Contributions to Zoology, 83 (4) 269-335 (2014) DNA barcoding, phylogeny and systematics of Golden-backed frogs (Hylarana, Ranidae) of the Western Ghats-Sri Lanka biodiversity hotspot, with the description of seven new species S.D. Biju1, 4, Sonali Garg1, Stephen Mahony1, 2, Nayana Wijayathilaka3, Gayani Senevirathne3, Madhava Meegaskum- bura3 1Systematics Lab, Department of Environmental Studies, University of Delhi, Delhi 110 007, India 2School of Biology and Environmental Science, UCD Science Centre (West), University College Dublin, Belfield, Dublin 4, Ireland 3Evolutionary Ecology and Systematics Lab, Faculty of Science & Postgraduate Institute of Science, University of Peradeniya, Sri Lanka 4 E-mail: [email protected] Key words: Amphibia, cryptic species, endemism, integrative taxonomy, lectotypification, multiple gene barcoding, neotypification, taxonomic revision Abstract Ghats; 3 — the Hylarana temporalis group, endemic to Sri Lanka; and 4 — the Hylarana malabarica group from Sri Lan- A systematic revision of the genus Hylarana in the Western ka and India. The discovery of numerous morphologically Ghats-Sri Lanka biodiversity hotspot is presented. Species de- cryptic Hylarana species in this region further emphasizes the lineation in Hylarana is complicated due to a lack of distinct benefits of utilizing an integrative taxonomic approach for un- colour differences or striking morphological characters, leading covering hidden diversity and highlighting local endemism in to potential misidentification. We conducted extensive surveys the Western Ghats-Sri Lanka biodiversity hotspot. throughout the Western Ghats-Sri Lanka biodiversity hotspot and performed multiple gene (16S, COI and Cytb) barcoding using 103 samples collected from cultivated land and natural Contents habitats. Genetic distance comparisons and Neighbor Joining trees indicated the presence of at least 14 candidate species in Introduction ................................................................................... 269 the region, supported by taxa groupings for all three genetic Material and methods .................................................................. 271 markers. Utilising a combination of molecular and morphologi- Field survey and specimen collection ............................... 271 cal data, we describe seven new species, doubling the number DNA extraction, PCR and sequencing .............................. 271 of Hylarana species previously known from this region. We DNA barcoding analysis ...................................................... 274 further demonstrate that H. temporalis, which was originally Phylogenetic analysis ............................................................ 274 described from Sri Lanka, was misidentified with the Western Morphology and recognition of new species ................... 274 Ghats endemic species for nearly 100 years. Conversely, H. Abbreviations .......................................................................... 275 auran tiaca was originally described from the Western Ghats Results ............................................................................................. 275 and misidentified in Sri Lanka. Our study confirms that the dis- DNA barcoding ....................................................................... 275 tribution of H. temporalis is restricted to Sri Lanka, while H. Morphological recognition of species ............................... 279 aurantiaca is endemic to the Western Ghats, and that there are Phylogenetic analysis ............................................................ 279 no shared Hylarana species between the two regions. Hylarana Generic level taxonomy ........................................................ 280 flavescens, H. intermedius and H. montanus, previously consid- Species level taxonomy and endemism ............................. 281 ered synonyms of H. temporalis are confirmed as valid species. Discussion ...................................................................................... 281 Hylarana bhagmandlensis is removed from the synonymy of Acknowledgements ...................................................................... 282 H. aurantiaca and placed as a junior subjective synonym of H. References ...................................................................................... 282 montanus. To establish nomenclatural stability, H. flavescens, Appendix ........................................................................................ 287 H. malabarica and H. temporalis are lectotypified andH . inter- medius is neotypified. Detailed descriptions, diagnosis, mor- phological and genetic comparisons, illustrations and data on distribution and natural history are provided for all species. Introduction Phylogenetic analyses based on three mitochondrial markers (16S, COI and Cytb) and a fragment of the nuclear Rag1 gene, The Western Ghats and Sri Lanka together form one show complete endemism of the Western Ghats-Sri Lankan species. Four major groups in this region are identified as: 1 — of the 34 global biodiversity hotspots with extraordi- the Hylarana aurantiaca group, endemic to the Western Ghats; nary amphibian diversity and high levels of endemism 2 — the Hylarana flavescens group, endemic to the Western (Myers et al., 2000; Meegaskumbura et al., 2002; Downloaded from Brill.com09/23/2021 09:55:29AM via free access 270 Biju et al. – Systematic revision of the genus Hylarana of Western Ghats-Sri Lanka Bossuyt et al., 2004; Mittermeier et al., 2004), but this Boulenger, 1920; Kirtisinghe, 1957; Inger et al., 1985; region still remains poorly understood with regard to Daniel and Sekar, 1989; Dutta and Manamendra- the species level identifications in many of its extant Arachchi, 1996; Dutta, 1997; Chanda, 2002; Daniels, amphibians (Biju, 2001; Meegaskumbura et al., 2002). 2005; Pethiyagoda et al., 2006; De Silva, 2009; Uku- However, recent herpetological studies involving sev- wela, 2009; Bopage et al., 2011; Reshmy et al., 2011). eral lineages in the Western Ghats and Sri Lanka have Phylogenetic relationships of taxa currently con- resulted in the discovery of many previously unde- tained in the genus Hylarana still remain unresolved scribed species of frogs (e.g. Pethiyagoda and Mana- at both species and generic level (Chen et al., 2005; mendra-Arachchi, 1998; Biju, 2001; Biju and Bossuyt, Matsui et al., 2005; Bossuyt et al., 2006; Che et al., 2003, 2005a, b, c, 2006, 2009; Biju et al., 2007, 2008a, 2007; Gawor et al., 2009; Inger et al., 2009). Within 2009, 2010, 2011, 2014; Meegaskumbura et al., 2002, the geographical region of interest to our study, Hy- 2010; Meegaskumbura and Manamendra-Arachchi, drophylax Fitzinger, 1843 and Sylvirana Dubois, 1992 2005, 2011; Zachariah et al., 2011; Abraham et al., are currently placed in the synonymy of Hylarana 2013). Despite being one of the largest genera of the (Che et al., 2007). At species level Hylarana auran- cosmopolitan anuran family Ranidae (Frost, 2014), tiaca contains Rana (Hylorana) bhagmandlensis Rao, Hylarana Tschudi, 1838, has not been subjected to any 1922 as a synonym (by Dubois, 1992), while Hylarana rigorous systematic analyses and presently has only temporalis contains three synonyms: Rana flavescens four known species from the entire Western Ghats-Sri Jerdon 1853, Rana (Hylorana) gracilis montanus Rao, Lanka biodiversity hotspot, which spans an area of 1922 and Rana (Hylorana) intermedius Rao, 1937 more than 227 600 km2. Globally, the genus Hylarana contains 84 nominal species distributed on three continents (Frost, 2014), among which several are widely distributed (Bain et al., 2003; Frost et al., 2006). Two such species are H. aurantiaca (Boulenger, 1904) and H. temporalis (Gün- ther, 1864), which are known as widely distributed species both in the Western Ghats and Sri Lanka (e.g. Daniel and Sekar, 1989; Dutta and Manamendra- Arachchi, 1996; Dutta, 1997; Chanda, 2002; Daniels, 2005; De Silva, 2009). The taxonomy of these com- monly occuring species is confusing and unstable (Biju, 2001; Biju et al., 2004a, b). Moreover, unlike the other genera from this region, Hylarana is the only widespread genus for which no new taxa have been described for the past 77 years. Günther (1864) described Hylorana [sic] temporalis from ‘Ceylon’ (= Sri Lanka) and this species was sub- sequently reported from the entire Western Ghats (e.g. Boulenger, 1920; Inger et al., 1985; Daniel and Sekar, 1989; Dutta and Manamendra-Arachchi, 1996; Dutta, 1997; Chanda, 2002; Daniels, 2005). On the other hand, a relatively smaller species, Rana aurantiaca was described by Boulenger (1904) from ‘near Trivan- drum, Travancore [Kerala]’, southern India, and later reported to occur in Sri Lanka (e.g. Dutta and Mana- mendra-Arachchi, 1996; Dutta, 1997; De Silva, 2009). Hylarana temporalis and H. aurantiaca have not been studied in any great detail from across their ranges and Fig. 1. Study area, the Western Ghats-Sri Lanka Global biodi- versity hotspot and distribution of the genus Hylarana. Closed many of the systematists working on amphibians of circles indicate the present study collections and open circles the Western Ghats and Sri Lanka continue to treat indicate specimens studied from museums. Coordinates are similar looking species from both regions as same (e.g. provided in Table 1. Downloaded from Brill.com09/23/2021 09:55:29AM via free access Contributions to Zoology, 83 (4) – 2014 271 (placed by Boulenger, 1882; Dutta, 1997; Dubois, 1992, during field trips from