ral
ssBioMed CentBMC Evolutionary Biology
Open AcceResearch article
Comparative phylogeography of Atlantic reef fishes indicates both 
origin and accumulation of diversity in the Caribbean
Luiz A Rocha*1, Claudia R Rocha1, D Ross Robertson2 and Brian W Bowen1
Address: 1Hawaii Institute of Marine Biology, University of Hawaii, P.O. 1346, Kaneohe, HI 96744, USA and 2Smithsonian Tropical Research 
Institute, Balboa, Republic of Panam?
Email: Luiz A Rocha* - rochal@hawaii.edu; Claudia R Rocha - crrocha@hawaii.edu; D Ross Robertson - drr@stri.org; 
Brian W Bowen - bbowen@hawaii.edu
* Corresponding author    
Abstract
Background: Two processes may contribute to the formation of global centers of biodiversity:
elevated local speciation rates (the center of origin hypothesis), and greater accumulation of species
formed elsewhere (the center of accumulation hypothesis). The relative importance of these
processes has long intrigued marine biogeographers but rarely has been tested.
Results: To examine how origin and accumulation affected the Greater Caribbean center of
diversity, we conducted a range-wide survey of mtDNA cytochrome b in the widespread Atlantic
reef damselfish Chromis multilineata (N = 183) that included 10 locations in all four tropical Atlantic
biogeographic provinces: the Greater Caribbean, Brazil, the mid-Atlantic ridge, and the tropical
eastern Atlantic. We analyzed this data and re-evaluated published genetic data from other reef fish
taxa (wrasses and parrotfishes) to resolve the origin and dispersal of mtDNA lineages. Parsimony
networks, mismatch distributions and phylogenetic analyses identify the Caribbean population of C.
multilineata as the oldest, consistent with the center of origin model for the circum-Atlantic
radiation of this species. However, some Caribbean haplotypes in this species were derived from
Brazilian lineages, indicating that mtDNA diversity has not only originated but also accumulated in
the Greater Caribbean. Data from the wrasses and parrotfishes indicate an origin in the Greater
Caribbean in one case, Caribbean origin plus accumulation in another, and accumulation in the
remaining two.
Conclusion: Our analyses indicate that the Greater Caribbean marine biodiversity hotspot did
not arise through the action of a single mode of evolutionary change. Reef fish distributions at the
boundaries between Caribbean and Brazilian provinces (the SE Caribbean and NE Brazil,
respectively) indicate that the microevolutionary patterns we detected in C. multilineata and other
reef fishes translate into macroevolutionary processes and that origin and accumulation have acted
in concert to form the Greater Caribbean biodiversity hotspot.
Background known as the Coral Triangle) in the Indo-Pacific, and the
Published: 22 May 2008
BMC Evolutionary Biology 2008, 8:157 doi:10.1186/1471-2148-8-157
Received: 23 September 2007
Accepted: 22 May 2008
This article is available from: http://www.biomedcentral.com/1471-2148/8/157
? 2008 Rocha et al; licensee BioMed Central Ltd. 
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), 
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 16
(page number not for citation purposes)
The extremely high biodiversity at the two global coral
reef hotspots ? the Indo-Malay Archipelago (IMA, also
Greater Caribbean (GC) region in the Atlantic ? has long
intrigued marine biologists [1-8]. Two primary hypothe-
BMC Evolutionary Biology 2008, 8:157 http://www.biomedcentral.com/1471-2148/8/157
ses have been proposed to explain such richness and the
corresponding biodiversity gradients moving away from
those regions: the center of origin (CO) hypothesis, intro-
duced by Darwin as "centers of creation" [9], proposes
that species originate in the center and disperse to the
periphery, and the high central diversity arises through in-
situ speciation [10]. According to Briggs, the most promi-
nent contemporary supporter of this hypothesis, centers
of diversity "act as centers of evolutionary radiation and
supply species to other areas that are less effective in an
evolutionary sense" [10-12]. Evidence from recent phylo-
genetic [13] and species-distribution surveys [10,14] sup-
port the CO model for the IMA. In contrast, the center of
accumulation (CA) hypothesis proposes that diversity
centers accumulate species that originated elsewhere. The
IMA lies on the western boundary of the Pacific and west-
ward flowing ocean currents could transport the pelagic
larvae of species originating anywhere in the Pacific to the
IMA [15,16]. A recent analysis of reef fish and coral distri-
butions in the Indian and Pacific Oceans concluded that
deviations from a random species-richness pattern pre-
dicted by a mid-domain model are consistent with this
hypothesis [17].
Genetic surveys of sea urchins, marine gastropods and
cowries in the Indo-Pacific indicate that species formation
has occurred throughout the region [4,18,19], both inside
the center of diversity (supporting the CO hypothesis)
and outside the IMA (supporting the CA hypothesis).
Likewise, the reef fish genera Thalassoma and Halichoeres
display no clear pattern, as ancient and recent species
occur both in the IMA and elsewhere in the Pacific
[20,21]. These and other studies have led to the proposi-
tion that both origin and accumulation of species contrib-
ute to the high diversity of the IMA [5,6,19,21].
Patterns of genetic variation within widely distributed
species can offer clues that may indicate how origin and
accumulation contribute to a center of diversity [22].
There are four ways in which intra-specific genetic varia-
tion can contribute pertinent information: (i) Resolution
of the geographic locations of both phylogenetically
ancestral or basal DNA sequences (haplotypes) and recent
or derived haplotypes. Under a CO scenario, basal line-
ages should be found at the center of diversity. In contrast,
the restriction of ancestral haplotypes to peripheral popu-
lations would support the CA model. (ii) Patterns of vari-
ation in genetic diversity throughout the species range
could also be informative. Under the CO hypothesis,
higher haplotype and nucleotide diversities should occur
in the diversity centers, but away from the center under
the CA hypothesis. Note, however, that the value of such
evidence is limited because at equilibrium, the largest
genetic analyses may offer other useful clues about the
geography of origination and subsequent dispersal: in
old, widely distributed species, basal haplotypes (which
assume an interior position in parsimony networks) and
derived haplotypes (peripherally located in parsimony
networks) may occur in all populations. However, if the
species recently expanded its range the younger popula-
tions would be less variable, may exhibit star-like parsi-
mony networks with a few very common haplotypes and
many rare haplotypes, and would have a Poisson-like mis-
match distribution [23-25]. Finally, (iv) direction of
migration can also be informative: gene flow away from
the center to the periphery would support the CO hypoth-
esis whereas the reverse flow would favor CA [26].
While attention has been focused on evolutionary mech-
anisms producing the Indo-Pacific center of diversity, the
CO and CA hypotheses have never been tested in the trop-
ical Atlantic Ocean. The tropical Atlantic is an appropriate
forum for such a test because geographically and oceano-
graphically it is a much simpler system than the western
Pacific, and, due to its isolation from the Indo-Pacific,
allows independent tests of the proposed mechanisms of
diversity production. This area comprises four tropical
biogeographic provinces: the Greater Caribbean (the Car-
ibbean itself, the Antilles, the Gulf of Mexico, Florida, the
Bahamas and Bermuda); Brazil (the coastline and oceanic
islands south of the Equator to 28?S); the mid-Atlantic
ridge (Ascension and St. Helena islands), and the tropical
eastern Atlantic (from Cape Verde to Angola, including
Cape Verde and the Islands in the Gulf of Guinea) [27,28]
(Fig. 1). Geologically the mid-Atlantic ridge includes St.
Paul's rocks (off northeastern Brazil) as well as Ascension
and St. Helena. However, effects of geographic proximity
mandate that the shore fishes of St. Paul's are more closely
related to the Brazilian fauna [29].
The biogeographic barriers separating these provinces
include vast geographic and oceanic distances lacking
suitable habitat: the northeastern South American coast is
heavily influenced by freshwater outflow, and there is no
coral reef development in the 2,300 km wide area
between the Amazon's mouth and Trinidad & Tobago; the
eastern and western Atlantic, as well as the central Atlantic
islands, are separated from the other provinces by thou-
sands of kilometers of deep open ocean [28,30]. Previous
mtDNA surveys of reef fishes and sea urchins have
revealed deep phylogenetic breaks among these four trop-
ical biogeographic provinces [20,31,32], as well as the
existence of some species that can apparently transcend
some of the barriers through dispersal of pelagic larval
and juvenile stages [33-38].Page 2 of 16
(page number not for citation purposes)
population will have the highest diversity, regardless of
age. Further, (iii) mismatch distributions and population
Here we analyze patterns of genetic diversity based on
mtDNA sequences in a common, widespread Atlantic reef
BMC Evolutionary Biology 2008, 8:157 http://www.biomedcentral.com/1471-2148/8/157
fish ? the brown chromis (Chromis multilineata) across all
four tropical Atlantic biogeographic provinces. The com-
bination of this transatlantic distribution and the lack of
a marked genetic break between populations in the two
western Atlantic provinces (see Results) makes it a good
candidate to study the roles of origin versus accumulation
in explaining high species diversity in the Greater Carib-
bean. Additionally, we re-evaluated data from four species
groups, published in two previous studies of Atlantic reef
fishes [36,38] that reported genetic lineages shared by Bra-
zil and the Caribbean. Our main objectives were: 1) to
search for signatures of origin vs. accumulation of genetic
diversity in the Greater Caribbean, which is the Atlantic
center of diversity for tropical reef organisms; and 2) to
assess how the barriers between major biogeographic
provinces influence the population structure of C. multi-
lineata, a widely distributed reef fish with a relatively short
pelagic larval stage.
Results
An 802 bp segment from the cytochrome b gene was ana-
lyzed for 183 individuals obtained from ten locations
(Fig. 1) spanning the entire geographical range of Chromis
multilineata on both sides of the tropical Atlantic. A total
of 121 polymorphic sites distributed among 132 haplo-
types were identified for those individuals. Mean nucle-
otide frequencies were A = 0.24, C = 0.35, G = 0.15, T =
0.26. The transition ? transversion ratio was 8.8:1. Aver-
age pairwise distances between populations ranged from
0.01 to 1.8 mean nucleotide substitutions (d = 0.001% to
0.22% sequence divergence) within the western/central
Atlantic, and from 7.1 to 7.9 mean nucleotide substitu-
tions (d = 0.88% to 0.98% sequence divergence) between
the eastern Atlantic and the other three provinces. Haplo-
type diversity (h) in C. multilineata was very high, with
unique haplotypes present in 71.8% of the individuals.
Populations in Caribbean, in Brazil and in the central
Sampling locations and Ocean currentsFigure 1
Sampling locations and Ocean currents. Sampling locations and major tropical Atlantic Ocean currents. Locations are: 1. 
St. Croix, USVI; 2. San Blas, Panama; 3. Grenada; 4. Paraiba; 5. Cabo Frio; 6. St. Paul's Rocks; 7. Ascension; 8. St. Helena; 9. 
Cape Verde; 10. Sao Tome. Current abbreviations are: GS, Gulf Stream; CarC, Caribbean Current; NEC, North Equatorial 
Current; NBC, North Brazil Current; CC, Canary Current; SEC, South Equatorial Current; ECC, Equatorial Counter Current; 
BrC, Brazil Current.
30?
15?N
0?
15?S
30?
90? 75? 60? 45? 30? 15?W 0? 15?E
BrC
CarC
SEC
South
Atlantic
Gyre
North
Atlantic
Gyre
ECC
NBC
NEC
GS
1
2
3
4
5
6
7
8
9
10
CCPage 3 of 16
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Atlantic each had slightly higher haplotype diversity (h =
0.92 ? 1.0), than the tropical eastern Atlantic populations
BMC Evolutionary Biology 2008, 8:157 http://www.biomedcentral.com/1471-2148/8/157
(h = 0.81 ? 0.92), although those differences are not sta-
tistically significant. Nucleotide diversity (?) was low in
all populations, but highest (although not significantly
higher) in the Caribbean (Table 1).
Within each of the four biogeographical provinces, pair-
wise population differentiation (?ST) values are very close
to zero and not significant indicating extensive gene flow
among locations (Table 2). Significant population separa-
tions (?ST = 0.033 ? 0.21) were observed between Carib-
bean and South Atlantic (Brazil and Central Atlantic
Islands) populations, whereas no significant differences
were observed between the Brazilian and Central Atlantic
provinces. Interestingly, connections between the south-
ern Caribbean locations (Panama and Grenada) and the
South Atlantic were stronger than those between St. Croix
(central Caribbean) and the South Atlantic (Table 2). All
comparisons between East and West/Central Atlantic pop-
ulations were significant (?ST = 0.621 ? 0.740), reflecting
an evolutionary genetic partition (sequence divergence d
= 0.88% ? 0.98%). AMOVA analysis (Table 3) shows that
divergence between the E Atlantic and the remaining
provinces accounted for 56.2% of the total molecular var-
iance. Phylogenetic analyses indicate that the ancestral
mtDNA lineages are located in the Caribbean (red
branches in Fig. 2, red circles in Fig. 3).
Demographic history was assessed through analysis of
pairwise mismatch distributions of all populations (Fig.
4). Caribbean populations have the wider mismatch dis-
tributions (13?15 mutations), followed by the Brazilian/
Central Atlantic populations (8?10 mutations), then the
eastern Atlantic populations (5?7 mutations). Pairwise
mismatch distributions (Fig. 5) were not significantly dif-
ferent from values expected under a model of population
expansion [39], except for the population at St. Paul's
Rocks. Fu's Fs and the Ramos-Onsins and Rozas's R2 also
yield a significant signal for population expansion at all
locations except St. Paul's Rocks (Table 1). The coales-
cence analysis revealed that mutational time (?) values
were similar for western and central Atlantic populations
and lower for the eastern Atlantic. Population sizes greater
than zero before expansion (?0) were only observed in the
Caribbean (Table 1), possibly indicating an older age for
this population.
Due to the lack of genetic differences among each of the
three Brazilian samples (Paraiba, Cabo Frio and St. Paul's
Rocks), among each of the three Caribbean samples (Pan-
ama, Grenada and St. Croix), between the two mid-Atlan-
tic locations (Ascension and St. Helena), and between the
two eastern Atlantic locations (Cape Verde and Sao
Tome), data from each group were combined to calculate
bidirectional migration rates among the four provinces.
Our analysis shows much migration from both Brazil and
the Central Atlantic to the GC, but little in the reverse
direction, and virtually none between the western/central
Atlantic and eastern Atlantic (the value for this last com-
parison in Fig. 4 is effectively zero).
In addition to the Chromis multilineata analysis, we built
maximum parsimony networks (Fig. 6) using previously
published data from the reef fish genera Halichoeres [36]
and Sparisoma [38]. In Fig. 6a, the network of H. bivittatus
indicates migration from Brazil to the Caribbean. In Fig.
6b, individuals of H. radiatus from the Brazilian oceanic
Table 1: Chromis multilineata populations data
N H h ? PD Fu's Fs R2 ? ?0 ?1
Greater Caribbean
Grenada 16 14 0.97 ? 0.03 0.009 ? 0.005 7.125 -5.631* (0.006) 0.0864* (0.026) 5.94 2.61 65.68
St. Croix (USVI) 25 23 0.99 ? 0.01 0.008 ? 0.004 6.203 -17.536* (0.000) 0.0475* (0.000) 6.84 0.10 122.97
Panama 19 19 1.00 ? 0.02 0.008 ? 0.005 6.602 -15.011* (0.000) 0.0632* (0.001) 4.33 2.70 3839.37
Central Atlantic Islands
Ascension 19 16 0.98 ? 0.02 0.007 ? 0.004 5.356 -8.637* (0.001) 0.0797* (0.024) 6.18 0.00 77.65
St. Helena 20 19 0.99 ? 0.02 0.007 ? 0.004 5.126 -15.492* (0.000) 0.0585* (0.001) 5.80 0.00 4001.25
Brazil
St. Paul's Rocks 17 10 0.92 ? 0.04 0.006 ? 0.003 4.500 -1.888 (0.163) 0.1091 (0.201) 5.40 0.00 47.07
Paraiba 11 11 1.00 ? 0.06 0.007 ? 0.004 5.145 -6.878* (0.000) 0.0613* (0.000) 5.32 0.00 6656.25
Cabo Frio 14 13 0.99 ? 0.02 0.007 ? 0.004 5.626 -7.041* (0.002) 0.0698* (0.001) 6.30 0.00 4680.00
Eastern Atlantic
Cape Verde 24 15 0.92 ? 0.03 0.003 ? 0.002 2.438 -10.252* (0.000) 0.0523* (0.000) 2.47 0.00 5453.12
Sao Tome 18 10 0.81 ? 0.09 0.002 ? 0.001 1.529 -6.567* (0.000) 0.0714* (0.004) 1.62 0.00 5754.27
Total 183 132 0.99 ? 0.03 0.010 ? 0.007 7.943 -24.555* (0.001) 0.0291* (0.003) 7.43 1.65 32.86
Sample size (N), number of haplotypes (H), haplotype diversity (h), nucleotide diversity (?), mean number of pairwise differences within populations 
(PD), Fu's Fs and Ramos-Onsins and Rozas' R2 tests followed by their respective P-values (in parenthesis), ? (units of mutational time), ? (function Page 4 of 16
(page number not for citation purposes)
0 
of population size before expansion), ?1 (function of population size after expansion) for all populations surveyed. Asterisks (*) indicate significance 
at a 95% confidence interval.
BMC Evolutionary Biology 2008, 8:157 http://www.biomedcentral.com/1471-2148/8/157
islands are derived from the Caribbean group, indicating
migration in the reverse direction, with the Caribbean
becoming a center of origin. The two Sparisoma networks
(Fig. 6c and 6d) show that populations and species
restricted to the SE corner of the Caribbean are closely
related to the Brazilian form.
Discussion
Phylogeography of the brown Chromis
Previous genetic surveys of reef organisms in the tropical
Atlantic have produced a mosaic of outcomes in terms of
the levels of separation among different provinces, with
virtually every conceivable pattern being evident [40]. The
short-spined sea urchin (genus Tripneustes), the red lip
blenny (genus Ophioblennius), and some wrasses of the
genera Thalassoma and Halichoeres show deep genetic
breaks (d = 2.0% ? 12.7% in mitochondrial DNA coding
regions) that correspond to the four major tropical Atlan-
tic biogeographic provinces [20,31,32,41]. In the ocean
surgeonfish (Acanthurus bahianus), GC populations are
separated from those in Brazil and the central Atlantic by
an mtDNA sequence divergence of d = 2.4%, but no dif-
ference is observed between populations at Brazil and the
mid-Atlantic islands [34]. Other species show little differ-
entiation throughout the tropical W Atlantic (doctorfish,
A. chirurgus; pygmy angelfish, genus Centropyge; goldspot
goby, Gnatholepis thompsoni [34,35,42]), or even through-
out the entire tropical Atlantic (blackbar soldierfish,
Myripristis jacobus [37]). In contrast, at the lower end of the
spatial scale, some species of wrasses (genus Halichoeres)
and cleaner gobies (genus Elacatinus) show deep genetic
breaks and reciprocally monophyletic groups on a scale of
tens of km within the GC [36,43,44].
In C. multilineata, three levels of genetic diversity were
observed: First, the tropical eastern Atlantic populations
formed a monophyletic group, separated from all other
populations by at least seven diagnostic mutations. Sec-
ond, there was a significant shift in haplotype frequencies
between the GC and the South Atlantic (Brazil + central
Atlantic islands) indicating population breaks between
the GC and those two provinces (Table 2). Third, there
was no detectable genetic difference between the Brazilian
and central Atlantic populations. The pattern that emerges
Table 2: Population pairwise ?ST for Chromis multilineata
Locations 1 2 3 4 5 6 7 8 9
1. Grenada -
2. St. Croix 0.023 -
3. Panama -0.011 0.039 -
4. Ascension 0.081* 0.173* 0.048 -
5. St. Helena 0.047* 0.147* 0.009 0.008 -
6. St. Paul's 0.165* 0.237* 0.108* 0.031 0.065 -
7. Paraiba 0.019 0.117* 0.005 -0.045 -0.018 0.025 -
8. Cabo Frio 0.058* 0.166* 0.033* -0.004 -0.022 0.036 -0.037 -
9. Cape Verde 0.636* 0.621* 0.621* 0.675* 0.675* 0.692* 0.710* 0.670* -
10. Sao Tome 0.640* 0.623* 0.624* 0.684* 0.685* 0.710* 0.740* 0.680* 0.004
Asterisks (*) indicate significance at a 99% confidence interval. Significance (P) is the probability of finding a variance component and F-statistic that 
are grater than or equal to the observed values and was tested using a non-parametric approach (Excoffier et al. 1992), with at least 3,000 
permutations of the dataset.
Table 3: Analysis of molecular variance (AMOVA) of Chromis multilineata
Source of variation Sum of Squares Percentage of variation Variance components
Between groups 239.036 56.65 3.575
Within groups 53.533 3.32 0.212
Within populations 439.042 39.99 2.523
Fixation Indices P-value
FCT 0.566 0.013
FSC 0.077 < 0.001
FST 0.600 < 0.001
Two groups were defined, western Atlantic, comprising the combination of the Brazilian, Caribbean and central Atlantic island populations, and 
eastern Atlantic, composed of Cape Verde and Sao Tome. Fixation indices are FCT: genetic variation between regions; FSC: genetic variation among 
populations within regions; and FST: genetic variation among all populations. Significance (P) is the probability of finding a variance component and Page 5 of 16
(page number not for citation purposes)
F-statistic that are greater than or equal to the observed values and was tested using a non-parametric approach (Excoffier et al. 1992), with at least 
3,000 permutations of the dataset.
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Page 6 of 16
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Phylogenetic tree of Chromis multilineata haplotypesFigure 2
Phylogenetic tree of Chromis multilineata haplotypes. The 50% majority-rule consensus tree from the Bayesian analysis 
of Chromis multilineata haplotypes. The alternative method of maximum parsimony resulted in almost identical topology. Num-
bers beside branches correspond to posterior probabilities estimated using the Bayesian approach, and numbers below 
branches refer to the bootstrap support calculated from the maximum likelihood analysis of 500 sequence replicates assuming 
model parameters values estimated from Modeltest (see methods). Color code as follows: red, Caribbean; blue, South Atlantic 
(Brazil + mid-Atlantic Islands); yellow, Africa (Cape Verde + Sao Tome). The branch leading to outgroup was reduced by 50%. 
Arrows indicate haplotypes shared by the South Atlantic and the Caribbean.
0.05
Chromis
atrilobata
0.60
0.99
0.98
0.91
0.91
0.85
0.84
0.81
0.78
0.78
0.76
0.82
0.75
0.76
0.65
0.95
0.61
0.87
0.76
0.57
Basal
haplotypes
African
lineage
Caribbean
lineage
South Atlantic
groups showing
migration towards
the Caribbean
Caribbean groups showing
migration towards
the South Atlantic
BMC Evolutionary Biology 2008, 8:157 http://www.biomedcentral.com/1471-2148/8/157
from most Atlantic phylogeography studies is that when
deep divergences are present, they generally correspond to
the major biogeographic provinces, although there are
notable instances of strong within-province differentia-
tion (e.g.: gobies and wrasses within both the GC and Bra-
zil [36,43,45]).
In the brown chromis and other reef organisms, the diver-
gence between the eastern Atlantic and the remaining
populations (Table 2) may be explained by 1,700 km or
more (the shorter straight line distances between the east-
ern Atlantic and the western/central Atlantic populations
are ~1,700 km between St. Paul's Rocks and Cape Verde,
~2,300 km between St. Helena and Sao Tome and ~2,500
km between Ascension and Sao Tome) of unsuitable
open-ocean distances, combined with the general east to
west direction of prevailing currents at low latitudes.
While low but significant pairwise comparisons were
observed between the two provinces of the South Atlantic
(Brazil and the central Atlantic) and the Caribbean, sepa-
rated by no more than 2,300 km, no difference was
observed between Brazil and the central Atlantic Islands,
separated by 2,200 km.
Low genetic structure, such as that seen in C multilineata
might be expected in organisms with a long pelagic larval
stage [34,37] and a similar signal has been observed in the
long-spined sea urchin, genus Diadema with a ~6 week lar-
val stage [33] and the ocean surgeonfish, Acanthurus bahi-
anus, which has a larval duration of ~60 days [34]. C.
multilineata, however, has a relatively short pelagic larval
stage averaging 27 days (33 days maximum [46]), which
is substantially less than the average time (48 days based
on average current velocity) to cross from Brazil to the
mid-Atlantic [47]. However, juvenile C. multilineata have
been observed in association with floating debris in Belize
(L. A. Rocha pers. obs.), and juvenile Chromis atrilobata (C.
multilineata's sister species in the eastern Pacific) have
been observed in open water under floating objects
[48,49]; D. R. Robertson pers. obs.). Thus, young C. mul-
tilineata juveniles survive in the open ocean long after
transforming from the larval stage, and relatively strong
tropical currents connecting the mid-Atlantic islands and
Brazil may transport juvenile Chromis between those loca-
tions [50,51]. Transport from the western and central
Atlantic to the eastern Atlantic evidently has been much
less frequent, probably due to larger distances involved in
Parsimony network of Chromis multilineata haplotypesFigure 3
Parsimony network of Chromis multilineata haplotypes. Statistical parsimony network representing relationships 
between Chromis multilineata haplotypes. Sizes of ovals are proportional to haplotype frequency. Small empty circles represent 
missing (extinct or unsampled) haplotypes. Colors as in Fig. 2.
Southwestern Atlantic
Caribbean
C. atrilobata
78 mutations
AfricaPage 7 of 16
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direct transport from W to E Atlantic and smaller source
BMC Evolutionary Biology 2008, 8:157 http://www.biomedcentral.com/1471-2148/8/157
populations for dispersal from the tiny central Atlantic
islands (Ascension, St. Helena) to the eastern Atlantic.
The mismatch distribution analysis (Fig. 5), Ramos-
Onsins and Rozas's R2 test for recent population expan-
sion and Fu's Fs neutrality test (Table 1) indicate that all
populations have undergone a recent expansion, except
for that at the diminutive St. Paul's Rocks. This expansion
was probably caused by the ~10 fold increase in suitable
shallow reef habitat associated with the sea-level rise of
~130 m since the last glacial maximum [52]. St. Paul's
Rocks is an exception: because there is no shelf at that
island, which is a pillar rising vertically from deep water
[29], available habitat probably hasn't increased with sea-
level rise. Thus the lack of an expansion signal may reflect
a relatively constant population size due to stability in
habitat availability. The lower number of pairwise differ-
ences, and haplotype and nucleotide diversities observed
at Cape Verde and Sao Tome in the eastern Atlantic, indi-
cate a more recent expansion at those locations than in the
the last glacial period in the eastern Atlantic, water tem-
peratures in that area were lowered more than in the west-
ern and central Atlantic by enhanced coastal upwelling
[53], a stress on tropical species that may have caused
strong fluctuations or reductions in population size.
Migration patterns among populations (Fig. 4) are con-
sistent with the general direction of surface current flows
(Fig. 1): the highest rates are from the South Atlantic to
the Caribbean (coinciding with the flow of the North Bra-
zil current) and from Brazil to the mid-Atlantic islands
(coinciding with the flow of the South Atlantic Gyre). In
accordance with the phylogenetic results, migration
between western and eastern Atlantic (a direction that
opposes major surface currents; Fig. 1) was effectively
zero.
The Greater Caribbean hotspot: center of origin and 
center of accumulation
Our phylogeographic analysis of C. multilineata provides
Migration rates between populations of Chromis multilineataFi ure 4
Migration rates between populations of Chromis multilineata. Migration rates between populations of Chromis multiline-
ata among the four tropical Atlantic biogeographical provinces, based on the program MIGRATE. Arrows indicate the direction 
of migration; arrow thickness is proportional to the amount of migration.
30?
15?N
0?
15?S
30?
90? 75? 60? 45? 30? 15?W 0? 15?E
76.8
20.7
0.34
6.15 x 10-9
27.8
1.75
x 10
-15
173.1
2.38
x 10
-13
Brazil
Mid-Atlantic
Islands
Africa
CaribbeanPage 8 of 16
(page number not for citation purposes)
western and central Atlantic. In addition to lower sea-lev-
els and corresponding reduced habitat available during
two lines of evidence relevant to the mechanism(s) that
produced the GC hotspot of diversity. First, supporting
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Page 9 of 16
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Mismatch distribution of Chromis multilineata haplotypesFigure 5
Mismatch distribution of Chromis multilineata haplotypes. Mismatch distribution of the pairwise number of nucleotide 
differences among pairs of individuals for the brown chromis (Chromis multilineata). Observed distribution represented by ver-
tical bars. Expected distribution as predicted by the sudden population expansion model of Rogers (1995) represented by solid 
line.
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Page 10 of 16
(page number not for citation purposes)
Parsimony networks of select Atlantic reef fishesFigure 6
Parsimony networks of select Atlantic reef fishes. Statistical parsimony networks representing relationships between: A. 
cytochrome b sequences of Halichoeres bivittatus haplotypes; B. cytochrome b of Halichoeres radiatus and H. brasiliensis; C. com-
bined 16s and 12s sequences of Sparisoma axillare and S. rubripinne; D. combined 16s and 12s sequences of S. chrysopterum, S. 
frondosum and S. griseorubra. Sizes of ovals are proportional to haplotype frequency. Small empty circles represent missing hap-
lotypes. Arrows indicate points of support for either the center of origin or the center of accumulation hypotheses. Colors as 
in Fig. 2.
Brazil
Caribbean
Floridian
H. bivittatus
H. bivittatus
H. radiatus
H. brasiliensis
S. chrysopterum
S. rubripinne
S. axillare
S. frondosum
S. griseorubra
2 4 mutations
9 mutations
15 mutations
15 mutations
A
B
C
D
BMC Evolutionary Biology 2008, 8:157 http://www.biomedcentral.com/1471-2148/8/157
accumulation, the short (i.e. relatively young) branches
are mostly in Brazil and the central Atlantic islands (in Fig.
2 ~75% of the short branches are blue, endemic to Brazil
and the two mid-Atlantic islands). However, the short
branches also include a few Caribbean haplotypes (~25%
of short branches in the mostly South Atlantic lineage in
Fig. 2), indicating that the lineages diversified in the South
Atlantic, and that individuals carrying those haplotypes
recently arrived in the Caribbean. This South to North pat-
tern of dispersal is consistent with the pattern of oceanic
current flow (Fig. 1) and migration rates (Fig. 4), and indi-
cates that the hotspot of diversity in the Caribbean has
acted recently as a CA.
Similarly to what we describe here for C. multilineata, the
Panamanian sample of the wrasse Halichoeres bivittatus
has one individual (among 23 sampled) with a haplotype
that is more similar to those found in Brazil than those in
other Caribbean locations (Fig. 6a), indicating the recent
arrival of a haplotype of South Atlantic origin [36]. Addi-
tionally, in the goby genus Gnatholepis, and the angelfish
genus Centropyge, the Atlantic species are recently derived
from much more diverse Indo-Pacific groups, and appar-
ently only recently arrived in the GC from the Indian
Ocean via the South Atlantic [35,42,54], supporting accu-
mulation of species at the GC hotspot.
The phylogeny of parrotfishes (genus Sparisoma) also sup-
ports accumulation at the Caribbean (Fig. 6c and 6d). The
parrotfish S. axillare is abundant and widely distributed
throughout the Brazilian coast, but known only from SE
Venezuela in the Caribbean, indicating that the popula-
tion there is probably a result of recent dispersal from Bra-
zil. Likewise, the parrotfish S. griseorubra is restricted to
the southern Caribbean (SE Venezuela) and its sister spe-
cies is S. frondosum (endemic to Brazil), indicating that S.
griseorubra likely originated from ancient dispersal by the
ancestor of the S. frondosum/S. griseorubra lineage. The
splitting of S. frondosum and S. griseorubra alternatively
could be explained by sympatric speciation in the Carib-
bean (the groups with Brazilian affinities co-occur with
their Caribbean counterparts in the southern Caribbean)
followed by dispersal towards Brazil. However, speciation
with gene flow in reef fishes has only been inferred when
there are strong ecological gradients [36], or where the
fish are strictly associated with coral hosts [55]. As this is
not the case for Sparisoma, this alternative is less likely
than an allopatric split between Brazil and the Caribbean
after dispersal northwards by a Brazilian lineage.
Supporting origin at the Caribbean, almost all basal hap-
lotypes of the C. multilineata tree are observed only in Car-
ibbean individuals (Figs. 2 and 3), which indicates that
(either separately or in combination) have the widest mis-
match distributions (spanning 13 to 15 mutations; Fig. 5)
indicating that this region hosts the oldest and most stable
population. We can also infer that, because all eastern
Atlantic haplotypes apparently have a single origin
(monophyly in Fig. 2), they most likely originated from a
single colonization event (Fig. 3) on the order of two hun-
dred thousand years ago (based on our trans-isthmian
molecular clock estimate of 4.4%/Myr). The recent find-
ing of Centropyge aurantonotus in the eastern Atlantic (pre-
viously known only from the western Atlantic, but
recently observed in low numbers in Sao Tome [56]) and
the general direction of trans-Atlantic range expansion in
the gold spot goby [35] support this rare west to east col-
onization route.
Lending further support to the CO hypothesis, popula-
tions of the wrasse H. radiatus in Brazilian oceanic islands
are much more closely related to Caribbean H. radiatus
than to populations of H. brasiliensis (its sister species) in
the adjacent coast line, indicating that these islands were
colonized by migrants of Caribbean origin (Fig. 6b). A
similar pattern of southward dispersal from the GC to the
offshore Brazilian islands has been observed in gobies of
the genus Bathygobius [45]. Colonization outward from
the Caribbean must be a rare event because it goes against
prevailing currents, but as our analysis indicates, it has
happened and can lead to the establishment of new pop-
ulations, supporting the CO hypothesis.
Support for origin of diversity in the GC comes from other
recent phylogenetic and biogeographic analyses. A survey
of seven-spined gobies [57] shows that two peripheral
species of Elacatinus (E. figaro from Brazil and E. puncticu-
latus from the eastern Pacific) are older species whereas
the 10 newest (youngest) Elacatinus species are found only
in the Caribbean biodiversity hotspot. That is, recent spe-
ciation that produced 83% of the species in this genus
occurred within the GC. Likewise, the diverse families
Chaenopsidae and Labrisomidae are represented in the
GC by 45 species each, but elsewhere in the Atlantic there
are only four and 11 Atlantic species in each of these fam-
ilies respectively [40,58,59]. Hamlets (genus Hypoplectrus)
include as many as a dozen or more closely related "spe-
cies" and are restricted entirely to the GC [60]. Thus, spe-
ciation leading to considerable faunal enrichment most
likely occurred in situ within the GC in those four taxa.
Recent phylogenetic analyses of large reef fish groups also
provide useful information to this debate. Among wrasses
(Labridae), the Caribbean and Eastern Pacific Halichoeres
are for the most part monophyletic, indicating that they
diversified in situ and supporting CO in a recent timePage 11 of 16
(page number not for citation purposes)
populations at the three other regions are derived from a
Caribbean ancestor. Moreover, the Caribbean samples
scale, but at the same time their diverse group of ancestors
is composed of Indo-Pacific species [21,61] supporting
BMC Evolutionary Biology 2008, 8:157 http://www.biomedcentral.com/1471-2148/8/157
CA deeper in time, on a scale of tens of millions of years.
Similarly to the wrasses, the Caribbean groupers of the
genera Epinephelus and Mycteroperca are monophyletic
groups derived from Indo-Pacific ancestors [62], support-
ing both ancient CA and more recent CO. The Caribbean
butterflyfishes are mostly a paraphyletic assemblage of
lineages derived from more diverse Indo-Pacific groups,
supporting CA [63]. Within the damselfishes, the Carib-
bean (and Atlantic) species Abudefduf saxatilis seems to be
a recent arrival from the Indo-Pacific (it is very closely
related to a group containing eight Indo-Pacific and one
eastern Pacific species), also supporting CA [64]. Even
though these large scale phylogenies are useful, most lack
peripheral (mostly Brazilian) endemics, making their
contribution to this debate limited. However they provide
an excellent framework that, with the addition of a few
key species, may become an important piece in the tropi-
cal biodiversity puzzle.
Conclusion
Our data indicates that the Greater Caribbean is both a
center of origin and accumulation for genetic lineages
within species (e.g. Chromis multilineata and Halichoeres
bivittatus) and for sister species within genera (e.g. Spari-
soma). Such bidirectional dispersal is also reflected in the
geographic distributions of tropical Atlantic fishes and
invertebrates: several species that are widely distributed in
the Brazilian coast are also recorded in the southeastern
corner of the Caribbean [30,65,66]. Likewise some widely
distributed Caribbean species also occur in northern Bra-
zil, sometimes on only a few reefs south of the Amazon
outflow, evidence of recent southward dispersal [30,40].
We conclude, on the basis of multiple lines of evidence,
that the GC marine biodiversity hotspot did not arise
through the action of a single mode of microevolutionary
change. This diversity is the product of a more complex
and idiosyncratic process in different taxa, and it is clear
from the accumulating data that several mechanisms have
contributed. The hypotheses of center of origin and center
of accumulation are not mutually exclusive, and acting in
concert, as they have done in the GC, origin and accumu-
lation can generate more diversity than either process act-
ing alone.
In closing we note that the IMA is a much larger hotspot
than the GC, in terms of both geography and biodiversity.
The IMA hotspot is flanked on both sides by the numer-
ous archipelagos of the Pacific and Indian Oceans, a fea-
ture nearly absent for the GC hotspot. Moreover, the GC
is marked by a turbulent history involving extinctions of
many reef-associated organisms in the past few million
years [67], a feature not yet detected at the IMA. We sug-
serves to strengthen the biodiversity feedback between
hotspots and other areas, and contribute to the global
center biodiversity in the IMA. Finally, it remains to be
seen whether the principles of origin and accumulation
apply to terrestrial biodiversity, or whether these evolu-
tionary mechanisms are restricted to high-dispersal
media, the exclusive domain of the world's oceans.
Methods
Sampling strategy
Chromis multilineata is a common reef fish that is widely
distributed on both sides of the tropical Atlantic, as well
as at the mid-Atlantic islands [68]. They have demersal
eggs that develop into pelagic larvae in about three days
[69,70]. Estimates of pelagic larval duration range from
24 to 33 days [46]. Adults can be locally very abundant
and are usually found in schools from a few to several
hundred individuals, swimming and feeding on plankton
above the reef [71].
A total of 183 specimens of Chromis multilineata were
obtained from 10 locations, which included at least two
locations within each of the four provinces (Table 1, Fig.
1). Specimens were collected with polespears while scuba
diving or snorkeling, between 1997 and 2002. Tissues
(muscle and/or gill) were stored in a saturated salt-DMSO
buffer (Amos and Hoelzel 1991). A recent phylogenetic
survey of damselfishes indicates that the eastern Pacific
species C. atrilobata is the sister of C. multilineata [64]. To
confirm this relationship we sequenced C. atrilobata from
Cocos Island (n = 3) and Panama (n = 3) and other species
of the genus in the Atlantic that were not included in the
phylogeny [64]: C. scotti from Florida (n = 2); C.
enchrysura from Florida (n = 2), C. limbata from the Azores
(n = 4) and C. lubbocki from Cape Verde (n = 3). The result-
ing tree (not shown) supports the conclusion that C. mul-
tilineata and C. atrilobata are sister species.
DNA Extraction and Sequencing
Total genomic DNA was extracted using QIAGEN (Valen-
cia CA) Dneasy extraction kits following the manufac-
turer's protocol. Extracted DNA was frozen in TE buffer
and archived at -20?C. Primer names indicate the DNA
strand (H = heavy and L = light strand) and the position
of the 3' end of the oligonucleotide primer relative to the
human mitochondrial DNA sequence. A segment of 802
base pairs of the mtDNA cytochrome b gene was ampli-
fied with the primers L14725 (5' GTG ACT TGA AAA ACC
ACC GTT G 3') and H15573 (5' AAT AGG AAG TAT CAT
TCG GGT TTG ATG 3') [72].
Thermal cycling in polymerase chain reactions (PCR) con-
sisted of an initial denaturing step at 94?C for 1 min 20Page 12 of 16
(page number not for citation purposes)
gest that the larger size and greater stability of the IMA,
combined with its extensive halo of peripheral habitats,
sec, then 35 cycles of amplification (40 sec of denatura-
tion at 94?C, 30 seconds of annealing at 52?C, and 55 sec
BMC Evolutionary Biology 2008, 8:157 http://www.biomedcentral.com/1471-2148/8/157
of extension at 72?C), and a final extension of 2 min 30
sec at 72?C. Each tube contained 15 ?l of water, 2.5 ?l of
MgCl2, Mg free buffer and DNTPs, 0.3 ?l of each primer,
0.4 ?l of Taq DNA polymerase and 1.5 ?l of purified DNA.
Excess primers were removed through simultaneous incu-
bation of PCR product with exonuclease I and shrimp
alkaline phosphatase (USB Corp., Cleveland OH).
Sequencing reactions with fluorescently-labeled dideoxy
terminators (BigDye) were performed according to manu-
facturer's recommendations, and analyzed with an ABI
377 automated sequencer (Applied Biosystems, Inc. Fos-
ter City, CA). All samples were sequenced in the forward
direction (with L14725 primer), but rare and questiona-
ble haplotypes were sequenced in both directions to
ensure accuracy of nucleotide designations. Representa-
tive haplotype sequences are deposited in GenBank under
accession numbers EU431997 ? EU432046. Copies of the
complete data set are available from LAR.
Phylogenetic and Population Analyses
Sequences were aligned and edited with Sequencher ver-
sion 3.0 (Gene Codes Corp., Ann Arbor, MI). Population
structure and gene flow were assessed with an analysis of
molecular variance AMOVA [73] in the program Arlequin
version 3.0, which generated ?ST values (a molecular ana-
log of FST that includes sequence divergence among haplo-
types as well as haplotype frequency shifts; [74]). Genetic
variation is described with nucleotide diversity (?; equa-
tion 10.19 [75]) and haplotype diversity (h; equation 8.5
[75]) within each location.
The computer program MODELTEST version 3.06 [76]
was used to determine the substitution model that best
fits the data through a minimal theoretical information
criterion (AIC). The model chosen was TRN+? [77] with a
gamma distribution of 0.97 to estimate sequence diver-
gences (d values) between haplotypes. Equal weighting of
all three codon positions was used. Relationships between
all haplotypes and closely related species were estimated
using a Bayesian phylogenetic analysis performed with
MrBayes 3.1 [78]. Preliminary runs were performed to
monitor the fluctuating value of the likelihoods of the
Bayesian trees, and all parameters appear to reach stability
before 250,000 generations. The Markov chain analysis
was run for 20 million generations. A burn-in period, in
which the initial 10,000 trees were discarded, was adopted
and the remaining tree samples were used to generate a
50% majority rule consensus tree. The posterior probabil-
ity of each clade was then provided by the percentage of
trees identifying the clade [79]. In addition, the software
PAUP 4.0b10 [80] was used to conduct maximum parsi-
mony (MP) and neighbor-joining analyses that were eval-
terion was used by applying maximum likelihood dis-
tances estimated with the model chosen by Modeltest in
the neighbor-joining analysis. The resulting Bayesian, par-
simony and neighbour joining trees were not significantly
different.
Departure from neutrality was tested using Fu's Fs [81]
and Ramos-Onsins and Rozas' R2 statistic [82]. The R2
measure is based on the difference between the number of
singleton mutations and the average number of nucle-
otide differences among sequences within a population
sample. Both Fs and R2 are powerful tests used to detect
recent population expansions under assumptions of neu-
trality [81,82]. Significance of R2 and Fs were evaluated by
comparing the observed value with a null distribution
generated by 10,000 replicates, using the empirical popu-
lation sample size and observed number of segregating
sites implemented by DnaSP version 4.10.9 [83]. Moreo-
ver, in order to estimate the demographic parameters of
past population expansions we calculated mismatch dis-
tributions (or the distribution of the pairwise genetic dis-
tances) for all populations using DnaSP. Time and
magnitude of the inferred population expansion were
determined by calculating ?0, ?1 and ?, where ?0 = 2N0?
(N0 = population size before expansion); ?1 = 2N1? (N1 =
population size after expansion); and ? = 2?t (? = muta-
tion rate per site per generation; t = time in generations).
The mutation rate per lineage per year (?) was estimated
by solving the formula ? = d/2T, where d is the genetic dis-
tance between C. multilineata and C. atrilobata, and T is the
time since divergence between the two species used. We
used a T of 3.5 million years (Myr), as that is the upper
limit age for the final closure of the Isthmus of Panama
[84]. The rate of 2.21%/Myr (within lineages) that we esti-
mated is very similar to that obtained in a recent survey of
Chromis using a slightly different portion of the cyt b gene
(2.36%/Myr; [85]. The closure of the Isthmus of Panama
was used in several other studies to estimate mutation
rates in reef fishes (e.g.: [35,86-88]).
Migration rates between the major biogeographical prov-
inces (Brazil, the Caribbean, central Atlantic islands and
eastern Atlantic islands) Nm (where N is effective female
population size and m is migration rate) were calculated
with the software MIGRATE version 1.7.6 [89], which uses
a maximum likelihood approach based on coalescence
theory [90]. Estimators of migration rates based on coales-
cence theory can detect asymmetries (directionality) in
migration rates and differences in population sizes, a con-
siderable advantage when testing migration rates in the
context of evolutionary theory [91]. Most of the default
settings for MIGRATE were used on the first run, but thePage 13 of 16
(page number not for citation purposes)
uated with 1000 bootstrap replicates implemented with
PAUP* version 4.0b10 [80]. The minimum evolution cri-
number of trees sampled was increased to 10,000 for the
short chains and 100,000 for the long chains to avoid
BMC Evolutionary Biology 2008, 8:157 http://www.biomedcentral.com/1471-2148/8/157
local maxima on the likelihood surface. A trial run was
used to generate the input parameters; the average of
results of 10 runs (number of trees sampled was progres-
sively increased from 10,000 to 300,000 for the short
chains and from 100,000 to 3,000,000 for the long
chains) is reported here. We also used the software IM to
estimate migration between populations of C. multiline-
ata; IM uses a Markov chain Monte Carlo approach [92].
The results from the IM analysis are not shown because
they were completely compatible with the results from
MIGRATE.
In addition, we re-analyzed data from four groups of reef
fish species (the wrasse genus Halichoeres [36], and the
parrotfish genus Sparisoma [38]) to specifically assess pre-
dictions from the center of origin and center of accumula-
tion hypotheses. To do so we constructed statistical
parsimony networks for those species as well as Chromis
multilineata using the computer program TCS version 1.21
[93] and analyzed the geographical distribution of
mtDNA haplotypes.
Authors' contributions
LAR obtained samples from the western Atlantic, carried
out the molecular genetic analyses and prepared the man-
uscript. CRR participated in DNA sequencing and popula-
tion genetic analysis. DRR collected samples at the Central
Atlantic Islands, the Caribbean, the Eastern Pacific and
Africa, and helped prepare the manuscript. BWB partici-
pated in the design and coordination of the study and
helped prepare the manuscript. All authors edited and
approved the final manuscript.
Acknowledgements
This research initiative began in discussions with John Briggs, and we wish 
to acknowledge his fundamental contribution to our research program. We 
thank B. M. Feitoza, B. P. Ferreira, C. E. Ferreira, S. R. Floeter, J. L. 
Gasparini, Z. Hillis-Starr, S. Karl, B. Luckhurst, O. Luiz-Junior, D. Murie, D. 
Parkyn, B. Philips, J. Pitt, I. L. Rosa, R. S. Rosa, C. L. S. Sampaio, S. Sponaugle, 
R. Toonen and B. Victor for help with sampling and laboratory work. Dis-
cussions with N. Knowlton, H. Lessios, G. Paulay, J. Randall and R. Toonen 
greatly improved the manuscript. Collection permits were provided by the 
Department of Agriculture and Fisheries of Bermuda (permit 01/12-2001), 
the Belize Fisheries Department (permit GEN/FIS/15/04/2002), the 
National Park Service at St. Croix, U.S. Virgin Islands (permit BUIS-2001-
SCI-0004), IBAMA Brazil, Departamento de Pesca de Sao Tome, Direccao 
das Pescas do Cabo Verde, the Administrator of Ascension Island, the Gov-
ernor of St. Helena Island. US Airforce for logistical support at Ascension 
Island. Fishes were collected following the guidelines of the University of 
Hawaii Institutional Animal Care & Use Committee. This work was finan-
cially supported by the Brazilian Navy, the Smithsonian Tropical Research 
Institute, the National Geographic Society (grant 7708-04 to L. A. Rocha), 
the US National Science Foundation (grants DEB 9727048 and OCE-
0453167 to B. Bowen), and the HIMB-NWHI Coral Reef Research Partner-
ship (NMSP MOA 2005-008/66882).
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