High cryptic diversity of endemic Indirana frogs in the Western Ghats biodiversity hotspot A. Nair 1 , S. V. Gopalan 2 , S. George 2 , K. S. Kumar 2 , A. G. F. Teacher 1,3 & J. Merilä 1 1 Ecological Genetics Research Unit, Department of Biosciences, University of Helsinki, Helsinki, Finland 2 Chemical Biology, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India 3 Centre for Ecology and Conservation, University of Exeter, Cornwall Campus, Tremough, Penryn TR10 4BE, UK Keywords amphibia; Ranixalidae; Indirana; cryptic diversity; biodiversity hotspot; Western Ghats. Correspondence Abhilash Nair, Ecological Genetics Research Unit, Department of Biosciences, University of Helsinki, PO Box 65, Biocenter 3, Viikinkaari, Helsinki FIN-00014, Finland. Tel: +358919157807; Fax: +358 919157694 Email: abhilash.nair@helsinki.fi Editor: Trent Garner Associate Editor: James Austin Received 6 November 2011; accepted 28 February 2012 doi:10.1111/j.1469-1795.2012.00539.x Abstract Amphibians are rapidly declining worldwide, but recent studies have shown that their diversity may be heavily underestimated, and many new species have been recently reported from biodiversity hotspots. For successful conservation and management strategies to be implemented within such hotspots, a better under- standing of the species diversity and their evolutionary relationships is required. We used three mitochondrial (16S, 12S and CO1) and two nuclear (rag1 and rhodopsin) gene fragments to investigate the genetic diversity within the endemic Indirana genus from the Western Ghats biodiversity hotspot. The species diversity within Indirana was found to be much higher than previously anticipated. Instead of the expected six species within this region, our analyses identified 11 clades with high (4.2–17.1%) sequence divergence. Each of these clades is likely to represent a distinct species. Particularly pronounced polyphyly was found within Indirana beddomii, which consisted of four highly supported monophyletic clades with high genetic divergence. Similarly, Indirana diplosticta was divided into two highly divergent monophyletic clades. We also report a new candidate species within the genus from Vellarimala in Kerala, which we believe is yet to be described. Our results suggest the existence of multiple unrecognized cryptic lineages within Indirana, all of which are likely to have more narrow distribution ranges and lower abundances than the taxonomic units into which they are currently assigned. Hence, the International Union for Conservation of Nature (IUCN) Red List statuses of Indirana frogs are likely to be in need of substantial revision, and detailed genetic studies across the Western Ghats might uncover additional new candidate species from this poorly studied endemic genus. Together with several earlier amphibian studies describing cryptic species from tropics, our results high- light the importance of proper species identification efforts before it is possible to reliably determine the IUCN conservation status of tropical amphibians described on the basis of morphological criteria. Introduction Species delimitation is a fundamental part of ecological and evolutionary studies, and also plays an important role in the development of conservation management programs. The methodological problem of identifying the boundaries between a set of species has recently attracted renewed attention (de Quieroz, 2007; Wiens, 2007). Cryptic species, which can be defined as two or more genetically distinct species that are (mistakenly) classified as single species on the basis of similar morphology, pose substantial taxonomic challenges as they are hard to describe on the basis of mor- phological or typological species concepts. The advent of relatively inexpensive and rapid DNA sequencing, and advances in molecular phylogenetic methods over the past decades have given biologists new tools for identifying pre- viously unrecognized cryptic species (Bickford et al., 2007). Amphibian systematics and taxonomy provide several examples of these and frequently highlight incongruence between morphology-based taxonomies and genetic line- ages (Hanken, 1999; Lemmon, Lemmon & Cannatella, 2007; Pauly, Piskurek & Shaffer, 2007). In recent years, many new cryptic amphibian lineages have been described from various biodiversity hotspots (e.g. Fouquet et al., 2007; Vieites et al., 2009), indicating that the amphibian diversity within these hotspots may be heavily under- estimated. If so, it follows that allocation of accurate International Union for Conservation of Nature (IUCN) conservation statuses and development of sound conserva- tion strategies in such regions is severely compromised. Therefore, there is an urgent need for efforts to identify cryptic species, especially given the increasing number of Animal Conservation. Print ISSN 1367-9430 Animal Conservation •• (2012) ••–•• © 2012 The Authors. Animal Conservation © 2012 The Zoological Society of London 1