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Phylogenetic Relationships of the Shafted Bonefish Albula Nemoptera (Albuliformes: Albulidae) from the Eastern Pacific Based on Cytochrome B Sequence Analyses

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  • Instituto Nacional de Pesca y Acuacultura de México. Centro Regional de Investigación Pesquera en Guaymas

Abstract and Figures

Nucleotide and amino acid sequence data from a 544-bp segment of the mitochondrial cytochrome b (Cytb) gene were used to examine phylogenetic relationships of the Shafted Bonefish, Albula (=Dixonina) nemoptera, a morphologically distinct bonefish limited to the coastal waters of the eastern Pacific and western Atlantic, among members of the widely distributed and cryptic A. vulpes species complex of Albula (Albuliformes: Albulidae). Phylogenetic trees based on Bayesian and parsimony methods indicated that A. nemoptera from the tropical eastern Pacific (Colima, México) nested within the A. vulpes complex, supporting the placement of the genus Dixonina Fowler, 1911 into the synonymy of Albula Scopoli, 1777. Phylogenetic analyses also showed that A. nemoptera is the sister taxon to the undescribed Albula sp. E from the western Atlantic (Bahia, Brazil), previously placed in the A. vulpes complex. This evidence, together with the presence of a diagnostic amino acid substitution in the translated Cytb segment in both species, suggested that Albula sp. E, identified solely from molecular data with no adult voucher available, should be provisionally assigned to A. nemoptera. © 2006 by the American Society of Ichthyologists and Herpetologists.
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Phylogenetic Relationships of the Shafted Bonefish Albula nemoptera
(Albuliformes: Albulidae) from the Eastern Pacific Based on
Cytochrome bSequence Analyses
EDWARD PFEILER,BEN G. BITLER,AND RAU
´LULLOA
Nucleotide and amino acid sequence data from a 544-bp segment of the
mitochondrial cytochrome b(Cytb) gene were used to examine phylogenetic relation-
ships of the Shafted Bonefish, Albula (5Dixonina)nemoptera, a morphologically distinct
bonefish limited to the coastal waters of the eastern Pacific and western Atlantic,
among members of the widely distributed and cryptic A. vulpes species complex of
Albula (Albuliformes: Albulidae). Phylogenetic trees based on Bayesian and parsimony
methods indicated that A. nemoptera from the tropical eastern Pacific (Colima, Me´xico)
nested within the A. vulpes complex, supporting the placement of the genus Dixonina
Fowler, 1911 into the synonymy of Albula Scopoli, 1777. Phylogenetic analyses also
showed that A. nemoptera is the sister taxon to the undescribed Albula sp. E from the
western Atlantic (Bahia, Brazil), previously placed in the A. vulpes complex. This
evidence, together with the presence of a diagnostic amino acid substitution in the
translated Cytb segment in both species, suggested that Albula sp. E, identified solely
from molecular data with no adult voucher available, should be provisionally assigned
to A. nemoptera.
Secuencias de nucleo´tidos y aminoa´cidos que provinieron de un segmento de 544 bp
del gen citocromo b(Cytb) de las mitocondrias fueron utilizadas para analizar las
relaciones filogene´ticas del macabı´ de hebra Albula (5Dixonina)nemoptera, un macabı´
morfolo´gicamente distinto que habita las aguas costeras del oce´ ano Pacifico Oriental y
el Atla´ntico Occidental, con las especies crı´pticas del complejo A. vulpes del ge´nero
Albula (Albuliformes: Albulidae) que tienen una amplia distribucio´n. Dos clases de
a´rboles filogene´ticos estaban de acuerdo y demostraron que el A. nemoptera del
Pacifico Oriental Tropical (Colima, Me´xico) se agruparon con las especies del
complejo A. vulpes, apoyando la conclusio´n de que el ge´nero Dixonina Fowler, 1911
deberı´a ser considerado un sino´nimo del ge´nero Albula Scopoli, 1777. Los ana´lisis
filogene´ticos tambie´n demostraron que la especie hermana a la A. nemoptera es la
especie no identificada Albula sp. E del Atla´ntico Occidental (Bahı´a, Brasil) que
previamente fue colocada en el complejo A. vulpes. Esta evidencia, ası´ como la
presencia de una sustitucio´ n diagno´stica de un aminoa´cido en la proteina de citocromo
ben ambas especies, sugirieron que el Albula sp. E, que fue descrito utilizando
solamente datos moleculares sin especimenes completos, deberı´a ser asignado
provisionalmente a la especie A. nemoptera.
THE bonefishes (Albuliformes, Albulidae,
Albula spp.) comprise a group of ancestral
teleost fishes belonging to the superorder (or
subdivision) Elopomorpha distributed worldwide
in coastal tropical and subtropical marine habi-
tats (Briggs, 1960; Hildebrand, 1963). With the
exception of the geographically restricted and
morphologically distinct Shafted Bonefish, Albula
(5Dixonina)nemoptera, the bonefishes had his-
torically been placed into a single taxon, Albula
vulpes. Allozyme and mitochondrial DNA (mt-
DNA) studies, however, have shown that A. vulpes
(sensu lato) represents a species complex pres-
ently thought to include at least eight genetically
distinct cryptic species (Shaklee and Tamaru,
1981; Pfeiler, 1996; Colborn et al., 2001), several
of which (e.g., Albula spp. A, B, C, D, and E) are
currently undescribed and unnamed.
Albula nemoptera inhabits the western Atlantic
and eastern Pacific (type locality: Santo Do-
mingo, Dominican Republic) and can be distin-
guished from members of the A. vulpes complex
by its larger mouth, longer and more conical
snout, prolonged last ray of the dorsal and anal
fins, and higher vertebral counts (Hildebrand,
1963; Rivas and Warlen, 1967). Nonetheless, A.
nemoptera is remarkably similar in general ap-
pearance to members of the A. vulpes complex
and can be easily confused with them on casual
observation (Myers, 1936; also see Kumada and
Copeia, 2006(4), pp. 778–784
#2006 by the American Society of Ichthyologists and Herpetologists
Hiyama [1937], who figured a specimen of A.
nemoptera labeled as A. vulpes). The phylogenetic
relationship of A. nemoptera to the A. vulpes
complex remains unresolved, mainly owing to
a lack of molecular data for the relatively
uncommon A. nemoptera. Although originally
assigned to the genus Dixonina Fowler, 1911,
the Shafted Bonefish is now generally recognized
as belonging to the genus Albula Scopoli, 1777,
following the work of Rivas and Warlen (1967).
Rivas and Warlen (1967), however, stated that
their grounds for placing Dixonina into the
synonymy of Albula were largely subjective. This
caveat, together with the morphological similar-
ity of members of the A. vulpes complex, has
raised the possibility of resurrecting the name
Dixonina for the Shafted Bonefish (Pfeiler et al.,
2002).
In the eastern Pacific, A. nemoptera is found in
coastal waters from Mexico to Panama (Allen and
Robertson, 1994; Castro-Aguirre et al., 1999).
Here we report results of analyses of cytochrome
b(Cytb) nucleotide and amino acid sequence
data from a sample of A. nemoptera collected
along the Pacific coast of central Mexico that
provide new insights into the phylogenetic
relationships and systematics of A. nemoptera
and the A. vulpes complex.
MATERIALS AND METHODS
Animals and source of sequences.—Seven specimens
of A. nemoptera were collected at Manzanillo,
Colima, Mexico during September 2004. Cytb
sequences from representatives of all described
and putative species of the A. vulpes complex
(Colborn et al., 2001) were obtained from
GenBank or from Pfeiler et al. (2002). The A.
vulpes complex species (with specimen identifi-
cation, geographic localities, and GenBank ac-
cession numbers) included Albula sp. A (ALB33;
Gulf of California, Mexico [Guaymas, Sonora];
AF311757), Albula sp. B (ALB10; western Atlantic
[Florida, USA]; AF311751), Albula sp. C (ALB40;
eastern Pacific [Gulf of Panama, Panama];
AF311760), Albula sp. D (ALB77; eastern Indian
Ocean [western Australia]; AF311770), Albula sp.
E (ALB22, ALB23; western Atlantic [Bahia,
Brazil]; AF311754 and AF311755), A. vulpes
(ALB7; western Atlantic [Belize]; AF311771), A.
glossodonta (ALB61; central Pacific [Hawaii,
USA]; AF311768), and A. forsteri (5A. neogui-
naica; central Pacific [ALB45; Hawaii, USA;
AF311763 and ALB54; Fiji; AF311765]). Se-
quences for Albula sp. A from southern California
(Ca-1; Pfeiler et al., 2002) and for a bonefish
identified as A. glossodonta (GenBank accession
number AP002973; Inoue et al., 2004) also were
incorporated into the data matrix. Representa-
tive new Cytb sequences for A. nemoptera (speci-
mens A12, A15, and A16) have been deposited in
GenBank (DQ272657–DQ272659). The seven
individuals of A. nemoptera (A11–A17), and
corresponding muscle tissue samples preserved
in 95%ethanol, have been deposited in the
fish collection at the Centro de Investigacio´n
en Alimentacio´n y Desarrollo in Guaymas,
Sonora (CIAD 04–100). We chose the albulid
Pterothrissus gissu (subfamily Pterothrissinae; Gen-
Bank accession no. AB051197; Inoue et al.,
2004), as one of the outgroup taxa. Elops
hawaiensis (Elopiformes, Elopidae; GenBank ac-
cession no. AB051070), an elopomorph ancestral
to the Albulidae (Inoue et al., 2004), was used as
an additional outgroup.
DNA extraction, gene amplification, and sequencing.—
Total genomic DNA was extracted from dorsal
white muscle using either the DNAzolH(Molec-
ular Research Center, Inc., Cincinnati, Ohio) or
the DNeasy
TM
(QIAGEN Inc., Valencia, Califor-
nia) protocol with proteinase K digestion. Poly-
merase chain reaction (PCR) was used to amplify
a 650-bp segment of the Cytb gene using primers
H14803 (59-TGCTAGGGTTGTGTTTAATT-39)
and L15526 (59-GTCTCCAAGAAGGTTAGG-
CGA-39). PCR was performed on a Perkin-Elmer
Thermal Cycler 480 in a reaction mixture
containing 1 ml template DNA, 5 ml103PCR
buffer (0.1 M Tris-HCl, 0.5 M KCl, pH 9.0),
1.25 ml of 10 mM dNTP, 2 ml of each 10 mM
primer, 10 ml 25 mM MgCl
2
, and 1.5–2.5 U Taq
DNA polymerase (Fisher Scientific, Fair Lawn,
NJ) and brought up to 50 ml with water. After
an initial denaturation at 94 C for 3 min, PCR
reaction conditions consisted of 30 cycles of
94 C for 1 min of denaturation, 45 C for
1 min of annealing, and 72 C for 1 min of
extension, followed by a final extension of
10 min. Verification of successful amplification
was assessed by agarose gel electrophoresis.
These PCR parameters consistently amplified
a single Cytb gene fragment with no secondary
bands seen.
Forward and reverse sequencing reactions
were performed on an Applied Biosystems
(Foster City, CA) ABI 3700 DNA sequencer at
the DNA Sequencing Facility, University of
Arizona, using the PCR primers. Alignments
were performed in ClustalX1.81 (Thompson et
al., 1997) followed by manual editing.
Data analyses.—Aligned DNA sequences were
imported into MEGA version 3.1 (Kumar et al.,
2004) for determination of genetic distances.
Calculations of genetic diversity indices were
PFEILER ET AL.—SHAFTED BONEFISH RELATIONSHIPS 779
performed in DnaSP version 3.51 (Rozas and
Rozas, 1999).
Phylogenetic relationships among bonefishes
were assessed using both Bayesian and maximum
parsimony (MP) methods. Analysis of the Cytb
data set with Modeltest 3.7 (Posada and Crandall,
1998; Posada and Buckley, 2004) indicated
that the model of nucleotide substitution that
best fit our data using the Akaike Information
Criterion was TN93+I+G(TamuraandNei,
1993). The parameters of this model were
then used in Bayesian analyses implemented
in MrBayes version 3.1 (Huelsenbeck and Ron-
quist, 2001). Analyses were run for 5,000,000
generations, sampled every 250
th
generation
(20,000 trees sampled), using the default ran-
dom tree option to begin the analysis and either
including or excluding third codon positions
(see below). Clade support was estimated utiliz-
ing a Markov chain Monte Carlo (MCMC)
algorithm. Log-likelihood values from four si-
multaneous MCMC chains (three hot and one
cold) stabilized at about 15,000 generations,
resulting in the first 60 trees being discarded
from the analysis (burnin 560). Maximum
parsimony analyses were carried out in
MEGA using the Max-mini branch-and-bound
algorithm (Kumar et al., 2004). Relative support
for MP tree topology was obtained by boot-
strapping (Felsenstein, 1985) using 1000
pseudoreplicates.
Nucleotide sequence divergence in the Cytb
gene segment can exceed 30%among bonefish
species (Colborn et al., 2001), suggesting that
transition substitutions are saturated, especially
at the third codon position. Plotting the number
of transitions and transversions at each codon
position against Tamura-Nei distances using the
computer program DAMBE (Xia and Xie, 2001)
revealed that third position transitions were
indeed saturated, whereas plots of all other
substitutions were linear (not shown). Phyloge-
netic relationships, therefore, were also exam-
ined with the two different tree-building algo-
rithms after deleting all third codon positions.
This resulted in both improved statistical support
for the deeper nodes and more consistent
topology among trees.
RESULTS
Sequence analysis and genetic diversity.—PCR
yielded a 608-bp segment of the Cytb gene in
the seven specimens of eastern Pacific A.
nemoptera, after deleting the primer sequences.
Average base frequencies (19%A, 19%C, 23%G,
and 39%T) were similar to those reported
previously for species of the A. vulpes complex
(Colborn et al., 2001). There were four variable
sites in the seven sequences, all third position
transitions. As expected for a protein-coding
gene, no stop codons, insertions, or deletions
were found.
For inferring phylogenetic relationships
within the genus Albula, we trimmed the
new sequences in A. nemoptera to 544 bp so
that they corresponded to the larger data set
in Colborn et al. (2001). Four haplotypes
were present in the seven trimmed sequences;
haplotype and nucleotide diversities (6SD)
were 0.81 60.13 and 0.003 60.001, respectively.
The four different CytbhaplotypesinA.
nemoptera are represented by specimens A12,
A15, A16, and A17 in the Bayesian phylogenetic
tree.
Phylogenetic relationships.—Phylogenetic relation-
ships among bonefishes revealed by Bayesian and
MP methods after deleting third codon positions
are shown in Figs. 1 and 2, respectively. Both
trees resolved two major lineages, one comprised
of Albula sp. D +A. forsteri +Albula sp. E +A.
nemoptera, and the other, which was only weakly
supported, containing Albula sp. A +sp. B +sp. C
+A. vulpes +A. glossodonta. The clustering of
A. glossodonta (GenBank accession number
AP002973) with Albula sp. D and A. forsteri with
high bootstrap support in Figs. 1 and 2 probably
resulted from specimen misidentification (see
Discussion). With the exception of the place-
ment of Albula sp. E, the relationships shown in
Figs. 1 and 2 were similar to those seen in the
neighbor-joining tree (tree C) of Colborn et al.
(2001), which was based on Kimura’s (1980) 2-
parameter (K2P) distances using all three codon
positions and midpoint rooting. The placement
of Albula sp. E, however, varied in the different
trees reported in Colborn et al. (2001), with the
MP tree (tree D) showing Albula sp. E clustering
with Albula sp. D and A. forsteri as shown here in
Figs. 1 and 2. In addition, the sister group
relationships previously found for Albula sp. A +
C, Albula sp. D +A. forsteri, and A. vulpes +A.
glossodonta (Colborn et al., 2001) were confirmed
in our analyses.
Figures 1 and 2 consistently showed that
A. nemoptera from the eastern Pacific nested
within the A. vulpes complex, forming a gener-
ally well-supported clade with the previously
unidentified Albula sp. E (Colborn et al., 2001)
from the western Atlantic. The average value
for both the uncorrected pdistance and
K2P distance between Albula sp. E and A.
nemoptera (all codon positions included) was
0.04, suggesting that the two species are very
similar genetically.
780 COPEIA, 2006, NO. 4
Translation of the Cytbgenesegmentin
Albula spp. yielded a protein containing 180
amino acids. Analysis of variable amino acid
positions in this segment (Table 1) revealed
a unique valine (V) to alanine (A) substitution
at site 145 in both Albula sp. E and A. nemoptera.
Albula sp. D and A. forsteri, which, together with A.
glossodonta (AP002973), clustered with high statis-
tical support in both phylogenetic trees, shared six
unique amino acid substitutions. The sister spe-
cies Albula sp. A from the Gulf of California and
Albula sp. C from the Gulf of Panama differed by
a single amino acid at site 147. The two separate
populations of Albula sp. A from the eastern
Pacific differed by two amino acids, as reported
earlier (Pfeiler et al., 2002).
DISCUSSION
Phylogenetic relationships.—Molecular data have
not been available previously to assess the
relationship of A. nemoptera to members of the
A. vulpes complex. It had been assumed that the
morphologically distinct A. nemoptera would form
the natural outgroup in phylogenetic studies of
the A. vulpes complex (Colborn et al., 2001).
However, phylogenetic analyses of mtDNA se-
quence data from a segment of the Cytb gene of
A. nemoptera from the Pacific coast of Mexico,
together with representative published se-
quences of all described and putative bonefish
species worldwide, have shown that A. nemoptera
clustered within the A. vulpes complex using both
Bayesian and parsimony methods, supporting the
placement of the genus Dixonina into the
synonymy of Albula (Rivas and Warlen, 1967).
Although the A. nemoptera +Albula sp. E clade
consistently resolved as sister to the Albula sp. D +
A. forsteri clade in the gene trees (Figs. 1 and 2),
inspection of Table 1 showed that the amino acid
composition of the translated Cytb gene segment
in A. nemoptera and Albula sp. E was more similar
to that of the remaining species (i.e., Albula sp.
A–C, A. vulpes,andA. glossodonta). Bayesian
phylogenetic analyses using amino acid se-
quences also revealed that the A. nemoptera +
Albula sp. E clade clustered with high support
with a clade consisting of Albula sp. A +sp. B +sp.
C+A. vulpes +A. glossodonta (not shown).
Albula forsteri and A. glossodonta, species that are
almost identical morphologically, are thought to
have split from a common ancestor roughly 20–
30 million years ago (Shaklee and Tamaru, 1981;
Colborn et al., 2001). The ancient split between
these two lineages, and the sister species relation-
Fig. 1. Fifty-percent majority rule consensus tree
showing relationships among representative indi-
viduals of Albula nemoptera from the eastern Pacific
and representative species of Albula from the A.
vulpes complex based on Bayesian analysis of a 544-
bp segment of Cytb sequences using first and second
codon positions only. Clade credibility values are
shown on branches. Scale represents expected
substitutions per site. Outgroup taxa in Figs. 1 and
2 are Elops hawaiensis and Pterothrissus gissu. Speci-
men identification or GenBank accession numbers
are shown in parentheses for each individual in
Figs. 1 and 2.
Fig. 2. Strict consensus of 12 equally-parsimoni-
ous trees obtained for representative individuals of
Albula spp. using the Max-mini branch-and-bound
algorithm implemented in MEGA (Length 573; CI
50.795; RI 50.800). Only first and second codon
positions in the Cytb gene segment (28 parsimony
informative sites) were used. Bootstrap support
values $50%are shown on branches. Single
representatives of A. nemoptera, Albula sp. A, and
Albula sp. E were used to reduce analysis time.
PFEILER ET AL.—SHAFTED BONEFISH RELATIONSHIPS 781
ship between Albula sp. D from the Indo-West
Pacific and the A. forsteri clade (K2P distance 5
0.083–0.133; Colborn et al., 2001), are further
supported by the amino acid data (Table 1). The
absence of easily identifiable external morpholog-
ical differences between the genetically distinct A.
forsteri and A. glossodonta, however, increases the
likelihood of misidentifications. The few morpho-
logical differences reported, mainly the shape of
the lower jaw and the length of the upper jaw
(Shaklee and Tamaru, 1981; Randall and Bau-
chot, 1999) are subtle and sometimes not easily
detected. Our results suggest that the GenBank
sequence for the complete mitochondrial ge-
nome of A. glossodonta (accession no. AP002973;
Inoue et al., 2004) came from a specimen of A.
forsteri. This conclusion is based on the observa-
tions that (a) both phylogenetic trees (Figs. 1 and
2) showed a highly-supported clustering of the
suspect A. glossodonta with A. forsteri; (b) the
544 bp Cytb segment of the suspect A. glossodonta
differed from that of A. forsteri from Fiji (ALB54)
by only three nucleotide substitutions (uncorrect-
ed pdistance 50.006); and (c) the amino acid
composition of the translated Cytb segment of the
suspect A. glossodonta showed the unique substitu-
tions of the A. forsteri +Albula sp. D clade
(Table 1).
Provisional identification of Albula sp. E as
A. nemoptera.—As mentioned earlier, although
there are several morphological differences be-
tween A. nemoptera and the species of the A. vulpes
complex, these can be easily overlooked on casual
observation, especially if the diagnostic prolonged
last rays of the dorsal and anal fins are not
examined carefully. Although no voucher speci-
mens of Albula sp. E from Bahia, Brazil are
available for confirmation (gill tissue was taken
from fish market specimens [Colborn et al.,
2001]), and we were unsuccessful in obtaining
new specimens of the relatively scarce western
Atlantic A. nemoptera, phylogenetic analyses based
on Cytb nucleotide sequences consistently showed
that A. nemoptera from the eastern Pacific and
Albula sp. E clustered as sister species. In addition,
the characteristic amino acid substitution in the
translated Cytb gene segment of both species was
not seen in any members of the A. vulpes complex
(Table 1). Based on these observations we suggest
that the two samples (ALB22 and ALB23)
originally assigned to Albula sp. E, and assumed
to be from the A. vulpes complex (Colborn et al.,
2001), should be provisionally identified as
A. nemoptera.
It is worth noting that the Cytb sequence
divergence of 4%between the putative A. nemop-
tera from the western Atlantic and A. nemoptera
from the eastern Pacific falls within the range of
values for the two lineages of A. forsteri from the
Indo-West Pacific (K2P distance 52.9–4.4%;
Colborn et al., 2001), and is also consistent with
expected divergences in tropical marine species
that have been geographically isolated by the
Isthmus of Panama for roughly 3.5 million years
(Coates et al., 1992), assuming a mtDNA molec-
ular clock of about 1.0–1.5%sequence divergence
per million years (Bermingham et al., 1997;
TABLE 1. VARIABLE AMINO ACID POSITIONS IN CONSENSUS SEQUENCES OF THE CytbGENE SEGMENT OF Albula spp.
a
Species Geographic Area
Position
1111111
3571144457
2 6325656707
Albula sp. A Gulf of California (Mexico) V I I I D A V V L T F
Albula sp. A
b
Southern California (USA) ? T T ........
Albula sp. C Gulf of Panama (Panama) . .... . . . I..
A. vulpes Western Atlantic (Belize) . .... . . . I..
Albula sp. B Western Atlantic (Florida) . .... . . . I..
A. glossodonta Central Pacific (Hawaii) . .... . . . I..
A. glossodonta
c
(locality not given) I ..TE S .IAV.
A. forsteri Central Pacific (Hawaii) I ..TE S .IAVV
A. forsteri Indo-West Pacific (Fiji) I ..TE S .IAV.
Albula sp. D Indo-West Pacific (W. Australia) I ..TE S .IAV.
Albula sp. E Western Atlantic (Brazil) I .... . A....
A. nemoptera Eastern Pacific (Mexico) I .... . A....
a
Translated from Cytb nucleotide sequences in Colborn et al. (2001) and the present study, unless indicated otherwise. Amino acid abbreviations: A,
alanine; D, aspartic acid; E, glutamic acid; F, phenylalanine; I, isoleucine; L, leucine; S, serine; T, threonine; V, valine. Dots indicate identical amino acids
relative to Albula sp. A from the Gulf of California.
b
from Pfeiler et al. (2002)
c
from Inoue et al. (2004)
782 COPEIA, 2006, NO. 4
Banford et al., 2004). Thus, it is possible that the
Atlantic and Pacific populations of the Shafted
Bonefishes could be considered separate species,
as originally proposed by Beebe (1942) and later
supported by Berry (1964). We suggest, however,
that further genetic studies of identified whole
specimens from the western Atlantic population
be conducted before considering the possibility of
resurrecting the name A. pacifica (Beebe, 1942)
for the eastern Pacific population of the Shafted
Bonefishes.
ACKNOWLEDGMENTS
We thank B. Bowen, J. Egido-Villarreal, E.
Espino-Barr, T. Markow, and W. Moore for their
help and comments. This research was supported
in part by NSF grant DEB-0346773 to T. Markow.
Travel funds were provided by the Centro de
Investigacio´n en Alimentacio´n y Desarrollo
(CIAD), A.C.
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(EP) CENTRO DE INVESTIGACIO
´NEN ALIMENTACIO
´NY
DESARROLLO, A.C., UNIDAD GUAYMAS,APARTADO
POSTAL 284, GUAYMAS,SONORA, C.P. 85480,
ME
´XICO ; (BGB) DEPARTMENT OF ECOLOGY AND
EVOLUTIONARY BIOLOGY,UNIVERSITY OF ARIZONA,
TUCSON,ARIZONA 85721-0088; AND (RU) COMU-
NIDAD Y BIODIVERSIDAD A.C., BOULEVARD AGUA
MARINA #297, COLONIA DELICIAS,GUAYMAS,
SONORA, C.P. 85420, ME
´XICO. E-mail: (EP)
epfeiler@asu.edu. Send reprint requests to
EP. Submitted: 2 Nov. 2005. Accepted: 6 June
2006. Section editor: D. Buth.
784 COPEIA, 2006, NO. 4
... Recently, Kwun et al. (2011) compared five specimens of Albula sp. collected from Korea and Taiwan with reference data (Pfeiler et al. 2006;Hidaka et al. 2008;Pfeiler 2008), using morphological and molecular analyses. In those analyses, Albula sp. from Korea and Taiwan did not correspond to any Albula species, suggesting that it was an undescribed species. ...
... Genomic DNA was extracted from the muscle tissues of the fish specimens using Chelex 100 resin (Bio-Rad, Hercules, CA). The polymerase chain reaction (PCR) was used to amplify a fragment of the cytochrome b gene, according to Kwun et al. (2011), using primers H14803 (5¢-TGCTAGGGTTGTGTTTAATT-3¢) and L15526 (5¢-GTCTCCAAGAAGGTTAGGCGA-3¢) (Pfeiler et al. 2006). The nucleotide sequences of the A. argentea specimens from Fiji were deposited in the DDBJ/EMBL/GenBank databases (accession numbers: HQ683755-HQ683761). ...
... Albula koreana sp. nov. is clearly distinguishable from A. esuncula, A. nemoptera, A. pacifica, and A. vulpes in its distribution (East China Sea, East Pacific Ocean, Atlantic Ocean, East Pacific Ocean, and Atlantic Ocean, respectively ;Smith 2002;Pfeiler et al. 2006;Pfeiler et al. 2008). Albula koreana sp. ...
Article
Full-text available
A new species of the genus Albula of the family Albulidae is described based on five specimens collected from Korea and Taiwan. Its new scientific name, Albula koreana sp. nov., is derived from its type locality (Korea). We compared Albula koreana sp. nov. with its similar species, A. argentea from Fiji (seven specimens), using morphological and molecular methods. Albula koreana sp. nov. differs morphologically from A. argentea in its tooth patch distributions on the mesopterygoids and parasphenoid. The tooth patches on the mesopterygoids are distributed more anteriorly than those on the parasphenoid in Albula koreana sp. nov. On the other hand, the anterior end of the tooth patches on the mesopterygoids almost corresponds to that of the tooth patches on the parasphenoid in A. argentea. The numbers of vertebrae also differ between the two species (77-78 in Albula koreana sp. nov. vs 72-73 in A. argentea). We analyzed 546 base pairs of the mitochondrial cytochrome b gene sequence, and the Albula koreana sp. nov. sequence differed considerably from that of A. argentea. Kimura's genetic distances between them were very large (15.9%-16.4%), robustly supporting the new species Albula koreana sp. nov.
... genomic DNA was extracted from a small piece of dorsal white muscle taken from the right side of the fish using the DNAzolH (Molecular Research Center, Inc., Cincinnati, OH) or the DNeasy TM (QIAGEN Inc., Valencia, CA) protocol. The polymerase chain reaction (PCR) was used to amplify a 650 base-pair (bp) segment of the cytb gene using primers and PCR conditions described previously (Colborn et al., 2001;Pfeiler et al., 2006). ...
... A and Albula sp. C (Pfeiler et al., 2006). For molecular identifications we trimmed the sequences to 544 bp so that they corresponded to the data set of eastern Pacific bonefishes given in Colborn et al. (2001). ...
... 441 in Albula sp. C, a first codon position transversion that results in a leucine to isoleucine substitution in the cytb protein (Pfeiler et al., 2006), was present in all five individuals from southern Mexico, and specimens AL12 and AL19 from Mazatlán, but was absent in all individuals from the Albula sp. A lineage. ...
Article
Full-text available
Mitochondrial DNA sequence data from a segment of the cytochrome b (cytb) gene were used to Infer geographic distributions of two unnamed putative species of eastern Pacific bonefishes, Albula sp. A and Albula sp. C, both members of the A. vulpes complex. The molecular data revealed that Albula sp. C, a lineage originally Identified from Panama, Is distributed north to the coastal waters of central Mexico and the southern Gulf of California (Sea of Cortez). Albula sp. A, originally Identified from the central Gulf of California and distributed to California, USA, Is shown here to range to Mazatlan, Sinaloa, In the southern gulf where It occurs sympatrically with Albuta sp. C. The distributions of the two putative species Inferred from the molecular data suggest that the single available name for eastern Pacific bonefishes, Albula esuncula (Garman, 1899), originally described from larvae (leptocephali) collected off Acapulco, Guerrero, should be assigned to Albula sp. C. Morphometric and meristic analyses of adults of A. esuncula and Albuta sp. A revealed no external characters that could be used reliably to distinguish between the two species.
... Most of these newly described or resurrected taxa are cryptic following the definition of Bickford et al. (2006), but the genus Albula also includes two species of the morphologically distinct shafted bonefishes, A. nemoptera (Fowler, 1911) from the western Atlantic and A. pacifica (Beebe, 1942) from the eastern Pacific. Phylogenetic analyses have shown that both sister species of shafted bonefishes, which were formerly placed in the genus Dixonina, nest within the cryptic species of Albula (Pfeiler et al. 2006), indicating that morphological stasis is not characteristic of the genus as a whole. ...
... GenBank accession numbers are as follows: A. gilberti from the Gulf of California, Mexico, JF803967 (holotype), JF803962-JF803966, JF803968 and EF602157 (paratypes) (Mazatlán, Sinaloa), AF311757-AF311759 and EF602155 (Guaymas, Sonora), EF602156 (Bahía de Kino, Sonora); A. gilberti from southern California, USA, JF803969-JF803971 (Newport Bay, Orange County); A. esuncula from the Gulf of Panama, AF311760-AF311762; A. esuncula from Mexico, EF602158 (San Dionisio del Mar, Oaxaca), EF602159 (near Acapulco, Guerrero), JF803972, JF803973 and EF602160 (Mazatlán, Sinaloa). Phylogenetic analyses showing the partitioning of the different genetic lineages of the Albulidae (Table 1) have been reported previously (Colborn et al. 2001;Pfeiler et al. 2006Pfeiler et al. , 2008aBowen et al. 2008;Valdéz-Moreno et al. 2010;. ...
... The cytb segment corresponds to nucleotide positions 14502-15045 in the complete mitochondrial genome of Albula glossodonta [GenBank accession no. AP002973; phylogenetic evidence suggests that the genome sequenced was actually from A. argentea (= A. forsteri) and not A. glossodonta (Pfeiler et al. 2006)]. In A. gilberti, the pelvic-fin tip may reach the posterior edge of the anus whereas in A. esuncula the tip does not surpass the anterior edge. ...
Article
A new species of bonefish (Albuliformes: Albulidae: Albulinae) from the eastern Pacific Ocean, Albula gilberti, is described. Previously referred to as Albula sp. A, A. gilberti is distributed throughout the Gulf of California, Mexico to the coastal waters of California, USA. Presently, it can be distinguished from its closest relative, A. esuncula (= Albula sp. C) from the tropical eastern Pacific, solely by differences in mitochondrial DNA (cytochrome b) gene sequences. No diagnostic external morphological characters have yet been found that can reliably separate the two distinct genetic lineages although some differences in placement of pelvic-fin tip relative to vent may exist. Differences detected in counts of meristic characters should be tested in larger series of specimens, especially of A. esuncula. Available evidence also suggests that A. gilberti and A. esuncula show a parapatric distribution, with an area of sympatry in the southern Gulf of California. We also provide a description of adults of A. esuncula (the original description was based solely on two larvae), review the current nomenclature of the subfamily Albulinae, and discuss the role and justification of using molecular data for diagnosing cryptic species.
... E from Colborn et al. [2001]) are further distinguished by shorter total length, elongated anal fin and caudal ray of the dorsal fin, mouth reaching a point below the eye, small scales, and a few differences in dentition and meristic characters (Rivas and Warlen 1967;Robins and Ray 1986). The western Atlantic Ocean form is designated A. nemoptera, and the Pacific Shafted Bonefish A. pacifica (Beebe 1942) is the eastern Pacific Ocean form (Pfeiler et al. 2006;Pfeiler 2008). Based on cytochrome b sequence data, they were designated sister species (Pfeiler 2008); additional nuclear gene sequence data supports this (Wallace 2014). ...
... Since relatively few morphological characters are capable of distinguishing between only some species, bonefish research will continue to require a large genetic component. Identification has routinely been accomplished based on mitochondrial cytochrome b sequence identity (Colborn et al. 2001;Pfeiler et al. 2002Pfeiler et al. , 2006Pfeiler 2008;Valdez-Moreno et al. 2010;Wallace 2014Wallace , 2015Díaz-Viloria et al. 2017), though some bonefishes may also be identified using microsatellite markers (Seyoum et al. 2008;Wallace 2015;Wallace and Tringali 2016). To resolve interspecific relationships, a robust phylogenetic analysis of the family will require more data because single-gene methods-especially from mtDNA-provide an incomplete picture of evolutionary history (Pamilo and Nei 1988;Nichols 2001;Song et al. 2008). ...
Article
Full-text available
Despite expanding research on the popular recreational fishery, bonefish taxonomy remains murky. The genus Albula, comprising these iconic circumtropical marine sportfishes, has a complex taxonomic history driven by highly conserved morphology. Presently, 12 putative species are spread among 3 species complexes. The cryptic morphology hinders visual identification, requiring genetic species identification in some cases. Unclear nomenclature can have unintended consequences, including exacerbating taxonomic uncertainty and complicating resolution efforts. Further, ignoring this reality in publications may erode management and conservation efforts. In the Indian and Pacific oceans, ranges and areas of overlap are unclear; precluding certainty about which species support the fishery and hindering conservation efforts. Species overlap, at both broad and localized spatial scales, may mask population declines if one is targeted primarily (as demonstrated in the western Atlantic fishery). Additional work is necessary, especially to increase our understanding of spatiotemporal ecology across life history stages and taxa. If combined with increased capacity to discern between cryptic species, population structure may be ascertained, and fisheries stakeholders will be enabled to make informed decisions. To assist in such efforts, we have constructed new range maps for each species and species complex. For bonefishes, conservation genomic approaches may resolve lingering taxonomic uncertainties, supporting effective conservation and management efforts. These methods apply broadly to taxonomic groups with cryptic diversity, aiding species delimitation and taxonomic revisions.
... The mitochondrial cytochrome b (cytb) gene was selected for species identification because that gene had the most reference sequences of Albula species in the GenBank. The partial sequence (650 bp) of the cytb was amplified by PCR with the primers H14803 (5 0 -TGC TAG GGT TGT GTT TAA TT-3 0 ) and L15526 (5 0 -GTC TCC AAG AAG GTT AGG CGA-3 0 ) (Colborn et al. 2001;Pfeiler et al. 2006). DNA amplifications were performed in a PCR thermal cycler (Bio-Rad Laboratories, Hercules, CA) under the following conditions: 2 min at 95 C, 35 cycles of 1 min at 95 C, 1 min at 45 C, 1 min at 72 C and a final extension of 4 min at 72 C. PCR products (20 ll, 100 ng ll À1 ) were sequenced in both directions in an automatic sequencer (ABI Prism 3730XL, Applied Biosystems, Carlsbad, CA) at Macrogen (Seoul, Korea). ...
... The comparison of the cytochrome b gene sequences of the 22 leptocephali of this study to existing sequences of 13 adults of the three other species of Albula present in the Northeastern Pacific region (Pfeiler et al. 2006;Pfeiler et al. 2008;Pfeiler et al. 2011) and three adults of A. vulpes from the Caribbean (Colborn et al. 2001) clearly showed that these larvae were those of A. gilberti. This was evident by their low genetic divergence values compared to adults of that species from the entrance of the Gulf. ...
Article
Bonefish leptocephali of the genus Albula are difficult to identify to the species level due to morphological similarities between two different species present in the Northeastern Pacific Ocean, A. esuncula and A. gilberti. In this study, 22 bonefish leptocephali (premetamorphic and early metamorphic), collected from two locations in the southern Gulf of California were identified as Albula gilberti by comparing 459 bp of their mitochondrial cytochrome b gene sequences to those of four other species of bonefish. The characteristics of these A. gilberti leptocephali were compared to those of previously described bonefish leptocephali in the region. No distinctive morphological characteristics (meristic and pigmentation) were found that differentiate premetamorphic leptocephali of A. gilberti from those of other Albula species, making species identification by molecular-genetics a necessity. In early metamorphic leptocephali some differences in horizontal eye diameter-head length ratio, number of rays of pelvic and anal fins and myomere of pelvic-fin origin may help to differentiate A. gilberti from A. esuncula.
... Bonefishes genus Albula Scopoli 1777 (Albulidae: Albuliformes) are distributed globally in tropical and subtropical coastal areas (Hildebrand 1963;Pfeiler et al. 2006;Randall 2005;Nelson 2006). Although Albula had been considered to contain only two species, A. vulpes (Linnaeus, 1758) and A. nemoptera (Fowler, 1911), eight valid species are recognized worldwide. ...
... Genomic DNAs were extracted from the muscle tissues of the fish using 10% Chelex 100 Resin (Bio-Rad, Hercules, CA). The polymerase chain reaction (PCR) was used to amplify the mitochondrial DNA cyt b gene using primers H14803 (5?-TGCTAGGGTTGTGTT-TAATT-3?) (Pfeiler et al. 2006) and albula-L (5?-GTCT CCAAGAAGGTTAGG-3?). PCR was conducted on a thermal cycler (ABI 2720 Thermal Cycler, Foster City, CA), with PCR solutions containing 5Á10 ml of genomic DNA, 2.5 ml of 10X PCR buffer, 2 ml of 2.5 mM dNTPs, 1 ml of each primer, 0.2 ml of FR Taq DNA polymerase (Biomedic, Korea) and 25 ml of distilled water. ...
Article
Full-text available
To clarify the taxonomic status of five specimens of Albula collected from Korea and Taiwan (Albula sp.), genetic differences among them and other congeneric species were investigated based on mitochondrial DNA cytochrome b sequences. All five specimens of Albula sp. showed identical haplotypes and strongly supported a monophyletic group on phylogenetic analysis. The genetic differences between Albula sp. and ten other species of Albula ranged from 0.100 to 0.164. These values were almost equal to or exceeded the interspecific differences among the latter ten species (0.038–0.206). Morphological comparisons of Albula sp. and another four Indo-Pacific species, A. argentea, A. glossodonta, A. oligolepis and A. virgata, are also presented. Albula sp. could be distinguished from the latter four species in their numbers of pored lateral-line scales, anal fin rays and vertebrae, as well as in several proportional measurements.
... Molecular identifications, based on cyt-b sequences, were determined by comparisons among the present samples and sequences published in INSDC. The published sequences used in this study were obtained from Colborn et al. (2001), Pfeiler et al. (2006), Pfeiler (2008, , and Wallace (2015) (see Table 1). The relationships between unidentified linage names (Albula spp. ...
Article
A taxonomic review of Albula Scopoli, 1777 (Albuliformes: Albulidae) in Japanese and adjacent waters, based on morphology and mtDNA cytochrome b sequence data, resulted in the recognition of four species: Albula argentea (Forster, 1801), Albula glossodonta (Forsskål, 1775), Albula koreana Kwun and Kim, 2011, and Albula oligolepis Hidaka, Iwatsuki, and Randall, 2008. Although Japanese ichthyologists have long considered A. glossodonta and a second Albula species (referred to by the Japanese name “Sotoiwashi”) to be distributed in Japanese waters, the latter having been reported as A. koreana or Albula sp. in recent literature, the present study revealed that, in fact, “Sotoiwashi” included three species, viz., A. argentea, A. koreana and A. oligolepis. Examined specimens of the latter three species represent the first reliable records of all three from Japanese waters, with comparative specimens of A. koreana from Vietnam and Malaysia also representing distributional range extensions (formerly known only from Korea and Taiwan). Albula koreana is readily distinguished from Japanese congeners by the striking yellow stripe on the cheek (just behind the mouth) in the former, a large dark blotch in front of the nostril, a dark oval blotch under an arc-shaped dark band on the snout tip, and greater numbers of body scales and vertebrae. As has been previously demonstrated, A. argentea and A. oligolepis are distinguished by pored lateral-line scale numbers (68–74 in the former vs. 61–67 in the latter) and total vertebrae (68–75 vs. 64–70). Updated distributional information shows A. argentea to be distributed from Indonesia east to French Polynesia, and north to Japan and Korea (there being no reliable records from Sri Lanka, Madagascar or the Hawaiian Islands); A. koreana in waters off Korea, Japan, Taiwan, Vietnam and Malaysia (east coast of Malay Peninsula); and A. oligolepis from the east coast of Africa to the Coral Sea, and north to Japan.
... Bonefish occur worldwide in tropical and subtropical areas and support valuable recreational fisheries (Pfeiler et al. 2006;Levesque 2011). These fisheries are usually characterised by catch-and-release fishing and are therefore considered to be a sustainable form of ecotourism (Holt and Holt 2005). ...
... cf. vulpes (Pfeiler et al. 2006;Bowen et al. 2008;Wallace and Tringali 2010;Wallace 2014). Of these, only A. nemoptera is visually distinct; the Abstract The distributions, genetic structure, and occurrence of hybrids were explored among three Atlantic bonefishes (Albula spp.), to determine recreational fishery composition, using 19 microsatellite loci. ...
Article
Full-text available
The distributions, genetic structure, and occurrence of hybrids were explored among three Atlantic bonefishes (Albula spp.), to determine recreational fishery composition, using 19 microsatellite loci. Between 1977 and 2012, study specimens were obtained from 14 locations across the Caribbean Sea and western Atlantic Ocean, with an external collection from the eastern Atlantic. The species were broadly sympatric across the region, though occurrence at the local level was variable, likely due to habitat partitioning. Albula vulpes appeared to be the primary contributor to the recreational fishery, which occurs predominantly in coastal flats habitats. Unexpected, non-geographic population partitioning was identified within all three species. Robust evidence for two genetic populations of A. vulpes was further supported by hypothesis testing for migration patterns between them. The potential existence of two quasi-discrete spawning groups (fall and spring) within species may explain the observed partitions. Patterns of interspecific hybridization were explored to further inform management and conservation efforts. Hybrids were observed at low frequency (40 individuals, equaling a 1.5 % hybridization rate), and most were collected from Florida and The Bahamas. Our results suggest that a combination of intrinsic and extrinsic semipermeable barriers to gene flow exists among these divergent species. These findings are congruent with previous studies that revealed substantial genetic divergence among these secondarily sympatric bonefishes. Further, clarification is provided on regional distributions and the composition of the economically and culturally important recreational fishery. On first order, management efforts would have the greatest impact if focused on A. vulpes populations and their preferred habitat.
Article
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The roundjaw bonefish, Albula glossodonta, is the most widespread albulid in the Indo-Pacific and is vulnerable to extinction. We assembled the genome of a roundjaw bonefish from Hawai‘i, USA, which will be instrumental for effective transboundary management and conservation when paired with population genomics datasets. The 1.05 gigabase pair (Gbp) contig-level assembly had a 4.75 megabase pair (Mbp) NG50 and a maximum contig length of 28.2 Mbp. Scaffolding yielded an LG50 of 20 and an NG50 of 14.49 Mbp, with the longest scaffold reaching 42.29 Mbp. The genome comprised 6.5% repetitive elements and was annotated with 28.3 K protein-coding genes. We then evaluated population genetic connectivity between six atolls in the Western Indian Ocean with 38,355 SNP loci across 66 A. glossodonta individuals. We discerned shallow population structure and observed genetic homogeneity between atolls in Seychelles and reduced gene flow between Seychelles and Mauritius. The South Equatorial Current might be the limiting mechanism of this reduced gene flow. The genome assembly will be useful for addressing taxonomic uncertainties of bonefishes globally.
Article
Full-text available
With its theoretical basis firmly established in molecular evolutionary and population genetics, the comparative DNA and protein sequence analysis plays a central role in reconstructing the evolutionary histories of species and multigene families, estimating rates of molecular evolution, and inferring the nature and extent of selective forces shaping the evolution of genes and genomes. The scope of these investigations has now expanded greatly owing to the development of high-throughput sequencing techniques and novel statistical and computational methods. These methods require easy-to-use computer programs. One such effort has been to produce Molecular Evolutionary Genetics Analysis (MEGA) software, with its focus on facilitating the exploration and analysis of the DNA and protein sequence variation from an evolutionary perspective. Currently in its third major release, MEGA3 contains facilities for automatic and manual sequence alignment, web-based mining of databases, inference of the phylogenetic trees, estimation of evolutionary distances and testing evolutionary hypotheses. This paper provides an overview of the statistical methods, computational tools, and visual exploration modules for data input and the results obtainable in MEGA.
Article
Shaklee and Tamaru (1981) identified two species of bonefishes (Albula) in the Hawaiian Islands that range elsewhere in the Indo-Pacific region as A. glossodonta (Forsskål, 1775) and A. neoguinaica Valenciennes, 1847. Albula forsteri Valenciennes, 1847, from Tahiti, the replacement name for Esox argenteus Forster & Schneider, 1801, is here regarded as the senior synonym of A. neoguinaica. Albula erythrocheilos Valenciennes, 1847 and A. seminuda Valenciennes, 1847 are junior synonyms of A. forsteri and A. glossodonta, respectively. The bonefish described as Butyrinus bananus by Lacepède is considered unidentifiable.
Chapter
This chapter highlights the biogeography of fishes with perspectives from the Panamanian isthmus as it provides a rich landscape to study the evolution of fish and molecules. It focuses on fish biogeography, particularly the geography of conspecific populations of tropical marine and freshwater fishes. It provides an insight into the mechanics and reliability of mitochondrial molecular clocks functioning across shallow spans of time. Conspecific populations, if differentiated, can provide historical information about a region. Molecules, particularly mtDNA, are well suited for reconstructing the evolutionary relationships among conspecific populations. For species or species groups demonstrating little or no phylogenetically informative morphological variation, molecules can provide a taxonomy that can be easily and immediately placed in a phylogenetic context. Tabular representations are provided for the geographic scaling of species groups like the Panama Isthmus, the circumtropical Abudefduf, and Neotropical freshwater fishes. A reasonable conclusion follows that molecular and genetic analysis can provide rapid means for surveying regional biotic diversity. Phylogenetic history and/or genetic diversity should be used in biodiversity indices to emphasize the phylogenetic and genetic distinctiveness of some groups compared to others.
Article
The final closure of the Isthmus of Panama at ∼3.5 Ma divided the American tropical ocean into two separate and different oceanographic regions. Consequences for the marine biota were profound, but, hitherto, correlation of the Pacific and Caribbean coastal sections has not been precise enough to track biologic patterns. We present here a correlation of 31 sections from the Pacific and Caribbean coasts of Costa Rica and western Panama. Using calcareous nannofossils and planktonic foraminifera at both the tops and bottoms of each formation, we estimate that the Caribbean section ranges from 8.2 Ma to 1.7 Ma; and the Pacific sequence, from 3.6 Ma to <1.7 Ma. These intervals bracket postulated dates for final closure of the Isthmus and provide the first well-dated record of middle and late Pliocene faunas from the region. The Caribbean and Pacific sections include very different environments of deposition, yet there is sufficient overlap and diversity of habitats to permit meaningful biological comparisons. On the Caribbean side, formations tied together by the overlap of the upper Pliocene markers Sphenolithus abies and Pseudoemiliana lacunosa (3.5 Ma to 3.6 Ma) range from very shallow to shallow inner shelf (<200 m) and upper slope (200-800 m). The Pacific coast sections were mostly deposited in a trench slope environment, which is absent on the Caribbean side. These sections fortuitously include abundant thick intra-formational slumps containing shallow-water fauna more appropriate for biological comparison with the Caribbean biota. Similarly, the ∼1.9 Ma to 1.5 Ma interval, well constrained by various taxa, includes middle- to outer-shelf, and inner-shelf to upper-slope deposits on the Caribbean side, and marginal-marine to inner-shelf deposits on the Pacific coast. Using our new biostratigraphic framework to correlate previously poorly constrained mollusc collections, we show that evolutionary divergence of the Pacific and Caribbean near-shore marine faunas had occurred by 3.5 Ma. This strongly suggests that the Isthmus was effectively closed by this time.
Article
Abstract Many examples of cryptic marine species have been demonstrated with biochemical and molecular studies. In most cases, a broadly distributed taxon is actually a group of sibling species that can be distinguished (upon closer examination) by ecological or morphological characters. Fishes of the family Albulidae constitute a notable exception. Bonefish (Albula spp.) morphology and ecology are highly conserved around the globe, and their extended pelagic larval stage could allow population connections on a vast geographic scale. Based on this perceived homogeneity, bonefishes were classified as a single pantropical species, A. vulpes. However, allozyme studies of Hawaiian populations indicated that two sympatric species (A. glossodonta and A. neoguinaica) are included in the synonymy of A. vulpes. To ascertain the number and distribution of evolutionary partitions in Albula, we surveyed 564 bp of mitochondrial DNA (mtDNA) cytochrome b from 174 individuals collected at 26 locations. Sequence comparisons reveal eight deep lineages (d= 5.56–30.6%) and significant population structure within three of the four lineages that could be tested (φST= 0.047–0.678). These findings confirm the genetic distinctiveness of the three species noted above and invoke the possibility of five additional species. Clock estimates for mtDNA indicate that these putative species arose 4–20 million years ago. Distinct evolutionary lineages coexist in several sample locations, yet show little morphological or ecological differentiation in sympatry. Thus, bonefish species seem to defy the evolutionary conventions of morphological differentiation over time and ecological displacement in sympatry. Despite multiple cases of sympatry, sister-taxa relationships inferred from mtDNA indicate that divergence in allopatry has been the predominant speciation mechanism in Albula. Stabilizing selection in the homogeneous habitat occupied by bonefishes (tropical sand flats) could promote the retention of highly conserved morphology and ecology.