Zootaxa 4254 (2): 221–239 ISSN 1175-5326 (print edition) http://www.mapress.com/j/zt/ Article ZOOTAXA Copyright © 2017 Magnolia Press ISSN 1175-5334 (online edition) https://doi.org/10.11646/zootaxa.4254.2.4 http://zoobank.org/urn:lsid:zoobank.org:pub:FA412A05-7C7E-40AA-9958-A54989248415 A new species of the genus Microhyla (Anura: Microhylidae) from Eastern Nepal JANAK RAJ KHATIWADA1,2, GUO CHENG SHU1,2, SHOU HONG WANG1,2, ARJUN THAPA2,3, BIN WANG1 & JIANPING JIANG1* 1Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China. E-mail: [email protected] 2University of Chinese Academy of Sciences, Beijing, 100049, P. R. China 3Institute of Zoology, Chinese Academy of Sciences, Beijing, P. R. China 4Corresponding author. E-mail: [email protected] Abstract A new species of the genus Microhyla is described from Jamun Khadi, Jhapa district of eastern Nepal, based on molecular and morphological comparisons. This species is the sister taxon of Microhyla ornata and can be distinguished by a unique vocalization, morphology and molecular phylogeny. The uncorrected genetic divergences based on rRNA gene between the new species and its closest congeners, M. nilphamariensis, M. ornata and M. rubra were 5.34%, 6.67%, and 8.31%, respectively. The new species, Microhyla taraiensis sp. nov., is distinguished from each other of Microhyla by a combi- nation of the following morphological characters: (1) relatively larger body size (SVL ranges 19.9–20.3 mm, n = 4 in the males and 22.1–24.9 mm, n = 3 in the females); (2) dorsal surface of head and body with light red dots; (3) toes webbing poorly developed or absent; (4) a large round inner metacarpal tubercle; and an (5) elongated outer metacarpal tubercle. In addition, our study also provides a new record of Microhyla nilphamariensis from Nepal. Key words: Microhyla taraiensis sp. nov., Range extension, Microhyla nilphamariensis, Molecular phylogeny, Taxono- my Introduction Eastern Nepal forms part of the Eastern Himalayan Mountains that represent one of the global biodiversity hotspots (Myers et al. 2000). The Himalaya is a mountain massif characterized by the largest elevation gradient in the world with diverse eco-climatic zones (Dobremez 1976) and supports a high cryptic anuran diversity (Schleich & Kästle 2002). Currently, Nepal harbors more than 52 species of amphibians (Shah & Tiwari 2004), but information about them is generally derived from relatively old studies (Smith & Battersby, 1951; Smith & Battersby, 1953; Nanhoe & Ouboter, 1987; Mitchell & Zug, 1995; Zug & Mitchell, 1995). The microhylid frog genus Microhyla Tschudi, 1838, contains approximately 40 species with a broad distribution in Asia, from Japan, China, South Asia and throughout Southeast Asia to Sumatra, Borneo, Java, and Bali (Frost 2017). At least 10 new species have been described in the last five years (Hasan, et al. 2014; Howlader et al. 2015a; Matsui, 2011; Matsui et al. 2013; Poyarkov Jr et al. 2014; Seshadri et al. 2016, Meegaskumburua et al. 2016). Members of this genus are characterized by a relatively small size (snout vent length smaller than 30 mm), skin warty or smooth, tympanum indistinct, vomerine teeth absent, and toes webbed or free of webbing (Malkmus 2002; Schleich & Kästle 2002; Fei et al. 2005, 2012). They reproduce in various habitats, including paddy fields, small ponds, within urban environments and puddles and in tree holes in tropical forests in South and East Asia (Frost 2016). In Nepal, only three microhylid genera have been reported, viz., Kaloula, Uperodon and Microhyla (Schleich & Kästle 2002; Shah & Tiwari 2004). There have been sporadic reports on the presence of the genus Microhyla in Nepal (Nanhoe & Ouboter, 1987; Schleich & Kästle 2002; Shah & Tiwari 20041); however, the genus remains poorly studied in this country. The available literature has suggested that this genus shows a wide distribution across elevations (70–2100 m above sea level) in Nepal. Recently, three new species of Microhyla have been Accepted by M. Vences: 6 Mar. 2017; published: 13 Apr. 2017 221 described from Bangladesh, representing populations that were formerly regarded as M. ornata (Dumeril and Bibron, 1841) (type locality Kerala, India) (Hasan, et al. 2014; Howlader et al. 2015a). This suggests that cryptic taxa of Microhyla also remain unexplored in other geographical locations. Also in Nepal, populations of the genus Microhyla have so far been assigned to Microhyla ornata in different publications, but without any detailed morphological or molecular comparisons (Schleich & Kästle 2002; Shah & Tiwari 2004). In the present study, we use molecular, morphological and bioacoustical approaches were used to investigate the taxonomic position of Microhyla populations from central and eastern Nepal. Our data provide evidence for the occurrence of M. nilphamariensis in Nepal, and suggest that a Nepalese Microhyla population from Jamun Khadi represents a new species that is described herein. Materials and methods Study area: Field work was conducted between May and August 2015 at several sites in agricultural lands in eastern and central Nepal (Figure 1). We used time-constrained visual encounter surveys at night from 8pm to 11pm as mentioned by Khatiwada et al. (2016). Opportunistic random searches and call survey methods were also used to maximize the species encounter rate in each survey locality. The climate varies greatly by elevation in Nepal Himalaya. A subtropical climate predominates at lower elevations, and a temperate climate predominates above 1000 m (Bhattarai & Vetaas 2003). The majority of monsoonal rainfall occurs between June and September. Sampling: Specimens were collected by hand and taken to a nearby dry place where animals were sexed, measured and toe clipped. Sex was determined by the observation of secondary sexual characteristics, such as vocal sacs and nuptial pads in males, and by direct inspection of the enlargement of the coelomic cavity and visible eggs around the groin region in gravid females. A total of 33 individuals (26 individuals from M. nilphamariensis and seven individual Microhyla of the Jamun Khadi population; for details see supplementary Table S1) were included in the morphological examination. Most of the individuals were released into the same habitat after measurements. Tissue samples were collected from only eight individuals (five individuals of M. nilphamariensis and three Microhyla from Jamun Khadi) by clipping toes; these samples were stored in 95% ethanol for molecular analysis. Representative voucher specimens were euthanized using 20% Benzocaine gel, fixed in 4% formalin for 24 hours and then transferred to 70% alcohol. The collected voucher specimens were deposited at the Tribhuvan University, Natural History Museum, Soyambhu, Kathmandu, Nepal (NHM-TU-17A-0110 to NHM-TU-17A 0114). Molecular analysis: Extraction of total genomic DNA was carried out from clipped toes preserved in 95% ethanol using the DNeasy Tissue Kit (QIAGEN). A DNA fragment of the mitochondrial 16s rRNA gene (hereafter 16s) was amplified with primers and polymerase chain reaction (PCR) conditions provided by Hasan et al. (2014). The amplified PCR products were purified using a Qiagen PCR purification kit, and sequences were obtained from an ABI 3100 automated sequencer. All newly determined sequences were deposited in GenBank under accession numbers (KY655947–KY655954). Nucleotide sequences of the 16s gene of species of Microhyla were downloaded from the NCBI GenBank database (Fig. 2) and aligned with ClustalW in BIOEDIT Version 7.1.9 (Thompson et al. 1994) using the default settings. Maximum likelihood (ML) and Bayesian inference (BI) analyses were conducted to reconstruct the phylogenetic relationships among the taxa based on the haplotypes of the 16s gene sequence dataset. The maximum likelihood analysis was conducted in MEGA7 with 1000 bootstraps (Kumar et al. 2016). The Bayesian analyses and the best-fit substitution model were selected under the Bayesian Information Criterion by the program jModeltest 2.1.4 (Darriba et al. 2012). The best-fit substitution model for the 16s dataset was GTR + I + G. Bayesian analyses were conducted in MrBayes 3.1.2 (Ronquist & Huelsenbeck 2003). Morphological measurements: The following morphological measurements were taken with a digital caliper (Ocean Premium measuring instruments) to the nearest 0.1 mm: snout-vent length (SVL, from tip of snout to posterior edge of vent); head length (HL, from angle of jaws and snout-tip); head width (HW, maximum head width); snout length (SL, from tip of snout to the anterior corner of eye); eye diameter (ED, horizontal diameter of the eye); eyelid-naris distance (ENL, minimum distance between eyelid and rim of naris); upper eyelid width (UEW, greatest width of the upper eyelid); inter orbital width (IOW, minimum distance between upper eyelids); internarial distance (IND, minimum distance between the external nares); length of arm (LA, distance from axilla to tip of elbow); LH (length of hand: distance from elbow to longest finger); first to fourth finger length (F1 to F4 222 · Zootaxa 4254 (2) © 2017 Magnolia Press KHATIWADA ET AL. maximum length from fingertip to the base of hand); femur length (FL, maximum length of femur); length of tibia (LT, maximum length of tibia); length of foot (LF, distance from knee to longest toe); and first to fifth toe length (T1 to T5 maximum length from toe tip to the base of foot). To reduce allometric bias in the SVL, twenty-four morphological measurements were converted into ratio values (morphological character/SVL*100). Morphological traits were compared among the new species and the closest congeners (M. ornata and M. nilphamariensis) identified by molecular analyses (see Figure 2). Due to the lack of voucher specimens, morphological description of Microhyla ornata was adopted from Howlader et al (2015a). Student's t-tests were used to determine the differences in SVL between new species and M. nilphamariensis. Principal component analysis (PCA) of size-corrected values was used to explore the morphometric difference between the new species and M. nilphamariensis. All statistical analyses were performed using R software 3.0.1 (R Core Team, 2013).