phylogenetic and taxonomic relationships of the...
TRANSCRIPT
Zoologica Scripta
Phylogenetic and taxonomic relationships of the Polypedatesleucomystax complex (Amphibia)NORIHIRO KURAISHI, MASAFUMI MATSUI, AMIR HAMIDY, DAICUS M. BELABUT, NORHAYATI AHMAD,SOMSAK PANHA, AHMAD SUDIN, HOI S. YONG, JIAN-PING JIANG, HIDETOSHI OTA, HO T. THONG &KANTO NISHIKAWA
Submitted: 7 April 2012Accepted: 3 July 2012doi:10.1111/j.1463-6409.2012.00562.x
ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian
Kuraishi, N., Matsui, M., Hamidy, A., Belabut, D. M., Ahmad, N., Panha, S., Sudin, A., Yong,
H. S., Jiang, J.-P., Ota, H., Thong, H. T. & Nishikawa, K. (2012). Phylogenetic and
taxonomic relationships of the Polypedates leucomystax complex (Amphibia). —Zoologica Scripta,
00, 000–000.
We investigated the phylogenetic and taxonomic relationships and estimated the history of
species diversification and biogeography in the Asian rhacophorid genus Polypedates, focus-
ing on the Polypedates leucomystax complex, whose members are notoriously difficult to clas-
sify. We first estimated phylogenetic relationships within the complex using 2005-bp
sequences of the mitochondrial 12S rRNA, tRNAval and 16S rRNA genes with maximum
parsimony, maximum likelihood (ML) and Bayesian methods of inference. Polypedates
exhibits well-supported monophyly, with distinct clades for P. otilophus, P. colletti, P. macula-
tus and the P. leucomystax complex, consisting of P. macrotis, and the Malay (Polypedates sp.
from Malay Peninsula), North China (P. braueri), South China (Polypedates cf. mutus 1),
Indochina (P. megacephalus), Sunda (P. leucomystax) and Laos (Polypedates cf. mutus 2) clades.
In a subsequent phylogenetic analysis of 4696-bp sequences of the nuclear brain-derived
neurotrophic factor (BDNF), sodium ⁄ calcium exchanger 1 (NCX), POMC, Rag-1, Rhod and
Tyr genes using Bayesian methods of inference, all of these clades were recovered. Some
clades of the P. leucomystax complex occur sympatrically and show high genetic diversity or
morphological and acoustic differences. Similar tendencies were observed between some
allopatric clades. Therefore, we consider each of these groups to be distinct specifically.
We also estimated absolute divergence times within the genus using Bayesian methods.
Divergence in Polypedates began with the divergence of a primarily South Asian Clade from
the common ancestor of secondarily South-East Asia P. maculatus and South-East Asian
members. The divergence between the latter occurred much later. The P. leucomystax com-
plex diverged in the Pliocene, much later than other congeners, and seems to have been
greatly affected by human-related dispersal after the Pleistocene.
Corresponding author: Masafumi Matsui, Graduate School of Human and Environmental
Studies, Kyoto University, Kyoto 606-8501, Japan. E-mail: [email protected]
Norihiro Kuraishi, Graduate School of Human and Environmental Studies, Kyoto University,
Kyoto 606-8501, Japan. E-mail: [email protected]
Amir Hamidy, Graduate School of Human and Environmental Studies, Kyoto University, Kyoto
606-8501, Japan; Museum Zoologicum Bogoriense (MZB), Research Center for Biology, Indone-
sian Institute of Sciences, Gd. Widyasatwaloka, Jl. Raya Jakarta Bogor km 46, Cibinong West
Java, Indonesia. E-mail: [email protected]
Daicus M. Belabut, Institute of Biological Sciences, Faculty of Science, University of Malaya,
50603 Kuala Lumpur, Malaysia; Institute for Environment and Development (LESTARI),
Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia. E-mail: daicus@
um.edu.my
Norhayati Ahmad, Institute for Environment and Development (LESTARI), Universiti Keb-
angsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia; Faculty of Science and Technology,
Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia. E-mail: yati_68
@yahoo.co.uk
Academy of Science and Letters 1
Somsak Panha, Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok
10330, Thailand. E-mail: [email protected]
Ahmad Sudin, Institute for Tropical Biology and Conservation, University Malaysia Sabah, Kota
Kinabalu 88999, Sabah, Malaysia. E-mail: [email protected]
Hoi Sen Yong, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603
Kuala Lumpur, Malaysia. E-mail: [email protected]
Jian-Ping Jiang, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041,
China. E-mail: [email protected]
Hidetoshi Ota, Institute of Natural and Environmental Sciences and Museum of Nature and
Human Activities, University of Hyogo, Yayoigaoka 6, Sanda, Hyogo 669-1546, Japan. E-mail:
Ho Trung Thong, Institute Hue University of Agriculture and Forestry, 102 Phung Hung, Hue,
Vietnam. E-mail: [email protected]
Kanto Nishikawa, Graduate School of Human and Environmental Studies, Kyoto University, Kyoto
606-8501, Japan. E-mail: [email protected]
Phylogenetic study on the Polypedates leucomystax complex d N. Kuraishi et al.
IntroductionRecent molecular phylogenetic analyses have revealed the
presence of cryptic species in many lineages of wide-rang-
ing animals, including frogs from South-East Asia, a cen-
tre of anuran diversity. Some examples of taxa found to
contain cryptic taxa include Fejervarya limnocharis (Toda
et al. 1998; Veith et al. 2001), Microhyla ornata (Matsui
et al. 2005), Staurois tuberilinguis (Matsui et al. 2007), Hyla-
rana chalconota (Stuart et al. 2006; Inger et al. 2009) and
Limnonectes kuhlii (Matsui et al. 2010c).
The Polypedates leucomystax complex (Matsui et al. 1986;
Dubois 1987 as Rhacophorus; Orlov et al. 2001) encom-
passes frogs that are notoriously difficult to identify and
distinguish from P. leucomystax. Frogs of this complex
occur widely from Nepal, through southern East Asia, to
all of South-East Asia. Several South-East Asian species
like P. macrotis and P. mutus and some South Asian (Indian
and Sri Lankan) species like P. maculatus were once associ-
ated with P. leucomystax, although all of them are currently
considered distinct species. Based on external morphology,
karyotype and mating calls, Matsui et al. (1986) separated
P. megacephalus as represented by the Taiwanese popula-
tions from P. leucomystax (see Kuraishi et al. 2011).
Subsequently, the presence of cryptic species in the
complex has been reported. The occurrence of two syn-
topic and surely independent species has been described
from Vietnam (Inger et al. 1999; Ziegler 2002) and the
Malay Peninsula (Narins et al. 1998). However, the taxo-
nomic relationships of these species pairs have never been
resolved, leaving the taxonomy of the P. leucomystax com-
plex unclear. Although the family Rhacophoridae, includ-
ing some species of Polypedates, has been included in
recent molecular studies on anuran phylogeny (e.g. Rich-
ards & Moore 1998; Marmayou et al. 2000; Wilkinson
et al. 2002; Delorme 2004; Frost et al. 2006; Li et al.
2008), few attempts have been made to elucidate phyloge-
2 ª 2
netic relationships among members of the P. leucomystaxcomplex.
Recently, Brown et al. (2010) analysed the phylogenetic
relationships among samples of the P. leucomystax complex
using mitochondrial DNA (mtDNA) genes. However, the
majority of their samples were from the Philippines and
Sulawesi, where frogs now identified as P. leucomystax pre-
dominate (see Riyanto et al. 2011 for the taxonomic status
of the Sulawesi population), and samples from the conti-
nent, where more taxa constitute difficult taxonomic prob-
lems, were limited. Specifically, no samples from Hong
Kong, the type locality of P. megacephalus (a key species
for understanding the relationships within the complex),
were included in their study. However, Brown et al. (2010)
did not resolve the phylogenetic and taxonomic problems
of the complex. More recently, Sheridan et al. (2010) com-
pared one population each of the P. leucomystax complex
from Thailand and Singapore using mtDNA and acoustic
characteristics, but reached no definite taxonomic
conclusions.
Some studies have suggested the sympatric occurrence
of two taxa of the P. leucomystax complex in Vietnam,
Malaysia and the Philippines (Narins et al. 1998; Inger
et al. 1999; Ziegler 2002; Brown et al. 2010). This leads to
the hypothesis that similar sympatric occurrences take
place at other localities within the wide range of this spe-
cies complex. The sympatric distribution of more than
one genetically distinct lineage can be strong evidence in
favour of their distinct specific status. Therefore, deter-
mining their distribution pattern would be useful for solv-
ing taxonomic problems of this complicated species
complex. In this study, by analysing mitochondrial and
nuclear DNA genes, we clarify the phylogenetic relation-
ships of samples of the P. leucomystax complex and allied
species collected over a wide range in South-East and
South Asia to obtain basic information for use in future
012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters
N. Kuraishi et al. d Phylogenetic study on the Polypedates leucomystax complex
taxonomic revision of the complex. We also discuss the
historical biogeography of the genus Polypedates in these
regions.
Materials and methodsSampling design
Our own samples of Polypedates consisted of 205 speci-
mens ⁄ tissues obtained from China, Indochina, Sundaland,
the Philippines, Sulawesi, Okinawa and India (Fig. 1),
including the following taxa: P. macrotis, P. maculatus,
P. colletti, P. otilophus and the P. leucomystax complex
(Table S1; P. iskandari synonymized with P. leucomystax
following Kurniati 2011). We used Rhacophorus norhayatii,
Buergeria buergeri and Rana japonica as out-group taxa. We
obtained mtDNA sequences for B. buergeri from GenBank
(AB127977). Rhacophorus is very close to Polypedates (Liem
1970), although their sister relationship has not yet been
demonstrated unambiguously (Li et al. 2008; Yu et al.
2009), whereas Buergeria is a sister genus to the clade con-
sisting of all other rhacophorids, including Rhacophorus and
Polypedates (Richards & Moore 1998; Li et al. 2008; Yu
et al. 2009), and Ranidae (represented by Rana) is the sister
Fig. 1 Map of South-East Asia showing the Polypedates sampling
localities included in this study. Sample numbers correspond to
those given Table S1.
ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters
group of Rhacophoridae plus its sister family Mantellidae
(Frost et al. 2006).
We first examined partial sequences of the 12S rRNA,
tRNAval and 16S rRNA mtDNA genes for all 208 samples
mentioned above (Table S1). Then, we analysed seven
nuclear (nu) DNA genes: BDNF, NCX, proopiomelano-
cortin A (POMC), recombination activating protein 1
(Rag1), rhodopsin (Rhod), solute carrier family 8 member
3 (SCF) and tyrosinase (Tyr) for 21 selected samples, rep-
resenting genetic groups recognized in the mtDNA genea-
logical tree and three out-group taxa (Table S2).
Voucher specimens ⁄ tissues are stored in the BORNE-
ENSIS collection, the University Malaysia Sabah (BORN),
Chengdu Institute of Biology (CIB), Kyoto University,
Graduate School of Human and Environmental Studies
(KUHE), Kyoto University, Department of Zoology
(KUZ), the Museum of Vertebrate Zoology, the Univer-
sity of California, Berkeley (MVZ), MZB, the United
States National Museum and the Zoological Institute,
Saint Petersburg.
DNA preparation, PCR and DNA sequencing
We obtained tissues from frozen or ethanol (95–99%)-
preserved specimens and extracted total genomic DNA
using a standard phenol ⁄ chloroform procedure (Hillis
et al. 1996). The tissues were homogenized in 0.6 mL of
STE buffer containing 10 mM Tris ⁄ HCl (pH 8.0),
100 mM NaCl and 1 mM EDTA (pH 8.0). Proteinase K
(0.1 mg ⁄ mL) was added to the homogenized solutions,
and the proteins were digested for 4–12 h at 55 �C. The
solution was treated with phenol and chloroform ⁄ isoamyl
alcohol, and DNA was precipitated with ethanol. The
DNA precipitates were dried and then resuspended in
0.6 mL TE (10 mM Tris ⁄ HCl, 1 mM EDTA, pH 8.0), and
1 lL was subjected to the polymerase chain reaction
(PCR).
For mtDNA, the PCR included an initial denaturation
for 5 min at 94 �C and 33 cycles of 15 s at 94 �C, 15 s at
53 �C and 2 min 40 s at 72 �C. The PCR primers are
shown in Table S3. The PCR products, purified using
polyethylene glycol (13%) precipitation, were used directly
as templates for cycle sequencing reactions with fluores-
cent dye-labelled terminator (BigDye Terminator v.3.1
Cycle Sequencing Kit; Applied Biosystems, Foster City,
CA, USA). The sequencing reaction products were puri-
fied by ethanol precipitation following the manufacturer’s
protocol and then run on an ABI PRISM 3130 Genetic Ana-
lyzer (Applied Biosystems). All samples were sequenced in
both directions using the same primers as for PCR and 10
additional sequencing primers (Table S3).
Sequence data obtained for each sample were adjusted
manually by eye using Chromas Pro (Technelysium,
3
Phylogenetic study on the Polypedates leucomystax complex d N. Kuraishi et al.
Tewantin, Australia). The resulting sequences were depos-
ited in GenBank (AB564278–AB564288, AB727997–
AB728192). The sequences of each gene region were
aligned using the ClustalW option of Bioedit (Hall 1999).
After testing consistency among gene partitions using
incongruence length difference tests with 1000 randomized
partitions (Farris et al. 1994) and confirming no significant
heterogeneity, we obtained an alignment matrix with 2450
nucleotide sites (919, 72 and 1459 for 12S rRNA, tRNAval
and 16S rRNA, respectively). To exclude gaps and ambig-
uous areas, we further revised the alignment with Gblocks
0.91b (Castresana 2000) using the default parameters. The
revised alignment was 2005 bp long (817, 58 and 1130 bp
for 12S rRNA, tRNAval and 16S rRNA, respectively).
For nuDNA, we extracted DNA, amplified it using the
primers shown in Table S3 and sequenced these regions
using a process similar to that used for mtDNA. The
obtained sequences were deposited in GenBank
(AB728193–AB728323). We obtained an alignment matrix
with 4696 nucleotide sites (508 sites for BDNF, 1000 sites
for NCX, 571 sites for POMC, 780 sites for Rag1, 316
sites for Rhod, 1076 sites for SCF and 445 sites for Tyr;
Table S4). Using these sequences, we conducted two anal-
yses. First, we estimated the phylogenetic relationships
among the groups detected using mtDNA (14 samples
from 10 in-group clades and three out-group samples). No
heterogeneous sites were found in these samples. We
reconstructed a phylogenetic tree based on the method
used for the mtDNA analysis (see below). Second, we
examined our data for evidence of reproductive isolation
(absence of common nuDNA haplotypes) between the two
mitochondrial lineages of the P. leucomystax complex
[Indochina Clade (five samples) and Sunda Clade (six sam-
ples), see below]. We obtained heterogeneous sites in the
genotypes of BDNF and Rag 1 and inferred a haplotype
pair from them using PHASE ver. 2.1 (Stephens et al. 2001).
Phylogenetic analysis
For the mtDNA data, we used three methods for estimat-
ing phylogenetic relationships: maximum parsimony (MP)
using a heuristic search (tree-bisection-reconnection
branch-swapping algorithm with 100 random addition rep-
licates) with an equal weighting option; ML based on the
substitution model for each partition (12S rRNA, tRNAval
and 16S rRNA) and phylogenetic parameters, chosen by
the program Kakusan3 (Tanabe 2007) based on the Akaike
information criterion (AIC); and Bayesian inference (BI;
Rannala & Yang 1996; Huelsenbeck et al. 2001) with the
models derived from Kakusan3 based on the AIC using
four simultaneous Metropolis-coupled Monte Carlo
Markov chains for 6 000 000 generations and sampling a tree
every 100 generations. Convergence of the log-likelihood
4 ª 2
scores was checked using TRACER ver. 1.4 (Rambaut &
Drummond 2007). As we found that the log-likelihood
scores stabilized after 3 000 000 generations, we discarded
the first 30 000 trees (3 000 000 generations) as burn-in
and used the remaining 30 001 trees to estimate the phy-
logeny and Bayesian posterior probabilities (BPPs).
MP analyses were conducted using PAUP*4.0b (Swof-
ford 2002). Pairwise comparisons of uncorrected sequence
divergences (p-distance) in 16S rRNA were also made
using PAUP. ML analysis was conducted using TREEFIND-
ER ver. Oct. 2008 (Jobb 2008). Bayesian analysis was con-
ducted using MRBAYES v3.1.2 (Huelsenbeck & Ronquist
2001). The robustness of the MP and ML trees was tested
using bootstrap analyses (Felsenstein 1985) with 1000 rep-
licates. We regarded tree topologies with bootstrap values
(BS) ‡ 70% as sufficiently supported (Hillis & Bull 1993).
For the Bayesian analysis, we considered BPP ‡ 0.95 as
significant support (Huelsenbeck et al. 2001; Leache &
Reeder 2002; Huelsenbeck & Rannala 2004).
For the nuDNA tree based on the first data set (see
above), we used BI with the optimum substitution model
for each codon position (1st, 2nd or 3rd position) for each
nucleotide site derived from Kakusan3, based on the AIC.
Estimating divergence time
To estimate the age of each genetic group, we prepared
three different data sets: (i) 2005 bp (12S and 16S rRNA,
and tRNAval) of 36 mtDNA sequences were selected from
our original data set, including 29 individuals from the
P. leucomystax complex, three other South-East Asian Po-
lypedates (P. macrotis, P. colletti and P. otilophus), one South
Asian Polypedates (P. maculatus), two other rhacophorid
frogs (R. norhayatii and B. buergeri) and one ranid (R. japon-
ica); (ii) 933 bp (12S and 16S rRNA) of 17 mtDNA
sequences were selected from the original data set and
GenBank, including 10 samples of Polypedates, each repre-
senting genetic groups obtained from phylogenetic analy-
ses, three South Asian Polypedates [P. fastigo
(Meegaskumbura et al. 2002: AY141802, AY141848),
P. eques (Meegaskumbura et al. 2002: AY141800,
AY141846), and P. cruciger (Bossuyt & Milinkovitch 2000:
AF240928, AF249045)] and four out-group taxa, adding
one mantellid [Mantella madagascariensis (Bossuyt & Milin-
kovitch 2000: AF249005, AF249049)] to those used in data
set #1; and (iii) 4696 bp (BDNF, NCX, POMC, Rag1,
Rhod, SCF and Tyr) of 16 nuDNA sequences selected
from our original data set for 14 samples of Polypedates,
R. norhayatii and B. buergeri.
For all data sets, we estimated the divergence times
using a Bayesian relaxed molecular clock calculated in
BEAST (Drummond & Rambaut 2007) using 100 million
generations, discarding the first 10 million generations as
012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters
N. Kuraishi et al. d Phylogenetic study on the Polypedates leucomystax complex
burn-in. Parameter values were sampled every 1000 gener-
ations, under a HKY substitution model and uncorrelated
lognormal ‘relaxed’ clock rate model (Drummond et al.
2006). Parameter estimates and convergence were checked
using TRACER ver. 1.4 (Rambaut & Drummond 2007).
For data set # 1, each external and internal calibration
point was used to estimate the dates of cladogenic events.
The divergence time Roelants et al. (2007) assumed to be
49.7 [95% credibility interval (CI) 36.6–57.6] million years
before present (MYBP) between Buergeria and other mem-
bers of Rhacophoridae was used as an external calibration
point, while an internal calibration point was set at the
divergence of the P. leucomystax complex populations from
Java and Sumatra as 2.8 (CI 1.4–4.5) MYBP, the values
estimated for the corresponding two populations of the
megophrid Leptobrachium hasseltii (Matsui et al. 2010b).
For data set #2, two external calibration points were used;
the divergence time of 73.1 (CI 53.6–92.6) MYBP between
Mantellidae and Rhacophoridae (Bossuyt & Milinkovitch
2000) was added to the divergence between Buergeria and
other members of the Rhacophoridae. For data set #3, the
divergence time between Buergeria and other members of
Rhacophoridae (Roelants et al. 2007) was used as an exter-
nal calibration point.
ResultsSequence statistics
Sequence statistics for the three mtDNA gene fragments
and for the combined alignment including all nucleotide
positions are provided in Table S4. The aligned 12S
rRNA, tRNAval and 16S rRNA data set consisted of 2005
characters, of which 823 sites were variable and 583 were
potentially phylogenetically informative. MP analysis
yielded 2400 most parsimonious trees of 2185 steps, a
consistency index of 0.543 and a retention index of 0.876.
The best substitution models estimated by Kakusan3 were
J2+ gamma shape parameter (G) of 0.245, J2 + G
(a = 0.269) and HKY (Hasegawa et al. 1985) + G
(a = 0.498) for the 12S rRNA, 16S rRNA and tRNAval,
respectively. For the Bayesian analyses, GTR (Tavare
1986) + G (a = 0.273) was selected as the best substitution
model for the 12S rRNA, GTR + G (a = 0.288) for the
16S rRNA and K80 + G (a = 0.535) for the tRNAval. The
ML and BI analyses produced topologies with lnL
)13536.09 and )13706.18, respectively (nucleotide fre-
quencies: A = 0.33670, C = 0.22394, G = 0.19895 and
T = 0.24042).
Sequence statistics for the seven nuDNA gene fragments
and for the combined alignment are given in Table S4.
For 24 samples including out-group taxa, we obtained
4696 bp of nuDNA, of which 746 were variable and 305
were parsimony informative. The best substitution models
ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters
selected for the Bayesian analyses by Kakusan3 were as
follows: K80 + G (a = 0.289), GTR and JC69 for
BDNF_p1, BDNF_p2 and BDNF_p3, respectively;
HKY85 + G (a = 1.296), HKY85 + G (a = 0.005) and
F81 + G (a = 0.005) for NCX_p1, NCX_p2 and NCX_p3,
respectively; HKY85 + G (a = 1.079), GTR + G
(a = 0.005) and HKY85 for POMC_p1, POMC_p2 and
POMC_p3, respectively; F81 + G (a = 0.046), F81 + G
(a = 0.005) and HKY85 + G (a = 1.778) for Rag1_p1,
Rag1_p2 and Rag1_p3, respectively; HKY85 + G
(a = 0.190), SYM and F81 for Rhod_p1, Rhod_p2 and
Rhod_p3, respectively; HKY85 + G (a = 1.597), GTR + G
(a = 0.005) and HKY85 for SCF_p1, SCF_p2 and
SCF_p3, respectively; GTR + G (a = 0.953), HKY85 + G
(a = 0.005) and HKY85 + G (a = 0.290) for Tyr_p1,
Tyr_p2 and Tyr_p3, respectively. The BI analyses of con-
catenated genes produced topologies with lnL –13706.18
(nucleotide frequencies: A = 0.23580, C = 0.22523,
G = 0.27249 and T = 0.26647).
Phylogenetic relationships
All mtDNA gene analyses resulted in essentially the same
topologies and differed only in associations with poorly
supported nodes. The Bayesian tree (Fig. 2) infers the fol-
lowing sets of relationships. Monophyly of Polypedates with
respect to Rhacophorus, Buergeria and Rana was supported
nearly fully in all trees (MPBS = 99%, MLBS = 100% and
BPP = 1.00). In the Polypedates clade, P. otilophus formed
the sister species to the clade of the remaining samples
(99%, 100%, 1.00). In the latter clade, P. colletti diverged
first, although the monophyly of the remaining samples
was not substantially supported by MLBS (71%, 55%,
0.96). Of the latter samples, P. maculatus diverged from
the clade of the P. leucomystax complex (84%, 83%, 1.00).
Within the P. leucomystax complex clade, 117 unique hapl-
otypes (Fig. 2) fell into three major clades with unresolved
relationships: P. macrotis, Polypedates sp. from the Malay
Peninsula (Malay Clade) and the remaining P. leucomystax
complex. The last major clade got substantial support for
monophyly only in MP (80%, 60%, 0.93) and split into three
clades: samples of the P. leucomystax complex from southern
China (Guangxi and Hainan) and Vietnam (Vinh Phu and
Kon Tum) (South China Clade: 100%, 100%, 1.00); samples
of the P. leucomystax complex from central, western and
southern China (Anhui, Zhejiang, Jiangxi, Sichuan, Guangxi
and Yunnan) and Taiwan (North China Clade: 100%,
100%, 1.00); and samples of the P. leucomystax complex
widely occurring from southern China (Hong Kong,
Guangxi and Hainan) to South-East Asia [Vietnam, Laos,
Myanmar, Thailand, the Malay Peninsula, Sumatra, Java,
Borneo, Sulawesi, the Philippines and Okinawa (intro-
duced)] (Indochina-Sunda-Laos Clade: 100%, 100%, 1.00).
5
Fig. 2 Bayesian tree using 2005 bp of the
mitochondrial 12S rRNA, tRNAval and
16S rRNA genes for the Polypedates
samples included in this study. The
sample numbers correspond to those
given in Fig. 1 and Table S1. The
numbers above the branches are the
bootstrap support for the MP and
maximum likelihood (ML) inferences and
the Bayesian posterior probability (MP-
BS ⁄ ML-BS ⁄ BPP). Asterisks indicate
nodes with MP-BS and ML-BS ‡ 70%
and BPP ‡ 0.95.
Phylogenetic study on the Polypedates leucomystax complex d N. Kuraishi et al.
Of these, the Indochina-Sunda-Laos Clade was split fur-
ther into a wide-ranging Indochina-Sunda Clade (99%,
100%, 1.00) and a Laos Clade (100%, 100%, 1.00). The
Laos Clade consisted solely of some samples from Laos,
while the Indochina-Sunda Clade consisted of Indochina
and Sunda Clades. The Indochina Clade (95%, 98%,
1.00) is found in southern China (Guangxi, Hong Kong
and Hainan), Vietnam (Vinh Phu, Thua Thien, Hue and
Kon Tun), Laos (Vientiane) and Thailand exclusive of
southern regions, while the Sunda Clade (73%, 96%,
1.00) occurs in Myanmar, southern Thailand, the Malay
Peninsula, Sundaland, Sulawesi, the Philippines and
Okinawa.
The sequence divergences (uncorrected p-distance in
16S rRNA) among these genetic groups are shown in
Table S5. The divergences between P. macrotis and the
four major clades of the P. leucomystax complex were large
6 ª 2
(8.2–10.8%), as were those between Polypedates sp. from
the Malay Peninsula (Malay Clade) and the remaining
three clades (7.8–10.2%). The divergences between the
South and North China Clades were moderate (4.3–
6.4%), but those between the Indochina-Sunda and Laos
Clades (3.3–5.1%), and between the Indochina and Sunda
Clades were lower (2.2–4.3%).
The Bayesian tree based on concatenated nuDNA genes
(Fig. 3) inferred relationships essentially similar to those
obtained from the mtDNA analyses, except for the order
of divergence in the South-East Asian P. leucomystax com-
plex. Unlike the relationships recovered with mtDNA, Po-
lypedates sp. from the Malay Peninsula (mtDNA Malay
Clade) diverged first, followed by P. macrotis. The mono-
phyly of all clades had significant support (BPP ‡ 0.96),
except for the sister group relationship of the mitochon-
drial Indochina and Sunda Clades (0.63).
012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters
Fig. 3 Bayesian trees using a 2005-bp
sequence of the mitochondrial 12S
rRNA, tRNAval and 16S rRNA genes
(left) and a 4696-bp sequence of the
nuclear brain-derived neurotrophic factor
(BDNF), sodium ⁄ calcium exchanger 1
(NCX), POMC, Rag1, Rhod, SCF and
Tyr genes (right) for selected samples of
Polypedates species. The sample numbers
correspond to those given in Fig. 1 and
Table S1. The numbers above the
branches are the bootstrap support for
the MP and maximum likelihood (ML)
inferences and the Bayesian posterior
probability (MP-BS ⁄ ML-BS ⁄ BPP) in the
mtDNA tree (left) and the BPP in the
nuDNA tree (right).
N. Kuraishi et al. d Phylogenetic study on the Polypedates leucomystax complex
We detected six variable sites from 508-bp fragments of
BDNF and 19 sites from 333 bp of Rag1, in five selected
samples from the Indochina Clade and six from the Sunda
Clade. From these fragments, we detected six haplotypes in
BDNF and nine in Rag1. The geographical distributions of
the haplotypes are shown in Table S6 and Fig. 4. The
mitochondrial Indochina and Sunda Clades did not have
identical haplotype compositions, although one individual
from the Indochina Clade from the base of the Malay Pen-
insula, southern Thailand (Sample 42), possessed a haplo-
type predominant in the Sunda Clade (BDNF-4).
Moreover, this individual shared a unique haplotype (Rag1-
5) with an individual from the Indochina Clade from near
the Isthmus of Kra, southern Thailand (Sample 48).
Divergence time
In all data sets, the divergence time estimations revealed
large degrees of overlap in the confidence intervals sur-
rounding the estimated values between many lineages, and
this precludes a confident conclusion regarding the timing
of colonization of the present areas of distribution. The
estimated divergence times of the two mtDNA data sets
(#1 and #2) were close to each other, while those of the
nuDNA (data set #3) were generally much younger.
ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters
Our data suggest that Polypedates diverged from Rhacopho-
rus between 42.5 (CI 28.6–56.4; data set #2) and 26.6 (CI
14.0–40.4; data set #3) MYBP. From the analysis of data set
#2, the first divergence within Polypedates was estimated to
have occurred 32.4 (CI 19.9–44.5) MYBP, when two major
clades split: the primarily South Asian Clade leading to the
group of Sri Lankan P. fastigo and P. eques, and the predomi-
nantly South-East Asian Clade, consisting of all of the other
lineages including the secondarily South Asian Clades. The
most recent common ancestor (MRCA) of the primarily
South Asian Clade is estimated at 6.1 (CI 1.3–12.2) MYBP,
while the secondarily South Asian Clade, P. cruciger and
P. maculatus, split earlier at 12.2 (CI 4.2–20.5) MYBP.
The divergence of the predominantly South-East Asian
Clade began with the separation of P. otilophus from the
remaining clades, including the South-East Asian and sec-
ondarily South Asian Clades, between 26.2 (CI 15.7–37.1;
data set #2) and 18.1 (CI 9.0–28.1; data set #3) MYBP. Sub-
sequent divergence occurred when P. colletti split from the
remaining clades between 18.4 (CI 10.6–26.6; data set #2)
and 11.4 (CI 5.4–17.8; data set #3) MYBP, and the second-
arily South Asian Clade (P. maculatus) split from the
remaining South-East Asian Clades between 18.3 (CI 10.6–
22.6; data set #2) and 8.4 (CI 3.9–13.0; data set #3) MYBP.
7
Fig. 4 Distributions of brain-derived neurotrophic factor (BDNF)
and Rag1 haplotypes for the mitochondrial groups of the
Indochina (P. megacephalus) and Sunda (Polypedates leucomystax)
Clades. Sample numbers correspond to those given in Fig. 1 and
Table S1. The white arrow indicates the Isthmus of Kra.
Phylogenetic study on the Polypedates leucomystax complex d N. Kuraishi et al.
In the remaining South-East Asian Clades, divergence
of P. macrotis from the others began between 14.1 (CI 7.4–
20.9; data set #2) and 4.8 (CI 2.4–7.6; data set #3) MYBP,
that of the Malay Clade from the others began between
12.8 (CI 6.9–19.2; data set #2) and 6.5 (CI 3.1–10.2; data
set #3) MYBP, and the divergence of the Indochina and
Sunda Clades began between 3.7 (CI 1.8–5.6; data set #1)
and 2.6 (CI 1.4–3.9; data set #3) MYBP, following the
split of their common ancestor from the Laos Clade
between 4.7 (CI 2.4–7.3; data set #1) and 2.8 (CI 1.5–4.4;
data set #3) MYBP.
DiscussionPhylogenetic relationships within the genus Polypedates
The genus Polypedates occurs in both South-East Asia and
South Asia (Inger 1999). However, the only the South
Asian member we could study was P. maculatus. To clarify
the patterns of differentiation across the extent of its geo-
8 ª 2
graphical range fully, we constructed a phylogenetic tree
using about 800 bp of 12S and 16S rRNA from 36
sequences representing each genetic group recognized in
this study and six sequences of South Asian taxa from
GenBank, in addition to the three sequences used for esti-
mating divergence time (P. maculatus: AF215184 and
AF215358, AY880607 and AY880518; P. cruciger:
AY141799 and AY141845; P. eques: AY141801 and
AY141847, AY880469 and AY920531; P. fastigo:
AY880604 and AY880518). As in several other studies (e.g.
Delorme et al. 2005; Frost et al. 2006; Li et al. 2008; Yu
et al. 2009), we recovered a monophyletic Polypedates. The
genus Polypedates was once treated as a part of Rhacophorus
(see Matsui & Wu 1994; Matsui & Panha 2006), but its
distinct generic status is now beyond doubt.
Our analysis suggests a primary division between a clade
including Sri Lankan P. eques and P. fastigo, and another
clade encompassing the remaining taxa from South-East
and South Asia. Within the latter clade, the phylogenetic
relationships among taxa did not differ from those
obtained in the analyses of a much larger mitochondrial
data set, except that Sri Lankan P. cruciger formed a group
with P. maculatus. From mtDNA and morphological evi-
dence, Delorme et al. (2005) recognized the P. eques group
(P. eques and P. fastigo) and the P. leucomystax group (all
other species) within Polypedates. Indeed, Meegaskumbura
et al. (2010) recently established a new genus, Taruga, to
accommodate the P. eques group of Delorme et al. (2005),
and our results support this division. These patterns sug-
gest that the common ancestor of the genus Taruga
diverged from the common ancestor of the remaining lin-
eages of Polypedates, probably in southernmost South Asia,
and that subsequent differentiation proceeded in more
northern latitudes.
In the course of this latter differentiation, the diver-
gence of South and South-East Asian lineages does not
seem to have been the first event. Rather, the divergence
of South-East Asian P. otilophus and P. colletti preceded the
divergence of South Asian P. maculatus and P. cruciger
from the mainly South-East Asian P. leucomystax complex.
Consequently, the group of P. maculatus and P. cruciger
likely invaded South Asia secondarily. As other species
exist that are apparently related to the P. leucomystax com-
plex in South Asia (e.g. P. zed and P. teraiensis; Dubois
1987), evolution of the genus in this region can be prop-
erly discussed only after sufficiently studying such taxa.
Differentiation of the genus Polypedates within South-East
Asia
Among South-East Asian Polypedates, P. otilophus differs
greatly in external morphology from the others (Inger
1966) and has never been confused taxonomically with
012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters
N. Kuraishi et al. d Phylogenetic study on the Polypedates leucomystax complex
them. In this study, that species proved to be markedly
different genetically from the other species and showed
basal divergence in both the mtDNA and nuDNA trees.
Conversely, P. colletti was once confused with P. leucomystax
(Wolf 1936), but then proved to be clearly differentiated
from P. leucomystax by external morphology (Inger 1966)
and overlaps the latter species in distribution (Inger &
Stuebing 1997). In both the mtDNA and nuDNA trees,
P. colletti showed basal divergence from the P. leucomystax
complex, indicating their remote genetic relationships.
Although P. macrotis has previously been treated as a
subspecies of P. leucomystax (Wolf 1936; Inger 1954), sub-
sequent studies demonstrated that it was distinct morpho-
logically (Inger 1966) and distributed sympatrically with
that taxon (Inger 1966; Inger & Stuebing 1997). The
genetic divergences between P. macrotis and the other six
clades of the P. leucomystax complex were as high as 8.7–
11.6%, and the species is also regarded as a valid species
genetically. Our mtDNA analyses failed to resolve the
phylogenetic relationships between P. macrotis and various
genetic groups within the P. leucomystax complex. Previ-
ously, Brown et al. (2010) demonstrated a close relation-
ship between P. macrotis and the P. leucomystax complex. In
our analyses, when the Polypedates sp. from the Malay Pen-
insula (Malay Clade, which was not included in Brown
et al. 2010) is removed, our Bayesian tree analyses suggest
the basal divergence of P. macrotis from the remaining spe-
cies. In addition, in our Bayesian analysis of nuDNA
sequences, P. macrotis was inferred to be sister to the P. leu-
comystax complex.
Phylogenetic relationships within the P. leucomystax
complex and the taxonomic assignment of each genetic
group
When the phylogenetic trees from 800 bp of mitochon-
drial 16S rRNA are compared between Brown et al. (2010)
and our study, Clades 1, 2, 3 plus 4, and 5, and the clade
of the remaining haplotypes in Brown et al. (2010) obvi-
ously corresponded to our South China Clade, Laos
Clade, Indochina Clade, North China Clade and Sunda
Clade, respectively, and their relationships were essentially
identical. The phylogenetic relationships estimated using
mtDNA and nuDNA, genetic divergences in 16S rRNA
and distribution pattern strongly supported the sympatric
occurrence of more than one taxon of the P. leucomystax
complex, and the complex is thought to encompass at least
the following four distinct taxa, in addition to P. macrotis:
South China Clade. This clade occurs in southern China
(Guangxi and Hainan) and Vietnam and is supposedly not
conspecific with the North China (P. braueri) or Indo-
china-Sunda-Laos Clades because they are sympatric. We
ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters
also consider the South China Clade to be heterospecific
with the allopatric Malay Clade because of the large
genetic divergence between these putative species. Unfor-
tunately, we could not examine male specimens of the
South China Clade. However, based on its distribution,
we consider it likely to be one of the two types of P. leuco-
mystax reported from Vietnam (Inger 1999; Ziegler 2002)
that are differentiated by the presence or absence of male
vocal sac openings.
Unfortunately, we could not examine the sequences cor-
responding to the South China Clade samples used for the
DNA analyses. However, the males of the Indochina
Clade possess vocal sac openings and are thought to be
the non-striped type of P. leucomystax of Inger et al. (1999)
or P. leucomystax of Ziegler (2002). Therefore, the South
China Clade seems to be another striped type of P. leuco-
mystax of Inger et al. (1999) or Polypedates sp. of Ziegler
(2002). Indeed, comparisons of our data with the GenBank
data on Ziegler’s Polypedates sp. (AF285221–4) revealed
that they are nearly identical. Similarly, the sequence of
P. leucomystax from Ha Tinh, Vietnam (DQ283048; Frost
et al. 2006), was identical to our South China Clade, indi-
cating an invalid original identification.
Ziegler (2002) did not refer to the presence or absence of
vocal openings in his Polypedates sp., but he later identified it
as P. mutus (Hendrix et al. 2008), suggesting that he inferred
the absence of vocal sac openings in this population. This
estimation has been confirmed by an examination of the
specimens used (T. Nguyen and T. Ziegler, personal com-
munication on 18 October 2010). Inger et al. (1999) also
noted that males of their ‘striped type’ lack vocal openings,
suggesting the absence of these structures in the South
China Clade. In the genus Polypedates, a lack of vocal open-
ings is known only in P. mutus from Myanmar (Smith
1940), but this name is not applied directly to the South
China Clade because the Laos Clade also lacks openings.
We believe that the South China and Indochina-Sunda-
Laos Clades are not conspecific, but must await application
of the name until P. mutus from the type locality can be
studied. Our sample from Hainan Island differed slightly
from samples from Guangxi and Vietnam. The morphology
of this population must also be studied in the future.
North China Clade. This clade occurs in Taiwan and
widely in continental China (Anhui, Zhejiang, Jiangxi,
Sichuan, Guangxi and Yunnan), but is absent from Hong
Kong. The Taiwan population was once assigned to
P. megacephalus, which was originally described from Hong
Kong (e.g. Matsui et al. 1986; Zhao & Adler 1993). How-
ever, the populations from Taiwan and Hong Kong clearly
differ in their morphometric and mating call characteris-
tics and are highly divergent genetically, as shown here,
9
Phylogenetic study on the Polypedates leucomystax complex d N. Kuraishi et al.
with high levels of divergence between the North China
and Indochina-Sunda-Laos Clades (the latter contains
samples from Hong Kong). Therefore, the Taiwan popu-
lation has been classified as a distinct species: P. braueri
(Kuraishi et al. 2011). Our results indicate that the North
China and Indochina-Sunda-Laos Clades likely overlap in
distribution within Guangxi, supporting their heterospeci-
fic relationships.
Pope (1931) considered the population of the P. leucomys-
tax complex from Fukien (=Fujian) to be P. leucomystax
megacephalus. Unfortunately, we could not examine sam-
ples from Fujian, but based on distribution, we consider it
likely that this lineage be assigned to P. braueri. Further-
more, Pope noted that skin in the Fujian specimens was
free from the skull, a trait found in the Taiwan P. braueri
samples, but not in the Hong Kong P. megacephalus sam-
ples (Kuraishi et al. 2011).
However, the calls noted by Pope (1931) seem to differ
from those of topotypic P. braueri from Taiwan (Kuramo-
to 1986; Matsui et al. 1986). Frogs of the P. leucomystax
complex within this wide range exhibit variation in their
acoustic and karyological characteristics (see Matsui et al.
1986), and taxonomic identification of the mainland Chi-
nese populations still requires caution. Detailed future
studies on adult and larval morphology and bioacoustic
characteristics might reveal the presence of cryptic taxa
within the populations presently assigned to P. braueri.
Yang & Rao (2008) described P. impresus and P. spinus
from Yunnan, southern China. Of these, P. spinus should
be placed in Rhacophorus because of its green body (see
Matsui & Panha 2006), while P. impresus surely represents
a member of the P. leucomystax complex. However, as
P. impresus is known only from the type locality and no
genetic information is available, we cannot discuss the
exact status of this species. The species was described
without comparison with other Polypedates species, render-
ing its taxonomic identification difficult. In our results, a
single sample of the North China Clade from Yunnan dif-
fered from the other samples with relatively high diver-
gences (2.0–2.7%). These values overlap those observed
between the heterospecific Indochina and Sunda Clades
(2.2–4.3%, see below). Unfortunately, our specimen is a
juvenile and lacks taxonomically important features,
including the distinct postero-cranial depression and white
lip that are said to be unique to P. impresus (Yang & Rao
2008). Clarifying the relationships of our Yunnan sample
and P. impresus would be highly desirable.
Indochina-Sunda-Laos Clade. The Indochina-Sunda-Laos
Clade contained samples that occurred widely in South-
East Asia from the Sunda region in the south to southern
China (Hong Kong, Guangxi and Hainan) in the north
10 ª 2
and Myanmar in the west. The clade was genetically dis-
tinct from the others and contained independent Indo-
china-Sunda and Laos Clades. The Laos Clade, whose
males lack vocal sac openings, is obviously not conspecific
with the Indochina-Sunda Clade, in which males possess
the openings. The Indochina-Sunda Clade is further split
into allopatric Indochina and Sunda Clades, occurring
north and south of the Isthmus of Kra in southern Thai-
land, respectively. Exceptionally, the sample from Myan-
mar was included in the Sunda Clade, despite its
geographical proximity to the Indochina Clade. This one
aberrant sample may indicate the presence of gene flow
along the west coast of the Malay Peninsula.
The Indochina Clade included samples from Hong
Kong, which is the type locality of P. megacephalus, while
the Sunda Clade contained samples from Java, the type
locality of P. leucomystax. Polypedates leucomystax and
P. megacephalus have long been distinguished chiefly on
morphological grounds (Pope 1931; Inger 1966; Matsui
et al. 1986), but these previous reports should be reas-
sessed carefully because specific identification of the popu-
lations, without consideration of P. braueri, is not always
correct. Both of them are variable morphologically and are
actually very similar to each other. Furthermore, exact
comparisons using topotypic specimens have rarely been
made (Kuraishi et al. 2011). However, they certainly differ
in their acoustic characteristics. Calls reported for males
assigned to the Indochina Clade are variable (Heyer 1971;
Trepanier et al. 1999; Ziegler 2002; Sheridan et al. 2010),
but never identical to that of the Sunda Clade. Although
the call of the Sunda Clade also shows slight variation, it
is essentially similar in different populations (e.g. Matsui
1982; Brzoska et al. 1986; Narins et al. 1998; Sheridan
et al. 2010).
Taylor (1962) recognized Rhacophorus (=Polypedates) l. leu-
comystax throughout Thailand and Rh. l. sexvirgatus from
the southern part of the country. Simultaneously, he sug-
gested that these subspecies differ only in colouration and
that Rh. l. sexvirgatus may be a colour variety of Rh. l. leuco-
mystax. According to our results, Taylor’s (1962) Rh. l. leu-
comystax and Rh. l. sexvirgatus should be treated as
P. megacephalus (Indochina Clade) and P. leucomystax (Sun-
da Clade), respectively. The Hong Kong samples of
P. megacephalus formed two mitochondrial haplotype
groups. Furthermore, as noted above, variation appears to
exist in the acoustic characteristics among populations of
the Indochina Clade. At present, however, collectively call-
ing this group P. megacephalus would be best.
Like P. megacephalus from Hong Kong, the P. leucomystax
samples from Java in the Sunda Clade were split into three
geographical mitochondrial phylogroups (western, central
and central ⁄ eastern groups), but these had low genetic
012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters
N. Kuraishi et al. d Phylogenetic study on the Polypedates leucomystax complex
diversities (0.0–2.1%). Moreover, because details of the
type locality within Java are difficult to trace and no infor-
mation on their variation other than genetic features
exists, the Sunda Clade is best called P. leucomystax. Brown
et al. (2010) briefly reviewed old names that possibly
applied to their clades, which correspond to our Sunda
Clade, and rejected splitting P. leucomystax taxonomically.
As they pointed out, no basis exists for splitting the species
at present, and further studies based on characters other
than genetic data will be necessary to arrive at a possible
future taxonomic revision.
One sample from the Indochina Clade (Sample 42)
shared nuclear BDNF and Rag1 haplotypes with one sam-
ple from the Sunda Clade (Sample 48; Table S6). This
suggests incomplete reproductive isolation of these two
clades at the boundary of their distributions and may be
interpreted in three possible ways: (i) introgression of
mtDNA genes from the Indochina Clade to the Sunda
Clade, (ii) introgression of nuDNA genes from the Sunda
Clade to the Indochina Clade and (iii) deep coalescence or
incomplete lineage sorting (Maddison 1997). Of these
three, the first and second hypotheses do not seem to be
supported by the distributions of the BDNF and Rag1
haplotypes. The BDNF-4 haplotype occurs widely in the
range of the Sunda Clade, and the first hypothesis requires
the occurrence of the Sunda Clade at the base of the
Malay Peninsula (near the locality of Sample 42), where
introgression of mtDNA genes from the Indochina Clade
occurred. However, despite intensive sampling efforts, we
failed to collect samples of the P. leucomystax complex in
the area between there and south of the Isthmus of Kra
(near the locality of Sample 48), and this hypothesis seems
implausible. From the nature of the transmission of nuD-
NA genes (e.g. Ballard & Whitlock 2004), the second
hypothesis also seems implausible. Therefore, we prefer
the deep coalescence hypothesis. Presumably, the Indo-
china and Sunda Clades split relatively recently and have
maintained ancient polymorphisms. From these lines of
evidence, the Indochina (P. megacephalus) and Sunda (P. leu-
comystax) Clades are again judged to represent different
species, although the degree of genetic divergence between
them (3.0%) is at the border of the proposed intraspecific
threshold for frogs (around 3% in 16S rRNA; Fouquet
et al. 2007).
The Laos Clade occurs in Laos, overlapping part of the
range of P. megacephalus (Indochina Clade). Our Laos
Clade corresponds to Clade 2 of Brown et al. (2010),
which contains samples from Guangxi in southern China.
Therefore, the sympatry of the Laos Clade and P. mega-
cephalus (Indochina Clade) does not seem to be limited to
Laos. Males of the Laos Clade match P. mutus in their
lack of vocal openings. Although the South China Clade
ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters
also lacks openings, as stated above, the Laos and South
China Clades are clearly different phylogenetically and are
undoubtedly heterospecific. As the sampling sites for the
Laos Clade are much nearer to Myanmar, where P. mutus
was described (Smith 1940), it seems more probable that
the Laos Clade represents P. mutus, although it is also
probable that neither the Laos Clade nor the South China
Clade actually corresponds to the true P. mutus. We tenta-
tively call the South China Clade Polypedates cf. mutus 1
and the Laos Clade Polypedates cf. mutus 2 to allude to
their lack of vocal openings in males.
Malay Clade. The Malay Clade (Polypedates sp.), which
occurs in the Malay Peninsula, first diverged from the
other genetic groups of the P. leucomystax complex in the
mtDNA tree and even faster than P. macrotis in the nuD-
NA tree. It is not conspecific with the South or North
China Clades because of the large genetic divergence, or
with the Sunda Clade because of the sympatric ⁄ syntopic
distribution. Narins et al. (1998) reported the syntopic
occurrence of two morphs (morphs A and B) of the P. leuco-
mystax complex in the Malay Peninsula that differ in mor-
phology, calls and allozyme characteristics. Based on
morphological and acoustic characteristics (M. Matsui, N.
Kuraishi & H. S. Yong, unpublished data), the Malay
Clade is thought to correspond to morph B of Narins et al.
(1998). Those authors found morph A in the range of our
Sunda Clade and reported that calls of morph A were simi-
lar to those of Philippine populations of P. leucomystax. As
our Sunda Clade occurs widely from the Malay Peninsula
to the Philippines, morph A of Narins et al. (1998) is most
probably identical to our Sunda Clade (P. leucomystax).
Evolutionary history
The evolutionary histories of various anuran lineages have
been studied in South-East Asia, including those of Eu-
phlyctis and Hoplobatrachus (Alam et al. 2008), Ansonia (Mat-
sui et al. 2010a), Leptobrachium (Matsui et al. 2010b) and
Paa (Che et al. 2010). However, only a few Polypedates have
been included as a part of large-scale divergence date esti-
mation of anuran lineages (e.g. Roelants et al. 2007; Wiens
et al. 2009), and few studies specifically estimate the age of
the genus (Brown et al. 2010).
Few stratigraphic events can be associated with the
divergence of the genetic groups recognized in Polypedates.
Therefore, to estimate divergence dates, we used the
divergence between Mantellidae and Rhacophoridae 73.1
MYBP (Bossuyt & Milinkovitch 2000) and between Buer-
geria and other Rhacophoridae 49.7 MYBP (Roelants et al.
2007) as external calibration points, and the divergence
between the Java and Sumatra populations of P. leucomystax
2.8 MYBP as an internal calibration point. This last
11
Phylogenetic study on the Polypedates leucomystax complex d N. Kuraishi et al.
calibration point was set following the divergence time
between the Java and Sumatra populations of L. hasseltii
(Matsui et al. 2010b). As Leptobrachium is a lotic breeder
and has an evolutionary history much different from that
of Polypedates, this calibration may be inappropriate and
should be treated with great caution, but no internal cali-
bration point is presently available within Polypedates.
Using mtDNA, the divergence between Polypedates and
Rhacophorus was estimated to have occurred in the late
Eocene (42.5 MYBP; data set #2 in Table S7), which is
similar to the date in Wiens et al. (2009), while that esti-
mated using nuDNA was much later in the late Oligocene
(26.6 MYBP; data set #3). All members of Polypedates
whose breeding habits are known invariably lay a foamy
egg nest on or near still water (Taylor 1962; Inger 1966;
Alcala 1986; Dutta 1997; Iskandar 1998; Maeda & Matsui
1999), a reproductive trait shared by many species of Rhac-
ophorus and Chiromantis. Therefore, formation of a foam
nest was likely acquired before the separation of Polypedates
and Rhacophorus. Some Rhacophorus species (e.g. Rh. anguli-
rostris, Rh. cyanopunctatus and Rh. gauni; Malkmus et al.
2002) have lotic breeding habits, while Polypedates lacks
this and is thought to be more conservative than Rhacopho-
rus with regard to breeding.
Our data suggest at least two dispersal events within
the South Asian Polypedates. Separation of the primarily
South Asian Clade and predominantly South-East Asian
Clade, including the secondarily South Asian Clade, was
Fig. 5 Estimated times million years before present (MYBP) of the m
those shown in Table S7. Dotted lines indicate divergences estimated o
12 ª 2
the first event in Polypedates evolution. Our estimated
mean date for this divergence was the early Oligocene
[32.4 (19.9–44.5) MYBP; data set #2; mtDNA]. Bocxlaer
et al. (2009) reported that some bufonids endemic to
Western Ghats in India and Sri Lanka arose as a result of
the ‘into India’ movement of Laurasian species after the
Indo-Asia collision. This also seems to be the case in our
primarily South Asian Clade of Polypedates, which also
occurs from Western Ghats to Sri Lanka. However,
Bocxlaer et al. (2009) estimated that specialized endemic
bufonids such as Ghatophryne (formerly part of Ansonia)
and Pedostibes on the Indian subcontinent arose
22.0–22.4 MYBP, after separation from South-East Asian
lineages 23.5–24.2 MYBP. These estimates are much
younger than our estimate from short mtDNA sequences
(data set #2). Unfortunately, we have no nuDNA data,
but extrapolation of the estimated dates in data set #3 for
events that occurred before and after this event (separa-
tion of Rhacophorus and Polypedates 26.6 MYBP and the
divergence of P. otilophus from the remaining clades 18.1
MYBP) yielded 21.3 MYBP for the divergence of the
primarily South Asian Clade of Polypedates (Fig. 5), which
agrees well with estimates for bufonids by Bocxlaer et al.
(2009). These events seem to correspond to putative geo-
logical movement in southern Tibet and the Himalayan
region (40–19 MYBP; Che et al. 2010; Matsui et al.
2010b), which also induced drastic climate change in the
region (An et al. 2001; Harris 2006).
ain divergences of Polypedates. The node numbers correspond to
nly from mtDNA and not from nuDNA data sets.
012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters
N. Kuraishi et al. d Phylogenetic study on the Polypedates leucomystax complex
The secondarily South Asian Clade should have sepa-
rated from the South-East Asian Clade much later, and
this was estimated to have occurred in the early Miocene
(18.3 MYBP) using data set #2 or the late Miocene (8.4
MYBP) using data set #3 (Fig. 5). As shown above, the
estimates from nuDNA (data set #3) seem to be more rea-
sonable than those from mtDNA (data sets #1 and #2) and
are consistent with the following scenarios. Even so, note
that the MRCA estimated from data set #2 was much
younger in the primary Clade (6.1 MYBP) than in the sec-
ondary Clade (12.2 MYBP), although the lack of other
South Asian samples prohibits further discussion.
Within the predominantly South-East Asian Clade,
P. otilophus first diverged in the early Miocene (18.1
MYBP; data set #3). This species occurs on Sumatra, Bor-
neo (Inger 1966) and Java (Riyanto et al. 2009), but is not
recorded from Peninsular Malaysia (Berry 1975). This sug-
gests the rapid dispersal of ancestral lineages and a present
relict distribution of P. otilophus. Later in the late Miocene,
P. colletti diverged from the remaining predominantly
South-East Asian Clade (5.4–17.8 MYBP), followed by the
divergence of the secondarily South Asian Clade (3.9–
13.0 MYBP, see above). Unlike P. otilophus, P. colletti
occurs in Borneo, Sumatra, Peninsular Malaysia and Thai-
land (Taylor 1962; Inger 1966; Berry 1975), but not in
Java (Iskandar 1998). Also, the species is also reported
from Vietnam (Orlov et al. 2002). This wide distribution
contrasts that of P. otilophus, but the known continental
localities are fragmented, suggesting a relict distribution
like P. otilophus. This wide distribution also suggests the
presence of intraspecific genetic divergence in P. colletti,
which is currently under study.
Although the order of divergence is ambiguous, Polype-
dates sp. from the Malay Peninsula (Malay Clade) and
P. macrotis diverged from the late Miocene to the early
Pliocene, earlier than the remaining members of the
P. leucomystax complex, which diverged nearly simulta-
neously in the Pliocene in wider regions of East and
South-East Asia. Polypedates macrotis occurs more widely
than P. colletti, being recorded from the Philippines, Bor-
neo, Sumatra, Peninsular Malaysia and Thailand, but not
from Java (Iskandar 1998). Although the species habitat
tends to be less widely disturbed than that of P. leucomystax
(see below), its occurrence in the Philippines may be
related to recent human activities. Future dense sampling
is required to test this hypothesis. Polypedates sp. from the
Malay Peninsula seems to have had a long history, at least
since the late Miocene, independent from P. leucomystax
with which it has long been confused (Narins et al. 1998).
This divergence is not very deep compared with the other
frog groups endemic to the Peninsula (e.g. Ansonia spp.;
Matsui et al. 2010a), but is regarded as sufficiently long
ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters
among members of the P. leucomystax complex. The spe-
cies is currently known only from central to southern Pen-
insular Malaysia and is less common than P. leucomystax
where they occur together. This restricted distribution and
the reduced intraspecific genetic divergence in this popula-
tion suggest that the species experienced past demographi-
cal or population reduction (e.g. Matsui et al. 2008).
From the current distribution, the ancestral lineage of
the remaining P. leucomystax complex seems to have occu-
pied much wider ranges in South-East Asia than Polype-
dates sp. from the Malay Peninsula or P. macrotis. This
ancestral lineage began to diverge in the northern regions
around 3.9 MYBP, which led to Polypedates cf. mutus 1.
Then, the ancestral lineage giving rise to P. braueri from
the northern regions split around 3.8 MYBP from the
common ancestor leading to Polypedates cf. mutus 2, P. leu-
comystax and P. megacephalus, which occupied more south-
ern regions. The absence of Polypedates cf. mutus 1 and
P. braueri in the southern Sundaland, which is thought to
have long been connected with the region including Indo-
china, suggests past ecological competition between these
clades and the clade ancestral to the remaining P. leucomys-
tax complex.
The divergence of Polypedates cf. mutus 2, which lacks
vocal sac openings, and the common ancestor of P. leuco-
mystax and P. megacephalus, both with vocal openings,
occurred in the southern region in the late Pliocene (1.5–
4.4 MYBP). The common ancestry of groups with and
without vocal openings indicates the importance of acous-
tic differentiation in speciation. Differentiation in vocaliza-
tion and acoustic characters may have facilitated rapid
diversification in the P. leucomystax complex.
The most recent divergence in the southern group
(P. leucomystax and P. megacephalus) occurred slightly later
in the late Pliocene or early Pleistocene (1.4–4.0 MYBP).
Their current allopatric distribution on both sides of the
Isthmus of Kra suggests that speciation was affected by a
short disjunction of the Malay Peninsula and Sunda
Islands by the South China Sea in the early Pliocene
(about 5 MYBP; Inger & Voris 2001). Although the evolu-
tionary history of P. megacephalus is short, the species
already had a wide range between the ranges of the more
northern P. braueri and more southern P. leucomystax.
Most of the intraspecific divergence in P. leucomystax
occurred very late, but divergence between Myanmar and
other samples seems to have occurred much earlier in the
evolution of this species, suggesting a relict status of the
Myanmar population. Furthermore, at least two groups
were recognized in the samples from Java, although the
degree of divergence is low. Notwithstanding such varia-
tion, a wide range of distribution, but low genetic diver-
gence, characterizes P. leucomystax. These traits seem to be
13
Phylogenetic study on the Polypedates leucomystax complex d N. Kuraishi et al.
related not only to very recent dispersal via land connec-
tions, such as is known to have occurred during the Last
Glacial Maximum (0.021 MYBP; Sathiamurthy & Voris
2006), but also to human activities.
For example, some samples from Sabah and Sarawak,
Borneo, had unique haplotypes, while others shared identi-
cal haplotypes with samples from the Malay Peninsula. In
contrast, the samples from Kalimantan, Borneo, had hapl-
otypes common to samples from central and southern
Sumatra. Inger & Voris (2001) suggested that P. leucomystax
now found in Borneo was introduced through human
activity in the last few thousand years. Frogs of the P. leuco-
mystax complex are notorious for being frequently trans-
ported artificially (e.g. Brown & Alcala 1970; Wiles 2000;
Kuraishi et al. 2009; Brown et al. 2010). Most probably,
P. leucomystax now found in Sabah and Sarawak is a mix-
ture of populations with two different origins. One has
origins at an older time, probably in the Pleistocene, and
has diversified within the island. The other has younger
origins and was transported from the Malay Peninsula via
human-mediated activity. In contrast, frogs found in Kali-
mantan seem to include descendents originally introduced
artificially from Sumatra (and vice versa).
The population genetic structure in commensal frogs
like P. leucomystax, P. megacephalus, Hylarana erythraea and
H. nicobariensis (Inger 1966) should be studied carefully; in
particular, frogs of the P. leucomystax complex require cau-
tion because these frogs, with strongly adhesive digital
disks and a high ability to resist desiccation, are more eas-
ily transported unintentionally than other anurans.
AcknowledgementsWe are grateful to the following for their encouragement,
permission to conduct research or companionship in the
field: L. Apin, K. Araya, Md. S. Azman, A. Bogadek, B.
Bounkhoun, S.-L. Chen, the late A.-A. Hamid, T. Hikida,
A. Iizuka, S. Iwanaga, T. Kusano, D. Labang, M.B.
Lakim, M.W. Lau, A.V. Le, J.-T. Lin, P. Lin, M. Maryati,
K. Mizuno, the late J. Nabitabhata, H. Nagaoka, Le.V.
Nguyen, Lu.V. Nguyen, T. Niizato, T. Shimada, T. Suga-
hara, T. Tachi, the late I. Takiguchi, U. Tanaka, M.
Toda, A. Tominaga, the late T. Utsunomiya, H. Wakaha-
ra, C.-H. Wang, G.-F. Wu and M. Yoshizumi. For tissue
samples, we are also indebted to M. Hori, M. Kato, N.
Orlov, T. Papenfuss (for the MVZ tissue collection) and
G.R. Zug. T. Nguyen and T. Ziegler kindly provided
information on Vietnamese specimens. The Economic-
Planning Unit (formerly the Socio-Economic Research
Unit) of Malaysia, the State Government of Sarawak,
Sabah Parks, The National Research Council of Thailand
and the Royal Forest Department of Thailand kindly
allowed MM to conduct the project, and the University
14 ª 2
Malaya, Universiti Malaysia Sabah, Universiti Kebangsaan
Malaysia (UKM), JICA, the Forest Department, Sarawak,
Center for Agriculture and Forestry Research and Devel-
opment of Hue University of Agriculture and Forestry,
and Chulalongkorn University kindly provided facilities
for conducting research. Field trips by MM were made
possible by grants from The Monbusho International Sci-
entific Research Program (Field Research, 01041051,
02041051, 04041068, 06041066, 08041144), The Mon-
bukagakusho through the Japanese Society for the Promo-
tion of Sciences (JSPS: Field Research, 10041166,
15370038, 20405013, 23405014), UKM (OUP-PLW-14-
59 ⁄ 2008) and TJTTP-OECF. AH thanks M.D. Kusrini, J.
McGuire and M.I. Setiadi for providing tissue samples and
the Monbukagakusho for scholarship funding. Research
export permits were obtained from the Forest Department,
Sarawak, and the Chinese Academy of Sciences.
ReferencesAlam, M. S., Igawa, T., Khan, M. R., Islam, M. M., Kuramoto,
M., Matsui, M., Kurabayashi, A. & Sumida, M. (2008). Genetic
divergence and evolutionary relationships in six species of
genera Hoplobatrachus and Euphlyctis (Amphibia: Anura) from
Bangladesh and other Asian countries revealed by
mitochondrial gene sequences. Molecular Phylogenetics and
Evolution, 48, 515–527.
Alcala, A. C. (1986). Guide to Philippine Flora and Fauna 10,
Amphibians and Reptiles. Quezon City: J. M. C. Press.
An, Z.-S., Kutzbach, J. E., Prell, W. L. & Porter, S. C. (2001).
Evolution of Asian monsoons and phased uplift of the
Himalaya-Tibet plateau since late Miocene times. Nature, 411,
62–66.
Ballard, J. W. O. & Whitlock, M. C. (2004). The incomplete
natural history of mitochondria. Molecular Ecology, 13, 729–744.
Berry, P. Y. (1975). The Amphibian Fauna of Peninsular Malaysia.
Kuala Lumpur: Tropical Press.
Bocxlaer, I. V., Biju, S. D., Loader, S. P. & Bossuyt, F. (2009).
Toad radiation reveals into-India dispersal as a source of
endemism in the Western Ghats-Sri Lanka biodiversity hotspot.
BMC Evolutionary Biology, 9, 131.
Bossuyt, F. & Milinkovitch, M. C. (2000). Convergent adaptive
radiations in Madagascan and Asian ranid frogs reveal
covariation between larval and adult traits. Proceedings of theNational Academy of Sciences of the United States of America, 97,
6585–6590.
Brown, W. C. & Alcala, A. C. (1970). The zoogeography of the
Philippine Islands, a fringing archipelago. Proceedings of theCalifornia Academy of Sciences, 38, 105–130.
Brown, R. M., Linkem, C. W., Siler, C. D., Sukumaran, J.,
Esselstyn, J. A., Diesmos, A. C., Iskandar, D. T., Bickford, D.,
Evans, B. J., McGuire, J. A., Grismer, L., Supriatna, J. &
Andayani, N. (2010). Phylogeography and historical
demography of Polypedates leucomystax in the islands of
Indonesia and the Philippines: evidence for recent human-
mediated range expansion? Molecular Phylogenetics and Evolution,
57, 598–619.
012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters
N. Kuraishi et al. d Phylogenetic study on the Polypedates leucomystax complex
Brzoska, J., Joermann, G. & Alcala, A. C. (1986). Structure and
variability of the calls of Polypedates leucomystax (Amphibia:
Rhacophoridae) from Negros, Philippines. Silliman Journal, 33,
87–103.
Castresana, J. (2000). Selection of conserved blocks form multiple
alignment for their use in phylogenetic analysis. Molecular
Biology and Evolution, 17, 540–552.
Che, J., Zhou, W.-W., Hu, J.-S., Yan, F., Papenfuss, T. J., Wake,
D. & Zhang, Y.-P. (2010). Spiny frogs (Paini) illuminate the
history of the Himalayan region and Southeast Asia. Proceedings
of the National Academy of Sciences of the United States of America,
107, 13765–13770.
Delorme, M. (2004). Phylogenie des Ranidae Rhacophorinae:confrontations des analyses moleculaires et morphologiques, et etude de
caracte‘res. Unpublished thesis, Museum National d’Histoire
Naturelle, Paris.
Delorme, M., Dubois, A., Grosjean, S. & Ohler, A. (2005). Une
nouvelle classification generique et subgenerique de la tribu des
Philautini (Amphibia, Anura, Ranidae, Rhacophorinae). BulletinMensuel de la Societe Linneenne de Lyon, 74, 165–171.
Drummond, A. J. & Rambaut, A. (2007). BEAST, BayesianEvolutionary Analysis Sampling Trees, Version 1.4.2. Available via
http://beast.bio.ed.ac.uk/.
Drummond, A. J., Ho, S. Y. W., Phillips, M. J. & Rambaut, A.
(2006). Relaxed phylogenetics and dating with confidence. PLoSBiology, 4, 699–710.
Dubois, A. (1987). Miscellanea taxinomica batrachologica (I).
Alytes, 5, 7–95.
Dutta, S. K. (1997). Amphibians of India and Sri Lanka (Checklist
and Bibliography). Bhubaneswar: Odyssey Publishing House.
Farris, J. S., Kallersjo, M., Kluge, A. G. & Bult, C. (1994).
Testing significance of incongruence. Cladistics, 10, 315–319.
Felsenstein, J. (1985). Confidence limits on phylogenies: an
approach using the bootstrap. Evolution, 39, 783–791.
Fouquet, A., Gilles, A., Vences, M., Marty, C., Blanc, M. &
Gemmell, N. J. (2007). Underestimation of species richness in
Neotropical frogs revealed by mtDNA analyses. PLoS ONE, 2,
e1109.
Frost, D. R., Grant, T., Faivovich, J. N., Bain, R. H., Haas, A.,
Haddad, C. F. B., De Sa, R. O., Channing, A., Wilkinson, M.,
Donnellan, S. C., Raxworthy, C. J., Campbell, J. A., Blotto, B. L.,
Moler, P., Drewes, R. C., Nussbaum, R. A., Lynch, J. D., Green,
D. M. & Wheeler, W. C. (2006). The amphibian tree of life.
Bulletin of the American Museum of Natural History, 297, 1–370.
Hall, T. A. (1999). BioEdit: a user-friendly biological sequence
alignment editor and analysis program for Windows
95 ⁄ 98 ⁄ NT. Nucleic Acids Symposium Series, 41, 95–98.
Harris, N. (2006). The elevation history of the Tibetan Plateau
and its implications for the Asian monsoon. Palaeogeography
Palaeoclimatology Palaeoecology, 241, 4–15.
Hasegawa, M., Kishino, H. & Yano, T. (1985). Dating of the
human-ape splitting by a molecular clock of mitochondrial
DNA. Journal of Molecular Evolution, 22, 160–174.
Hendrix, R., Nguyen, Q. T., Bohme, W. & Ziegler, T. (2008).
New anuran records from Phong Nha – Ke Bang National
Park, Truong Son, Central Vietnam. Herpetology Notes, 1, 23–
31.
Heyer, W. R. (1971). Mating calls of some frogs from Thailand.
Fieldiana: Zoology, 58, 61–82.
ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters
Hillis, D. M. & Bull, J. J. (1993). An empirical test of
bootstrapping as a method for assessing confidence in
phylogenetic analysis. Systematic Biology, 42, 182–192.
Hillis, D. M., Mable, B. K., Larson, A., Davis, S. K. & Zimmer,
E. A. (1996). Nucleic acids IV: sequencing and cloning. In D.
M. Hillis, C. Moritz & B. K. Mable (Eds) Molecular Systematics
(pp. 321–378). Sunderland: Sinauer.
Huelsenbeck, J. P. & Rannala, B. (2004). Frequentist properties
of Bayesian posterior probabilities of phylogenetic trees under
simple and complex substitution models. Systematic Biology, 53,
904–913.
Huelsenbeck, J. P. & Ronquist, F. (2001). MRBAYES: Bayesian
inference of phylogeny. Bioinformatics, 17, 754–755.
Huelsenbeck, J. P., Ronquist, F., Nielsen, R. & Bollback, J. P.
(2001). Bayesian inference of phylogeny and its impact on
evolutionary biology. Science, 294, 2310–2314.
Inger, R. F. (1954). Systematics and zoogeography of Philippine
Amphibia. Fieldiana: Zoology, 33, 183–531.
Inger, R. F. (1966). The systematics and zoogeography of the
Amphibia of Borneo. Fieldiana: Zoology, 52, 1–402.
Inger, R. F. (1999). Distribution of amphibians in southern Asia
and adjacent islands. In W. E. Duellman (Ed.) Patterns of
Distribution of Amphibians: A Global Perspective (pp. 445–482).
Baltimore: Johns Hopkins University Press.
Inger, R. F. & Stuebing, R. B. (1997). A Field Guide to the Frogs ofBorneo. Kota Kinabalu: Natural History Publications.
Inger, R. F. & Voris, H. K. (2001). The biogeographical relations
of the frogs and snakes of Sundaland. Journal of Biogeography,
28, 863–891.
Inger, R. F., Orlov, N. & Darevsky, I. (1999). Frogs of Vietnam: a
report on new collections. Fieldiana: Zoology, New Series, 92, 1–46.
Inger, R. F., Stuart, B. L. & Iskandar, D. T. (2009). Systematics
of a widespread Southeast Asian frog, Rana chalconota
(Amphibia: Anura: Ranidae). Zoological Journal of the LinneanSociety, 155, 123–147.
Iskandar, D. T. (1998). The Amphibians of Java and Bali. Bogor:
Puslitbang Biologi, LIPI.
Jobb, G. (2008). TREEFINDER Version of October 2008. Available
via http://www.treefinder.de.
Kuraishi, N., Matsui, M. & Ota, H. (2009). Estimation of the
origin of Polypedates leucomystax (Amphibia: Anura:
Rhacophoridae) introduced to the Ryukyu Archipelago, Japan.
Pacific Science, 63, 317–325.
Kuraishi, N., Matsui, M., Ota, H. & Chen, S.-L. (2011). Specific
separation of Polypedates braueri (Vogt, 1911) from
P. megacephalus (Hallowell, 1861) (Amphibia: Anura:
Rhacophoridae). Zootaxa, 2744, 53–61.
Kuramoto, M. (1986). Call structures of the rhacophorid frogs
from Taiwan. Scientific Report of the Laboratory for Amphibian
Biology, Hiroshima University, 8, 45–68.
Kurniati, H. (2011). Vocalization of Asian striped tree frogs,
Polypedates leucomystax (Grevenhorst, 1829) and P. iskandariRiyanto, Mumpuni & McGuire, 2011. Treubia, 38, 1–13.
Leache, A. D. & Reeder, T. W. (2002). Molecular systematics of
the eastern fence lizard (Sceloporus undulatus): a comparison of
parsimony, likelihood, and Bayesian approaches. SystematicBiology, 51, 44–68.
Li, J.-T., Che, J., Bain, R. H., Zhao, E.-M. & Zhang, Y.-P.
(2008). Molecular phylogeny of Rhacophoridae (Anura): a
15
Phylogenetic study on the Polypedates leucomystax complex d N. Kuraishi et al.
framework of taxonomic reassignment of species within the
genera Aquixalus, Chiromantis, Rhacophorus, and Philautus.
Molecular Phylogenetics and Evolution, 48, 302–312.
Liem, S. S. (1970). The morphology, systematics, and evolution
of the Old World treefrogs (Rhacophoridae and Hyperoliidae).
Fieldiana: Zoology, 57, 1–145.
Maddison, W. P. (1997). Gene trees in species trees. SystematicBiology, 46, 523–536.
Maeda, N. & Matsui, M. (1999). Frogs and Toads of Japan, Revised
Edition. Tokyo: Bun-ichi Sogo Shuppan.
Malkmus, R., Manthey, U., Vogel, G., Hoffman, P. & Kosuch, J.
(2002). Amphibians and Reptiles of Mount Kinabalu (North Borneo).
Koenigstein: A.R.G. Gantner K.G., Koeltz Scientific Books.
Marmayou, J., Dubois, A., Ohler, A., Pasquet, E. & Tillier, A.
(2000). Phylogenetic relationships in the Ranidae (Amphibia,
Anura). Independent origin of direct development in the genera
Philautus and Taylorana. Comptes Rendus de l’Academie desSciences. Serie III, Life Sciences, 323, 287–297.
Matsui, M. (1982). Amphibians from Sabah II. Acoustic
characteristics of three common anuran species. Contributions
from the Biological Laboratory, Kyoto University, 26, 123–129.
Matsui, M. & Panha, S. (2006). A new species of Rhacophorus from
eastern Thailand (Anura: Rhacophoridae). Zoological Science, 23,
477–481.
Matsui, M. & Wu, G.-F. (1994). Acoustic characteristics of
treefrogs from Sichuan, China, with comments on systematic
relationship of Polypedates and Rhacophorus (Anura,
Rhacophoridae). Zoological Science, 11, 485–490.
Matsui, M., Seto, T. & Utsunomiya, T. (1986). Acoustic and karyotypic
evidence for specific separation of Polypedates megacephalus from
P. leucomystax. Journal of Herpetology, 20, 483–489.
Matsui, M., Ito, H., Shimada, T., Ota, H., Saidapur, S. K.,
Khonsue, W., Tanaka-Ueno, T. & Wu, G.-F. (2005).
Taxonomic relationships within the Pan-Oriental narrow-mouth
toad Microhyla ornata as revealed by mtDNA analysis (Amphibia,
Anura, Microhylidae). Zoological Science, 22, 489–495.
Matsui, M., Maryati, M., Shimada, T. & Sudin, A. (2007).
Resurrection of Staurois parvus from S. tuberilinguis from
Borneo (Amphibia, Ranidae). Zoological Science, 24, 101–106.
Matsui, M., Tominaga, A., Liu, W.-Z. & Tanaka-Ueno, T.
(2008). Reduced genetic variation in the Japanese giant
salamander, Andrias japonicus (Amphibia: Caudata). MolecularPhylogenetics and Evolution, 49, 318–326.
Matsui, M., Tominaga, A., Liu, W.-Z., Khonsue, W., Grismer, L.
L., Diesmos, A. C., Das, I., Sudin, A., Yambun, P., Yong, H.-
S., Sukumaran, J. & Brown, R. M. (2010a). Phylogenetic
relationships of Ansonia from Southeast Asia inferred from
mitochondrial DNA sequences: systematic and biogeographic
implications (Anura: Bufonidae). Molecular Phylogenetics and
Evolution, 54, 561–570.
Matsui, M., Hamidy, A., Murphy, R. W., Khonsue, W., Yambun,
P., Shimada, T., Norhayatii, A., Daicus, B. M. & Jiang, J.-P.
(2010b). Phylogenetic relationships of megophryid frogs of the
genus Leptobrachium (Amphibia, Anura) as revealed by mtDNA
gene sequences. Molecular Phylogenetics and Evolution, 56, 259–
272.
Matsui, M., Panha, S., Khonsue, W. & Kuraishi, N. (2010c). Two
new species of the ‘‘kuhlii’’ complex of the genus Limnonectesfrom Thailand (Anura: Dicroglossidae). Zootaxa, 2615, 1–22.
16 ª 2
Meegaskumbura, M., Bossuyt, F., Pethiyagoda, R.,
Manamendra-Arachchi, K., Bahir, M., Milinkovitch, M. C. &
Schneider, C. J. (2002). Sri Lanka: an amphibian hotspot.
Science, 298, 379.
Meegaskumbura, M., Meegaskumbura, S., Bowatte, G.,
Manamendra-Arachchi, K., Pethiyagoda, R., Hanken, J. &
Schneider, C. J. (2010). Taruga (Anura: Rhacophoridae), a new
genus of foam-nesting tree frogs endemic to Sri Lanka. Ceylon
Journal of Science: Biological Science, 39, 75–94.
Narins, P. M., Feng, A. S., Yong, H. S. & Christensen-Dalsgard,
J. (1998). Morphological, behavioural and genetic divergence of
sympatric morphotypes of Polypedates leucomystax in Peninsular
Malaysia. Herpetologica, 54, 129–142.
Orlov, N. L., Lathrop, A., Murphy, R. W. & Ho, T. C. (2001).
Frogs of the family Rhacophoridae (Anura: Amphibia) in the
northern Hoang Lien Mountains (Mount Fan Si Pan, Sa Pa
District, Lao Cai Province), Vitenam. Russian Journal ofHerpetology, 8, 17–44.
Orlov, N. L., Murphy, R. W., Ananjeva, N. B., Ryabov, S. A. &
Ho, T. C. (2002). Herpetofauna of Vietnam. A checklist. Part
I. Amphibia. Russian Journal of Herpetology, 9, 81–104.
Pope, C. H. (1931). Notes on amphibians from Fukien, Hainan,
and other parts of China. Bulletin of the American Museum ofNatural History, 61, 397–611.
Rambaut, A. & Drummond, A. J. (2007). Tracer v1.4. Available
via http://beast.bio.ed.ac.uk/Tracer.
Rannala, B. & Yang, Z. (1996). Probability distribution of
molecular evolution trees: a new method of phylogenetic
inference. Journal of Molecular Evolution, 43, 304–311.
Richards, C. M. & Moore, W. S. (1998). A molecular
phylogenetic study of the Old World treefrog family
Rhacophoridae. The Herpetological Journal, 8, 41–46.
Riyanto, A., Kusrini, M. D., Lubis, M. I. & Darmawan, B. (2009).
Preliminary comparison of file-eared tree frogs, Polypedatesotilophus (Boulenger, 1893) (Anura: Rhacophoridae) from Java
and other Sundaic islands, Indonesia. Russian Journal ofHerpetology, 16, 217–220.
Riyanto, A., Mumpuni & McGuire, J. A. (2011). Morphometry of
striped tree frogs, Polypedates leucomystax (Gravenhorst, 1829)
from Indonesia with description of a new species. RussianJournal of Herpetology, 18, 29–35.
Roelants, K., Gower, D. J., Wilkinson, M., Loader, S. P., Biju, S.
D., Guillaume, K., Moriau, L. & Bossuyet, F. (2007). Global
patterns of diversification in the history of modern amphibians.
Proceedings of the National Academy of Sciences of the United States
of America, 104, 887–892.
Sathiamurthy, E. & Voris, H. K. (2006). Maps of Holocene sea level
trangression and submerged lakes on the Sunda Shelf. The NaturalHistory Journal of Chulalongkorn University, Supplement, 2, 1–43.
Sheridan, J. A., Bickford, D. & Su, K. F.-Y. (2010). An
examination of call and genetic variation in three wide-ranging
Southeast Asian anuran species. The Raffles Bulletin of Zoology,
58, 369–379.
Smith, M. A. (1940). The amphibians and reptiles obtained by
Mr. Ronald Kaulback in upper Burma. Records of the Indian
Museum, 42, 465–486.
Stephens, M., Smith, N. J. & Donnelly, P. (2001). A new
statistical method for haplotype reconstruction from population
data. The American Journal of Human Genetics, 68, 978–989.
012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters
N. Kuraishi et al. d Phylogenetic study on the Polypedates leucomystax complex
Stuart, B. L., Inger, R. F. & Voris, H. K. (2006). High level of
cryptic species diversity revealed by sympatric groups of
Southeast Asian forest frogs. Biology Letters, 2, 470–474.
Swofford, D. (2002). PAUP*: Phylogenetic Analysis Using Parsimony
(*and Other Methods), Version 4. Sunderland, MA: Sinauer
Associates.
Tanabe, A. S. (2007). Kakusan: a computer program to automate
the selection of a nucleotide substitution model and the
configuration of a mixed model on multilocus data. MolecularEcology Notes, 7, 962–964.
Tavare, S. (1986). Some probabilistic and statistical problems in
the analysis of DNA sequence. Lectures on Mathematics in the
Life Sciences, 17, 57–86.
Taylor, E. H. (1962). The amphibian fauna of Thailand. The
University of Kansas Science Bulletin, 63, 265–599.
Toda, M., Matsui, M., Nishida, M. & Ota, H. (1998). Genetic
divergence among Southeast and East Asian populations of
Rana limnocharis (Amphibia: Anura), with special reference to
sympatric cryptic species in Java. Zoological Science, 15, 607–613.
Trepanier, T. L., Lathrop, A. & Murphy, R. M. (1999).
Rhacophorus leucomystax in Vietnam with acoustic analyses of
courtship and territorial calls. Asiatic Herpetological Research, 8,
102–106.
Veith, J., Kosuch, M., Ohler, A. & Dubois, A. (2001). Systematics
of Fejervarya limnocharis (Gravenhorst, 1829) (Amphibia, Anura,
Ranidae) and related species. 2. Morphological and molecular
variation in frogs from the Greater Sunda Islands (Sumatra, Java,
Borneo) with the definition of two species. Alytes, 19, 5–28.
Wiens, J. J., Sukumaran, J., Pyron, R. A. & Brown, R. M. (2009).
Evolutionary and biogeographic origins of high tropical diversity
in Old World frogs (Ranidae). Evolution, 63, 1217–1231.
Wiles, G. J. (2000). Recent records of reptiles and amphibians
accidentally transported to Guam, Mariana Islands. Micronesica,
32, 285–287.
Wilkinson, J. A., Drewes, R. C. & Tatum, O. L. (2002). A
molecular phylogenetic analysis of the family Rhacophoridae
with an emphasis on the Asian and African genera. Molecular
Phylogenetics and Evolution, 24, 265–273.
Wolf, S. (1936). Revision der Untergattung Rhacophorus
(ausschliesslich der madagaskar-Formen). Bulletin of the RafflesMuseum, 12, 137–217.
Yang, D.-T. & Rao, D. (Eds) (2008). Amphibia and Reptilia ofYunnan. Kunming: Yunnan Science and Technology Press.
Yu, G.-H., Rao, D.-Q., Zhang, M.-W. & Yang, J.-X. (2009).
Re-examination of the phylogeny of Rhacophoridae (Anura)
ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters
based on mitochondrial and nuclear DNA. MolecularPhylogenetics and Evolution, 50, 571–579.
Zhao, E. M. & Adler, K. (1993). Herpetology of China.
Contributions to Herpetology, 10, 1–522.
Ziegler, T. (2002). Die Amphibien und Reptilien einesTieflandfeuchtwald-Schutzgebietes in Vietnam. Munster: Natur und
Tier-Verlag.
Supporting InformationAdditional Supporting Information may be found in the
online version of this article:
Table S1. Samples used in this study. L, larva; UN,
unnumbered. See text for voucher abbreviations.
Table S2. Samples used for the nuDNA gene analyses.
Sample numbers correspond to those shown in Fig. 1.
Table S3. Primers used in this study.
Table S4. Alignment statistics for fragments of the
mitochondrial 12S rRNA, tRNAval, and 16S rRNA genes
and the nuDNA genes; number of base pairs (bp); number
of variable sites (vs.); number of parsimony informative
sites (pi); and the transition ⁄ transversion ratio given for in-
groups only (ti ⁄ tv).
Table S5. Genetic distances (mean p-distance in %, fol-
lowed by ranges) in 16S rRNA between genetic clades of
Polypedates.
Table S6. A list of the Rag1 and BDNF haplotypes
observed in the mitochondrial Indochina (P. megacephalus)and Sunda (P. leucomystax) Clades. Only the variable posi-
tions are shown. For the sample numbers, refer to
Table S1.
Table S7. Estimated divergence times (MYBP) of main
divergences within Polypedates. Node numbers are shown
in Fig. 5. See text for additional details and description of
the data sets.
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supporting materials sup-
plied by the authors. Any queries (other than missing
material) should be directed to the corresponding author
for the article.
17