specimen shares a cyt b haplotype with two specimens of V. louti.
One of the V. louti shares a LDHA6 allele with one specimen of Plectropomus laevis,
while differing from the V. albomarginata matching its haplotype by a single transition
and 18 indels. This LDHA6 allele was present in several species of Epinephelus, causing
one V. louti specimen to be removed from basal epinephelines in the intron gene tree
62
(Figure 3-4). Thus both gene trees detected deep lineage splits within this genus, but the
splits were not consistent among specimens at the different loci.
The Plectropomus mtDNA haplotypes were monophyletic, and shared a common
ancestry with the Saloptia powelli haplotype observed in two specimens from the
Marshall Islands (western central Pacific). LDHA6 alleles were not available from either
specimen, but the strong support (93% probability) for this relationship at one locus
concurs with other evidence. Saloptia and Plectropomus also possess a set of osteologic
and meristic features that led Smith (1964) and Leis (1986) to suggest that these IndoPacific fishes are sister taxa. Both genera are restricted to the Indo-Pacific, although the
depth range (140 to 367 m) could allow Saloptia to have a distribution broader than
currently recorded. Another deepwater monotypic genus, Gonioplectrus, was not
available for analysis, but larval morphology placed this genus intermediate between
Plectropomus and Cephalopholis. Further analyses of DNA sequences and larval
morphology are necessary to resolve the evolutionary history of these genera with
soapfishes and groupers.
Conclusions and Recommendations
The cytochrome b locus, and to a lesser extent the LDHA6 intron, contained
enough variation to evaluate relationships among epinepheline genera and thereby test
the monophyly of tribe Epinephelini. A parallel exists between molecular and
morphological data at many points in the gene trees. In addition to the agreements
between alternate sources of data, both types of characters present problems for highly
divergent taxa. Anyperodon leucogrammicus and Epinephelus itajara are notable
examples of extreme morphological and ecological divergence within lineages
considered here. A. leucogrammicus is extremely compressed and arguably the ultimate
63
morphological adaptation to exclusive piscivory. In contrast, most Epinephelus spp.
consume some combination of fish, crabs, and small crustaceans. The gigantic size of the
broad-headed E. itajara may restrict larger specimens to a diet comprised almost entirely
of lobsters (Randall 1967). Both of these taxa are or have been in a separate genus of
uncertain affinity, and this study has done little to resolve the situation. In the case of E.
itajara, however, reconsideration of the subgenus Promicrops requires a study be made
of E. lanceolatus, the Indo-Pacific member of the former genus. Two genera not
examined at both loci deserve mention. Leis (1986) believed the larval morphology of
Gonioplectrus to be intermediate between Plectropomus and Cephalopholis; making the
placement of this genus within a tribe uncertain. The relative position of genus
Paranthias may be easier to predict: these two species were noted by Craig et al. (2001)
to have little genetic divergence at 16S from Cephalopholis fulvus. Given the uncommon
but well-documented hybridization between these species (Bostrom et al. 2000), the
inclusion of Paranthias within Cephalopholis may not be surprising. The strength of
intrageneric relationships varies widely and species phylogenies are discussed in the
Appendix. Obviously more data should be collected from both taxa and as many other
species as possible to narrow the level of divergence and reconstruct their evolutionary
history.
The large number of species not included in this dataset could reveal polyphyletic
and paraphyletic groups within the gene trees as currently described. However, some
taxonomic changes may be warranted with the accumulation of a consistent pattern from
independent sources (Table 3-1, Figure 3-5). The consistent failure of three loci to
construct a monophyletic Epinephelini warrants removal of the genera Plectropomus,
64
Saloptia, and Variola from the tribe. Evidence has been presented in this and other
studies for a lack of monophyly for the tribe Epinephelini and the genera Epinephelus,
Cephalopholis, Dermatolepis, and Plectropomus. The ecological and morphological
differences that set Dermatolepis, Alphestes, Anyperodon, Saloptia, and E. itajara apart
from their closest ancestors are extensive; their taxonomic status as genera and subgenera
may require an evaluation of not only other genetic loci but a detailed study of life history
behavior, ecology, and morphology. Such tests of the evolutionary hypothesis proposed
here (a tribe Plectropomini ancestral to the soapfish tribes and the Epinephelini; Figure 35) provides a framework for testing diversification mechanisms in the marine tropics.
A well-described evolutionary history affects more than taxonomic practice
(Collette and Vecchione 1995). Evaluations of global fishery resources and ecosystemwide effects of fishing (e.g., Pauly et al. 1998, Sadovy 2001, Vecchione et al. 2000) are
based in part upon agency reports of “grouper” catch data. Designation of a
Plectropomini could remove exploited coralgroupers and lyretails (both easily
distinguished from epinephelins) to a separate category, refining the accuracy and
relevance of fishery statistics in the Indo-Pacific. The quantification of biodiversity
patterns has increased in popularity and scope, resulting in fine-scale maps of habitat and
faunal diversity (e.g., Bellwood and Hughes 2001, McAllister et al. 1994, Moritz and
Faith 1998, Roberts et al. 2001). The taxonomic unit considered for tropical reef
assessment is not only species but families and subfamilies as well, providing criteria for
identifying areas of endemism (De Grave et al. 2001, Gill and Kemp 2002, Knowlton
2001, Roberts et al. 2002). Phylogenetic accuracy in taxonomic schema is the
prerequisite for accurate evaluation of such ‘biodiversity hotspots’ and an understanding
65
of the evolutionary ecology of coral reef fishes (Bellwood and Wainwright 2002, Choat
and Bellwood 1991).
66
Table 3-1. Four alternative classifications of grouper and coralgrouper genera
Smith (1971)
Johnson (1983)
Heemstra and
Randall (1991)c
SERRANIDAE
SERRANIDAE
SERRANIDAE
SERRANINAE
ANTHIINAE
SERRANINAE
ANTHIINAE
SERRANINAE
ANTHIINAE
SERRANIDAE
SERRANINAE
Serranini
Anthiini
NIPHONINAE
EPINEPHELINAE
Niphonini
NIPHONINAE
EPINEPHELINAE
Niphonini
Epinephelini
EPINEPHELINAE
incertae sedis
Anyperodon
Cromileptes
Epinephelus
(Alphestes)
(Epinephelus)
(Cephalopholisb)
(Dermatolepis)
(Promicrops)
Gonioplectrus
Gracila
Mycteropercaa
Paranthias
Plectropomus
Variola
incertae sedis
Plectropomus
Saloptia
Gonioplectrus
Cephalopholis
Aethaloperca
Gracila
Mycteroperca
Epinephelus
(Promicrops)
Alphestes
Dermatolepis
Triso
Paranthias
Cromileptes
Variola
EPINEPHELINAE
Epinephelini
Paranthias
Mycteropercaa
Epinephelus
(Cephalopholisb)
(Promicrops)
(Epinephelus)
(Alphestes)
(Dermatolepis)
incertae sedis
Aethaloperca
Anyperodon
Cromileptes
Gonioplectrus
Gracila
Plectropomus
Saloptia
Variola
this study
Epinephelini
Cephalopholis
Aethaloperca
Paranthias
Epinephelus
Alphestes
Dermatolepis
Mycteroperca
Epinephelusd
(Promicrops)
Anyperodon
Cromileptes
tribe nov.e
Plectropomus
Saloptia
Variola
incertae sedis
Gonioplectrus
Gracila
Triso
GRAMMISTINAE
Diploprionini
Diploprionini
Diploprionini
Diploprionini
Liopropomini
Liopropomini
Liopropomini
Liopropomini
Grammistini
Grammistini
Grammistini
Grammistini
Note: Names are listed in phylogenetic order (most ancestral to most derived). Parenthetical
names indicate subgenera. a, The genus Mycteroperca included Mycteroperca (=Trisotropis)
dermopterus until removed to the monotypic genus Triso by Randall et al. (1989). Cephalopholis
(Aethaloperca) rogaa. b, The genus Cephalopholis included C. albomarginatus until its removal
to the monotypic genus Gracila by Smith-Vaniz et al. (1988). c, Although inset generic names
indicate hypotheses of relationship, authors did not perform phylogenetic analyses. d, This
Epinephelus clade is the same as the Epinephelus (Hyporthodus) clade of Craig et al. (2001)
including E. (Promicrops) itajara. The subgeneric status of Promicrops may be in question (see
text). e, The sister clade to Epinephelini and ancestral to the soapfish tribes would be named as a
tribe after its oldest member genus, Plectropomus Oken 1817 (ICZN 2001).
Table 3-2. Nucleotide composition of DNA sequence alignments from percoid fishes sampled at cytochrome b (cyt b) and LDHA
intron 6
Locus
Alignment
Taxa
m
Base Composition
%A
%C
%G
%T
P
Ti:Tv
Phylogenetic Signal
Variable
Pars Inf
Cyt b (n = 665)
All sequences
Epinephelinae
(Epinephelini)
87
65
57
24.41
24.34
24.27
29.98
30.36
30.64
15.54
15.41
15.72
30.07
29.89
29.72
1.000
1.000
1.000
1.61
2.19
2.61
67:18:221
50:11:220
42: 9:219
61
57
44
23.66
23.61
23.05
23.84
23.82
24.05
23.80
23.87
24.41
28.70
28.69
28.49
1.000
1.000
1.000
1.16
1.23
1.44
25
21
01
13
12
0
61
57
44
24.23
25.31
24.78
23.40
23.40
24.82
23.36
23.20
23.44
29.01
28.08
26.95
1.000
1.000
1.000
—
—
—
5
1
2
8
7
1
61
57
44
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
3
9
0
6
4
9
67
LDHA6
No Indels (n = 116)
All sequences
Epinephelinae
(Epinephelini)
All Sites (n = 186)
All sequences
Epinephelinae
(Epinephelini)
Indels Only (n = 70)
All sequences
Epinephelinae
(Epinephelini)
55:11:221
45: 7:219
35: 6:216
68
Table 3-3. Results of a posteriori topology tests among 50% majority-rule consensus
trees generated from epinephelin cytochrome b
Cytochrome b Tree
Method
Minimum Evolution
SH Likelihood Tests
Model
−lnL
P
δ
p-distance
13816.760
59.054
0.075
p BC
13959.037
201.331
0.000*
Maximum Parsimony
equal weighting
14093.645
335.939
0.000*
Weighted Parsimony
4:1 tv:ti
14066.530
308.825
0.000*
4:1 tv:ti, 3:5:1 codon
13816.760
59.054
0.075
Bayesian Likelihood
chain heat = 0.2
13764.102
6.396
0.710
chain heat = 0.4
13757.706
(best)
Note: BC = bootstrap consensus after 500 iterations. Bayes consensus = consensus of all
Bayesian trees generated at different powers. δ = difference between given topology and shortest
tree. P-values with an asterisk (*) indicate a tree significantly different by ShimodairaHasegawa testing from the most likely tree at a significance of α = 0.05.
69
Table 3-4. Results of a posteriori topology tests among 50% majority-rule consensus
trees generated from epinephelin LDHA6 introns
LDHA6 Intron Tree
SH Likelihood Tests
Method
Model
Gaps
−lnL
P
δ
Minimum
p-distance
N
1277.604
5.011
0.789
Evolution
p BC
N
1354.903
82.310
0.078
mean NJ
O
1409.880
137.288
0.001*
mean NJ BC
O
1515.243
242.650
0.000*
Maximum
equal weighting
N
1276.975
4.382
0.902
Parsimony
equal weighting
E
1274.885
2.293
0.912
equal weighting
O
1742.813
470.220
0.000*
Weighted
2:1 tv:ti
N
1364.899
92.307
0.702
Parsimony
2:1 tv:ti
E
1272.593
(best)
2:1 tv:ti, 5:1 indel
W
1284.157
11.565
0.702
Bayesian
chain heat = 0.2
N
1316.618
44.025
0.354
Likelihood
chain heat = 0.4
N
1475.678
44.959
0.351
Note: BC = bootstrap consensus after 500 iterations. NJ = Neighbor-joining. Bayes consensus =
consensus of all Bayesian trees generated at different powers. Treatment of gaps in the alignment
is encoded as: N: not included in analysis, E: treated as 5th base with equal weighting as
transitions, W: treated as 5th base weighted 5 times transitions, O: only gaps were analyzed
(coded as presence/absence characters). δ: Difference between given topology and shortest tree.
P-values with an asterisk (*) indicate a tree significantly different by Shimodaira-Hasegawa
testing from the most likely tree at a significance of α = 0.05.
Table 3-5. Results of topology tests among unconstrained gene trees and gene trees with a priori topological constraints
Locus
Enforced Constraint
Cyt b mtDNA
monophyletic Epinephelini
5 subgenera within Epinephelus
Source
KH Tests
Length
Johnson (1983)
Smith (1971)
13914.356
13977.082
Diff
P
Result
156.649
219.376
<0.001*
<0.001*
H0 rejected; Epinephelini paraphyletic
H0 rejected; Smith’s Epinephelus is
paraphyletic
H0 rejected; rejection due to paraphyletic
Epinephelus
H0 rejected; data not explained by 16S
rRNA tree
H0 rejected; one or both gene trees do not
equal the species tree
best explains cyt b data
Plectropomus is ancestral to
Mycteroperca + Epinephelus
16SrRNA mtDNA gene tree
Leis (1986)
13834.189
76.483
0.001*
Craig et al. (2001)
14150.717
393.011
<0.001*
LDHA6 intron tree
this study
15577.693
1819.987
<0.001*
none
this study
13757.706
(best)
Smith (1971)
Leis (1986)
958
865
176
83
<0.001*
<0.001*
Craig et al. (2001)
this study
967
997
185
215
<0.001*
<0.001*
H0 rejected as above
H0 rejected as above
H0 rejected as above
H0 rejected; one or both gene trees do not
equal the species tree
none
this study
782
(best)
best explains intron data
Note: Cyt b trees were generated by Bayesian analysis, LDHA6 intron trees were maximum parsimony cladograms including gaps as a fifth base.
Lengths and differences in lengths (Diff) were calculated using likelihood scores (cyt b) or parsimony scores (LDHA6). The Johnson (1983)
hypothesis (i.e., Epinephelini is monophyletic) could not be tested at LDHA6 intron due to lack of sequences from outgroup taxa. Asterisked
values in table indicate significant difference at P < 0.05.
70
LDHA6 intron
5 subgenera within Epinephelus
Plectropomus is ancestral to
Mycteroperca + Epinephelus
16SrRNA mtDNA gene tree
cyt b mtDNA gene tree
71
Figure 3-1. Phylogenetic hypotheses of epinephelin evolution. Hypotheses are based
upon (A) adult meristics and body shape (Smith 1971), (B) larval
development (Leis 1986), and (C) a 16S gene tree (Craig et al. 2001).
Triangles indicate unresolved intrageneric relationships.
72
Figure 3-2. The most likely gene tree for cytochrome b, showing derived species only.
Numbers indicate node probability (shown above nodes when >50%) or
numbers of specimens (shown right of taxon name when n > 1). Gray vertical
bars indicate groups of intraspecific haplotypes. Abbreviations: Alp =
Alphestes, Any = Anyperodon, Cep = Cephalopholis, Cro = Cromileptes, Der
= Dermatolepis, Epi = Epinephelus, Myc = Mycteroperca.
73
Figure 3-3. The most likely gene tree for cytochrome b, showing ancestral species only.
Abbreviations follow those in Figure 3-2 and: Aet = Aethaloperca, Apo =
Aporops, Cep = Cephalopholis, Dic = Dicentrarchus, Epi = Epinephelus, Hyp
= Hypoplectrus, Lat = Lateolabrax, Par = Paralabrax, Ple = Plectropomus,
Ryp = Rypticus, Sal = Saloptia, Ser = Serranus, Ste = Stereolepis, Var =
Variola.
74
Figure 3-4. The most parsimonious epinephelin gene tree at LDHA6. Numbers at nodes
indicate per cent node retention after resampling by 500 bootstrap iterations.
Gray vertical bars indicate intraspecific haplotypes. Abbreviations are
explained in Figures 3-2 and 3-3.
75
Figure 3-5. Hypothetical phylogeny of the groupers and their allies. Tree is an Adams
consensus of the cytochrome b gene tree (Figures 3-2 and 3-3), the LDHA6
gene tree (Figure 3-4) and the 16S rRNA gene tree of Craig et al. (2001).
Numbered nodes indicate the taxonomic limits of: 1 = Family Serranidae, 2 =
Subfamily Serraninae, 3 = Subfamily Epinephelinae, 4 = Tribe Epinephelini, 5
= Tribe Plectropomini, 6 = Tribe Grammistini. All other abbreviations follow
those in Figures 3-2 and 3-3.
76
CHAPTER 4
CONCLUSIONS
The groupers and coralgroupers exhibit a wide array of molecular evolutionary
patterns. Gene flow in epinephelines has been described among genera (Bostrom et al.
2000) and species within genera (E. nigritus and E. merra, this study; Glamuzina et al.
2001, van Herwerden et al. 2002) while other species show a distinct genetic break
among regional samples (E. adscensionis, E. fasciatus, R. saponaceous, Plectropomus;
this study). The consistent failure of three loci to construct a monophyletic Epinephelini
warrants creation of the tribe Plectropomini, including at least the genera Plectropomus,
Saloptia, and Variola. Evidence has been presented in this and other studies for a lack of
monophyly for the tribe Epinephelini and the genera Epinephelus, Cephalopholis,
Dermatolepis, and Plectropomus. The epinepheline species E. adscensionis and the
grammistine Rypticus saponaceous were each monophyletic, but with deep divergences
between allopatric lineages.
The molecular genealogies described thus far are typified by having a few ancient
lineages with recent and extensive cladogenesis (i.e., Category I phylogeographic pattern;
Avise 2000). Thus, the epinephelines may be an example of episodic, or ‘punctuated,’
speciation, with speciation rates varying between clades (e.g., between E. itajara and
other groupers) and time periods (Jackson and Cheetham 1999, Schluter 2000).
Examples of unequal evolutionary rates within monophyletic clades are common in
speciose marine taxa (Caldara et al. 1996, Johns and Avise 1998, McMillan and Palumbi
76
77
1997, Streelman et al. 2002). However, the same patterns could arise under a model of
constant diversification but with high extinction rates, with extinction by chance or
selection of incipient lineages (Avise 2000).
Future investigations of grouper larval morphology, dispersal ability, adult life
history characteristics, and habitat requirements could utilize these data to address the
role of the Indo-Pacific triangle in reef fish evolution (Bellwood and Hughes 2001,
Bernardi et al. 2000, Santini and Winterbottom 2002); the open population paradigm of
coral reef fishes (Leis 2002, Mora and Sale 2002); the relationship among genetic stocks,
fishery practice, and effective population size (Baker et al. 1995, Smith 1994, Smith et al.
1991); the evolution and retention of microsatellite and intron sequences (Dimmick 2001,
Orti et al. 1997, Rico et al. 1996); and creation of species/region forensic database for
monitoring trade in grouper products (Baker et al. 1996, Sweijd et al. 2000).
78
APPENDIX
COMMENTS ON GROUPER SPECIES RELATIONSHIPS
Species Relationships within a Reduced Epinephelini
The de facto incomplete taxon sampling involved when delineating a large species
complex prevents rigorous testing of species limits in this study. However, some
relationships among haplotypes and genotypes are well supported and concordant with
other lines of evidence. Perhaps more interesting to the evolutionary biologist are those
cases in which no concordance among mitochondrial, nuclear, larval or adult
morphological data was present. Detailed accounts of major groupings and problematic
taxa are given below in order of divergence from outgroup taxa.
The Hind Clade
Five species of Cephalopholis were examined, and haplotypes from each formed
monophyletic groups in two distinct and divergent lineages. A well-resolved cyt b clade
comprised of the East Atlantic C. taeniops as ancestral sister taxon of the West Atlantic
C. fulvus and the Indo-West Pacific C. sexmaculata. Cephalopholis fulvus and C.
taeniops were sister taxa in the LDHA6 gene tree as well. A second lineage within
Cephalopholis observed at both cyt b and LDHA6 contains C. nigri and C. cruentata as
sister taxa. At 16S, C. cruentata was moderately supported (76% bootstrap support) as
ancestral to a clade including C. fulvus. A sample of A. rogaa from Hong Kong was
placed into the Cephalopholis clade, but at the LDHA6 locus, this specimen was placed
into an unresolved polytomy with Plectropomus, thus the evolutionary relationships of
this genus remains uncertain.
78
79
The True Grouper Clade
The data presented in this study identify a sharp genetic divergence among the
coralgroupers, lyretails, and hinds from other sampled epinephelin genera, confirming
general hypotheses based upon larval morphology and 16S gene trees. Leis (1986) noted
an increase in dorsal-fin spine number from 8 directly forming spines (Plectropomus), to
8+1=9 directly+indirectly forming spines (Cephalopholis) to the 9 to 11 spines of
Epinephelus and Mycteroperca. The node is well supported at cyt b (P=98%), LDHA6
(P~100%), and 16S (87% bootstrap support; Craig et al. 2001). The more ancestral
genera of Cephalopholis, Saloptia, and Variola can be generalized as oblong piscivores
with a distribution nearly restricted to Indo-Pacific waters. The remaining genera
sampled in this study have a relatively deeper body form and considerable representation
in the Atlantic. Weaver (1996) suggested that serranid body form may be the result of a
coupling between prey size and energy yield with gape limitation and predator avoidance.
Smaller size (like the 30 to 40 cm Cephalopholis) increases reliance upon sprint speed,
visual acuity, and dependence upon reefs for shelter and predator avoidance. An
increased body size means a lessening of gape limitation, perhaps resulting in capture of
prey items with higher energy yield. While it is difficult to distinguish if the inclusion of
more demersal, hard-shelled prey was a result or a progenitor of an adaptive radiation, the
mtDNA data lend some support to the existence of such a morphological spectrum in
epinephelins. Of course, exceptions can be named: the derived Anyperodon’s elongate
body form would have to be encoded as a derived compression while the crustaceaneating C. sonnerati, whose body depth is nearly equal to its standard length, would
represent convergence upon an Epinephelus-like form from within a more primitive
lineage.
80
The Haifa and Leather Bass Clades
The Haifa grouper E. haifensis unambiguously roots (P = 100%) a monophyletic
group of five groupers in the cyt b gene tree. Epinephelus flavolimbatus is a sister taxon
to E. niveatus, E. niphobles, and a haplotype shared between the west Atlantic E. nigritus
and the Indo-Pacific E. merra. The relationship among E. nigritus, E. niphobles and E.
niveatus is mirrored at 16S (Craig et al. 2001). Members of the Haifa clade differed from
each other by 1 to 7 substitutions at cyt b, but all shared seven deletions in LDHA6
relative to all other sequences, creating a strongly divergent clade intermediate between
Cephalopholis intron alleles and true grouper intron alleles. However, two other E.
merra and one other E. nigritus did not possess these deletions and differed by only 1 to 3
substitutions.
Smith (1971) synonymized E. niphobles and E. niveatus because they have
identical juvenile coloration and adult meristics. Heemstra and Randall (1993) disputed
the taxonomic decision, citing slight differences in eye and pectoral fin morphology, but
did not deny a potentially close relationship. The close relationship between the E.
niphobles/E. niveatus pair and the west Atlantic E. nigritus is more unexpected.
Epinephelus nigritus is the only member of the Haifa clade to have X instead of XI
dorsal-fin spines, and to have 4 to 5 rows of teeth instead of 2 to 3 rows on the midlateral
of the lower jaw. Leis (1986) commented upon the confusing nature of dorsal-fin spine
counts, noting that direct formation of 8 or 9 spines are reliable characters for unifying
epinephelin genera, but that spines beyond the count of 9 may form directly (orthologous)
or form by fusion of fin rays (paralogous). The number of tooth rows is notable because
juvenile E. nigritus possess 2 to 3 midlateral tooth rows and develop additional rows
upon settlement.
81
The closest sister taxon to the Haifa clade in this study is a strongly supported (at
cyt b and 16S) clade including the leather bass Dermatolepis dermatolepis and two of the
three described species of Alphestes. Both genera possess morphological novelties that
have made past attempts at phylogenetic placement problematic (Heemstra and Randall
1993). The genetic divergence among Alphestes, Dermatolepis, and Epinephelus is large,
thus the leather bass clade may represent early specializations outside most species in the
true grouper complex.
The Mycteroperca Clade
The species of Mycteroperca form a well-supported monophyletic group at cyt b
and at least partially at LDHA6. The genus is restricted to the Atlantic and East Pacific,
and surely represents a radiation from a single lineage predating the closure of the
Panamanian Seaway 3.1 to 3.7 MYA (Coates and Obando 1996). Only one East Pacific
species, M. rosacea, was available for analysis. The cyt b M. rosacea haplotype differed
from M. bonaci by a single transition; these differed from two M. venenosa haplotypes by
28 to 29 substitutions. The placement of M. bonaci and M. venenosa as sister taxa is
supported at 16S, whereas M. tigris intron alleles are sister taxa to M. venenosa intron
alleles. Unlike 16S, the cyt b Mycteroperca clade did not include Anyperodon (see
discussion below). The most divergent Mycteroperca sequence of those sampled in this
study was M. fusca. Specimens of M. fusca from the Gulf of Mexico and the Atlantic
coast of Florida shared a haplotype with a specimen from the Azores, the first record of
M. fusca in these Mid-Atlantic islands (Morato et al., in review). The cyt b haplotype and
two sister LDHA6 alleles were most closely related to specimens of Epinephelus
marginatus from the Azores in the central Atlantic. The 56 ti and 16 tv difference at cyt
b resulted in a single nonsynonymous amino acid substitution, and the LDHA6 alleles
82
differed by 1 to 2 ti, 2-4 tv, and 0 indels. With such sequence similarity, E. marginatus
and M. fusca specimens were sister taxa in NJ, MP, WP, and Bayesian likelihood trees at
both loci despite meristic and morphological differences between the two genera
(Heemstra and Randall 1993). The E. marginatus and M. fusca lineage may also include
the Mediterranean species E. caninus, based upon analyses of 431 bp of cyt b (T.
Maggio, Univ. de Palermo, pers. comm.).
The American Clade
Although Smith’s (1971) assumption of monophyly among all American groupers
is not supported (this study, Craig et al. 2001), six Epinephelus species from the West
Atlantic and East Pacific form a well-supported (P=94%) monophyletic lineage in the cyt
b gene tree. Epinephelus striatus, E. morio, and E. guttatus show identical relationships
at cyt b, LDHA6, and 16S. Johnson and Keener (1984) were unable to distinguish the
larvae of E. morio and E. guttatus from each other or E. drummondhayi. These three
species have identical historical distributions except that E. guttatus is absent from
southern Brazil, and form a sister clade to a clade comprised of Epinephelus
adscensionis, E. labriformis, and E. analogus. Although E. adscensionis and E. analogus
have been proposed as geminate species across the Panamanian Isthmus (Heemstra and
Randall, 1993), it is the East Pacific E. labriformis that separates the West Atlantic E.
adscensionis from the East Pacific by 9.5% sequence divergence. Given the total overlap
among geographic and depth distributions (Heemstra and Randall 1993), nearly identical
diets (Randall 1967), and microsympatry at the same fishing grounds (Heemstra and
Randall 1993), the American clade therefore presents an opportunity for studying
speciation tempo and mode.
83
The Pacific Grouper and Promicrops Group
The West Pacific species E. awoara, one of the relatively few groupers in the East
China Sea and a species of increasing importance in grouper aquaculture (Sadovy 2000,
Sadovy and Cornish 2000), could not be unambiguously assigned a position among true
grouper clades. A set of cyt b sequences was aligned with moderate support at cyt b (P =
79%) that includes a strongly supported set of four species and two highly divergent taxa.
Epinephelus caerulopunctatus and E. tauvina positioned as sister taxa to E.
polyphekadion and C. altivelis. The pairs are joined with strong support in the cyt b
DNA alignment (P = 96%) but are not joined upon conversion to an amino acid NJ
phylogram. The monotypic genus Cromileptes has a highly specialized cranial
morphology with a dorsal-fin shape, position, and spine count unique in Indo-Pacific
groupers. The pairing of E. polyphekadion as a sister taxon of Cromileptes may be the
result of long-branch attraction, although the relationship is supported after alignment
translation. The same might be said for the strong support for joining the elongate
exclusive piscivore E. tauvina and the deeper-bodied and crustacean-feeding E.
caerulopunctatus. The monotypic genus Anyperodon was placed as a sister taxon of E.
itajara at cyt b, a sister of Cromileptes at LDHA6, and within the Mycteroperca clade at
16S. The three loci, together with an advanced pterygiophore arrangement (Smith-Vaniz
et al. 1988), are concordant with a generic placement as a derived true grouper, but
otherwise its phylogenetic position cannot be determined with reliability.
The genus Promicrops included two species with a unique arrangement of lateral
line tubules and gigantic size (up to at least 2.5 m and 320 kg). Smith (1971) reduced
Promicrops to a subgenus of Epinephelus, a decision supported by later workers,
although Heemstra and Randall (1993) noted the extreme specialization present in the
84
genus. Johnson and Keener (1984) noted that E. itajara larvae are characterized by
having pigmentation recorded from Mycteroperca and a Cephalopholis species. Two
samples of Atlantic E. itajara shared identical LDHA6 alleles with three unique deletions
and a mean sequence divergence of 3.7% from various true grouper lineages (including
Anyperodon and E. polyphekadion). Epinephelus itajara intron allele was the ancestral
sequence to most Epinephelus and Mycteroperca sequences. The divergence between E.
itajara at cyt b, however, was one of the greatest seen within the true groupers (mean
d=15.7% between E. itajara and E. tauvina). The cyt b divergence was overwhelmingly
comprised of nonsynonymous substitutions, and translated to 15 to 21 amino acid
substitutions between either E. itajara haplotype and any other amino acid sequence in
the Epinephelinae. Obviously additional loci and morphological characters are needed
before the systematic relationships of the subgenus Promicrops, or the systematic status
of Epinephelus (Promicrops) itajara can be determined.
Final Thoughts
Taxonomies may reflect ease of diagnosis, an attempt to reconstruct phylogenetic
history, or a need for stability. Usually in practice, taxonomies are some combination of
all three (Stevens 1992). The de facto incomplete taxon sampling involved when
delineating a large species complex prevents rigorous testing of species limits in this
study. At first glance many binomials could be questioned on the basis of the mtDNA
gene trees alone. Gilles et al. (2000) suggested that E. marginatus is comprised of cryptic
species, and Carlin et al. (in press) identified a similar genetic divergence within E.
adscensionis. Yet it is unknown whether the alternate phylogeographic units with
symparapatric distribution fit definitions as different species. Alternate species concepts
may change the taxonomic label of a unit, but this does not change its cladistic
85
relationship. Even as phylogenetic species, confirmation at multiple loci is preferable to
reliance upon a single locus (Slowinski and Page 1999). Are these organisms failing to
interbreed entirely? Do they form cohesive lineages at multiple loci across their range
and across generations? Dupré (1992) has noted that evolutionary biologists historically
state belief in a species concept without providing the means to test its assumptions. The
groupers and coralgroupers possess a wide range of features which, examined in the
context of an accurate genealogical framework, can provide the means of testing
speciation processes.
86
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BIOGRAPHICAL SKETCH
Joel Carlin was born in Kalamazoo, Michigan in 1969; and was raised in Indiana.
He earned a B.Sc. in marine biology from the University of North Carolina at
Wilmington in 1991; and a M.Sc. in zoology from Louisiana State University in 1995.
Since 1995 he has co-published six peer-reviewed articles and two reports on genetic
variability in aquatic organisms and received ten competitive grants or awards. He
currently resides in Gainesville, Florida and continues to develop as a teacher and
researcher of marine science, evolution, and natural history.
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