Short Communication Mitochondrial phylogeny of an Asian tree frog genus Theloderma (Anura: Rhacophoridae) Tao Thien Nguyen a , Masafumi Matsui b,⇑ , Koshiro Eto b,c a Vietnam National Museum of Nature, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Hanoi, Viet Nam b Graduate School of Human and Environmental Studies, Kyoto University, Sakyo, Kyoto 606-8501, Japan c Kyoto University Museum, Yoshida-Honmachi, Sakyo, Kyoto 606-8501, Japan article info Article history: Received 1 November 2014 Revised 3 February 2015 Accepted 4 February 2015 Available online 12 February 2015 Keywords: Biogeography Character evolution mtDNA phylogeny Nyctixalus Southeast Asia abstract We assessed phylogenetic and systematic relationships among 17 out of 23 species of Theloderma and all three species of Nyctixalus from 2412 bp sequences of the mitochondrial DNA genes of 12S rRNA, tRNA val , and 16S rRNA using maximum likelihood and Bayesian inference methods. With the exception of T. moloch, Theloderma and Nyctixalus are confirmed to form a clade, in which each genus also forms a clade. Theloderma moloch is phylogenetically outside these clades and closer to samples from Chiromantis, Fei- hyla, Gracixalus, Kurixalus, Philautus, Polypedates, Raorchestes, and Rhacophorus. Within Theloderma, T. hor- ridum and T. stellatum form the sister taxon to a clade comprising the remaining species. The basal split within the latter clade groups T. asperum, T. licin, T. petilum, and T. ryabovi as the sister to a clade com- prising T. bicolor, T. chuyangsinense, T. corticale, T. gordoni, T. laeve, T. lateriticum, T. nebulosum, T. rhododis- cus, and T. truongsonense. Our phylogenetic results indicate homoplastic evolution of four morphological characters: small vs. large body size, presence of vomerine teeth, presence of a vocal opening in males, and interdigital webbing on hands. The common ancestor of Theloderma and Nyctixalus is inferred to have arisen in the area including the current Sunda region. Ó 2015 Elsevier Inc. All rights reserved. 1. Introduction The Southeast Asian rhacophorid genus Theloderma was once restricted to several species with numerous large or small, and sometimes calcified, warts on body and limbs, and fingers webbed at their base or up to half their length in metamorphs. Also they breed in water-filled tree cavities or holes (Taylor, 1962; Liem, 1970). However, from the results of more recent extensive field- work, several new species were added to the genus (e.g., Orlov et al., 2006). Furthermore, from the results of molecular analyses, some species with smooth skin or unwebbed fingers, and once placed in various genera such as Philautus, Chirixalus, and Aquixalus have been transferred to this genus (e.g., Yu et al., 2007; Rowley et al., 2011; Nguyen et al., 2014a). Also, breeding habits proved to have some variation from those typical of the original species of the genus (Orlov and Ho, 2005). These new findings yield 23 rec- ognized species in the genus at present (Frost, 2014). Additionally, one phylogenetically enigmatic species from India and China (T. moloch) has been included in this genus. Some newly discovered small-sized species are very similar in morphology, mainly differentiated by coloration, and are consid- ered to form a clade (Rowley et al., 2011; Orlov et al., 2012). DNA barcoding has been used to determine distinct specific status of some of these species, but overall phylogenetic relationships within the genus are poorly known. In only one available study (Rowley et al., 2011), relationships among taxa studied were poor- ly resolved and paraphyly with respect to genus Nyctixalus was suggested. This is probably due to limited taxon sampling and use of short DNA fragments (see Section 4). Thus, more extensive studies employing larger numbers of taxa and longer sequences are presented here to elucidate overall relationships among var- ious taxa of Theloderma, and to confirm relationship of the genus with Nyctixalus. With this in mind, we present in this paper, a molecular phylogeny of Theloderma and Nyctixalus, sampling 17 species of Theloderma and all three known species of Nyctixalus. We also tried to estimate ancestral states of four morphological characters in common ancestors of the genus and clades/subclades recovered. http://dx.doi.org/10.1016/j.ympev.2015.02.003 1055-7903/Ó 2015 Elsevier Inc. All rights reserved. ⇑ Corresponding author. Fax: +81 75 753 6846. E-mail addresses: [email protected](T.T. Nguyen), [email protected]. kyoto-u.ac.jp (M. Matsui), [email protected](K. Eto). Molecular Phylogenetics and Evolution 85 (2015) 59–67 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev
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Molecular Phylogenetics and Evolution 85 (2015) 59–67
Tao Thien Nguyen a, Masafumi Matsui b,⇑, Koshiro Eto b,c
a Vietnam National Museum of Nature, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Hanoi, Viet Namb Graduate School of Human and Environmental Studies, Kyoto University, Sakyo, Kyoto 606-8501, Japanc Kyoto University Museum, Yoshida-Honmachi, Sakyo, Kyoto 606-8501, Japan
a r t i c l e i n f o
Article history:Received 1 November 2014Revised 3 February 2015Accepted 4 February 2015Available online 12 February 2015
Keywords:BiogeographyCharacter evolutionmtDNA phylogenyNyctixalusSoutheast Asia
a b s t r a c t
We assessed phylogenetic and systematic relationships among 17 out of 23 species of Theloderma and allthree species of Nyctixalus from 2412 bp sequences of the mitochondrial DNA genes of 12S rRNA, tRNAval,and 16S rRNA using maximum likelihood and Bayesian inference methods. With the exception of T.moloch, Theloderma and Nyctixalus are confirmed to form a clade, in which each genus also forms a clade.Theloderma moloch is phylogenetically outside these clades and closer to samples from Chiromantis, Fei-hyla, Gracixalus, Kurixalus, Philautus, Polypedates, Raorchestes, and Rhacophorus. Within Theloderma, T. hor-ridum and T. stellatum form the sister taxon to a clade comprising the remaining species. The basal splitwithin the latter clade groups T. asperum, T. licin, T. petilum, and T. ryabovi as the sister to a clade com-prising T. bicolor, T. chuyangsinense, T. corticale, T. gordoni, T. laeve, T. lateriticum, T. nebulosum, T. rhododis-cus, and T. truongsonense. Our phylogenetic results indicate homoplastic evolution of four morphologicalcharacters: small vs. large body size, presence of vomerine teeth, presence of a vocal opening in males,and interdigital webbing on hands. The common ancestor of Theloderma and Nyctixalus is inferred to havearisen in the area including the current Sunda region.
� 2015 Elsevier Inc. All rights reserved.
1. Introduction
The Southeast Asian rhacophorid genus Theloderma was oncerestricted to several species with numerous large or small, andsometimes calcified, warts on body and limbs, and fingers webbedat their base or up to half their length in metamorphs. Also theybreed in water-filled tree cavities or holes (Taylor, 1962; Liem,1970). However, from the results of more recent extensive field-work, several new species were added to the genus (e.g., Orlovet al., 2006). Furthermore, from the results of molecular analyses,some species with smooth skin or unwebbed fingers, and onceplaced in various genera such as Philautus, Chirixalus, and Aquixalushave been transferred to this genus (e.g., Yu et al., 2007; Rowleyet al., 2011; Nguyen et al., 2014a). Also, breeding habits provedto have some variation from those typical of the original speciesof the genus (Orlov and Ho, 2005). These new findings yield 23 rec-ognized species in the genus at present (Frost, 2014). Additionally,
one phylogenetically enigmatic species from India and China (T.moloch) has been included in this genus.
Some newly discovered small-sized species are very similar inmorphology, mainly differentiated by coloration, and are consid-ered to form a clade (Rowley et al., 2011; Orlov et al., 2012).DNA barcoding has been used to determine distinct specific statusof some of these species, but overall phylogenetic relationshipswithin the genus are poorly known. In only one available study(Rowley et al., 2011), relationships among taxa studied were poor-ly resolved and paraphyly with respect to genus Nyctixalus wassuggested. This is probably due to limited taxon sampling anduse of short DNA fragments (see Section 4). Thus, more extensivestudies employing larger numbers of taxa and longer sequencesare presented here to elucidate overall relationships among var-ious taxa of Theloderma, and to confirm relationship of the genuswith Nyctixalus. With this in mind, we present in this paper, amolecular phylogeny of Theloderma and Nyctixalus, sampling 17species of Theloderma and all three known species of Nyctixalus.We also tried to estimate ancestral states of four morphologicalcharacters in common ancestors of the genus and clades/subcladesrecovered.
60 T.T. Nguyen et al. / Molecular Phylogenetics and Evolution 85 (2015) 59–67
2. Materials and methods
2.1. Sampling design
We examined partial DNA sequences of the mitochondrial DNAgenes encoding 12S rRNA, tRNAval, and 16S rRNA from the newlycollected specimens and sequences available from GenBank,including 17 species of Theloderma and three of Nyctixalus: T. corti-cale, T. bicolor, T. rhododiscus, T. chuyangsinense, T. nebulosum, T.truongsonense, T. laeve, T. lateriticum, T. leporosum, T. gordoni, T.asperum, T. petilum, T. licin, T. ryabovi, T. horridum, T. stellatum, T.moloch, Nyctixalus margaritifer, N. spinosus, and N. pictus. Speciesidentification for T. chuyangsinense and T. laeve simply followedOrlov et al. (2012). Specimens of other rhacophorid genera, Chiro-mantis doriae, Feihyla kajau, Gracixalus sp., Kurixalus eiffingeri, Phi-lautus aurifasciatus, Polypedates leucomystax, Raorchestes parvulus,
Table 1Samples of Theloderma and reference species used for mtDNA analysis in this study togspecimen deposited at the National Museum of the Philippines], AMNH [American MuseumMalaysia Sabah], CIB [Chengdu Institute of Biology], HNUE [Hanoi National University of EdUniversity, Graduate School of Human and Environmental Studies], NAP [Field ID of ZoologOntario Museum], MZB [Museum Zoologicum Bogoriense], VNMN [Vietnam National Mus
and Rhacophorus borneensis, were also used for comparisons, andLiuixalus romeri and Buergeria buergeri, as outgroups. Voucherspecimens/tissues are as shown in Table 1.
2.2. Preparation of DNA, PCR, and DNA sequencing
The total genomic DNA from a small amount of tissue from fro-zen or ethanol (95–99%) preserved specimens was extracted usingstandard Phenol–Chloroform extraction procedure (Hillis et al.,1996). Amplifications were done by polymerase chain reaction(PCR) with primers followed Kuraishi et al. (2013). The PCR cycleincluded an initial denaturation step of 2 min at 94 �C; 33 cyclesof denaturation for 15 s at 94 �C, primer annealing for 15 s at53 �C, and extension for 2 min 30 s at 72 �C; and a final extensionfor 7 min at 72 �C. The PCR products purified using polyethyleneglycol (PEG, 13%) precipitation procedures were used directly as
ether with the information on voucher specimens (ACD [Arvin Diesmos field series,of Natural History], BORN [Institute for Tropical Biology and Conservation, University
ucation], IABHU [Institute for Amphibian Biology, Hiroshima University], KUHE [Kyotoical Museum of Moscow University], RAO [field number of Ding-Qi Rao], ROM [Royaleum of Nature]), collection locality, and GenBank accession numbers.
ality Genbank References
nam, Vinh Phuc, Tam Dao KJ802916 Nguyen et al. (2014a)nam, Tuyen Quang, Na Hang KJ802917 Nguyen et al. (2014a)nam, Vinh Phuc, Tam Dao LC012841 This studynam, Lao Cai, SaPa KJ802915 Nguyen et al. (2014a)
na, Guangxi LC012842 This studynam, Daklac LC012843 This studynam, Daklac LC012844 This studynam LC012845 This studynam, Gia Lai, Mang Yang LC012846 This studynam, Khanh Hoa, Hon ba LC012847 This studynam, Bac Giang, Yen Tu LC012851 This studynam, Bac Giang, Yen Tu LC012850 This studynam, Son La, Ta Sua LC012849 This studynam, Lao Cai, SaPa LC012848 This studyaysia, Negeri Sembilan, Kenaboi AB847128 Matsui et al. (2014)s, Houapan KJ802919 Nguyen et al. (2014a)nam, Son La, Ta Sua KJ802918 Nguyen et al. (2014a)nam, Kon Tum, Ngoc Linh LC012852 This studynam, Vinh Phuc, Tam Dao KJ802914 This studynam, Vinh Phuc, Tam Dao LC012853 This studynam, Lao Cai, SaPa KJ802913 Nguyen et al. (2014a)nam, Kon Tum, Ngoc Linh LC012854 This studynam, Gia Lai, Kon Ka Kinh LC012855 This studynam, Thanh Hoa, Xuan Lien LC012856 This studynam, Son La, Ta LC012857 This studyiland, Doi Changdao LC012858 This studynam, Dien Bien, Muong Nhe KJ802925 Nguyen et al. (2014a)iland, Nakon Sri Tamarat LC012859 This studyaysia, Selangor KJ802920 Nguyen et al. (2014a)nam, Kon Tum, Mang Canh LC012860 This studyaysia, Negeri Sembilan, Kenaboi LC012861 This studynam, Phu Yen, Krong Chai KJ802923 Nguyen et al. (2014a)nam, Phu Yen, Krong Chai KJ802922 Nguyen et al. (2014a)iland, MaeYom LC012862 This studyneo, Sarawak, Bario LC012863 This studyippines, Mindanao DQ283114 Frost et al. (2006)
onesia, Java LC012864 This studyneo, Sabah, Maliau Basin AB781693 Matsui et al. (2013)neo, Sarawak AB847122 Matsui et al. (2014)iland, Kanchanaburi LC011932 Matsui et al. (2015)onesia, Java, Depok AB564285 Kuraishi et al. (2013)iland, Loei AB813159 Matsui et al. (2014)na, Xizang GQ285679 Li et al. (2009)iland, Khao Sabap LC012865 This studyonesia, Java, Central Java KJ802924 Nguyen et al. (2014a)n, Iriomote Is. AB933305 Nguyen et al. (2014b)
na, Hong Kong AB871412 Nguyen et al. (2014c)n, Hiroshima AB127977 Sano et al. (2004)
T.T. Nguyen et al. / Molecular Phylogenetics and Evolution 85 (2015) 59–67 61
templates for Cycle Sequencing Reactions with fluorescent-dye-la-beled terminator (ABI Big Dye Terminators v.3.1 cycle sequencingkit). We purified the sequencing reaction products by ethanol pre-cipitation following the manufacturer’s protocol and then ran themon an ABI PRISM 3130 genetic analyzer. We sequenced all samplesin both directions using the same primers as for PCR and 10 addi-tional primers used by Kuraishi et al. (2013). The resultantsequences were deposited in GenBank (Accession numbersLC012841–012865: Table 1).
2.3. Phylogenetic analysis
Aligned, combined sequences of 12S rRNA, tRNAval, and 16S rRNAyielded a total 2412 bp positions. Chromas Pro software (Technely-sium Pty Ltd., Tewantin, Australia) was used to edit the sequences,which were aligned using the MAFFT version 7 (Katoh andStandley, 2013) with default setting (FFT-NS-2 algorithm). We thenchecked the initial alignments by eye and adjusted slightly. Weconstructed phylogenetic trees using maximum likelihood (ML)and Bayesian inference (BI). Prior to ML and Bayesian analyses,we first divided the dataset into three partitions, 12S rRNA, tRNAval,and 16S rRNA. The optimum substitution models for each partitionwere selected by Kakusan4 (Tanabe, 2011), based on the Akaikeinformation criterion (AIC). We performed ML analyses byTreefinder version March 2011 (Jobb, 2011), while we estimatedBI and Bayesian posterior probabilities (BPP) by MrBayes v.3.2.1(Ronquist and Huelsenbeck, 2003). The best model for eachpartition of 12S and 16S rRNA in ML and Bayesian analyses wasthe general time reversible model (GTR: Tavaré, 1986) with a gam-ma shape parameter (G: 0.317 in ML and 0.340 in BI for 12S; 0.323and 0.335 for 16S). For tRNAval gene, the optimum model for MLanalysis was J2ef (Jobb, 2011) + G (0.313), while Hasegawa–Kishi-no–Yano-1985 (HKY85: Hasegawa et al., 1985) + G (0.375) wasselected as the best model for Bayesian analysis. The BI summa-rized two independent runs of four Markov Chains for 10,000,000generations. A tree was sampled every 100 generations, and a con-sensus topology was calculated for 70,000 trees after discardingthe first 30,001 trees (burn-in = 3,000,000). We checked parameterestimates and convergence using Tracer version 1.5 (Rambaut andDrummond, 2009).
Strength of nodal support in the ML tree was analyzed usingnon-parametric bootstrapping (MLBS) with 1000 replicates. We apriori regarded tree nodes in ML with bootstrap value 70% orgreater as sufficiently resolved (Huelsenbeck and Hillis, 1993),and we considered nodes with a BPP of 0.95 or greater significantin the BI analysis (Leaché and Reeder, 2002). Pairwise comparisonsof uncorrected sequence divergences (p-distance) were alsocalculated.
2.4. Reconstruction of character evolution
Evolution of four characters (body size, vomerine teeth, handwebbing, and male vocal opening) was reconstructed using a Baye-sian approach implemented in the software package BayesTraits(PC version 1.0: Pagel et al., 2004) and based on the consensus phy-logenetic tree derived from Bayesian inference analyses. Rev-ersible-jump Markov chain Monte Carlo (MCMC) methods wereused to derive posterior probabilities of values of traits at ancestralnodes of phylogenies. A total of 10,000,000 iterations were run foreach analysis with the first 100,000 samples discarded as burn-in.Since posterior probabilities for ancestral states of the single runspartly varied, we calculated the arithmetic mean of all samplesthree times for reconstruction of the ancestral condition. Statesof four characters were taken from our own examination of speci-mens and published data.
3. Results
3.1. Sequence and statistics
Of 2412 nucleotide sites, 1238 were variable and 1011 wereparsimony informative within the ingroup. The ML and Bayesiananalyses produced topologies with �lnL = 32640.096 and32693.581, respectively.
3.2. Phylogenetic relationships
Phylogenetic analyses employing ML and BI methods yieldedslightly different topologies only among referenced species, andonly the ML tree is presented in Fig. 1. Monophyly of target taxa(Theloderma species, excluding T. moloch, and Nyctixalus species)and referenced taxa (Chiromantis, Feihyla, Gracixalus, Kurixalus, Phi-lautus, Polypedates, Raorchestes, and Rhacophorus) with respect tothe outgroup Liuixalus and Buergeria was not strongly supported(MLBS = 70%, BPP = 0.90). However, monophyly of the generaTheloderma, excluding T. moloch, and Nyctixalus was strongly sup-ported (98%, 1.00), and the sister-taxon relationship of Theloderma(83%, 0.99) and Nyctixalus (100%, 1.00) was also supported. Thelo-derma moloch was nested among the referenced rhacophorid taxa,and its relationships to other taxa were unresolved.
In Theloderma, other than T. moloch, two major clades (Clades Aand B in Fig. 1) were recovered with substantial support (Clade A:88%, 1.00; Clade B: 100%, 1.00). The first of these (Clade A) consist-ed of two subclades: Subclade A1 (83%, 1.00) with 10 species andSubclade A2 (95%, 1.00) with four species. The other main clade(Clade B) consisted of two species.
In the Subclade A1, six lineages with unresolved relationshipstended to form two poorly supported groups. One of these groups(61%, 0.96) contained (1) T. corticale and its sister species T. bicolor(100%, 1.00), (2) T. rhododiscus, (3) T. chuyangsinense, and the cladeof (4) T. nebulosum, T. truongsonense, and T. laeve (100%, 1.00). Inthe last clade, T. nebulosum and T. laeve tended to form a clade(79%, 0.94), which is the sister group to T. truongsonense. Anotherpoorly supported group in the Subclade A1 (48%, 0.60) contained(5) T. lateriticum and (6) T. gordoni and its sister species T. leporo-sum (100%, 1.00). In the Subclade A2, T. ryabovi was the sister spe-cies to the clade containing T. licin, T. petilum, and T. asperum (100%,1.00), in which T. licin was the sister species to the clade of T. peti-lum and T. asperum (86%, 1.00). The Clade B of Theloderma consistedof T. horridum and its sister species T. stellatum, and in the genusNyctixalus, N. pictus was the sister species to the clade of N. spinosusand N. margaritifer (100%, 1.00).
Uncorrected p-distance within a species of Theloderma varied0–7.7% in T. asperum (Table 2). The distances between Thelodermaand Nyctixalus varied from 16.5% to 21.9%, which values were equalto or even smaller than those observed between Clades and Sub-clades of Theloderma (18.6–22.4% between Clade A and B, 14.0–21.1% between Subclades A1 and A2, 18.6–21.9% between SubcladeA1 and Clade B, and 19.5–22.4% between Subclade A2 and Clade B).Between species of Theloderma, the distances varied from 8.7–9.1%(between T. leporosum and T. gordoni) to 17.2–18.3% (between T.lateriticum and T. nebulosum) in Subclade A1, from 13.0–13.3% (be-tween T. asperum and T. petilum) to 18.3–19.2% (between T. aspe-rum and T. ryabovi) in Subclade A2, and 12.1–12.9% in Clade B(between T. horridum and T. stellatum). Thus the minimum inter-species distance 8.7% (between T. leporosum and T. gordoni) wasonly slightly larger than the maximum intraspecies distance 7.7%(in T. asperum). In Nyctixalus, variation was consistently 13.2–13.3% among three species. Finally, between T. moloch, species ofTheloderma and Nyctixalus differed 17.7–21.8% and 18.5–19.8%,respectively, in uncorrected p-distances.
62 T.T. Nguyen et al. / Molecular Phylogenetics and Evolution 85 (2015) 59–67
3.3. Reconstruction of character evolution
Our molecular phylogenetic analyses unambiguously revealedmonophyly of Theloderma (excluding T. moloch); thus, we tracedcharacter evolution for all lineages detected in our molecular sys-tematic studies. The results are given as pie charts displaying dif-ferent fractions (calculated as posterior probabilities) of thereconstructed character states and mapped onto the phylogenetictree in Fig. 2.
The reconstruction of character evolution of Theloderma sug-gested that in the ancestral state, body size was either large (pos-terior probability = 0.44) or small (0.56), vomerine teeth wereabsent (0.98), vocal slit was either absent (0.40) or present(0.60), and hand web was also either absent (0.50) or present(0.50). In Clade A, body size was either large (0.63) or small(0.37), vomerine teeth were absent (0.98), vocal slit was eitherabsent (0.69) or present (0.31), and hand web was more likelyabsent (0.79), while in Clade B, body size was either large (0.63)or small (0.37) and vomerine teeth were absent (0.98) like CladeA, but vocal slit and hand web were present (both 0.98). In bothSubclades A1 and A2, body size was either large or small, andvomerine teeth were more likely absent. However, vocal slit andhand web were absent (0.99 and1.00, respectively) in SubcladeA1, but they were present (0.98 and 0.89, respectively) in SubcladeA2.
Fig. 1. Maximum likelihood (ML) phylogram of 2412 bp of 12S rRNA, tRNAval, and 16S rnumbers are included in Table 1. Numbers above or below branches represent bootstra
4. Discussion
4.1. Systematic problem of T. moloch
In our phylogenetic tree, T. moloch was not imbedded within theTheloderma clade, and its position was unresolved. This conformsto previous reports by Li et al. (2009), Rowley et al. (2011), andPyron and Wiens (2011), but disagrees with the report of Li et al.(2013), who showed T. moloch to be nested in Theloderma. Howev-er, T. moloch is actually not listed in Li et al.’s (2013) Supplemen-tary Table 1, where T. leporosum (KC465841) is listed, instead.Thus, it is most probable that figure (Fig. S3) of Li et al. (2013) isincorrect, and inclusion of moloch in Theloderma is not accepted.
Theloderma moloch was originally described as Phrynodermamoloch (Annandale, 1912), but was later moved to Rhacophorus(as a subgenus: Ahl, 1931), Nyctixalus (Dubois, 1981), and finallyto Theloderma (Inger in Frost, 1985). The genus Phrynoderma is syn-onymized with Theloderma (Taylor, 1962), and the name of themonotypic species of the genus, P. asperum, is now T. phrynoderma(Inger in Frost (1985)). Because phylogenetic position of T.phrynoderma has never been elucidated, it is not appropriate torevive the name of Phrynoderma to accommodate T. moloch. Inorder to determine taxonomic status of T. moloch, future studiesof additional molecular phylogeny and morphology of this species,as well as of T. phrynoderma are necessary.
RNA mitochondrial genes for samples of Theloderma and reference species. Samplep support for ML, and Bayesian posterior probabilities (MLBS/BPP).
Table 2Uncorrected p-distances (%) in 16S rRNA among species of Theloderma and Nyctixalus. Values in bold face show intraspecific variation.
Fig. 2. Results of reconstruction of character evolution mapped onto the tree coded as pie charts displaying color coded fractions for the four morphological characters(inferred from posterior probabilities). A: body size (black = small, white = large); B: vomerine teeth (black = present, white = absent); C: vocal opening (black = present,white = absent, gray = unknown); D: hand web (black = present, white = absent).
64 T.T. Nguyen et al. / Molecular Phylogenetics and Evolution 85 (2015) 59–67
4.2. Relationships of Nyctixalus and Theloderma
Prior authors (Wilkinson et al., 2002; Frost et al., 2006; Li et al.,2008, 2009, 2013; Yu et al., 2009; Pyron and Wiens, 2011), exceptfor Rowley et al. (2011) and Nguyen et al. (2014a), invariablyreported Theloderma and Nyctixalus as sister genera. Rowley et al.(2011) obtained phylogenetic trees in which N. pictus was nestedin species of Theloderma they studied, and Nguyen et al. (2014a)obtained a similar result. However, these are surely results ofinsufficient taxon sampling and short sequences (see below). Closerelationship of Theloderma and Nyctixalus has long been suggestedfrom morphological and ecological evidence (Taylor, 1962; Liem,1970). Liem (1970) presented characteristics common to Theloder-ma and Nyctixalus (as Hazelia): warty skin of the body and limbsand the skin of the head, which is co-ossified to the skull; similarreproductive behavior, i.e., small numbers of eggs are placed abovewater-filled cavities or holes in tree trunks, and the tadpolesdevelop in these water pockets.
The two genera are differentiated by several morphologicalcharacteristics, such as shape of canthus rostralis (rounded inTheloderma vs. sharp in Nyctixalus), bony ridges from canthus
rostralis to occiput (absent in Theloderma vs. sharp in Nyctixalus),and skin of head (not co-ossified to the skull in Theloderma vs.co-ossified to skull in Nyctixalus) (Liem, 1970; Rowley et al.,2011). Whether or not these differences warrant generic separa-tion requires future consideration. This is because current genericallocation based on results of molecular phylogeny is sometimesarbitrary, and involves many problems. For example, amongrhacophorids, recent inclusion of several morphologically differentspecies in Feihyla strongly contrasts to the case of Theloderma andNyctixalus.
4.3. Phylogeny of Theloderma
Previous authors examined only a few species, such as T. aspe-rum, T. bicolor, T. corticale, and T. rhododiscus (Wilkinson et al.,2002; Frost et al., 2006; Yu et al., 2007, 2009; Li et al., 2009;Pyron and Wiens, 2011). Yu et al. (2007) synonymized T. albopunc-tatus (Liu and Hu, 1962) and T. asperum, because of small geneticdivergence found between them, and transferred Philautusrhododiscus to Theloderma because of its sister-species relationshipto T. corticale. The root of the tree usually separates T. asperum from
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a group containing T. rhododiscus, T. corticale and T. bicolor (Yuet al., 2007, 2009; Li et al., 2009), but Pyron and Wiens (2011)found T. corticale outside a group of T. rhododiscus, T. bicolor, andT. asperum. It is to be noted that Li et al. (2009), who added T.moloch in their analyses, found it be the sister species to a cladeof Philautus and not to Theloderma. In contrast, Li et al. (2013)showed that T. moloch grouped with T. asperum and the clade ofT. corticale and T. rhododiscus in their Fig. S3, but this must be incor-rect as discussed above.
Rowley et al. (2011) analyzed short sequences (ca. 550 bp of 16SrRNA) of 10 species of Theloderma, including T. moloch and N. pictus,using ML and BI methods. In ML, no clear relationships wereresolved except for sister-species relationships of T. bicolor and T.corticale, and T. nebulosum and T. truongsonense (T. laeve, accordingto Orlov et al. (2012); see below). Moreover, N. pictus showed tri-chotomous relationships with T. asperum and others. In the BI tree,resolution was better, and T. stellatum was the sister species to theclade of other species of Theloderma and N. pictus. In addition, sis-ter-species relationships of T. rhododiscus and T. palliatum, T. bicolorand T. corticale, and of these two clades, as well as sister speciesrelationships of T. nebulosum and T. truongsonense clade, were sup-ported. However, N. pictus was again embedded in these clades.Recently, Nguyen et al. (2014a) examined nine species usingsimilarly short sequences (ca. 640 bp of 16S rRNA) but obtainedbetter results than Rowley et al. (2011), although the position ofN. pictus was similar. Thus, the phylogenetic relationships amongspecies of Theloderma, as well as between Nyctixalus, have neverbeen well understood.
Unfortunately, sequences of T. andersoni, T. baibengensis, T. bam-busicolum, T. kwangsiense, T. nagalandense, and T. phrynoderma arecompletely absent, and only short sequences are available for T.palliatum. Although we lack information about these species, ourtaxon sampling, including more than two-thirds of named species,and use of longer sequences than many previous authors wouldstrengthen resolution of the phylogeny of Theloderma. In ourresults, Theloderma and Nyctixalus each formed a clade, and CladeA of Theloderma contained Subclade A1 with 10 species and Sub-clade A2 with four species, while Clade B consists of two species.Remote relationships of T. stellatum in Clade B from the other con-geners agree with Rowley et al. (2011) and Nguyen et al. (2014a),while its sister-species relationship with T. horridum is demon-strated here for the first time. Separation of Subclade A1, includingT. corticale, T. bicolor, T. rhododiscus, T. gordoni, and T. leporosum,and A2, including T. licin, T. petilum, and T. asperum, also agreeswith Nguyen et al. (2014a), and is consistent with Rowley et al.(2011). Sister-species relationships of T. corticale and T. bicolor, T.gordoni, and T. leporosum, and T. petilum and T. asperum, which isthe sister group to T. licin, also completely agreed with Nguyenet al. (2014a). Rowley et al.’s (2011) clade of T. nebulosum and T.truongsonense was supported, too. Sister-species relationship of T.ryabovi to the clade of T. licin, T. petilum, and T. asperum, and inclu-sion of T. laeve in the clade of T. nebulosum and T. truongsonense arealso newly elucidated. In the genus Nyctixalus, the sister-speciesrelationship of N. pictus to the clade of N. spinosus and N. mar-garitifer was confirmed.
In their phylogenetic analyses, Rowley et al. (2011) included T.truongsonensis (as Philautus) and reported that the species wasmost closely related to T. nebulosum, with the p-distance of 8.5%.However, Orlov et al. (2012) noted that T. truongsonensis shownby Rowley et al. (2011) was a misidentification of T. laeve (as Phi-lautus). In our results, however, T. truongsonense was the sister spe-cies to a weakly supported clade of T. nebulosum and T. laeve.Unfortunately we could obtain long sequence of only one sample,but together with two shorter sequences of T. truongsonense,Rowley et al.’s (2011) T. truongsonensis (Genbank: JN688174)formed a clade in short fragment (340 bp) of 16S rRNA with
p-distances of 5.1–5.9%. Thus, Rowley et al.’s (2011) placementof Philautus truongsonensis within Theloderma is supported, andinclusion of Philautus laevis within Theloderma suggested byOrlov et al. (2012) from morphology was also supportedmolecularly.
Unfortunately we failed to obtain long sequences for our T. pal-liatum samples, but short sequences (410 bp of 16S rRNA) of thespecies were nearly identical not only with Genbank sequencesof T. palliatum (JN688172 and JN688173: Rowley et al., 2011),but also with those of our T. chuyangsinense (p-distance 0–7.6%).In the original description (Orlov et al., 2012), T. chuyangsinensewas differentiated from T. palliatum by belly coloration (blackblotches are present only in the posterior part of the belly andinternal surface of hindlimb in T. chuyangsinense vs. black spotsand blotches almost uniformly cover the ventral surface of thebody and inner surfaces of the thigh and forelimb in T. palliatum),and the relative length of the toes (III < V in T. chuyangsinense vs.III = V in T. palliatum). Unfortunately, both species were describedon the basis of a few specimens (two males and one juvenile inT. palliatum and only one male in T. chuyangsinense), and it isunknown how extensively the characteristics shown in the originaldescriptions vary among individuals. Thus, at the moment, weretain the two names until longer sequences are compared anddegree of morphological variation is clarified.
P-distances obtained by us from 1358 bp of 16S rRNA were12.5% between T. nebulosum and T. truongsonense, and 13.9–14.1% between T. palliatum (as T. chuyangsinense) and T. rhododis-cus, both much larger than values reported by Rowley et al.(2011: 8.5% and 8.5–8.8%, respectively). This difference surelyarose from our longer sequences. Short sequences of a single genemay be useful for species barcoding (Vences et al., 2005), butwould mislead phylogenetic relationships. This may have causedthe inclusion of N. pictus among species of Theloderma (Rowleyet al., 2011; Nguyen et al., 2014a).
4.4. Evolutionary trends in some characters
Although we could not employ all species from China and India,our taxonomic sampling encompasses more than two-thirds ofnamed species, and the following evolutionary trends are estimat-ed in some characters. Species of Theloderma have been classifiedby their body size. Orlov (1997) once proposed two different sizegroups in Theloderma: small species with SVL 28–35 mm and largespecies with SVL 48–75 mm. However, in order to encompass T.ryabovi (44 mm in a single male described), Orlov et al. (2006)added the third size group with SVL 40–45 mm. Actually, SVL inT. ryabovi varies from 44 to 59 mm in males and 64 mm in a female(Matsui, unpublished), and simple recognition of two size groups,small (<40 mm) and large (P40 mm) would suffice to separateTheloderma species by body size. In our phylogeny, species withsimilar body size did not form groups. In the Subclade A1, four spe-cies are large and six are small, but neither large nor small speciesforms a clade. In the Subclade A2, one large species (T. ryabovi) isthe sister taxon to the clade of three small species. In Clade B,one is small and another is large. Because Nyctixalus, the sisterclade of Theloderma, is small, the common ancestor of the two gen-era would be inferred to have had small body size, although recon-struction of character evolution does not support such ahypothesis. Large body size in several species would have beenattained independently.
A similar trend was found in the vomerine teeth in Subclade A1,four large species had teeth, but the small species lacked them.However, in Subclade A2, the vomerine teeth were absent exceptfor a small species (T. petilum), which had weak teeth. Furthermore,the teeth are absent in both small and large species in Clade B.Thus, the correlation of body size and presence or absence of
66 T.T. Nguyen et al. / Molecular Phylogenetics and Evolution 85 (2015) 59–67
vomerine teeth is rejected. Liem (1970) estimated presence of theteeth as a primitive state in Rhacophoridae (and Hyperoliidae).However, the vomerine teeth are absent in Nyctixalus, and thereconstruction of character evolution of Theloderma also suggeststhe ancestral state to be lack of teeth. The vomerine teeth seemto have been acquired independently in large species of SubcladeA1 and T. petilum.
The presence or absence of a vocal slit in males is not alwayseasy to identify. Theloderma lateriticum has been reported to lackit (Bain et al., 2009), but we confirmed the presence in our sample.We have no information about a vocal opening in T. nebulosum,which was described from a female (Rowley et al., 2011), but theopening is present in T. laeve and T. lateriticum in Subclade A1. Incontrast, a vocal opening is present in all species in Subclade A2and Clade B. The opening is absent in Nyctixalus, and the ancestralstate of this character is unknown, as shown by the character evo-lution reconstruction. The absent condition seems to be succeededby Clade A and Subclade A1, but is estimated to characterize ances-tral Subclade A2. The slit is also thought to be present in Clade Bancestor.
Webbing on hands varies in degree of development, but wasabsent in Subclade A1, and present in Subclade A2 and Clade B,except for T. petilum in Subclade A2. Theloderma asperum has beenreported to lack webbing (Rowley et al., 2011), but we confirmedthe presence of a web remnant, supporting Taylor (1962). BecauseNyctixalus lacks webbing, state of this character in the commonancestor of the two genera is unknown, and was not clarified inancestral Theloderma by character evolution reconstruction. Thereconstruction of character evolution of Theloderma suggests thatClade A more likely lacked webbing, while it was possessed byClade B. Hand web is estimated to be absent in Subclade A1, butwas present in Subclade A2 in their ancestral states. The webbingseems to have been attained independently in Clade B andSubclade A2, except for T. petilum.
4.5. Biogeography
Liem (1970) proposed that the center of radiation of Orientalrhacophorids was the Indochinese region, which may be applicableto the genus Kurixalus (Nguyen et al., 2014b). Nyctixalus, withundoubtedly a close sister-taxon relationship with Theloderma, iscurrently distributed in the Philippines, Malay Peninsula, Sumatra,Java, Borneo, and possibly in southern Vietnam, while Thelodermaoccupies wider regions from northeastern India to Myanmar andsouthern China through Indochina to Malaya, Sumatra, and Borneo(Frost, 2014; Sri Lankan T. schmarda (Kelaart, 1854) now moved toPseudophilautus). Thus the areas now occupied by both genera aremainly the Malay Peninsula, Sumatra, and Borneo, and it is possiblethat the common ancestor of Theloderma and Nyctixalus arose inthe Sunda region. Currently, Theloderma has its greatest speciesdiversity in Indochina, and this region seems to be apparently asecondary center of speciation.
Acknowledgments
We are grateful to K. Araya, A. Hamidy, J.-P. Jang, K. Nishikawa,and N. Orlov for providing tissue samples, and N. Kuraishi for helpin the laboratory. MM thanks the National Research Council ofThailand, the Royal Forest Department of Thailand, the Eco-nomic-Planning Unit (former Socio-Economic Research Unit) ofMalaysia, and the State Government of Sarawak for permitting toconduct the project, Chulalongkorn University, University Malaya,Universiti Kebangsaan Malaysia (UKM), and the Forest Depart-ment, Sarawak for kindly providing all the facilities, and the fol-lowing for their companionship: N. Ahmad, K. Araya, D. Belabut,K.-O. Chan, T. Hikida, J.-P. Jang, W. Khonsue, the late J. Nabitabhata,
K. Nishikawa, H. Ota, S. Panha, and M. Toda. We are also grateful toA. Larson and anonymous reviewers for valuable comments on themanuscript. This research was partly supported by the JSPS RON-PAKU Program the Project TN3/T13 of the National Program TayNguyen III and STMVQG. 06G14-16 to T.T. Nguyen, and Grants-inAid from the Monbusho through JSPS (Field Research, Nos.06041066, 08041144, 20405013, and 20405014), UKM (OUP-PLW-14-59/2008), and TJTTP-OECF to M. Matsui. T.T. Nguyen isgrateful to M. Motokawa for support of work in Japan.
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