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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