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

A systematic revision of the genus Hylarana in the Western Ghats-Sri Lanka biodiversity hotspot is presented. Species delineation in Hylarana is complicated due to a lack of distinct colour differences or striking morphological characters, leading to potential misidentification. We conducted extensive surveys 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 habitats. Genetic distance comparisons and Neighbor Joining trees indicated the presence of at least 14 candidate species in the region, supported by taxa groupings for all three genetic markers. Utilising a combination of molecular and morphological data, we describe seven new species, doubling the number of Hylarana species previously known from this region. We further demonstrate that H. temporalis, which was originally described from Sri Lanka, was misidentified with the Western Ghats endemic species for nearly 100 years. Conversely, H. aurantiaca was originally described from the Western Ghats and misidentified in Sri Lanka. Our study confirms that the distribution of H. temporalis is restricted to Sri Lanka, while H. aurantiaca is endemic to the Western Ghats, and that there are no shared Hylarana species between the two regions. Hylarana flavescens, H. intermedius and H. montanus, previously considered synonyms of H. temporalis are confirmed as valid species. Hylarana bhagmandlensis is removed from the synonymy of H. aurantiaca and placed as a junior subjective synonym of H. montanus. To establish nomenclatural stability, H. flavescens, H. malabarica and H. temporalis are lectotypified and H. intermedius is neotypified. Detailed descriptions, diagnosis, morphological and genetic comparisons, illustrations and data on distribution and natural history are provided for all species. Phylogenetic analyses based on three mitochondrial markers (16S, COI and Cytb) and a fragment of the nuclear Ragl gene, show complete endemism of the Western Ghats-Sri Lankan species. Four major groups in this region are identified as: 1 the Hylarana aurantiaca group, endemic to the Western Ghats; 2 the Hylarana flavescens group, endemic to the Western Ghats; 3 the Hylarana temporalis group, endemic to Sri Lanka; and 4 the Hylarana malabarica group from Sri Lanka and India. The discovery of numerous morphologically cryptic Hylarana species in this region further emphasizes the benefits of utilizing an integrative taxonomic approach for uncovering hidden diversity and highlighting local endemism in the Western Ghats-Sri Lanka biodiversity hotspot.