Amphibia: Anura: Rhacophoridae

Zootaxa 3702 (2): 101–123 www.mapress.com /zootaxa / Copyright © 2013 Magnolia Press

Article

ISSN 1175-5326 (print edition)

ZOOTAXA

ISSN 1175-5334 (online edition)

http://dx.doi.org/10.11646/zootaxa.3702.2.1 http://zoobank.org/urn:lsid:zoobank.org:pub:539F4210-B601-4CBB-9297-951DE26846EF

Re-evaluating the taxonomic status of Chiromantis in Thailand using multiple lines of evidence (Amphibia: Anura: Rhacophoridae) ANCHALEE AOWPHOL1,4, ATTAPOL RUJIRAWAN1, WUT TAKSINTUM1, SUTIPONG ARSIRAPOT2 & DAVID S. MCLEOD3 1

Department of Zoology, Faculty of Science, Kasetsart University, Bangkok 10900 Thailand Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok 10330 Thailand 3 University of Kansas Biodiversity Institute, 1345 Jayhawk Boulevard, Lawrence, KS 66045-7561, USA 4 Corresponding author. Email: [email protected] 2

Abstract Because of general phenotypic similarities and distribution of species across two continents, the genus Chiromantis has proven somewhat enigmatic. Among Indochinese species, the validity of C. hansenae has been questioned by some who consider it a junior synonym of C. vittatus. We employ three lines of evidence to elucidate the taxonomic status and phylogenetic relationships of four congeneric species of Chiromantis frogs from Thailand. Results of molecular, morphological, and bioacoustic data analyses support at least four evolutionarily distinct and monophyletic clades: C. doriae, C. nongkhorensis, C. vittatus and C. hansenae. Genetic divergence between C. vittatus and C. hansenae is >10%, significantly greater than C. doriae and C. nongkhorensis (4.5%). Our results support the taxonomic validity of C. hansenae and suggest that there may be more diversity within C. hansenae and C. vittatus than is currently recognized. Key words: Advertisement call; Morphometrics; mtDNA sequences; bioacoustics

Introduction The genus Chiromantis Peters, 1854 comprises 15 recognized species (Frost 2013) and is disjunctly distributed, occurring in the African tropics, south of the Sahara, and northeastern India to Southeast Asia (Frost 2013). This widespread yet disjunctive distribution across diverse climatic zones, in combination with evidence from molecular phylogenetic studies has called into question the monophyly of this genus (Wilkinson et al. 2002; Frost et al. 2006; Li et al. 2009; Wiens et al. 2009). Four species of these small rhacophorid tree frogs have been recorded from Thailand (Taylor 1962; Chan-ard 2003): Chiromantis doriae (Boulenger 1853), C. hansenae (Cochran 1927), C. nongkhorensis (Cochran 1927) and C. vittatus (Boulenger 1887). These frogs occur in forested and non-forested habitats across a range of elevation from low elevation to >1000 m ASL. Breeding occurs in small ponds where females deposit eggs in gelatinous masses (C. hansenae and C. vittatus) or foam-nests (C. doriae and C. nongkhorensis) on natural structures (e.g., tree branches, shrubs, and herbaceous vegetation) above water level (Taylor 1962; Sheridan & Ocock 2008). Little is known of the natural history and ecology of these tree frogs, and only recently was C. hansenae reported to demonstrate parental care behavior (Sheridan & Ocock 2008). Considerable morphological similarity among some Asian members of this genus has caused several authors to question the taxonomic validity of C. hansenae or treat it as a junior synonym of C. vittatus (Wilkinson et al. 2003; Stuart & Emmett 2006; Chan et al. 2011). Different authors have employed traditional morphological characters (e.g., body size, digital webbing, and size of the tympanum) to distinguish C. vittatus from C. hansenae (Taylor 1962; Wilkinson et al. 2003), but these characters have been found to be inconsistently reliable when examining specimens from across the distributional ranges of these species. Resolution of the standing taxonomic confusion between C. vittatus and C. hansenae is particularly relevant in light of recent discoveries of new species of Chiromantis that are morphologically similar to these taxa (e.g., Chan et al. 2011).

Accepted by J. Rowley: 8 Aug. 2013; published: 26 Aug. 2013

101

To date, the geographic distribution of Chiromantis in Thailand has not been extensively studied. Current understanding of distributions in Thailand is that C. doriae has a patchy distribution encompassing areas of northern, western and northeastern Thailand; C. nongkhorensis is broadly distribution across western, central and southeastern Thailand; C. vittatus occurs in the northern (Chiang Mai) and western regions; and C. hansenae is restricted to the east-central region but also found in Chiang Mai, northern Thailand (Taylor 1962; Chan-ard, 2003; Nabhitabhata & Chan-ard 2005). Chan-ard (2003) considered C. hansenae and C. vittatus as allopatric, reporting the latter only from northwestern Thailand and the former only in eastern Thailand. Several recent studies have reported C. vittatus from Lao PDR, Cambodia and Vietnam (Stuart & Emmett 2006; Grismer et al. 2008; Rowley & Srei 2010). This brings two issues to light: either C. hansenae is being treated as a junior synonym of C. vittatus by these authors, and/or there is evidence for rejecting the East-West distributional pattern presented by Chan-ard (2003). It is generally recognized that acoustic communication of anurans plays an important role in reproductive biology (Gerhardt 1991; Ryan 2001) and that call data can be an effective tool for species identification (e.g., Köhler et al. 2005; Meegaskumbura & Manamendra-Arachchi 2005; Kuraishi et al. 2011). To date, however, much remains unknown about the bioacoustics signals in frogs of the genus Chiromantis, particularly those in Thailand. We present the first bioacoustic data sampled from the four species of Chiromanits known to occur in Thailand. The purpose of this study is to use acoustic data from advertisement calls, in combination with morphological and molecular data to assess the taxonomic status of the Chiromantis species in Thailand. Using these lines of evidence we address the taxonomic validity of C. hansenae and the combination of characters that distinguish it from C. vittatus.

Materials and methods Sampling Chiromantis doriae, C. nongkhorensis, C. hansenae and C. vittatus were sampled from 10 localities in Thailand (Fig. 1 and Table 1) by opportunistic searching and by locating calling males. Sampling localities were selected based on published distributions, type localities, and collecting opportunity. Field identification of C. hansenae and C. vittatus was based on published distributions, collector’s perception of call differentiation, and gross morphology (e.g., slender body in C. vittatus). Whole voucher specimens were fixed in 10% formalin and preserved in 70% ethanol. Tissue samples (liver or muscle) were taken in the field immediately following euthanasia and preserved in 95% ethyl alcohol. All specimens were deposited in the herpetological collection, Zoological Museum, Kasetsart University, Bangkok, Thailand (ZMKU; Table 2). Tissue samples of C. vittatus from Myanmar, deposited at The California Academy of Sciences (CAS), were included in the molecular analysis (Table 2). DNA extraction, amplification and sequencing Mitochondrial DNA (mtDNA) data were selected for use in this study to take advantage of abundant comparative material already available from previous work (Delorme 2004; Delorme et al. 2004; Faivovich et al. 2005; Frost et al. 2006; Li et al. 2008; Yu et al. 2008; Li et al. 2009; Meegaskumbura et al. 2010; Kuraishi et al. 2011; Li et al. 2012). Whereas the utility of mtDNA in phylogenetic studies of amphibians has been debated (e.g., Hertwig et al. 2004) it seems clear that mtDNA is useful to identify candidate species (Vences et al. 2005a; Vences et al. 2005b; Fouquet et al. 2007; Vieites et al. 2009). Genomic DNA was extracted from liver tissues preserved in 95% ethanol using the Dneasy kit (QIAGEN, Inc.). A fragment of the mitochondrial DNA gene, 16S rRNA was amplified via PCR (94 °C, 45 s; 52 °C, 30 s; 72 °C 1 min) for 35 cycles using primers 16Sc and 16Sd (Moriarty & Cannatella 2004). All PCR products were purified using the Qiagen PCR purification kit (QIAGEN, Inc.). Sequencing was performed using a 3730 Analyzer (Applied Biosystems). Sequences were edited using Geneious v. 5.6.3 (Biomatter, Ltd.) and deposited in GenBank (Accession numbers KC357597–KC357669; KC692874– KC692883).

102 · Zootaxa 3702 (2) © 2013 Magnolia Press

AOWPHOL ET AL.

FIGURE 1. Distribution of species and sampling localities in this study. New samples collected within Thailand for this study represented by numbers within symbols: 1 = Mae Hong Sorn; 2 = Loei; 3 = Nakhon Ratchasima; 4 = Chon Buri (Type locality of C. hansenae and C. nongkhorensis); 5 = Chanthaburi; 6 = Nakhon Na Yok; 7 = Thong Pha Phum, Kanchanaburi; 8 = Sangkhla Buri, Kanchanaburi; 9 = Prachuap Khiri Khan; 10 = Surat Thani. Small black square represents the type locality for Chiromantis vittatus.

TAXONOMIC STATUS OF CHIROMANTIS IN THAILAND

Zootaxa 3702 (2) © 2013 Magnolia Press ·

103

TABLE 1. Localities and sample sizes of Chiromantis in morphological study. Species

Locality

Sample size

Total sample size

Prachuap Khiri Khan

8

20

Loei

12

Nakhon Ratchasima

22

Chon Buri**

9

Chanthaburi

23

Nakorn Na Yok

2

Thong Pha Phum, Kanchanaburi

1

C. vittatus Group II

Sangkhla Buri, Kanchanaburi

8

8

C. hansenae Group I

Nakhon Ratchasima

19

27

Loei

1

Chon Buri**

4

Surat Thani

3

Mae Hong Sorn

13


9

C. doriae

C. nongkhorensis

C. hansenae Group II

57

24

** Specimens of C. hansenae and C. nongkhorensis were collected from the type locality (Nong Khor, Chon Buri Province).

Phylogenetic analyses To increase the breadth of our sampling both geographically and taxonomically, we added available sequence data from GenBank of congeners (Chiromantis xerampelina and C. rufensens) and conspecifics (C. doriae, C. nongkhorensis, C. vittatus, and C. hansenae from Lao PDR, China and Vietnam; Table 3). Based on therelationship between Chiromantis and other rhacophorids evidenced by previous studies (Li et al. 2008, 2009; Yu et al. 2009) we used closely related taxa (Feihyla palpebralis, Rhacophorus bipunctatus, R. kio, Polypedates cruciger, P. leucomystax and P. megacephalus) to form the out-group. Buergeria buergeri was used to root the tree (Table 3). Sequences were aligned using MUSCLE (Edgar 2004) and then adjusted by eye in Se-Al Carbon version 2.0a11 (Rambaut 2002). Uncorrected pair-wise distances were calculated using MEGA 5.05 (Tamura et al. 2011). Maximum likelihood analyses were performed using RAxML-HPC Blackbox version 7.3.2 on the CIPRES Science Gateway (Stamatakis 2006; Miller et al. 2010) via 1000 non-parametric rapid bootstrap replicates. A thorough maximum likelihood search with bootstrap scores was mapped onto the best-scoring maximum likelihood tree. Bayesian analyses were conducted using MrBayes version 3.1.2 (Ronquist & Huelsenbeck 2003) on XSEDE accessed through the CIPRES Science Gateway (Miller et al. 2010). Four independent analyses were run with four Metropolis-coupled Markov chains each. All Markov chains were run for 10 million generations, sampling every 1000 generations. To assess convergence between chains, we verified that the average standard deviation of split frequencies approached zero, the potential scale reduction factor approached 1 and that the log likelihood scores had reached stationarity. The output files were examined in Tracer version 1.5 (Rambaut & Drummond 2007) to determine the number of generations to exclude as burn-in and as a final check for convergence (ensuring that all parameters and statistics had reached stationarity and sufficient (>100) effective sample sizes).


AOWPHOL ET AL.

TABLE 2. Specimens used in (A) morphological analysis and/or (B) molecular analysis. Species C. doriae

GenBank

Type of analysis

ZMKU AM 00600

–

A A

Locality

Field no.

Museum No.

Hua Hin, Prachuap Khiri Khan

19837

C. doriae


19838

ZMKU AM 00601

–

C. doriae


19839

ZMKU AM 00602

–

A

C. doriae


19840

ZMKU AM 00603

–

A

C. doriae


19841

ZMKU AM 00604

KC357618

A,B

C. doriae


19842

ZMKU AM 00605

KC357617

A,B

C. doriae


19843

ZMKU AM 00606

KC357619

A,B

C. doriae


19844

ZMKU AM 00607

–

A

C. doriae

Phu Ruea, Loei

AA00344

ZMKU AM 00691

–

A A

C. doriae

Phu Ruea, Loei

AA00345

ZMKU AM 00692

–

C. doriae

Phu Ruea, Loei

AA00346

ZMKU AM 00693

–

A

C. doriae

Phu Ruea, Loei

AA00347

ZMKU AM 00694

–

A

C. doriae

Phu Ruea, Loei

AA00349

ZMKU AM 00696

–

A A

C. doriae

Phu Ruea, Loei

AA00350

ZMKU AM 00697

–

C. doriae

Phu Ruea, Loei

AA00351

ZMKU AM 00698

–

A

C. doriae

Phu Ruea, Loei

AA00352

ZMKU AM 00699

–

A

C. doriae

Phu Ruea, Loei

AA00353

ZMKU AM 00700

KC357611

A,B

C. doriae

Phu Ruea, Loei

AA00355

ZMKU AM 00701

KC357613

A,B

C. doriae

Phu Ruea, Loei

AA01007

ZMKU AM 00788

KC357612

B

C. doriae

Phu Ruea, Loei

AA01008

ZMKU AM 00789

KC357615

B

C. doriae

Phu Ruea, Loei

AR00042

ZMKU AM 00964

KC357614

A,B

C. doriae

Phu Ruea, Loei

AR00045

ZMKU AM 00967

KC357616

A,B

C. nongkhorensis

Kang Hang Meaw, Chanthaburi

20191

ZMKU AM 00631

–

A

C. nongkhorensis


20197

ZMKU AM 00637

–

A

C. nongkhorensis


20198

ZMKU AM 00638

–

A A A

C. nongkhorensis


20199

ZMKU AM 00639

–

C. nongkhorensis


20200

ZMKU AM 00640

–

C. nongkhorensis


20201

ZMKU AM 00641

–

A

C. nongkhorensis


20202

ZMKU AM 00642

–

A

C. nongkhorensis


20203

ZMKU AM 00643

–

A

C. nongkhorensis


20204

ZMKU AM 00644

–

A

C. nongkhorensis


20205

ZMKU AM 00645

–

A

C. nongkhorensis


20206

ZMKU AM 00646

–

A

C. nongkhorensis


20209

ZMKU AM 00649

KC357607

A,B

C. nongkhorensis


20210

ZMKU AM 00650

KC357608

A,B

C. nongkhorensis


20211

ZMKU AM 00651

–

A

C. nongkhorensis


20212

ZMKU AM 00652

–

A

C. nongkhorensis


20213

ZMKU AM 00653

–

A

C. nongkhorensis


20214

ZMKU AM 00654

–

A

C. nongkhorensis


20215

ZMKU AM 00655

–

A

C. nongkhorensis


20216

ZMKU AM 00656

–

A

C. nongkhorensis


20217

ZMKU AM 00657

–

A

C. nongkhorensis


AA00418

ZMKU AM 00715

KC357603

A,B

C. nongkhorensis


AA00419

ZMKU AM 00716

KC357605

A,B

......continued on next the page



105

TABLE 2. (Continued) Locality

Field no.

Museum No.

GenBank

Type of analysis

C. nongkhorensis


AA00420

ZMKU AM 00717

KC357606

A,B

C. nongkhorensis

Mueang, Nakhon Nayok

AA00309

ZMKU AM 00686

KC357602

A,B

C. nongkhorensis

Mueang, Nakhon Nayok

AR00027

ZMKU AM 00963

–

A

C. nongkhorensis

Sriracha, Chonburi

20180

ZMKU AM 00620

KC357604

A,B

C. nongkhorensis

Sriracha, Chonburi

20181

ZMKU AM 00621

–

A

C. nongkhorensis

Sriracha, Chonburi

20182

ZMKU AM 00622

–

A

C. nongkhorensis

Sriracha, Chonburi

20183

ZMKU AM 00623

KC357609

A,B

C. nongkhorensis

Sriracha, Chonburi

20184

ZMKU AM 00624

KC357610

A,B

C. nongkhorensis

Sriracha, Chonburi

20185

ZMKU AM 00625

–

A

C. nongkhorensis

Sriracha, Chonburi

20186

ZMKU AM 00626

–

A

C. nongkhorensis

Sriracha, Chonburi

20187

ZMKU AM 00627

–

A

Species

C. nongkhorensis

Sriracha, Chonburi

20188

ZMKU AM 00628

–

A

C. nongkhorensis

Wang Nam Khieo, Nakhon Ratchasima

20117

ZMKU AM 00610

–

A

C. nongkhorensis


20118

ZMKU AM 00611

–

A

C. nongkhorensis


20282

ZMKU AM 00658

–

A

C. nongkhorensis


20283

ZMKU AM 00659

–

A

C. nongkhorensis


20284

ZMKU AM 00660

–

A

C. nongkhorensis


20285

ZMKU AM 00661

–

A

C. nongkhorensis


20286

ZMKU AM 00662

–

A

C. nongkhorensis


AA00438

ZMKU AM 00719

–

A

C. nongkhorensis


AA00472

ZMKU AM 00734

–

A

C. nongkhorensis


AA00473

ZMKU AM 00735

–

A

C. nongkhorensis


AA00474

ZMKU AM 00736

–

A

C. nongkhorensis


AA00475

ZMKU AM 00737

–

A

C. nongkhorensis


AA00476

ZMKU AM 00738

–

A

C. nongkhorensis


AA00478

ZMKU AM 00740

–

A

C. nongkhorensis


AA00479

ZMKU AM 00741

–

A

C. nongkhorensis


AA00481

ZMKU AM 00743

–

A

C. nongkhorensis


AA00482

ZMKU AM 00744

–

A

C. nongkhorensis


AA00599

ZMKU AM 00764

KC357599

A,B

C. nongkhorensis


AR00078

ZMKU AM 00970

KC357600

A,B

C. nongkhorensis


AR00084

ZMKU AM 00973

–

A

C. nongkhorensis


AR00087

ZMKU AM 00976

–

A

C. nongkhorensis


AR00120

ZMKU AM 00982

KC357601

A,B

C. nongkhorensis


20367

ZMKU AM 00684

–

A

C. nongkhorensis


AA01076

ZMKU AM 00813

KC357597

A,B

C. nongkhorensis


AA01097

ZMKU AM 00834

KC357598

A,B

C. hansenae Group I

Sriracha, Chonburi

20179

ZMKU AM 00619

KC357638

B

C. hansenae Group I

Kang Hang Meaw, Chantaburi

20193

ZMKU AM 00633

KC357639

B

C. hansenae Group I


20194

ZMKU AM 00634

KC357641

B

C. hansenae Group I


AA00415

ZMKU AM 00712

KC357647

B

C. hansenae Group I

Sriracha, Chonburi

AA00387

ZMKU AM 00707

KC357642

B

C. hansenae Group I

Sriracha, Chonburi

AA00392

ZMKU AM 00710

KC357644

B

C. hansenae Group I

Sriracha, Chonburi

AA00393

ZMKU AM 00711

KC357643

A,B

C. hansenae Group I

Sriracha, Chonburi

AA00421

ZMKU AM 00718

KC357645

B

C. hansenae Group I

Sriracha, Chonburi

AA00586

ZMKU AM 00763

KC357646

B



AOWPHOL ET AL.


Field no.

Museum No.

GenBank

Type of analysis

C. hansenae Group I

Sriracha, Chonburi

AA01195

ZMKU AM 00870

KC357650

A,B

C. hansenae Group I

Sriracha, Chonburi

AA01197

ZMKU AM 00872

KC357632

A,B

C. hansenae Group I

Sriracha, Chonburi

AA01198

ZMKU AM 00873

KC357651

A,B

C. hansenae Group I

Sriracha, Chonburi

AA01199

ZMKU AM 00874

KC357640

B

C. hansenae Group I

Sriracha, Chonburi

AA01200

ZMKU AM 00875

KC357652

B

Species

C. hansenae Group I

Sriracha, Chonburi

AA01204

ZMKU AM 00876

KC357648

B

C. hansenae Group I

Sriracha, Chonburi

AA01205

ZMKU AM 00877

KC357649

B

C. hansenae Group I


20120

ZMKU AM 00613

–

A

C. hansenae Group I


20121

ZMKU AM 00614

–

A

C. hansenae Group I


20122

ZMKU AM 00615

–

A

C. hansenae Group I


20123

ZMKU AM 00616

–

A

C. hansenae Group I


20124

ZMKU AM 00617

–

A

C. hansenae Group I


20125

ZMKU AM 00618

–

A

C. hansenae Group I


AA00324

ZMKU AM 00689

–

A

C. hansenae Group I


AA00460

ZMKU AM 00722

–

A

C. hansenae Group I


AA00461

ZMKU AM 00723

–

A

C. hansenae Group I


AA00462

ZMKU AM 00724

–

A

C. hansenae Group I


AA00463

ZMKU AM 00725

–

A

C. hansenae Group I


AA00465

ZMKU AM 00727

–

A

C. hansenae Group I


AA00468

ZMKU AM 00730

–

A

C. hansenae Group I


AA00471

ZMKU AM 00733

–

A

C. hansenae Group I


AR00079

ZMKU AM 00971

KC357631

A,B

C. hansenae Group I


AR00083

ZMKU AM 00972

KC357637

A,B

C. hansenae Group I


AR00085

ZMKU AM 00974

KC357635

A,B

C. hansenae Group I


AR00086

ZMKU AM 00975

KC357634

A,B

C. hansenae Group I


AR00088

ZMKU AM 00977

KC357633

A,B

C. hansenae Group I

Phu Ruea, Loei

AR00044

ZMKU AM 00966

–

A

C. hansenae Group I

Phu Ruea, Loei

AR00046

ZMKU AM 00968

KC357636

B



20368

ZMKU AM 00685

–

A



AA01062

ZMKU AM 00799

KC357661

B



AA01063

ZMKU AM 00800

KC357662

A,B



AA01064

ZMKU AM 00801

–

A



AA01065

ZMKU AM 00802

KC357658

A,B



AA01066

ZMKU AM 00803

KC357664

A,B



AA01067

ZMKU AM 00804

–

A



AA01068

ZMKU AM 00805

KC357660

A,B



AA01069

ZMKU AM 00806

KC357659

A,B



AA01081

ZMKU AM 00818

KC357665

A,B



AA01082

ZMKU AM 00819

KC357668

A,B



AA01091

ZMKU AM 00828

KC357663

A,B



AA01094

ZMKU AM 00831

KC357657

B



AA01095

ZMKU AM 00832

KC357669

B



AR00167

ZMKU AM 00985

KC357667

A,B



AR00168

ZMKU AM 00986

KC357666

A,B

C. hansenae Group I

Ban Ta Khun, Surat Thani

AA00767

ZMKU AM 00781

KC357625

A,B

C. hansenae Group I


AA00768

ZMKU AM 00782

KC357628

B




107


Field no.

Museum No.

GenBank

Type of analysis

C. hansenae Group I


AA00769

ZMKU AM 00783

KC357627

A,B

C. hansenae Group I


AA00770

ZMKU AM 00784

KC357626

A,B

C. hansenae Group I


AA00795

ZMKU AM 00785

KC357630

B

C. hansenae Group I


AA00796

ZMKU AM 00786

KC357629

B


Mueang, Mae Hong Son

20310

ZMKU AM 00668

KC357654

A,B



20312

ZMKU AM 00670

KC357653

A,B



20313

ZMKU AM 00671

KC357655

A,B

Species



20314

ZMKU AM 00672

KC357656

A,B



20315

ZMKU AM 00673

–

A



20316

ZMKU AM 00674

–

A



20317

ZMKU AM 00675

–

A



20318

ZMKU AM 00676

–

A



20319

ZMKU AM 00677

–

A



20320

ZMKU AM 00678

–

A



20322

ZMKU AM 00680

–

A



20324

ZMKU AM 00682

–

A



20328

ZMKU AM 00683

–

A



AA00544

ZMKU AM 00749

KC357621

A,B



AA00545

ZMKU AM 00750

–

A



AA00553

ZMKU AM 00758

–

A



AA00554

ZMKU AM 00759

–

A



AR00110

ZMKU AM 00978

KC357623

A,B



AR00111

ZMKU AM 00979

KC357624

A,B



AR00112

ZMKU AM 00980

KC357622

A,B



AR00113

ZMKU AM 00981

KC357620

A,B

C. vittatus Group I

Putao District, Kachin State, Myanmar

–

CAS 221212

KC692874

B

C. vittatus Group I

Putao District, Kachin State, Myanmar

–

CAS 221213

KC692876

B


Myitkyina District, Kachin State, Myanmar

–

KC692882

B

Myitkyina District, Kachin State, Myanmar

–

KC692881

B

Myitkyina District ,Kachin State, Myanmar

–

KC692882

B

C. vittatus Group I

Putao District ,Kachin State, Myanmar

–

KC692875

B


Dewei District ,Tanintharyi Division, Myanmar

–

KC692878

B


–

KC692879

B


–

KC692877

B


–

KC692880

B

C. vittatus Group II C. vittatus Group II

C. vittatus Group II C. vittatus Group II C. vittatus Group II


CAS 241113 CAS 241274 CAS 241280 CAS 244065 CAS 245724 CAS 245915 CAS 245916 CAS 245922

AOWPHOL ET AL.

TABLE 3. Mitochondrial DNA sequences (16S rRNA) downloaded from GenBank and used in molecular analysis. Species

Locality

Accession no.

Reference

Chiromantis doriae

Huaphahn, Lao PDR

DQ283135

Frost et al. (2006)

C. doriae

Huaphahn, Lao PDR

GQ204721

Meegaskumbura et al. (2010)

C. doriae

Simao, Yunnan, China

GQ285682

Li et al. (2009)

C. doriae

China

EF564517

Yu et al. (2008)

C. doriae

Hainan, China

EU215527

Li et al. (2008)

C. doriae

Binh Thuan, Vietnam

GQ285683

Li et al. (2009)

C. nongkhorensis

Champasak, Laos

GQ204723


C. vittatus

Gia Lai, Vietnam

DQ283134

Frost et al. (2006)

C. vittatus

Huaphahn, Lao PDR

GQ204722


C. vittatus

Simao, Yunnan, China

EF564519

Yu et al. (2008)

C. vittatus

Simao,Yunnan, China

GQ285684

Li et al. (2009)

C. xerampelina

Africa

GQ204734


C. xerampelina

Africa

AY880495

Delorme (2004)

C. rufensens

Africa

AY880494

Delorme (2004)

Feihyla palpebralis

Mt. Dawei, Yunnan, China

EU215546

Li et al. (2008)

Ha Tinh,Vietnam

AY843750

Faivovich et al. (2005)

Chin State, Myanmar

JX219444

Li et al. (2012)

Xishuangbanna, Yunnan, China

EU215532

Li et al. (2008)

Rhacophorus bipunctatus R. bipunctatus R. kio Polypedates cruciger

Sri Langka

GQ204692


P. leucomystax

Depok, Java, Indonesia

AB564288

Kuraishi et al. (2011)

P. leucomystax

Huidong, Guangdong, China

EU215550

Li et al. (2008)

Hong Kong, China

AB564284

Kuraishi et al. (2011)

Japan

AY880444

Delorme et al. (2004)

P. megacephalus Buergeria buergeri

TABLE 4. Estimates of evolutionary divergence based on uncorrected mean pairwise distances between and within groups (shown in grey) using 16S rRNA data. GROUP ID

#

1

C. doriae

1

0.019

C. nongkhorensis

2

0.045

2

3

4

5

6

7

8

0.006

C. vittatus Group I

3

0.161

0.150

0.034


4

0.163

0.149

0.085

0.007

C. hansenae Group I

5

0.175

0.159

0.088

0.106

0.012


6

0.184

0.170

0.087

0.115

0.047

0.012

Non-Thai Chiromantis

7

0.106

0.108

0.158

0.168

0.173

0.172

0.092

Outgroups

8

0.145

0.135

0.184

0.176

0.189

0.192

0.149

0.113

Morphological analyses Morphological measurements (to nearest 0.1 mm) of adult specimens (determined by gonadal examination) were made with Mitutoyo digital calipers and an Olympus SZ40 dissecting microscope. Only specimens collected in Thailand were used for morphological analyses. Abbreviations follow those of Grismer et al. (2007): SVL = snout-vent length, measured from tip of snout to vent; HL = head length, measured from posterior margin of lower jaw to tip of snout; HW = head width, measured at posterior to eyes; ELW = width of upper eyelid, measured from medial base of upper eyelid to lateral edge at its widest point; ED = eye diameter, measured distance between anterior and posterior corners of upper and lower eyelids; IND = internarial distance, measured between nostrils; IOD = interorbital distance, measured across top of head between medial margins of orbits at their closest points; SNL = snout length, measured from anterior corner of eye to tip of snout; DNE = distance from nostril to eye,



109

measured from anterior corner of eye to posterior edge of nostril; TD = tympanum diameter, measured as horizontal width of tympanum at its widest point; FLL = forelimb length, measured from elbow to tip of third finger; HLT = hand length, measured from proximal edge of palmar tubercle to tip of third finger; THL = thigh length, measured from center of knee to center of vent; TIL = tibia length, measured from center of knee to center of ankle; FL = Foot length, measured from proximal edge of inner metatarsal tubercle to tip of fourth toe; 3FDW = width of disk of third finger; 4TDW = width of disk of fourth toe. Individuals were sexed by examination of gonads and external morphology. Males with large, developed testes and females with convoluted oviduct or developed eggs were considered mature. Only a few females were collected and sex bias in body size was detected. Therefore, only adult males were used in the morphometric analyses.

FIGURE 2. Color and body pattern in 70 % ethanol. (A) Chiromantis doriae (AR00043; Loei); (B) C. nongkhorensis (AR00078; Nakhon Ratchasima); (C) C. hansenae Group I (AA00421; Chonburi); (D) C.vittatus Group II (AR00110; Sangkhla Buri, Kanchanaburi). Scale bar = 5 mm.

Morphological statistical analyses Normality of the data was determined using the Shapiro-Wilk’s W-test (Zar 1984). When non-normally distributed, data were log-transformed in order to meet the assumption of normality more closely and sizecorrected values were used in subsequent multivariate analyses. Table 5 shows the mean and standard deviation of SVL and the ratios of each character to SVL (expressed in percentage ratios) for each Chiromatis species. For


AOWPHOL ET AL.

comparison of SVL, we analyzed untransformed SVL data of C. vittatus and C. hansenae using Kruskal-Wallis test (Zar 1984). To investigate morphological differentiation among groups, principal component analysis (PCA) was performed. Discriminant function analysis (DFA) was conducted to calculate the probability of correct classification of samples to their original group. The analysis was based on a stepwise discriminant model. In addition to quantitative morphological measurements, some qualitative characters (i.e., dorsal pattern and tympanum character) were also recorded, but were not used in the analyses. Statistical analyses were performed in Stata (Release 12, StataCorp, College Station, TX, USA), and XLStat-Pro (Addinsoft 2013). TABLE 5. Morphological measurement of specimens examined in this study. Comparison of snout-vent length (SVL; in mm) and percentage ratio of each morphological character to SVL are shown as mean and standard deviation, followed by the range in parentheses. C. doriae (n=20)

C. nongkhorensis (n=57)

C. vittatus Group II (n=8)

C. hansenae Group I (n=27)

C. hansenae Group II (n=24)

SVL

24.5±1.4 (21.8–26.5)

28.5±1.3 (25.6–31.1)

24.7±0.4 (24.0–25.2)

21.3±0.7 (19.1–22.8)

22.3±1.3 (20.4–25.5)

HL/SVL

33.7±2.0 (30.6–39.0)

31.7±1.3 (28.3–35.3)

32.6±1.7 (30.5–35.1)

34.1±1.1 (32.2–36.2)

32.7±1.1 (30.3–35.5)

HW/SVL

31.8±1.1 (30.1–33.9)

31.2±1.1 (28.3–35.6)

29.6±1.2 (27.5–31.3)

29.8±0.8 (27.7–31.7)

29.9±0.9 (26.4–30.7)

ELW/SVL

9.4±0.8 (8.0–11.4)

9.4±0.8 (7.7–11.3)

7.5±0.6 (6.4–8.4)

7.6±1.4 (4.9–9.7)

7.3±1.2 (4.9–10.5)

ED/SVL

12.2±0.8 (10.6–13.3)

12.2±0.8 (10.6–14.3)

10.5±0.5 (9.7–11.4)

11.8±0.8 (9.1–13.1)

11.4±1.1 (9.7–14.5)

IND/SVL

9.9±0.5 (8.8–11.1)

9.4±1.4 (7.9–11.8)

10.0±0.4 (9.3–10.8)

10.2±0.6 (9.2–12.0)

10.1±0.6 (7.8–11.4)

IOD/SVL

20.0±1.0 (18.1–22.1)

19.7±1.4 (11.3–22.1)

19.8±1.0 (18.5–21.6)

20.5±1.0 (18.7–22.8)

18.9±1.3 (16.2–21.6)

SNL/SVL

15.5±0.8 (13.8–16.8)

13.7±0.9 (11.8–16.2)

15.7±0.8 (14.8–17.0)

16.1±0.8 (14.2–17.5)

15.6±0.7 (14.1–17.0)

DNE/SVL

8.0±0.5 (7.1–9.1)

7.6±0.5 (6.2–9.1)

8.4±0.5 (7.7–9.3)

8.2±0.6 (6.8–9.8)

8.2±0.6 (7.3–10.3)

TD/SVL

6.7±0.5 (5.7–8.3)

7.1±0.5 (5.9–8.3)

*

*

*

FLL/SVL

44.8±2.2 (40.3–49.0)

45.1±2.0 (41.4–50.3)

45.9±2.2 (42.6–48.7)

42.8±1.7 (38.4–45.5)

44.6±2.7 (40.4–53.5)

HLT/SVL

28.3±0.7 (27.0–29.6)

28.5±1.3 (26.9–32.7)

30.7±1.9 (27.9–34.1)

27.6±1.5 (24.4–31.6)

28.3±2.0 (20.4–31.4)

THL/SVL

50.1±1.9 (45.5–54.2)

49.5±2.4 (42.0–55.3)

48.9±4.8 (38.0–53.1)

48.8±2.0 (45.8–53.5)

48.7±2.2 (43.0–53.0)

TIL/SVL

51.9±1.4 (49.3–54.7)

50.8±1.9 (46.4–54.8)

48.8±3.4 (43.5–53.8)

48.2±2.2 (44.5–52.3)

49.1±1.8 (44.1–53.1)

FL/SVL

41.2±1.6 (38.8-–44.2)

41.4±2.1 (37.4–47.1)

44.4±2.1 (40.7–47.5)

41.6±2.4 (37.9–47.9)

42.5±2.0 (38.1–47.9)

3FDW/SVL

5.2±0.4 (4.3–6.1)

5.1±0.5 (3.8–6.0)

5.1±0.2 (4.6–5.5)

4.6±0.4 (3.4–5.6)

4.8±0.5 (3.0–5.9)

4TDW/SVL

4.2±0.4 (4.2–5.7)

4.6±0.5 (3.5–5.7)

4.7±0.3 (4.4–5.2)

4.2±0.5 (3.0–5.2)

4.2±0.4 (3.2–4.9)

* Percentages of the ratio of TD/SVL were not shown for C. vittatus Group II, C. hansenae Groups I and II because this character was indistinct for some individuals.



111

FIGURE 3. Phylogram of 16S rRNA gene for samples of Chiromantis and its related species. Nodal Numbers above the branches represent Bayesian posterior probability support values and numbers below the branches represent ML bootstrap support. The clade colors refer to Figure 1 map.


AOWPHOL ET AL.

Call recording Calls of Chiromantis species were recorded in the field between May 2011 and February 2012. We recorded calls for at least 5 minutes per individual. Recordings were made using a R09 Edirol recorder with a tripod mounted Røde NTG-2 condenser shotgun microphone (44.1 kHz sampling rate and 24-bit encoding) at a distance of approximately 30–50 cm. Air temperature near the frog was recorded during each call recording. Bioacoustic analyses Audiospectrograms and oscillograms were quantified with Raven Software 1.4 (Cornell Laboratory of Ornithology, Ithaca, NY, USA). Audiospectrograms were calculated with a fast-Fourier transform (FFT) and 50% overlap using Hanning windows function with 256 band-resolution. Mean and standard deviation of call properties of each species were computed. Due to the diversity of call structures among amphibian species, call terminology in this study follows the definitions of Cocroft and Ryan (1995), and Meegaskumbura and Manamendra-Arachchi (2005). Six call properties were compared among species: Call length (ms), number of pulses per call, pulse length (ms), Inter-pulse duration (ms), pulse rate (pulses/s) and dominant frequency (kHz).

FIGURE 4. Discriminant function analysis depicting morphological differentiation within the C. vittatus-hansenae group. Close grey circle indicates C. vittatus Group II. Open circle denotes C. hansenae Group I. Closed black circle represents C. hansenae Group II.



113

Results Molecular analyses Based on our analyses, two samples identified on GenBank as Chiromantis doriae (GQ285683) and C. vittatus (DQ283134), both from Vietnam were found to consistently align with topotypic C. nongkhorensis and C. hansenae (from Thailand), respectively. Without the opportunity to observe the voucher specimens and confirm their identities we excluded them from further analysis. Two well-supported clades containing the focal taxa were recovered in both Maximum likelihood analyses and Bayesian Inference (Fig. 3). Clade A comprises Feihyla palpebralis and two sub-clades of specimens genetically allied to Chiromantis vittatus and C. hansenae. Each sub-clade comprises two groups: C. vittatus Group I includes specimens from Putao (Kachin, Myanmar), Lao PDR and China; C. vittatus Group II contains specimens from Sangkhla Buri (Thailand), Myitkyina (Kachin, Myanmar) and Dewei (Tanintharyi, Myanmar); C. hansenae Group I contains specimens from central, eastern, and peninsular Thailand, and C. hansenae Group II from northwestern Thailand. Clade B comprises C. nongkhorensis from Thailand, and Lao PDR; C. doriae from Thailand, China and Lao PDR; C. xerampelina and C. rufescens from Africa. Well-supported sister relationships between C. hansenae and C. vittatus and between C. doriae and C. nongkhorensis were recovered in both analyses. Feihyla palpebralis is the sister taxon to the clade containing C. hansenae and C. vittatus. In the ML analysis, species of Chiromantis from Africa (C. xerampelina and C. rufescens) comprise a weakly supported monophyletic clade (69% BSS) that is most closely related to the clade containing C. doriae and C. nongkhorensis. In both analyses monophyletic Rhacophorus (R. bipunctatus and R. kio) and Polypedates (P. cruciger, P. leucomystax and P. megacephalus) clades were recovered but the relationship of these groups to each other and to Clades A and B is unresolved because of weak support. Comparisons of within group uncorrected mean pair-wise distance data is quite low among the four focal taxa: C. nongkhorensis (0.6%), C. doriae (1.9%), C. vittatus Group I (3.4 %), C. vittatus Group II (0.7 %), C. hansenae Group I (1.2%), C. hansenae Group II (1.2%). In contrast, between group distances are considerably higher (8.5– 18.4%) with the exception of the C. nongkhorensis-C. doriae pairing (4.5%). Between group means for C. vittatus (Groups I and II) was 8.5% and C. hansenae (Groups I and II) was 4.7% (Table 4).

Morphological comparison Groups used for morphological comparisons correspond to clades identified in molecular analyses (C. doriae, C. nongkhorensis, C. vittatus Group II and C. hansenae Groups I and II). Individuals comprising C. vittatus Group I were represented only by molecular data and thus were not included in the morphological analyses. In some individuals of C. vittatus Group II and C. hansenae Groups I and II, the tympanum (tympanic annulus) was indistinct as reported by Wilkinson et al. (2003); therefore the TD character was excluded from statistical analysis. Table 5 shows the mean, standard deviation and ranges for the morphological variables. Results from MannWhitney U test of the ratio variables indicated significant differences between C. vittatus Group II and C. hansenae in ED, FLL, HTL, FL, FWD3 and TDW4 (P