Research Article
Print
Research Article
Description of a new Megophrys Kuhl & Van Hasselt, 1822 (Anura, Megophryidae) from Guizhou Province, China
expand article infoShi-Ze Li§, Ning-Ning Lu§, Jing Liu, Bin Wang§
‡ Moutai Institute, Renhuai, China
§ Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, China
Open Access

Abstract

A new species of the genus Megophrys is described from Guizhou Province, China. Molecular phylogenetic analyses based on mitochondrial DNA indicated the new species as a clade clustered into the Megophrys clade. The new species can be distinguished from its congeners by a combination of the following characters: body size moderate (SVL 40.0–45.5 mm in males and 48.9–51.2 mm in females); vomerine teeth absent; tongue not notched behind; tympanum distinctly visible, oval; a small horn-like tubercle at the edge of each upper eyelid; two metacarpal tubercles in hand; toes with rudimentary webbing; heels overlapping when thighs are positioned at right angles to the body; tibiotarsal articulation reaching the level of mid-eye when leg stretched forward; in breeding males, an internal single subgular vocal sac present and brownish nuptial pads, made up of black nuptial spines, present on the dorsal base of the first two fingers.

Keywords

Molecular phylogenetic analysis, morphology, new species, taxonomy

Introduction

The Asian horned toad Megophrys Kuhl & Van Hasselt, 1822 (Anura: Megophryidae Bonaparte, 1850) is widely distributed in eastern and central China, throughout southeastern Asia, and extending to the islands of the Sunda Shelf and the Philippines (Frost 2020). This group was indicated to be a monophyletic group by most molecular phylogenetic studies (e.g., Chen et al. 2017; Mahony et al. 2017; Liu et al. 2018; Li et al. 2018a; Liu et al. 2020; Wang et al. 2020) though the taxonomic profiles especially on generic assignments of species in the group are still on debate (e.g., Tian and Hu 1983; Dubois 1987; Rao and Yang 1997; Lathrop 1997; Jiang et al. 2003; Delorme et al. 2006; Fei et al. 2009; Fei and Ye 2016; Chen et al. 2017; Deuti et al. 2017; Mahony et al. 2017; Liu et al. 2018; Frost 2020). Currently, the genus Megophrys contains 106 species, of which, 49 species were described in the last ten years (Frost 2020). Molecular phylogenetic frameworks even still proposed many cryptic species in the genus (e.g., Chen et al. 2017; Liu et al. 2018). In Guizhou Province, China, in recent five years, four Megophrys species have been described, and they are, M. liboensis Zhang, Li, Xiao, Li, Pan, Wang, Zhang & Zhou, 2017, M. leishanensis Li, Xu, Liu, Jiang, Wei & Wang, 2018, M. jiangi Liu, Li, Wei, Xu, Cheng, Wang & Wu, 2020, and M. chishuiensis Xu, Li, Liu, Wei & Wang, 2020.

During field surveys in Anlong County, Guizhou Province, China, we collected eight Megophrys specimens. Molecular phylogenetic analyses and morphological comparisons supported it as an undescribed species and it is described herein as a new species.

Materials and methods

Sampling

Three adult females and five adult males of the undescribed species were collected from Anlong County, Guizhou Province, China (Fig. 1; Table 1). The toads were firstly euthanised using isoflurane, and then the specimens were fixed in 75% ethanol for preservation. Tissue samples were taken and preserved separately in 95% ethanol prior to fixation. The specimens were deposited in Chengdu Institute of Biology, Chinese Academy of Sciences (CIB, CAS).

Figure 1. 

Geographical location of the type locality of Megophrys anlongensis sp. nov., Anlong County, Guizhou Province, China.

Table 1.

Measurements of the adult specimens of Megophrys anlongensis sp. nov. Units given in mm. See abbreviations for the morphological characters in Materials and methods section.

Voucher Sex SVL HDL HDW SL IND IOD UEW ED TYD LAL LW HLL THL TL TW TFL FL
CIBAL20190531021 45.5 12.3 15.9 6.0 5.6 4.1 4.0 4.7 3.1 19.9 5.0 70.8 18.8 22.5 5.2 33.1 22.7
CIBAL20190531019 41.1 12.4 14.0 5.0 4.5 3.2 4.3 4.8 2.7 18.8 4.3 65.4 17.7 22.3 5.1 29.4 19.9
CIBAL20190531017 42.5 11.5 14.4 5.1 4.7 4.1 3.7 4.4 3.1 19.7 4.2 67.7 20.2 22.1 5.5 31.1 21.5
CIBAL20190531020 42.5 11.6 14.5 6.0 5.2 3.4 4.3 5.1 3.5 19.4 4.5 63.9 19.9 20.9 5.2 29.2 19.0
CIBAL20190531018 40.0 13.0 14.5 5.6 4.7 4.0 3.8 4.6 2.7 19.1 3.8 65.2 19.7 21.2 5.0 29.6 20.0
Range of males 40.0–45.5 11.5–13.0 14.0–15.9 5.0–6.0 4.5–5.6 3.2–4.1 3.7–4.3 4.4–5.1 2.7–3.5 18.8–19.9 3.8–5.0 63.9–70.8 17.7–20.2 20.9–22.5 5.0–5.5 29.2–33.1 19.0–22.7
Mean ± SD of males 42.3 ± 2.04 12.1 ± 0.62 14.6 ± 0.71 5.5 ± 0.48 4.9 ± 0.45 3.8 ± 0.41 4.0 ± 0.28 4.7 ± 0.25 3.0 ± 0.36 19.4 ± 0.44 4.34 ± 0.44 66.6 ± 2.70 19.3 ± 1.03 21.8 ± 0.73 5.2 ± 0.18 30.3 ± 1.64 20.6 ± 1.47
CIBAL20190531022 51.2 12.9 17.4 6.5 5.7 4.2 4.7 4.9 3.3 23.6 4.0 83.7 26.0 27.5 6.0 38.6 25.6
CIBAL20190811015 48.9 13.1 16.1 5.5 5.1 3.1 4.1 4.9 2.9 23.8 3.7 83.4 24.7 26.2 5.3 38.0 26.0
CIBAL20190811014 49.4 13.2 16.5 6.0 5.3 4.0 4.5 5.1 3.1 24.5 3.3 88.2 25.4 28.0 5.0 40.4 20.5
Range of females 48.9–51.2 12.9–13.2 16.0–17.0 5.5–6.5 5.1–5.7 3.1–4.2 4.1–4.7 4.9–5.1 2.9–3.3 23.6–24.5 3.3–4.0 83.4–88.2 24.7–26.0 26.2–28.0 5.0–6.0 38.0–40.4 20.5–26.0
Mean ± SD of females 49.8 ±1.21 13.1 ±0.17 16.7 ±0.66 6.0 ±0.50 5.4 ±0.31 3.8 ±0.59 4.4 ±0.31 5.0 ±0.12 3.1 ±0.21 24.0 ±0.48 3.7 ±0.35 85.1 ±2.68 25.4 ±0.64 27.2 ±0.92 5.4 ±0.51 39.0 ±1.24 24.0 ±3.06

Phylogenetic analyses

Six specimens of the undescribed species were included in the molecular analyses (Table 2). Total DNA was extracted using a standard phenol-chloroform extraction protocol (Sambrook et al. 1989). Two fragments of the mitochondrial 16S rRNA (16S) and cytochromeoxidase subunit I (COI) genes were amplified. For 16S gene, the primers P7 (5’-CGCCTGTTTACCAAAAACAT-3’) and P8 (5’-CCGGTCTGAACTCAGATCACGT-3’) were used following Simon et al. (1994), and for COI gene, Chmf4 (5’-TYTCWACWAAYCAYAAAGAYATCGG-3’) and Chmr4 (5’-ACYTCRGGRTGRCCRAARAATCA-3’) were used following Che et al. (2012). The fragments were amplified under the following conditions: an initial denaturing step at 95 °C for 4 min; 36 cycles of denaturing at 95 °C for 30 s, annealing at 52 °C (for 16S)/47 °C (for COI) for 40 s and extending at 72 °C for 70 s. Sequencing was conducted using an ABI3730 automated DNA sequencer in Shanghai DNA BioTechnologies Co., Ltd. New sequences were deposited in GenBank (for GenBank accession numbers see Table 2).

For molecular analyses, the available sequence data for congeners of Megophrys were downloaded from GenBank (Table 2), primarily from previous studies (Chen et al. 2017; Liu et al. 2018). For phylogenetic analyses, corresponding sequences of one Leptobrachella oshanensis (Liu, 1950) and one Leptobrachium boringii (Liu, 1945) were also downloaded from GenBank (Table 2), and used as outgroups according to Mahony et al. (2017). Sequences were assembled and aligned using the Clustalw module in BioEdit v.7.0.9.0 (Hall 1999) with default settings. Alignments were checked by eye and revised manually if necessary. For phylogenetic analyses of mitochondrial DNA, the dataset concatenated with 16S and COI gene sequences. To avoid under- or over-parameterisation (Lemmon and Moriarty 2004; McGuire et al. 2007), the best partition scheme and the best evolutionary model for each partition were chosen for the phylogenetic analyses using PARTITIONFINDER v. 1.1.1 (Robert et al. 2012). In this analysis, 16S gene and each codon position of COI gene were defined, and Bayesian Inference Criteria was used. As a result, the analysis suggested that the best partition scheme is16S gene/each codon position of COI gene, and selected GTR + G + I model as the best model for each partition. Phylogenetic analyses were conducted using maximum likelihood (ML) and Bayesian Inference (BI) methods, implemented in PhyML v. 3.0 (Guindon et al. 2010) and MrBayes v. 3.12 (Ronquist and Huelsenbeck 2003), respectively. For the ML tree, branch supports were drawn from 10,000 nonparametric bootstrap replicates. In BI, two runs each with four Markov chains were simultaneously run for 50 million generations with sampling every 1,000 generations. The first 25% trees were removed as the “burn-in” stage followed by calculations of Bayesian posterior probabilities and the 50% majority-rule consensus of the post burn-in trees sampled at stationarity. Finally, genetic distance between species based on uncorrected p-distance model was estimated on each gene using MEGA v. 6.06 (Tamura et al. 2013).

Table 2.

Information for samples used in molecular phylogenetic analyses in this study.

ID Species Voucher number Locality GenBank accession number
16S COI
1 Megophrys anlongensis sp. nov. CIBAL20190531018 Anlong County, Guizhou, China MT823184 MT823261
2 CIBAL20190531017 Anlong County, Guizhou, China MT823185 MT823262
3 CIBAL20190531022 Anlong County, Guizhou, China MT823186 MT823263
4 CIBAL20190811014 Anlong County, Guizhou, China MT823187 MT823264
5 CIBAL20190811015 Anlong County, Guizhou, China MT823188 MT823265
6 CIBAL20190531019 Anlong County, Guizhou, China MT823189 MT823266
7 Megophrys nankunensis SYS a004498 Nankun Shan, Guangdong, China MK524108 MK524139
8 Megophrys dongguanensis SYS a001972 Yinping Shan, Guangdong, China MK524098 MK524129
9 Megophrys cheni SYS a001427 Jinggang Shan, Jiangxi, China KJ560391
10 Megophrys obesa SYS a002272 Heishiding Nature Reserve, Guangdong, China KJ579122
11 Megophrys ombrophila KRM18 Wuyishan, Fujian, China KX856404
12 Megophrys wugongensis SYS a002610 Wugongshan Scenic Area, Jiangxi, China MK524114 MK524145
13 Megophrys lini SYS a002370 Suichuan, Jiangxi, China KJ560412
14 Megophrys xiangnanensis SYS a002874 Yangming Shan, Hunan, China MH406713 MH406165
15 Megophrys nanlingensis SYS a001959 Nanling Nature Reserve, Guangdong, China MK524111 MK524142
16 Megophrys kuatunensis SYS a001579 Wuyi Shan, Fujian, China KJ560376
17 Megophrys jinggangensis KIZ07132 Chashan Forest Farm, Jiangxi, China KX811840 KX812108
18 Megophrys xianjuensis CIBXJ190505 Xianju, Zhejiang, China MN563753 MN563769
19 Megophrys lishuiensis WYF00169 Lishui, Zhejiang, China KY021418
20 Megophrys huangshanensis KIZ022004 Huang Shan, Anhui, China KX811821 KX812107
21 Megophrys boettgeri Tissue ID: YPXJK033 Wuyi Shan, Fujian, China KX811814 KX812104
22 Megophrys liboensis GNUG:20160408003 Libo, Guizhou, China MF285262
23 Megophrys mufumontana SYS a006391 Mufu Shan, Hunan, China MK524105 MK524136
24 Megophrys mirabilis SYS a002192 Huaping Nature Reserve, Guangxi, China MH406669 MH406109
25 Megophrys shunhuangensis HNNU16SH02 Shunhuang Mountains, Hunan, China MK836037
26 Megophrys acuta SYS a001957 Heishiding Nature Reserve, Guangdong, China KJ579118
27 Megophrys leishanensis CIBLS20171101001 Leigong Shan, Guizhou, China MK005310 MK005306
28 Megophrys shimentaina SYS a002077 Shimentai Nature Reserve Guangdong, China MH406655 MH406092
29 Megophrys yangmingensis SYS a002877 Yangming Shan, Hunan, China MH406716 MH406168
30 Megophrys jiulianensis SYS a002107 Jiulian Shan, Jiangxi, China MK524099 MK524130
31 Megophrys wushanensis KIZ045469 Guangwu Shan, Sichuan, China KX811838 KX812094
32 Megophrys baolongensis KIZ019216 Baolong, Chongqing, China KX811813 KX812093
33 Megophrys tuberogranulata Tissue ID: YPX10987 Badagongshan Nature Reserve, Hunan, China KX811823 KX812095
34 Megophrys binchuanensis KIZ019441 Jizu Shan, Yunnan, China KX811849 KX812112
35 Megophrys sangzhiensis SYSa004307 Zhangjiajie, Hunan, China MH406798 MH406260
36 Megophrys spinata SYSa002227 Leigong Shan, Guizhou, China MH406676 MH406116
37 Megophrys binlingensis SYSa005313 Wawu Shan, Sichuan, China MH406892 MH406354
38 Megophrys angka KIZ040591 Kiew Mae Pan nature trail, Chiang Mai, Thailand MN508052
39 Megophrys omeimontis KIZ025765 Emei Shan, Sichuan, China KX811884 KX812136
40 Megophrys palpebralespinosa KIZ011603 Pu Hu Nature Reserve, Thanh Hoa, Vietnam KX811888 KX812137
41 Megophrys jingdongensis KIZ-LC0805067 Huanglianshan National Nature Reserve, Yunnan, China KX811872 KX812131
42 Megophrys daweimontis KIZ048997 Dawei Shan, Yunnan, China KX811867 KX812125
43 Megophrys wuliangshanensis KIZ046812 Huangcaoling, Yunnan, China KX811881 KX812129
44 Megophrys fansipanensis VNMN 2018.01 Lao Cai, Sa Pa, Vietnam MH514886
45 Megophrys hoanglienensis VNMN 2018.02 Lao Cai, Sa Pa, Vietnam MH514889
46 Megophrys minor KIZ01939 Qingcheng Shan, Sichuan, China KX811896 KX812145
47 Megophrys jiangi CIBKKS20180722006 Kuankuosui Nature Reserve, Guizhou, China MN107743 MN107748
48 Megophrys chishuiensis CIBCS20190518031 Chishui Nature Reserve, Guizhou, China MN954707 MN928958
49 Megophrys brachykolos ROM 16634 Hong Kong, China KX811897 KX812150
50 Megophrys elfina ZMMU ABV-00454 Bidoup Mountain, Lam Dong, Vietnam KY425379
51 Megophrys gerti ITBCZ 1108 Nui Chua National Park, Ninh Thuan, Vietnam KX811917 KX812161
52 Megophrys synoria FMNH 262778 O’Reang, Mondolkiri, Cambodia KY022198
53 Megophrys microstoma KIZ048799 Xiaoqiaogou Nature Reserve, Yunnan, China KX811914 KX812156
54 Megophrys hansi KIZ010360 Phong Dien Nature Reserve, Thua Thien Hue, Vietnam KX811913 KX812155
55 Megophrys pachyproctus KIZ010978 Beibeng, Xizang, China KX811908 KX812153
56 Megophrys baluensis ZMH A13125 Gunung Kinabalu National Park, Kogopan Trail, Malaysia KJ831310
57 Megophrys stejnegeri KU 314303 Pasonanca Natural Park, Zamboanga, Philippines KX811922 KX812052
58 Megophrys ligayae ZMMU NAP-05015 Palawan, Philippines KX811919 KX812051
59 Megophrys nasuta KIZ019419 Malaysia KX811921 KX812054
60 Megophrys kobayashii UNIMAS 8148 Gunung Kinabalu National Park, Sabah, Malaysia KJ831313
61 Megophrys edwardinae FMNH 273694 Bintulu, Sarawak, Malaysia KX811918 KX812050
62 Megophrys aceras KIZ025467 Khao Nan National Park, Nakhon Si Thammarat, Thailand KX811925 KX812159
63 Megophrys zhangi KIZ014278 Zhangmu, Xizang, China KX811765 KX812084
64 Megophrys sanu K5198/ZSI11393 KX894679
65 Megophrys katabhako ZSIA11799 KX894669
66 Megophrys periosa BNHS 6061 West Kameng dist., Arunachal Pradesh, India KY022309 MH647528
67 Megophrys himalayana SDBDU2009.75 East Siang dist., Arunachal Pradesh, India KY022311
68 Megophrys glandulosa KIZ048439 Husa, Yunnan, China KX811762 KX812075
69 Megophrys medogensis KIZ06621 Beibeng, Xizang, China KX811767 KX812082
70 Megophrys flavipunctata SDBDU2009.297 East Khasi Hills dist., Meghalaya, India KY022307 MH647536
71 Megophrys maosonensis KIZ016045 Xiaoqiaogou Nature Reserve, Yunnan, China KX811780 KX812080
72 Megophrys mangshanensis KIZ021786 Nanling National Forest Park, Guangdong, China KX811790 KX812079
73 Megophrys oreocrypta BNHS 6046 West Garo Hills dist., Meghalaya, India KY022306
74 Megophrys major SYSa002961 Zhushihe, Yunnan, China MH406728 MH406180
75 Megophrys parva SYSa003042 Zhushihe, Yunnan, China MH406737 MH406189
76 Megophrys auralensis NCSM 79599 Aural, Kampong Speu, Cambodia KX811807
77 Megophrys dringi UNIMAS 8943 Gunung Mulu National Park, Sarawak, Malaysia KJ831317
78 Megophrys gigantica SYSa003933 Wuliang shan, Yunnan, China MH406775 MH406235
79 Megophrys shapingensis KIZ014512 Liziping Nature Reserve, Sichuan, China KX811904 KX812060
80 Megophrys wawuensis KIZ025799 Wawu Shan, Sichuan, China KX811902 KX812062
81 Megophrys nankiangensis CIB ZYC517 Nanjiang, Sichuan, China KX811900
82 Megophrys lancip MZB:Amp:22233 KY679891
83 Megophrys montana LSUMZ 81916 Sukabumi, Java, Indonesia KX811927 KX812163
84 Megophrys popei SYS a000589 Naling Nature Reserve, Guangdong, China KM504251
85 Megophrys carinense Tissue ID: YPX20455 Dayao Shan, Guangxi, China KX811811 KX812057
86 Megophrys feae KIZ046706 Huangcaoling, Yunnan, China KX811810 KX812056
87 Megophrys chuannanensis CIB20050081 Hejiang, Sichuan, China KM504261
88 Megophrys intermedia ZFMK 87596 U Bo, Phong Nha-Ke Bang NP, Vietnam HQ588950
89 Leptobrachium boringii Tissue ID: YPX37539 Emei Shan, Sichuan, China KX811930 KX812164
90 Leptobrachella oshanensis KIZ025778 Emei Shan, Sichuan, China KX811928 KX812166

Morphological comparisons

All eight adult specimens of the undescribed species were measured (Table 1). The terminology and methods followed Fei et al. (2009). Measurements were taken with a dial caliper to 0.1 mm. Seventeen morphometric characters of adult specimens were measured:

ED eye diameter (distance from the anterior corner to the posterior corner of the eye);

FL foot length (distance from tarsus to the tip of fourth toe);

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

HDW maximum head width (greatest width between the left and right articulations of jaw);

HLL hindlimb length (maximum length from the vent to the distal tip of the Toe IV);

IND internasal distance (minimum distance between the inner margins of the external nares);

IOD interorbital distance (minimum distance between the inner edges of the upper eyelids);

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

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

SVL snout-vent length (distance from the tip of the snout to the posterior edge of the vent);

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

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

THL thigh length (distance from vent to knee);

TL tibia length (distance from knee to tarsus);

TW maximal tibia width;

TYD maximal tympanum diameter;

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

The undescribed species was also compared with all other congeners on morphology. Comparative data were obtained from related species as described in literature (Table 3).

Table 3.

Bibliographic references for morphological characters for congeners of the genus Megophrys.

Species Literature obtained
M. aceras Boulenger, 1903 Boulenger 1903
M. acuta Wang, Li & Jin, 2014 Li et al. 2014
M. ancrae Mahony, Teeling & Biju, 2013 Mahony et al. 2013
M. angka Wu, Suwannapoom, Poyarkov, Chen, Pawangkhanant, Xu, Jin, Murphy & Che, 2019 Wu et al. 2019
M. auralensis Ohler, Swan & Daltry, 2002 Ohler et al. 2002
M. baluensis (Boulenger, 1899) Boulenger 1899a
M. baolongensis Ye, Fei & Xie, 2007 Ye et al. 2007
M. binchuanensis Ye & Fei, 1995 Ye and Fei 1995
M. binlingensis Jiang, Fei & Ye, 2009 Fei et al. 2009
M. boettgeri (Boulenger, 1899) Boulenger 1899b
M. brachykolos Inger & Romer, 1961 Inger and Romer 1961
M. carinense (Boulenger, 1889) Boulenger 1889
M. caobangensis Nguyen, Pham, Nguyen, Luong, and Ziegler, 2020 Nguyen et al. 2020
M. caudoprocta Shen, 1994 Shen 1994
M. cheni (Wang & Liu, 2014) Wang et al. 2014
M. chishuiensis Xu, Li, Liu, Wei & Wang, 2020 Xu et al. 2020
M. chuannanensis (Fei, Ye & Huang, 2001) Fei et al. 2001
M. damrei Mahony, 2011 Mahony 2011
M. daweimontis Rao & Yang, 1997 Rao and Yang 1997
M. dongguanensis Wang & Wang, 2019 Wang et al. 2019b
M. dringi Inger, Stuebing & Tan, 1995 Inger et al. 1995
M. edwardinae Inger, 1989 Inger 1989
M. elfina Poyarkov, Duong, Orlov, Gogoleva, Vassilieva, Nguyen, Nguyen, Nguyen, Che & Mahony, 2017 Poyarkov et al. 2017
M. fansipanensis Tapley, Cutajar, Mahony, Nguyen, Dau, Luong, Le, Nguyen, Nguyen, Portway, Luong & Rowley, 2018 Tapley et al. 2018
M. feae Boulenger, 1887 Boulenger 1887
M. feii Yang, Wang & Wang, 2018 Yang et al. 2018
M. flavipunctata Mahony, Kamei, Teeling & Biju, 2018 Mahony et al. 2018
M. gerti (Ohler, 2003) Ohler 2003
M. gigantica Liu, Hu & Yang, 1960 Liu et al. 1960
M. glandulosa Fei, Ye & Huang, 1990 Fei et al. 1990
M. hansi (Ohler, 2003) Ohler 2003
M. himalayana Mahony, Kamei, Teeling & Biju, 2018 Mahony et al. 2018
M. hoanglienensis Tapley, Cutajar, Mahony, Nguyen, Dau, Luong, Le, Nguyen, Nguyen, Portway, Luong & Rowley, 2018 Tapley et al. 2018
M. huangshanensis Fei & Ye, 2005 Fei and Ye 2005
M. insularis (Wang, Liu, Lyu, Zeng & Wang, 2017) Wang et al. 2017a
M. intermedia Smith, 1921 Smith 1921
M. jiangi Liu, Li, Wei, Xu, Cheng, Wang & Wu, 2020 Liu et al. 2020
M. jingdongensis Fei & Ye, 1983 Fei et al. 1983
M. jinggangensis (Wang, 2012) Wang et al. 2012
M. jiulianensis Wang, Zeng, Lyu & Wang, 2019 Wang et al. 2019b
M. kalimantanensis Munir, Hamidy, Matsui, Iskandar, Sidik & Shimada, 2019 Munir et al. 2019
M. kobayashii Malkmus & Matsui, 1997 Malkmus and Matsui 1997
M. koui Mahony, Foley, Biju & Teeling, 2017 Mahony et al. 2017
M. kuatunensis Pope, 1929 Pope 1929
M. lancip Munir, Hamidy, Farajallah & Smith, 2018 Munir et al. 2018
M. leishanensis Li, Xu, Liu, Jiang, Wei & Wang, 2018 Li et al. 2018a
M. lekaguli Stuart, Chuaynkern, Chan-ard & Inger, 2006 Stuart et al. 2006
M. liboensis (Zhang, Li, Xiao, Li, Pan, Wang, Zhang & Zhou, 2017) Zhang et al. 2017
M. ligayae Taylor, 1920 Taylor 1920
M. lini (Wang & Yang, 2014) Wang et al. 2014
M. lishuiensis (Wang, Liu & Jiang, 2017) Wang et al. 2017b
M. longipes Boulenger, 1886 Boulenger 1886
M. major Boulenger, 1908 Boulenger 1908
M. mangshanensis Fei & Ye, 1990 Fei et al. 2012
M. maosonensis Bourret, 1937 Bourret 1937
M. medogensis Fei, Ye & Huang, 1983 Fei et al. 1983
M. megacephala Mahony, Sengupta, Kamei & Biju, 2011 Mahony et al. 2011
M. microstoma (Boulenger, 1903) Boulenger 1903
M. minor Stejneger, 1926 Stejneger 1926
M. mirabilis Lyu, Wang & Zhao Lyu et al. 2020
M. montana Kuhl & Van Hasselt, 1822 Kuhl and Van Hasselt 1822
M. monticola (Günther, 1864) Günther 1864
M. mufumontana Wang, Lyu & Wang, 2019 Wang et al. 2019b
M. nankiangensis Liu & Hu, 1966 Hu and Liu 1966
M. nankunensis Wang, Zeng &. Wang, 2019 Wang et al. 2019b
M. nanlingensis Lyu, Wang, Liu & Wang, 2019 Wang et al. 2019b
M. nasuta (Schlegel, 1858) Schlegel 1858
M. obesa Wang, Li & Zhao, 2014 Wang et al. 2014
M. ombrophila Messenger & Dahn, 2019 Messenger et al. 2019
M. omeimontis Liu, 1950 Liu 1950
M. oreocrypta Mahony, Kamei, Teeling & Biju, 2018 Mahony et al. 2018
M. oropedion Mahony, Teeling & Biju, 2013 Mahony et al. 2013
M. orientalis Li, Lyu, Wang & Wang, 2020 Li et al. 2020
M. pachyproctus Huang, 1981 Huang and Fei 1981
M. palpebralespinosa Bourret, 1937 Bourret 1937
M. parallela Inger & Iskandar, 2005 Inger and Iskandar 2005
M. parva (Boulenger, 1893) Boulenger 1893
M. periosa Mahony, Kamei, Teeling & Biju, 2018 Mahony et al. 2018
M. popei (Zhao, Yang, Chen, Chen & Wang, 2014) Zhao et al. 2014
M. robusta Boulenger, 1908 Boulenger 1908
M. rubrimera Tapley, Cutajar, Mahony, Chung, Dau, Nguyen, Luong & Rowley, 2017 Tapley et al. 2017
M. sangzhiensis Jiang, Ye & Fei, 2008 Jiang et al. 2008
M. serchhipii (Mathew & Sen, 2007) Mathew and Sen 2007
M. shapingensis Liu, 1950 Liu 1950
M. shimentaina Lyu, Liu & Wang Lyu et al. 2020
M. shuichengensis Tian & Sun, 1995 Tian and Sun 1995
M. shunhuangensis Wang, Deng, Liu, Wu & Liu, 2019 Wang et al. 2019a
M. spinata Liu & Hu, 1973 Hu et al. 1973
M. stejnegeri Taylor, 1920 Taylor 1920
M. synoria (Stuart, Sok & Neang, 2006) Stuart et al. 2006
M. takensis Mahony, 2011 Mahony 2011
M. tuberogranulata Shen, Mo & Li, 2010 Mo et al. 2012
M. vegrandis Mahony, Teeling, Biju, 2013 Mahony et al. 2013
M. wawuensis Fei, Jiang & Zheng, 2001 Fei et al. 2012
M. wugongensis Wang, Lyu & Wang, 2019 Wang et al. 2019b
M. wuliangshanensis Ye & Fei, 1995 Ye and Fei 1995
M. wushanensis Ye & Fei, 1995 Ye and Fei 1995
M. xianjuensis Wang, Wu, Peng, Shi, Lu & Wu, 2020 Wang et al. 2020
M. xiangnanensis Lyu, Zeng & Wang Lyu et al. 2020
M. yangmingensis Lyu, Zeng & Wang Lyu et al. 2020
M. zhangi Ye & Fei, 1992 Ye and Fei 1992
M. zunhebotoensis (Mathew & Sen, 2007) Mathew and Sen 2007

Bioacoustics data

The advertisement calls of the undescribed species were recorded from the holotype specimen CIBAL20190531018 in the field on 31 May 2019 in Anlong County, Guizhou Province, China. The advertisement call of the undescribed species was recorded in the stream at ambient air temperature of 18.5 °C and air humidity of 83%. SONY PCM-D50 digital sound recorder was used to record within 30 cm of the calling individual. The sound files in wave format were resampled at 48 kHz with sampling depth 24 bits. The sonograms and waveforms were generated by WaveSurfer software (Sjöander and Beskow 2000) from which all parameters and characters were measured. Ambient temperature was taken by a digital hygrothermograph.

Results

Phylogenetic analyses

Aligned sequence matrix of 16S+COI contains 1104 bp. ML and BI trees of the mitochondrial DNA dataset presented almost consistent topology (Fig. 2). In mitochondrial DNA trees, all samples of the undescribed species were clustered into one clade which was nested into the Megophrys clade. However, the relationships between the undescribed species and its related species were not resolved though it was likely sister to M. binchuanensis in topology.

Figure 2. 

Maximum likelihood (ML) tree of the genus Megophrys reconstructed based on the 16S rRNA and COI gene sequences. Bayesian posterior probability/ML bootstrap supports were denoted beside each node. Samples 1–90 refer to Table 2.

Genetic distances between samples of the undescribed species either on 16S or on COI genes were below 0.2% much lower than the interspecific genetic distance between recognised Megophrys species (Suppl. materials 1, 2). The genetic distance between the undescribed species and its closest related species M. binchuanensis were 2.3% and 10.0% on 16S and COI, respectively, which was higher than or at the same level with those among many pairs of sister species, such as, 2.1% and 6.3% on 16S and COI respectively between M. wushanensis and M. baolongensis, 1.7% and 3.8% on 16S and COI respectively between M. spinata and M. sangzhiensis (Suppl. materials 1, 2).

Morphological comparisons

The new species could be identified from its congeners in a series of morphological characters (Suppl. material 3). The detailed demonstration based on morphological comparisons see the following section on describing the new species.

Taxonomic account

Megophrys anlongensis sp. nov.

Holotype

CIBAL20190531018 (Figs 3, 4), adult male, from Anlong County, Guizhou Province, China (24.9899277°N, 105.5990611°E, ca. 1290 m a. s. l.), collected by Jing Liu on 31 May 2019.

Paratype

Four adult males and three females from the same place as holotype collected by Shi-Ze Li and Jing Liu. CIBAL20190531017, CIBAL20190531019, CIBAL20190531021 and CIBAL20190531022 collected on 31 May 2019 by Jing Liu, and CIBAL20190811014 and CIBAL20190811015 collected by Shi-Ze Li on 11 August 2019.

Diagnosis

Megophrys anlongensis sp. nov. is assigned to the genus Megophrys based on molecular phylogenetic analyses and the following generic diagnostic characters: snout shield-like, projecting beyond the lower jaw; canthus rostralis distinct; chest glands small and round, closer to the axilla than to midventral line; femoral glands on rear part of thigh; vertical pupils.

Megophrys anlongensis sp. nov. could be distinguished from its congeners by a combination of the following morphological characters: (1) body size moderate (SVL 40.0–45.5 mm in males and 48.9–51.2 mm in females); (2) vomerine teeth absent; (3) tongue not notched behind; (4) a small horn-like tubercle at the edge of each upper eyelid; (5) tympanum distinctly visible, oval; (6) two metacarpal tubercles on hand; (7) toes with rudimentary webbing; (8) heels overlapping when thighs are positioned at right angles to the body; (9) tibiotarsal articulation reaching the level of mid-eye when leg stretched forward; (10) an internal single subgular vocal sac in male; (11) in breeding males, brownish nuptial pads, made up of black nuptial spines, present on the dorsal base of the first two fingers.

Figure 3. 

Photographs of the holotype CIBAL20190531018 of Megophrys anlongensis sp. nov. in life A dorsal view B ventral view C dorsal view of hand D ventral view of hand. E ventral view of foot.

Description of holotype

(Figs 3, 4). SVL 40.0 mm; head width larger than head length slightly (HDW/HDL ratio about 1.1); snout obtusely pointed, protruding well beyond the margin of the lower jaw in ventral view; loreal region vertical and concave; canthus rostralis well-developed; top of head flat in dorsal view; eye large, eye diameter 35.4% of head length; pupils vertical; nostril orientated laterally, closer to snout than eye; tympanum distinct, 60% of eye diameter; vomerine ridges present and vomerine teeth absent; margin of tongue smooth, not notched behind.

Forelimbs slender, the length of lower arm and hand 47.9% of SVL; fingers slender, relative finger lengths: I < II < V < III; tips of digits globular, without lateral fringes; subarticular tubercle distinct at the base of each finger; two metacarpal tubercles, prominent, oval-shaped, the inner one bigger than the outer one.

Hindlimbs slender; heels overlapping when thighs are positioned at right angles to the body; tibiotarsal articulation reaching the middle eye when leg stretched forward; tibia length longer than thigh length; relative toe lengths I < II < V < III < IV; tips of toes round, slightly dilated; subarticular tubercles present on each toes; toes with rudimentary webbing and narrow lateral fringe; inner metatarsal tubercle oval-shaped; outer metatarsal tubercle absent.

Dorsal skin rough, several large warts scattered on flanks; a small horn-like tubercle at the edge of each upper eyelid; tubercles on the dorsum forming a weak X-shaped ridge, two dorsolateral parallel ridges on either side of the X-shaped ridges; an inverted triangular brown speckle between two upper eyelids; several tubercles on the flanks and dorsal surface of thighs and tibias; supratympanic fold distinct.

Ventral surface smooth; numerous granules scattered on flanks; glands on chest indistinct; numerous white granules on outer thighs and posterior end of the body distinctly protruding and forming an arc-shaped swelling above the anal region.

Colouration of holotype in life

(Fig. 3). Dorsal brown, an inverted triangular brown speckle between the eyes; X-shaped ridges on the dorsum, four dark transverse bands on the dorsal surface of the thigh and shank; ventral surface of body brown with white spots; several dark brown and white vertical bars on the lower and upper lip; ventral surface of anterior limb orange, with some brown spots and posterior limb orange with numerous white granules; tip of digits pale grey; inner metatarsal tubercle and two metacarpal tubercles pinkish; soles uniform black; pectoral glands white.

Colouration of holotype in preservation

(Fig. 4). Colour of dorsal surface fades to taupe; the inverted triangular brown speckle between the eyes and X-shaped ridges on dorsum are more distinct; ventral surface greyish white; creamy white substitutes the purple-grey on tip of digits; the posterior of ventral surface of body, inner of thigh and upper of tibia fades to creamy white.

Figure 4. 

Photographs of the holotype specimen CIBAL20190531018 of Megophrys anlongensis sp. nov. A dorsal view B ventral view C lateral view D ventral view of hand E ventral view of foot.

Variation

In CIBAL20190531017 the inverted triangular brown speckle is connected to the X-shape ridge (Fig. 5A), and the ventral surface is reddish brown with creamy white in the posterior of belly (Fig. 5B); in CIBAL20190531022 an X-shaped marking on the dorsum (Fig. 5C), and anterior of ventral surface is brownish (Fig. 5D); in CIBAL20190811014 dorsal skin more rough, some black warts scattered on dorsal (Fig. 5E), and the white spots on ventral surface are less numerous and some black spots are mixed with the white spots or brown spots on ventral surface (Fig. 5F).

Figure 5. 

Colour variation in Megophrys anlongensis sp. nov. A dorsolateral view of the specimen CIBAL20190531017 B ventral view of the male specimen CIBAL20190531017 C dorsolateral view of the specimen CIBAL20190531022 D ventral view of the specimen CIBAL20190531022 E dorsolateral view of the specimen CIBAL20190811014 F ventral view of the specimen CIBAL20190811014.

Advertisement call

The call description is based on recordings of the holotype CIBAL20190531018 (Fig. 6) calling from a shrub leaf near a streamlet, and the ambient air temperature was 18.5 °C. Each call consists of 14–26 (mean 22.5 ± 4.4, N = 6) notes. Call duration was 2832–5621 ms (mean 4413 ± 972, N = 6). Call interval was 6812–14387 ms (mean 10878 ± 2701, N = 5). Each note had a duration of 129–211 ms (mean 167 ± 0.02, N = 135) and the intervals between notes 34–94 ms (mean 57 ± 0.01, N = 128). Amplitude modulation within note was apparent, beginning with moderately high energy pulses, increasing slightly to a maximum by approximately mid note, and then decreasing towards the end of each note. The average dominant frequency was 2469 ± 197.47 (2250–3000 Hz, N = 6).

Figure 6. 

Visualisation of advertisement calls of Megophrys anlongensis sp. nov. A waveform showing one note B sonogram showing one note C waveform showing 20 notes of one call D sonogram showing 20 notes of one call.

Secondary sexual characters

Adult males have a single subgular vocal sac. In breeding males, brownish nuptial pads, made up of black nuptial spines, present on the dorsal bases of the first two fingers (Fig. 3C).

Comparisons

By body size medium, Megophrys anlongensis sp. nov. differs from M. aceras, M. acuta, M. angka, M. auralensis, M. binchuanensis, M. boettgeri, M. caobangensis, M. cheni, M. daweimontis, M. dringi, M. elfina, M. feii, M. gerti, M. jinggangensis, M. jiulianensis, M. kuatunensis, M. leishanensis, M. lishuiensis, M. microstoma, M. mufumontana, M. nankunensis, M. nanlingensis, M. obesa, M. ombrophila, M. oropedion, M. pachyproctus, M. palpebralespinosa, M. rubrimera, M. serchhipii, M. shimentaina, M. shunhuangensis, M. vegrandis, M. wugongensis, M. wuliangshanensis, M. wushanensis, M. xianjuensis, M. yangmingensis, M. zhangi, and M. zunhebotoensis (SVL > 40.0 mm in the new species vs. maximum SVL < 39.0 mm in the latter), and differs from M. carinense, M. caudoprocta, M. chuannanensis, M. damrei, M. feae, M. flavipunctata, M. gigantica, M. glandulosa, M. himalayana, M. kalimantanensis, M. kobayashii, M. lekaguli, M. ligayae, M. mangshanensis, M. medogensis, M. mirabilis, M. nasuta, M. omeimontis, M. orientalis, M. periosa, M. platyparietus, M. popei, M. sangzhiensis, M. shapingensis, and M. shuichengensis (maximum SVL < 52.0 mm in the new species vs. minimum SVL > 54.0 mm in the latter), and differs from M. edwardinae and M. monticola (SVL 48.9–51.2 mm in female in the new species vs. 69–82 mm in M. edwardinae and 40.5 mm in M. monticola).

By vomerine teeth absent, Megophrys anlongensis sp. nov. differs from M. ancrae, M. baluensis, M. carinense, M. caudoprocta, M. chuannanensis, M. damrei, M. daweimontis, M. dongguanensis, M. fansipanensis, M. feae, M. flavipunctata, M. glandulosa, M. himalayana, M. hoanglienensis, M. insularis, M. intermedia, M. jingdongensis, M. jinggangensis, M. jiulianensis, M. kalimantanensis, M. kobayashii, M. lancip, M. lekaguli, M. liboensis, M. ligayae, M. longipes, M. mangshanensis, M. maosonensis, M. medogensis, M. megacephala, M. montana, M. nankunensis, M. nanlingensis, M. nasuta, M. omeimontis, M. oreocrypta, M. orientalis, M. oropedion, M. pachyproctus, M. palpebralespinosa, M. parallela, M. parva, M. periosa, M. platyparietus, M. popei, M. robusta, M. rubrimera, M. serchhipii, M. shimentaina, M. stejnegeri, M. takensis, M. zhangi, and M. zunhebotoensis (vs. present in the latter).

By a small horn-like tubercle at the edge of each upper eyelid, Megophrys anlongensis sp. nov. differs from M. aceras, M. acuta, M. carinense, M. caudoprocta, M. chuannanensis, M. feae, M. gerti, M. hansi, M. intermedia, M. intermedia, M. jinggangensis, M. kalimantanensis, M. koui, M. lancip, M. liboensis, M. microstoma, M. montana, M. nasuta, M. orientalis, M. palpebralespinosa, M. platyparietus, M. popei, M. shuichengensis, M. stejnegeri, and M. synoria (vs. having a prominent and elongated tubercle in the latter).

By tongue not notched behind, Megophrys anlongensis sp. nov. differs from M. ancrae, M. baolongensis, M. binlingensis, M. boettgeri, M. carinense, M. cheni, M. chuannanensis, M. damrei, M. dringi, M. fansipanensis,M. feae, M. feii, M. flavipunctata, M. gerti, M. glandulosa, M. hoanglienensis, M. huangshanensis, M. insularis, M. jiulianensis. M. jingdongensis, M. kalimantanensis , M. kuatunensis, M. liboensis, M. mangshanensis, M. maosonensis, M. medogensis, M. minor, M. nankiangensis, M. nanlingensis, M. omeimontis, M. oropedion, M. pachyproctus, M. parallela, M. popei, M. robusta, M. sangzhiensis, M. shapingensis, M. shuichengensis, M. spinata, M. vegrandis, M. wawuensis, M. zhangi, and M. zunhebotoensis (vs. tongue notched behind in the latter).

By toes with narrow lateral fringes, Megophrys anlongensis sp. nov. differs from M. angka, M. baolongensis, M. brachykolos, M. caobangensis, M. chishuiensis, M. damrei, M. daweimontis, M. dongguanensis, M. fansipanensis, M. feae, M. himalayana, M. hoanglienensis, M. huangshanensis, M. insularis, M. jiangi, M. jiulianensis, M. kalimantanensis, M. koui, M. lekaguli, M. lishuiensis, M. major, M. mangshanensis, M. medogensis, M. megacephala, M. microstoma, M. minor, M. nankunensis, M. obesa, M. ombrophila, M. oreocrypta, M. oropedion, M. pachyproctus, M. parva, M. periosa, M. shunhuangensis, M. takensis, M. tuberogranulata, M. wawuensis, M. wugongensis, M. wuliangshanensis, and M. xianjuensis (vs. lacking lateral fringes on toes in the latter), and differs from M. binchuanensis, M. boettgeri, M. carinense, M. cheni, M. chuannanensis, M. dringi, M. feii, M. gigantica, M. glandulosa, M. intermedia, M. jingdongensis, M. liboensis, M. lini, M. orientalis, M. palpebralespinosa, M. platyparietus, M. shapingensis, M. shuichengensis, M. spinata, and M. xiangnanensis (vs. with wide lateral fringes in the latter).

By toes with rudimentary webbing, Megophrys anlongensis sp. nov. differs from M. brachykolos, M. carinense, M. flavipunctata, M. jingdongensis, M. jinggangensis, M. lini, M. major, M. palpebralespinosa, M. popei, M. shuichengensis, and M. spinata (vs. at least one-fourth webbed in the latter).

By heels overlapping when thighs are positioned at right angles to the body, Megophrys anlongensis sp. nov. differs from M. acuta, M. brachykolos, M. dongguanensis, M. huangshanensis, M. kuatunensis, M. nankunensis, M. obesa, M. ombrophila, and M. wugongensis (vs. not meeting in the latter).

By tibiotarsal articulation reaching to the level of mid-eye when leg stretched forward, Megophrys anlongensis sp. nov. differs from M. daweimontis, M. glandulosa, M. lini, M. major, M. medogensis, M. obesa, and M. sangzhiensis (vs. reaching the anterior corner of the eye or beyond eye or nostril or tip of snout in the latter), differs from M. mufumontana (vs. reaching tympanum in males and to the eye in females in the latter), and differs from M. chishuiensis (vs. reaching the level between tympanum and eye in the latter).

By having an internal single subgular vocal sac in male, Megophrys anlongensis sp. nov. differs from M. caudoprocta, M. shapingensis, and M. shuichengensis (vs. vocal sac absent in the latter).

Megophrys anlongensis sp. nov. is genetically closest to M. binchuanensis. The new species could be identified from M. binchuanensis distinctly by having a bigger body size (SVL 40.0–45.5 mm in males and 48.9–51.2 in females in the new species vs. SVL 32.0–36.0 mm in males and 40.2–42.5 mm in females in the latter), having narrow lateral fringes on toes (vs. wide in the latter), and heels overlapping when thighs are positioned at right angles to the body (vs. just meeting in the latter).

Distribution and habitats

Megophrys anlongensis sp. nov. is known only from the type locality, Anlong County, Guizhou Province, China at elevations between 1400–1600 m. The individuals were frequently found near the streams surrounded by evergreen broadleaved forests (Fig. 7).

Figure 7. 

Habitats of Megophrys anlongensis sp. nov. in the type locality, Anlong County, Guizhou Province, China A landscape of montane forests in the type locality B a mountain stream where toads of the new species live (insert the holotype standing on the leaf beside the stream).

Etymology

The specific name anlongensis refers to the known distribution of this species, Anlong County, Guizhou Province, China. We propose the common English name “Anlong horned toad”, and Chinese name “An Long Jiao Chan” (安龙角蟾).

Discussion

Southwestern China was proposed as biodiversity hotspot (Myers et al. 2000). Guizhou Province, China is an important part of southwestern China, especially concerning the particular environments of karst rocky desertification, and knowledge of biodiversity levels and/or patterns are still seriously lacking in this region. Recently, a series of new amphibian species were described from Guizhou Province (Zhang et al. 2017; Li et al. 2018a, b, 2019a, b; Lyu et al. 2019; Wang et al. 2019c; Luo et al 2020; Liu et al 2020; Wei et al, 2020; Xu et al, 2020, Li et al, 2020), highlighting the underestimation of the species diversity of this province. For the genus Megophrys, molecular phylogenetic differences still suggested some cryptic species in or near this region (Liu et al. 2018), but Megophrys anlongensis sp. nov. was not found before. This indicates that more work should focus on detailed information for describing such species, and additionally, comprehensive and in-depth surveys should be led to discover more cryptic species of the genus in this province. According to our surveys, habitat degradation due to construction and human activities are impacting the population of Megophrys anlongensis sp. nov. Hence, it is urgent for us to understand its population status and suggest strategies for supplying conservation needs of the species.

Acknowledgements

We are grateful to editors and reviewers for their working on the manuscript. This work was supported by National Natural Sciences Foundation of China (Grant Nos.: 32070426 and 31960099), Basic research project of science and technology department of Guizhou Province (Nos. [2020] 1Y083), Science and technology support project of science and technology department of Guizhou Provincial (No. [2020] 4Y029) and Guizhou Provincial Department of Education Youth Science and Technology Talents Growth Project (Nos. KY[2018]455 and KY[2018]468).

References

  • Boulenger GA (1887) Description of a new frog of the genus Megalophrys. Annali del Museo Civico di Storia Naturale di Genova Serie 2, 4: 512–513.
  • Boulenger GA (1889) Description of a new batrachian of the genus Leptobrachium, obtain by M. L. Burma. Annali del Museo Civico di Storia Naturale di Genova, Serie 2, 7: 748–750.
  • Boulenger GA (1899a) Descriptions of three new reptiles and a new batrachian from Mount Kina Balu, North Borneo. Annals and Magazine of Natural History, Series 7(4): 1–453. https://doi.org/10.1080/00222939908678228
  • Boulenger GA (1899b) On a collection of reptiles and batrachians made by Mr. J. D. La Touche in N.W. Fokien, China. Proceedings of the Zoological Society of London 1899: 159–172.
  • Boulenger GA (1903) Report on the batrachians and reptiles. Annandale, N., and H. C. Robinson eds., Fasciculi Malayenses. Anthropological and Zoological Results of an Expedition to Perak and the Siamese Malay States 1901–1903 undertaken by Nelson Annandale and Herbert C. Robinson under the auspecies of the University of Edinburgh and the University of Liverpool. Volume 2, Zoology, Part 1: 131–176.
  • Bourret R (1937) Notes herpétologiques sur l’Indochine française. XIV. Les batraciens de la collection du Laboratoire des Sciences Naturelles de l’Université. Descriptions de quinze especes ou variétés nouvelles. Annexe au Bulletin Général de l’Instruction Publique Hanoi, 5–56.
  • Chen JM, Zhou WW, Nikolay A, Poyarkov Jr, Stuart BL, Brown RM, Lathrop A, Wang YY, Yuan ZL, Jiang K, Hou M, Chen HM, Suwannapoom C, Nguyen SN, Duong TV, Papenfuss TJ, Murphy RW, Zhang YP, Che J (2017) A novel multilocus phylogenetic estimation reveals unrecognized diversity in Asia toads, genus Megophrys sensu lato (Anura: Megophryidae). Molecular Phylogenetics and Evolution 106: 28–43. https://doi.org/10.1016/j.ympev.2016.09.004
  • Delorme M, Dubois A, Grosjean S, Ohler A (2006) Une nouvelle ergotaxinomie des Megophryidae (Amphibia, Anura). Alytes 24: 6–21.
  • Deuti K, Grosjean S, Nicolas V, Vasudevan K, Ohler A (2017) Nomenclatural puzzle in early Megophrys nomina (Anura, Megophryidae) solved with description of two new species from India (Darjeeling hills and Sikkim). Alytes 34: 20–48.
  • Dubois A (1987) Miscellanea taxinomica batrachologica (I). Alytes 1987 [1986]: 7–95.
  • Fei L, Hu SQ, Ye CY, Huang YZ (2009) Fauna Sinica. Amphibia. Volume 2. Anura. Science Press, Beijing. [In Chinese]
  • Fei L, Ye CY (2005) Two new species of Megophryidae from China. In: Fei et al. (Ed.) The Key and Illustration of Chinese. Sichuan Publishing House of Science and Technology, Chongqing, China. [In Chinese]
  • Fei L, Ye CY (2016) Genus Liuophrys Fei, Ye and Jiang, new genus; Subgenus Atympanophrys (Borealophrys) Fei, Ye and Jiang, new subgenus; Subgenus Atympanophrys (Gigantophrys) Fei, Ye and Jiang, new subgenus; Genus Boulenophrys Fei, Ye and Jiang, 2016, new genus; Subgenus Xenophrys (Tianophrys) Fei, Ye and Jiang, new subgenus. In Fei L, Ye CY (Eds) Amphibians of China. Volume (I). Science Press, Beijing.
  • Fei L, Ye CY, Huang YZ (1983) Two new subspecies of Megophrys omeimontis Liu from China (Amphibia, Pelobatidae). Acta Herpetologica Sinica. New Series, Chengdu, 2(2): 49–52. [In Chinese with English abstract]
  • Fei L, Ye CY, Huang YZ (1983) Two new subspecies of Megophrys omeimontis Liu from China (Amphibia, Pelobatidae). Acta Herpetologica Sinica. New Series, Chengdu 2(2): 49–52. [In Chinese with English abstract]
  • Fei L, Ye CY, Huang YZ (1990) Key to Chinese Amphibians. Publishing House for Scientific and Technological, Chongqing, China. [In Chinese]
  • Fei L, Ye CY, Huang YZ (2001) Colour Handbook Amph. Sichuan, Science Press, Beijing, China. [In Chinese]
  • Fei L, Ye CY, Jiang JP (2012) Colored atlas of Chinese Amphibians and their distributions. Sichuan Publishing House of Science and Technology, Chengdu, China. [In Chinese]
  • Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Systematic Biology 59(3): 07–321. https://doi.org/10.1093/sysbio/syq010
  • Günther ACLG (1864) The reptiles of British India. London: Ray Society by R. Hardwicke.
  • Hall TA (1999) BIOEDIT: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41(41): 95–98.
  • Hu SX, Zhao EM, Liu CZ (1966) A herpetological survey of the Tsinling and Ta-pa shan region. Acta Zoologica Sinica 18(1): 57–89.
  • Hu SX, Zhao EM, Liu CZ (1973) A survey of amphibians and reptiles in Kweichow province, including a herpetofauna analysis. Acta Zoologica Sinica, 19(2): 149–171.
  • Huang YZ, Fei L (1981) Two new species of amphibians from Xizang. Acta Zootaxonomica Sinica 6: 211–215.
  • Inger RF (1989) Four new species of frogs from Borneo. Malayan Nature Journal. Kuala Lumpur 42: 229–243.
  • Inger RF, Iskandar DT (2005) A collection of amphibians from west Sumatra, with description of a new species of Megophrys (Amphibia: Anura). Raffles Bulletin of Zoology, Singapore 133–142.
  • Inger RF, Stuebing RB, Lian TF (1995) New species and new records of Anurans from Boreno. Raffles Bulletin of Zoology, Singapore 43(1): 115–131.
  • Jiang JP, Ye CY, Fei L (2008) A new horn toad Megophrys sangzhiensis from Hunan, China (Amphibia, Anura). Zoological Research 29(2): 219–222. https://doi.org/10.3724/SP.J.1141.2008.00219 [In Chinese with English abstract]
  • Mathew R, Sen N (2007) Description of two new species of Megophrys (Amphibia: Anura: Megophryidae) from North-east India. Cobra 1: 18–28.
  • Jiang JP, Yuan FR, Xie F, Zheng ZH (2003) Phylogenetic relationships of some species and genera in megophryids inferred from partial sequences of mitochondrial 12S and 16S rRNA genes. Zoological Research 24: 241–248. [In Chinese with English abstract]
  • Kuhl H, Van Hasselt JC (1822) Uittreksels uit breieven van de Heeren Kuhl en van Hasselt, aan de Heeren C. J. Temminck, Th. van Swinderen en W. de Haan. Algemeene Konst-en Letter-Bode 7: 99–104.
  • Li SZ, Liu J, Wei G, Wang B (2020) A new species of the Asian leaf litter toad genus Leptobrachella (Amphibia, Anura, Megophryidae) from southwest China. ZooKeys 943: 91–118. https://doi.org/10.3897/zookeys.943.51572
  • Li SZ, Wei G, Xu N, Cui JG, Fei L, Jiang JP, Liu J, Wang B (2019a) A new species of the Asian music frog genus Nidirana (Amphibia, Anura, Ranidae) from Southwestern China. PeerJ 7: e7157. https://doi.org/10.7717/peerj.7157
  • Li SZ, Xu N, Liu J, Jiang JP, Wei G, Wang B (2018a) A new species of the Asian toad genus Megophrys sensu lato (Amphibia: Anura: Megophryidae) from Guizhou Province, China. Asian Herpetological Research 9(4): 224–239.
  • Li SZ, Xu N, Lv JC, Jiang JP, Wei G, Wang B (2018b) A new species of the odorous frog genus Odorrana (Amphibia, Anura, Ranidae) from southwestern China. PeerJ 6(e5695): 1–28. https://doi.org/10.7717/peerj.5695
  • Li SZ, Zhang MH, Xu N, Lv JC, Jiang JP, Liu J, Wei G, Wang B (2019b) A new species of the genus Microhyla (Amphibia: Anura: Microhylidae) from Guizhou Province, China. Zootaxa 4624: 551–575. https://doi.org/10.11646/zootaxa.4624.4.7
  • Li Y, Zhang DD, Lyu ZT, Wang J, Li YL, Liu ZY, Chen HH, Rao DQ, Jin ZF, Zhang CY, Wang YY (2020) Review of the genus Brachytarsophrys (Anura: Megophryidae), with revalidation of Brachytarsophrys platyparietus and description of a new species from China. Zoological Research 41(2): 105–122. https://doi.org/10.24272/j.issn.2095-8137.2020.033
  • Li YL, Jin MJ, Zhao J, Liu ZY, Wang YY, Pang H (2014) Description of two new species of the genus Megophrys (Amphibia: Anura: Megophryidae) from Heishiding Natural Resreve, Fengkai, Guangdong, China, based on molecular and morphological data. Zootaxa 3795(4): 449–471. https://doi.org/10.11646/zootaxa.3795.4.5
  • Liu CZ, Hu SQ, Yang HH (1960) Amphibian of Yunnan collected in 1958. Acat Zoological Sinica 12(2): 149–174.
  • Liu J, Li SZ, Wei G, Xu N, Cheng YL, Wang B, Wu J (2020) A new species of the Asian toad genus Megophrys sensu lato (Anura: Megophryidae) from Guizhou Province, China. Asian Herpetological Research 11(1): 1–18.
  • Liu ZY, Zhu TQ, Zeng ZC, Lyu ZT, Wang J, Messenger K, Greenberg A J, Gou ZX, Yang ZH, Shi SH, Wang YY (2018) Prevalence of cryptic species in morphologically uniform taxa – Fast speciation and evolutionary radiation in Asian frogs. Molecular Phylogenetics and Evolution 127: 723–731. https://doi.org/10.1016/j.ympev.2018.06.020
  • Lyu ZT, Li YQ, Zeng ZC, Zhao J, Liu ZL, Guo GX, Wang YY (2020) Four new species of Asian horned toads (Anura, Megophryidae, Megophrys) from southern China. ZooKeys 942: 105–140. https://doi.org/10.3897/zookeys.942.47983
  • Lyu ZT, Zeng ZC, Wan H, Yang JH, Li YL, Pang H, Wang YY (2019) A new species of Amolops (Anura: Ranidae) from China, with taxonomic comments on A. liangshanensis and Chinese populations of A. marmoratus. Zootaxa 4609: 247–268. https://doi.org/10.11646/zootaxa.4609.2.3
  • Mahony S, Kamei RG, Teeling EC, Biju SD (2018) Cryptic diversity within the Megophrys major species group (Amphibia: Megophryidae) of the Asian Horned Frogs: Phylogenetic perspectives and a taxonomic revision of South Asian taxa, with descriptions of four new species. Zootaxa 4523: 1–96. https://doi.org/10.11646/zootaxa.4523.1.1
  • Mahony S, Nicole MF, Biju SD, Teeling EC (2017) Evolutionary history of the Asian Horned Frogs (Megophryinae): integrative approaches to timetree dating in the absence of a fossil record. Molecular Phylogenetics and Evolution 34(3): 744–771. https://doi.org/10.1093/molbev/msw267
  • Mahony S, Sengupta S, Kamei RG, Biju S D (2011) A new low altitude species of Megophrys Kuhl and van Hasselt (Amphibia: Megophryidae), from Assam, Northeast India. Zootaxa 3059: 36–46. https://doi.org/10.11646/zootaxa.3059.1.2
  • Mahony S, Teeling EC, Biju SD (2013) Three new species of horned frogs, Megophrys (Amphibia: Megophryidae), from northeast India, with a resolution to the identity of Megophrys boettgeri populations reported from the region. Zootaxa 3722(2): 143–169. https://doi.org/10.11646/zootaxa.3722.2.2
  • Malkmus R, Matsui M (1997) Megophrys kobayashii, ein neuer pelobatider Frosch vom Mount Kinabalu. Sauria, Berlin 19: 31–37.
  • McGuire JA, Witt CC, Altshuler DL, Remsen JV (2007) Phylogenetic systematics and biogeography of hummingbirds: Bayesian and maximum likelihood analyses of partitioned data and selection of an appropriate partitioning strategy. Systematic Biology 56(5): 837–856. https://doi.org/10.1080/10635150701656360
  • Messenger KR, Dahn HA, Liang YR, Xie P, Wang Y, Lu CH (2019) A new species of the genus Megophrys Gunther, 1864 (Amphibia: Anura: Megophryidae) from Mount Wuyi, China. Zootaxa 4554(2): 561–583. https://doi.org/10.11646/zootaxa.4554.2.9
  • Mo XY, Shen YH, Li HH, Wu MS (2010) A new species of Megophrys (Amphibia: Anura: Megophryidae) from the northwestern Hunan Province, China. Current Zoology, 56(4): 432–436. https://doi.org/10.1093/czoolo/56.4.432
  • Munir M, Hamidy A, Farajallah A, Smith EN (2018) A new Megophrys Kuhl and Van Hasselt (Amphibia: Megophryidae) from s https://doi.org/10.11646/zootaxa.4442.3.3 outhwestern Sumatra, Indonesia. Zootaxa 4442: 389–412.
  • Munir M, Hamidy A, Matsui M, Iskandar DT, Sidik I, Shimada T (2019) A new species of Megophrys Kuhl & Van Hasselt (Amphibia: Megophryidae) from Borneo allied to M. nasuta. Zootaxa 4679: 1–24. https://doi.org/10.11646/zootaxa.4679.1.1
  • Myers N, Mittermeier RA, Mittermeier CG, Da Fonseca GAB, Kent J (2000) Biodiversity hotspots for conservation priorities. Nature 403: 853–858 https://doi.org/10.1038/35002501
  • Ohler A (2003) Revision of the genus Ophryophryne Boulenger, 1903 (Megophryidae) with description of two new species. Alytes 21(1): 23–44.
  • Ohler A, Swan SR, Daltry JC (2002) A recent survey of the amphibian fauna of the Cardamom Mountains, Southwest Cambodia with descriptions of three new species. Raffles Bulletin of Zoology, Singapore 50(2): 465–481.
  • Pope CH (1929) Four new frogs from Fukien Province, China. American Museum Novitates 352: 1–5.
  • Poyarkov NA, Duong Jr TV, Orlov NL, Gogoleva SI, Vassilieva AB, Nguyen LT, Nguyen VDH, Nguyen SN, Che J, Mahony S (2017) Molecular, morphological and acoustic assessment of the genus Ophryophryne (Anura, Megophryidae) from Langbian Plateau, southern Vietnam, with description of a new species. ZooKeys 672: 49–120. https://doi.org/10.3897/zookeys.672.10624
  • Rao DQ, Yang DT (1997) The karyotypes of Megophryinae (Pelobatidae) with a discussion on their classification and phylogenetic relationships. Asian Herpetological Research 7: 93–102. https://doi.org/10.5962/bhl.part.18858
  • Robert L, Brett C, Simon YWH., Stephane G (2012) PartitionFinder: combined selection of partitioning schemes and substitution models for phylogenetic analyses. Molecular Phylogenetics and Evolution 29(6): 1695–1701. https://doi.org/10.1093/molbev/mss020
  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York, America.
  • Shen YH (1994) A new pelobatid toad of the genus Megophrys from China (Anura: Pelobatidae). In: Zoological Society of China Editor. The 60th Anniversary of the Foundation of the Zoological Society of China, Nanking (China), September 1994. China Scienc and Technology Publishing House, 603–606.
  • Simon C, Frati F, Beckenbach A, Crespi B, Liu H, Flook P (1994) Evolution, weighting and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Annals of the Entomological Society of America 87(6): 651–701. https://doi.org/10.1093/aesa/87.6.651
  • Sjöander K, Beskow J (2000) Wavesurfer (Anura: Pelobatidae). In: Acoustical society of China Editor name (Ed.) The International Conference on Spoken Language Processing, Beijing (China), October 2000. Military Yiwen Publishing House, city, 464–467.
  • Stejneger L (1926) Two new tailless amphibians from western China. Proceedings of the Biological Society of Washington 39: 53–54.
  • Tapley B, Cutajar T, Mahony S, Nguyen CT, Dau VQ, Nguyen TT, Luong HV, Rowley JJL (2017) The Vietnamese population of Megophrys kuatunensis (Amphibia: Megophryidae) represents a new species of Asian horned frog from Vietnam and southern China. Zootaxa 4344(3): 465–492. https://doi.org/10.11646/zootaxa.4344.3.3
  • Tapley B, Cutajar TP, Mahony S, Nguyen CT, Dau VQ, Luong AM, Le DT, Nguyen TT, Nguyen TQ, Portway C, Luong HV, Rowley JJL (2018) Two new and potentially highly threatened Megophrys Horned frogs (Amphibia: Megophryidae) from Indochina’s highest mountains. Zootaxa 4508: 301–333. https://doi.org/10.11646/zootaxa.4508.3.1
  • Tian WS, Hu QX (1983) Taxonomic study on genus Megophrys, with descriptions of two genera. Acta Herpetologica Sinica 2: 41–48.
  • Tian YZ, Sun A (1995) A new species of Megophrys from China (Amphibia: Pelobatidae). Journal of Liupanshui Teachers College 52(3): 11–15. [In Chinese]
  • Wang B, Wu YQ, Peng JW, Shi SC, Lu NN, Wu J (2020) A new Megophrys Kuhl and Van Hasselt (Amphibia: Megophryidae) from southeastern China. ZooKeys 851: 113–164. https://doi.org/10.3897/zookeys.851.29107
  • Wang J, Li YL, Li Y, Chen H-H, Zeng YJ, Shen JM, Wang Y-Y (2019c) Morphology, molecular genetics, and acoustics reveal two new species of the genus Leptobrachella from northwestern Guizhou Province, China (Anura, Megophryidae). ZooKeys 848: 119–154. https://doi.org/10.3897/zookeys.848.29181
  • Wang J, Liu ZY, Lyu ZT, Wang YY (2017a) A new species of the genus Megophrys (Amphibia: Anura: Megophryidae) from an offshore island in Guangdong Province, southeastern China. Zootaxa 4324(3): 541–556. https://doi.org/10.11646/zootaxa.4324.3.8
  • Wang J, Lyu ZT, Liu ZY, Liao CK, Zeng ZC, Li YL, Wang YY (2019b) Description of six new species of the subgenus Panophrys within the genus Megophrys (Anura, Megophryidae) from southeastern China based on molecular and morphological data. ZooKeys 851: 113–164. https://doi.org/10.3897/zookeys.851.29107
  • Wang YE, Liu BQ, Jiang K, Jin W, Xu JN, Wu CH (2017b) A new species of the Horn toad of the genus Xenophrys from Zhejiang, China (Amphibia: Megophryidae). Chinese Journal of Zoology 52: 19–29. [In Chinese with English abstract]
  • Wang YY, Zhang TD, Zhao J, Sung YH, Yang JH, Pang H, Zhang Z (2012) Description of a new species of the genus Megophrys Günther, 1864 (Amphibia: Anura: Megophryidae) from Mount Jinggang, China, based on molecular and morphological data. Zootaxa 3546: 53–67. https://doi.org/10.11646/zootaxa.3546.1.4
  • Wang YY, Zhao J, Yang JH, Zhou ZM, Chen GL, Liu Y (2014) Morphology, molecular genetics, and bioacoustics support two new sympatric Megophrys (Amphibia: Anura Megophryidae) species in Southeast China. PLOS ONE 9: e93075. https://doi.org/10.1371/journal.pone.0093075
  • Wu YH, Suwannapoom C, Jr. Poyarkov NA, Chen JM, Pawangkhanant P, Xu K, Jin JQ, Murphy RW, Che J (2019) A new species of the genus Xenophrys (Anura: Megophryidae) from northern Thailand. Zoological Research 40: 564–574. https://doi.org/10.24272/j.issn.2095-8137.2019.032
  • Xu N, Li SZ, Liu J, Wei G, Wang B (2020) A new species of the horned toad Megophrys Kuhl & Van Hasselt, 1822 (Anura, Megophryidae) from southwest China. ZooKeys 943: 119–144. https://doi.org/10.3897/zookeys.943.50343
  • Ye CY, Fei L (1992) A new Pelobatid toda of the genus Megophrys from Xizang, China. Acta Herpetologica Sinica 1–2: 50–52. [In Chinese]
  • Ye CY, Fei L (1995) Taxonomic studies on the small type Megophrys in China including descriptions of the new species (subspecies) (Pelobatidae: genus Megophrys). Herpetologica Sinica 4–5: 72–81. [In Chinese]
  • Ye CY, Fei L (1995) Taxonomic studies on the small type Megophrys in China including descriptions of the new species (subspecies) (Pelobatidae: genus Megophrys). Herpetologica Sinica 4–5: 72–81. [In Chinese]
  • Ye CY, Fei L, Xie F (2007) A new species of Megophryidae Megophrys baolongensis from China (Amphibia, Anura). Herpetologica Sinica 11: 38–41. [In Chinese]
  • Zhang Y, Li G, Xiao N, Li J, Pan T, Wang H, Zhang B, Zhou J (2017) A new species of the genus Xenoophrys (Amphibia: Anura: Megophryicae) from Libo County, Guizhou, China. Asian Herpetological Research 8: 75–85.
  • Zhao J, Yang JH, Chen GL, Chen CQ, Wang YY (2014) Description of a new species of the genus Brachytarsophrys Tian and Hu, 1983 (Amphibia: Anura: Megophryidae) from Southern China based on molecular and morphological data. Asian Herpetological Research 5(3): 150–160. https://doi.org/10.3724/SP.J.1245.2014.00150

Supplementary materials

Supplementary material 1 

Table S1. Uncorrected p-distances between the Megophrys species based on the 16S gene sequences.

Shi-Ze Li, Ning-Ning Lu, Jing Liu, Bin Wang

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 (42.13 kb)
Supplementary material 2 

Table S2. Uncorrected p-distances between the Megophrys species based on the COI gene sequences

Shi-Ze Li, Ning-Ning Lu, Jing Liu, Bin Wang

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 (23.51 kb)
Supplementary material 3 

Table S3. Diagnostic characters separating the new species described in this study from other species of Megophrys

Shi-Ze Li, Ning-Ning Lu, Jing Liu, Bin Wang

Data type: species 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 (18.21 kb)