ARTICLE IN PRESS Organisms, Diversity & Evolution 8 (2008) 267–276 www.elsevier.de/ode Phylogenetic position of the montane treefrog Polypedates variabilis Jerdon, 1853 (Anura: Rhacophoridae), and description of a related species S.D. Bijua,b, Kim Roelantsb, Franky Bossuytb,Ã aCentre for Environmental Management of Degraded Ecosystems, School of Environmental Studies, University of Delhi, Delhi 110007, India bBiology Department, Unit of Ecology & Systematics, Vrije University Brussel (VUB), Pleinlaan 2, 1050 Brussels, Belgium Received 3 October 2006; accepted 29 November 2007 Abstract Since its original description, the Indian treefrog species Polypedates variabilis Jerdon, 1853 has been assigned variously to one of the widespread genera Polypedates Tschudi, 1832, Rhacophorus Kuhl & van Hasselt, 1822, and Philautus (Kirtixalus) Dubois, 1987. Here we present phylogenetic analyses based on 1.4 kb of mitochondrial DNA showing that P. variabilis and a previously undescribed relative are not nested within any of those genera, but stem from a lineage that originated relatively early in the rhacophorid radiation. We propose the name Ghatixalus gen. n. for this lineage, whose known members are restricted to high altitudes in the Western Ghats of India. The sister species of G. variabilis (Jerdon), comb. n. is described as Ghatixalus asterops sp. n. The morphological and ecological features of both species are discussed. r 2008 Gesellschaft fu¨r Biologische Systematik. Published by Elsevier GmbH. All rights reserved. Keywords: Biodiversity; India; Molecular phylogenetics; New genus; New species; Old world treefrogs Introduction The generic allocation of many rhacophorid species is complicated by the poor definition of the recognized The Rhacophoridae constitute a radiation of over genera, which generally lack distinct morphological 270 treefrog species (Frost 2006), whose divergence from synapomorphies or show a high degree of homoplasy. the Madagascan Mantellidae has been associated with A particularly problematic species is Polypedates the breakup of Gondwana (Bossuyt and Milinkovitch variabilis Jerdon, 1853, described from the Nilgiri Hills 2001; Van Bocxlaer et al. 2006). Primary centres of in the Indian Western Ghats. Since its original descrip- rhacophorid diversity are now located in Southeast Asia tion, P. variabilis (or its junior synonym, P. pleurostictus and the Indian subcontinent (Inger 1999), the latter Gu¨nther, 1864) has been assigned variously to Rhaco- centre being characterised by remarkable species phorus (e.g. Inger and Dutta 1986; Daniel and Sekar abundance and endemism (Dutta 1997; Biju 2001; 1989; Dutta 1997; Bossuyt and Dubois 2001), to Kuramoto and Joshy 2003; Biju and Bossuyt 2003, Philautus (Kirtixalus)(Dubois 1987)ortoPolypedates 2005a–c; Das and Kunte 2005; Meegaskumbura and (Ravichandran 1997; Biju 2001). This instability was due Manamendra-Arachchi 2005; Das and Dutta 2006). to the fact that none of those assignments is supported by convincing morphological or ecological characters. ÃCorresponding author. To clarify the evolutionary position of Polypedates E-mail address: [email protected] (F. Bossuyt). variabilis and an undescribed relative, we performed 1439-6092/$ - see front matter r 2008 Gesellschaft fu¨r Biologische Systematik. Published by Elsevier GmbH. All rights reserved. doi:10.1016/j.ode.2007.11.004 ARTICLE IN PRESS 268 S.D. Biju et al. / Organisms, Diversity & Evolution 8 (2008) 267–276 molecular phylogenetic analyses of approximately for among-site rate heterogeneity and an estimated 1.4 kilobases (kb) of mitochondrial DNA from both proportion of invariable sites. Clade confidence was species, as well as from representatives of the major assessed by non-parametric bootstrap analyses, under rhacophorid lineages. MP using PAUP* and under ML using PHYML 2.4.4 (Guindon and Gascuel 2003), with 1000 sampling replicates in both cases. Material and methods Morphology Field survey and specimen collection Measurements (in mm) and terminology follow Ecological surveys and collection of specimens were Bossuyt and Dubois (2001). performed during field trips in the Western Ghats The following measurements were taken to the nearest between 1997 and 2000. Specimens were preserved 0.1 mm, using a digital slide-calliper or a binocular in 5% formaldehyde for 2 days, then transferred to microscope with a micrometre ocular: EL=eye length, 70% ethanol. Samples for molecular analyses were horizontal distance between bony orbital borders of eye; taken from muscle tissue, preserved in 100% ethanol EN=eye to nostril distance, i.e. from nostril to anterior and stored at –20 1C. orbital border of eye; FDI–IV=disk width on fingers I–IV; FFTF=distance from maximum incurvation of Phylogenetics web between fourth and fifth toe to tip of fourth toe; FLL=forelimb length, from elbow to base of outer For phylogenetic inference, we assembled a mito- palmar tubercle; FOL=foot length, from base of inner chondrial DNA matrix of approximately 2000 base metatarsal tubercle to tip of fourth toe; FTL=length of pairs (bp), covering part of the 12S RNA gene, the fourth toe, from base of first subarticular tubercle to tip Val complete tRNA gene, and part of the 16S RNA gene. of fourth toe; FWI–IV=width of fingers I–IV, at base of The relevant sequences were compiled for Polypedates disk; HAL=hand length, from base of outer palmar variabilis, and 32 other rhacophorid ingroup species tubercle to tip of third finger; HL=head length, from (Appendix A). This taxon set comprises all genera rear of mandible to tip of snout; HW=head width, at recognized in Dubois (2005), and includes 11 type angle of jaw; IBE=internal back of eyes, shortest species. Because the phylogenetic position of Polype- distance between posterior orbital borders of eyes; dates variabilis is particularly relevant with respect to IFE=internal front of eyes, shortest distance between Polypedates and Rhacophorus, these genera are repre- anterior orbital borders of eyes; IMT=inner metatarsal sented by an increased number of taxa (five and nine tubercle length; IN=internarial distance, i.e. between species, respectively). Five species from families closely internal borders of nostrils; ITL=inner toe length; related to Rhacophoridae served as outgroup taxa. For IUE=inter upper eyelid width, the shortest distance most species, DNA sequences were retrieved from between the upper eyelids; MBE=distance from rear of previous studies (Richards and Moore 1998; Meegas- mandible to posterior orbital border of eye; MFE= kumbura et al. 2002; Wilkinson et al. 2002); others were distance from rear of mandible to anterior orbital newly obtained by whole-genome extraction (Sambrook border of eye; MN=distance from rear of mandible to et al. 1989), PCR amplification, and cycle sequencing nostril; MTFF=distance from distal edge of metatarsal along both strands. The new sequences are deposited in tubercle to maximum incurvation of web between fourth GenBank under accession numbers EU178086– and fifth toe; MTTF=distance from distal edge of EU178099. Alignments were created using the program metatarsal tubercle to maximum incurvation of web ClustalX 1.81 (Thompson et al. 1997); minor corrections between third and fourth toe; NS=distance from nostril were made with MacClade 4.06 (Maddison and to tip of snout; ShL=shank length; ShW=maximum Maddison 2000). shank width; SL=snout length, from tip of snout to Phylogenetic relationships were estimated using anterior orbital border of eye; SVL=snout-vent length; heuristic maximum-parsimony (MP) and maximum- TDI–V=disk width on toes I–V; TFL=third finger likelihood (ML) searches, both executed with the length, from base of first subarticular tubercle; program PAUP* 4.0b10 (Swofford 2002). The MP TFOL=distance from heel to tip of fourth analysis involved equal character weighting, 1000 toe; TFTF=distance from maximum incurvation of replicates of random taxon addition and tree-bisection- web between third and fourth toe to tip of fourth toe; reconnection branch swapping. The ML search included TL=thigh length; TWI–V=width of toes I–V, at base of 10 replicates of random taxon addition and was disk; TYD=largest tympanum diameter; TYE=tympanum performed using a general time-reversible (GTR) to eye distance, i.e. from posterior orbital border of eye to model of DNA evolution, with gamma correction tympanum; UEW=maximum upper eyelid width. ARTICLE IN PRESS S.D. Biju et al. / Organisms, Diversity & Evolution 8 (2008) 267–276 269 Live colouration was recorded for individual category of genus, though artificial, should reflect animals within 1 h after collection. Drawings of evolutionary history and be ‘‘a monophyletic group the holotype were made using a stereomicro- composed of one or more species separated from other scope with camera lucida. Museum abbreviations: generic taxa by a decided gap’’ (Mayr and Ashlock 1991). BNHS=Bombay Natural History Society, Bombay, Although we cannot reject a sister-clade relationship to Maharashtra, India; IRSNB=Institut Royal des Polypedates or Rhacophorus, our analyses indicate that Sciences Naturelles de Belgique, Brussels, Belgium; Polypedates variabilis and its relative form a distinct clade TNHC=Texas Natural History Collections, University that is not nested among species currently allocated to of Texas, Austin, USA; VUB=Vrije Universiteit these genera. Recognition of a new genus for species that Brussel, Brussels, Belgium. are morphologically and phylogenetically distinct, though subjective, is consistent with the generic status