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A NEW CRYPTIC SPECIES of BUSH FROG (AMPHIBIA: ANURA: Raorchestes) from the SOUTHERN EASTERN GHATS, INDIA

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A NEW CRYPTIC SPECIES of BUSH FROG (AMPHIBIA: ANURA: Raorchestes) from the SOUTHERN EASTERN GHATS, INDIA MAJOR ARTICLE TAPROBANICA, ISSN 1800–427X. November, 2020. Vol. 09, No. 02: pp. 164–173, pl. 49–50. © Research Center for Climate Change and Department of Biology, Faculty of Mathematics & Natural Sciences, University of Indonesia, Depok 16424, INDONESIA. http://www.taprobanica.org/ https://doi.org/10.47605/tapro.v9i2.229 urn:lsid:zoobank.org:pub:A1E14F8A-48AB-4F0A-B2EC-FB2850BF42F3 A NEW CRYPTIC SPECIES OF BUSH FROG (AMPHIBIA: ANURA: Raorchestes) FROM THE SOUTHERN EASTERN GHATS, INDIA Section Editor: Suranjan Karunarathna Submitted: 30 April 2020, Accepted: 31 October 2020 Gaurang G. Gowande1,2, S.R. Ganesh3 & Zeeshan A. Mirza4 1 Department of Biodiversity, Abasaheb Garware College, Pune 411004, Maharashtra, India 2 Department of Biotechnology, Fergusson College, Pune 411004, Maharashtra, India 3 Chennai Snake Park, Guindy, Chennai 600022, Tamil Nadu, India 4 National Centre for Biological Sciences, TIFR, Bengaluru 560065, Karnataka, India 4 Corresponding author. E-mail: [email protected] Abstract A new cryptic lineage of bush frogs of the genus Raorchestes from the understudied Eastern Ghats of India is described. Molecular phylogenetic reconstructions using mitochondrial 16S rRNA gene sequences suggest that the new species belongs to the Raorchestes charius clade, is sister to the allopatric R. charius of the Western Ghats and support the species-level distinctiveness of the lineage. The new species morphologically resembles R. charius from which it can be distinguished using the following characters: narrower head (HW/SVL 0.31 vs 0.34–0.36), wider inter upper eyelid (IUE/SVL 0.14–0.16 vs 0.11–0.13), greater maximum upper eyelid width (UEW/SVL 0.10–0.12 vs 0.06–0.09) and shorter thigh (TL/SVL 0.32–0.43 vs 0.44–0.51). Acoustic observations from the breeding males further corroborate the distinct specific status of the new species. The discovery of this new species of bush frog from an understudied landscape suggests that additional extensive surveys in the Eastern Ghats would yield several more amphibian lineages yet unknown to science. Key words: arboreal frog, cryptic species, Kollimalai Massif, Philautus, Rhachophoridae, systematics Introduction 2010). Currently, more than 50 species of Bush Frogs, genus Raorchestes Biju, Shouche, Raorchestes are known from India, primarily Dubois et al., 2010, belong to the arboreal frog from the Western Ghats and north-east India. On family Rhachophoridae. These frogs were earlier the other hand, bush frogs of the drier Eastern placed in the genus Philautus Gistel, 1848, Ghats have received comparatively little however molecular phylogenetic reconstruction taxonomic attention. About five species of bush revealed the polyphyly in the genus (Biju et al. frogs were previously described from the 2010, Vijayakumar et al. 2014). The Peninsular Eastern Ghats: Philautus sanctisilvaticus Das & Indian and Sri Lankan representatives were Chanda, 1998; P. terebrans Das & Chanda, consequently shifted to the genera Raorchestes 1998; P. similipalensis Dutta, 2003; R. cf. and Pseudophilautus Laurent, 1943 (Biju et al. leucolatus Vijaykumar, Dinesh, Prabhu et al., 164 TAPROBANICA VOL. 09: NO. 02 A NEW BUSH FROG (Raorchestes) FROM THE EASTERN GHATS, INDIA 2014 (Ganesh & Arumugam 2016) and males, from Chikmagalur, Karnataka. Pseudophilautus cf. wynaadensis (Jerdon, 1853). Acoustic data and analysis: Calls of a However, Mirza et al. (2019) showed that the breeding male were recorded in the field using first three species also belong to the genus the voice recorders loaded on an android mobile Raorchestes and represent not three, but a single phone, and were analyzed later using RavenPro species, R. sanctisilvaticus. The taxonomic v1.5. Range, mean and standard deviation were status of the population inhabiting the Kolli calculated from five individual notes of a call. Hills of R. cf. leucolatus and P. cf. wynaadensis Calls were compared with the calls of breeding in the Eastern Ghats remains unstudied. During males of other species that showed genetic a recent survey of the herpetofauna in the affinity. Published acoustic data of R. charius Kollimalai Massif, southern Eastern Ghats, we were used to make these comparisons (Priti et al. encountered and collected bush frog specimens 2016). of the genus Raorchestes. We tentatively Molecular analysis: Complete genomic identified the specimens as R. charius, due to DNA was isolated from the preserved tissues the overall morphological similarity. Ganesh & using Qiagen DNAeasy kits following protocols Arumugam (2016) identified this population as directed by the manufacturer. A fragment of the Raorchestes cf. leucolatus. However, detailed mitochondrial 16S rRNA gene was amplified morphological, molecular and acoustic analyses using the primers 16SarL and 16Sbr (Yu et al. revealed that these specimens represent an 2009). A 12µl reaction was set containing 5µl of undescribed, cryptic species of the R. charius QiagenTaq PCR Master Mix, 4µl of water, 0.5µl clade. Here we describe and present a detailed of each primer and 2µl template DNA, carried morphological, molecular and acoustic account out with an Eppendorf Mastercycler Nexus to diagnose the new species and distinguish it GSX1. Thermo-cycle profile used for from its congeners. amplification were as follows: 94°C for 15 minutes, (denaturation temperature 94°C for 50 Material and Methods seconds, annealing temperature 45°C for 50 Fieldwork and specimen acquisition: One of us seconds, elongation temperature 72°C for 2 (SRG) undertook fieldwork throughout the Kolli minutes) x 30 cycles, 72°C for 15 minutes, hold Hills during 2011–2012 and observations on this at 4°C. PCR product was cleaned using species from those surveys are summarized in QIAquick PCR Purification Kit and sequenced Ganesh & Arumugam (2016). However, no with an AB 3730 DNA Analyzer. Sequences for specimens were collected during this period. In 16s rRNA gene of selected bush frog species October 2018, GG & ZM conducted a short were downloaded following (Vijayakumar et al. survey during which Raorchestes sp. specimens 2016). Downloaded sequences were aligned in were located in the field by the calls of the males MegaX (Kumar et al. 2018) using ClustalW at night. Specimens were collected by hand and (Thompson et al. 2003) with default settings. subsequently euthanized with Tricaine Maximum Likelihood method for describing the Methanesulfonate (MS 222) following standard phylogeny was implemented using W-IQ-TREE euthanasia protocols for amphibians. Thigh (Trifinopoulus et al. 2016), the web muscle tissue was collected and preserved in implementation of IQ-TREE (Nguyen et al. >95% ethanol for DNA extraction purposes. The 2015). Branch support was tested using ultrafast specimens were subsequently fixed in 4% bootstrap (UFboot) analysis (Minh et al. 2013), formaldehyde buffer for 24 hours, after which performing 1000 bootstrap alignments. TIM2e+I they were washed in water for five hours and model of sequence evolution was used for ML transferred to 70% ethanol solution for long- analysis, which was selected following the term preservation. Photographs were taken in model test run using ModelFinder preservative (70% ethanol) with a Canon 70D (Kalyaanamoorthy et al. 2017). For optimal illuminated with a Canon MT-24EX flash. partitioning strategy and evolutionary The specimens have been deposited at the substitution model for BI analysis, aligned data Bombay Natural History Society, Mumbai were analyzed using PartitionFinder v.1.1.1. (BNHS) and the collection facility of the (Lanfear et al. 2012). Bayesian Inference National Centre for Biological Sciences, (Appendix III) was implemented in MyBayes Bangalore (NCBS). Comparison of the new 3.2.2 (Ronquist & Huelsenbeck 2003) and was species was largely based on the topotypes of R. run for 10 million generations and sampled charius BNHS 4420 and BNHS 4421, two adult every 1000 generations, under GTR model of 165 TAPROBANICA VOL. 09: NO. 02 GOWANDE ET AL. 2020 sequence evolution. BI run included five parallel Results chains, three hot and tow cold chains. The Molecular phylogenetic reconstruction based on standard deviation of split frequencies of the the ~515bp long fragment of 16S rRNA analysis reached were below 0.01, and the mitochondrial gene (Fig. 1) suggests that the analysis was not continued further. Twenty-five new species belongs to the R. charius clade percent of trees generated were discarded as (Vijayakumar et al. 2014, Priti et al. 2016) and burn-in. Un-corrected p-distance (divergence) is a sister to R. charius with high support (ML was calculated in MegaX and a pairwise deletion 100, BI 1.0). The clade containing the new was adopted to deal with missing data and gaps. species and R. charius is a sister to R. Data for NADH dehydrogenase 1 (ND1) honnametti with high support (ML 100, BI 1.0). generated by Vijaykumar et al. (2014) and p- The intra-specific genetic p-distance is 0–0.02% distance for the same is also presented (Table 1). (n=4), whereas the species is 1.2–1.9% Morphological analysis: The new species divergent from R. charius; for 580bp of was compared with other members of the R. mitochondrial ND1 the new species shows a charius clade using morphometric data from divergence of 5% from R. charius (Tables 1, 2). Biju & Bossuyt (2009) and Bossuyt & Dubois The interspecific p-distance obtained between (2001) for R. charius, and from Priti et al. the new species and R. charius is reasonably (2016) for R. honnametti Gururaja, Priti, shallow for its recognition as a distinct species. Roshmi et al., 2016. Nonetheless, morphology and acoustics, in The following morphometric data were addition with geography, establish its collected using digital calipers, to the nearest distinctness, which is here described as a cryptic 0.01 mm: snout–vent length (SVL), head width species of the R. charius clade. A sequence from (HW), head length (HL), inner upper eyelid unknown locality generated by Vijayakumar et width (IUE), maximum upper eyelid width al. (2014), labelled as an unidentified species of (UEW), snout length (SL), eye length (EL), bush frogs was found to be conspecific with the internarial distance (IN), nostril–snout distance new species.
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