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2004 by the American Society of Ichthyologists and Herpetologists
Copeia, 2004(4), pp. 770–782
Mitochondrial DNA and Color Pattern Variation in Three Western
Atlantic Halichoeres (Labridae), with the Revalidation of Two Species
L
UIZ
A. R
OCHA
Genetic surveys of widely distributed marine species often find previously unde-
tected biodiversity. In the present study, populations of three species of Halichoeres
were sampled across their entire geographical ranges: Halichoeres cyanocephalus and
Halichoeres maculipinna were sampled on both sides of the Amazon freshwater out-
flow, the main biogeographic barrier in the tropical western Atlantic; and Halichoeres
garnoti was sampled in the Caribbean and Bermuda. Genetic divergences between
populations separated by the Amazon ranged from 2.3% in H. cyanocephalus to 6.5%
in H. maculipinna. There is inconsistency between color differences and genetic
partitions in the species surveyed. The color differences between populations of H.
cyanocephalus and H. maculipinna correspond to deep genetic partitions at the cy-
tochrome b locus. However, genetic similarity at this same locus was observed be-
tween populations of H. garnoti with striking color differences. Based on the com-
bination of the observed genetic differences with diagnostic color differences, the
Brazilian species Halichoeres dimidiatus (Agassiz) and Halichoeres penrosei Starks,
1913 are revalidated. In addition, a neotype is designated to H. cyanocephalus (Bloch,
1791), to clarify its taxonomic status and type locality. All species analyzed have a
similar larval dispersal potential, but varying degrees of genetic divergences were
observed, indicating that benthic stage ecology may also play a role in speciation in
this group.
T
HE presence of potentially widely dispers-
ing planktonic larva in most marine fishes
has led to the assumption that such species are
composed of genetically homogeneous popula-
tions (Warner, 1997). However, recent studies
on larval behavior (Leis and McCormick, 2002)
and oceanographic processes (Cowen, 2002) in-
dicate that larvae often disperse far less than
their potential. Moreover, habitat selection by
larvae can also overcome the effects of long-dis-
tance dispersal and give rise to genetic differ-
ences in geographically close populations (Bier-
ne et al., 2003). Consequently, genetic studies
of widely distributed taxa often reveal the pres-
ence of previously overlooked species (Knowl-
ton, 2000).
Early genetic surveys within the Caribbean re-
gion found no evidence of cryptic speciation in
reef fishes (Shulman and Bermingham, 1995).
However, when broader geographic areas were
sampled, analyses of DNA variation demonstrat-
ed the presence of deeply divergent evolution-
ary partitions in several species, such as bone-
fishes (Colborn et al., 2001), blennies (Muss et
al., 2001), surgeonfishes (Rocha et al., 2002),
groupers (Carlin et al., 2003), and wrasses (Ro-
cha, 2003a). Some of these genetic divergences
are accompanied by slight color differences.
In reef fish taxonomy, color pattern is consis-
tently used as a useful diagnostic character.
When color differences are observed between
populations that also show slight morphological
differences, those populations are usually ele-
vated to specific status (Randall, 1998). Howev-
er, color alone is rarely used as a character to
define species, especially in groups with highly
variable intraspecific color patterns such as
wrasses (Labridae). Despite the presence of
bright color patterns and their wide variation
observed in reef fishes, little is known about its
evolutionary significance (McMillan et al.,
1999). Sexual selection related to coloration ap-
pears to be present in damselfishes (Pomacen-
tridae; Thresher and Moyer, 1983), but no cor-
relation between mating success and color pat-
tern was observed in the blue-head wrasse (La-
bridae; Warner and Schultz, 1992). In closely
related species, coloration has been proposed
to be useful in mate recognition. The sharpnose
pufferfishes are an example of a group with lit-
tle or no diagnostic external morphological
characters, where color has been proposed as
important in speciation and is routinely used as
a species defining character (Allen and Randall,
1977; Moura and Castro, 2002).
Wrasses of the genus Halichoeres in the west-
ern Atlantic provide an excellent system to ex-
amine the relationship between genetic struc-
ture and color differentiation. The populations
surveyed in this study are present on both sides
of recognized biogeographic barriers, have a
similar dispersal potential (pelagic larval phase
771ROCHA—MITOCHONDRIAL DNA VARIATION IN HALICHOERES
Fig. 1. Tropical western Atlantic. Numbers repre-
sent sampled locations: 1. Bermuda; 2. Bahamas; 3.
Florida Keys; 4. Belize; 5. St. Croix; 6. Venezuela; 7.
northeast Brazil; 8. southeast Brazil; 9. Trindade Is-
land. Solid arrows indicate direction of mean surface
oceanic currents; broken lines indicate subsurface
currents.
of 25–30 days; Sponaugle and Cowen, 1997;
Wellington and Robertson, 2001) and exhibit
slight color differences. Populations of Halicho-
eres garnoti in the Caribbean and Bermuda are
separated by at least 1400 km of open-ocean
(Smith-Vaniz et al., 1999), and populations of
Halichoeres cyanocephalus and Halichoeres maculi-
pinna in Brazil and the Caribbean are separated
by the Amazon barrier ( Joyeux et al., 2001; Ro-
cha, 2003b). These populations have been con-
sidered to be the same species, but they show
consistent color differences in three instances:
(1) the juveniles of H. cyanocephalus in Brazil
lack a blue spot on the midventral portion of
the soft dorsal fin, which is present on juveniles
of the Caribbean population; (2) terminal-
phase males of H. garnoti at Bermuda have a red
band on the upper posterior portion of the
body, whereas males from the Caribbean have a
black band at the same place; (3) the upper
body of individuals from the Brazilian popula-
tions of H. maculipinna is usually brownish-pink,
whereas individuals from the Caribbean have a
yellowish-green upper body.
Samples of the above-mentioned species were
collected across their entire geographical range
in the tropical western Atlantic, and an analysis
of mitochondrial DNA sequences was carried
out aiming to assess the following questions: (1)
Does genetic differentiation parallel the ob-
served patterns of color differentiation in west-
ern Atlantic Halichoeres? (2) Do biogeographic
barriers (the Amazon freshwater outflow and
vast open-ocean distances) equally affect species
with similar dispersal potential?
M
ATERIALS AND
M
ETHODS
Specimens were collected with pole spears or
hand nets while scuba diving or snorkeling in
Bermuda, the Bahamas, Florida, Belize, St.
Croix (U.S. Virgin Islands), Venezuela, Paraı´ba
(northeastern Brazil), Espı´rito Santo (south-
eastern Brazil) and Trindade Island (Fig. 1) be-
tween 1997 and 2002. Tissue (muscle and/or
gill) was stored in a saturated salt-DMSO buffer
(Amos and Hoelzel, 1991).
Total genomic DNA was extracted using QIA-
GEN DNeasy extraction kits following the man-
ufacturer’s protocol. Extracted DNA was frozen
in TE buffer (10 mM Tris-HCl, pH 7.5 and 1
mM EDTA, pH 8.0, diluted in water) and ar-
chived at
2
20 C. Primer names indicate the
DNA strand (H
5
heavy and L
5
light strand)
and the position of the 3
9
end of the oligonu-
cleotide primer relative to the human mito-
chondrial DNA sequence. A fragment of ap-
proximately 800 base pairs of the mtDNA cyto-
chrome b gene was amplified with the primers
L14725 (5
9
GTG ACT TGA AAA ACC ACC GTT
G3
9
) and H15573 (5
9
AAT AGG AAG TAT CAT
TCG GGT TTG ATG 3
9
; Meyer, 1993). Based on
initial sequences, the internal primers L14768
(5
9
ACC CAC CCA CTC CTT AAA ATC 3
9
), and
H15496 (5
9
TTG GAG ACC CAG ATA ATT TCA
C3
9
) were designed and worked consistently
among Halichoeres species, yielding a product of
690–709 base pairs.
Thermal cycling in polymerase chain reac-
tions (PCR) consisted of an initial denaturing
step at 94 C for 1 min 20 sec, then 35 cycles of
amplification (40 sec of denaturation at 94 C,
30 sec of annealing at 52 C, and 55 sec of ex-
tension at 72 C), and a final extension of 2 min,
30 sec at 72 C. Excess primers were removed by
incubation of PCR products with exonuclease I
and shrimp alkaline phosphatase (USB Corp.,
Cleveland OH).
Sequencing reactions with fluorescently la-
beled dideoxy terminators (BigDye, Applied
Biosystems, Inc., Foster City, CA) were per-
formed according to manufacturer’s recom-
mendations and analyzed with an ABI 377 au-
tomated sequencer (Applied Biosystems, Inc.,
Foster City, CA). All samples were sequenced in
the forward direction (with L14725 or L14768
primers), and rare or questionable haplotypes
were sequenced in both directions to ensure ac-
curacy of nucleotide designations. Haplotypes
of all species were deposited at GenBank (see
Material Examined section).
772 COPEIA, 2004, NO. 4
Fig. 2. Phylogenetic tree of Halichoeres cyanocephal-
us (Belize) and Halichoeres dimidiatus (Brazil) unique
haplotypes. Neighbor-joining bootstrap support (
.
50%) indicated on nodes, maximum-parsimony sup-
port below nodes. Branch lengths are according to
indicated scale; the branch leading to the outgroup
(Halichoeres garnoti) was reduced by 50%.
Sequences were aligned and edited with Se-
quencher version 3.0 (Gene Codes Corp., Ann
Arbor, MI). The computer program MODEL-
TEST version 3.06 (Posada and Crandall, 1998)
was used to determine the substitution model
and the gamma distribution shape parameter
that best fit the data through hierarchical like-
lihood ratio tests (hLRTs). The HKY (Hasegawa
et al., 1985) substitution model was chosen for
all datasets and the gamma distribution shape
parameter varied from 0 in H. cyanocephalus to
0.1 in H. maculipinna to 0.4 in H. garnoti. The
analyses were also run using the Tamura-Nei
substitution model (Tamura and Nei, 1983)
with no significant changes in tree topology. Re-
lationships between unique haplotypes were re-
constructed based on starting trees calculated
using the neighbor-joining method (Nei, 1987)
and further searched by 10
7
tree bisection and
reconnection (TBR) iterations under the mini-
mum evolution criterion with the software
PAUP* version 4.0b10 (D. L. Swofford, Sinauer,
Sunderland, MA, 2002, unpubl.). Additionally,
maximum parsimony analyses were performed
on datasets with deep phylogenetic breaks. To-
pological confidence was evaluated with 1000
bootstrap replicates (Felsenstein, 1985). Equal
weighting of all three codon positions was used.
Population structure and gene flow were as-
sessed with the program Arlequin v2.0 (S.
Schneider, D. Roessli, and L. Excoffier, Univer-
sity of Geneva, Switzerland, 2000, unpubl.),
which generated F
ST
-values (a molecular analog
of F
ST
that takes into consideration sequence di-
vergence among haplotypes). Genetic variation
is described with nucleotide diversity (
p
, equa-
tion 10.19; Nei, 1987) and haplotype diversity
(h, equation 8.5; Nei, 1987) within each loca-
tion.
In addition to the genetic analysis, morpho-
logical comparisons were also carried out.
Counts and measurements follow Randall and
Lobel (2003). Measurements were taken with a
digital caliper and recorded to the nearest tenth
millimeter. Unless otherwise stated, institutional
abbreviations follow Leviton et al. (1985). Spec-
imens from the following museums were ex-
amined: MBML (Museu de Biologia Melo Lei-
ta˜o, Espı´rito Santo, Brazil), MCZ, MZUSP, SU,
UF, UFES (Colec¸a˜o Ictiolo´gica, Universidade
Federal do Espı´rito Santo, Brazil), UFPB (Co-
lec¸a˜o Ictiolo´gica, Universidade Federal da Pa-
raı´ba, Brazil), USNM and ZUEC (Museu de His-
toria Natural, Universidade Estadual de Cam-
pinas, Brazil).
R
ESULTS
Halichoeres cyanocephalus.—A 701 bp fragment
from the cytochrome b gene was analyzed from
17 individuals. A total of 26 polymorphic sites
distributed among 10 haplotypes were identi-
fied. Mean nucleotide frequencies were A
5
0.22, C
5
0.31, G
5
0.16, T
5
0.31. The tran-
sition:transversion ratio was 5.5:1. The phylo-
genetic analysis (Fig. 2) assigned the haplotypes
of H. cyanocephalus into two lineages (Brazil and
Caribbean). Pairwise distances within lineages
ranged from 1.17–2.83 nucleotide substitutions
(d
5
0.002–0.004). Although sample sizes are
low, Brazilian and Caribbean populations are
separated by at least 16 mutations (d
5
0.023)
and the F
ST
between Brazil and Belize was 0.91
(P
,
0.01). No morphological difference was
detected between the lineages; however, a slight
color difference is present (Fig. 5C–D).
Halichoeres garnoti.—A 704 bp fragment from
the cytochrome b gene was analyzed from 116
individuals. A total of 66 polymorphic sites were
distributed among 55 haplotypes. Mean nucle-
otide frequencies were A
5
0.22, C
5
0.30, G
5
0.16, T
5
0.32. The transition:transversion ra-
tio was 10.16:1. No geographic signal was de-
tected in the phylogenetic analysis of H. garnoti
(Fig. 3). Individuals with a red dorsum (indi-
cated by arrows and the letter B in Fig. 3) were
nested within the Caribbean lineage. Genetic
distances among populations ranged from 0.06–
2.45 nucleotide substitutions (d
5
0.001–0.004).
Haplotype diversity was relatively high and over-
all nucleotide diversity (
p
) was low in all pop-
ulations (Table 1). No significant population
structure was observed across the species’ entire
range from Bermuda to Venezuela (Table 2).
773ROCHA—MITOCHONDRIAL DNA VARIATION IN HALICHOERES
Fig. 3. Phylogenetic tree of all (55) Halichoeres gar-
noti unique haplotypes. No geographical signal was
detected in the phylogeny. Arrows indicate haplotypes
shared between Bermuda and one or more Caribbean
location; haplotypes only found in Bermuda are
marked with a B. Bootstrap support (
.
50%) is in-
dicated on nodes. Branch lengths are according to
indicated scale; the branch leading to the outgroup
(Halichoeres poeyi) was reduced by 50%.
Halichoeres maculipinna.—A 691 bp fragment
from the cytochrome b gene was analyzed from
86 individuals. A total of 71 polymorphic sites
distributed among 44 haplotypes were identi-
fied. Mean nucleotide frequencies were A
5
0.22, C
5
0.29, G
5
0.17, T
5
0.32. The tran-
sition:transversion ratio was 5.91:1. The phylo-
genetic analysis (Fig. 4) assigned the haplotypes
of Halichoeres maculipinna into two lineages (Bra-
zil and the North Atlantic) separated by 45.2–
49.0 nucleotide substitutions (d
5
0.065–0.071
sequence divergence). Genetic distances within
lineages ranged from 0.01–2.05 nucleotide sub-
stitutions (d
5
0.001–0.003). Haplotype diversity
was relatively high and overall nucleotide diver-
sity (
p
) was low in all populations of both line-
ages (Table 1).
The two lineages were allopatric, one north-
ern (Florida, Bahamas, St. Croix, Belize, and Ve-
nezuela) and the other southern (coastal Brazil
and Trindade Island), being separated by highly
significant F
ST
-values (F
ST
5
0.958–0.984). No
significant pairwise F
ST
-values were observed
within either lineage (Table 3). No morpholog-
ical difference was detected between the line-
ages; however, a slight color difference is pre-
sent (Fig. 5E–F).
D
ISCUSSION
Halichoeres cyanocephalus.—The genetic diver-
gence (d
5
2.3%) between the Brazilian and
Caribbean populations of H. cyanocephalus (Fig.
2) coincides with a subtle but consistent color
difference: individuals in Belize, Florida, and
the Virgin Islands have the color pattern shown
in Figure 5D, whereas individuals in north and
south Brazil have the pattern shown in Figure
5C. The Amazon is unlikely to be an effective
barrier between Brazilian and Caribbean line-
ages, because the Brazilian lineage occurs over
deep sponge bottoms off Brazil (Rocha et al.,
2000) and French Guiana (Uyeno et al., 1983),
the latter under the Amazon plume.
If not the Amazon barrier, what is causing ge-
netic differentiation between these lineages?
Rocha (2003b) suggests that it may be ecologi-
cal speciation or speciation driven by ‘‘divergent
selection on traits between populations or sub-
populations in contrasting environments’’
(Schluter, 2001). Environmental differences be-
tween locations in the Caribbean Sea (charac-
terized by clear waters all year round, relatively
stable environmental conditions, and bottom
sediments largely composed of calcium carbon-
ate) and the Brazilian coast (a typical continen-
tal environment, with terrigenous substrates in-
fluenced by run off from rivers, and high tur-
bidity caused by wind driven suspension of bot-
tom sediments) may generate divergent
selection pressures in the insular versus conti-
nental habitats, potentially promoting ecologi-
cal speciation, even in the presence of small mi-
gration between populations.
Halichoeres garnoti.—As pointed out by Smith-
Vaniz et al. (1999, pl. 12, figs. 78–80), there are
noticeable color differences between Caribbean
and Bermuda populations of this species (Fig.
774 COPEIA, 2004, NO. 4
T
ABLE
1. S
AMPLE
S
IZE
,N
UMBER OF
H
APLOTYPES
,H
APLOTYPE
D
IVERSITY
(h)
AND
N
UCLEOTIDE
D
IVERSITY
(
p
)
OF
THE
P
OPULATIONS
S
URVEYED
.
N Haplotypes h
p
Halichoeres cyanocephalus
Belize
NE Brazil
Total
12
5
17
6
4
10
0.68
6
0.15
0.90
6
0.16
0.002
6
0.001
0.004
6
0.003
Halichoeres garnoti
Bahamas
Belize
Bermuda
Florida Keys
St. Croix
21
21
27
3
23
17
15
20
1
18
0.97
6
0.03
0.95
6
0.03
0.97
6
0.02
0.00
6
0.00
0.98
6
0.02
0.008
6
0.005
0.009
6
0.005
0.008
6
0.004
0.00
6
0.00
0.009
6
0.005
Venezuela
Total
22
117
16
55
0.97
6
0.02 0.009
6
0.005
Halichoeres maculipinna
Bahamas
Belize
NE Brazil
Florida Keys
St. Croix
Trindade
19
19
5
3
18
5
12
12
4
2
9
3
0.90
6
0.06
0.87
6
0.07
0.90
6
0.16
0.67
6
0.31
0.80
6
0.09
0.70
6
0.21
0.003
6
0.002
0.002
6
0.001
0.002
6
0.001
0.001
6
0.001
0.002
6
0.001
0.001
6
0.001
Venezuela
Total
17
86
11
44
0.88
6
0.07 0.003
6
0.002
T
ABLE
2. P
OPULATION PAIRWISE
F
ST FOR
Halichoeres garnoti. No comparisons are significant at P
5
0.05.
Locations 123456
1. Bermuda
2. Florida
3. Bahamas
4. Belize
5. St. Croix
5. Venezuela
0
0.001
2
0.023
0.011
2
0.006
2
0.013
0
0.041
2
0.026
0.141
2
0.078
0
2
0.004
2
0.024
2
0.016
0
2
0.005
0.018
0
2
0.017 0
5A–B). Unlike what occurs in other Halichoeres
species (color differences are matched by fixed
genetic differences in the pair Halichoeres radia-
tus/brasiliensis and the Brazilian and Caribbean
lineages of H. cyanocephalus and H. maculipinna;
Rocha and Rosa, 2001b; Rocha, 2003a) color
differences are not accompanied by fixed ge-
netic differences at the cytochrome b of H. gar-
noti. The population of H. garnoti in Bermuda
shares 12 of its 20 haplotypes with one or more
of the Caribbean locations (Fig. 3). Moreover,
haplotypes endemic to Bermuda are randomly
distributed across the tree on Figure 3.
Three hypothesis may explain the absence of
genetic differentiation despite the presence of
color differences: (1) the population at Ber-
muda is exposed to some environmental con-
dition that causes their color to change phe-
notypically without an underlying genetic basis;
(2) color differences are associated with differ-
ences at loci other than the cytochrome b; (3)
the population at Bermuda is young, possibly
postdating the last glacial maximum, such that
color differences that appeared as a result of
strong selection, drift or founder effect are not
accompanied by fixed genetic differences at the
cytochrome b.
The first two hypotheses remain untested;
however, there is evidence favoring the third.
During the last glacial maximum (25,000 to
15,000 yr B.P.), temperatures at Bermuda were
3–5 C lower than today (Sachs and Lehman,
1999), bringing mean annual temperatures be-
low the 21 C (with much lower temperatures
during the winter) threshold necessary for reef
coral survival and leading to the likely exter-
775ROCHA—MITOCHONDRIAL DNA VARIATION IN HALICHOERES
Fig. 4. Phylogenetic tree of Halichoeres maculipinna
(Caribbean) and Halichoeres penrosei (Brazil) unique
haplotypes. Neighbor-joining bootstrap support (
.
50%) indicated on nodes, maximum-parsimony sup-
port below nodes. Branch lengths are according to
indicated scale; the branch leading to the outgroup
(Halichoeres poeyi) was reduced by 50%.
T
ABLE
3. P
OPULATION PAIRWISE
F
ST FOR
Halichoeres maculipinna. Asterisks indicate significance at P
5
0.05.
Locations 1 2 3 4 5 6 7
1. Florida
2. Bahamas
3. Belize
4. St. Croix
5. Venezuela
6. Coastal Brazil
7. Trindade
0
2
0.101
2
0.075
2
0.105
2
0.100
0.973*
0.984*
0
0.006
0.010
2
0.002
0.958*
0.961*
0
2
0.014
0.013
0.969*
0.972*
0
0.009
0.971*
0.974*
0
0.959*
0.963*
0
0.001 0
mination of most of the tropical reef fish fauna
(Smith-Vaniz et al., 1999). Assuming that H. gar-
noti is a strictly tropical species (it does not oc-
cur in the northern Gulf of Mexico or in any
coastal location north of Florida), it is probable
that the Bermuda population is less than 15,000
years old. Thus, color differences may have a
genetic basis but were not detected in neutral
genes (such as the cytochrome b) because of
insufficient time for lineage sorting (Avise,
2000).
Halichoeres maculipinna.—The pelagic larval du-
ration (PLD) of H. maculipinna (30 days; Spon-
augle and Cowen, 1997) is the longest among
the Atlantic Halichoeres. However, this species
showed the deepest genetic divergence ob-
served in this study (d
5
6.5%) between Brazil-
ian and Caribbean populations, demonstrating
the poor value of PLD as a predictor of genetic
divergence. Despite the deep genetic split be-
tween Brazil and the Caribbean, careful exami-
nation of several individuals of H. maculipinna
from locations at the two regions revealed no
morphological differences. However, slight dif-
ferences in coloration were detected (Fig. 5E–
F).
The divergence observed between Caribbean
and Brazilian lineages of H. maculipinna is the
highest among all species examined. One pos-
sible explanation is that this high divergence is
a result of strict habitat preferences. There are
no field survey data that indicates that H. ma-
culipinna crosses the Amazon barrier; it is a shal-
low water species (20–30 m maximum depth;
Randall and Bo¨hlke, 1965; Humann and De-
loach, 2002) and was not collected under the
Amazon plume (Collette and Ru¨tzler, 1977) or
on deep sponge bottoms off northeastern Brazil
(Rocha et al., 2000). Moreover, H. maculipinna
has the weakest jaw musculature, the smallest
mouth gap and the most specialized diet (fewer
and smaller species of benthic invertebrates
compared to congeners) among all Atlantic
Halichoeres (Randall, 1967; Wainwright, 1988).
As a specialized predator, H. maculipinna may be
more sensitive to habitat differences than the
other more generalist species surveyed here. In
this case, stronger natural selection may lead to
earlier and faster differentiation, possibly gen-
erating the higher observed divergences in this
species.
Taxonomic implications.—The genetic divergence
between the Caribbean and Brazilian popula-
tions observed in this study equals or exceeds
divergences between recognized sister species
pairs of other wrasses (Rocha, 2003a; Bernardi
et al., 2004). In addition, there are diagnostic
color characters that distinguish the Brazilian
populations from their Caribbean counterparts
776 COPEIA, 2004, NO. 4
Fig. 5. Halichoeres garnoti at the Bahamas (A) and Bermuda (B). Halichoeres dimidiatus at northeastern Brazil
(C) and Halichoeres cyanocephalus at Belize (D) Halichoeres penrosei at northeastern Brazil (E) and Halichoeres
maculipinna the Bahamas (F). All photos taken by Luiz Rocha, except 5C (by Gerald Allen) and 5D (by Jack
Randall).
(Fig. 5). Randall (1998) suggested that the im-
portance of diagnostic color characters in reef
fish systematics is dramatically increased when
those characters are observed in combination
with (even minor) diagnostic counts or mea-
surements. Here I propose that this principle
should be extended to genetics: the coinci-
dence of diagnostic genetic and color charac-
ters should warrant elevation of evolutionary
lineages to specific status. Thus, the Brazilian
populations of H. cyanocephalus and H. maculi-
pinna should be considered valid species.
Bloch (1791) gave no type locality for H. cyan-
ocephalus, and no types are known (Randall and
Bo¨hlke, 1965; Eschmeyer, 1998; Paepke, 1999;
Parenti and Randall, 2000). The original de-
scription (Bloch, 1791:140) states [my transla-
tion]: ‘‘The residence of this fish is for me un-
known. The original drawing is kept in the cab-
inet of Linkeschen (Heinrich Linck).’’ More-
over, the original description is inaccurate and
incomplete because it is based on a single, ap-
parently adult individual. Nonetheless, this
name has been associated with the Caribbean
species since at least the end of the 19th century
( Jordan and Evermann, 1898).
To clarify the taxonomic status and the type
locality of H. cyanocephalus (Bloch, 1791), and
777ROCHA—MITOCHONDRIAL DNA VARIATION IN HALICHOERES
Fig. 6. Neotype of Halichoeres cyanocephalus (UF 224284). Photo by Luiz Rocha.
in accordance with article 75(3) of the Inter-
national Code of Zoological Nomenclature
(ICZN, 1999), I designate UF 224284 (202.2
mm SL) as a neotype. Detailed descriptions of
H. cyanocephalus are available in the scientific lit-
erature (Randall and Bo¨hlke, 1965) and in most
Caribbean reef fish guides (Randall, 1996; Hu-
mann and Deloach, 2002).
Halichoeres cyanocephalus (Bloch, 1791)
Figures 5D, 6
Neotype.—UF 224284, adult from Haulover Cut,
North Miami Beach, Florida, collected on 8 Au-
gust 1967 by W. Zeiller, at a depth between 18
and 27 m. This specimen was chosen because it
has the typical characters described by Randall
and Bo¨hlke (1965) and because it is the best
preserved specimen of this taxon at the Univer-
sity of Florida.
Description of the neotype.—Dorsal IX, 12; anal III,
12; pectoral ii,11; total gill rakers on first arch
19 (seven on the upper limb); lateral-line scales
27 (20
1
2
1
5); anterior lateral-line scales with
three pores; two pairs of enlarged canine teeth
anteriorly in lower jaw, one pair on upper jaw.
Standard length 202.2 mm; total length 227.5
mm; body depth 29.1% SL; caudal peduncle
depth 14.6% SL; head length (HL) 28.3% SL;
snout length 37.8% HL; upper jaw length 22%
HL; eye diameter 14.1% HL; interorbital width
22.3% HL.
Color in alcohol.—Body light brown with a broad
dark brown stripe on upper half, beginning at
pectoral fin and extending into middle of cau-
dal fin; a narrower stripe of lighter brown be-
tween the broad stripe and the dorsal fin. Faint
indication of a dark bar on head, extending up-
ward diagonally from upper posterior half of
eye to upper portion of head. Dorsal fin dark
brown on base, pale on margin; upper base of
pectoral fin with a dark spot.
Distribution, habitat, and ecology.—Caribbean is-
lands, North American coast from Florida to
North Carolina and Central American coast
from Colombia to Belize. Usually solitary, rare
in shallow waters, but juveniles and small adults
frequently observed between 5 and 15 m at Be-
lize; mostly observed at depths over 30 m at the
remaining locations.
The Brazilian species should now be consid-
ered valid as follows.
Halichoeres dimidiatus (Agassiz, in Spix and
Agassiz, 1831)
Figures 5C, 7
This species was originally described as Julis
dimidiatus by Agassiz, in Spix and Agassiz, 1831,
and later placed in the synonymy of Halichoeres
cyanocephalus (Bloch, 1791) by (Jordan and Ev-
ermann, 1898). Agassiz states that several spec-
imens were preserved, but only one (MHNN
563, 153 mm SL) still exists (Kottelat, 1988).
The type locality is given as Atlantic, off Brazil
(Spix and Agassiz, 1831).
Diagnosis.—Dorsal IX, 12 (IX, 11 on remaining
Atlantic Halichoeres, except H. cyanocephalus);
anal III, 12; pectoral 13; total gill rakers 18 to
21; two pairs of enlarged canine teeth anteriorly
in lower jaw. Juveniles and females blue with a
bright yellow region dorsally from mouth to
posterior base of dorsal fin; a single dark spot
on caudal (a small ocellated dark spot at rear
base of dorsal fin in addition to the caudal spot
in H. cyanocephalus). Adults with a broad blue
stripe on upper half of body ending at the be-
ginning of the caudal fin (black and narrowing
as it passes to end of caudal fin in H. cyanoce-
phalus); lower half of body light blue; a diagonal
dark band from eye to nape.
Distribution, habitat, and ecology.—From French
Guyana (Uyeno et al., 1983) to the State of Sa˜o
778 COPEIA, 2004, NO. 4
Fig. 7. Adult Halichoeres dimidiatus, approximately 150 mm SL, Guarapari, southeast Brazil. Photo by Joa˜o
Luiz Gasparini.
Fig. 8. Halichoeres penrosei, approximately 50 mm SL, Guarapari, southeast Brazil. Photo by Joa˜o Luiz Gas-
parini.
Paulo (24
8
S, 46
8
W), in southeastern Brazil (Me-
nezes and Figueiredo, 1985). It is also present
at Fernando de Noronha and Atol das Rocas
(20
8
30
9
S, 29
8
20
9
W) but apparently absent from
the remaining Brazilian oceanic islands of Trin-
dade and St. Paul’s Rocks (Gasparini and Floe-
ter, 2001; Feitoza et al., 2003). Usually observed
solitary; juveniles relatively common in shallow
waters (3–20 m), adults in deeper waters (30–
60 m).
Halichoeres penrosei Starks, 1913
Figures 5E, 8
The Brazilian form of H. maculipinna was de-
scribed as Halichoeres penrosei by Starks (1913);
the 55.7 mm SL holotype (SU 22211) is well
preserved and was collected in tide pools at Na-
tal, northeastern Brazil (5
8
S, 35
8
W), located at
the middle of the range of the species. Digital
photographs and a radiograph of the holotype
are available for download at the California
Academy of Sciences website (http://www.
calacademy.org/research/ichthyology/Types/
index.html).
Diagnosis.—Dorsal IX, 11; anal III, 11; pectoral
14; gill rakers 13–15; one pair of enlarged ca-
nine teeth anteriorly in lower jaw (two pair of
canines on remaining Atlantic Halichoeres except
H. maculipinna). Juveniles and females with
wide black stripe through eye to base of tail,
bordered above by prominent pinkish-brown
line (bright yellow in H. maculipinna). White
scales with orange spots on lower half of body
(no spots on H. maculipinna) Adult males are
primarily green with scales bordered by pink
and a black spot on anterior portion of the dor-
sal fin. Narrow (
,
0.5 mm width) orange stripes
on lower half of head, from mouth to gill open-
ing (stripes are
.
1 mm wide in H. maculipin-
na).
Distribution, habitat, and ecology.—From Parcel
Manuel Luiz (0
8
52
9
S, 44
8
15
9
W; Rocha and Rosa,
2001a) to the State of Sa˜o Paulo (24
8
S, 46
8
W),
in southeastern Brazil (Carvalho-Filho, 1999). It
is present at Trindade Island (20
8
30
9
S, 29
8
20
9
W;
Gasparini and Floeter, 2001), but apparently ab-
sent from the remaining Brazilian oceanic is-
lands (Atol das Rocas, Fernando de Noronha,
779ROCHA—MITOCHONDRIAL DNA VARIATION IN HALICHOERES
and St. Paul’s Rocks; Feitoza et al., 2003). Com-
mon in coral and rocky reef tops to depths of
30 m and often observed solitary or in pairs.
C
ONCLUSIONS
Contrary to expectations of congruent pop-
ulation structure due to similar dispersal poten-
tial (25–30 days; Sponaugle and Cowen, 1997;
Wellington and Robertson, 2001), the genetic
effects of biogeographical barriers varied
among species of Atlantic Halichoeres. Genetic
divergences between Brazil and the North At-
lantic ranged from 2.3% in H. cyanocephalus/H.
dimidiatus to 6.5% in the pair H. maculipinna/
H. penrosei. There is inconsistency between color
differences and genetic partitions in the species
surveyed. The color differences between the
species pairs H. cyanocephalus/H. dimidiatus and
H. maculipinna/H. penrosei correspond to deep
genetic partitions at the cytochrome b gene.
However, genetic similarity at this same gene
was observed between populations of H. garnoti
with striking color differences. Thus, caution
against the use of color differences (when not
supported by genetics and/or morphology) in
the alpha systematics of fishes is herein suggest-
ed, a conclusion also reached by others (Mc-
Millan et al., 1999; Bernardi et al., 2004).
Genetic divergences ranged from 2.3–6.5% in
lineages in Brazil and the Caribbean. A conven-
tional molecular clock of 2% per million years
for the cytochrome b gene (Avise, 2000) sug-
gests that this divergence is between 0.6 and
1.65 millions of years (myr) old, much younger
than the estimated age of the initial connection
of the Amazon with the Atlantic, 10–12 myr old
(Hoorn, 1994; Nittrouer et al., 1996; Costa et
al., 2001). Thus, vicariance alone cannot ex-
plain the observed pattern, and dispersal from
one area to the other after the formation of the
Amazon is probably involved in speciation of
western Atlantic Halichoeres.
The species pair with the highest divergence
(6.5 %, H. maculipinna/H. penrosei) is also the
more reef specialized and the one with the nar-
rowest diet width. Although apparently being
able to cross the Amazon barrier, a deep phy-
logenetic break (2.3 %) between Brazil and the
Caribbean is present in the pair H. cyanocephal-
us/H. dimidiatus. These observations indicate
that divergent environmental conditions (con-
tinental sediment-rich Brazilian waters versus in-
sular, clear Caribbean waters) can be as impor-
tant as vast distances of unsuitable habitat (ei-
ther open ocean waters or the area influenced
by the Amazon) in producing genetic partitions
and that benthic-stage habitat preferences may
be strongly influencing the biogeography of
western Atlantic reef fishes.
M
ATERIAL
E
XAMINED
Voucher catalog numbers followed by num-
ber of specimens and GenBank accession num-
bers in parentheses (when available).
Halichoeres cyanocephalus: Cuba: MCZ 14252 (3,
syntypes of Julis internasalis Poey 1861); Flor-
ida: UF 65506 (1), UF 224284 (1, neotype of
H. cyanocephalus); North Carolina: UF 88359
(2); Belize: UF 126324 (1, AY591376); St.
John, U.S. Virgin Islands: UF 206250 (1); Ja-
maica: UF 229817 (1); Colombia: UF 231301
(1). Sequences without museum voucher:
AY591377–AY591379.
Halichoeres dimidiatus: (all from Brazil); Parcel
Manoel Luiz, Maranha˜o: MZUSP 53066 (2),
UFPB 3890 (1), UFPB 3977 (1); Fernando de
Noronha: MZUSP 14626 (1); Mamanguape,
Paraı´ba: UFPB 4312 (1, AY591380); Cabedelo,
Paraı´ba: UFPB 4325 (2); Joa˜o Pessoa: UFPB
3790 (1), UFPB 4354 (2, AY591381,
AY591382); Recife: MZUSP 47482 (1); Salva-
dor, Bahia: USNM 357718 (1); Guarapari, Es-
pı´rito Santo: MZUSP 51543 (1), ZUEC 3184
(1), ZUEC 3196 (1), MBML 354 (28); Angra
dos Reis, Rio de Janeiro: MZUSP 66256 (2);
Alcatrazes, Sa˜o Paulo: MZUSP 47146 (1),
USNM 357717 (1), ZUEC 2793 (1). Sequenc-
es without museum voucher: AY591383.
Halichoeres garnoti: Bermuda: UF 119713 (11,
AY591372, AY591374), UF 119725 (1,
AY591375), USNM 348309 (1); Bahamas: UF
13485 (5); Cayman Islands: UF 17620 (3); Co-
lombia: UF 18830 (2); St. John U.S. Virgin
Islands: UF 204900 (2); Belize: UF 209277
(4); Cuba: USNM 343625; Tobago: USNM
318896 (7). Sequences without museum
voucher: AY591366–AY591371.
Halichoeres maculipinna: Bermuda: UF 119714
(2), UF 119731 (9, AY591359); St. John, U.S.
Virgin Islands: UF 203752 (4); Bahamas: UF
206337 (5); Florida Keys: UF 219121 (12); Co-
lombia: UF 223079 (12); Belize: USNM
329838 (1); Tobago: USNM 318864 (4). Se-
quences without museum voucher:
AY591360–AY591365.
Halichoeres penrosei: (all from Brazil); Natal: SU
22211 (1, holotype); Espı´rito Santo: MZUSP
52303 (1), MBML 223 (2), 318(1), 395(1,
AY591354), UFES 037 (1), 208(1); Ceara´:
MZUSP 65172 (1); Arembepe, Bahia: MZUSP
66259 (3); Salvador, Bahia: MZUSP 66268 (10
of 25). Sequences without museum voucher:
AY591355–AY591358.
780 COPEIA, 2004, NO. 4
A
CKNOWLEDGMENTS
I thank my Ph.D. advisor, B. Bowen, for pro-
viding scientific guidance, laboratory space, and
financial support. I also thank A. L. Bass, A. Cas-
tro, H. Choat, B. B. Collette, M. Coura, T. L.
Dias, B. M. Feitoza, C. E. Ferreira, S. R. Floeter,
J. L. Gasparini, Z. Hillis-Starr, S. Karl, B. Luck-
hurst, O. Luiz-Junior, D. Murie, D. Parkyn, B.
Philips, J. Pitt, C. Rocha, D. R. Robertson, I. L.
Rosa, R. S. Rosa, S. Sponaugle, B. Victor, and D.
Weaver for help with sampling and laboratory
work. Discussions with C. Gilbert, S. A. Karl, N.
Knowlton, G. Paulay, and M. Westneat greatly
improved the manuscript. S. Floeter, H. Lessios,
and W. F. Smith-Vaniz critically read the manu-
script. G. Allen, J. L. Gasparini, and J. Randall
provided excellent photographs. Specimen
loans, digital photographs of type specimens,
and access to fish collections for morphological
analyses were made available by G. Burgess, D.
Catania, W. Eschmeyer, K. Hartel, S. Jewett, N.
Menezes, R. Robins, R. Rosa, I. Sazima, A. Var-
andas, and J. Williams. 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), and Instituto Brasileiro do Meio Am-
biente e dos Recursos Naturais Renova´veis, Bra-
zil (general collecting permit for nonprotected
areas). Fishes were collected following the
guidelines of the University of Florida Institu-
tional Animal Care and Use Committee. This
work was financially supported by CAPES Bra-
zilian Ministry of Education, the Brazilian Navy,
PADI Project Aware, the Smithsonian Tropical
Research Institute, the University of Florida,
and the National Science Foundation through
a grant to B. Bowen (DEB 9727048).
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