A New Species of the Genus Microhyla (Anura: Microhylidae) from Eastern Nepal

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). Call character: Advertisement calls were recorded with a Marantz PMD670 recorder using a Sennheiser ME 66 shotgun microphone (16-bit resolution, sampling rate 44.1 kHz). Microhyla species in the area were detected by the loud and sharp calls of males at night (19:00-23:00). After locating a calling male, calls were recorded from approximately 50 cm. Two calls were recoded and examined as described by Wijayathilaka & Meegaskumbura (2016). Call recordings were visualized and edited with SoundRuler 0.9.6.0 (Gridi-Papp 2003–2007) and Raven Pro 1.5 software (Cornell Laboratory of Ornithology, Ithaca, NY, USA).

FIGURE 1. Map showing the sampling localities: the plus sign denotes the type locality of the new species from Jamun Khadi and asterisk signs denote the collection sites of M. nilphamariensis.

Results

Molecular analyses: The aligned dataset of 16s contained 548 bps, including 337 variable sites and 162 parsimony informative sites. The molecular data analysis suggested that the three populations of Nepalese Microhyla from Jhuwani, Budhabare and Hangdewa were genetically similar to M. nilphamariensis (type locality Bangladesh) and clustered together in an ML tree (Figure 2). The uncorrected genetic divergence between Nepalese and type species

A NEW SPECIES OF MICROHYLA FROM NEPAL Zootaxa 4254 (2) © 2017 Magnolia Press · 223 FIGURE 2. Maximum likelihood tree based on DNA sequences of the 16s rRNA gene showing the phylogenetic relationships among 27 species of the genus Microhyla. Numbers present on branches of ML tree are bootstrap support values for Maximum likelihood (above) and Bayesian posterior probabilities (below) respectively. ML and BI values lower than 50% were represented with dash (–). Chaperina fusca was selected as an outgroup. Genbank accession number are presented in parenthesis. JRK refers to field collection number of Janak Raj Khatiwada and species with bold letters refer to Holotype.

224 · Zootaxa 4254 (2) © 2017 Magnolia Press KHATIWADA ET AL. of M. nilphamariensis in the 16s gene was 0.50%, whereas the Jamun Khadi specimens formed a distinct clade with high bootstrap and posterior probabilities values and were suggested to form a monophyletic group. The genetic divergences between the Jamun Khadi population and its three closest relatives (M. nilphamariensis, M. ornata and M. rubra) were 5.34%, 6.67% and 8.31%, respectively. Intraspecific genetic divergence within the Jamun Khadi Microhyla specimens was 0.40%. Based on the phylogenetic results discussed above, the three populations of Microhyla (Jhuwnai, Chitwan district, Hangdewa, Taplejung district and Budhabare, Jhapa district) represent a new record of M. nilphamariensis in Nepal, while the Jamun Khadi population revealed a new species that is described below.

Microhyla taraiensis sp. nov.

Holotype: NHM-TU-17A-0110 (Fig. 3 A, B), an adult male collected from a paddy field in Jamun Khadi, Jhapa district, Nepal, 26.65358°N & 87.91161°E; elevation 119 m asl; collected by Janak Raj Khatiwada between 19:30 and 20:00 h on 15th May 2015 and deposited in the collection of Tribuvan University, Natural History Museum, Soyambhu, Kathmandu, Nepal. Suggested common name. Tarai narrow-mouthed frog Etymology. The species name is derived from the noun “Tarai,” which refers to the flat southern plains of Nepal composed of alluvial soil, where the new species was collected. Paratypes. NHM-TU-17A-0112 (adult male) and NHM-TU-17A-0111 (adult female) from the same location as the holotype; specimens were deposited in the collection of Tribuvan University, Natural History Museum, Soyambhu, Kathmandu, Nepal. Diagnosis. The new species is assigned to the genus Microhyla by the following morphological characters: absence of vomerine teeth, hidden tympanum, elliptical tongue, short snout, small eyes not protuberant and invisible when viewed from the ventral side, indistinct canthus rostralis, fingers free of webbing, single outer palmar tubercle, and skin with small tubercles. Microhyla taraiensis sp. nov. is distinguished from other Microhyla species by the following morphological characters: (1) relatively larger body size (SVL = 20.5 mm in male, SVL = 23.7 mm in female); (2) head relatively broad (HL/HW = 0.96–0.83); (3) head length is approximately 79% of head width; (4) short and round snout; (5) hidden tympanum; (6) wider inter-orbital distance approximately 1.5 times greater than the internarial distance; (7) flat and larger inner metacarpal tubercle two times greater than the outer metacarpal; (8) elongated and bean shaped inner metatarsal tubercle; (9) rounded outer metatarsal tubercle (10) toes webbing poorly developed or absent ; and (11) light red dots present all over the dorsal surface. Description of holotype. A medium sized male (SVL = 20.3 mm). Head relatively broad (HW 31.23% of SVL and HL 30% of SVL), snout truncate, eyes not protuberant and not visible when viewed from the ventral side. Canthus rostralis indistinct, nostril closer to the tip of the snout than to the eye (nostril 4.82% and eye 7.19% of SVL), tympanum hidden, supratympanic fold indistinct, moderate eye size (ED 22.80% of HL and ED 8.81% of SVL), pupil round, inter-orbital distance (12.21% of SVL) is greater than the inter-narial distance (8.47% of SVL), elliptical tongue, maxillary and vomerine teeth absent. LA shorter (length of arm 17% of SVL) than LH (length of hand 27% of SVL), fingers thin, free of webbing, finger tips round and not dilated, relative length of fingers from shortest to longest F1 < F2 < F4 < F3. Rounded inner metacarpal tubercle two times greater than outer metacarpal; nuptial pads absent. Subarticular tubercles small, round, formula 1, 2, 3, and 2. Tibiotarsal articulation reached the nostril when the hindlimb is kept parallel to the body. Hindlimbs muscular and slender; length of femur 40.83% of SVL; length of tibia 31.13% of SVL; length of foot 57.33% of SVL. Toes thin and small, toe tips rounded, relative length of toes T1 < T2 < T5 < T3< T4, webbing weakly developed. Elongated and bean-shaped inner metatarsal measuring almost half the length of the first toe. Rounded outer metatarsal tubercle. All five toes had small and round subarticular tubercles, formula 1, 1, 2, 3, and 2. Skin texture in preservation. Skin on the dorsal and lateral of head and body granular. Skin around the head region smooth, small tubercles present all over the dorsum surface. Small granules present in the cloacal and foot region. Color in preservative. Skin on the dorsal and lateral surface light brownish-gray; snout gray; arm, femur and tibia brownish-grey; hand and foot (meta-tarsus) creamy white; pupil turned to white from black; iris grey. Small light red spots present all over the dorsal body surface. A rectangular light black mark is present between

A NEW SPECIES OF MICROHYLA FROM NEPAL Zootaxa 4254 (2) © 2017 Magnolia Press · 225 interorbital spaces; two long light black stripes extend from the orbital region up to the groin. Irregular dark bands are present: one band on the arm, two on the hand, one on the femur and three on the tibia. Belly creamy white; throat light brown in females and light grey in males.

FIGURE 3. Morphological illustration of specimens used. A. Dorsal view of male holotype (NHM–TU–17A–0110) of Microhyla taraiensis sp. nov. B. Ventral view of the male holotype C. Dorsal view of M. nilphamariensis collected from Jhuwani, Chitwan, Nepal D. Ventral view of M. nilphamariensis E. Lateral view of head of holotype F. Lateral view of head of M. nilphamariensis G. Ventral view of hand of holotype H. Ventral view of foot of holotype I. Line drawing of hand of holotype showing metacarpal tubercles J. Line drawing of foot of holotype showing metatarsal tubercles K. Ventral view of hand of M. nilphamariensis L. Ventral view of foot of M. nilphamariensis.

226 · Zootaxa 4254 (2) © 2017 Magnolia Press KHATIWADA ET AL. Color in life. Skin light brown with small red spots present all over the dorsal surface of the body, forelimbs and hindlimbs, except on the hand, meta-tarsus and foot. A rectangular black marking was present in the interorbital region; two long black stripes extended from the orbital region to the groin. Irregular dark bands are present: one band on the arm, two on the hand, one on the femur and three on the tibia. Pupil black; iris golden yellow. Belly creamy white; throat brown in the female and blackish gray in males. Variations and sexual dimorphism. Morphometric variation of type specimens is presented in Table 1. Female body size larger than male body size (t = 4.42, df = 5, p = 0.007). Males had a sub-gular vocal sac, and gravid females contained un-pigmented eggs, which were visible in the belly near the groin. Morphological comparisons. Based on phylogenetic analysis, Microhyla taraiensis sp. nov. was closely related to M. nilphamariensis and M. ornata. Principal component analysis based on size-corrected values was used to examine the overall morphological variation between Microhyla taraiensis sp. nov. and the M. nilphamariensis population. PCA extracted seven principal component axes with eigenvalues greater than one, and of these, the first two component axes explained 42.47% of the variation (Table 2). The first two principal component axes radially separated the new species from M. nilphamariensis based on hand, finger and toe length (Figure 4). Species with a larger and positive score on PC1 reflected shorter HW, SL, UEW, IOW, IND, LA, LH, F1, F2, F3, F4, FL, LT, LF, T1, T2, T3, T4 and T5, while a negative score signified larger HL, ED and ENL. The second PC with negative scores were associated with species having shorter HL, SL, UEW, ENL, IOW, IND and FL, whereas positive scores were associated with species with larger morphological traits such as HW, ED, LA, LH, F1, F2, F3, F4, LT, LF, T1, T2, T3, T4 and T5. The SVL of Microhyla taraiensis sp. nov. was significantly larger than that of M. nilphamariensis (t = -5.65, df = 30, P <0.001). In Microhyla taraiensis sp. nov., the head length is approximately 79% of the head width (vs. roughly equal in M. nilphamariensis and M. ornata), the eye diameter is 35% of the head length (vs. approximately 30% in M. nilphamariensis and 48% in M. ornata), the upper eyelid is 83% of the eye diameter (vs. 82% in M. nilphamariensis and 39% in M. ornata), the nostril to eye distance is > 1.5 times that of the nostril to snout distance (vs. < 1.5 times in M. nilphamariensis and similar in M. ornata), and the interorbital distance is < 1.5 times that of the internarial distance (vs. >1.5 times in M. nilphamariensis and approximately 3 times in M. ornata). Microhyla taraiensis sp. nov. has a round inner metacarpal tubercle (vs. oval shape in M. nilphamariensis and goblet-shape in M. ornata), an elongated outer metacarpal tubercle (vs. small and round in M. nilphamariensis and prominent and heart-shaped in M. ornata), a round inner metatarsal tubercle (vs. similar in M. nilphamariensis and elongated and very prominent in M. ornata), and a large and elongated outer metatarsal tubercle (vs. ovoid-shaped, minute, and indistinct in M. nilphamariensis and compressed and large in M. ornata). Furthermore, a detailed morphometric comparison between the Jamun Khadi population and M. nilphamariensis is presented in Table 3.

FIGURE 4. Plots of the first principal component (PC 1) versus the second (PC 2) of the Microhyla taraiensis sp. nov. versus M. nilphimarensis from a Principal Component Analysis based on size corrected morphometric variables in Appendix S1.

A NEW SPECIES OF MICROHYLA FROM NEPAL Zootaxa 4254 (2) © 2017 Magnolia Press · 227 M. nilphamariensis M. Microhyla taraiensis taraiensis Microhyla Microhyla nilphamariensis nilphamariensis Microhyla Microhyla taraiensis Microhyla

228 · Zootaxa 4254 (2) © 2017 Magnolia Press KHATIWADA ET AL. TABLE 2. Variable loadings for principal components with Eigen value greater than, from size corrected morphological traits. Morphological traits PC 1 PC 2 PC 3 PC 4 PC 5 PC 6 PC 7 HL -0.05 -0.01 0.81 -0.19 0.01 0.37 -0.04 HW 0.46 0.58 0.10 -0.10 0.18 0.26 0.40 SL 0.12 -0.35 0.65 0.06 -0.35 0.03 -0.43 ED -0.13 0.10 0.77 -0.19 0.00 0.06 0.14 ENL -0.24 -0.07 -0.70 0.20 -0.35 0.14 0.23 UEW 0.31 -0.21 -0.67 0.06 0.32 0.25 -0.09 IOW 0.06 -0.11 0.20 -0.10 -0.83 0.01 0.00 IND 0.00 -0.07 -0.03 0.06 0.03 0.02 0.90 LA 0.02 0.16 0.03 -0.13 0.05 0.90 0.02 LH 0.80 0.22 0.26 0.20 -0.10 0.17 -0.09 F1 0.34 0.30 0.29 0.21 0.73 0.01 0.06 F2 0.69 0.13 -0.01 0.21 0.49 -0.02 0.20 F3 0.75 0.10 -0.29 -0.17 0.22 -0.11 0.03 F4 0.60 0.35 -0.28 0.09 0.17 0.13 -0.29 FL 0.24 -0.02 0.07 0.16 -0.06 0.84 0.05 LT 0.62 0.37 -0.07 0.23 -0.20 0.20 0.34 LF 0.59 0.17 -0.02 -0.04 -0.06 0.41 -0.08 T1 0.04 0.20 -0.26 0.89 0.27 0.02 0.01 T2 0.10 0.02 -0.20 0.95 0.03 -0.02 0.05 T3 0.17 0.89 0.03 0.27 0.22 0.01 -0.09 T4 0.35 0.87 0.09 -0.07 0.07 0.12 -0.08 T5 0.11 0.91 0.02 0.07 0.06 0.03 0.04 Eigenvalues 6.12 3.63 2.16 1.69 1.63 1.47 1.27 Cumulative percentage of 27.80 44.29 54.09 61.79 69.18 75.87 81.64 variance

In Microhyla taraiensis sp. nov., the interorbital distance was 0.5 times greater than the internarial distance, while it was < 1.3 in M. rubra (Table 3, Howlader et al. 2015). Furthermore, M. rubra has a smaller body size, a shovel-shaped inner metatarsal tubercle, one-third webbed toes, and a black spot on the flanks (Hasan et al. 2014; Howlader et al. 2015; Wijayathilaka et al. 2016). Likewise, Microhyla taraiensis sp. nov. differs from M. mukhlesuri, M. mymensinghensisn and M. laterite by lacking round expanded discs on the toe tips (Seshadri et al. 2016; Hasan et al. 2014). Moreover, the new species is morphologically distinct from the other species of the genus Microhyla by lacking toe webbing and toe tips with digital discs ( versus presence of these characters in M. achatina, M. annamensis, M. annectens, M. arboricola, M. berdmorei, M. borneensis, M. butleri, M. chakrapanii, M. darevskii, M. fissipes, M. fusca, M. heymonsi, M. karunaratnei, M. laterite, M. maculifera, M. malang, M. mantheyi, M. marmorata, M. mihintalei, M. minuta, M. mixtura, M. nanapollexa, M. okinavensis, M. orientalis, M. palmipes, M. perparva, M. petrigena, M. picta, M. pineticola, M. pulchella, M. pulchra, M. pulverata, M. rubra, M. sholigari, M. superciliaris, and M. zeylanica) by lacking toe webbing and toe tips with digital discs (Tschudi 1838; Andersson 1942; Bain & Nguyen 2004; Blyth 1856; Boulenger 1884; Boulenger 1897; Boulenger 1900; Dutta & Ray 2000; Fernando & Siriwardhane 1996; Hallowell 1861; Hu et al. 1966; Inger & Frogner 1979; Inger 1989; Jerdon 1853; Matsui 2011; Parker & Osman-Hill 1948; Parker 1928; Pillai 1977; Poyarkov Jr et al. 2014; Schenkel 1901; Seshadri et al. 2016; Smith 1923; Stejneger 1901; Vogt 1911; Wijayathilaka et al. 2016). Distribution and habitat. Microhyla taraiensis sp. nov is currently known only from its type locality in Jamun Khadi, Jhapa district, Eastern Nepal (26.65358oN & 87.91161oE; 119 m asl). Jamun Khadi is an artificial wetland with a few scattered Sal trees (Shorea robusta). This wetland is surrounded by agricultural lands. Rice is

A NEW SPECIES OF MICROHYLA FROM NEPAL Zootaxa 4254 (2) © 2017 Magnolia Press · 229 planted twice in a year in the area. During our survey work, most of the areas were fallow and only a few plots were planted with rice. Microhyla taraiensis sp. nov. was collected from fallow land near a rice plantation area. We propose to list this species as Data Deficient (DD) under the IUCN Red List criteria (IUCN 2001). Most of the males were detected by the loud calls. Microhyla taraiensis sp. nov. is sympatric with Fejervarya sp., Duttaphrynus sp. and Hoplobatrachus sp. Vocalization. Advertisement calls of Microhyla taraiensis sp. nov. (NHM-TU-17A-0110) were recorded in Jamun Khadi, Nepal on 15th May 2015 between 19:00–23:00. Males start calling at dust and can be heard from approximately 50 m away. The characteristics and structure of a call are shown in Fig. 5A and 5B. The call sounds were ‘karr…karr…karr….’. Each call consists of several notes, and a total of 10 notes were analyzed from two males. Each note had 13–14 pulses. The intervals between notes and pulses were 0.985±0.106 (1.151 ̶ 0.818 sec.) and 0.022±0.003 (0.027̶ 0.015 sec.), respectively. Call duration was 0.75 ± 0.12 (0.688 ̶ 0.911 sec.). The average dominant frequency was 3305.50 ± 95.46 (3433 ̶ 3101 Hz). The advertisement call of Microhyla taraiensis sp. nov. was recorded at a water temperature of 25.6°C, a water pH of 4.6, a soil temperature of 28.6°C, a soil moisture of 45%, a soil pH of 6.6, an air temperature of 28.6°C and a relative humidity of 63%. Range extension of M. nilphamariensis. The molecular analysis revealed that Microhyla species collected from the three populations of central and eastern Nepal formed a single clade in the ML tree. The genetic divergence between Nepalese and type specimens from Nilphamari, Bangladesh is 0.40%. M. nilphamariensis in this study were characterized by the following characters: head length and width almost equal, eye diameter 35% of the head length, upper eyelid 82% of the eye diameter, nostril to eye distance less than one and half times greater than the nostril to snout distance, inter-orbital distance more than one and half times greater than the internarial distance, small oval inner metacarpal, small rounded outer metacarpal tubercle, rounded inner metatarsal tubercle, elongated outer metatarsal tubercle, toes with poorly developed webbing, the absence of digital discs, and irregular back spots present all over the belly (Fig. 3D).

FIGURE 5. Advertisement call structure of Microhyla taraiensis sp. nov. recorded in Jamun Khadi, Jhapa district, Nepal. A. Oscillogram of a male advertisement call B. Spectrogram showing the dominant frequency.

230 · Zootaxa 4254 (2) © 2017 Magnolia Press KHATIWADA ET AL. et al M. ornata M. M. ornata. M. M. nilphamariensis nilphamariensis Microhyla ornata Microhyla 3=3 (33=3 33 (333 Microhyla. Taraiensis Taraiensis Microhyla. Microhyla taraiensis taraiensis Microhyla

A NEW SPECIES OF MICROHYLA FROM NEPAL Zootaxa 4254 (2) © 2017 Magnolia Press · 231 Advertisement calls of M. nilphamariensis were recorded in Jhuwani, Nepal on 13th May 2015 between 19:00– 23:00. Males start calling after sunset. The characteristics and structure of a call are shown in Fig. 6A and 6B. The call sounds were ‘karr…karr…karr….’ A total of 10 notes were analyzed from two males. Each note had 19–20 pulses. The interval between notes and pulses were 2.039±0.212 (2.119 ̶ 1.718 sec.) and 0.016±0.0008 (0. 016– 0.015 sec.), respectively. Each note call duration was 0.357±0.211 (0.506 ̶ 0.384 sec.). The average dominant frequency was 2595.32±689.85 (3569 ̶2261 Hz). The advertisement call of nilphamariensis was recorded at a water temperature of 25.4°C, a water pH of 4.7, a soil temperature of 27.8°C, a soil moisture of 85%, a soil pH of 6.2, an air temperature of 28.2°C and a relative humidity of 75%. M. nilphamariensis showed a wide range of elevational distribution from 120 to 1690 m in Nepal. All specimens of M. nilphamariensis were observed near rice fields and were sympatric with Fejervarya sp., Duttaphrynus sp., Polypedates sp. and Hoplobatrachus sp.

FIGURE 6. Advertisement call structure of M. nilphamarensis recorded in Jhuwani, Chitwan district, Nepal. A. Oscillogram of a male advertisement call B. Spectrogram showing the dominant frequency.

Discussion

The Eastern Himalaya is one of the biodiversity hotspots of Asia and harbors a large number of endemic species (Olson et al. 2001). In recent years, many new species of amphibians have been described from this region (Al Haidar et al. 2014; Biju et al. 2016; Dutta et al. 2015; Howlader et al. 2015b; Khatiwada et al. 2015; Mahony et al. 2013). Continuous descriptions of new species from eastern Nepal indicate that the biodiversity in the region remains imperfectly studied. With the advancement of molecular technology in taxonomic research, diagnosing cryptic taxa has become easier (Matsui et al. 2005; Hajibabaei et al. 2007; Matsui et al. 2011; Peloso et al. 2015). However, small-bodied microhylid frogs are extremely difficult to diagnose using only morphological information (Matsui et al. 2005). Asian microhylid frogs possess high morphological similarity and are found in various

232 · Zootaxa 4254 (2) © 2017 Magnolia Press KHATIWADA ET AL. habitats (Frost 2017). Because of these uncertainties, microhylid frogs are often misidentified in the field. Using morphological, acoustic and molecular comparisons, we have described a new species and provided an additional new record for Nepal. The morphological and molecular data analysis confirmed that the individuals of the genus Microhyla found in eastern and central Nepal include two species, i.e., M. nilphamariensis and M. taraiensis sp. nov. The Jamun Khadi area is characterized by an agricultural landscape with scattered settlements. The area lies outside the protected area system of Nepal, and agricultural lands are being converted to settlements. Furthermore, agricultural intensification and the extensive use of chemical fertilizers and pesticides could be existing threats for this species in the area. However, agriculture lands in Nepal are regarded as the major habitat of amphibians and support higher diversity in low-land Nepal (Khatiwada et al. 2016). Our study further highlighted the need to formulate appropriate conservation strategies in these landscapes. Species inventories from the paddy fields in lowland Nepal are easier to perform than inventories in forest and rugged mountainous areas. New species descriptions from agricultural landscapes further indicate the possibilities of finding additional new species from other habitats; for example, urban areas, forests and protected areas should be examined. The information on the geographic distribution and the taxonomy of Nepalese amphibians is still vague. M. taraiensis sp.nov. was found in highly disturbed and fragmented habitats and was relatively close to the highway in the eastern region of Nepal. Furthermore, the population status and distributional range of M. taraiensis sp.nov. is only known from the type locality. This work was limited to some selected regions of Nepal. Therefore, further extensive field work and research is needed.

Acknowledgements

We are grateful to Miguel Vences and three anonymous reviewers for providing critical suggestions that greatly improved the quality of our manuscript. We are thankful to Subarna Ghimire, Purnman and Bibas Shrestha for their assistance during field work. We are grateful to Jian Li for drawings. We are grateful to the Department of National Park and Wildlife Conservation (DNPWC) and the Department of Forest, Government of Nepal for providing the necessary research permit for amphibian study in the Nepal Himalaya. This work was supported by the National Natural Sciences Foundation of China (NSFC-31471964 granted to Jianping Jiang), the World Academy of Sciences (TWAS), and CAS-TWAS President Fellowship Program.

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