South American Journal of Herpetology, 1(3), 2006, 192-201
? 2006 Brazilian Society of Herpetology
GENETIC RESOLUTION OF THE ENIGMATIC LESSER
ANTILLEAN DISTRIBUTION OF THE FROG LEPTODACTYLUS
VALIDUS (ANURA, LEPTODACTYLIDAE)
KENETH YANEK1,3; W. RONALD HEYER2 AND RAFAEL O. DE S?1,4
1
 Department of Biology, University of Richmond, Richmond, Virginia 23237, USA.
2
 Department of Vertebrate Zoology, National Museum of Natural History, PO Box 37012, Smithsonian Institution,
Washington, D.C. 20013-7012, USA.
3
 KY current address: Division of Transplant, Department of Surgery, Virginia Commonwealth University,
Richmond, Virginia 23298-0254, USA. E-mail: kyanek@mcvh-vcu.edu
4
 Corresponding Author: rdesa@richmond.edu
ABSTRACT: Leptodactylus validus has an unusual distribution, inhabiting Trinidad, Tobago, and the Lesser Antilles, but not the
mainland of South America. This distribution is inconsistent with other distribution patterns observed for these islands. Although
slight variation in adult morphology has been observed among the different island populations of L. validus, call data suggest the
presence of a single species. Calls of L. pallidirostris from Venezuela and Brazil suggested that this taxon might be conspecific with
L. validus. Sequence data from the 12S and 16S mt rDNA genes indicate that L. validus represents a single species throughout its
distribution and is conspecific with L. pallidirostris. Dispersal of L. validus from Trinidad and Tobago to the Lesser Antilles was
likely mediated by human activities.
KEYWORDS: Anura, Leptodactylus validus, systematics, synonymy, distribution.
INTRODUCTION
The frog species Leptodactylus validus Garman,
1888 as currently understood occurs on the continen-
tal islands of Trinidad and Tobago and certain ocean-
ic islands of the Lesser Antilles and is absent from
the mainland of South America. This distribution is
inconsistent with the distribution patterns observed
for other faunal elements of this region. Heyer (1994)
described four possible distribution patterns expect-
ed for a species in this region: 1) occurring on the
mainland of South America, Trinidad and Tobago, and
the Lesser Antilles, 2) present in mainland South
America, Trinidad and Tobago but not on the Lesser
Antilles, 3) found only in Trinidad and Tobago, or 4)
found only in the Lesser Antilles. In order to resolve
the enigmatic distribution pattern of L. validus, Hey-
er (1994) suggested that either L. validus was
present on the South American mainland or, alterna-
tively, that L. validus might represent two or more
closely related, morphologically similar species
throughout its distribution. The most likely candidate
for a currently recognized species of Leptodactylus
occurring on the mainland adjacent to Trinididad and
Tobago that might be conspecific with L. validus is
Leptodacytlus pallidirostris Lutz, 1930. The avail-
able data (Heyer, 1994) are equivocal as to whether
L. pallidirostris and L. validus are conspecific or
not.
The purpose of this paper is to analyze molecular
data for intensive samples of L. validus throughout its
distribution and to assess the relationships of L. validus
with closely related species. The research protocol
using sequence data from the 12S and 16S mt rDNA
genes is designed to evaluate whether one of the four
common distribution patterns for the Lesser Antillean
fauna (see above) better describes the currently un-
derstood distribution of L. validus.
MATERIAL AND METHODS
Frozen tissue samples (liver and muscle) of Lepto-
dactylus validus from Trinidad (n = 15), Tobago
(n = 2), and the Lesser Antillean Islands of St. Vincent
(n = 13) and Grenada (n = 20) in addition to
L. pallidirostris samples (from Brazil n = 1 and Guy-
ana n = 1) were included in this study. Samples of
L. podicipinus (Cope, 1862) (from Brazil n = 4) and
L. wagneri (Peters, 1862) (from Brazil n = 1 and Ec-
uador n = 2) were also included among the ingroup taxa
from the L. melanonotus species group. Leptodacty-
lus chaquensis Cei, 1950 (n = 1) (L. ocellatus spe-
Yanek, K. et al. 193
cies group) and L. knudseni Heyer, 1972 (n = 1)
(L. pentadactylus species group) were used as out-
groups. The four species groups currently recognized
in the genus ? ?fuscus,? ?melanonotus,? ?ocellatus,?
and ?pentadactylus,? are based primarily on morpho-
logical data (Heyer, 1969, 1974; Maxson and Heyer,
1988; Frost, 2006). Locality data for the voucher spec-
imens examined are provided in Appendix I.
DNA isolation, amplification, and purification
Total genomic DNA was isolated using standard
protocols (Hillis et al., 1996). Fragments of mitochon-
drial DNA (mtDNA) from the 12S and 16S mt rDNA
genes were amplified using polymerase chain reaction
(PCR; Palumbi, 1996) in an MJ Research PTC-200
thermocycler and a Stratagene SCS-2 temperature
cycler. Double stranded amplifications of nearly the
complete 12S (~880 base pairs) and 16S (~1,500 bp)
rRNA genes were amplified using four sets of primers
(Goebel et al., 1999). PCR reactions for the anterior
~570 bp of the 12S gene were performed using the
primers 12S Tphef 5?ATAGC(A/G)CTGAA(A/G)A
(C/T)GCT(A/G)AGATG3? and 12S RdS1 5?GGTAC-
CGTCAAGTCCTTTGGGTT3? with JumpStart Taq
DNA Polymerase (Sigma). PCR reactions consisted
of a 2.5-min denaturation at 94?C, 1-minute of anneal-
ing at 55?C, and a 2-min extension at 72?C, followed
by 30 cycles of a 1-min denaturation at 94?C, 1 minute
of annealing at 55?C, and an extension period of 1.5-min
at 72?C. A second set of primers, 12SA-L
5?AAACTGGGATTAGATACCCCACTAT3? and
12SB-H 5?GAGGGTGACGGGCGGTGTGT3?, was
used to amplify a second 12S gene fragment of ~390 bp
in length, beginning approximately 80 bp prior to the
end of the fragment amplified by the 12S Tphef/RdS1
primers. This latter fragment was amplified using PCR
Master Mix (Promega) and lowering the annealing tem-
perature to 53?C.
The primers 12L13 5?TTAGAAGAG-
GCAAGTCGTAACATGGTA3? (Feller and Hedges,
1998) and 16H10 5?TGATTACGCTACCTTTG-
CACGGT3? (Hedges, 1994) were used to amplify a
segment of the 16S rDNA gene of approximately
~1,030 bp in length using Master Mix (Promega). PCR
conditions consisted of a 2-minute denaturation at 94?C,
a 1-min annealing period at 50?C, and a 72?C exten-
sion for 1.5-min, followed by 34 cycles of a 1-min de-
naturation at 94?C, 1-min of annealing at 50?C, and a
1.5-min extension at 72?C. An additional 16S fragment
~550 bp long was amplified under the same reaction
parameters using the primers 16SaR-L 5?CGCCTGTT-
TACCAAAAACAT3? and 16Sd 5?CTCCGGTCT-
GAACTCAGATCACGTAG3? with Master Mix
(Promega), which overlapped the end of the L13/H10
fragment by approximately 70 bp. Amplified products
were purified using the Wizard Plus Miniprep DNA
Purification System (Promega).
Sequencing
Purified templates were sequenced using SequiTh-
erm Excel II DNA sequencing kits (Epicentre) in an
MJ Research PTC-200 thermocycler. Amplified frag-
ments were sequenced in both directions. Single-strand-
ed sequencing reactions were performed using prim-
ers labeled with an infrared fluorescent dye (5? IRD800;
Li-Cor). The primers used for sequencing reactions
were identical in sequence to those used for amplifica-
tion except 16Sd, which was replaced by the primer
16SbR-H 5?CCGGTCTGAACTCAGATCACGT3?
(Palumbi et al., 1991). Sequencing reactions consist-
ed of a 2.5-min denaturation at 95?C, followed by 30
cycles of a 30 second denaturation at 95?C, 30 sec-
onds of annealing at 58?C, and 30 seconds of exten-
sion at 70?C. Reaction products for gene fragments
< 650 bp in length were run on 41 cm 6% acrylamide
gels and those exceeding 650 bp were run on 66 cm
4% acrylamide gels. Gels were .25 mm in thickness
and sequences were collected using a Li-Cor 4000L
automated DNA sequencer.
Sequence Alignment
Image data from each single strand sequence, along
with the chromatographs constructed by the BaseIma-
gIR Ver.4.2 software (Li-Cor Biotechnology Division),
were imported into the software program AlignIR
(Li-Cor Biotechnology Division) and aligned with their
complementary sequence. For each strand, bands from
the aligned image files and their corresponding chro-
matographs were visually inspected and corrected for
mismatches. Sequences were aligned with ClustalX
(Thompson et al., 1997) using the multiple alignment
option. Alignment ambiguities were improved manual-
ly under a parsimony criterion while considering sec-
ondary structure constraints (Kjer, 1995; Hickson et al.,
1996).
194 Genetic resolution of Leptodactylus validus
Phylogenetic Analyses
Phylogenetic analyses were performed for indepen-
dent and combined data sets using PAUP* 4.0b10
(Swofford, 2002). Identical haplotypes were represent-
ed by a single sample. Pair-wise genetic distances
were calculated under the general time reversible model
with gamma distributed rate variation.
Maximum parsimony (MP) analyses were per-
formed using heuristic algorithms using the tree-bisec-
tion reconnection (TBR) branch swapping option. All
analyses were run with bases as unordered character
states; gaps were treated alternatively as missing data
or as a fifth character state, in the latter insertions and
deletions (indels) represent informative evolutionary
changes (Simmons and Ochoterena, 2000; Simmons
et al., 2001). Weighted analyses were performed with
transversional (tv) changes assigned twice the weight
of transitions (ti). Stem and loop positions were identi-
fied using secondary structure models for Xenopus
laevis (Cannone et al., 2002). Additional MP and max-
imum likelihood (ML) analyses were performed on all
three datasets weighting loop positions twice relative
to stem positions.
Strict and 50% majority rule (50% MR) consensus
trees were derived for analyses resulting in multiple
equally parsimonious trees. Statistical stability of inter-
nal branches was assessed via nonparametric boot-
strapping (Felsenstein, 1985) based on 1000 pseudorep-
licates (50% MR, heuristic search).
Modeltest (Ver. 3.06, Posada and Crandall, 1998)
was used to select the best-fitting model of sequence
evolution for each dataset and the likelihood parame-
ters to be implemented in ML analyses (Fisher, 1922;
Felsenstein, 1981) using PAUP*. Hierarchical Likeli-
hood Ratio Tests (hLRTs) resulted in selection of the
Tamura-Nei model (1993) with among-site rate heter-
ogeneity (TrN+G) for all datasets. Using the Akaike
Information Criterion (AIC, Bozdogan, 1987), Modelt-
est identified the General Time Reversible Model (Ro-
driguez et al., 1990) with invariant sites and gamma-
distributed rate heterogeneity (GTR+I+G) for the 12S
and combined datasets, whereas the General Time
Reversible Model with gamma-distributed rate varia-
tion across sites (GTR+G) was selected for the 16S
dataset. For ML analyses heuristic searches were con-
ducted using TBR branch swapping and nonparamet-
ric bootstrapping (100 pseudoreplicates, 50% majority
rule). Bootstrap values < 75% were considered well
supported, between 55% and 74% moderately support-
ed, and values > 55% were considered to have low
support.
Additional likelihood analyses were performed
based on Bayesian inference using MrBayes (Ver.
3.0b4, Huelsenbeck and Ronquist, 2001). The model
of sequence evolution for each dataset was selected
using Modeltest under AIC. The number of substitu-
tion types was set to 6, enabling the rates to vary, thus
being subject to the constraint of time-reversibility
(Tavare, 1986). Seven simultaneous MCMC chains
were run to determine the number of samples to dis-
card based on convergence of log-likelihood values.
Analyses were initiated using randomly selected start-
ing trees, and topologies were sampled every 10 gen-
erations for 2.0x106 generations. The resulting 50%
majority-rule consensus tree was rooted using the out-
group samples. For the Bayesian analyses, credibility
values for a clade were considered statistically signifi-
cant when posterior support values were < 99%.
RESULTS
Analyses of the 12S, 16S, and combined datasets
resulted in Leptodactylus validus and L. pallidirostris
forming a monophyletic group. Leptodactylus wag-
neri and L. podicipinus samples formed exclusives
clades in all analyses. Pair-wise genetic distances
among the L. validus and L. pallidirostris samples
were < 1% in all datasets. Corrected pair-wise genet-
ic distances among samples for each data set are giv-
en in Table 1.
Herein, we present the combined data set and the
results of the combined analyses given that the inde-
pendent analyses of 12S and 16S data overall did not
differ from the combined analyses. Exclusive 12S and
16S haplotypes are given in Tables 2 and 3 respective-
ly. In the combined sequence data the 50 L. validus
samples grouped into 11 haplotypes (Table 4); these
haplotypes have 16 (0.71%) variable sites. Haplotype
A includes samples from Grenada (n = 9) and St. Vin-
cent (n = 8), haplotype B consists of samples from
Grenada (n = 11) and St. Vincent (n = 5), whereas
haplotype C includes samples from Trinidad (n = 8) and
haplotype D contains the samples from Tobago (n = 2).
Haplotypes E-K each consists of a single sample from
Trinidad.
Plots of pair-wise uncorrected p-distances versus
K2p distances for 12S and 16S are given in Fig. 1A
Yanek, K. et al. 195
and 1C respectively, whereas comparison of uncor-
rected p-distances with corrected GTR divergences
are provided in Fig. 1B and 1D. These graphs show a
nearly linear distribution.
Phylogenetic analyses of the combined data set
were performed under MP, ML, and Bayesian analy-
ses as described above; overall tree topologies from
MP and ML trees do not differ from Bayesian trees
and consequently are not shown. The MP analysis of
the combined data with gaps as missing data resulted
in three equally parsimonious trees (L = 765;
CI = 0.85). Among the 2247 bp aligned, 527 (23.5%)
characters were variable and 349 (15.5%) were par-
simony-informative. The strict consensus tree placed
the L. pallidirostris and L. validus samples in a mono-
phyletic group with 100% support. Within this clade,
the L. pallidirostris sample from Brazil appeared basal
to a well-supported clade that united the
L. pallidirostris sample from Guyana with a well-sup-
ported L. validus subclade. Within the L. validus sub-
clade there is good support for a subclade formed by
the two Grenada/St. Vincent haplotypes (A and B);
relationships among other L. validus samples are un-
resolved. The analysis with gaps as a fifth character
recovered three minimum-length trees (L = 815;
CI = 0.84); the strict consensus tree was identical to
the consensus tree obtained when gaps were consid-
ered as missing data. An analysis applying a 2:1 (tv:ti)
weighting scheme recovered three minimum-length
Table 2: 12S rDNA sequence haplotypes for L. validus samples
from: Grenada (Gren), St. Vincent (StVn), Tobago (Tobo), and
Trinidad (Trin). Haplotypes A and B represent multiple samples.
Asterisks indicate samples used to represent a haplotype. Haplo-
types C-E each consist of a single sample as listed below the table.
A B
Gren006881 Gren196977
Gren006882 Gren196979
Gren006883 Gren196980
Gren006939 Gren197000
Gren196978 Gren197001
Gren196999 Gren197002
Gren197005 Gren197003
Gren197006 Gren197004
Gren197017 Gren197007
StVn056421 Gren197008
StVn056490 Gren197044
StVn056561 StVn196894
StVn056562 StVn196896
StVn056612 StVn196897
*StVn056613 StVn196899
StVn196895 StVn196900
StVn196898 Trin175410
Tobo186596 Trin175620
Tobo186597 *Trin196726
Trin196729 Trin196727
Trin196730
Trin196731
Trin196732
Trin196733
Trin196734
Trin196735
Trin196888
(C) Trin175424
(D) Trin196728
(E) Trin196886
Table 1: Percentage values of pair-wise genetic distances for: the 12S data set (top graph), 16S data set (middle graph), and combined data set
(bottom graph). Distances corrected using the general time-reversible model with gamma distributed rates for variable sites.
12S Data L. validus L. pallidirostris L. podicipinus L. wagneri L. chaquensis
L. validus 0-0.49
L. pallidirostris 0.12-0.62 0.25
L. podicipinus 10.51-11.15 10.52-10.85 0.12-0.88
L. wagneri 5.04-5.69 5.04-5.72 10.53-11.21 0.24-1.52
L. chaquensis 12.23-12.81 12.19-12.68 12.18-12.72 10.95-11.81
L. knudseni 16.93-17.50 17.00-17.01 15.11-15.57 15.84-16.60 12.10
16S Data L. validus L. pallidirostris L. podicipinus L. wagneri L. chaquensis
L. validus 0-0.36
L. pallidirostris 0.14-0.73 0.51
L. podicipinus 15.22-16.79 15.34-16.43 0-2.1
L. wagneri 9.12-9.92 8.84-9.57 13.98-15.39 0.22-1.73
L. chaquensis 20.43-20.96 20.20-20.79 17.66-17.99 21.25-22.31
L. knudseni 26.10-26.75 25.93-26.62 26.02-26.91 28.39-28.76 20.70
Combined Data L. validus L. pallidirostris L. podicipinus L. wagneri L. chaquensis
L. validus 0-0.32
L. pallidirostris 0.14-0.64 0.41
L. podicipinus 13.5-14.54 13.45-14.21 0.05-1.64
L. wagneri 7.64-8.04 7.41-7.78 12.79-13.59 0.23-1.65
L. chaquensis 17.21-17.55 16.93-17.50 15.50-15.86 16.91-17.90
L. knudseni 22.44-22.74 22.27-22.66 21.57-22.18 23.18-23.57 17.20
196 Genetic resolution of Leptodactylus validus
trees (L = 1041; CI = 0.856). Weighting loop positions
(n = 1135) relative to stem positions (n = 1112) with
gaps as missing data also resulted in three minimum-
length trees (L = 1269; CI = 0.850). The strict con-
sensus trees from these analyses were identical to the
strict consensus trees obtained from the unweighted
combined dataset. Using gaps as a fifth character un-
der the same weighting scheme resulted in a single
tree (L = 1379; CI = 0.839) identical to the consensus
tree of the unweighted analyses. The ML analysis of
the combined data under the TrN+G model parame-
ters resulted in a bootstrap 50% MR consensus tree
similar to the one from the MP analyses, with slight
differences in relationships among samples within the
L. podicipinus clade. A similar tree was obtained us-
ing the GTR+I+G model parameters, with better reso-
lution demonstrated among samples within the
L. podicipinus clade. ML analyses considering sec-
ondary structure and weighting loop:stem positions (2:1),
using both evolutionary models resulted in bootstrap
50% MR consensus trees identical to the tree from
the analysis using the GTR+I+G model.
The Bayesian analysis was performed under the
GTR+I+G model settings. Convergence of the log like-
lihood values among the seven MCMC chains occurred
within 40,000 generations of sampling, consequently
the first 4,000 trees sampled were discarded. The re-
sulting consensus tree from MrBayes (Fig. 2) was sim-
ilar to the unweighted ML tree obtained using the
GTR+I+G model parameters, with a better resolution
of relationships among L. validus samples. However,
with the exception of the subclade consisting of sam-
ples from Grenada/St. Vincent (haplotypes A and B),
there was little posterior support for relationships among
other L. validus samples in the clade.
Table 4: Combined sequence haplotypes for L. validus samples
from: Grenada (Gren), St. Vincent (StVn), Tobago (Tobo), and
Trinidad (Trin). Haplotypes A-D represent multiple samples. Sam-
ples used to represent each haplotype are indicated by an asterisk.
Haplotypes E-K each consist of a single sample as listed below the
table.
A B C D
Gren006881 Gren196977 *Trin196726 *Tobo186596
Gren006882 Gren196979 Trin196727 Tobo186597
Gren006883 Gren196980 Trin196730
Gren006939 Gren197000 Trin196731
Gren196978 Gren197001 Trin196732
Gren196999 Gren197002 Trin196733
Gren197005 Gren197003 Trin196734
Gren197006 Gren197004 Trin196888
Gren197017 Gren197007
StVn056421 Gren197008
StVn056490 Gren197044
StVn056561 StVn196894
StVn056562 *StVn196896
StVn056612 StVn196897
*StVn056613 StVn196899
StVn196895 StVn196900
StVn196898
(E) Trin175410
(F) Trin175424
(G) Trin175620
(H) Trin196728
(I) Trin196729
(J) Trin196735
(K) Trin196886
Table 3: 16S rDNA sequence haplotypes for L. validus samples
from: Grenada (Gren), St. Vincent (StVn), Tobago (Tobo), and
Trinidad (Trin). Samples A-C represent multiple samples. Aster-
isks indicate samples used to represent a haplotype. Haplotypes
D-G each consist of a single sample as listed below the table.
A B C
Gren006881 Trin175424 *Tobo186596
Gren006882 *Trin196726 Tobo186597
Gren006883 Trin196727
Gren006939 Trin196728
Gren196977 Trin196729
Gren196978 Trin196730
Gren196979 Trin196731
Gren196980 Trin196732
Gren196999 Trin196733
Gren197000 Trin196734
Gren197001 Trin196888
Gren197002
Gren197003
Gren197004
Gren197005
Gren197006
Gren197007
Gren197008
Gren197017
Gren197044
StVn056421
StVn056490
StVn056561
StVn056562
StVn056612
*StVn056613
StVn196894
StVn196895
StVn196896
StVn196897
StVn196898
StVn196899
StVn196900
(D) Trin196886
(E) Trin175410
(F) Trin175620
(G) Trin196735
Yanek, K. et al. 197
DISCUSSION
Morphological characters and call data have been
previously used to assess variation among species within
the Leptodactylus podicipinus-wagneri complex
(Heyer, 1994). However, morphological data were in-
sufficient to resolve all species boundaries in this com-
plex. The status of L. pallidirostris and L. validus
within this complex remained one of the major unre-
solved problems (Heyer, 1994).
Slight adult morphological variation exists between
populations from the Lesser Antilles and those from
Trinidad and Tobago. The overall morphologies of
L. pallidirostris and L. validus are very similar to each
other. Leptodactylus pallidirostris is distributed
throughout Venezuela, Guyana, Suriname, French Gui-
ana, and northern Brazil. Available call data for
L. validus from Trinidad and Tobago are similar to calls
analyzed from Brazilian and Venezuelan populations
of L. pallidirostris (Heyer, 1994). The overall data
presented in Heyer (1994) were equivocal as to wheth-
er L. pallidirostris and L. validus were conspecific
or represented distinct species.
All analyses performed in this study strongly sup-
port a monophyletic group consisting of the
L. pallidirostris and L. validus samples; moreover,
L. pallidirostris samples did not cluster as an exclu-
sive monophyletic clade. These results support the
conspecificity of the two taxa. However, saturation of
nucleotide substitutions among samples can affect the
estimation of evolutionary distances and potentially
result in misleading tree topologies (Swofford et al.,
1996; Page and Holmes, 1998; Nei and Kumar, 2000;
de Peer et al., 2002). Our assessment of sequence
saturation indicates a low degree of both transitional
and overall substitutional saturation for these sequenc-
es (Fig. 1).
The combined analyses reveal some genetic struc-
turing among populations within this clade, i.e., genet-
ically distant samples are also geographically distant.
For example, closer relationships are demonstrated
among samples of L. validus from the Lesser Anti-
lles. Likewise, the L. pallidirostris sample from Guy-
ana appears more closely related to the L. validus
samples than the L. pallidirostris sample from Brazil.
The following maximum genetic distances were ob-
tained for L. validus and L. pallidirostris samples:
< 0.37% with the sample from Guyana and < 0.73%
for the sample from Brazil. Also, less than 0.5% se-
quence divergence is observed among the L. validus
samples. Levels of sequence divergence are not abso-
lute predictors of species diversity; however, these low
levels of sequence divergence are consistent with
L. pallidirostris and L. validus being conspecific. In
this scenario, L. validus is a single species distributed
throughout the Lesser Antilles, Trinidad and Tobago,
and adjacent mainland South America.
Advertisement calls are commonly analyzed in anu-
ran systematic studies. These calls are almost always
Figure 1. Plots depicting relative rates of transitional saturation for
the 12S (A) and 16S (C) Gene sequences; and substitutional satu-
ration for 12S (B) and 16S (D) using pair-wise genetic distances
corrected under the GTR evolutionary model.
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
0 0.02 0.04
Kimura 2-parameter distance
Un
co
rr
ec
te
d 
s
eq
u
en
ce
 d
iv
er
ge
n
ce
0.06 0.08 0.1
(A)
0.12 0.14
Corrected GRT+G distance
Un
co
rr
ec
te
d 
se
qu
en
ce
 
di
v
er
ge
n
ce
0.14
0.12
0.08
0.06
0.04
0.02
0
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14
0.1
(B)
Un
c
o
rr
ec
te
d 
se
qu
en
ce
 
di
v
er
ge
n
ce
Kimura 2-parameter distance
(C)
0.2
0.18
0.14
0.16
0.12
0.1
0.08
0.06
0.04
0.02
0
0 0.20.180.160.140.120.10.080.060.040.02
Un
co
rr
ec
te
d 
se
qu
en
ce
 d
iv
er
ge
n
ce
Corrected GTR+G distance
(D)
0.22
0.2
0.18
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
0 0.02 0.04 0.06 0.08 0.1 0.12 0.220.14 0.16 0.18 0.2
198 Genetic resolution of Leptodactylus validus
species-specific in Leptodactylus (see Heyer et al.,
2005 for an exception) and in anurans in general, and
usually serve as a reliable indicator of species bound-
aries in frogs (Heyer and Straughan, 1976; Heyer, 1978,
1979, 1994; Heyer et al., 1996; Wieczorek and Chan-
ning, 1997; Camargo et al., 2006). The available call
data from Brazilian and Venezuelan populations of
L. pallidirostris are very similar to the advertisement
call of L. validus (Heyer, 1994), providing additional
support for the conspecific status of the two species.
Figure 2. Bayesian consensus tree of combined dataset. Posterior probability values > 50% are shown above branches. See text for clade
descriptions.
100
100
100
100
100
100
100
62
100
100
75
58
L. validus B
L. validus A
L. validus C
L. validus G
L. validus H
L. validus J
L. validus E
L. validus I
L. validus K
L. validus F
L. validus D
L. pallidirostris (Guyana)
L. pallidirostris (Brazil)
wagneri 10616
wagneri 177351
wagneri (Brazil)
L. podicipinus UC243
L. podicipinus UC244
L. podicipinus UC278
L. podicipinus 53124
L. knudseni
L. chaquensis
Yanek, K. et al. 199
Heyer (1994) also indicated that some of the Venezue-
lan samples examined and assigned to L. pallidirostris
closely resembled Trinidad and Tobago samples desig-
nated as L. validus, supporting their conspecificity.
Leptodactylus pallidirostris was described by Lutz
in 1930 from Kartabo, Guyana, who repeatedly referred
to the species? resemblance to L. validus. This is in
agreement with the present study which proposes that
L. pallidirostris and L. validus represent a single tax-
on. Leptodactylus validus was described by Garman
in 1888 ?1887?, therefore this name has priority over
L. pallidirostris. Consequently, L. pallidirostris Lutz,
1930 is placed in the synonymy of L. validus Garman,
1888.
The taxonomic resolution of L. pallidirostris as a
synonym of L. validus results in the resolution of the
previous distributional enigma involving L. validus sen-
su Heyer, 1994. Murphy (1997) considered all five of
the Lesser Antillean amphibians and reptiles that oc-
cur in Trinidad and Tobago to have been recent intro-
ductions from the Lesser Antilles. Murphy (1997) indi-
cated that most, if not all, of the introductions were the
result of human activity. The distribution and minimal
genetic variation of L. validus is consistent with hu-
man mediated introductions, but in this case the direc-
tion was most likely from Trinidad and Tobago to the
Lesser Antilles. As Trinidad and Tobago are continen-
tal islands, gene flow in L. validus from Trinidad and
Tobago with the mainland populations likely occurred
as late as about 20,000 years ago (Murphy, 1997).
RESUMEN
Leptodactylus validus tiene una distribuci?n parti-
cular, encontrandose en Trinidad, Tobago, y las Antillas
Menores, pero no en Am?rica del Sur. Esta distribuci?n
es inconsistente con los patr?nes de distribuci?n para
otros grupos en estas islas. A pesar que se ha obervado
variaci?n en la morfolog?a adulta de L. validus en dife-
rentes islas, los datos de canto sugieren la presencia de
una sola especie. Cantos de L. pallidirostris de Vene-
zuela y Brasil sugieren que esta especie podr?a ser co-
espec?fica con L. validus. Datos moleculares de se-
cuencias de los gene mt 12S y 16S sugieren que
L. validus consiste de una sola especie en su distribu-
ci?n y que esta especie es coespec?fica con
L. pallidirostris. La dispersi?n de L. validus de Trini-
dad y Tobago a las Antillas Menores podr?a haber ocu-
rrido atraves de actividades humanas.
ACKNOWLEDGMENTS
We acknowledge funding support for this study through
National Science Foundation Awards #9815787 and #0342918 to
RdS and WRH. KY thanks the Graduate School of Arts and Sci-
ences, University of Richmond, for financial support. We thank
the colleagues and institutions that kindly provided tissue speci-
mens and/or helped in collecting samples.
LITERATURE CITED
BOZDOGAN, H. 1987. Model selection and Akaike?s information
criterion (AIC): The general theory and its analytical
extensions. Psychometrica 52(3):345-370.
CAMARGO, A., R.O. DE S? AND W.R. HEYER. 2006. Phylogenetic
analyses of mtDNA sequences reveal three cryptic lineages in
the widespread neotropical frog Leptodactylus fuscus
(Schneider, 1799) (Anura, Leptodactylidae). Biological Journal
of the Linnean Society 87:325-341.
CANNONE J.J., S. SUBRAMANIAN, M.N. SCHNARE, J.R. COLLETT, L.M.
D?SOUZA, Y. DU, B. FENG, N. LIN, L.V. MADABUSI, K.M. MULLER,
N. PANDE, Z. SHANG, N. YU AND R.R. GUTELL. 2002. The
comparative RNA web (CRW) site: an online database of
comparative sequence and structure information for ribosomal,
intron, and other RNAs. BioMed Central Bioinformatics 3:15.
URL:http://www.rna.icmb.utexas.edu/.
FELSENSTEIN, J. 1981. Evolutionary trees from DNA sequences: a
maximum likelihood approach. Journal of Molecular Evolution
17(6):368-376.
FELSENSTEIN, J. 1985. Confidence limits on phylogenies: An approach
using the bootstrap. Evolution 39:783-791.
FELLER, A.E. AND S.B. HEDGES. 1998. Molecular evidence for the
early history of living amphibians. Molecular Phylogenetics
and Evolution 9(3):509-516.
FISHER, R.A. 1922. On the mathematical foundations of theoretical
statistics. Philosophical Transactions of the Royal Society
A222:309-368.
FROST, D.R. 2006. Amphibian Species of the World: an Online
Reference. Version 4 (17 August, 2006). Electronic Database
accessible at http://research.amnh.org/herpetology/amphibia/
index.php. American Museum of Natural History, New York,
USA.
GARMAN, S. 1888 ?1887?. West Indian Batrachia in the Museum
of Comparative Zoology. Bulletin of the Essex Institute
19:13-16.
GOEBEL, A.M., J.M. DONNELLY AND M.E. ATZ. 1999. PCR primers
and amplification methods for 12S ribosomal DNA, the control
region, cytochrome oxidase I, and cytochrome b in bufonids
and other frogs, and an overview of PCR primers which have
amplified DNA in amphibians successfully. Molecular
Phylogenetics and Evolution 11(1):163-199.
HEDGES, S.B. 1994. Molecular evidence for the origin of birds.
Proceedings of the National Academy of Sciences of the United
States of America 91:2621-2624.
HEYER, W.R. 1969. The adaptive ecology of the species groups of
the genus Leptodactylus (Amphibia, Leptodactylidae).
Evolution 23:421-428.
HEYER, W.R. 1974. Relationships of the marmoratus species group
(Amphibia, Leptodactylidae) within the subfamily
Leptodactylinae. Contributions in Science, Los Angeles County
Museum 253:1-45.
200 Genetic resolution of Leptodactylus validus
HEYER, W.R. 1978. Systematics of the fuscus group of the frog
genus Leptodactylus (Amphibia: Leptodactylidae). Science
Bulletin, Natural History Museum of Los Angeles County
29:1-85.
HEYER, W.R. 1979. Systematics of the pentadactylus species group
of the frog genus Leptodactylus (Amphibia: Leptodactylidae).
Smithsonian Contributions to Zoology 301:1-43.
HEYER, W.R. 1994. Variation within the Leptodactylus podicipinus-
wagneri complex of frogs (Amphibia: Leptodactylidae).
Smithsonian Contributions to Zoology 546:1-24.
HEYER, W.R. AND I.R. STRAUGHAN. 1976. A functional analysis of
the mating calls of the Neotropical frog genera of Leptodactylus
complex (Amphibia: Leptodactylidae). Pap?is Avulsos de
Zoologia 29:221-245.
HEYER, W.R., J.M. GARC?A-LOPEZ AND A.J. CARDOSO. 1996.
Advertisement call variation in the Leptodactylus mystaceus
species complex (Amphibia: Leptodactylidae) with a
description of a new sibling species. Amphibia-Reptilia 17:7-31.
HEYER, W.R., R.O. DE S? AND A. RETTIG. 2005. Sibling species,
advertisement calls, and reproductive isolation in frogs of the
Leptodactylus pentadactylus species cluster (Amphibia,
Leptodactylidae). Russian Journal of Herpetology,
12(Supplement):35-39.
HICKSON, R.E., C. SIMON, A. COOPER, G.S. SPICER, J. SULLIVAN AND D.
PENNY. 1996. Conserved sequence motifs, alignment, and
secondary structure for the third domain of animal 12S rRNA.
Molecular Biology and Evolution 13(1):150-169.
HILLIS, D.M., C. MORITZ AND B.K. MABLE (EDS.). 1996. Molecular
Systematics, Second Edition. Sinauer Associates Inc.,
Sunderland, Massachusetts.
HUELSENBECK, J.P. AND F. RONQUIST. 2001. MRBAYES: Bayesian
inference of phylogeny. Bioinformatics 17:754-755.
KJER, K.M. 1995. Use of rRNA secondary structure in phylogenetic
studies to identify homologous positions: an example of
alignment and data presentation from the frogs. Molecular
Phylogenetics and Evolution 4(3):314-330.
LUTZ, A. 1930. Segunda memoria sobre especies brasileiras do genero
Leptodactylus, incluindo outras alliadas. Mem?rias do Instituto
Oswaldo Cruz 23(1):1-20.
MAXSON, L.R. AND W.R. HEYER. 1988. Molecular systematics of
the frog genus Leptodactylus (Amphibia: Leptodactylidae).
Fieldiana: Zoology, New Series 41:1-13.
MURPHY, J.C.E. 1997. Amphibians and Reptiles of Trinidad and
Tobago, Krieger Publishing Co, Malabar, Florida, 245 pp.
NEI, M. AND S. KUMAR (EDS.). 2000. Molecular Phylogenetics and
Evolution, Oxford University Press, Oxford, UK.
PAGE, R.D.M. AND E.C. HOLMES (EDS.). 1998. Molecular Evolution,
A Phylogenetic Approach, Blackwell Science, Oxford, UK.
PALUMBI, S.R. 1996. Nucleic Acids II: The polymerase chain reaction.
In Hillis, D.M., Moritz, C. and Mable, B.K. (eds.), Molecular
Systematics, Second Edition, pp. 205-221. Sinauer Associates
Inc., Sunderland Massachusetts.
PALUMBI, S., A. MARTIN, S. ROMANO, W.O. MCMILLAN, L. STICE AND
G. GRABOWSKI. 1991. The simple fool?s guide to PCR, version
2.0. Privately published document compiled by S. Palumbi,
Department of Zoology and Kewalo Marine Laboratory.
University of Hawaii, Honolulu, HI.
POSADA, D. AND CRANDALL, K.A. 1998. Modeltest: testing the model
of DNA substitution. Bioinformatics 14(9):817-818.
RODRIGUEZ, F., J.L. OLIVER, A. MARTIN AND J.R. MEDINA. 1990. The
general stochastic model of nucleotide substitution. Journal of
Theoretical Biology 142:485-501.
SIMMONS, M.P. AND H. OCHOTERENA. 2000. Gaps as characters in
sequence-based phylogenetic analyses. Systematic Biology
49:369-381.
SIMMONS, M.P., H. OCHOTERENA AND T. CARR. 2001. Incorporation,
relative homoplasy, and effect of gap characters in sequence-
based phylogenetic analyses. Systematic Biology 50:454-462.
SWOFFORD, D.L. 2002. PAUP*. Phylogenetic Analysis Using
Parsimony (*and Other Methods). Version 4.0b10. Sinauer
Associates, Sunderland, Massachusetts.
SWOFFORD, D.L., G.J. OLSEN, P.J. WADDELL AND D.M. HILLIS. 1996.
Phylogenetic Inference. In Hillis, D.M., Moritz, C. and Mable,
B.K. (eds.), Molecular Systematics, Second Edition,
pp. 407-514. Sinauer Associates Inc., Sunderland,
Massachusetts.
TAMURA, K. AND M. NEI. 1993. Estimation of the number of
nucleotide substitutions in the control region of mitochondrial
DNA in humans and chimpanzees. Molecular Biology and
Evolution 10:512-526.
TAVARE, S. 1986. Some probabilistic and statistical problems in the
analysis of DNA sequences. Lectures on Mathematics in the
Life Sciences 17:57-86.
THOMPSON, J.D., T.J. GIBSON, F. PLEWNIAK, F. JEANMOUGIN AND D.G.
HIGGINS. 1997. The ClustalX windows interface: flexible
strategies for multiple sequence alignment aided by quality
analysis tools. Nucleic Acid Research 25:4876-4882.
WIECZOREK, A.M. AND A. CHANNING. 1997. The taxonomic status of
Broadley?s reed frog. African Journal of Herpetology
46(2):110-116
Received 01 August 2006
Accepted 01 October 2006
Yanek, K. et al. 201
APPENDIX I
Museum catalogue numbers, field numbers, and locality data for the samples utilized in this study. Museum abbreviations:
BWMC = Bobby Witcher Memorial Collection, Avila University, LSUMZ = Louisiana State University, Museum of Natural Science,
QCAZ = Museo de Zoolog?a de la Pontificia Universidad Cat?lica del Ecuador, Quito, USNM National Museum of Natural History,
Smithsonian Institution.
Species Museum Number Field Number Locality
L. chaquensis USNM 319708 USFS 186524 Argentina: Tucum?n; ca. 40 km SE San Miguel de Tucum?n.
L. knudseni QCAZ 13244 Ecuador: Provincia de Orellana; Parque Nacional Yasun?.
L. pallidirostris USNM 535774 USFS 207682 Guyana: Northwest District; Baramita.
L. pallidirostris USNM 302408 WRH 8626 Brazil: Roraima; Igarap? Cocal..
L. podicipinus UC 243 Brazil: Mato Grosso do Sul; Est?ncia Mimosa; Mun. de Bonito.
L. podicipinus UC 244 Brazil: Mato Grosso do Sul; Est?ncia Mimosa; Mun. de Bonito.
L. podicipinus UC 278 Brazil: S?o Paulo; Ch?cara Renascer; Bauru.
L. podicipinus USNM 053124 USFS 303207 Brazil: S?o Paulo; Fazenda Jatai.
L. validus BWMC 06881 Grenada: St. Andrew; Spring Gardens Estate.
L. validus BWMC 06882 Grenada: St. Andrew; Birch Grove.
L. validus BWMC 06883 Grenada: St. Andrew; Birch Grove.
L. validus BWMC 06939 Grenada: St. George; Beausejour.
L. validus USNM 314793 USFS 196977 Grenada: St. George; Grand Anse Bay.
L. validus USNM 314794 USFS 196978 Grenada: St. George; Grand Anse Bay.
L. validus USNM 314795 USFS 196979 Grenada: St. George; Grand Anse Bay.
L. validus USNM 314796 USFS 196980 Grenada: St. George; Grand Anse Bay.
L. validus USNM 314798 USFS 197044 Grenada: St. George; Grand Anse Bay.
L. validus USNM 314813 USFS 196999 Grenada: St. George; Grand Anse Bay.
L. validus USNM 314814 USFS 197000 Grenada: St. George; inland from Grand Anse Bay.
L. validus USNM 314815 USFS 197001 Grenada: St. George; inland from Grand Anse Bay.
L. validus USNM 314816 USFS 197002 Grenada: St. George; inland from Grand Anse Bay.
L. validus USNM 314817 USFS 197003 Grenada: St. George; inland from Grand Anse Bay.
L. validus USNM 314818 USFS 197004 Grenada: St. George; inland from Grand Anse Bay.
L. validus USNM 314819 USFS 197005 Grenada: St. George; inland from Grand Anse Bay.
L. validus USNM 314820 USFS 197006 Grenada: St. George; inland from Grand Anse Bay.
L. validus USNM 314821 USFS 197007 Grenada: St. George; inland from Grand Anse Bay.
L. validus USNM 314822 USFS 197008 Grenada: St. George; inland from Grand Anse Bay.
L. validus USNM 314831 USFS 197017 Grenada: St. George; inland from Grand Anse Beach.
L. validus USNM 314512 USFS 56421 St. Vincent: St. Andrew; near Vermont.
L. validus USNM 314513 USFS 56490 St. Vincent: St. George; Arnos Vale.
L. validus USNM 314514 USFS 56612 St. Vincent: St. George; Arnos Vale.
L. validus USNM 314515 USFS 56613 St. Vincent: St. George; Arnos Vale.
L. validus USNM 314718 USFS 196894 St. Vincent: St. George; Arnos Vale.
L. validus USNM 314719 USFS 196895 St. Vincent: St. George; Arnos Vale.
L. validus USNM 314720 USFS 196896 St. Vincent: St. George; Arnos Vale.
L. validus USNM 314721 USFS 196897 St. Vincent: St. George; Arnos Vale.
L. validus USNM 314722 USFS 196898 St. Vincent: St. George; Arnos Vale.
L. validus USNM 314723 USFS 196899 St. Vincent: St. George; Arnos Vale.
L. validus USNM 314714 USFS 196900 St. Vincent: St. George; Arnos Vale.
L. validus USNM 314516 USFS 56561 St. Vincent: St. George; Rose Cottage.
L. validus USNM 314517 USFS 56562 St. Vincent: St. George; Rose Cottage.
L. validus USNM 523940 USFS 186596 Tobago: St. Paul; Delaford, Louis d?Or River.
L. validus USNM 523941 USFS 186597 Tobago: St. Paul; Delaford, Louis d?Or River.
L. validus USNM 286948 USFS 175410 Trinidad: St. George; Simla Research Station.
L. validus USNM 286959 USFS 175424 Trinidad: St. George; north of Simla Research Station.
L. validus USNM 306105 USFS 175620 Trinidad: St. George; near Brasso Seco Village.
L. validus USNM 314671 USFS 196886 Trinidad: St. George; west of Carapo.
L. validus USNM 314672 USFS 196888 Trinidad: St. George; west of Carapo.
L. validus USNM 314627 USFS 196726 Trinidad: St. Patrick; near Chatham Beach.
L. validus USNM 314628 USFS 196727 Trinidad: St. Patrick; near Chatham Beach.
L. validus USNM 314629 USFS 196728 Trinidad: St. Patrick; near Chatham Beach.
L. validus USNM 314630 USFS 196729 Trinidad: St. Patrick; near Chatham Beach.
L. validus USNM 314631 USFS 196730 Trinidad: St. Patrick; near Chatham Beach.
L. validus USNM 314632 USFS 196731 Trinidad: St. Patrick; near Chatham Beach.
L. validus USNM 314633 USFS 196732 Trinidad: St. Patrick; near Chatham Beach.
L. validus USNM 314634 USFS 196733 Trinidad: St. Patrick; near Chatham Beach.
L. validus USNM 314635 USFS 196734 Trinidad: St. Patrick; near Chatham Beach.
L. validus USNM 314636 USFS 196735 Trinidad: St. Patrick; near Chatham Beach.
L. wagneri LSUMZ H-13653 JPC 12969 Brazil: Acre; ca. 5km N Porto Walter, inland from the Rio Juru?.
L. wagneri LSUMZ H-12885 JPC 10616 Ecuador: Sucumbios; Estaci?n Cient?fica near Cuyabeno.
L. wagneri USNM 320988 USFS 177351 Ecuador: Pastaza; Coca, 1km ENE of Tiguino.