DNA barcoding reveals the first record of the Burmese Whipping Frog, Polypedates mutus (Anura: Rhacophoridae), from India

Abstract

Copyright is held by the authors. Articles in R&A are made available under a Creative Commons Attribution-NonCommercial 4.0 International license.was originally described by Smith (1940) from N' Chang Yang, Kachin, northern Myanmar (25.833313 N, 94.799994 E; elev. 789 m asl). The species belongs to the family Rhacophoridae, consisting of 434 species in 22 genera. The genus Polypedates Tschudi (1838) currently includes 25 nominal species and has a distribution ranging from eastern India throughout much of southeastern Asia (Frost 2022). According to Dinesh et al. (2020) and Frost (2022), the genus includes 13 species from India, two of which (P. teraiensis and P. braueri) occur in Mizoram.Polypedates mutus is a cryptic species that was removed from the synonymy of the P. leucomystax complex by Liu and Hu (1961). These treefrogs inhabit broadly disturbed habitats with shrub or tree borders (Zug 2022). Their distribution ranges from mainland (Bago, Kachin, Shan) and peninsular (Tanitharyi) Myanmar through adjacent southern China, northern Laos, northern Thailand, and Vietnam (Frost 2022;Kuraishi et al. 2012). Liu et al. ( 2018) described call characteristics that differ from its congener, P. megacephalus. The species is listed by the International Union for Conservation of Nature (IUCN) Red List of Threatened Species as being of Least Concern (LC) (IUCN SSC Amphibian Specialist Group 2022). The species is diagnosable based on the combination of characters provided by Smith (1940), Pham et al. (2017), and Ziegler et al. (2006. MethodsAt 1315 h on 18 September 2021, we collected a subadult Polypedates sp. (Fig. 1) that was not readily identifiable to species on damp ground surrounded by vegetation near the human settlement of Sialhawk, Khawzawl District, Mizoram (23.291645 N, 93.095818 E; elev. 1,300 m asl) (Fig. 2). It was euthanized following methods of Conroy et al. (2009), preserved in 70% ethanol, and catalogued in the Departmental Museum of Zoology, Mizoram University (MZMU2612). Measurements to the nearest 0.1 mm were taken with Mitutoyo dial Vernier Callipers (Model 505-671).We extracted genomic DNA from liver tissue using DNeasy Blood and Tissue Kit (QiagenTM, Valencia, California, USA) following the standard protocol provided by the manufacturer. We amplified and sequenced the partial 16S rRNA gene using a pair of primers: forward L02510-CGCCTGTTTATCAAAAACAT (Palumbi 1996) and reverse H03063-CTCCGGTTTGAACTCAGATC (Rassmann 1997) before performing a PCR reaction for a 20-μL reaction mixture containing 1X amplification buffer, 2.5 mMMgCl 2 , 0.25 mM dNTPs, 0.2 pM each forward and reverse primers, 1 μL genomic DNA, and 1 U Taq DNA polymerase with a pair of partial 16S rRNA primers (above). The PCR thermal regime for amplification was 5 min at 95 °C for initial denaturation, followed by 35 cycles of 1 min at