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Twistedsisterspeciesofpygmyangelfishes:Discordancebetweentaxonomy,coloration,andphylogenetics

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Twisted sister species of pygmy angelfishes: discordancebetween taxonomy, coloration, and phylogenetics

Joseph D. DiBattista • Ellen Waldrop • Brian W. Bowen •

Jennifer K. Schultz • Michelle R. Gaither • Richard L. Pyle •

Luiz A. Rocha

Received: 19 November 2011 / Accepted: 9 April 2012 / Published online: 5 May 2012

� Springer-Verlag 2012

Abstract The delineation of reef fish species by colora-

tion is problematic, particularly for the pygmy angelfishes

(genus Centropyge), whose vivid colors are sometimes the

only characters available for taxonomic classification. The

Lemonpeel Angelfish (Centropyge flavissima) has Pacific

and Indian Ocean forms separated by approximately

3,000 km and slight differences in coloration. These

disjunct populations hybridize with Eibl’s Angelfish

(Centropyge eibli) in the eastern Indian Ocean and the

Pearl-Scaled Angelfish (Centropyge vrolikii) in the western

Pacific. To resolve the evolutionary history of these species

and color morphs, we employed mitochondrial DNA

(mtDNA) cytochrome b and three nuclear introns (TMO,

RAG2, and S7). Phylogenetic analyses reveal three deep

mtDNA lineages (d = 7.0–8.3 %) that conform not to

species designation or color morph but to geographic

region: (1) most Pacific C. flavissima plus C. vrolikii, (2) C.

flavissima from the Society Islands in French Polynesia,

and (3) Indian Ocean C. flavissima plus C. eibli. In con-

trast, the nuclear introns each show a cluster of closely

related alleles, with frequency differences between the

three geographic groups. Hence, the mtDNA phylogeny

reveals a period of isolation (ca. 3.5–4.2 million years) typical

of congeneric species, whereas the within-lineage mtDNA

UST values and the nuclear DNA data reveal recent or ongoing

gene flow between species. We conclude that an ancient

divergence of C. flavissima, recorded in the non-recombining

mtDNA, was subsequently swamped by introgression and

hybridization in two of the three regions, with only the Society

Islands retaining the original C. flavissima haplotypes among

our sample locations. Alternatively, the yellow color pattern

of C. flavissima may have appeared independently in the

central Pacific Ocean and eastern Indian Ocean. Regardless of

how the pattern arose, C. flavissima seems to be retaining

species identity where it interbreeds with C. vrolikii and

C. eibli, and sexual or natural selection may help to maintain

color differences despite apparent gene flow.

Keywords Centropyge � Color variation � Coral reef fish �Hybridization � Incomplete lineage sorting �Mitochondrial DNA � Nuclear introns

Introduction

The delineation of species is important for preserving bio-

diversity, and yet, this process is fraught with difficulties

(Coyne and Orr 2004). The problem is particularly acute in

reef fishes, whose conspicuous colors are often the sole

character available to distinguish taxa. Coloration in fishes

serves various functions, including social communication,

camouflage, and mimicry, but it can also indicate repro-

ductive isolation if it serves as a cue in mate recognition

(Randall 1998; McMillan et al. 1999). If sexual selection is

strong (Puebla et al. 2007) or ecological differentiation is

rapid (Ramon et al. 2003; Choat 2006; Rocha and Bowen

2008), color polymorphisms in incipient reef fish species

Communicated by Biology Editor Prof. Philip Munday

J. D. DiBattista (&) � E. Waldrop � B. W. Bowen � J. K. Schultz

Hawai’i Institute of Marine Biology, P.O. Box 1346, Kane’ohe,

HI 96744, USA

e-mail: joseph99@hawaii.edu

M. R. Gaither � L. A. Rocha

Section of Ichthyology, California Academy of Sciences,

55 Music Concourse Dr, San Francisco, CA 94118, USA

R. L. Pyle

Bernice P. Bishop Museum, 1525 Bernice St., Honolulu,

HI 96817, USA

123

Coral Reefs (2012) 31:839–851

DOI 10.1007/s00338-012-0907-y

may accumulate faster than genetic differences at neutral

markers, including mitochondrial DNA (mtDNA) genes

(Bowen et al. 2006). Therefore, as a taxonomic tool, color is

much more reliable when coupled with biogeographic,

genetic, or additional morphological data (Rocha 2004).

To explore the relationship between coloration, taxonomy,

and phylogeographic patterns, we surveyed the Lemonpeel

Angelfish (Centropyge flavissima [Cuvier 1831]) throughout

its Indo-Pacific range using mtDNA cytochrome b (cyt b) and

three nuclear introns (TMO, RAG2, and S7). C. flavissima

inhabits shallow lagoons or seaward reefs (Allen et al. 1998)

and has a long pelagic larval duration (PLD) relative to

other genera of angelfish (PLD = 28–32 days; Thresher and

Brothers 1985). C. flavissima is found at Christmas (S10�300,E105�400) and Cocos-Keeling (S12�100, E96�520) Islands in

the Indian Ocean and in the Western and Central Pacific

Ocean (east to the Marquesas Islands and north to the Line

Islands but not Hawaii; see Fig. 1), but despite its high dis-

persal potential, it is not found in most of the species rich

Coral Triangle region between the extremes of its range. This

disjunct distribution is coupled with subtle differences in

coloration; the blue ring around the eye characteristic of

Pacific C. flavissima is notably absent from Indian Ocean

populations, and a distinct black bar on the posterior margin of

the operculum is only present in the Pacific fish (Fig. 2; Allen

et al. 1998). A blue-rimmed black ocellate spot on the side of

the body during the juvenile stage of this species also appears

to persist in larger juveniles of the Indian Ocean form, com-

pared with the Pacific Ocean form (see photo on p. 131 of

Allen and Steene 1987). Our first aim is therefore to determine

whether these color differences represent separate popula-

tions or deeper evolutionary lineages.

Evidence from a molecular phylogeny of angelfishes

(Bellwood et al. 2004) indicates that the genus Centropyge is

not monophyletic. However, apart from coloration, C. fla-

vissima is indistinguishable from two other species within

this genus, Eibl’s Angelfish (Centropyge eibli Klausewitz

1963) and the Pearl-Scaled Angelfish (Centropyge vrolikii

[Bleeker 1853]). Although previous research focused on the

number of lateral-line or vertical scales as diagnostic char-

acters, these often vary with size of the specimen and are

therefore unreliable (Pyle 2003). C. eibli is found exclu-

sively in the eastern Indian Ocean (including parts of Indo-

nesia) and Northwestern Australia, whereas C. vrolikii is

distributed throughout the tropical western Pacific, from the

Coral Triangle east to the Marshall Islands and south to

Vanuatu and the Great Barrier Reef (Fig. 1). C. flavissima

regularly hybridizes with these two species in regions of

overlap (Pyle and Randall 1994; Hobbs et al. 2009).

The propensity of species in this genus to hybridize in

areas of sympatry, along with their outlined distributions and

morphological similarity, led Pyle (1992) to suggest that

they may form a single monophyletic group of recently

diverged species. In addition, a molecular phylogeny of the

Centropyge genus (M.R. Gaither et al. pers. comm.) unam-

biguously supports sister relationships (monophyly) among

these three species. Therefore, our second goal is to test

whether these three Indo-Pacific angelfish species represent

old lineages that have secondary contact or whether they

form a cluster of species that diverged recently.

Materials and methods

Sample collection

A total of 271 C. flavissima were collected at nine sites

while scuba diving or snorkeling between 2005 and 2010

(Fig. 1). Specimens of C. eibli (N = 7) and C. vrolikii

(N = 14) were also collected from several locations

(Fig. 1). Tissue samples were preserved in saturated salt-

DMSO (Seutin et al. 1991). Total genomic DNA was

extracted using the ‘‘HotSHOT’’ protocol (Meeker et al.

2007) and subsequently stored at -20 �C.

Mitochondrial DNA analysis

A 594-base pair (bp) segment of the mtDNA cyt b

gene was amplified using modified primers, which were

KIR

MORNUK

PAL

TOK

FIJ

= C. flavissima= C. vrolikii

= C. eibli

XMACOC

Species distribution

INDI

PAUPHO

GBR

OKI

CAR

Fig. 1 Scaled map indicating collection sites (site abbreviations are

provided in Table 1) for Centropyge flavissima (open circles) in the

Indo-Pacific. Additional specimens from two related angelfish

species, Centropyge eibli (filled black circles) and Centropygevrolikii (filled gray circles), which have been shown to hybridize in

areas of overlap with C. flavissima, were collected opportunistically

(additional site abbreviations include the Caroline Islands [CAR], the

Great Barrier Reef, Australia [GBR], Indonesia [IND], Okinawa,

Japan [OKI], and the Republic of Palau [PAU])

840 Coral Reefs (2012) 31:839–851

123

designed for these species (CFLM_FOR: 50-TCCCTCC

AACATTTCAGCAT-30; CFLM_REV: 50-TCTGGATCTC

CAAGCAGGTT-30). Polymerase chain reaction (PCR)

mixes contained 7.6 ll of BioMix solution (BioMix Red;

Bioline Ltd., London, UK), 0.26 lM of each primer, and

5–50 ng template DNA in 15 ll total volume. PCRs also

used an initial denaturing step at 95 �C for 3 min, then 35

cycles of amplification (30 s of denaturing at 94 �C, 45 s of

annealing at 62 �C, and 45 s of extension at 72 �C), fol-

lowed by a final extension at 72 �C for 10 min.

PCR products were visualized through 1.5 % agarose gel

electrophoresis and purified by incubating with exonuclease

I and shrimp alkaline phosphatase (ExoSAP; USB, Cleve-

land, OH, USA) at 37 �C for 60 min, followed by 85 �C for

15 min. All samples were sequenced in the forward direction

(and reverse direction for questionable cyt b haplotypes

[N = 6]) with fluorescently labeled dye terminators (Big-

Dye version 3.1, Applied Biosystems Inc., Foster City, CA,

USA) and analyzed using an ABI 3130XL Genetic Analyzer

(Applied Biosystems). The sequences were aligned, edited,

and trimmed to a uniform length using Geneious Pro vers.

4.8.4 (Drummond et al. 2009); unique mtDNA cyt b haplo-

types were deposited in GenBank (accession numbers:

JQ914310–JQ914394). jModelTest vers. 1.0.1 (Posada 2008;

but also see Guindon and Gascuel 2003) was used with an

Akaike information criterion (AIC) test to determine the

best nucleotide substitution model for our dataset; the

TIM1 ? G (Posada 2003) model with a gamma parameter

of 0.12 was here selected.

To evaluate phylogenetic relationships among cyt b

haplotypes, we constructed neighbor-joining (NJ) and

maximum-likelihood (ML) trees using PAUP* vers. 4.0

(Swofford 2000). For comparison, we constructed a Bayes-

ian tree using MRBAYES (Ronquist and Huelsenbeck 2003)

implemented in Geneious Pro. Bootstrap support values for

NJ and ML trees were calculated using default settings with

10,000 replicates; only nodes with bootstrap values[50 %

were considered. The Bayesian MCMC search strategy con-

sisted of four heated, 1 million step chains with an initial

burn-in of 100,000 steps. A single Centropyge bicolor sample

(Genbank accession number: JQ914309) was used to root the

tree. We calculated divergence between mitochondrial lin-

eages (here denoted d) as the uncorrected pairwise sequence

distance between lineages minus the pairwise sequence dis-

tance within a lineage using ARLEQUIN vers. 3.1 (Excoffier

et al. 2005). The evolutionary relationship among all angelfish

haplotypes was further explored with an unrooted network

constructed with NETWORK vers. 4.5.1.0 (www.fluxus-

engineering.com/network_terms.htm) using a median-join-

ing algorithm and default settings (as per Bandelt et al. 1999).

ARLEQUIN was used to calculate haplotype (h) and

nucleotide diversity (p), as well as to test for range-wide

patterns of population structure for C. flavissima. Initially,

global UST, which incorporates sequence divergence in

Fig. 2 ‘‘Pure’’ Centropyge flavissima (a Pacific Ocean morph, Mar-

shall Islands; d Indian Ocean morph, Christmas Island, Australia),

Centropyge vrolikii (c Indonesia), and Centropyge eibli (f Indonesia),

in addition to some hybrids between these species (b C. flavissima 9 C.

vrolikii, Guam; e C. vrolikii 9 C. eibli, Indonesia). Note the charac-

teristic blue ring around the orbit of the Pacific Ocean C. flavissimaspecimen (a), which is absent from the Indian Ocean C. flavissimaspecimen (d). Photo credit: Luiz Rocha

Coral Reefs (2012) 31:839–851 841

123

addition to allele frequency differentiation, was estimated

using analysis of molecular variance (AMOVA; Excoffier

et al. 1992); deviations from null distributions were tested

with nonparametric permutation procedures (N = 99,999).

Subsequently, pairwise UST statistics were generated to

identify particular sites associated with genetic partition-

ing. We controlled for false discovery rate using the

method of Narum (2006).

Deviations from neutral sequence evolution were asses-

sed with Fu’s FS (Fu 1997) for each population using

ARLEQUIN; significance was tested with 99,999 permuta-

tions. Each site (and lineage) was also fitted with the popu-

lation parameter s in order to estimate the time since the most

recent population (or lineage) expansion (as per Rogers and

Harpending 1992). Time since expansion was estimated

using the equation s = 2lt, where t is the age of the popu-

lation (or lineage) in generations, and l is the mutation

rate per generation for the sequence (l = number of

bp 9 divergence rate within a lineage 9 generation time in

years). We used a range of cyt b mutation rates, available

from previous fish studies: 1 % per million years (MY)

within lineages (Bowen et al. 2001; Reece et al. 2010) or

1.55 % per MY within lineages (Lessios 2008). While

generation time is unknown for our study species, we con-

ditionally used the equation T = (a ? x)/2, where a is the

age at first reproduction, and x is the age at last reproduction

(or longevity; Pianka 1978). We therefore obtained a gen-

eration time of 4 years for C. flavissima based on the study

of larval stages in captivity for this species (20 months to

reproductive maturity and 7-year longevity; Frank Baensch,

Reef Culture Technologies, LLC, Oahu, HI). Although

absolute expansion times should be interpreted with caution

owing to mutation rate and generation time estimates, rela-

tive comparisons are robust to such approximations.

Nuclear gene analysis

To provide independent estimates of phylogenetic rela-

tionships and ensure genealogical concordance across

multiple loci, we sequenced a subset of the specimens at

three nuclear genes. Individuals sequenced included

specimens from all species and across the three observed

mtDNA lineages (Lineage 1, N = 50; Lineage 2, N = 24;

Lineage 3, N = 34). Approximately 254 bp of the TMO

4C4 gene was amplified using the modified primers TMO

F1 (50-ACCTCTCATTAAGAAAMGAGTGTTTG-30) and

TMO R2 (50-TGCTTCTCAAATTCTTTMACCTS-30),122 bp of the recombination-activating gene 2 (RAG2) was

amplified using the modified primers RAG2 2F (50-SAC-

CTTGTGCTGCAAAGAGA-30) and RAG2 2R (50-GG

ATCCCCTTBTCATCCAGA-30), and 120 bp of the first

intron of the S7 ribosomal protein (S7) gene was amplified

using the primers S7RPEX1F (50-TGGCCTCTTCCT

TGGCCGTC-30) and S7RPEX2R (50-AACTCGTCTGGCT

TTTCGCC-30; Chow and Hazama 1998).

Sequences for each nuclear locus were aligned and edited

using Geneious Pro. In all cases, the alignment was unam-

biguous with no frameshift mutations or indels; unique geno-

types were deposited in GenBank (accession numbers:

JQ914560–JQ914585 [TMO], JQ914395–JQ914456 [RAG2],

JQ914457–JQ914559 [S7]). Allelic states of nuclear sequen-

ces trimmed to a uniform length, with more than one hetero-

zygous site, were estimated using the Bayesian program

PHASE vers. 2.1 (Stephens and Donnelly 2003) as imple-

mented in the software DnaSP vers. 5.0 (Librado and Rozas

2009). We conducted 3 runs in PHASE for each dataset with

a burn-in of 10,000, and 100,000 or 200,000 iterations. All

runs returned consistent allele identities, and PHASE was

able to differentiate most alleles with [85 % probability

except at single nucleotide positions in 3 individuals at

the TMO locus, 19 individuals at the RAG2 locus, and 28

individuals at the S7 locus (or 3, 17, and 26 % of the samples,

respectively), which were excluded from further analysis.

Unrooted median-joining networks were produced for

each nuclear dataset as outlined above. Although the S7

network was further simplified by removing all singleton

alleles (N = 4) to minimize circularity between closely

related alleles, this did not influence our overall interpre-

tation. ARLEQUIN was used to test for genetic structure

between lineages, color morphs, and previously recognized

species. Because jModelTest did not converge on a model

of sequence evolution for any nuclear locus, we calculated

global and pairwise FST values based on conventional

allele frequencies only.

Results

Mitochondrial DNA analysis

Phylogenetic analysis revealed that samples partitioned into

three reciprocally monophyletic lineages, based on cyt

b sequence data: (1) C. flavissima and C. vrolikii sampled

at most Pacific Ocean sites, which shared six haplotypes

between the species, (2) C. flavissima sampled at Moorea, and

(3) C. flavissima and C. eibli sampled at Indian Ocean sites,

which shared the most common haplotype (Fig. 3). Lineage 1

was 7.2 and 7.0 % divergent from Lineage 2 and Lineage 3,

respectively (which represents ca. 3.6 or 3.5 MY of separa-

tion, based on 2 % per MY between lineages), whereas

Lineage 2 was 8.3 % divergent from Lineage 3 (ca. 4.2 MY of

separation). Sequence divergence within each lineage, on the

other hand, was low (Lineage 1, d = 0.17–0.26 %; Lineage 2,

d = 0.20–0.31 %; Lineage 3, d = 0.15–0.22 %). The out-

group, C. bicolor, was 12.0–14.0 % divergent from the

C. flavissima complex.

842 Coral Reefs (2012) 31:839–851

123

Median-joining haplotype networks were consistent

with a scenario of minimal genetic differentiation within

each lineage and no shared haplotypes between geographic

groupings (Fig. 4). The only exception was Nuku Hiva in

the Marquesas Archipelago (within Lineage 1), which did

not share a haplotype with any other Pacific location and

was distinguished by two diagnostic mutations (at bp 300

and bp 567).

C. flavissima cyt b sequence data revealed 43, 19, and 17

haplotypes for Lineages 1, 2, and 3, respectively, with

h = 0.22 to 0.94 and p = 0.0004 to 0.0043 for individual

sample locations (Table 1). Nucleotide diversity was

almost twice as high in Lineage 2 (Moorea) versus Lineage

3 (Indian Ocean), which cannot be explained by a greater

sampling effort in the former (N = 45 vs. N = 66).

Tests of neutrality revealed negative and significant Fu’s

FS values at all sample sites (Fu’s FS = -18.28 to -2.58;

Table 1). Our estimates of s yielded time of expansion

approximations for each lineage, with Lineage 3

(63,000–97,600 years) expanding more recently than

either Lineage 1 (100,400–155,600 years) or Lineage 2

(131,700–204,200 years), although there was variability

among individual sites within lineages (Table 1).

AMOVA confirmed the geographic grouping based

on mtDNA lineages (overall UST = 0.98, P \ 0.001;

Table 2), with 97 % of the variation in haplotype diversity

explained by these three groups. Population pairwise tests

also revealed that mtDNA haplotypes were significantly

different in 25 of 36 comparisons (all P \ 0.001; Table 3),

with estimates of UST ranging from -0.01 to 0.99. All UST

values were significant between geographic regions, but

Nuku Hiva (Marquesas Islands) was the only site that was

significantly different from other sites within regions

(UST = 0.70 to 0.81, all P \ 0.001; Table 3).

Nuclear gene analysis

Based on nuclear sequences from 108 angelfish specimens,

15 variable sites yielded 18 alleles at the TMO locus, 8

variable sites yielded 13 alleles at the RAG2 locus, and 7

variable sites yielded 12 alleles at the S7 locus. Median-

joining networks for all three nuclear genes revealed

common alleles at each locus that were shared among

every sampling site or putative species (Fig. 5). We did,

however, detect significant shifts in allele frequencies

among samples from Indian Ocean sites, Moorea, and

all other Pacific Ocean sites (TMO, FST = 0.062,

P \ 0.001; RAG2, FST = 0.024, P = 0.028; S7, FST =

0.062, P = 0.0013). We found few allele frequency dif-

ferences, however, among recognized species (TMO,

FST = 0.26, P \ 0.001; RAG2, FST = 0.040, P = 0.084;

S7, FST = 0.025, P = 0.12), and none between C. fla-

vissima color morphs (TMO, FST = 0.029, P = 0.33;

RAG2, FST = 0.028, P = 0.29; S7, FST = 0.041,

P = 0.22), indicating that these markers may be in the

process of segregating by location, albeit much more

slowly than the mtDNA.

Discussion

Our genetic survey of C. flavissima color morphs and two

closely related species (C. vrolikii and C. eibli) indicates

that previous taxonomic divisions are not compatible with

a molecular phylogenetic hypothesis. The mtDNA analyses

identified three monophyletic lineages that grouped by

sampling location rather than coloration or species desig-

nation. C. flavissima and C. vrolikii sampled at Pacific

Ocean sites grouped together in Lineage 1, C. flavissima

sampled at Moorea grouped in Lineage 2, and C. flavissima

and C. eibli sampled at Indian Ocean sites grouped in

Lineage 3. Our mtDNA results confirm that the two

C. flavissima color morphs, separated by approximately

3,000 km in adjacent ocean basins, are following inde-

pendent evolutionary trajectories with an approximate

divergence time of 3.5 MY. Genetic differentiation at the

three nuclear genes indicates concordant separation

between these three regional groups, although at the level

of allele frequency differences only. This is consistent with

recent work in other reef fish systems (e.g., Cleaner

Wrasse, Labroides dimidiatus, Drew et al. 2008; Brown-

cheek Blenny, Acanthemblemaria crockery, Lin et al.

2009), where location but not phenotype dictates the

genetic affinity of color morphs.

Discordance between genetic divergence and coloration

is well documented in butterflyfishes (family Chaetodonti-

dae: McMillan and Palumbi 1995), the sister family to

marine angelfishes, and in hamlets (Ramon et al. 2003;

Garcia-Machado et al. 2004); both groups having bright

coloration. Counter examples exist as well, wherein similar

coloration masks evolutionary divergence in damselfishes

(Pomacentridae: Planes and Doherty 1997a, b; Bernardi

et al. 2002; McCafferty et al. 2002; Rocha 2004; Drew et al.

2010; Leray et al. 2010), groupers (Serranidae: McCartney

et al. 2003; Craig et al. 2009), and wrasses (Labridae: Rocha

et al. 2005). In most cases, however, color morphs do cor-

respond to genetic partitions (Randall and Rocha 2009).

Centropyge angelfishes stand out as an evolutionary

enigma, even against this backdrop of discordant taxon-

omy, coloration, and genetics in reef fishes. All phy-

logeographic studies published on Centropyge to date

(including this one) show discordance between color

morphs (or coloration-based taxonomy) and genetic parti-

tions. In the Atlantic, color differences separate three

described species inhabiting the Caribbean, Brazil, and

mid-Atlantic ridge, but these species share mtDNA

Coral Reefs (2012) 31:839–851 843

123

haplotypes, and the Brazilian and Caribbean species are not

distinguishable even at the population level (Bowen et al.

2006). The Flame Angelfish (Centropyge loricula) in the

central Pacific maintains distinct color morphs in different

parts of its range yet shows high gene flow between these

regions (Schultz et al. 2007). These observations may be

0.4 substitutions/site

Centropyge bicolor

100/100/1.00

73/67/0.91

100/100/1.00

100/100/1.00

cflm1105 (1)cflm1123 (5)

cflm1 (1)cflm1104 (17)

cflm1110 (1)cflm1111 (3)

cflm1112 (1)cflm1113 (1)

cflm1116 (2)cflm1117 (1)cflm1121 (1)

cflm1124 (2)cflm1131 (2)

cflm1133 (1)cflm1135 (1)

cflm1138 (1)cflm13 (2)cflm26 (1)cflm31 (1)

cflm1225 (10)

cflm1226 (1)

cflm1237 (1)cflm1239 (19)

cflm1248 (1)cflm1179 (1)

cflm1230 (3)cflm12 (1)

cflm1224 (19) + cei1 (IND/XMA; 2)

cflm1234 (2)cflm1227 (1)

cflm1236 (1)cflm1240 (1)cflm1188 (1)cflm1194 (2)

cflm1200 (1)cei1001 (XMA; 1)

cei1003 (XMA; 1)

cei3 (IND; 1)

cflm9 (1)cei1002 (XMA; 1)

cei2 (IND; 1)

Lineage 1(Pacific Ocean & C. vrolikii)

Lineage 2(Moorea, French Polynesia)

Lineage 3(Indian Ocean and C. eibli)

cflm1139 (31)

cvr1005 (CAR; 1) + cflm1207 (5)

cflm1151 (1)cflm1154 (1)cflm1157 (1)cflm1171 (1)

cflm15 (1)cflm1093 (1)cflm1251 (1)

cflm1203 (4)cflm1216 (1)

cflm1040 (2)cflm1221 (1)

cflm1050 (2)cflm1257 (1)

cvr4 (CAR/GBR/OKI; 8) + cflm1206 (54)cvr3 (GBR; 1) + cflm1047 (1)

cvr1002 (CAR; 1) + cflm1103 (4)

cvr1006 (CAR; 1) + cflm1058 (1)cvr7 (OKI; 1)

cvr1001 (PAU; 1) + cflm1205 (6)

cflm1211 (6)

cflm1212 (1)

cflm1214 (3)

cflm1215 (1)cflm1217 (3)

cflm17 (2)cflm1095 (1)cflm1097 (1)cflm1100 (4)cflm1101 (2)cflm1102 (1)

cflm1045 (1)

cflm1053 (3)cflm1063 (1)

cflm1065 (1)

cflm1066 (1)cflm1220 (1)cflm21 (2)

cflm1090 (1)cflm1076 (1)

cflm1254 (1)cflm1258 (1)

cflm1272 (1)

844 Coral Reefs (2012) 31:839–851

123

the key to understanding the triad of putative species

examined here, as apparently members of this genus tend

to preserve color differences despite gene flow.

What conditions can explain the maintenance of color

differences in the face of gene flow? The most likely

explanation is that natural or sexual selection is acting to

conserve these color differences. For many reef fishes,

coloration is a key character involved in mate recognition

and therefore subject to strong sexual selection (Seehausen

et al. 1997, 1999). As one example, strong preference for

mating with their own morphotype (assortative mating)

appears to have played a role in maintaining evolutionarily

stable color polymorphisms in Caribbean hamlets (Hypo-

plectrus complex; Puebla et al. 2008; Holt et al. 2011).

Selective pressures that are not directly related to mate

choice (i.e., predation, habitat preference, or territoriality)

can also reinforce differences in color patterns among

butterflyfishes in the face of ongoing gene flow (McMillan

et al. 1999). Indeed, predation against novel color types is

an important factor in reducing hybrid success in other

vertebrate systems (Langham 2007) and may factor into the

maintenance of regional color morphs here. Although our

three study species inhabit similar depths and habitats

(Hobbs et al. 2010), C. flavissima is found almost

X-mas Island, Indian Ocean

Cocos-Keeling

Moorea

Phoenix Islands

Palmyra

X-mas Island, Pacific Ocean

Nuku Hiva

Fiji

Tokelau Islands

Centropyge eibli (cei)

Centropyge vrolikii (cvr)

c

Cyt b - Lineage 1(Pacific Ocean & C. vrolikii)

a

Cyt b - Lineage 2(Moorea, French Polynesia)

b

Cyt b - Lineage 3(Indian Ocean & C. eibli )

Fig. 4 Median-joining

networks showing relationships

among mitochondrial DNA,

non-singleton cytochrome

b haplotypes for Centropygeflavissima specimens (N = 271)

collected in the Indo-Pacific.

Additional sequences from two

closely related angelfishes,

Centropyge eibli (cei: Christmas

Island, Australia [XMA],

N = 4; Indonesia [IND],

N = 3) and Centropyge vrolikii(cvr: Caroline Islands [CAR],

N = 6; Great Barrier Reef,

Australia [GBR], N = 4;

Okinawa, Japan [OKI], N = 3;

Republic of Palau [PAU],

N = 1), were also included.

Each circle represents a

haplotype and its size is

proportional to its total

frequency. Branches or blackcrossbars represent a single

nucleotide change, open circlesrepresent unsampled

haplotypes, and colors denote

collection location as indicated

by the embedded key. The

network was separated into the

three distinct lineages

(a Lineage 1; b Lineage 2;

c Lineage 3)

Fig. 3 Phylogenetic relationship among mitochondrial DNA cyto-

chrome b haplotypes (594 base pairs) for Centropyge flavissimaspecimens (cflm; N = 271) collected in the Indo-Pacific based on

neighbor-joining (NJ), maximum-likelihood (ML), and Bayesian

(BA) inference. Additional sequences from two closely related

angelfish, Centropyge eibli (cei: Christmas Island, Australia

[XMA], N = 4; Indonesia [IND], N = 3) and Centropyge vrolikii(cvr: Caroline Islands [CAR], N = 6; Great Barrier Reef, Australia

[GBR], N = 4; Okinawa, Japan [OKI], N = 3; Republic of Palau

[PAU], N = 1) were also included. Branch support values ([50 %,

based on 10,000 replicates) for NJ and ML analysis, and posterior

probabilities for BA analysis are shown above the nodes (NJ/ML/

BA). All analyses resulted in identical lineages, and so the BA

topology is presented here. Branch lengths are according to indicated

scale but the branch leading to the outgroup species, Centropygebicolor, was here reduced by 50 %. Values in parentheses represent

the number of samples for each haplotype, and colors denote

collection location as indicated by the embedded key. Each haplotype

in the tree was therefore assigned a corresponding rectangle to the

right of the figure, and each color denoting a location within that

rectangle was proportional to its total frequency for that haplotype

b

Coral Reefs (2012) 31:839–851 845

123

exclusively on reefs around low oceanic islands, whereas

C. vrolikii and C. eibli predominate at high islands or

continental shelves, indicating that there may be some

niche partitioning in areas of overlap.

Our findings include deep (reciprocally monophyletic)

differentiation in mtDNA, coupled with weak population-

level differentiation at nuclear loci, which can certainly be

explained by the non-recombining nature of mtDNA

inheritance and the slower mutation rate at introns relative

to mtDNA. Two additional (not mutually exclusive) factors

require consideration here: (1) incomplete lineage sorting

and (2) hybridization and introgression. These two expla-

nations are difficult to tease apart but we discuss each

possibility. It should be noted that natural selection for

particular mtDNA types can also produce such a pattern,

although such selective sweeps are thought to be rare (Karl

et al. 2012).

Incomplete lineage sorting occurs during the transitional

stage when evolutionary lineages begin to diverge (Avise

2004). The haploid inheritance of mtDNA yields a fourfold

lower effective population size (Ne) relative to nuclear DNA,

such that isolated mtDNA lineages are expected to drift to

reciprocal monophyly in Ne generations on average, whereas

diploid nuclear loci will attain this evolutionary divergence

in 4Ne generations on average (Tavare 1984). A divergence

time of 4 MY (based on our mtDNA cyt b molecular clock)

seems sufficient for these lineages to completely sort at both

mtDNA and nuclear genes, although no comparable

Table 1 Molecular diversity indices for Centropyge flavissima based on mitochondrial DNA (cytochrome b) sequence data for all sampling

locations, including the three identified Indo-Pacific lineages (also see Fig. 3)

Collection locality N HN HU Time since

expansion (Years)

Haplotype diversity

(h ± SD)

Nucleotide diversity

(p ± SD)

Fu’s FS

Pacific Ocean

Christmas Island, Line Islands (KIR) 43 19 8 69,700–108,000 0.78 ± 0.066 0.0026 ± 0.0018 218.28a

Fiji (FIJ) 19 12 4 106,500–165,100 0.94 ± 0.035 0.0043 ± 0.0027 26.97

Nuku Hiva, Marquesas Islands (NUK) 35 5 5 126,300–195,700 0.22 ± 0.092 0.0004 ± 0.0005 24.74

Palmyra Atoll, Line Islands (PAL) 30 11 5 73,700–114,200 0.67 ± 0.093 0.0023 ± 0.0016 26.96

Phoenix Islands (PHO) 24 15 6 89,400–138,600 0.87 ± 0.067 0.0035 ± 0.0023 212.26

Tokelau Islands (TOK) 9 6 1 82,900–128,400 0.83 ± 0.130 0.0030 ± 0.0022 22.58

All samples—Lineage 1 160 43 43 100,400–155,600 0.85 ± 0.023 0.0035 ± 0.0022 227.30

Moorea, French Polynesia (MOR)

All samples—Lineage 2 45 19 19 131,700–204,200 0.85 ± 0.049 0.0043 ± 0.0026 211.93

Indian Ocean

Christmas Island, Aus. (XMA) 27 11 6 70,100–108,600 0.85 ± 0.042 0.0027 ± 0.0018 26.32

Cocos-Keeling Islands, Aus. (COC) 39 11 6 56,600–87,700 0.78 ± 0.047 0.0027 ± 0.0018 -5.01

All samples—Lineage 3 66 17 17 63,000–97,600 0.82 ± 0.029 0.0026 ± 0.0017 211.13

Time since the most recent population expansion was calculated using a range of mutation rates (1–1.55 % per million years within lineages;

Bowen et al. 2001; Lessios 2008; Reece et al. 2010) and a generation time of four years (see ‘‘Methods’’)

N sample size, HN number of haplotypes, HU number of unique haplotypesa Numbers in bold are significant, P \ 0.02 (see Fu 1997)

Table 2 Results of the analysis of molecular variance (AMOVA) based on mitochondrial DNA cytochrome b sequence data for Centropygeflavissima (N = 271)

Source df SS Variance

components

% Variation UCT

USC

P-value UST P-value

Among groups 2 7147.24 46.51 97.43 0.97 0.004 0.98 \0.001

0.32 \0.001

Among populations (within groups) 6 68.79 0.40 0.83

Within populations 262 217.36 0.83 1.74

Groups were based on the three Indo-Pacific lineages identified by phylogenetic reconstruction (see Fig. 3). UCT is the group variance component

relative to total variance, USC is the between sample within group variance component divided by the sum of itself and within sample variance,

and UST is the sum of the variance due to group and sample within group divided by the total variance

df degrees of freedom, SS sum of squares

846 Coral Reefs (2012) 31:839–851

123

molecular clock is available for the latter. That said, the

introns in this study show weak population structure between

regions and extensive sharing of alleles, indicating that

nuclear lineage sorting is not proceeding toward fixation of

alternate states. While incomplete lineage sorting almost

certainly contributes to our findings, it is not sufficient

to explain the discordance between mtDNA and nuclear

introns.

The alternate explanation, hybridization and intro-

gression, can explain the observed discordance between

mtDNA and nuclear DNA. Because pygmy angelfish are

primarily distinguished on the basis of coloration and are

also highly prized in the aquarium trade, hybrids between

these species tend to be both noticed and documented.

Hence, these hybrids are among the best characterized for

any tropical reef fish family (Pyle and Randall 1994; but

also see Yaakub et al. 2006; see Fig. 2). C. flavissima 9

C. vrolikii hybrids (documented genetically by L.A. Rocha)

are regularly exported through the aquarium trade from the

Marshall Islands, Pohnpei, Guam, Kosrae, the Ryukyu

Islands, and Vanuatu (Takeshita 1976) and have been

reported from almost all locations where these two species

co-occur. At these overlapping sites, individual fish range

from nearly ‘‘pure’’ C. flavissima to nearly ‘‘pure’’

C. vrolikii. No hybrids have been reported from New

Caledonia, the Solomon Islands, and Palau (where only

C. vrolikii is present), or the Society Islands, Line Islands,

Phoenix Islands, and Marquesas Islands (where only

C. flavissima is present). Although hybrids have not been

reported (to our knowledge) from areas of sympatry in the

Ogasawara Islands and the Great Barrier Reef, this could

be attributed to insufficient sampling effort or very low

abundance of one or the other species (Pyle and Randall

1994; D. Bellwood pers. comm.).

The two species that hybridize with C. flavissima,

C. eibli, and C. vrolikii, also hybridize with each other in

Indonesia (Pyle and Randall 1994), and C. flavissima 9

C. eibli hybrids are common at Christmas and Cocos-

Keeling Islands (400 and 1,000 km southwest of Indonesia;

Hobbs et al. 2009), which is at the boundary of the Western

Indo-Pacific and the Central Indo-Pacific ecoregions

(Spalding et al. 2007). The sharing of mtDNA haplotypes

between recognized species (this study), the spectrum of

intermediate color forms (Pyle and Randall 1994), and the

presence of areas where hybrids outnumber parental forms

(Pyle and Randall 1994) are evidence for hybridization that

extends beyond the F1 generation. Moreover, rarity of

conspecific partners and a polygynous mating system (i.e.,

harems with a single male and two to seven or more

females; Moyer and Nakazono 1978; Moyer 1990), likely

promote hybridization in these fish. We therefore conclude

that hybridization has played a major role in shaping

the genetic architecture of the three angelfish lineagesTa

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Coral Reefs (2012) 31:839–851 847

123

observed in this study, although further research on the

fertility and viability of hybrids is needed to confirm this

conclusion.

We here suggest a scenario where the species complex

was divided three to four MY ago into ancestral C. eibli in

the Indian Ocean, C. vrolikii in the western Pacific, and

C. flavissima in the central South Pacific. C. flavissima

subsequently extended its range to the western Pacific,

where it hybridized with C. vrolikii, and the eastern Indian

Ocean where it hybridized with C. eibli. The end result is

three lineages: C. eibli found in the Indian Ocean (whose

haplotypes are mixed with Indian Ocean C. flavissima due

to introgression), C. vrolikii found everywhere in the

western Pacific (whose haplotypes are mixed with western

Pacific C. flavissima, also due to introgression), and

C. flavissima found in the Society Islands, which may be

the last refugium of pure individuals in this lineage. The

fact that C. flavissima currently inhabits the Phoenix

Islands without C. vrolikii, yet still has C. vrolikii mtDNA,

supports a scenario of introgression. Even though precise

reasons for this ancient isolation could not be identified,

similarly deep mtDNA divergences have been observed in

other reef fish: two lineages coalescing between 2.9 and 5.5

MY ago were identified for Naso vlamingii (Klanten et al.

2007), and three lineages coalescing between 2.0 and 5.0

MY ago were observed in Naso brevirostris (Horne et al.

2008). In both cases, the authors invoke ancient periods of

isolation likely caused by sea level fluctuations to explain

the observed genetic divergences. Indeed, the intensifica-

tion of glaciation 2.7 MY ago is one of several factors that

altered and accelerated surface currents in the Indo-Pacific

(Ivanova 2009). We suggest that these processes started the

ancient differentiation in our study group, with one key

distinction: the Naso lineages show no color or morpho-

logical difference and are completely mixed in all locali-

ties, whereas in our case, the lineages are still segregated

geographically, but they do not match the color (and spe-

cies) boundaries.

A second and perhaps equally plausible (although hard

to test) scenario is the possibility that the yellow coloration

of C. flavissima originated independently in the Indian and

Pacific oceans (Pyle 2003). In this scenario, the ancestral

a

X-mas Island, Indian Ocean

Cocos-Keeling

Moorea

Phoenix Islands

Palmyra

X-mas Island, Pacific Ocean

Nuku Hiva

Fiji

Tokelau Islands

Centropyge eibli (cei)

Centropyge vrolikii (cvr)

TMO

c S7

b RAG2

Fig. 5 Median-joining

networks showing relationships

among nuclear DNA alleles

(a TMO, 254 base pairs,

N = 145; b RAG2, 122 base

pairs, N = 160; c S7, 120 base

pairs, N = 138) based on a

subset of all angelfish samples

collected in this study. Each

circle represents an allele and its

size is proportional to its total

frequency. Branches or blackcrossbars represent a single

nucleotide change, open circlesrepresent unsampled alleles, and

colors denote collection

location as indicated by the

embedded key. All singleton

alleles (N = 4) were removed

from the S7 analysis in order to

minimize circularity between

closely related alleles

848 Coral Reefs (2012) 31:839–851

123

C. eibli in the Indian Ocean and the ancestral C. vrolikii in

the Pacific Ocean each gave rise to a xanthic form. Indeed,

the aquarium trade has documented a long list of xanthic

fish variants that are cultivated for their beauty and novelty,

including a wild-caught xanthic strain of the Dusky

Angelfish, Centropyge multispinis (http://www.reefs.org/

forums/topic120708.html). Slight color differences between

the Indian and Pacific Ocean ‘‘C. flavissima’’ lend further

support to this hypothesis, however, the presence of three

genetic lineages instead of two (one C. eibli ? Indian Ocean

xanthics and one C. vrolikii ? Pacific Ocean xanthics), and

the lack of xanthic C. vrolikii and C. eibli inside their

respective ranges, weaken it.

All but one of our sampling sites is located in the vast

Indo-Polynesian biogeographic province, which stretches

from Polynesia to the central Indian Ocean (Briggs and

Bowen 2012). Our sample from Nuku Hiva in the Marquesas

Archipelago (east of the Society Islands; Fig. 1) represents a

distinct biogeographic province with a depauperate fish

fauna and high rates of endemism (11.6 % in fishes; Randall

1998). Recent phylogeographic surveys have found genetic

differentiation between Society Island and Marquesan

populations in several groups of fishes, including snappers

(genus Lutjanus; Gaither et al. 2010), surgeonfishes (genus

Acanthurus; Planes and Fauvelot 2002), and wrasses (genus

Halichoeres; W.B. Ludt et al. pers. comm.). The distinc-

tiveness of Marquesan reef fishes is attributed to a combi-

nation of geographic isolation owing to the westerly South

Equatorial Current, limited coral reef development, and

variable water temperatures due to major upwelling events

(Randall 1998; Gaither et al. 2010).

Our results reinforce the genetic uniqueness of fishes in

the Marquesas Islands, with C. flavissima sampled at

Nuku Hiva not sharing haplotypes with any other location

(Table 1, Fig. 4). In phylogenetic analyses, however, the

Marquesas individuals grouped with the widespread Pacific

lineage and not the nearby Society Islands lineage (Fig. 3).

A relatively recent colonization event is consistent with the

low genetic variation detected at Nuka Hiva despite good

sampling effort (N = 35); haplotype or nucleotide diversity

was at least three or six times lower here than at any other

location. Further sampling is needed at other reef systems

in the Society Islands, Tuamotu Islands, and surrounding

islands chains (such as Samoa or Tonga) to test our

hypotheses of a recent colonization event of the Marquesas

Islands by introgressed western Pacific C. flavissima, in

addition to the rest of French Polynesia acting as a refu-

gium for the ‘‘pure’’ C. flavissima. Indeed, we identified

a single C. flavissima specimen from Tonga (2,000 km

west of the Society Islands; GenBank Accession Number:

FJ582964.1) that groups with our Moorea samples based on

the mtDNA barcoding gene (COI; data not shown), indi-

cating that there are other places in the Central Pacific

where C. flavissima and C. vrolikii have not yet fully

introgressed.

Taxonomic implications

Since our phylogeny seems to contradict recognized spe-

cies boundaries, are these angelfishes valid species? While

the recognized angelfish species in the complex studied

here might not represent reciprocally monophyletic

mtDNA or nuclear intron lineages, they represent stable

color forms and the presence of deeply separated mtDNA

lineages indicate that those forms have existed for millions

of years.

The solution proposed by de Queiroz (2007) for

dilemmas like the one presented here is simple; use the

common element to define the species, and one or more

secondary properties as qualifiers to support this designa-

tion. In our case, despite the apparent gene flow, these

species maintain unique color characteristics and are par-

titioned into cohesive geographic regions. We therefore

suggest that these angelfish remain recognized as taxo-

nomically diagnosable species.

In conclusion, the emerging picture of evolution in the

pygmy angelfishes (genus Centropyge) includes a number

of factors known from other organisms, but which combine

here into a unique synthesis of dispersal, hybridization,

natural (or sexual) selection, and speciation. First, this

group contains members that are good dispersers, with low

or no population structure recorded across entire ocean

basins (Bowen et al. 2006; Schultz et al. 2007; present

study). This is almost certainly due to a pelagic larval stage

that readily traverses oceans; Centropyge larvae have been

detected in mid-oceanic trawls (MCZ 73476, 73518,

73521, 73531–73532, 73546, 735554–735556, 81683,

82468, 158311, 163525; http://www.mcz.harvard.edu/

Departments/Fish/), notably to the exclusion of many

other common and abundant reef fishes (D. Smith and

K. Hartel, pers. comm.). Second, this group is known for

extensive natural hybridization (Pyle and Randall 1994), a

phenomenon that is rare in other groups of reef organisms

(Hubbs 1955; Gardner 1997; but see Hobbs et al. 2009).

Third, all seven species examined with molecular data

retain regional color morphs in the face of gene flow, a

signature of natural or sexual selection. At least some

species are also sexually dimorphic for color patterns

(Allen et al. 1998), adding greater weight to the interpre-

tation that coloration influences mate choice. Fourth, the

three species (C. flavissima, C. eibli, and C. vrolikii) would

probably be regarded as a single species if coloration

was omitted from taxonomic consideration (Pyle 2003).

In these circumstances, it is tempting (and defensible) to

conclude that these are emerging species, but this is con-

tradicted by the ancient history inscribed in mtDNA, which

Coral Reefs (2012) 31:839–851 849

123

is also concordant with biogeographic partitions observed

in other reef fish species (Rocha et al. 2007). The evidence

therefore indicates that at one time three species existed but

that extensive dispersal and hybridization has rearranged

formerly isolated species into semi-isolated color morphs.

Like the cichlids of Africa’s rift lakes, the novel aspects of

pygmy angelfish evolution will continue to provide insights

about the ragged edge of speciation in the oceans.

Acknowledgments This research was supported by the National

Science Foundation grants OCE-0453167 and OCE-0929031 to

BWB, NOAA National Marine Sanctuaries Program MOA No.

2005-008/66882 to R.J. Toonen, and by a Natural Sciences and

Engineering Research Council of Canada (NSERC) postgraduate

fellowship to JDD. For specimen collections, we thank Kim Ander-

sen, Paul Barber, Larry Basch, David Bellwood, J. Howard Choat,

Matthew Craig, Joshua Drew, John Earle, Jeff Eble, Brian Greene,

Matthew Iacchei, Stephen Karl, Randall Kosaki, David Pence, and

Ross Robertson. We thank Sue Taei at Conservation International,

Graham Wragg of the RV Bounty Bay, the Government of Kiribati,

including Tukabu Teroroko and the Phoenix Island Protected Area

who assisted with Kiribati collections. We also thank Robert Toonen,

Serge Planes, Stephen Karl, John Randall, Joann Leong, Patrick

Colin, Laura Colin, the Coral Reef Research Foundation, and mem-

bers of the ToBo lab for their logistic support; we thank the Center

for Genomics, Proteomics, and Bioinformatics at the University of

Hawaii for their assistance with DNA sequencing. This is contribution

no. 1492 from the Hawai’i Institute of Marine Biology and no. 8605

from the School of Ocean and Earth Science and Technology.

References

Allen GR, Steene R (1987) Reef fishes of the Indian Ocean. T.F.H.

Publications Inc., Neptune City

Allen GR, Steene R, Allen M (1998) A guide to angelfishes and

butterflyfishes. Odyssey Publishing/Tropical Reef Research,

Perth, Australia

Avise JC (2004) Molecular markers, natural history, and evolution,

2nd edn. Sinauer Associates, Sunderland, MA

Bandelt HJ, Forster P, Rohl A (1999) Median-joining networks for

inferring intraspecific phylogenies. Mol Biol Evol 16:37–48

Bellwood DR, Van Hewerden L, Konow N (2004) Evolution and

biogeography of marine angelfishes (Pisces: Pomacanthidae).

Mol Phylogenet Evol 33:150–155

Bernardi G, Holbrook SJ, Schmitt RJ, Crane NL, DeMartini E (2002)

Species boundaries, populations and colour morphs in the coral

reef three-spot damselfish (Dascyllus trimaculatus) species

complex. Proc R Soc Lond B Biol Sci 269:599–605

Bowen BW, Bass AL, Rocha LA, Grant WS, Robertson DR (2001)

Phylogeography of the trumpetfishes (Aulostomus): Ring species

complex on a global scale. Evolution 55:1029–1039

Bowen BW, Muss A, Rocha LA, Grant WS (2006) Shallow mtDNA

coalescence in Atlantic pygmy angelfishes (genus Centropyge)

indicates a recent invasion from the Indian Ocean. J Hered

97:1–12

Briggs JC, Bowen BW (2012) A realignment of marine biogeographic

provinces with particular reference to fish distributions. J Bioge-

ogr 39:12–30

Choat JH (2006) Phylogeography and reef fishes: bringing ecology

back into the argument. J Biogeogr 33:967–968

Chow S, Hazama K (1998) Universal PCR primers for S7 ribosomal

protein gene introns in fish. Mol Ecol 7:1255–1256

Coyne JA, Orr HA (2004) Speciation. Sinauer Associates, Sunder-

land, MA

Craig MT, Graham RT, Torres RA, Hyde JR, Freitas MO, Ferreira

BP, Hostim M, Gerhardinger LC, Andrade AB, Robertson DR

(2009) How many species of goliath grouper are there?

Cryptic genetic divergence in a threatened marine fish and the

resurrection of a geopolitical species. Endanger Species Res

7:167–174

de Queiroz K (2007) Species concepts and species delimitation. Syst

Biol 56:879–886

Drew JA, Allen GR, Kaufman L, Barber PH (2008) Endemism and

regional color and genetic differences in five putatively cosmo-

politan reef fishes. Conserv Biol 22:965–975

Drew JA, Allen GR, Erdmann MV (2010) Congruence between

mitochondrial genes and color morphs in a coral reef fish:

population variability in the Indo-Pacific damselfish Chrysipterarex (Snyder, 1909). Coral Reefs 29:439–444

Drummond AJ, Ashton B, Cheung M, Heled J, Kearse M, Moir R,

Stones-Havas S, Thierer T, Wilson A (2009) Geneious v4.8,

Available from http://www.geneious.com

Excoffier R, Smouse P, Quattro J (1992) Analysis of molecular

variance inferred from metric distances among DNA haplotypes:

Application to human mitochondrial DNA restriction data.

Genetics 131:479–491

Excoffier R, Laval LG, Schneider S (2005) Arlequin ver. 3.0: An

integrated software package for population genetics data anal-

ysis. Evol Bioinform Online 1:47–50

Fu YX (1997) Statistical tests of neutrality of mutations against

population growth, hitchhiking and background selection.

Genetics 147:915–925

Gaither MR, Toonen RJ, Robertson DR, Planes S, Bowen BW (2010)

Genetic evaluation of marine biogeographic barriers: perspec-

tives from two widespread Indo-Pacific snappers (Lutjanus spp.).

J Biogeogr 37:133–147

Garcia-Machado E, Chevalier Monteagudo PP, Solignac M (2004)

Lack of mtDNA differentiation among hamlets (Hypoplectrus,

Serranidae). Mar Biol 144:147–152

Gardner JPA (1997) Hybridization in the sea. Adv Mar Biol 31:1–78

Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm

to estimate large phylogenies by maximum likelihood. Syst Biol

52:696–704

Hobbs JPA, Frisch AJ, Allen GR, Van Herwerden L (2009) Marine

hybrid hotspot at Indo-Pacific biogeographic border. Biol Lett

5:258–261

Hobbs JPA, Jones GP, Munday PL (2010) Rarity and extinction risk

in coral reef angelfishes on isolated islands: interrelationships

among abundance, geographic range size and specialization.

Coral Reefs 29:1–11

Holt BG, Cote IM, Emerson BC (2011) Searching for speciation

genes: Molecular evidence for selection associated with colour

morphotypes in the Caribbean reef fish genus Hypoplectrus. PloS

ONE [doi:10.1371/journal.pone.0020394.g001]

Horne JB, van Herwerden L, Choat JH, Robertson DR (2008) High

population connectivity across the Indo-Pacific: Congruent lack

of phylogeographic structure in three reef fish congeners. Mol

Phylogenet Evol 49:629–638

Hubbs CL (1955) Hybridization between fish species in nature. Syst

Zool 4:1–20

Ivanova EV (2009) The global thermohaline circulation. Springer,

New York

Karl SA, Toonen RJ, Grant WS, Bowen BW (2012) Common

misconceptions in molecular ecology: Echos of the modern

synthesis. Mol Ecol (in press)

Klanten OS, Choat JH, van Hewerden L (2007) Extreme genetic

diversity and temporal rather than spatial partitioning in a widely

distributed coral reef fish. Mar Biol 150:659–670

850 Coral Reefs (2012) 31:839–851

123

Langham GM (2007) Specialized avian predators repeatedly attack

novel color morphs of Heliconius butterflies. Evolution 58:

2783–2787

Leray M, Beldade R, Holbrook SJ, Schmitt RJ, Planes S, Bernardi G

(2010) Allopatric divergence and speciation in coral reef fish:

The Three-Spot Dascyllus, Dascyllus trimaculatus, species

complex. Evolution 64:1218–1230

Lessios HA (2008) The great American schism: Divergence of marine

organisms after the rise of the Central American isthmus. Annual

Reviews of Ecology Evolution and Systematics 39:63–91

Librado P, Rozas J (2009) DnaSP v5: A software for comprehensive

analysis of DNA polymorphism data. Bioinformatics 25:1451–1452

Lin H-C, Sanchez-Ortiz C, Hastings PA (2009) Colour variation is

incongruent with mitochondrial lineages: cryptic speciation and

subsequent diversification in a Gulf of California reef fish

(Teleostei: Blennioidei). Mol Ecol 18:2476–2488

McCafferty S, Bermingham E, Quenouille B, Planes S, Hoelzer G,

Asoh K (2002) Historical biogeography and molecular system-

atics of the Indo-Pacific genus Dascyllus (Teleostei: Pomacen-

tridae). Mol Ecol 11:1377–1392

McCartney MA, Acevedo J, Heredia C, Rico C, Quenouille B,

Bermingham E, McMillan WO (2003) Genetic mosaic in a

marine species flock. Mol Ecol 12:2963–2973

McMillan WO, Palumbi SR (1995) Concordant evolutionary patterns

among Indo-West Pacific butterflyfishes. Proc R Soc Lond B

Biol Sci 260:229–236

McMillan WO, Weigt LA, Palumbi SR (1999) Color pattern evolution,

assortative mating, and genetic differentiation in brightly colored

butterflyfishes (Chaetodontidae). Evolution 53:247–260

Meeker ND, Hutchinson SA, Ho L, Trede NS (2007) Method for

isolation of PCR-ready genomic DNA from zebrafish tissues.

Biotechniques 43:610–614

Moyer JT (1990) Social and reproductive behavior of Chaetodonto-plus mesoleucus (Pomacanthidae) at Bantayan Island, Philip-

pines, with notes on pomacanthid relationships. Japan J Ichthyol

36:459–467

Moyer JT, Nakazono A (1978) Population structure, reproductive

behavior, and protogynous hermaphroditism in the angelfish

Centropyge interruptus at Miyake-jima, Japan. Japan J Ichthyol

25:25–39

Narum SR (2006) Beyond Bonferroni: Less conservative analyses for

conservation genetics. Conserv Genet 7:783–787

Pianka ER (1978) Evolutionary ecology. Harper and Row, New York,

USA

Planes S, Doherty PJ (1997a) Genetic relationships of the colour

morphs of Acanthochromis polyacanthus (Pomacentridae) on the

northern Great Barrier Reef. Mar Biol 130:109–117

Planes S, Doherty PJ (1997b) Genetic and color interactions at a

contact zone of Acanthochromis polyacanthus: A marine fish

lacking pelagic larvae. Evolution 51:1232–1243

Planes S, Fauvelot C (2002) Isolation by distance and vicariance drive

genetic structure of a coral reef fish in the Pacific Ocean.

Evolution 56:378–399

Posada D (2003) Using Modeltest and PAUP* to select a model of

nucleotide substitution. pp. 6.5.1–6.5.14. In: Baxevanis AD,

Davison DB, Page RDM, Petsko GA, Stein LD, Stormo GD (eds)

Current protocols in bioinformatics. John Wiley & Sons, Inc

Posada D (2008) jModelTest: Phylogenetic model averaging. Mol

Biol Evol 25:253–1256

Puebla O, Bermingham E, Guichard F, Whiteman E (2007) Colour

pattern as a single trait driving speciation in Hypoplectrus coral

reef fishes? Proc R Soc Lond B Biol Sci 274:1265–1271

Puebla O, Bermingham E, Guichard F (2008) Population genetic

analyses of Hypoplectrus coral reef fishes provide evidence that

local processes are operating during the early stages of marine

adaptive radiations. Mol Ecol 17:1405–1415

Pyle RL (1992) Rare and unusual marines: A hybrid angelfish

Centropyge flavissimus x eibli. Freshw Mar Aquar 15(98–110):212

Pyle RL (2003) A systematic treatment of the reef-fish family

Pomacanthidae (Pisces: Perciformes). Ph.D. Dissertation,

University of Hawai’i, Honolulu, p 422

Pyle RL, Randall JE (1994) A review of hybridization in marine

angelfishes (perciformes: Pomacanthidae). Environ Biol Fish

41:127–145

Ramon ML, Lobel PS, Sorenson MD (2003) Lack of mitochondrial

genetic structure in hamlets (Hypoplectrus spp.): recent speci-

ation or ongoing hybridization? Mol Ecol 12:2975–2980

Randall JE (1998) Zoogeography of shore fishes of the Indo-Pacific

region. Zool Stud 37:227–268

Randall JE, Rocha LA (2009) Chaetodontoplus poliourus, a new

angelfish (Percirformes: Pomacanthidae) from the tropical

western Pacific. Raffles Bull Zool 57:511–520

Reece JS, Bowen BW, Joshi K, Goz V, Larson L (2010) Phyloge-

ography of two moray eels indicates high dispersal throughout

the Indo-Pacific. J Hered 101:391–402

Rocha LA (2004) Mitochondrial DNA and color pattern variation in

three western Atlantic Halichoeres (Labridae), with the reval-

idation of two species. Copeia 2004:770–782

Rocha LA, Bowen BW (2008) Speciation in coral reef fishes. J Fish

Biol 72:1101–1121

Rocha LA, Robertson DR, Roman J, Bowen BW (2005) Ecological

speciation in tropical reef fishes. Proc R Soc Lond B Biol Sci

272:573–579

Rocha LA, Craig MT, Bowen BW (2007) Phylogeography and the

conservation genetics of coral reef fishes. Coral Reefs 26:

501–512

Rogers AR, Harpending H (1992) Population growth makes waves in

the distribution of pairwise genetic differences. Mol Biol Evol

9:552–569

Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic

inference under mixed models. Bioinformatics 19:1572–1574

Schultz JK, Pyle RL, DeMartini E, Bowen BW (2007) Genetic

connectivity among color morphs and Pacific archipelagos for the

flame angelfish, Centropyge loriculus. Mar Biol 151:167–175

Seehausen O, Van Alphen JJM, Witte F (1997) Cichlid fish diversity

threatened by eutrophication that curbs sexual selection. Science

277:1808–1811

Seehausen O, Mayhew PJ, Van Alphen JJM (1999) Evolution of

colour patterns in East African cichlid fish. J Evol Biol 12:

514–534

Seutin G, White BN, Boag PT (1991) Preservation of avian and blood

tissue samples for DNA analyses. Can J Zool 69:82–92

Spalding MD, Fox HE, Allen GR, Davidson N, Ferdana ZA,

Finlayson M, Halpern BS, Jorge MA, Lombana A, Lourie SA,

Martin KD, McManus E, Molnar J, Recchia CA, Robertson J

(2007) Marine ecoregions of the world: A bioregionalization of

coastal and shelf areas. Bioscience 57:573–583

Stephens M, Donnelly P (2003) A comparison of Bayesian methods

for haplotype reconstruction from population genotype data. Am

J Hum Genet 73:1162–1169

Swofford DL (2000) PAUP*: Phylogenetic analysis by parsimony,

version 4.0. Sinauer Associates. Sunderland, MA

Takeshita GY (1976) An angel hybrid. Mar Aquarist 7:27–35

Tavare S (1984) Line-of-descent and genealogical processes, and

their applications in population genetics models. Theor Popul

Biol 26:119–164

Thresher RE, Brothers EB (1985) Reproductive ecology and bioge-

ography of Indo-West Pacific angelfishes (Pisces: Pomacanthi-

dae). Evolution 39:878–887

Yaakub SM, Bellwood DR, Van Herwerden L (2006) A rare

hybridization event in two common Caribbean wrasses (genus

Halichoeres; family Labridae). Coral Reefs 26:597–602

Coral Reefs (2012) 31:839–851 851

123