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Research Article
A species of the genus Panophrys (Anura, Megophryidae) from southeastern Guizhou Province, China
expand article infoTao Luo, Yali Wang, Siwei Wang, Xueli Lu, Weifeng Wang, Huaiqing Deng, Jiang Zhou
‡ Guizhou Normal University, Guiyang, China
Open Access

Abstract

Herein, we describe Panophrys congjiangensis sp. nov. obtained from the Yueliangshan Nature Reserve, Congjiang County, Guizhou Province, China. Phylogenetic analyses based on the mitochondrial genes 16S rRNA and COI indicated that this new species represented an independent lineage, closely related to P. leishanensis. The uncorrected genetic distances between the new species and its closest congener, P. leishanensis, were 3.0% for 16S rRNA and 8.4% for COI. The new species is distinguished from its congeners by a combination of the following morphological characteristics (1) medium body size (SVL 28.6–33.4 mm in males and 38.4–40.2 mm in females); (2) a small horn-like tubercle at the edge of each upper eyelid; (3) the tympanum distinctly visible (TD/ED ratio 0.47–0.66); (4) vomerine teeth absent; (5) the tongue not notched behind; (6) a narrow and unobvious lateral fringe on toes; (7) relative finger lengths II < I < V < III; (8) rudimentary webs on toes; (9) hindlimbs slender, heels overlapping when thighs are positioned at right angles to the body; (10) two metacarpal tubercles on the palm, with the inner metatarsal tubercle long and oval-shaped; (11) the tibiotarsal articulation reaching the nostril when the leg is adpressed and stretched forward; (12) dorsal skin rough with numerous orange–red granules, ventral surface smooth; (13) a single internal subgular vocal sac present in males; and (14) in breeding males, weak gray-black nuptial pads with black nuptial spines present on the dorsal surface of the bases of the first and second fingers. To date, the new species is only known from the type locality.

Keywords

Morphology, new species, Panophrys, southwest China, taxonomy

Introduction

The Asian horned toad subfamily Megophryinae (Bonaparte, 1850) is widely distributed in southern China, the southern and eastern Himalayas, and across Indochina to the islands of the Sunda Shelf and the Philippines (Fei and Ye 2016; Mahony et al. 2017; Liu et al. 2018; Frost 2021). The widespread distributions and morphological similarities of subfamily Megophryinae species have long made the discrimination of species in this subfamily controversial (Dubois 1987 “1986”; Rao and Yang 1997; Dubois and Ohler 1998; Jiang et al. 2003; Zheng et al. 2004; Frost et al. 2006; Li and Wang 2008; Fei et al. 2009; Fei and Ye 2016; Chen et al. 2017; Mahony et al. 2017). In recent phylogenetic analyses, subfamily Megophryinae has been recognized as a broadly monophyletic genus (i.e., Megophrys sensu lato; Chen et al. 2017; Mahony et al. 2017; Liu et al. 2018). These revisions have recommended that subfamily Megophryinae be divided into seven subgenera under the genus Megophrys sensu lato: Atympanophrys Tian & Hu, 1983; Brachytarsophrys Tian & Hu, 1983; Megophrys s.s Kuhl & Van Hasselt, 1822; Ophryophryne Boulenger, 1903; Pelobatrachus Beddard, 1908; Panophrys Rao & Yang, 1997; and Xenophrys Günther, 1864. To resolve these classification conflicts, Li et al. (2020a) suggested to elevate the seven monophyletic subgenera by Mahony et al. (2017) to the level of genera, namely: Atympanophrys, Brachytarsophrys, Megophrys, Ophryophryne, Panophrys, Pelobatrachus, and Xenophrys. In this study, we have followed this recommendation (Li et al. 2020a; Frost 2021).

In the most recent revision, 59 species were assigned to the genus Panophrys (Frost 2021), 40 of these species were described in the past decade. In total, 55 Panophrys species have been described from China (see Panophrys species list in Frost 2021), and 11 are known specifically from Guizhou Province, i.e., Panophrys anlongensis (Li, Lu, Liu & Wang, 2020), Panophrys binlingensis (Ye & Fei, 1995), Panophrys chishuiensis (Xu, Li, Liu, Wei & Wang, 2020), Panophrys jiangi (Liu, Li, Wei, Xu, Cheng, Wang & Wu, 2020), Panophrys leishanensis (Li, Xu, Liu, Jiang, Wei & Wang, 2018), Panophrys liboensis (Zhang, Li, Xiao, Li, Pan, Wang, Zhang & Zhou, 2017), Panophrys omeimontis (Liu, 1950), Panophrys shuichengensis (Tian & Sun, 1995), Panophrys spinata (Liu & Hu, 1973), Panophrys platyparietus (Rao & Yang, 1997), and Panophrys qianbeiensis (Su, Shi, Wu, Li, Yao, Wang & Li, 2020). All of these species inhabit isolated mountain streams in evergreen broadleaf forests in Guizhou Province. Such isolated conditions may be favorable for species formation. For example, P. spinata has historically been recorded from several counties in Guizhou Province (Dafang, Jinsha, Suiyang, Jiangkou, Yinjiang, and Leishan; Wu et al. 1986; Fei et al. 2009). A recent phylogenetic analysis showed that the Suiyang population, originally recorded as P. spinata, was genetically closer to P. spinata and Panophrys sangzhiensis (Jiang, Ye & Fei, 2008). Thus, the Suiyang population was described as a new species, P. qianbeiensis (Su et al. 2020). Therefore, the diversity of Panophrys may be greater in Guizhou Province than is currently assumed.

During herpetological surveys conducted between 2019 and 2020 in Yueliangshan Nature Reserve, Congjiang County, Guizhou Province, China (Fig. 1), we captured several specimens of an unknown anuran species. Based on morphological characteristics, including body size (i.e., body length < 45 mm) and a small horn-like tubercle at the middle edge of each upper eyelid, these specimens were identified as a species of Panophrys, initially P. minor (Fei et al. 2009; Fei and Ye 2016). However, subsequent observation indicated that these newly collected specimens differed from any currently described Panophrys species. Indeed, molecular phylogenetics, comparative morphology, and bioacoustics data suggest that these specimens represent a previously unknown species. This new species is described herein.

Material and methods

Sampling

A total of 25 specimens were collected in this study: 22 were collected in Congjiang County, Guizhou Province, China, and were identified as an unknown species. The remaining 3 specimens, collected in Kuankuoshui National Nature Reserve, Suiyang County, Guizhou Province, China, were identified as P. jiangi. All specimens were fixed in 10% buffered formalin and later transferred to 75% ethanol for preservation. The muscle samples used for molecular analysis were preserved in 95% alcohol and stored at -20 °C. All specimens are housed at Guizhou Normal University (GZNU), Guiyang City, Guizhou Province, China.

DNA extraction, PCR, and sequencing

Genomic DNA was extracted from the muscle tissue samples using DNA extraction kits (Tiangen Biotech (Beijing) Co., Ltd.). We amplified and sequenced two mitochondrial genes from each DNA sample: partial 16S ribosomal RNA (16S rRNA), using primers L3975 (5’-CGCCTGTTTACCAAAAACAT-3’) and H4551 (5’-CCGGTCTGAACTCAGATCACGT-3’) following Simon et al. (1994); and cytochrome C oxidase I (COI), using primers Chmf4 (5’-TYTCWACWAAYCAYAAAGAYATCGG-3’) and Chmr4 (5’-ACYTCRGGRTGRCCRAARAATAATCA-3’) following Che et al. (2012). PCR amplifications were performed in 25 μL reaction volumes with the following cycling conditions: an initial denaturing step at 95 °C for five min; 36 cycles of denaturation at 95 °C for 40 s, annealing at 52 °C (for 16S rRNA) or 47 °C (for COI) for 40 s, then extension at 72 °C for 1 min; and a final extension step at 72 °C for 10 min. The purified PCR products were sequenced with both forward and reverse primers using BigDye Terminator Cycle Sequencing Kits, following the manufacturer’s instructions, on an ABI Prism 3730 automated DNA sequencer by Tsing KE Biological Technology Co. Ltd. (Chengdu, China). All sequences have been deposited in GenBank (Table 1).

Table 1.

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

ID Species Locality Voucher number Genbank accession No.
16S rRNA COI
1 Panophrys obesa Heishiding Nature Reserve, Guangdong, China SYS a002271 KJ579121 MH406123
2 Panophrys ombrophila Wuyi Shan, Fujian, China WUYI2015101 KX856397
3 Panophrys cheni Taoyuandong Nature Reserve, Hunan, China SYS a002123 KJ560396 MF667904
4 Panophrys dongguanensis Yinping Shan, Guangdong, China SYS a002007 MH406654 MH406090
5 Panophrys nankunensis Nankun Shan, Guangdong, China SYS a004498 MK524108 MK524139
6 Panophrys wugongensis Wugongshan Scenic Area, Jiangxi, China SYS a002610 MK524114 MK524145
7 Panophrys insularis Nan’ao Island, Guangdong, China SYS a002169 MF667887 MF667924
8 Panophrys lini Nanfengmian Nature Reserve, Jiangxi, China SYS a002128 KJ560416 MF667907
9 Panophrys nanlingensis Nanling Nature Reserve, Guangdong, China SYS a001959 MK524111 MK524142
10 Panophrys xiangnanensis Yangming Shan, Hunan, China SYS a002875 MH406714 MH406166
11 Panophrys baishanzuensis Baishanzu National Park, Qingyuan,
Zhejiang, China
CIBQY20200719001 MW001150 MT998291
12 Panophrys brachykolos Hongkong, China SYS a005563 MK524122 MK524153
13 Panophrys kuatunensis Wuyi Shan, Jiangxi, China SYS a003449 MF667881 MF667916
14 Panophrys lishuiensis Lishui City, Zhejiang, China CIBWYF00169 KY021418
15 Panophrys xianjuensis Xianju, County, Zhejiang, China CIBXJ20190801 MN563754 MN563770
16 Panophrys jinggangensis Jinggang Shan, Jiangxi, China SYS a004028 MH406780 MH406239
17 Panophrys liboensis Libo Country, Guizhou, China GZNU20150813001 MF285253 MW959767
18 Libo Country, Guizhou, China GZNU20160408007 MF285258 MW959768
19 Libo Country, Guizhou, China GZNU20160408006 MF285257 MW959769
20 Libo Country, Guizhou, China GZNU20160408004 MF285256 MW959770
21 Panophrys boettgeri Longhu Forest Station, Fujian, China SYS a004126 MH406785 MH406245
22 Panophrys huangshanensis Huang Shan, Anhui, China SYS a002702 MF667882 MF667919
23 Panophrys congjiangensis sp. nov. Yueliangshan Nature Reserve, Congjiang, Guizhou, China GZNU20200706003 MW959773 MW959761
24 Yueliangshan Nature Reserve, Congjiang, Guizhou, China GZNU20200706004 MW959774 MW959762
25 Yueliangshan Nature Reserve, Congjiang, Guizhou, China GZNU20200706005 MW959775 MW959763
26 Yueliangshan Nature Reserve, Congjiang, Guizhou, China GZNU20200706000 MW959776 MW959764
27 Panophrys leishanensis Leigong Shan, Guizhou, China CIBLS20141004003 MK005308 MK005304
28 Leigong Shan, Guizhou, China SYSa002213 MH406673 MH406113
29 Leigong Shan, Guizhou, China CIBLS20160610005 MK005309 MK005305
30 Panophrys baolongensis Baolong, Chongqing, China KIZ019216 KX811813 KX812093
31 Panophrys wushanensis Shennongjia Nature Reserve, Hubei, China SYS a003008 MH406732 MH406184
32 Panophrys tuberogranulata Badagong Shan, Hunan, China SYS a004310 MH406801 MH406263
33 Panophrys shimentaina Shimentai Nature Reserve, Guangdong, China SYS a002078 MH406656 MH406093
34 Panophrys yangmingensis Yangming Shan, Hunan, China SYS a002889 MH406720 MH406172
35 Panophrys jiulianensis Nankun Shan, Guangdong, China SYS a003623 MK524103 MK524134
36 Panophrys mirabilis Huaping Nature Reserve, Guangxi, China SYS a002193 MH406670 MH406110
37 Panophrys shunhuangensis Nanshan National Forest Park, Hunan, China HNNU18NS01 MK836023 MK977594
38 Panophrys acuta Heishiding Nature Reserve, Guangdong, China SYS a002159 MF667869 MF667899
39 Panophrys mufumontana Mufu Shan, Hunan, China SYS a006390/CIB110012 MK524104 MK524135
40 Panophrys caudoprocta Badagong Shan, Hunan, China SYS a004281 MH406795 MH406257
41 Panophrys sangzhiensis Badagong Shan, Hunan, China SYS a004307 MH406798 MH406260
42 Panophrys spinata Leigong Shan, Guizhou, China SYS a002226 MH406675 MH406115
43 Panophrys qianbeiensis Huanglian Nature Reserve, Guizhou, China CIBTZ20190608015 MT651553 MT654520
44 Panophrys binlingensis Wawu Shan, Sichuan, China SYS a005313 MH406892 MH406354
45 Panophrys binchuanensis Jizu Shan, Yunnan, China KIZ019441 KX811849 KX812112
46 Panophrys angka Kiew Mae Pan nature trail,
Chiang Mai, Thailand
KIZ040591 MN508052
47 Panophrys anlongensis Anlong County, Guizhou, China CIBAL20190531018 MT823184 MT823261
48 Panophrys omeimontis Laojun Shan, Sichuan, China SYS a002741 MH406710 MH406162
49 Panophrys palpebralespinosa Pu Hu Nature Reserve, Thanh Hoa, Vietnam KIZ011603 KX811888 KX812137
50 Panophrys caobangensis Nguyen Binh, Cao Bang,Vietnam IEBR 4385 LC483945
51 Panophrys daweimontis Dawei Shan, Yunnan, China KIZ048997 KX811867 KX812125
52 Panophrys jingdongensis Wuliang Shan, Yunnan, China SYS a003928 MH406773 MH406232
53 Panophrys rubrimera Lao Cai, Sa Pa, Vietnam AMSR177676 MF536419
54 Panophrys wuliangshanensis Wuliang Shan, Yunnan, China SYS a003924 MH406771 MH406230
55 Panophrys fansipanensis Lao Cai, Sa Pa, Vietnam VNMN 2018.01 MH514886
56 Panophrys hoanglienensis Lao Cai, Sa Pa, Vietnam VNMN 2018.02 MH514889
57 Panophrys jiangi Huoqiuba Nature Reserve, Guizhou, China GZNU20180606020 MW959777 MW959765
58 Kuankuosui Nature Reserve, Guizhou, China GZNU20070712001 MW959778 MW959766
59 Kuankuosui Nature Reserve, Guizhou, China CIBKKS20180722006 MN107743 MN107748
60 Panophrys minor Qingcheng Shan, Sichuan, China SYS a003209 MF667862 MF667891
61 Panophrys chishuiensis Chishui, County, Guizhou, China CIBCS20190518031 MN954707 MN928958
62 Ophryophryne pachyproctus Beibeng, Xizang, China KIZ010978 KX811908 KX812153
63 Panophrys yeae Didong, Medog, Tibet, China CIB201706MT01 MN963217 MN964312
64 Panophrys zhoui Renqinbeng, Medog, Tibet, China CIBMT171053 MN963207 MN964322
65 Xenophrys vegrandis West Kameng district, Arunachal Pradesh, India ZSI A 11605 KY022305 MH647530
66 Ophryophryne elfina Bidoup Mountain, Lam Dong, Vietnam ZMMU ABV000454 KY425379
67 Ophryophryne gerti Nui Chua National Park, Ninh Thuan, Vietnam ITBCZ 1108 KX811917 KX812161
68 Ophryophryne synoria O’Reang, Mondolkiri, Cambodia FMNH 262779 MN629394
69 Ophryophryne hansi Phong Dien Nature Reserve, Thua Thien Hue, Vietnam KIZ010360 KX811913 KX812155
70 Ophryophryne microstoma Wuhuang Shan,, Guangxi, China SYS a003492 MK524125 MK524156
71 Megophrys montana Sukabumi, Java, Indonesia LSUMZ 81916 KX811927 KX812163
72 Megophrys parallela RMAS 021 KY679897
73 Megophrys lancip Ulubelu, Ngarip, Indonesia MZB:Amp:22233 KY679891
74 Xenophrys medogensis Medog County, Tibet, China SYS a002932 MH406725 MH406177
75 Xenophrys robusta Darjeeling dist, West Bengal, India SDBDU 2011.1057 KY022314 MH647535
76 Xenophrys glandulosa Gaoligong Shan,, Yunnan, China SYS a003758 MH406755 MH406214
77 Xenophrys himalayana East Siang dist, Arunachal Pradesh, India SDBDU2009.75 KY022311
78 Xenophrys periosa East Siang dist, Arunachal Pradesh, India BNHS 6061 KY022309 MH647528
79 Xenophrys monticola Darjeeling dist, West Bengal, India SDBDU 2011.1047 KX894679
80 Xenophrys zhangi Zhangmu, Xizang, China KIZ014278 KX811765 KX812084
81 Xenophrys flavipunctata Hills dist, East Khasi, Meghalaya SDBDU2009.297 KY022307 MH647536
82 Xenophrys mangshanensis Longtou glandulosa, Guangdong, China SYS a002750 MF667866 MF667895
83 Xenophrys maosonensis Xiaoqiaogou Nature Reserve, Yunnan, China KIZ016045 KX811780 KX812080
84 Xenophrys oreocrypta West Garo Hills dist, Meghalaya BNHS 6046 KY022306
85 Xenophrys major Zhushihe, Yunnan, China SYSa002961 MH406728 MH406180
86 Xenophrys awuh SDBDU2007.161 KY022319
87 Xenophrys serchhipii North dist, Tripura, India SDBDU 2009.612 KY022323 MH647532
88 Xenophrys zunhebotoensis SDBDU 2009.110 KY022321
89 Xenophrys ancrae Changlang dist, Arunachal Pradesh, India SDBDU 2009.727 KY022318 MH647531
90 Xenophrys numhbumaeng SDBDU 2007.041 KY022316
91 Xenophrys oropedion Mawphlang Sacred Forest, Meghalaya, India SDBDU 2009.299 KY022317 MH647534
92 Xenophrys megacephala ZSI A 11213 KY022315 MH647533
93 Xenophrys dzukou SDBDU2007.106 KY022324
94 Xenophrys lekaguli Pang Si Da National Park, Sa Kaeo, Thailand FMNH 265955 KY022214
95 Xenophrys takensis FMNH 261711 KY022215
96 Xenophrys auralensis Aural, Kampong Speu, Cambodia NCSM 79599 KX811807
97 Xenophrys parva Zhushihe, Yunnan, China SYSa003042 MH406737 MH406189
98 Xenophrys aceras Khao Nan National Park, Nakhon Si Thammarat, Thailand KIZ025467 KX811925 KX812159
99 Xenophryslongipes Genting highland, Malaysia IABHU 21101 AB530656
100 Atympanophrys gigantica Ailao Shan, Yunnan, China SYS a003883 MH406766 MH406225
101 Atympanophrys shapingensis Wawu Shan, Sichuan, China SYS a005310 MH406890 MH406352
102 Atympanophrys nankiangensis Nanjiang, Sichuan, China CIB ZYC517 KX811900
103 Atympanophrys wawuensis Wawu Shan, Sichuan, China KIZ025799 KX811902 KX812062
104 Brachytarsophrys feae Huangcaoling, Yunnan, China KIZ046706 KX811810 KX812056
105 Brachytarsophrys platyparietus W01395 AY526206
106 Brachytarsophrys chuannanensis Hejiang County, Sichuan, China SYS a004926 MH406901 MH406364
107 Brachytarsophrys carinense Dayao Shan, Guangxi, China Tissue ID: YPX20455 KX811811 KX812057
108 Brachytarsophrys popei Jinggang Shan, Jiangxi, China SYS a004209 MK524124 MK524155
109 Brachytarsophrys intermedia Phong Nha0Ke Bang NP, U Bo, Vietnam ZFMK 87596 HQ588950
110 Megophrysdringi Mulu National Park, Sarawak Gunung, Malaysia UNIMAS 8943 KJ831317
111 Pelobatrachus baluensis Gunung Kinabalu National Park, Kogopan Trail, Malaysia ZMH A13125 KJ831310
112 Pelobatrachus stejnegeri Pasonanca Natural Park, Zamboanga, Philippines KU 314303 KX811922 KX812052
113 Pelobatrachus kobayashii Gunung, Sabah, Meghalaya UNIMAS 8148 KJ831313
114 Pelobatrachus ligayae Palawan, Philippines ZMMU NAP005015 KX811919 KX812051
115 Pelobatrachus kalimantanensis Kalimantan Selatan, Borneo, Indonesia MZB. Amph 21482 MG993554
116 Pelobatrachus nasuta Sabah, Lahad Datu District, Malaysia FMNH 231281 KY022186
117 Pelobatrachus edwardinae Bintulu, Sarawak, Malaysia FMNH 273694 KX811918 KX812050
118 Leptobrachium boringii Emei Shan, Sichuan, China Tissue ID: YPX37539 KX811930 KX812164
119 Leptobrachella oshanensis Emei Shan, Sichuan, China KIZ025778 KX811928 KX812166

Phylogenetic analyses

We used a total of 194 gene sequences (112 16S rRNA sequences and 82 COI sequences) for the molecular analyses, representing 102 species of subfamily Megophryinae. Two mitochondrial genes were sequenced in 10 muscle tissue samples from the specimens collected in this study, and 178 sequences were downloaded from GenBank. Samples included those from the undescribed species collected and named in this study (Fig. 1). Following Mahony et al. (2017), we selected Leptobrachium boringii (Liu, 1945) and Leptobrachella oshanensis (Liu, 1950) as outgroups. The two outgroup sequences were obtained from GenBank. Details of the sequences used for phylogenetic analysis are given in Table 1.

Figure 1. 

Sampling collection localities and distribution of the Panophrys congjiangensis sp. nov., P. leishanensis, P. liboensis, and P. jiangi in Guizhou Province, China 1 Yueliangshan Nature Reserve, Congjiang County, Guizhou Province 2 Leigongshan National Nature Reserve, Leishan County, Guizhou Province 3 Maolan National Nature Reserve, Libo County, Guizhou Province 4, 5 Huoqiuba Nature Reserve, Suiyang County, Guizhou Province 6 Xingxiu Township, Dafang County, Guizhou Province 7 Fenghuang Township, Shuicheng County, Guizhou Province. The base maps are from Standard Map Service website (http://bzdt.ch.mnr.gov.cn/index.html).

All sequences were assembled and aligned using the MUSCLE (Edgar 2004) module in MEGA 7.0 (Kumar et al. 2016) with default settings. Alignments were checked by eye and revised manually if necessary. Trimming, with gaps partially deleted, was performed using GBLOCKS 0.91b (Castresana 2000). The best-fit partitioning schemes and corresponding substitution models for the concatenated-sequence supermatrix were selected in PartitionFinder 2.1.1 using the Bayesian information criterion (Lanfear et al. 2016). As a result, the analysis suggested that the best partition scheme 16S rRNA gene/each codon position of COI gene, and selected GTR+I+G model as the best model for 16S rRNA gene, and TRNEF+G, HKY+I+G, and TIM+G model as the best model for first, second and third codons position of COI gene, respectively. Phylogenetic analysis of the concatenated-sequence matrix was performed using maximum likelihood (ML) and Bayesian inference (BI). ML and BI phylogenies based on the concatenated-sequence matrix were constructed using both IQ-tree 2.0.4 (Nguyen et al. 2015) and MrBayes 3.2.1 (Ronquist et al. 2012), according to the best-fit partitioning schemes and the corresponding substitution models. The ML analysis was performed using the best-fit model for each partition with 2000 ultrafast bootstrap (UFB) replicates (Minh et al. 2013); the analysis was continued until a correlation coefficient of at least 0.99 was reached (Hoang et al. 2018). We performed two independent BI runs using four Markov chains (three heated chains and a single cold chain). The best-fit partitioning schemes and corresponding substitution models were selected. The BI analysis started with a random tree; each run consisted of 2 × 107 generations, sampled every 1000 generations. Runs were considered to have converged when the average standard deviation of split frequencies (ASDSF) was less than 0.01, and the effective sample sizes (ESS) in Tracer 1.7.1 (Rambaut et al. 2018) was greater than 200. Nodes in the trees were considered well-supported when Bayesian posterior probabilities (BPP) were ≥ 0.95 and ML ultrafast bootstrap values (UFB) were ≥ 95%. The phylogenetic trees were visualized using FigTree 1.4.3 (Rambaut 2016). The uncorrected p-distance model in MEGA 7.0 (Kumar et al. 2016) was used to calculate average genetic distances among species based on 16S rRNA and COI.

Species delimitation

To assess whether new species represent a valid species, two different methods were executed. We chose to include new species in the phylogenetic tree as well as several closely related species for species delimitation analysis. First, a Bayesian hypothesis-testing approach (Bayes Factor Delimitation, BFD) was implemented to statistically test alternate hypotheses of species delimitation (Gummer et al. 2014). Two species models were tested: 11 species (contains new species) and 10 species (lump new species with P. leishanensis). All analyses were performed in *BEAST using BEAST 1.8.2 (Drummond et al. 2012) under an uncorrelated lognormal relaxed molecular clock. A Yule process was used for the species tree prior, and the piecewise linear and constant root was used for the population size model. Two independent runs for each model were performed in BEAST 1.8.2 to assess convergence of the MCMC runs. *BEAST was run each time for 1×107 generations of the MCMC algorithm sampling every 1000 generations and discarding the first 25% of the iterations as “burn-in”. After *BEAST analyses, two methods of marginal-likelihood estimation, including path-sampling (PS; Baele et al. 2012) and stepping-stone analysis (SS; Xie et al. 2011), were performed. PS and SS analyses were each run for a chain length of 1×106 generations for 100 path steps. We followed the suggestions provided by Gummer et al. (2014) to assess the strength of support for a particular species delimitation hypothesis.

In addition to the Bayesian methods tested, we also applied three tree-based species-delimitation methods, i.e., Bayesian implementation of the Poisson Tree Processes model (bPTP; Zhang et al. 2013). The parameters of these three analyses were set as follows: 1×105 generations, a thinning of 100 and burn-in of 10%. Convergence of models were assessed by visualizing plots of the MCMC iteration vs. the Log likelihood results. The bPTP analysis was conducted on the bPTP web server (http://species.h-its.org/ptp/) using mtDNA-based BI gene tree as input.

Morphological comparisons

Table 2.

Measurements of the adult specimens of Panophrys congjiangensis sp. nov. All units in mm. See abbreviations for the morphological characters in the Materials and Methods section. (M = male, F = female, other abbreviations defined in text), * for the holotype.

Voucher number Sex SVL HDL HDW SNT ED IOD IND TD UEW NED TED HND LAHL LW FIL FIIL FIIIL FIVL TL THL FL TFL HLL TW IPTL OPTL IMTL
GZNU20200706010* M 33.4 11.3 11.1 4.2 3.8 3.1 3.7 2.4 2.9 2.3 2.0 8.6 14.7 2.3 4.5 4.1 6.3 5.5 17.5 17.8 15.7 24.9 60.2 3.5 1.8 1.6 2.2
GZNU20200706001 M 33.1 11.2 11.4 4.2 3.5 3.2 3.7 2.3 2.8 2.5 1.9 8.3 14.3 2.1 4.7 4.2 6.1 5.2 16.7 16.5 15.6 23.6 56.8 3.2 2.1 1.5 2.3
GZNU20200706002 M 30.1 11.2 10.8 3.9 3.5 3.5 3.4 2.1 2.4 1.9 1.9 7.9 13.6 2 4.6 4.2 5.8 5.2 14.9 14.6 14.7 20.8 50.3 3.1 1.9 1.3 2.3
GZNU20200706003 M 30.6 10.8 10.3 4.0 3.6 3.7 3.1 2.3 2.7 2.4 1.8 7.7 13.9 2.1 3.9 3.3 6.2 4.9 15.3 14.6 13.4 20.8 50.7 2.6 2.0 1.2 1.8
GZNU20200706004 M 32.3 11.2 11.1 4.3 3.3 4.3 3.4 2.0 2.8 2.2 2.0 8.3 14.5 2.3 4.3 4.5 6.1 4.7 16.5 15.4 15.1 22.5 54.4 3.3 2.1 1.2 2.0
GZNU20200706005 M 29.8 11.0 11.2 4.4 3.7 3.7 3.3 2.0 2.6 2.5 2.2 8.2 14.3 2 3.7 5.7 7.7 5.9 15.9 15.3 14.6 22.8 54.0 3.1 1.6 1.2 1.7
GZNU20200706006 M 30.4 11.2 10.6 4.2 3.3 3.7 3.3 1.7 2.5 2.7 1.7 7.3 13.2 1.9 4.3 4.0 5.6 4.4 15.6 15.1 13.7 21.1 51.8 3.2 2.1 1.7 1.9
GZNU20200706007 M 28.6 10.2 9.1 3.9 3.4 3.9 2.8 1.7 2.7 2.3 2.1 7.5 13.9 1.9 3.9 3.6 5.8 4.1 14.6 13.8 13.5 20.9 49.3 2.8 1.8 1.6 1.9
GZNU20200706008 M 31.4 10.8 10.4 4.9 3.6 3.2 3.3 2.1 2.9 2.2 2.0 8.23 14.7 1.7 4.4 4.3 6.3 4.5 16.2 16.3 13.8 22.1 54.6 2.9 2.3 1.4 1.8
GZNU20200706009 M 31.1 10.7 11.2 3.4 3.9 3.2 3.5 2.4 2.9 1.9 1.9 7.9 14.8 2.4 4.4 3.9 6.3 5.1 15.6 15.2 14.5 22.9 53.7 3.1 1.7 1.4 1.9
GZNU20200706012 M 33.3 11.5 10.9 4.3 3.6 3.8 3.3 2.2 2.6 1.9 2.1 8.1 14.6 2.1 4.3 4.9 6.1 4.8 16.2 16.1 15.3 23.5 55.8 3.4 1.8 1.2 2.1
GZNU20200706013 M 30.2 10.6 9.8 4.1 3.6 3.5 3.1 1.7 3.0 1.9 1.9 7.9 13.8 2.1 4.5 3.9 5.7 4.9 14.8 13.4 13.5 22.1 50.3 3.1 2.3 1.4 2.2
GZNU20200707001 M 31.2 11.4 10.4 4.1 3.9 3.9 3.4 2.3 2.9 2.4 1.9 8.5 15.2 2.0 4.5 4.5 6.9 4.9 16.9 15.5 15.1 23.1 55.5 3.4 1.7 1.4 2.0
GZNU20200707002 M 30.1 11.2 10.8 3.9 3.5 3.5 3.4 2.1 2.4 1.9 1.9 7.9 13.6 2 4.1 3.6 5.7 4.9 14.9 14.6 14.7 20.8 50.3 3.1 2.2 1.6 1.9
GZNU20200707003 M 31.8 11.4 11.1 4.5 3.5 3.7 3.3 2.3 2.9 2.3 1.9 8.5 14.9 2.4 4.7 3.8 6.5 4.8 16.6 15.6 15.1 22.4 54.6 3.2 2.5 1.5 2.6
GZNU20200706011 F 38.4 12.1 11.8 4.5 4.8 3.8 3.5 2.5 3.7 2.5 1.8 8.9 16.2 2.0 5.1 4.3 6.6 5.7 19.1 18.6 16.6 25.7 63.4 3.9 2.8 2.1 2.2
GZNU20200706004 F 39.2 13.2 11.7 4.3 4.3 3.7 3.9 2.5 3.9 2.3 1.9 9.5 16.3 1.9 4.8 5.9 7.5 6.4 19.2 19.5 17.8 26.3 65 3.6 2.9 2.3 2.4
GZNU20200706005 F 39.5 13.3 11.5 4.4 4.2 3.8 3.5 2.4 3.8 2.6 1.9 9.1 16.2 2.1 4.8 5.9 7.7 6.4 19.3 19.2 18.2 26.2 64.7 3.5 2.8 2.4 2.6
GZNU20200706006 F 40.2 14.5 13.2 5.1 5.2 4.5 4.8 3.4 3.9 2.5 2.2 9.8 17.4 2.0 4.8 6.0 7.7 6.8 20 19.9 18.9 26.8 66.7 3.8 2.9 2.9 2.9

Morphometric data were collected from 19 well-preserved adult specimens (voucher information given in Table 2). Measurements were recorded to the nearest 0.1 mm with digital calipers by Tao Luo following Fei et al. (2009). A total of 27 morphological features were measured in each well-preserved specimen. These following measurements were taken:

ED eye diameter (diameter of exposed portion of eyeball);

FIL first finger length;

FIIL second finger length;

FIIIL third finger length;

FIVL fourth finger length;

FL foot length (distance from distal end of tibia to the tip of the distal phalanx of toe IV);

HDL head length (from tip of snout to the articulation of the jaw);

HDW head width (head width at the commissure of the jaws);

HLL hindlimb length (distance from tip of fourth toe to vent);

HND hand length (from the proximal border of the outer palmar tubercle to the tip of digit III);

IMTL inner metatarsal tubercle length (taken as maximal length of inner metatarsal tubercle);

IND internasal distance (distance between nares);

IOD interorbital distance (minimum distance between upper eyelids);

IPTL inner palmar tubercle length (measured as maximal distance from proximal to distal ends of the inner palmar tubercle);

LAHL length of lower arm and hand (distance from the elbow to the distal end of finger IV);

LW lower arm width (maximum width of the lower arm);

NED nasal to eye distance (distance between the nasal and the anterior corner of the eye);

OPTL outer metacarpal tubercle length (measured as maximal diameter of outer metacarpal tubercle);

SNT snout length (from tip of snout to the anterior corner of the eye);

SVL snout-vent length (from tip of snout to posterior margin of vent);

TD tympanum diameter (horizontal diameter of tympanum);

TED tympanum to eye distance (distance from anterior edge of tympanum to posterior corner of eye);

TFL length of foot and tarsus (distance from the tibiotarsal articulation to the distal end of toe IV);

THL thigh length (distance from vent to knee);

TL tibia length (distance from knee to heel);

TW tibia width (maximum width of the tibia);

UEW upper eyelid width (greatest width of the upper eyelid margins measured perpendicular to the anterior-posterior axis).

To reduce allometric effects, all measurements were size-corrected with respect to SVL prior to morphometric analysis. Principal component analyses (PCAs) of size-corrected measurements and simple bivariate scatterplots were used to explore and characterize the morphometric differences between the new species and P. leishanensis. Mann–Whitney U tests were conducted to determine the significance of differences in morphometric characters between the new species and P. leishanensis. Mann–Whitney U tests also were used to test the significance of morphometric differences between males and females of the new species. All statistical analyses were performed using SPSS 21.0 (SPSS, Inc., Chicago, IL, USA), and differences were considered statistically significant at P < 0.05. Sex was determined based on male secondary sexual characters: the presence of a vocal sac and nuptial pads/spines (Fei and Ye 2016).

We compared the morphological characters of the new species with literature data for 59 other species in the Panophrys (Table 3). We also examined the type and/or topotype materials for P. jiangi, P. liboensis, P. shuichengensis, and P. spinata (see Appendix 1).

Table 3.

References for morphological characters for congeners of the genus Panophrys.

ID Species Literature consulted
1 Panophrys acuta (Wang, Li & Jin, 2014) Li et al. 2014
2 Panophrys angka (Wu, Suwannapoom, Poyarkov, Chen, Pawangkhanant, Xu, Jin, Murphy & Che, 2019) Wu et al. 2019
3 Panophrys anlongensis (Li, Lu, Liu & Wang, 2020) Li et al. 2020b
4 Panophrys baishanzuensis (Wu, Li, Liu, Wang & Wu, 2020) Wu et al. 2020
5 Panophrys baolongensis (Ye, Fei & Xie, 2007) Ye et al. 2007
6 Panophrys binchuanensis (Ye & Fei, 1995) Ye and Fei 1995
7 Panophrys binlingensis (Jiang, Fei & Ye, 2009) Fei et al. 2009
8 Panophrys boettgeri (Boulenger, 1899) Fei et al. 2012
9 Panophrys brachykolos (Inger & Romer, 1961) Inger and Romer 1961
10 Panophrys caobangensis (Nguyen, Pham, Nguyen, Luong & Ziegler, 2020) Nguyen et al. 2020
11 Panophrys caudoprocta (Shen, 1994) Fei et al. 2012
12 Panophrys cheni (Wang & Liu, 2014) Wang et al. 2014
13 Panophrys chishuiensis (Xu, Li, Liu, Wei & Wang, 2020) Xu et al. 2020
14 Panophrys daiyunensis Lyu, Wang & Wang, 2021 Lyu et al. 2021
15 Panophrys daoji Lyu, Zeng, Wang & Wang, 2021 Lyu et al. 2021
16 Panophrys daweimontis (Rao & Yang, 1997) Fei et al. 2012
17 Panophrys dongguanensis (Wang & Wang, 2019) Wang et al. 2019a
18 Panophrys fansipanensis (Tapley, Cutajar, Mahony, Nguyen, Dau, Luong, Le, Nguyen, Nguyen, Portway, Luong & Rowley, 2018) Tapley et al. 2018
19 Panophrys frigida (Tapley, Cutajar, Nguyen, Portway, Mahony, Nguyen, Harding, Luong & Rowley, 2021) Tapley et al. 2021
20 Panophrys hoanglienensis (Tapley, Cutajar, Mahony, Nguyen, Dau, Luong, Le, Nguyen, Nguyen, Portway, Luong & Rowley, 2018) Tapley et al. 2018
21 Panophrys huangshanensis (Fei & Ye, 2005) Fei et al. 2012
22 Panophrys insularis (Wang, Liu, Lyu, Zeng & Wang, 2017) Wang et al. 2017a
23 Panophrys jiangi (Liu, Li, Wei, Xu, Cheng, Wang & Wu, 2020) Liu et al. 2020
24 Panophrys jingdongensis (Fei & Ye, 1983) Fei et al. 2012
25 Panophrys jinggangensis (Wang, 2012) Wang et al. 2012
26 Panophrys jiulianensis (Wang, Zeng, Lyu & Wang, 2019) Wang et al. 2019a
27 Panophrys kuatunensis (Pope, 1929) Fei et al. 2012
28 Panophrys leishanensis (Li, Xu, Liu, Jiang, Wei & Wang, 2019 «2018») Li et al. 2018b
29 Panophrys liboensis (Zhang, Li, Xiao, Li, Pan, Wang, Zhang & Zhou, 2017) Zhang et al. 2017
30 Panophrys lini (Wang & Yang, 2014) Wang et al. 2014
31 Panophrys lishuiensis (Wang, Liu & Jiang, 2017) Wang et al. 2017b
32 Panophrys lushuiensis (Shi, Li, Zhu, Jiang, Jiang & Wang, 2021) Shi et al. 2021
33 Panophrys minor (Stejneger, 1926) Wang et al. 2017a
34 Panophrys mirabilis (Lyu, Wang & Zhao, 2020) Lyu et al. 2020
35 Panophrys mufumontana (J. Wang, Lyu & Y.Y. Wang, 2019) Wang et al. 2019a
36 Panophrys nankunensis (Wang, Zeng &. Wang, 2019) Wang et al. 2019a
37 Panophrys nanlingensis (Lyu, J. Wang, Liu & Y.Y. Wang, 2019) Wang et al. 2019a
38 Panophrys obesa (Wang, Li & Zhao, 2014) Li et al. 2014
39 Panophrys ombrophila (Messenger & Dahn, 2019) Messenger et al. 2019
40 Panophrys omeimontis (Liu, 1950) Fei et al. 2009
41 Panophrys palpebralespinosa (Bourret, 1937) Fei et al. 2012
42 Panophrys qianbeiensis (Su, Shi, Wu, Li, Yao, Wang & Li, 2020) Su et al. 2020
43 Panophrys rubrimera (Tapley, Cutajar, Mahony, Chung, Dau, Nguyen, Luong & Rowley, 2017) Tapley et al. 2017
44 Panophrys sangzhiensis (Jiang, Ye & Fei, 2008) Jiang et al. 2008
45 Panophrys sanmingensis Lyu & Wang, 2021 Lyu et al. 2021
46 Panophrys shimentaina (Lyu, Liu & Wang, 2020) Lyu et al. 2021
47 Panophrys shuichengensis (Tian & Sun, 1995) Tian et al. 2000
48 Panophrys shunhuangensis (Wang, Deng, Liu, Wu & Liu, 2019) Wang et al. 2019b
49 Panophrys spinata (Liu & Hu, 1973) Fei et al. 2012
50 Panophrys tongboensis Wang & Lyu, 2021 Lyu et al. 2021
51 Panophrys tuberogranulata (Shen, Mo & Li, 2010) Mo et al. 2010
52 Panophrys wugongensis (J. Wang, Lyu & Y.Y. Wang, 2019) Wang et al. 2019a
53 Panophrys wuliangshanensis (Ye & Fei, 1995) Fei et al. 2012
54 Panophrys wushanensis (Ye & Fei, 1995) Fei et al. 2012
55 Panophrys xianjuensis (Wang, Wu, Peng, Shi, Lu & Wu, 2020) Wang et al. 2020
56 Panophrys xiangnanensis (Lyu, Zeng & Wang, 2020) Lyu et al. 2021
57 Panophrys yangmingensis (Lyu, Zeng & Wang, 2020) Lyu et al. 2020
58 Panophrys yeae (Shi, Zhang, Xie, Jiang, Liu, Ding, Luan & Wang, 2020) Shi et al. 2020
59 Panophrys zhoui (Shi, Zhang, Xie, Jiang, Liu, Ding, Luan & Wang, 2020) Shi et al. 2020

Bioacoustics analyses

The advertisement calls of the new species were recorded from the holotype specimen (voucher number GZNU20200706010) in the field on 5 July 2020 at the Yueliangshan Nature Reserve, Congjiang County, Guizhou Province, China. The advertisement calls were recorded in a stream, using a digital sound recorder (TASCAM DR-40) at an ambient air temperature of 25 °C and 92% humidity. Sounds were recorded within 5 cm of the calling individual. The wave-format sound files were sampled at 44 kHz, with sampling depth 24 bits. Praat 6.1.16 (Boersma 2001) was used to obtain oscillograms, sonograms, and power spectra at a window length of 0.005 s. The ambient temperature at the type locality was measured using a digital hygrothermograph (ECOFIVE MS6508).

Results

Phylogenetic analyses and genetic divergence

ML and BI phylogenies were constructed based on two concatenated mitochondrial gene sequences: 16S rRNA (548 bp) and COI (672 bp). The ML and BI topologies were largely identical (Fig. 2). Panophrys (except for P. yeae and P. zhoui) was strongly supported as monophyletic by both phylogenetic analyses. The phylogenetic trees also supported the monophyly of four of the seven genera of subfamily Megophryinae proposed in the revision of Li et al. (2020a): Ophyrophryne, Atympanophrys, Brachytarsophrys, Panophrys (except for P. yeae and P. zhoui), and Pelobatrachus; the monophyly of Xenophrys and Ophyrophryne was not supported. In both analyses, the new species formed a lower supported clade (0.59 in BI and 56% in ML) with P. leishanensis, P. baolongensis, P. wushanensis, P. tuberogranulata, P. shimentaina, P. yangmingensis, P. jiulianensis, P. mirabilis, P. shunhuangensis, and P. acuta. However, relationships among species in this clade were not well resolved except for the following well-supported sister relationships: P. baolongensis and P. wushanensis; P. shimentaina and P. yangmingensis; and P. mirabilis and P. shunhuangensis. The new species was recovered in a relatively poorly-supported sister relationship with P. leishanensis (0.60 in BI and 79% in ML; Fig. 2).

Figure 2. 

Phylogenetic tree based on mitochondrial 16S+COI genes. In this phylogenetic tree, ultrafast bootstrap supports (UFB) from ML analyses/Bayesian posterior probabilities (BPP) from BI analyses were noted beside nodes. The symbol “-” represents value below 0.60/60. Photos of new collections and 11 of 4 Panophrys species in Guizhou Province. The scale bar represents 0.08 nucleotide substitutions per site. The numbers at the tip of branches corresponds to the ID numbers in Table 1.

The smallest p-distance between this lineage and any other species of Panophrys was 1.2% in 16S rRNA (with P. huangshanensis) and 6.5% in COI (with P. wushanensis). These levels of divergence were similar to those between other pairs of recognized congeners. For example, the 16S rRNA p-distance was 1.2% between P. leishanensis and P. huangshanensis, 1.2% between P. jingdongensis and P. binchuanensis, while the COI p-distance was 5.9% between P. lini and P. nanlingensis, 3.6% between P. spinata and P. sangzhiensis, and 4.5% between Brachytarsophrys carinense and B. popei (Suppl. material 1: Table S1; Suppl. material 2: Table S2). These results suggested that this population, from the Yueliangshan Nature Reserve, Congjiang County, Guizhou Province, China, represented an independent evolutionary lineage.

Species delimitation

The results of the *BEAST analysis for the alternative species model are provided in Table 4. Both SS and PS estimations based on 16S rRNA+COI datasets had the largest values for the 11 species taxonomy, indicating that it was supported in favor of the currently accepted 11 species model. In addition, the results of the maximum likelihood solution of the bPTP analysis supported 11 species taxonomy model (Appendix 1). Thus, the results of the BFD and bPTP analyses suggest support for treating the new species as a single valid species.

Table 4.

The species delimitation results of new species and several closely related species in BF method.

Model Species delimitation MLE Path Sampling (PS) MLE Stepping Stone (SS) Rank BF (PS) BF (SS)
M1 11 species: -4011.49 -4011.48 1 14.14 14.02
AC+BA+JU+MI+SI+SU+TU+WU+YA+LE+CO
M2 10 species: -4018.56 -4018.49 2
AC+BA+JU+MI+SI+SU+TU+WU+YA+{LE+CO}

Morphological analyses

The results of the Mann–Whitney U tests indicated that males of the new species differed significantly from P. leishanensis males based on several morphometric characters (all p-values < 0.05; Table 5). Using PCA, we extracted two and three principal component factors with Eigenvalues greater than two for males and females, respectively (Suppl. material 3: Table S3). The first two principal components explained 67.23% and 80.68% of the total variation in males and females, respectively. The variances in the data were mainly associated with limb and head characters, including TW, THL, HDL, LW, HDW, LAHL, HLL, FIIIL, FIL, FIIL, TFL, TL, IND, and IOD (Table 5). The characters of the new species were distinct from those of P. leishanensis on two-dimensional plots of PC1 and PC2 for both males and females (Fig. 3).

Figure 3. 

Plots of the first principal component (PC1) versus the second (PC2) for Panophrys congjiangensis sp. nov. and P. leishanensis from a principal component analysis A male B female.

Table 5.

Morphological comparison of Panophrys congjiangensis sp. nov. (PC) and P. leishanensis (PL). All units in mm. P-values are at 95% significance. Morphometric characters are explained in the methods section. CM and CF are the abbreviations of male and female from Panophrys congjiangensis sp. nov.

Measurements Panophrys congjiangensis sp. nov. Panophrys leishanensis# P-value from Mann-Whitney U test
Male (N=15) Female (N=4) Male (N=10) Female (N=2) Male Female CM vs.CF
Range Mean ± SD Range Mean ± SD Range Mean ± SD Range PC vs. PL PC vs. PL
SVL 28.6–33.4 31.2 ± 1.4 38.4–40.2 39.3 ± 0.7 30.4–38.7 34.3 ± 2.7 42.3–42.3 0.000 0.133 0.003
HDL 10.2–11.5 11.0 ± 0.4 12.1–14.5 13.3 ± 1.0 9.1–11.0 10.1 ± 0.7 11.3–11.7 0.000 0.267 0.002
HDW 9.1–11.4 10.7 ± 0.6 11.5–13.2 12.1 ± 0.8 10.5–12.0 11.4 ± 0.5 12.1–12.4 0.014 0.133 0.003
SNT 3.4–4.9 4.2 ± 0.3 4.3–5.1 4.6 ± 0.4 3.6–4.5 4.2 ± 0.3 4.5–5.0 0.007 0.267 0.027
ED 3.3–3.9 3.6 ± 0.2 4.2–5.2 4.6 ± 0.5 3.3–4.3 3.9 ± 0.3 4.1–4.8 0.643 1.000 0.002
IOD 3.1–4.3 3.6 ± 0.3 3.7–4.5 4.0 ± 0.4 3.3–4.3 3.7 ± 0.3 3.9–4.2 0.062 0.267 0.084
IND 2.8–3.7 3.3 ± 0.2 3.5–4.8 3.9 ± 0.6 3.5–4.7 4.0 ± 0.4 4.1–4.3 0.392 0.267 0.011
TD 1.7–2.4 2.1 ± 0.2 2.4–3.4 2.7 ± 0.5 2.0–2.6 2.3 ± 0.2 2.5–2.8 0.461 1.000 0.003
UEW 2.4–3.0 2.7 ± 0.2 3.7–3.9 3.8 ± 0.1 / / / / / 0.002
NED 1.9–2.7 2.2 ± 0.3 2.3–2.6 2.5 ± 0.1 / / / / / 0.059
TED 1.7–2.2 1.9 ± 0.1 1.8–2.2 2.0 ± 0.2 / / / / / 0.750
HND 7.3–8.6 8.1 ± 0.4 8.9–9.8 9.3 ± 0.4 / / / / / 0.003
LAHL 13.2–15.2 14.3 ± 0.6 16.2–17.4 16.5 ± 0.6 14.4–16.3 15.3 ± 0.6 18.1–18.4 0.036 0.133 0.003
LW 1.7–2.4 2.1 ± 0.2 1.9–2.1 2.0 ± 0.1 2.7–3.9 3.2 ± 0.5 2.8–2.9 0.001 1.000 0.355
TL 14.6–17.5 15.9 ± 0.9 19.1–20 19.4 ± 0.4 16.2–18.6 17.5 ± 0.9 19.2–19.2 0.129 0.800 0.003
THL 13.4–17.8 15.3 ± 1.1 18.6–19.9 19.3 ± 0.5 14.4–16.8 15.4 ± 0.8 17.6–17.7 0.000 1.000 0.003
FL 13.4–15.7 14.6 ± 0.8 16.6–18.9 17.9 ± 1.0 14.9–17.3 15.9 ± 1.0 18.1–19.0 0.129 0.800 0.003
TFL 20.8–24.9 22.3 ± 1.2 25.7–26.8 26.3 ± 0.5 21.1–25.9 23.5 ± 0.5 27.5–27.9 0.004 1.000 0.003
HLL 49.3–60.2 53.5 ± 3.0 63.4–66.7 65.0 ± 1.4 50.3–60.2 54.2 ± 3.0 49.3–50.3 0.023 0.533 0.003
TW 2.6–3.5 3.1 ± 0.2 3.5–3.9 3.7 ± 0.2 3.6–4.7 4.2 ± 0.3 4.8–5.1 0.000 0.133 0.003
IPTL 1.6–2.5 2.0 ± 0.3 2.8–2.9 2.9 ± 0.1 / / / / / 0.003
OPTL 1.2–1.7 1.4 ± 0.2 2.1–2.9 2.4 ± 0.3 / / / / / 0.002
IMTL 1.7–2.6 2.0 ± 0.2 2.2–2.9 2.5 ± 0.3 / / / / / 0.014
FIL 3.7–4.7 4.3 ± 0.3 4.8–5.1 4.9 ± 0.2 3.2–3.9 3.5 ± 0.2 4.0–4.3 0.000 0.133 0.003
FIIL 3.3–5.7 4.2 ± 0.6 4.3–6 5.5 ± 0.8 2.8–3.5 3.2 ± 0.3 3.8–4.1 0.000 0.133 0.011
FIIIL 5.6–7.7 6.2 ± 0.5 6.6–7.7 7.4 ± 0.5 4.2–5.4 4.8 ± 0.4 5.4–5.8 0.000 0.133 0.009
FIVL 4.1–5.9 4.9 ± 0.4 5.7–6.8 6.3 ± 0.5 3.4–4.1 3.7 ± 0.2 4.2–4.3 0.000 0.133 0.004

Taxonomic account

Panophrys congjiangensis sp. nov.

Table 2, Figs 4, 5, 6

Type material

Holotype. GZNU20200706010 (Figs 4, 5), adult male, collected by Tao Luo on 6 May 2020 in the Yueliangshan Nature Reserve, Congjiang County, Guizhou Province, China (25.614417°N, 108.410076°E; ca. 730 m a.s.l.).

Figure 4. 

Morphological features of the live adult male holotype GZNU20200706010 of Panophrys congjiangensis sp. nov. A single subgular vocal sac B dorsal view C dorsolateral view D ventral view E ventral view of hand F ventral view of foot G dorsal view of hand (1 indicates villiform gray-black nuptial spines) H iris. A was photographed at about 9 p.m., and B to H during the day, respectively.

Figure 5. 

Morphological features of the preserved adult male holotype GZNU20200706010 of Panophrys congjiangensis sp. nov. A dorsal view B ventral view C lateral view D view of oral cavity E tongue F ventral view of hand G ventral view of foot.

Paratypes. Nineteen adult specimens (15 males and 4 females) from the same locality. Eleven males (GZNU20200706001–06009, GZNU20200706012–06013) collected with the holotype on 6 July 2020 by Tao Luo, Xueli Lu, and Weifeng Wang. One female (GZNU20200706011) collected with the holotype by Tao Luo. Three males (GZNU200707001–07003) collected on 7 July 2020 by Tao Luo. Three females (GZNU20200706004, GZNU20200706005, and GZNU20200706006) collected on 7 July 2020 by Tao Luo.

Etymology

The specific epithet “congjiangensis” refers to the holotype locality, which is Yueliangshan Nature Reserve, located in Congjiang County, Guizhou Province, China. We propose the English common name “Congjiang Horned Toad” and the Chinese common name “Cong Jiang Jiao Chan (从江角蟾)”.

Differential diagnosis

Panophrys congjiangensis sp. nov. is assigned to the genus Panophrys based on molecular phylogenetic analyses and the following characteristics, which are diagnostic for this genus: (1) snout shield-like; (2) snout projecting beyond the lower jaw; (3) tympanum distinct (4) canthus rostralis distinct; (5) chest gland small and round, closer to axilla than to midventral line; (6) femoral gland on rear of thigh; (7) vertical pupils (Fei et al. 2006; Fei and Ye 2016; Su et al. 2020).

Panophrys congjiangensis sp. nov. is distinguished from its congeners by a combination of the following characters: (1) medium body size (SVL: 28.6–33.4 mm in males and 38.4–40.2 mm in females); (2) single small horn-like tubercle at edge of each upper eyelid; (3) tympanum distinctly visible (TD/ED ratio 0.47–0.66); (4) vomerine teeth absent; (5) tongue not notched behind; (6) a narrow and unobvious lateral fringe on toes; (7) relative finger lengths II < I < V < III; (8) rudimentary webs on toes; (9) hindlimbs slender, heels overlapping when thighs are positioned at right angles to body; (10) two metacarpal tubercles on palm, with inner metatarsal tubercle long and oval-shaped; (11) tibiotarsal articulation reaching nostril when leg is stretched forward; (12) dorsal skin rough, with numerous orange-red granules, ventral surface smooth; (13) single internal subgular vocal sac present in males; (14) in breeding males, weak gray-black nuptial pads with black nuptial spines present on dorsal surfaces of bases of first and second fingers.

Description of holotype

GZNU20200706010 (Figs 4, 5), adult male. Medium body size, SVL 33.4 mm; head length slightly larger than head width (HDL/HDW ratio 1.02); snout short, rounded and projecting beyond the lower jaw in dorsal view, longer than eye diameter (SNT/ED ratio 1.11); nostril rounded, distinct, and closer to the tip of the snout than to the eye (SNT/NED ratio 1.83); internasal distance greater than interorbital distance (IND/IOD ratio 1.19); internasal distance greater than upper eyelid width (IND/UEW ratio 1.28); region vertical and concave; canthus rostralis well-developed; top of head slightly concave in dorsal view; a small horn-like tubercle at the edge of the upper eyelid; eyes large, slightly protuberant in dorsal view, eye diameter 34% of head length, pupils vertical (Fig. 4H); tympanum distinct, tympanum diameter less than eye diameter (TD/ED ratio 0.63); vomerine ridges and vomerine teeth absent; tongue is melon seed-shaped and not notched behind (Fig. 5E).

Forelimbs slender and comparatively short, the length of lower arm and hand 44.01% of SVL; fingers slender, relative finger lengths: II < I < IV < III; tips of fingers slightly dilated, round, without lateral fringes; one distinct subarticular tubercle at the base of each finger; two metacarpal tubercles on the palm; prominent, the outer one long and thin, the inner one oval-shaped, inner metacarpal tubercles longer than outer metacarpal tubercles (IPTL/OPT ratio 1.13).

Hindlimbs slender (HLL/SVL ratio 1.80); heels slightly overlapping when thighs are positioned at right angles to the body; tibiotarsal articulation reaching the nostril when leg stretched forward; foot length less than tibia length (FL/TL ratio 0.90); relative toe lengths I < II < V < III < IV; tips of toes round and slightly dilated; toes with narrow and unobvious lateral fringes and rudiment webs; one subarticular tubercle at the base of each toe; inner metatarsal tubercle long oval-shaped and the outer one absent.

Dorsal skin rough with numerous orange-red granules; several large warts scattered on flanks and dorsal limbs; several tubercles on upper eyelid, including a small horn-like prominent tubercle on the edge (Fig. 4H); supratympanic fold distinct; tubercles on the dorsum forming a discontinuous X-shaped ridge, the V-shaped ridges disconnected; two discontinuous dorsolateral parallel ridges on either side of the X-shaped ridges; an inverted triangular brown speckle between two upper eyelids; four transverse skin ridges on the dorsal shank and thigh; ventral surface smooth; chest with small, round glands, closer to the axilla than to midventral line; femoral glands on rear of thigh; numerous white granules on ventral surface of thigh; posterior end of body distinctly protruding, forming an arc-shaped swelling above anal region.

Coloration of holotype in life

(Fig. 4). Dorsal surfaces of head and trunk brownish gray; triangular marking with light edge between eyes; dark X-shaped marking with light edge on central dorsum; supratympanic fold light brown; four dark brown transverse bands on dorsal surfaces of thigh and shank; 2–4 dark brown and white vertical bars on lower and upper lip; dark vertical band below eye; iris copper-brown; throat and anterior chest light purple-brown; belly light orange-red with large white blotch and small grey blotch in belly center, and small white blotches and large black patches on belly sides, forming a discontinuous line; ventral surfaces of forelimbs purplish brown; some white spots on the ventral surfaces of hindlimbs; palms orange-red with a small black-brown blotch; ventral surfaces of first and second toes orange-red, ventral surfaces of remaining three toes black-brown; soles black-brown; tips of digits grey-white; pectoral and femoral glands white.

Preserved holotype coloration

(Fig. 5). After preservation in ethanol, dorsal surfaces light brownish grey; dorsal surface of head dark gray; X-shaped ridges on dorsum indistinct and transverse bands on limbs and digits distinct, coloration lighter; throat dark black-brown; chest light black-brown; belly light gray-white with large black-brown blotches on sides and a small gray-brown blotch in center; posterior ventral body surface, inner thigh, and upper part of tibia milky yellow; palms and metatarsal tubercle milky yellow with a small gray-brown blotch; ventral surfaces of soles and toes dark black-brown; inner metatarsal tubercle milky yellow.

Variations

Measurements of the type series are shown in Tables 2, 4. Females (SVL 39.3 ± 0.7 mm, N = 4) had larger bodies than males (SVL 31.2 ± 1.4 mm, N = 15). In life, the diagnostic morphological characters of all paratypes were identical to those of the holotype. However, coloration and stripe patterns differed among individuals (Fig. 6). For example, male GZNU20200706007 (Fig. 6A) had a brown-black back and a black-brown belly with some large white patches, as well as two V-shaped markings that were virtually connected. This specimen also had warts on both sides of the body, forming a transverse skin ridge that almost connected to the second V-shaped marking. In contrast, male GZNU20200706008 (Fig. 6B) had a large black spot between the upper eyelids. The throat and anterior belly of this specimen were purple–brownish, while the belly was light milky yellow, with two large black blotches and a small white blotch on the body sides. In specimens GZNU20200706009 and GZNU20200706012 (Fig. 6C, E), the warts on both sides of the body formed transverse skin ridges connected to the second V-shaped marking and extending behind the tympanum; three white small blotches were present on the body sides. In specimens GZNU20200706013 and GZNU20200706012 (Fig. 6D, E), the back was light reddish brown.

Figure 6. 

Paratypes of Panophrys congjiangensis sp. nov. in life A GZNU20200706007, adult male B GZNU20200706008, adult male C GZNU20200706009, adult male D GZNU2020706013, adult male E GZNU20200706012, adult female. So, the images were all taken at 8 am.

Advertisement call

The call description is based on recordings of the holotype GZNU 20200706010 (Fig. 7) from the bamboo forest near the streamlet. The ambient air temperature during the recording was 25.3 °C. Each call contains 9–14 syllables (mean 11.60 ± 2.07, N = 5). The call consists of a few strophes, each 2.41–3.43 s in duration (mean 2.75 ± 0.46, N = 4). Each syllable has a duration of 0.05–0.09 s (mean 0.07 ± 0.06, N = 58). The interval between syllables has a duration of 0.10–0.31 s (mean 0.167 ± 0.042, N = 53).

Figure 7. 

Visualization of advertisement calls of Panophrys congjiangensis sp. nov. A waveform showing 12 notes of one call B waveform showing one note C sonogram showing 12 notes of one call D sonogram showing one note.

Sexual dimorphism

Adult males (SVL 28.6–33.4 mm) smaller than adult females (SVL 38.4–40.2 mm). Adult males with single internal subgular vocal sac (Fig. 4A). Breeding males with grey-black nuptial pads with obvious black nuptial spines on dorsal surfaces of bases of first and second fingers.

Comparisons

Comparative data of Panophrys congjiangensis sp. nov. with 59 recognized congeners of Panophrys are given in Suppl. material 4: Table S4.

By having small body size, SVL 30.4–34.1 mm in males, Panophrys congjiangensis sp. nov. differs from P. baolongensis (42.0–45.0 in males), P. binlingensis (45.1–51.0 in males), P. boettgeri (34.5–37.8 in males), P. caobangensis (34.9–38.9 in males), P. caudoprocta (81.3 in single male), P. hoanglienensis (37.4–47.6 in males), P. huangshanensis (36.0–41.6 in males), P. insularis (36.8–41.2 in males), P. jingdongensis (53.0–56.5 in males), P. mirabilis (55.8–61.4 in males), P. obesa (35.6 in single male), P. palpebralespinosa (36.2–38.0 in males), P. sangzhiensis (54.7 in single male), and P. xiangnanensis (38.6–42.0 in males). By having larger body size, SVL 30.4–34.1 mm in males, Panophrys congjiangensis sp. nov. differs from P. cheni (26.2–29.5 in males), P. daiyunensis (27.6–28.7 in males), P. kuatunensis (26.2–29.6 in males), P. sanmingensis (27.0–29.5 in males), P. yeae (23 in single male), and P. zhoui (23.8–29.1 in males). By having small body size, SVL 38.9–40.2 mm in females, Panophrys congjiangensis sp. nov. differs from P. fansipanensis (41.7–42.5 in females), P. minor (42.0–48.2 in females), P. tuberogranulata (50.5 in single female), P. wuliangshanensis (41.3 in single female), P. xianjuensis (41.6 in single female), and P. yangmingensis (45.2 in single female).

Nine Panophrys species were previously recorded from the Guizhou Province, namely P. anlongensis, P. chishuiensis, P. jiangi, P. leishanensis, P. liboensis, P. omeimontis, P. shuichengensis, P. spinata, and P. qianbeiensis. Panophrys congjiangensis sp. nov. differs from P. anlongensis by having small body size, SVL 30.4–34.1 mm in males and 38.9–40.2 mm in females (vs. 40.0–45.5 mm in males and 48.9–51.2 in females), tibiotarsal articulation reaching the nostril when leg stretched forward (vs. reaching to the level of mid-eye). Panophrys congjiangensis sp. nov. differs from P. chishuiensis by having small body size, SVL 30.4–34.1 mm in males and 38.9–40.2 mm in females (vs. 43.4–44.1 mm in males and 44.8–49.8 in females), rudimentary webs on toes (vs. lacking webs), subarticular tubercles present on each toes (vs. absent), tibiotarsal articulation reaching the nostril when leg stretched forward (vs. reaching the level between tympanum and eye). Panophrys congjiangensis sp. nov. differs from P. jiangi by having slightly small body size, SVL 30.4–34.1 mm in males (vs. 34.4–39.2 mm in males), relative finger lengths are II < I < V < III (vs. I < II < V < III), tibiotarsal articulation reaching the nostril when leg stretched forward (vs. reaching forward to the region between tympanum and eye). Panophrys congjiangensis sp. nov. differs from P. liboensis, P. omeimontis, P. qianbeiensis, P. shuichengensis, and P. spinata by having small body size, SVL 30.4–34.1 mm in males and 38.9–40.2 mm in females (vs. SVL>40 mm in males in P. liboensis, P. omeimontis, P. qianbeiensis, P. shuichengensis, and P. spinata; vs. SVL>50 mm in females in P. liboensis, P. omeimontis, P. shuichengensis, and P. spinata), small horn-like tubercle at the edge of each upper eyelid (vs. slightly large in P. liboensis and P. shuichengensis; absence in P. qianbeiensis), absence of vomerine teeth (vs. present in P. liboensis, P. omeimontis, and P. qianbeiensis), tongue not notched behind (vs. notched in P. liboensis, P. omeimontis, P. qianbeiensis, P. shuichengensis, and P. spinata), lateral fringes on toes narrow and unobvious (vs. wide in P. liboensis, P. qianbeiensis, P. shuichengensis, and P. spinata), rudimentary webs on toes (vs. more than one-fourth webs in P. qianbeiensis, P. shuichengensis, and P. spinata), subarticular tubercles present on each toes (vs. absent in P. liboensis and P. shuichengensis), tibiotarsal articulation reaching the nostril when leg stretched forward (vs. reaching to ocular region in P. liboensis, P. omeimontis, P. shuichengensis, and P. spinata; reaching to the level between tympanum and eye in P. qianbeiensis). Panophrys congjiangensis sp. nov. differs from P. leishanensis by having slightly small body size, SVL 38.9–40.2 mm in females (vs. 42.3 in single female), having narrow and unobvious lateral fringes on toes (vs. lacking), tibiotarsal articulation reaching the nostril when leg stretched forward (vs. reaching between tympanum to eye). The mean SVL of male Panophrys congjiangensis sp. nov. was significantly greater than that of P. leishanensis. In addition, the ratios of HDL, HDW, SNT, LAHL, LW, THL, TFL, HLL, and TW to SVL were all significantly greater in male Panophrys congjiangensis than in male P. leishanensis (all p-values < 0.05; Table 4). Panophrys congjiangensis sp. nov. also differs from P. leishanensis by having one call 9–14 syllables (vs. calls of P. leishanensis, which are 12–14 syllables long), shorter call intervals between syllables (0.167 ± 0.042 s, N = 53 in the new species vs. 0.409 ± 0.075 s, N = 36 in P. leishanensis), and shorter call syllables (0.07 ± 0.06 s, N = 58 in the new species vs. 0.105 ± 0.003 s, N = 37 in P. leishanensis).

From the remaining 24 species occurring in Panophrys, Panophrys congjiangensis sp. nov. can be distinguished by the absence of vomerine teeth (vs. present in P. daweimontis, P. dongguanensis, P. frigida, P. jinggangensis, P. jiulianensis, P. nankunensis, P. nanlingensis, P. rubrimera, P. shimentaina, and P. tongboensis), by the unnotched tongue (vs. tongue notched in P. binchuanensis, P. cheni, P. kuatunensis, and P. lushuiensis), by the small horn-like tubercle at edge of upper eyelid (vs. slightly large in P. acuta), by the absence of lateral fringes on toes (vs. lacking in P. angka, P. brachykolos, P. lishuiensis, P. ombrophila, P. shunhuangensis, and P. wugongensis; vs. wide in P. lini; vs. lacking in males in P. wushanensis, wide in females in P. wushanensis), by the subarticular tubercles present (vs. absent in P. baishanzuensis and P. mufumontana), tibiotarsal articulation reaching the nostril when leg stretched forward (vs. reaching to ocular region in P. acuta, P. baishanzuensis, P. binchuanensis, P. jiulianensis, P. lini, P. nanlingensis, P. ombrophila, and P. wushanensis; vs. reaching to the level between tympanum and eye in P. angka, P. dongguanensis, P. kuatunensis, P. lishuiensis, and P. nankunensis; vs. reaching to the level between eye and snout in P. cheni, P. daweimontis, and P. shunhuangensis; vs. reaching to the level behind the eye in P. brachykolos, P. mufumontana, P. shimentaina, and P. wugongensis; vs. reaching to the level at center of tympanum P. daoji).

Distribution and ecology

Panophrys congjiangensis sp. nov. is only known from the type locality, Yueliangshan Nature Reserve, Congjiang County, Guizhou Province, China at elevations of 1142–1206 m. Individuals of the new species were frequently found in bamboo forests, grasses, and shrubberies near streams. Plants in the type locality predominantly fall into the families Urticaceae, Gramineae, Cyperaceae, Rosaceae, Dryopteridaceae, Polygonaceae, Aquifoliaceae, and Fagaceae. In the Yueliang Mountains, Panophrys congjiangensis sp. nov. is sympatric with Pachytriton inexpectatus Nishikawa, Jiang, Matsui & Mo, 2010; Amolops sinensis Lyu, Wang & Wang, 2019; Nidirana leishanensis Li, Wei, Xu, Cui, Fei, Jiang, Liu & Wang, 2019; Hylarana latouchii (Boulenger, 1899); Quasipaa boulengeri (Günther, 1889); Hyla annectans (Jerdon, 1870); Opisthotropis zhaoermii Ren, Wang, Jiang, Guo & Li, 2017; Trimeresurus stejnegeri (Schmidt, 1925); and Rhabdophis tigrinus (Boie, 1826). These species were often found in the same streams as Panophrys congjiangensis sp. nov.

Discussion

Phylogenetic analyses based on two mitochondrial genes suggested that the specimens collected in this study fell into the Panophrys, but were distinct from all previously described species in this genus. Genetic distances between Panophrys congjiangensis sp. nov. and its sister species P. leishanensis were 3.0% for 16S rRNA and 8.4% for COI, within the ranges expected for interspecific divergences in amphibians (Fouquet et al. 2007; Che et al. 2012). Indeed, other species have been distinguished and recognized based on much lower genetic distances. For example, the p-distance is 1.2% between P. angka and P. anlongensis for 16S rRNA, and 3.6% between P. sangzhiensis and P. spinata for COI (Suppl. material 1: Table S1; Suppl. material 1: Table S2). Panophrys congjiangensis sp. nov. is morphologically similar to P. leishanensis, but Panophrys congjiangensis sp. nov. is smaller, has a narrow and unobvious lateral fringe on the toes, and the tibiotarsal articulation of the hindlimb reaches the nostril when the leg is adpressed and stretched forward. The two species can also be distinguished based on bioacoustics characters: the call of Panophrys congjiangensis sp. nov. had fewer syllables than that of P. leishanensis, and the call intervals were shorter. Without phylogenetic, morphological, and bioacoustics data, it is difficult to determine the taxonomic status of new species. In this study, these multiple pieces of evidence supported the validity of Panophrys congjiangensis sp. nov. The new species described in this study increases the number of species assigned to Panophrys to 60, with 56 recorded from China (Fei and Ye 2016; AmphibiaChina 2020; Frost 2021).

Climatic fluctuations, habitat heterogeneity, habitat diversity, and the dynamics of montane forests may play important roles in driving diversification in the Panophrys (Chen et al. 2017; Liu et al. 2018). These factors may have led to the development of complex phenotypes in this genus. Recent studies have revealed high levels of species diversity in the Panophrys (Frost 2021). However, Panophrys congjiangensis sp. nov. does not belong to any of the clades identified by Chen et al. (2017) and Liu et al. (2018), suggesting that Panophrys diversity may remain severely underestimated, even where Panophrys species are sympatric ally distributed (Li et al. 2018; Lyu et al. 2020; Su et al. 2020). Until recently, it was difficult to perform taxonomic and phylogenetic studies of the Panophrys because many species in this genus are morphologically similar and have sympatric distributions; the many possible cryptic species in the Panophrys may have hindered our understanding of diversity in this genus (Chen et al. 2017; Liu et al. 2018; Li et al. 2018; Wang et al. 2019a, b; Mahony et al. 2020; Lyu et al. 2020; Liu et al. 2020; Xu et al. 2020). The high species diversity, strong forest dependence, and sympatric distributions in the Panophrys indicate that speciation patterns, niche differentiation, and coexistence mechanisms in this genus require further study.

Biodiversity conservation in southwestern China is a priority of the Chinese government (Ministry of Environmental Protection 2015). Biodiversity conservation programs in this region play an important role in maintaining the stability of mountain ecosystems as well as protecting biodiversity (Körner and Spehn 2002; Tang et al. 2006). Mountain ecosystems are characterized by high biodiversity, with species tending to exhibit a wide range of evolutionary adaptations (McCain and Colwell 2011; Elsen and Tingley 2015). Mountain ecosystems also serve as sanctuaries for many endemic and threatened species, and thus play a major role in the maintenance of biodiversity (Favre et al. 2016). Mountains ecosystems provide key ecological service functions and provide important natural resources that are utilized by local human populations (Körner and Spehn 2002; Grêt-Regamey et al. 2012). Thus, mountain species face a higher risk of extinction due to their limited range, unique environmental adaptations, and geographic isolation, rendering mountain taxa among the most likely to be threatened by climate change.

In the past three years alone, 11 new amphibian species have been described from Guizhou Province, China (Zhang et al. 2017; Li et al. 2018a, b; Li et al. 2019a, b; Lyu et al. 2019b; Wang et al. 2019c; Wei et al. 2020; Luo et al. 2020; Liu et al. 2020; Su et al. 2020). The discovery of these new species suggests that amphibian species diversity in this region is severely underestimated. In the context of global warming, there is an urgent need for a comprehensive, systematic, and in-depth survey of the impacts of climate change on terrestrial vertebrates to provide a basis for scientific decisions regarding amphibian conservation (IPCC 2014).

Acknowledgements

This work was supported by the programs of the Ministry of Science and Technology of the People’s Republic of China major project “Protect technology and endangered mechanism research on rare animals” (No. 2016YFC0503200), the Strategic Priority Research Program B of the Chinese Academy of Sciences(CAS) (No. XDB31000000), the National Animal Collection Resource Center, China (Grant No. 2005DKA21402), the Application of Amphibian Natural Antioxidant Peptides as Cosmetic Raw Material Antioxidants (QKZYD [2020]4002). We thank Professor Paul A. Garber for editing assistance during the preparation of this manuscript. We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.

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Appendix 1

Specimens examined

Panophrys jiangi (N=3): China: Guizhou Province: Suiyang County: Kuankuosui National Nature Reserve (type locality): GZNU20070712001. China: Guizhou: Suiyang: Huoqiuba Nature Reserve (topotype locality): GZNU20180606020–606022.

Panophrys liboensis (N=5): China: Guizhou Province: Libo County (type locality): GNUG20150813001, 408002, 408004, 408006–408008.

Panophrys shuichengensis (N=7): China: Guizhou Province: Shuicheng County (type locality): 944001, 98001, 98002, 945005, 2007030, 2007031, 2007032.

Panophrys spinata (N=6): China: Guizhou Province: Dafang County (topotype locality): GZNU201707015011–606016.

Supplementary materials

Supplementary material 1 

Table S1. Uncorrected p-distance between Megophryinae species based on 16s rRNA sequences

Tao Luo, Yali Wang, Siwei Wang, Xueli Lu, Weifeng Wang, Huaiqing Deng, Jiang Zhou

Data type: molecular data

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
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Supplementary material 2 

Table S2. Uncorrected p-distance between Megophryinae species based on COI gene sequences

Tao Luo, Yali Wang, Siwei Wang, Xueli Lu, Weifeng Wang, Huaiqing Deng, Jiang Zhou

Data type: molecular data

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (68.73 kb)
Supplementary material 3 

Table S3. Variable loadings for principal components with Eigenvalues greater than 2, from morphometric characters corrected by SVL

Tao Luo, Yali Wang, Siwei Wang, Xueli Lu, Weifeng Wang, Huaiqing Deng, Jiang Zhou

Data type: statistical data

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (14.68 kb)
Supplementary material 4 

Table S4. Diagnostic characters separating all 59 species of the Panophrys

Tao Luo, Yali Wang, Siwei Wang, Xueli Lu, Weifeng Wang, Huaiqing Deng, Jiang Zhou

Data type: species data

Explanation note: Data modified from Wang et al. 2020; Li et al. 2020; Lyu et al. 2020; Nguyen et al. 2020.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (34.50 kb)