Asian Herpetological Research 2019, 10(4): 219–229 ORIGINAL ARTICLE DOI: 10.16373/j.cnki.ahr.190016

Description of a New of (Anura: Ranidae) from Tibet,

Shuo QI1#, Zhengyan ZHOU1#, Zhitong LYU2, Yuyan LU1*, Han WAN2, Mian HOU1,3, Keji GUO4 and Pipeng LI1*

1 Institute of herpetology, Shenyang Normal University, Shenyang 110034, Liaoning, China 2 The Museum of Biology, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, Guangdong, China 3 College of Continuing (Online) Education, Sichuan Normal University, Chengdu 610068, Sichuan, China 4 Central South Inventory and Planning Institute of National Forestry and Grassland Administration, Changsha 410014, Hunan, China

Abstract A new species, Amolops pallasitatus sp. nov. is described based on specimens collected from Chentang Town, Dinggyê County, southern Tibet, China. The new species can be distinguished from other known congeners by mitochondrial divergence and morphological characteristics including: (1) medium body size, SVL 70.6–72.3 mm in adult females; (2) skin smooth over the entire body; (3) absence of dorsolateral fold; (4) tympanum small, edge indistinct, less than half of eye diameter; (5) vomerine teeth in two short oblique; (6) circummarginal and transverse grooves absent on disk of the first finger; (7) presence of inner metacarpal tubercle; (8) toes fully webbed, webbing formula I 0 - 0- II 0 - ½ III0 - 1+ IV 1+ - 0 V; (9) absence of outer metatarsal tubercle and tarsal glands; (10) tibio- tarsal articulation of the hind limb reaches posterior corners of the eye; (11) dorsum yellow-green, with irregular dark brown blotches without margins; (12) blotches concentrated on the dorsum, less on the flanks. In morphology, Amolops pallasitatus sp. nov. is similar to A. himalayanus and A. formosus, the difference between them is length of hind limbs, web of toe and dorsal colour pattern. The systematic placement of the new species within the is unresolved and it is not assigned to any recognized species group, for the lack of convictive evidences.

Keywords Amolops pallasitatus sp. nov., mitochondrial, morphology, unknown species group, torrent

1. Introduction adults, which lead to their common name “torrent ” or “sucker frogs”. The ranid genus Amolops Cope, 1865, as a typical Tibet is an important element across two biodiversity representative for Oriental fauna, is widespread from hotspots of the world, the Himalaya Hotspot and Indo- and northern , eastward to southeastern Burma Hotspot, which is well-known as the hottest of mainland China, and southward to Malaya Peninsula the hotspots (Tordoff et al., 2012). The mountains rise (Frost, 2019). These frogs inhabit swift, rocky streams, abruptly, resulting in a diversity of ecosystems that including torrents, rapids and waterfalls, enabled by range from alluvial grassland and subtropical broadleaf abdominal suckers in larvae and enlarged digital pads in forests to alpine meadows above the tree line. This area also holds remarkable endemism and is the home to # Both authors contributed equally to this paper. *Corresponding authors: Prof. Pipeng LI, from Shenyang Normal the important populations of numerous and University, China, with his research focusing on biodiversity and reptiles. evolution of amphibians and reptiles. Prof. Yuyan LU, Shenyang Normal University, China, with his research focusing on biodiversity Previous studies have reported eight species of genus and evolution of amphibians and reptiles.. Amolops in this region: A. aniqiaoensis Dong, Rao, and E-mail: [email protected] (Pipeng LI); [email protected] (Yuyan Lü, 2005, A. chayuensis Sun, Luo, Sun, and Zhang, 2013, LU) Received: 11 March 2019 Accepted: 24 September 2019 A. formosus (Günther, 1876), A. gerbillus (Annandale, 220 Asian Herpetological Research Vol. 10

1912), A. marmoratus (Blyth, 1855), A. medogensis Li 2016; Schleich and Kästle, 2002; Subba et al., 2016). In and Rao, 2005, A. monticola (Anderson, 1871) and A. addition, a total of 25 museum specimens of eight species nyingchiensis Jiang, Wang, Xie, Jiang, and Che, 2016. of Amolops were examined for comparison, which are During our herpetological surveys in Chentang Town, listed in Appendix I. Dinggyê County, southern Tibet, China (Figure 1), two 2.2. Taxon sampling A total of 29 samples of genus specimens of Amolops were collected in June 2015. Amolops were used for molecular analysis in this study. The morphological examinations and further molecular All samples were attained from euthanasia specimens and analysis supported the two specimens represented an then preserved in 95% ethanol and stored at –40°C. In independent evolutionary lineage of genus Amolops addition, 43 sequences were obtained from GenBank and and can be consistently distinguished from other known incorporated into our dataset for phylogenetic analysis. molecular data congeners. Therefore in this study we Detail information of these materials is shown in Table 1. describe them as a new species, whose phylogenetic placement is also discussed. 2.3. Extraction, PCR amplification, and sequencing Genomic DNA was extracted from muscular sample 2. Materials and Methods by using a DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd. Partial 16S ribosomal RNA gene 2.1. Sampling Two individuals of the new species were (16S) and partial cytochrome C oxidase 1 gene (CO1) collected from Chentang Town, Dinggyê County, southern were amplified. Primers used for 16S were L3975 Tibet, China. Following euthanasia, all specimens were (5'-CGCCTGTTTACCAAAAACAT-3') and H4551 fixed in 10% buffered formalin solution for preservation (5'-CCGGTCTGAACTCAGATCACGT-3'), and L2A after sampling of liver tissues from the adults in 95% (5'-CCAAACGAGCCTAGTGATAGCTGGTT-3') and ethanol, and subsequently transferred to 75% ethanol. All H10 (5'-TGATTACGCTACCTTTGCACGGT-3'), and for specimens were deposited in The Institute of herpetology, CO1 were Chmf4 (5'-TYTCWACWAAYCAYAAAGAYA SYNU. We obtained diagnostic characters of A. formosus TCGG-3') and Chmr4 (5'-ACYTCRGGRTGRCCRAAR (Günther, 1876), A. himalayanus (Boulenger, 1900), A. AATCA-3'), and dgLCO (5'-GGTCAACAAATCATAAA medogensis and A. viridimaculatus (Jiang, 1983) from GAYATYGG-3') and dgHCO (5'-AAACTTCAGGGTGA literature (Fei et al., 2012; Li et al., 2010; Nidup et al., CCAAARAAYCA-3') following Lyu et al., (2019). PCR

Figure 1 Map showing the collecting location of Amolops pallasitatus sp. nov. indicated by red star. No. 4 Shuo QI et al. A New Species of Amolops from Tibet, China 221

Table 1 Localities, voucher information and GenBank numbers for all samples used in this study.

ID Species Localities Voucher 16S CO1 Reference Amolops pallasitatus sp. 1 China: Dinggyê County, Tibet SYNU 1507034 MK573816 / This study nov. Amolops pallasitatus sp. 2 China: Dinggyê County, Tibet SYNU 1507035 MK573817 / This study nov. 3 Amolops medogensis China: Mêdog County, Tibet SYS a006657 MK573813 MK568328 This study 4 Amolops medogensis China: Mêdog County, Tibet SYNU 0400II145 MK573819 MK568332 This study 5 Amolops medogensis China: Mêdog County, Tibet SYNU 0400II140 MK573818 MK568331 This study 6 Amolops medogensis China: Mêdog County, Tibet KIZ 06635 / KU243076 Jiang et al., (2016) 7 Amolops viridimaculatus China: Mt. Gaoligong, Yunnan SYS a003753 MK573793 MK568310 This study 8 Amolops viridimaculatus China: Mt. Gaoligong, Yunnan SYS a003754 MK573794 MK568311 This study 9 Amolops viridimaculatus China: Tenchong City, Yunnan KIZ 930501 DQ204490 / Ngo et al., (2006) 10 Amolops viridimaculatus China: Yunnan C-green 05 AB211480 / Matsui et al., (2006) 11 Amolops afghanus China: Yingjiang County, Yunnan SYS a003872 MK573795 MK568312 This study 12 Amolops afghanus China: Yingjiang County, Yunnan SYS a003873 MK573796 MK568313 This study 13 Amolops afghanus : Kachin CAS 224449 JF794464 / Dever et al., (2012) 14 Amolops afghanus Myanmar: Kachin CAS 224451 JF794433 / Dever et al., (2012) Amolops 15 Myanmar: Twi Rein, Chin CAS 235070 JF794446 / Dever et al., (2012) indoburmanensis Amolops 16 Myanmar: Twi Rein, Chin CAS 235071 JF794442 / Dever et al., (2012) indoburmanensis 17 Myanmar: Mon CAS 240593 JF794456 / Dever et al., (2012) 18 Amolops marmoratus Myanmar: Mon CAS 240594 JF794453 / Dever et al., (2012) : Kao Chan water fall, 19 Amolops panhai 0332Y KR827705 KR087620 Grosjean et al., (2015) Ratchaburi Thailand: Suan Phueng Dist., 20 Amolops panhai 0868Y KR827706 KR087621 Grosjean et al., (2015) Ratchaburi 21 Amolops granulosus China: Mt. Guangwu, Sichuan SYS a005399 MK573811 MK568326 This study 22 Amolops granulosus China: Mt. Guangwu, Sichuan SYS a005400 MK573812 MK568327 This study 23 Amolops granulosus China: Mt. Wawu, Sichuan SYS a005318 MK573802 MK568317 This study 24 Amolops granulosus China: Mt. Wawu, Sichuan SYS a005319 MK573803 MK568318 This study 25 Amolops jinjiangensis China: Mt. Gaoligong, Yunnan SYS a004571 MK573801 MK568316 This study 26 Amolops jinjiangensis China: Tengchong City, Yunnan CAS 234058 FJ417162 / Stuart et al., (2010) 27 Amolops jinjiangensis China: Deqing County, Yunnan SCUM050435CHX EF453741 / Cai et al., (2007) 28 Amolops lifanensis China: Lixian County, Sichuan SYS a005374 MK573809 MK568324 This study 29 Amolops lifanensis China: Lixian County, Sichuan SYS a005375 MK573810 MK568325 This study 30 Amolops lifanensis China: Lixian County, Sichuan KIZ C93156 DQ204482 / Ngo et al., (2006) 31 Amolops lifanensis China: Sichuan KIZ C93150 AB211482 / Matsui et al., (2006) 32 Amolops loloensis China: Zhaojue City, Sichuan SYS a005350 MK573805 MK568320 This study 33 Amolops loloensis China: Zhaojue City, Sichuan SYS a005351 MK573806 MK568321 This study 34 Amolops loloensis China: Sichuan JF101 AF206493 / Chen et al., (2005) 35 Amolops loloensis China: Sichuan KIZ C18 AB211478 / Matsui et al., (2006) 36 Amolops mantzorum China: Fengtongzhai, Sichuan SYS a005365 MK573808 MK568323 This study 37 Amolops mantzorum China: Kangding City, Sichuan SYS a005360 MK573807 MK568322 This study 38 Amolops mantzorum China: Mt. Wawu, Sichuan SYS a005336 MK573804 MK568319 This study 39 Amolops mantzorum China: Maoxian County, Sichuan SCUM030014GP DQ360000 / Che et al., (2007) 40 Amolops tuberodepressus China: Mt. Ailao, Yunnan SYS a003900 MK573797 MK568314 This study 41 Amolops tuberodepressus China: Mt. Ailao, Yunnan SYS a003901 MK573798 MK568315 This study This study; Lyu et al., 42 Amolops tuberodepressus China: Mt. Wuliang, Yunnan SYS a003931 MK573799 MG991933 (2018) This study; Lyu et al., 43 Amolops tuberodepressus China: Mt. Wuliang, Yunnan SYS a003932 MK573800 MG991934 (2018) Laos: Vieng Phou Kha, Luang 44 Amolops akhaorum FMNH 271355 FJ417158 / Stuart et al., (2010) Namtha Laos: Vieng Phou Kha, Luang 45 Amolops akhaorum FMNH 271406 FJ417159 / Stuart et al., (2010) Namtha 222 Asian Herpetological Research Vol. 10

Continued Table 1

ID Species Localities Voucher 16S CO1 Reference 46 Amolops aniqiaoensis China: Mêdog County, Tibet KIZ 07364 / KU243072 Jiang et al., (2016) 47 Amolops aniqiaoensis China: Mêdog County, Tibet KIZ 011138 / KU243073 Jiang et al., (2016) 48 Amolops archotaphus Thailand: Doi Inthanon, Chiang Mai CUMZ A 2000.62 FJ417124 / Stuart et al., (2010) 49 Amolops archotaphus Thailand: Doi Inthanon, Chiang Mai FMNH 261712 FJ417125 / Stuart et al., (2010) 50 Amolops bellulus China: Lushui County, Yunnan KIZ 9810021 DQ204473 / Ngo et al., (2006) 51 Amolops chayuensis China: Baxoi County, Tibet SYNU 1608048 MK573820 MK568333 This study 52 Amolops chayuensis China: Baxoi County, Tibet SYNU 1608049 MK573821 MK568334 This study 53 Amolops chayuensis China: Baxoi County, Tibet KIZ 014016 / KU243074 Jiang et al., (2016) 54 Amolops chayuensis China: Baxoi County, Tibet KIZ 014022 / KU243075 Jiang et al., (2016) 55 Amolops chunganensis China: Mt. Jinggang, Jiangxi SYS a004212 MK263263 MG991914 Lyu et al., (2019) 56 Amolops chunganensis China: Mt. Jinggang, Jiangxi SYS a004213 MK263264 MG991915 Lyu et al., (2019) Vietnam: A Luoi Dist., Thua Tien 57 Amolops compotrix AMNH 169315 FJ417132 / Stuart et al., (2010) Hue 58 Amolops compotrix Vietnam: Dak Glei Dist., Kon Tum ZISP A7367 FJ417142 / Stuart et al., (2010) 59 Amolops cucae Vietnam: Van Ban Dist., Lao Cai AMNH 168727 FJ417144 / Stuart et al., (2010) 60 Amolops cucae Vietnam: Van Ban Dist., Lao Cai AMNH 168729 FJ417145 / Stuart et al., (2010) 61 Amolops daorum Vietnam: Sa Pa, Lao Cai ROM 38501 FJ417150 / Stuart et al., (2010) 62 Amolops daorum Vietnam: Sa Pa, Lao Cai ROM 38503 FJ417151 / Stuart et al., (2010) 63 Amolops iriodes Vietnam: Vi Xuyen Dist., Ha Giang AMNH 163926 FJ417152 / Stuart et al., (2010) 64 Amolops iriodes Vietnam: Vi Xuyen Dist., Ha Giang AMNH 163928 FJ417153 / Stuart et al., (2010) 65 Amolops nyingchiensis China: Lhünzê County, Tibet SYS a007508 MK573815 MK568330 This study 66 Amolops nyingchiensis China: Mêdog County, Tibet SYS a006679 MK573814 MK568329 This study 67 Amolops nyingchiensis China: Mêdog County, Tibet KIZ016416 / KU243068 Jiang et al., (2016) 68 Amolops vitreus Laos: Phongsaly Dist., Phongsaly FMNH 258183 FJ417163 / Stuart et al., (2010) 69 Amolops vitreus Laos: Phongsaly Dist., Phongsaly FMNH 258187 FJ417164 / Stuart et al., (2010) 70 Amolops wenshanensis China: Jingxi City, Guangxi KU 292045 FJ417129 / Stuart et al., (2010) 71 Amolops ricketti China: Mt. Wuyi, Fujian SYS a004141 MK263259 MG991927 Lyu et al., (2019) 72 Amolops ricketti China: Mt. Wuyi, Fujian SYS a004142 MK263260 MG991928 Lyu et al., (2019)

Museum abbreviations: AMNH: American Museum of Natural History, New York, USA; CAS: California Academy of Sciences, San Francisco, USA; CUMZ: Cameron University Museum of Zoology, Lawton, USA; FMNH: Field Museum of Natural History, Chicago, USA; KIZ: Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China; ROM: Royal Ontario Museum, Toronto, Canada; SCUM: Zoological Museum of the School of Life Science, Sichuan University, Chengdu, China; SYNU: The Institute of herpetology, Shenyang Normal University, Shenyang, China; SYS: The Museum of Biology, Sun Yat-sen University, Guangzhou, China; ZISP: Zoological Institute, Russian Academy of Sciences, St. Petersburg, Russia. amplifications were processed in a 20 reaction volume 2.4. Phylogenetic analysis DNA sequences were aligned with the cycling conditions with an initial denaturing step by the Clustal W algorithm with default parameters at 95°C for 4 min, 35 cycles of denaturing at 94°C for 40 (Thompson et al., 1997) and trimmed with the gaps s, annealing at 53°C (for 16S) / 48°C (for CO1) for 40 s partially deleted in MEGA 6 (Tamura et al., 2013), and extending at 72°C for 1 min, and final extending step while within highly variable regions, all gaps were of 72°C for 10 min. PCR products were purified with spin removed. Two genes, 1080 base pairs (bp) of 16S and columns. The purified products were sequenced with both 639 bp of CO1, were concatenated seriatim into a 1719- forward and reverse primers using a BigDye Terminator bp sequence, and further divided into two partitions by Cycle Sequencing Kit per the guidelines, on an ABI Prism gene. Partitions were tested respectively in jmodeltest 3730 automated DNA sequencer by Shanghai Majorbio v2.1.2 with Akaike and Bayesian information criteria, Bio-pharm Technology Co., Ltd and Beijing Genomics all resulting in the best-fitting nucleotide substitution Institute. All sequences were deposited in GenBank models of GTR + I + G. Sequenced data were analyzed (Table 1). using maximum likelihood (ML) implemented in No. 4 Shuo QI et al. A New Species of Amolops from Tibet, China 223

RaxmlGUI 1.3 (Silvestro and Michalak, 2012) with A. iriodes, A. jaunsari Ray, 1992, A. jinjiangensis Su, the sequences of A. ricketti as out-group. The bootstrap Yang, and Li, 1986, A. kohimaensis Biju, Mahony, and consensus tree inferred from 1000 replicates was used to Kamei, 2010, A. longimanus (Andersson, 1939), A. represent the evolutionary history of the taxa analyzed. mengyangensis Wu and Tian, 1995, A. minutus Orlov and The phylogenetic tree was displayed and annotated using Ho, 2007, A. monticola, A. nyingchiensis, A. vitrea and A. iTOL v3 (Letunic and Bork, 2016). Because most of the wenshanensis]; (2) presence of vomerine teeth [vs. absent GenBank sequences lacked CO1 gene data, pairwise in A. daiyunensis (Liu and Hu, 1975), A. hainanensis distances (p-distance) were calculated only for 16S in (Boulenger, 1900), A. hongkongensis (Pope and Romer, MEGA 6, using the uncorrected p-distance model. 1951), A. torrentis (Smith, 1923) and A. wuyiensis (Liu 2.5. Morphology and morphometrics Measurements and Hu, 1975)]; (3) dorsal and lateral skin absolutely follow Fei et al., (2009) and Lyu et al., (2019), webbing smooth in adult females [vs. dorsal and lateral rough in formula followed Savage and Heyer (1997) and were A. albispinus Sung, Hu, Wang, Liu, and Wang, 2016, A. taken with digital calipers (Mitutoyo 500-752-20 australis Chan, Abraham, Grismer, and Grismer, 2018, A. Stainless Steel Digital Caliper, Japan) to the nearest gerutu Chan, Abraham, Grismer, and Grismer, 2018, A. 0.1 mm, including snout–vent length (SVL) from tip of larutensis (Boulenger, 1899), A. ricketti, A. sinensis Lyu, snout to posterior margin of vent, head length (HDL) Wang, and Wang, 2019, A. yatseni Lyu, Wang, and Wang, from tip of snout to the articulation of the jaw, head 2019 and A. yunkaiensis Lyu, Wang, Liu, Zeng, and width (HDW) at the commissure of the jaws, snout Wang, 2018, or dorsal and lateral granular in A. afghanus, length (SNT) from tip of snout to the anterior corner of A. marmoratus, A. panhai and A. spinapectoralis Inger, the eye, internasal distance (IND), interorbital distance Orlov, and Darevsky, 1999, or smooth middorsally but (IOD), eye diameter (ED) from the anterior corner of the tuberculate laterally in A. assamensis Sengupta, Hussain, eye to posterior corner of the eye, tympanum horizontal Choudhury, Gogoi, Ahmed, and Choudhury, 2008, diameter (TD), tympanum–eye distance (TED) from A. indoburmanensis, A. kaulbacki (Smith, 1940), A. anterior edge of tympanum to posterior corner of the lifanensis, A. loloensis, A. mantzorum, A. tuberodepressus eye, hand length (HND) from distal end of radioulna to and A. xinduqiao Fei, Ye, Wang, and Jiang, 2017)]; tip of distal phalanx III, radioulna length (RAD) from (4) Tibio-tarsal articulation of the hind limb reaches the flexed elbow to the base of the outer palmar tubercle, posterior corners of the eye [vs. reaches the nostril or the foot length (FTL) from distal end of tibia to tip of distal tip of the snout in A. formosus]; (5) tibia significantly phalanx IV, tibial length (TIB) from the outer surface of shorter than the trunk [vs. tibia as long as the trunk in the flexed knee to the heel, hindlimb length (HLL) from A. himalayanus]; (6) dorsum with irregular dark brown the vent to tip of distal phalanx IV, width of finger III blotches without margins [vs. dorsum with irregular dark digital disc (F3W) and width of toe IV digital disc (T4W). brown blotches with black margins or entirely black in A. We determined sex by observation of secondary sexual formosus, or dorsum with numerous small round light- characters, i.e. the presence of nuptial pads in males. In yellow spots in A. caelumnoctis Rao and Wilkinson, 2007 the webbing formula, Roman numbers refer to the fingers and A. splendissimus Orlov and Ho, 2007, or dorsum and toes, and the Arabic number refer to the number of with small irregularly arranged cobalt green spots in A. subarticular tubercles. nidorbellus Biju, Mahony, and Kamei, 2010]; (7) blotches concentrated on the dorsum, less on the flanks [vs. 3. Results blotches covered the dorsum and flanks in A. medogensis and A. viridimaculatus]; (8) webbing formula I 0 - 0- II 0 - 3.1. Morphological comparison The unnamed Amolops ½ III0 - 1+ IV 1+ - 0 V [vs. I 0 – 1½ II 0 – 2+ III 0 - 2- IV 2- specimens from Dinggyê County, southern Tibet were - 0 V in front in A. formosus]. compared with all recognized species of the genus, and In morphology, these unnamed specimens are similar they can significantly differ from the congeners by the to A. himalayanus and A. formosus, more principal following characteristics: (1) absence of dorsolateral morphological differences between the three taxa are fold [ vs. present in A. akhaorum, A. aniqiaoensis, given in Table 2. The detailed morphological comparisons A. archotaphus, A. bellulus, A. chakrataensis Ray, indicated that the unnamed specimens are significantly 1992, A. chayuensis, A. chunganensis, A. compotrix, different from A. formosus by its shorter hindlimb, tibio- A. cremnobatus Inger and Kottelat, 1998, A. cucae, A. tarsal articulation reaches posterior corners of the eye daorum, A. gerbillus, A. granulosus (Liu and Hu, 1961), (vs. reaches the nostril or the tip of the snout), webbing 224 Asian Herpetological Research Vol. 10

Table 2 The principal morphological differences between A. himalayanus (Boulenger, 1888), A. formosus (Günther, 1875) and A. pallasitatus sp. nov., reference from Nidup et al., (2016).

Species A. himalayanus A. formosus A. pallasitatus sp. nov.

Tibia as long as the trunk considerably shorter than the trunk considerably shorter than the trunk Tibio-tarsal articulation of reaches the nostril or the tip of the reaches beyond the tip of the snout reaches posterior corners of the eye the hind limb snout Web of toes feebly notched deeply notched deeply notched Webbing formula I 0 - 1½ II 0 - 2+ III 0 – 2- IV 2- - 0 V I 0 - 0- II 0 - ½ III0 - 1+ IV 1+ - 0 V Nostril nearer to eye than to tip of snout midway between eye and tip of snout nearer to eye than to tip of snout Tympanum indistinct distinct indistinct small granules on side of body and small granules on side of temples; belly granulate Skin temples; belly and posterior half of posterior half of lower surface of thighs lower surface of thighs granulate granulate granules on side of head absent only a few granules on side of temples spinules limited to side of the head and without spinules base of the forelimb yellow-green above with irregular dark bright green above with sharply defined olive or greyish above with indistinct brown blotches without margins on the Coloration of dorsum black or blackish spots on the head and darker spots on the body head and body; blotches concentrated body on the dorsum, less on the flanks Coloration of hinder side of purplish brown marbled with black banded with dark brown thighs Coloration of lower parts brownish or pale olive brown or marbled brown and whitish light yellow and whitish Snout rounded in front obtusely pointed in front rounded in front SVL in males 74–75 mm (n = 2) 53 mm (n = 1) SVL in females 80–83 mm (n = 2) 53–75 mm (n = 6) 70–72­­ mm (n = 2) Literature cited Boulenger (1888, 1920), Yang (1991) Boulenger (1888, 1920), Yang (1991) This study

Figure 2 Maximum-likelihood phylogenic tree. Numbers in parentheses coresponding to the ID in Table 1. No. 4 Shuo QI et al. A New Species of Amolops from Tibet, China 225

taxonomic placement of this lineage was unresolved for the relatively insignificant bootstrap supports of < 75. The lowest p-distances between this lineage and a known congener (A. akhaorum) was 2.4%–2.4%, which was significant when compared to the values among described species, for example, between A. tuberodepressus and A. jinjiangensis (p-distances of 1.7%–2.0%). These results indicate that there are substantial genetic divergences between the recognized Amolops species and the lineage from Dinggyê County, Tibet, indicating that these two Figure 3 Holotype (SYNU 1507035, female) of Amolops specimens represented a separately evolving lineage pallasitatus sp. nov. in life. Photo by Zhengyan ZHOU. within the genus.

+ + Based on the molecular and morphological evidences, formula I 0 - 0- II 0 - ½ III0 - 1 IV 1 - 0 V (vs. I 0 - we hereby describe the specimens from Dinggyê County, 1½ II 0- 2+ III 0 – 2- IV 2- - 0 V), skin without spinules Tibet as a new species, Amolops pallasitatus sp. nov. (vs. spinules limited to side of the head and base of the forelimb), dorsum yellow-green above with irregular dark 3.3. Taxonomic account brown blotches without margins on the head and body; Amolops pallasitatus Qi, Zhou, Lyu, Lu & Li, sp. nov. blotches concentrated on the dorsum, less on the flanks Holotype: SYNU 1507035 (Figures 3, 4, 5), adult female, (vs. dorsum olive or greyish above with indistinct darker collected by Zhengyan ZHOU on June 2015 from a spots on the body), lower parts light yellow and whitish stream close to the China-Nepal border (27.92° N, 87.47° (vs. brownish or pale olive). E; 3270 m a.s.l.), Chentang Town, Dinggyê County, Tibet Autonomous Region, China. 3.2. Phylogenetic analyses The ML phylogenetic tree Paratype: SYNU 1507034 (Figure 6), adult female, the was shown in Figure 2. According to the phylogenetic same collection information as the holotype. results, the Amolops specimens from Dinggyê County, Diagnosis. Amolops pallasitatus sp. nov. is distingusished Tibet, gathered together in a lineage with strong support from its congeners by a combination of the following value and without genetic distance (bootstrap support morphological characteristic: (1) medium body size, = 100, p-distance = 0). Despite of the lineage being a SVL 70.6–72.3 mm in adult females; (2) skin smooth sister taxon to (A. medogensis + A. viridimaculatus), the over the entire body; (3) absence of dorsolateral fold; (4)

Figure 4 Holotype of Amolops pallasitatus sp. nov. in preservative. A and B: Dorsal and ventral view of the holotype; C: Ventral view of the left hand; D: Ventral view of the left foot; E: Lateral view of the head. Photos by Shuo QI. 226 Asian Herpetological Research Vol. 10

the first finger; (7) presence of inner metacarpal tubercle; (8) toes fully webbed, webbing formula I 0 - 0- II 0 - ½ III0 - 1+ IV 1+ - 0 V; (9) absence of outer metatarsal tubercle and tarsal glands; (10) tibio-tarsal articulation of the hind limb reaches posterior corners of the eye; (11) dorsum yellow-green, with irregular dark brown blotches without margins; (12) blotches concentrated on the dorsum, less on the flanks. Description of holotype Adult female, body stout, SVL 72.3 mm, medium size in the genus Amolops. Head flattened, width slightly smaller than length (HDW/HDL = 0.91); snout short (SNT/HDL = 0.40) and rounded in profile, projecting beyond lower jaw; nostril closer to eye than tip of snout; eye large (ED/HDL = 0.29) and convex; canthus rostralis rounded; pineal body invisible; tympanum small, edge indistinct, less than half of eye diameter (TD/ED = 0.42); tympanum-eye distance larger than tympanum, TED/TD 1.10; supratympanic fold distinct; vomerine teeth distinct, on two short oblique between choanae, converging posteriorly, teeth ridges longer than space between them; tongue cordiform, deeply notched posteriorly. Forelimbs moderately long; hands moderately long (HND/SVL = 0.36); relative finger lengths I < II = IV < III; tip of the first finger slightly dilated, large disks with Figure 5 Shows the tibio-tarsal articulation of the hind limb reaches circummarginal grooves on tips of three outer fingers, posterior corners of the eye (SYNU 1507035). Photo by Zhengyan relative width of finger disks I < II < III = IV; subarticular ZHOU. tubercles prominent, rounded; supernumerary tubercles indistinct below the base of fingers II, III and IV, but

Table 3 Measurements (in mm) of the type series of Amolops pallasitatus sp. nov.

Collection Number SYNU1507035holotype SYNU1507034 paratype

Sex F F SVL (mm) 72.3 70.6 HDL (mm) 25.4 24.4 HDW (mm) 23.1 24.2 SNT (mm) 10.2 10.2 IND (mm) 7.2 7.4 IOD (mm) 6.5 6.2 ED (mm) 7.3 7.2 TD (mm) 3.0 3.0 TED (mm) 3.4 3.2 HND (mm) 25.0 27.1 Figure 6 Paratype (SYNU 1507034, female) of Amolops pallasitatus RAD (mm) 38.4 43.0 sp. nov. in life. Photo by Zhengyan ZHOU. FTL (mm) 38.2 39.6 TIB (mm) 38.1 38.7 tympanum small, edge indistinct, less than half of eye HLL (mm) 119.3 112.2 diameter; (5) vomerine teeth in two short oblique; (6) F3W (mm) 4.3 4.3 circummarginal and transverse grooves absent on disk of T4W (mm) 3.0 3.1 No. 4 Shuo QI et al. A New Species of Amolops from Tibet, China 227

oval and distinct; inner metatarsal tubercle prominent, elongated; outer metatarsal tubercle and tarsal glands absent; toes fully webbed, webbing formula is I 0 - 0- II 0 - ½ III 0 - 1+ IV 1+ - 0 V; lateral fringes of toes I and V developed. Skin dorsally and ventrally smooth, including throat, chest, abdomen, ventral surface of limbs, and flanks; dorsolateral fold absent; only a few raised warts on both sides of temporal region; rictal gland prominent and ellipsoidal, posterior to corner of mouth; a few warts surrounding cloaca. Measurements of holotype (in mm) SVL 72.3; HDL 25.4, HDW 23.1; SNT 10.2; IND 7.2; IOD 6.5; ED 7.3; TD 3.1; TED 3.4; HND 25.0; RAD 38.4; FTL 36.9; TIB 38.1; F3W 4.1; T4W 3.0. Color of holotype in life Dorsum yellow-green, with irregular dark brown blotches without margins; blotches concentrated on the dorsum, less on the flanks; irregular dark brown patches on dorsal surface of upper arms, distinct dark brown transverse bars on dorsal surface of lower arms and hindlimbs; a dark brown stripe along lower edge of the canthus rostralis extending from the snout tip across the eyes, to the anterior edge of supratympanic fold; ventral surfaces of the forelimbs light yellow; hinder side of thighs banded with dark brown. Color of holotype in preservative After preservation, dorsal surface fades to dark blue gray with irregular dark taupe blotches; ventral surface of body and limbs beige; transverse bars on limbs blur; webs on toes grayish white. Variations Measurements of type series specimens are given in Table 3. Two specimens are very similar in morphology, but paratype shows more irregularly blotches on the dorsum than holotype. Etymology The specific name pallasitatus means “pallasite like”, which derived from meteoritics term pallasite (a class of stony–iron meteorite). The name refers to the numerous irregular dark brown blotches on Figure 7 Habitat of Amolops pallasitatus sp. nov. in the type locality. Photo by Zhengyan ZHOU. the dorsal background resembling olivine crystals in an iron-nickel matrix. According to the type locality, we absent below the base of fingers I; inner metacarpal suggest the English common name as “Chentang cascade tubercles oval, outer metacarpal tubercle separated with frog”, and the Chinese common name as “chén ­­­­táng tuān the outer part flat and barely visible; absence of webbing wā” ( 陈塘湍蛙 ). and lateral fringes on fingers. Distribution Currently, this species is only known Hindlimbs long and robust (TIB/SVL = 0.53, TIB/ from Chentang Town, Dinggyê County, Tibet, China, HLL=0.32); tibio-tarsal articulation reaching posterior presumably also distributed in Nepal. corners of the eye when hindlimb stretched alongside of Ecology and habitat All the specimens were caught body; heels overlapping when hindlimbs flexed at right at night, living in high-altitude (3270 m a.s.l.) streams angles to axis of body; relative toe lengths I < II < III < within moist forest and grassland habitats (Figure 7). The V < IV; tips of all toes expanded to well-developed oval frog is hidden underwater during the day. When disturbed, discs with circummarginal grooves; subarticular tubercles it jumped into the water immediately. 228 Asian Herpetological Research Vol. 10

4. Discussion providing tissue samples, specimens and literature. We thank Y. Y. Wang for provide valuable suggestions. Currently, Amolops contains 58 recognized species, making it one of the largest Asian frog genera. Because References of the large number of these frogs, six species groups based on morphological comparisons were proposed AmphibiaChina. 2019. The database of Chinese amphibians. for the Chinese congeners (Pang 1992; Fei et al.,; 2005; Kunming Institute of Zoology (CAS), Kunming, Yunnan, China. Retrieved from http://www.amphibiachina.org Fei et al., 2009), namely A. marmoratus group, A. Ao J. M., Bordoloi S., Ohler A. 2003. fauna of monticola group, A. mantzorum group, A. ricketti group, Nagaland with nineteen new records from the state including A. daiyunensis group and A. hainanensis group. This five new records for India. Zoos’ Print J, 18(6): 1117–1125 species group division were followed by subsequent Biju S. D., Mahony S., Kamei R. G. 2010. Description of two investigations and further applied to the species out of new species of torrent frog, Amolops Cope (Anura: Ranidae) China (Matsui et al., 2006; Ngo et al., 2006; Cai et al., from a degrading forest in the northeast Indian state of 2007; Stuart et al., 2010; Dever et al., 2012; Lyu et al., Nagaland. Zootaxa, 2408(1): 31–46 Bhattarai S., Pokheral C. P., Lamichhane B. R., Regmi U. R., 2018; Lyu et al., 2019). The respective monophylies of A. Ram A. K., Subedi N. 2018. Amphibians and reptiles of Parsa ricketti group, A. daiyunensis group and A. hainanensis National Park, Nepal. Amphib Reptile Conse, 12(1): 35–48 group were well supported by mitochondrial data (Lyu Boulenger G. A. 1888. Description of two new Indian species of et al., 2019), while the monophylies of A. marmoratus . Ann Mag nat Hist, 6(2): 506–508 group, A. monticola group and A. mantzorum group have Boulenger G. A. 1920. A monograph of the south Asian, Papuan, not been tested, despite that the studies on each species Melanesian and Australian frogs of the genus Rana. Rec Indian group were conducted (Stuart et al., 2010; Dever et al., Museum, 20: 1–226 2012; Lu et al., 2014). Cai H. X., Che J., Pang J. F., Zhao E. M., Zhang Y. P. 2007. Paraphyly of Chinese Amolops (Anura, Ranidae) and Morphologically, Amolops pallasitatus sp. nov. can be phylogenetic position of the rare Chinese frog, Amolops steadily distinguished from all congeners but more similar tormotus. Zootaxa 1531: 49–55 to A. himalayanus and A. formosus whose molecular Che J., Pang J., Zhao H., Wu G.F., Zhao E.M., Zhang Y.P. data are unavailable. They differ in length of hind limbs, 2007. Phylogeny of Raninae (Anura: Ranidae) inferred from web of toe and dorsal colour pattern. In the molecular mitochondrial and nuclear sequences. Mol Phylogenet Evol. 43 analysis of this study, we used as many samples from A. (1): 1–13 marmoratus group, A. monticola group and A. mantzorum Chen L.Q., Murphy R. W., Lathrop A., Ngo A., Orlov N. L., Ho C. T., Somorjai I. L. M. 2005. Taxonomic chaos in Asian ranid group as we can, to reconstruct the phylogenetic frogs: An initial phylogenetic resolution. Herpetol J, 15 (4): relationship of these three species group from 231–243 southwestern China. The phylogenetic result disagreed Das I., Palden J. 2000. A herpetological collection from , with the monophylies of these three morphological with new country records. Herpetol Rev, 31(4): 256–258 species groups. The placements of A. chayuensis from Dever J. A., Fuiten A. M., Konu Ö., Wilkinson J. A. 2012. Cryptic A. monticola group, A. medogensis from A. marmoratus torrent frogs of Myanmar: an examination of the Amolops group, A. lifanensis and A. viridimaculatus from A. marmoratus species complex with the resurrection of Amolops afghanus and the identification of a new species. Copeia, (1): mantzorum group, and the newly described species 57–76 Amolops pallasitatus sp. nov. were unresolved, because Fei L., Ye C. Y., Huang Y. Z., Jiang J. P., Xie F. 2005. An of the relatively insignificant supports. illustrated key to Chinese amphibians. Chengdu, China: Sichuan Amolops pallasitatus sp. nov. forms the sister taxon Publishing House of Science and Technology, 1–340 (In to (A. medogensis + A. viridimaculatus), while with Chinese) relatively insignificant supports. Therefore in this study, Fei L., Hu S. Q., Ye C. Y., Huang Y. Z. 2009. Fauna Sinica. the new species is not assigned to any recognized species Amphibia Vol. 3 Anura Ranidae. Beijing, China: Science Press, group and further study on this topic will be explored. 1491–1585 (In Chinese) Fei L., Ye C. Y., Jiang J. P. 2012. Colored atlas of Chinese Acknowledgements This study is supported by Ministry amphibians and their distributions. Chengdu, China: Sichuan of Science and Technology of China (2014FY210200), Publishing House of Science and Technology, 1–619 (In Chinese) The Second National Survey of Terrestrial Wildlife Frost D. 2019. Amphibian Species of the World: an Online Resources in China and The Second National Survey Reference. Version 6.0. American Museum of Natural History, of Terrestrial Wildlife Resources in Tibet of China. We New York, USA. Retrieved from http://research.amnh.org/ would like to thank K. JIANG and Y. T. WANG for herpetology/amphibia/index.html No. 4 Shuo QI et al. A New Species of Amolops from Tibet, China 229

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Appendix I. List of specimens examined in this study. Amolops aniqiaoensis (n = 1): China, Tibet, Mêdog County (type locality): SYNU 1607058 Amolops chayuensis (n = 6): China, Tibet, Baxoi County: SYNU 1608048–54 Amolops mantzorum (n = 1): China, Sichuan, Tianquan County: SYNU 1807049 Amolops medogensis (n = 5): China, Tibet, Mêdog County (type locality): SYNU 1607034, 1607040–41, 1607068; China, Tibet, Mêdog County: SYS a006657 Amolops nyingchiensis (n = 2): China, Tibet, Zayü County: SYNU 1608023; Lhünzê County: SYNU 1807055 Amolops sp. (n = 3): China, Tibet, Mêdog County: SYNU 1607069; China, Tibet, Mêdog County, Beibeng Village: SYS a006638–39 Amolops viridimaculatus (n = 4): China, Yunnan, Tengchong County, Gaoligong Nature Reserve (type locality): SYS a003753–3754, 3812–3813 Amolops xinduqiao (n = 3): China, Sichuan, Xinduqiao Town (type locality), SYNU 1506002–04