A New Species of the Asian Toad Genus Megophrys Sensu Lato (Anura: Megophryidae) from Guizhou Province, China

ORIGINAL Asian Herpetological Research 2020, 11(1): 1–18 ARTICLE DOI: 10.16373/j.cnki.ahr.190041 A New Species of the Asian Toad Genus Megophrys sensu lato (Anura: Megophryidae) from Guizhou Province, China Jing LIU1, Shize LI1, 2, Gang WEI3, Ning XU3, Yanlin CHENG1, Bin WANG1, 4* and Jun WU2* 1 Department of Resources and Environment, Moutai Institute, Renhuai 564500, China 2 Nanjing Institute of Environmental Sciences, Ministry of Ecology and Environment of China, Nanjing 210042, China 3 Biodiversity Conservation Key Laboratory, Guiyang College, Guiyang 550002, China 4 Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China Abstract We describe a new species of the genus 1. Introduction Megophr ys from Guizhou Province, China. Molecular phylogenetic analyses based on mitochondrial DNA The Asian Horned toads of the subfamily Megophryinae and nuclear DNA sequences all strongly supported the (Amphibia: Anura: Megophryidae) is widely distributed in new species as an independent clade nested into the tropical and subtropical regions of Asia from India and Bhutan Megophr ys clade and sister to M. minor. On morphology, to China and south to the Sundas and the Philippines (Fei et the new species is distinguished from its congeners by al., 2012; Frost, 2019). Taxonomic assignments especially generic a combination of the following characters: (1) small arrangements in the group have been controversial (Dubois, body size with SVL < 39.2 mm in male and SVL < 40.4 1986; Dubois a nd Ohler, 1998; La throp, 1997; Tia n a nd Hu, 1983; mm in female; (2) vomerine teeth absent; (3) tongue Fei et al., 2009; Fei et al., 2012). Fei et al. (2016) classified 36 Chinese not notched behind; (4) a small horn-like tubercle at species of the subfamily Megophryinae into six genera, i.e. the edge of each upper eyelid; (5) tympanum distinctly Liuophr ys Fei, Ye and Jiang, 2016, Atympanophr ys Tian and Hu, visible, rounded; (6) two or three metacarpal tubercles 1983 containing three subgenera (Atympanophr ys, Borealophrys in hand; (7) relative finger lengths: I < II < V < III; (8) toes Fei, Ye and Jiang, 2016 and Gigantophr ys Fei, Ye and Jiang, without webbing; (9) heels overlapped when thighs are 2016), Boulenophr ys Fei, Ye and Jiang, 2016, Xenophr ys Günther, positioned at right angles to the body; (10) tibiotarsal 1864 containing two subgenera (Tianophr ys Fei and Ye, 2016 and articulation reaching tympanum to eye when leg Xenophr ys), Ophyrophr yne Boulenger, 1908 and Brachytarsophr ys stretched forward; (11) an internal single subgular vocal Tian and Hu, 1983. Chen et al. (2017) classified the Asian sac in male; (12) in breeding male, the nuptial pads with horned toads into the genera Atympanophr ys, Megophr ys black nuptial spines on the dorsal bases of the first and Kuhl and Van Hasselt, 1822 and Xenophr ys, Ophyrophr yne, second fingers. and Brachytarsophrys. Mahony et al. (2017) classified all members of Megophryinae into a single genus Megophr ys sensu lato including seven subgenera (Megophr ys, Xenophr ys, Keywords Taxonomy, new species, molecular phylogenetic Panophr ys Rao and Yang, 1997, Atympanophr ys, Ophyrophr yne, analysis, morphology, China Pelobatrachus Bedda rd, 1908 “1907” a nd Brachytarsophrys) based on molecular phylogenetics and morphological comparisons. *Corresponding author: Dr. Jun WU, from Nanjing Institute of Megophr ys s. l. currently contains 91 species (Frost, 2019), and Environmental Sciences, Ministry of Ecology and Environment of China, as note, of which, 37 species were described in the last decade Nanjing, China, with his research focusing on consevation management and biodiversity of amphibians; Dr. Bin WANG, from Chengdu Institute (Deuti et al., 2017; Fei et al., 2009; Li et al., 2014; Li et al., 2018a ; of Biology, Chinese Academy of Sciences, Chengdu, China, with his Mahony, 2011; Mahony et al., 2011; Mahony et al., 2013; Mahony research focusing on taxonomy and biodiversity of amphibians. E-mail: [email protected] (Jun WU); [email protected] (Bin WANG) et al., 2017; Mahony et al., 2018; Messenger et al., 2019; Mo et al., Received: 8 August 2019 Accepted: 29 October 2019 2010; Munir et al., 2018; Orlov et al., 2015; Tapley et al., 2017; Asian Herpetological Research 2 Vol. 11 Tapley et al., 2018; Wa ng et al., 2012; Wa ng et al., 2014; Wa ng in a mountain stream where the new taxon was found in the et al., 2017a, b; Wang et al., 2019; Ya ng et al., 2018; Zha ng et al., Kuankuoshui National Nature Reserve. They were identified 2017; Zhao et al., 2014). What’s more, molecular phylogenetic as the new taxon because they were almost identical in studies still surprisingly indicated mass of cryptic species in the morphology and one representative of them was genetically group, for example, 20 cryptic species were suggested by Chen close to the adult specimens of the new taxon (see the results). et al. (2017), and 41 cryptic species were proposed just only in The stages of tadpoles were identified following Gosner (1960). subgenus Megophr ys (Panophr ys) by Liu et al. (2018). Obviously, All specimens were fixed in 10% buffered formalin for one day, detailed investigations need to be conducted for describing the and then later transferred to 70% ethanol. Tissue samples were cryptic species. taken and preserved separately in 95% ethanol prior to fixation. During field surveys in the Kuankuoshui National Nature The specimens were deposited in Chengdu Institute of Biology, Reserve, Suiyang County, Guizhou Province, China and Chinese Academy of Sciences (CIB, CAS). Fanjing Mountain, Jiangkou County, Guizhou Province, China, 2.2. Molecular data and phylogenetic analyses Four adult we collected some Megophr ys s. l. specimens from the montane specimens and one tadpole of the new taxon were included forests. Our molecular phylogenetic analyses and morphological in molecular analyses (for voucher information see Table 1). comparisons indicated it as a new taxon of Megophrys s. l. Total DNA was extracted using a standard phenol-chloroform Herein, we describe it as a new species. extraction protocol (Sambrook et al., 1989). Two fragments of the mitochondrial genes encoding 16S rRNA and cytochrome 2. Materials and Methods oxidase subunit I (COI) were amplified using the primers in Simon et al. (1994) and Che et al. (2012), respectively. They 2.1. Specimen Two adult females and nine adult males of the were amplified under the following conditions: 35 cycles new taxon (for voucher numbers see Table S1) were collected at 95 °C for 4 min, 95 °C for 1 min, 52 °C (for 16S rRNA)/ from the mountain streams in the Kuankuoshui National 46 °C (for COI) for 40 s, and 72 °C for 1 min followed by a 10 Nature Reserve, Suiyang County, Guizhou Province, China and min extension at 72 °C. The nuclear gene fragments encoding Fanjing Mountain, Jiangkou County, Guizhou Province, China brain-derived neurotrophic factor (BDNF) and recombination (Figure 1). Two tadpoles (voucher numbers: CIBKKS20180426001 activating gene 1 (RAG1) were amplified using the primers and and CIBKKS20180426002) of the new taxon were also collected protocols in Vieites et al. (2007) and Shen et al. (2013). All primers Figure 1 Sampling localities of Megophrys jiangi sp. nov. in this study. The type locality is Kuankuoshui National Nature Reserve, Suiyang County, Guizhou Province, China and another locality is Fanjing Mountain, Jiangkou County, Guizhou Province, China. Jing LIU et al. A New Species of Megophrys No. 1 Table 1 Information of samples used in the molecular analyses in this study. Sample GenBank accession number Species Voucher number Locality Number 16S COI RAG1 BDNF 1 Megophrys jiangi sp. nov. CIBKKS20180722006 Kuankuosui Nature Reserve, Guizhou, China MN107743 MN107748 MN107757 MN107753 2 Megophrys jiangi sp. nov. CIBKKS20180426001 Kuankuosui Nature Reserve, Guizhou, China MN107744 MN107749 MN107758 MN107754 3 Megophrys jiangi sp. nov. CIBKKS20180723007 Kuankuosui Nature Reserve, Guizhou, China MN107745 MN107750 MN107759 – 4 Megophrys jiangi sp. nov. CIBFJS20150719009 Fanjingshan, Guizhou, China MN107746 MN107751 – MN107755 5 Megophrys jiangi sp. nov. CIBFJS20150720004 Fanjingshan, Guizhou, China MN107747 MN107752 MN107760 MN107756 6 Megophrys jiangi sp. nov. KIZ021889 Fanjingshan, Guiz`hou, China KX811912 – – – 7 Megophrys spinata KIZ016100 Leigong Shan, Guizhou, China KX811864 KX812119 KX812228 KX811963 8 Megophrys sangzhiensis Tissue ID: YPX11006 Badagongshan Nature Reserve, Hunan, China KX811856 KX812117 KX812226 KX811961 9 Megophrys binlingensis KIZ025807 Wawu Shan, Sichuan, China KX811852 KX812115 KX812221 KX811970 10 Megophrys binchuanensis KIZ019441 Jizu Shan, Yunnan, China KX811849 KX812112 KX812219 KX811952 11 Megophrys palpebralespinosa KIZ011603 Pu Hu Nature Reserve, Thanh Hoa, Vietnam KX811888 KX812137 KX812236 KX811994 12 Megophrys jingdongensis KIZ-LC0805067 Huanglianshan National Nature Reserve, Yunnan, China KX811872 KX812131 KX812232 KX811973 13 Megophrys daweimontis KIZ048997 Dawei Shan, Yunnan, China KX811867 KX812125 KX812248 KX811969 14 Megophrys wuliangshanensis KIZ046812 Huangcaoling, Yunnan, China KX811881 KX812129 KX812258 KX811958 15 Megophrys omeimontis KIZ025765 Emei Shan, Sichuan, China KX811884 KX812136 KX812223 KX811967 16 Megophrys fansipanensis VNMN 2018.01 Lao Cai, Sa Pa, Vietnam MH514886 – – – 17 Megophrys hoanglienensis VNMN 2018.02 Lao Cai, Sa Pa, Vietnam MH514889 – – – 18 Megophrys minor KIZ01939 Qingcheng Shan, Sichuan, China KX811896 KX812145 KX812253 KX812045 19 Megophrys minor Tissue ID: YPX37544

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