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Research Article
Description of a new horned toad of Megophrys Kuhl & Van Hasselt, 1822 (Anura, Megophryidae) from southwest China
expand article infoHaijun Su‡, Shengchao Shi§, Yanqing Wu|, Guangrong Li, Xiaogang Yao, Bin Wang§, Shize Li§
‡ Guizhou University, Guiyang, China
§ Chinese Academy of Sciences, Chengdu, China
| Nanjing Institute of Environmental Sciences, Nanjing, China
¶ Kuankuoshui National Nature Reserve Administration, Suiyang, China

Corresponding author: Bin Wang ( wangbin@cib.ac.cn )

Corresponding author: Shize Li ( 976722439@qq.com )

Academic editor: Anthony Herrel
© 2020 Haijun Su, Shengchao Shi, Yanqing Wu, Guangrong Li, Xiaogang Yao, Bin Wang, Shize Li.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Su H, Shi S, Wu Y, Li G, Yao X, Wang B, Li S (2020) Description of a new horned toad of Megophrys Kuhl & Van Hasselt, 1822 (Anura, Megophryidae) from southwest China. ZooKeys 974: 131-159. https://doi.org/10.3897/zookeys.974.56070
ZooBank: urn:lsid:zoobank.org:pub:42BA600C-5E98-4408-8C4E-2CC8339D582A
Open Access

Abstract

A new species of the genus Megophrys is described from Guizhou Province, China. Molecular phylogenetic analyses supported the new species as an independent clade nested into the Megophrys. The new species could be distinguished from its congeners by a combination of the following characters: body size moderate (SVL 49.3–58.2 mm in males); vomerine ridges present distinctly, vomerine teeth present; tongue feebly notched behind; tympanum distinctly visible, oval; two metacarpal tubercles in hand; toes with one-third webbing and wide lateral fringes; heels overlapped when thighs are positioned at right angles to the body; tibiotarsal articulation reaching the level between tympanum and eye when leg stretched forward; an internal single subgular vocal sac present in male; in breeding male, the nuptial pads with large and sparse black nuptial spines present on the dorsal bases of the first two fingers.

Keywords

China, molecular phylogenetic analysis, morphology, new species, taxonomy

Introduction

The toad genus Megophrys Kuhl & Van Hasselt, 1822 (Anura; Megophryidae) 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). The taxonomic assignments of the taxa in this group have been debated for a long time (e.g., Tian and Hu 1983; Dubois 1987; Lathrop 1997; Rao and Yang 1997; Jiang et al. 2003; Delorme et al. 2006; Fei et al. 2009; Chen et al. 2016; Fei and Ye 2016; Deuti et al. 2017; Mahony et al. 2017; Frost 2020). Regardless, molecular phylogenetic studies indicate the group as a monophyletic group (Chen et al. 2017; Mahony et al. 2017; Li et al. 2018b; Liu et al. 2018, 2020; Wang et al. 2020), and thus some studies regarded it as a large genus, Megophrys sensu lato (Mahony et al. 2017; Li et al. 2018b; Liu et al. 2018, 2020; Wang et al. 2020; Frost 2020) while other studies divided the taxon into different genera and subgenera (Chen et al. 2017; Fei and Ye 2016; Liu et al. 2018).

The genus Megophrys currently contains 106 species, of which 50 species have been described in the last decade (Frost 2020). Many cryptic species were indicated in the genus by molecular phylogenetic frameworks (Chen et al. 2017; Liu et al. 2018). In recent years, four species were described from Guizhou Province, China: Megophrys liboensis Zhang, Li, Xiao, Li ,Pan, Wang, Zhang & Zhou, 2017, Megophrys leishanensis Li, Xu, Liu, Jiang, Wei & Wang, 2018, Megophrys jiangi Liu, Li, Wei, Xu, Cheng, Wang & Wu, 2020, and Megophrys chishuiensis Xu, Li, Liu, Wei, & Wang, 2020. However, many areas have not been well investigated in this province, and it is expected that there are still cryptic species of the toads in the region.

During field surveys in the Huanglian Nature Reserve, Tongzi County, and Kuankuoshui National Nature Reserve, Suiyang County in Guizhou Province, China, we collected a number of Megophrys specimens. Molecular phylogenetic analyses, morphological comparisons, and bioacoustics data support it as an undescribed species.

Materials and methods

Sampling

A total of nine molecular samples were collected in this study: five adult males of the undescribed species from two localities of Guizhou Province, China, two M. sangzhiensis and two M. spinata (Table 1; Fig. 1). In the field, the toads were euthanized using isoflurane, and the specimens were fixed in 75 % ethanol. Tissue samples were taken and preserved separately in 99% ethanol prior to fixation. The specimens were deposited in Chengdu Institute of Biology, Chinese Academy of Sciences (CIB, CAS).

Figure 1. 

Sampling localities of Megophrys qianbeiensis sp. nov., M. sangzhiensis and M. spinata in China. 1. Huanglian Nature Reserve, Tongzi County, Guizhou Province; 2. Kuankuoshui National Nature Reserve, Suiyang County, Guizhou Province; 3. Badagong Mountain, Hunan Province, China; 4. Leigong Mountain, Guizhou Province.

Molecular data and phylogenetic analyses

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, the primers P7 (5’-CGCCTGTTTACCAAAAACAT-3’) and P8 (5’-CCGGTCTGAACTCAGATCACGT-3’) were used following Simon et al. (1994), and for COI, Chmf4 (5’-TYTCWACWAAYCAYAAAGAYATCGG-3’) and Chmr4 (5’-ACYTCRGGRTGRCCRAARAATCA-3’) were used following Che et al. (2012). Gene 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. (Shanghai, China). New sequences were deposited in GenBank (for accession numbers see Table 1).

Table 1.
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Information for samples used in molecular phylogenetic analyses in this study.

ID Species Voucher number Locality GenBank accession number
16S COI
1 Megophrys qianbeiensis sp. nov. CIBTZ20190608015 Huanglian Nature Reserve, Guizhou, China MT651553 MT654520
2 Megophrys qianbeiensis sp. nov. CIBTZ20190608017 Huanglian Nature Reserve, Guizhou, China MT651554 MT654521
3 Megophrys qianbeiensis sp. nov. CIBTZ20160715003 Huanglian Nature Reserve, Guizhou, China MT651555 MT654522
4 Megophrys qianbeiensis sp. nov. CIBKKS20180722002 Huanglian Nature Reserve, Guizhou, China MT651556 MT654523
5 Megophrys qianbeiensis sp. nov. CIBKKS20180722001 Kuankuoshui Nature Reserve, Guizhou, China MT651557 MT654524
6 Megophrys sangzhiensis CIBSZ2012062005 Badagongshan Nature Reserve, Hunan, China MT651558 MT654525
7 Megophrys sangzhiensis CIBSZ2012062008 Badagongshan Nature Reserve, Hunan, China MT651559 MT654526
8 Megophrys sangzhiensis SYSa004307 Zhangjiajie, Hunan, China MH406798 MH406260
9 Megophrys spinata CIBLS20190801001 Leigong Shan, Guizhou, China MT651551 MT654518
10 Megophrys spinata CIBLS20190801002 Leigong Shan, Guizhou, China MT651552 MT654519
11 Megophrys spinata SYSa002227 Leigong Shan, Guizhou, China MH406676 MH406116
12 Megophrys binlingensis KIZ025807 Wawu Shan, Sichuan, China KX811852 KX812115
13 Megophrys binlingensis SYSa005313 Wawu Shan, Sichuan, China MH406892 MH406354
14 Megophrys binlingensis SYSa005314 Wawu Shan, Sichuan, China MH406893 MH406355
15 Megophrys binchuanensis KIZ019441 Jizu Shan, Yunnan, China KX811849 KX812112
16 Megophrys palpebralespinosa KIZ011603 Pu Hu Nature Reserve, Thanh Hoa, Vietnam KX811888 KX812137
17 Megophrys omeimontis KIZ025765 Emei Shan, Sichuan, China KX811884 KX812136
18 Megophrys angka KIZ040591 Kiew Mae Pan nature trail, Chiang Mai, Thailand MN508052 –
19 Megophrys wuliangshanensis KIZ046812 Huangcaoling, Yunnan, China KX811881 KX812129
20 Megophrys daweimontis KIZ048997 Dawei Shan, Yunnan, China KX811867 KX812125
21 Megophrys jingdongensis KIZ-LC0805067 Huanglianshan National Nature Reserve, Yunnan, China KX811872 KX812131
22 Megophrys fansipanensis VNMN 2018.01 Lao Cai, Sa Pa, Vietnam MH514886 –
23 Megophrys hoanglienensis VNMN 2018.02 Lao Cai, Sa Pa, Vietnam MH514889 –
24 Megophrys minor KIZ01939 Qingcheng Shan, Sichuan, China KX811896 KX812145
25 Megophrys jiangi CIBKKS20180722006 Kuankuosui Nature Reserve, Guizhou, China MN107743 MN107748
26 Megophrys chishuiensis CIBCS20190518031 Chishui Nature Reserve, Guizhou, China MN954707 MN928958
27 Megophrys dongguanensis SYS a001972 Yinping Shan, Guangdong, China MK524098 MK524129
28 Megophrys nankunensis SYS a004498 Nankun Shan, Guangdong, China MK524108 MK524139
29 Megophrys cheni SYS a001427 Jinggang Shan, Jiangxi, China KJ560391 –
30 Megophrys obesa SYS a002272 Heishiding Nature Reserve, Guangdong, China KJ579122 –
31 Megophrys ombrophila KRM18 Wuyishan, Fujian, China KX856404 –
32 Megophrys wugongensis SYS a002610 Wugongshan Scenic Area, Jiangxi, China MK524114 MK524145
33 Megophrys lini SYS a002370 Suichuan, Jiangxi, China KJ560412 –
34 Megophrys xiangnanensis SYS a002874 Yangming Shan, Hunan, China MH406713 MH406165
35 Megophrys nanlingensis SYS a001959 Nanling Nature Reserve, Guangdong, China MK524111 MK524142
36 Megophrys kuatunensis SYS a001579 Wuyi Shan, Fujian, China KJ560376 –
37 Megophrys jinggangensis KIZ07132 Chashan Forest Farm, Jiangxi, China KX811840 KX812108
38 Megophrys lishuiensis WYF00169 Lishui, Zhejiang, China KY021418 –
39 Megophrys xianjuensis CIBXJ190505 Xianju, Zhejiang, China MN563753 MN563769
40 Megophrys wushanensis KIZ045469 Guangwu Shan, Sichuan, China KX811838 KX812094
41 Megophrys baolongensis KIZ019216 Baolong, Chongqing, China KX811813 KX812093
42 Megophrys leishanensis CIBLS20171101001 Leigong Shan, Guizhou, China MK005310 MK005306
43 Megophrys yangmingensis SYS a002877 Yangming Shan, Hunan, China MH406716 MH406168
44 Megophrys shimentaina SYS a002077 Shimentai Nature Reserve, Guangdong, China MH406655 MH406092
45 Megophrys jiulianensis SYS a002107 Jiulian Shan, Jiangxi, China MK524099 MK524130
46 Megophrys shunhuangensis HNNU16SH02 Shunhuang Mountains, Hunan, China MK836037 –
47 Megophrys mirabilis SYS a002192 Huaping Nature Reserve, Guangxi, China MH406669 MH406109
48 Megophrys tuberogranulata Tissue ID: YPX10987 Badagongshan Nature Reserve, Hunan, China KX811823 KX812095
49 Megophrys huangshanensis KIZ022004 Huang Shan, Anhui, China KX811821 KX812107
50 Megophrys boettgeri Tissue ID: YPXJK033 Wuyi Shan, Fujian, China KX811814 KX812104
51 Megophrys liboensis GNUG:20160408003 Libo, Guizhou, China MF285262 –
52 Megophrys mufumontana SYS a006391 Mufu Shan, Hunan, China MK524105 MK524136
53 Megophrys brachykolos ROM 16634 Hong Kong, China KX811897 KX812150
54 Megophrys acuta SYS a001957 Heishiding Nature Reserve, Guangdong, China KJ579118 –
55 Megophrys gerti ITBCZ 1108 Nui Chua National Park, Ninh Thuan, Vietnam KX811917 KX812161
56 Megophrys elfina ZMMU ABV-00454 Bidoup Mountain, Lam Dong, Vietnam KY425379 –
57 Megophrys synoria FMNH 262778 O’Reang, Mondolkiri, Cambodia KY022198 –
58 Megophrys hansi KIZ010360 Phong Dien Nature Reserve, Thua Thien Hue, Vietnam KX811913 KX812155
59 Megophrys microstoma KIZ048799 Xiaoqiaogou Nature Reserve, Yunnan, China KX811914 KX812156
60 Megophrys pachyproctus KIZ010978 Beibeng, Xizang, China KX811908 KX812153
61 Megophrys baluensis ZMH A13125 Gunung Kinabalu National Park, Kogopan Trail, Malaysia KJ831310 –
62 Megophrys stejnegeri KU 314303 Pasonanca Natural Park, Zamboanga, Philippines KX811922 KX812052
63 Megophrys ligayae ZMMU NAP-05015 Palawan, Philippines KX811919 KX812051
64 Megophrys kobayashii UNIMAS 8148 Gunung Kinabalu National Park, Sabah, Malaysia KJ831313 –
65 Megophrys nasuta KIZ019419 Malaysia KX811921 KX812054
66 Megophrys edwardinae FMNH 273694 Bintulu, Sarawak, Malaysia KX811918 KX812050
67 Megophrys aceras KIZ025467 Khao Nan National Park, Nakhon Si Thammarat, Thailand KX811925 KX812159
68 Megophrys dringi UNIMAS 8943 Gunung Mulu National Park, Sarawak, Malaysia KJ831317 –
69 Megophrys maosonensis KIZ016045 Xiaoqiaogou Nature Reserve, Yunnan, China KX811780 KX812080
70 Megophrys mangshanensis KIZ021786 Nanling National Forest Park, Guangdong, China KX811790 KX812079
71 Megophrys flavipunctata SDBDU2009.297 East Khasi Hills dist., Meghalaya KY022307 MH647536
72 Megophrys glandulosa KIZ048439 Husa, Yunnan, China KX811762 KX812075
73 Megophrys medogensis KIZ06621 Beibeng, Xizang, China KX811767 KX812082
74 Megophrys periosa BNHS 6061 West Kameng dist., Arunachal Pradesh, IN KY022309 MH647528
75 Megophrys himalayana SDBDU2009.75 East Siang dist., Arunachal Pradesh, IN KY022311 –
76 Megophrys sanu K5198/ZSI11393 – KX894679 –
77 Megophrys zhangi KIZ014278 Zhangmu, Xizang, China KX811765 KX812084
78 Megophrys katabhako ZSIA11799 – KX894669 –
79 Megophrys major SYSa002961 Zhushihe, Yunnan, China MH406728 MH406180
80 Megophrys oreocrypta BNHS 6046 West Garo Hills dist., Meghalaya KY022306 –
81 Megophrys auralensis NCSM 79599 Aural, Kampong Speu, Cambodia KX811807 –
82 Megophrys parva SYSa003042 Zhushihe, Yunnan, China MH406737 MH406189
83 Megophrys nankiangensis CIB ZYC517 Nanjiang, Sichuan, China KX811900 –
84 Megophrys wawuensis KIZ025799 Wawu Shan, Sichuan, China KX811902 KX812062
85 Megophrys gigantica SYSa003933 Wuliang shan, Yunnan, China MH406775 MH406235
86 Megophrys shapingensis KIZ014512 Liziping Nature Reserve, Sichuan, China KX811904 KX812060
87 Megophrys montana LSUMZ 81916 Sukabumi, Java, Indonesia KX811927 KX812163
88 Megophrys lancip MZB:Amp:22233 – KY679891 –
89 Megophrys feae KIZ046706 Huangcaoling, Yunnan, China KX811810 KX812056
90 Megophrys chuannanensis CIB20050081 Hejiang, Sichuan, China KM504261 –
91 Megophrys carinense Tissue ID: YPX20455 Dayao Shan, Guangxi, China KX811811 KX812057
92 Megophrys popei SYS a000589 Naling Nature Reserve, Guangdong, China KM504251 –
93 Megophrys intermedia ZFMK 87596 U Bo, Phong Nha-Ke Bang NP, Vietnam HQ588950 –
94 Leptobrachium boringii Tissue ID: YPX37539 Emei Shan, Sichuan, China KX811930 KX812164
95 Leptobrachella oshanensis KIZ025778 Emei Shan, Sichuan, China KX811928 KX812166

For molecular analyses, the available sequence data for congeners of Megophrys were downloaded from GenBank (Table 1), 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 (Table 1), 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-parameterization (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.

Morphological comparisons

In total, 16 specimens including six males of the undescribed species, five males of M. sangzhiensis, and five males of M. spinata were measured (for voucher information see Table 2). The terminology and methods followed Fei et al. (2009). Measurements were taken with a dial caliper to 0.1 mm. Twenty-one 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);

IAE distance between posterior corner of eyes;

IFE distance between anterior corner of eyes;

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);

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

NSD nasal to snout distance (distance between the nasal the posterior edge of the vent);

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).

Table 2.
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Measurements of the adult specimens of Megophrys qianbeiensis sp. nov., M. spinata, and M. sangzhiensis. Units are given in mm. See abbreviations for the morphological characters in Materials and methods section.

Species Voucher number Sex SVL HDL HDW SL NED NSD IND IOD ED UEW LAL LW HLL THL TL TW TFL FL TYD IFE IAE
Megophrys qianbeiensis sp. nov. CIBTZ20190608016 male 58.2 16.5 21.0 6.7 2.7 3.9 7.5 5.1 6.9 6.2 25.0 6.7 89.3 27.9 32.4 8.2 40.7 28.2 4.3 10.1 16.4
Megophrys qianbeiensis sp. nov. CIBTZ20190608018 male 55.1 14.9 20.6 6.9 3.0 4.0 7.0 4.2 6.5 5.1 25.0 6.5 93.0 28.4 30.6 8.7 44.0 29.1 3.5 9.0 16.0
Megophrys qianbeiensis sp. nov. CIBTZ20190608017 male 56.3 14.6 19.2 7.2 3.2 3.6 6.7 4.3 6.5 5.3 24.0 6.9 87.0 27.6 28.0 7.8 38.3 25.7 3.2 10.9 15.0
Megophrys qianbeiensis sp. nov. CIBTZ20160715003 male 54.1 17.0 20.8 6.9 3.4 3.7 6.3 4.5 6.9 6.0 25.4 5.7 93.7 27.0 30.8 7.8 43.7 29.4 3.5 10.4 15.8
Megophrys qianbeiensis sp. nov. CIBTZ20190608015 male 52.6 15.3 19.4 6.8 2.4 4.3 6.9 3.7 5.9 5.5 24.1 7.4 86.9 24.1 28.3 8.3 41.4 28.0 3.3 10.0 15.7
Megophrys qianbeiensis sp. nov. CIBKKS20180722001 male 49.3 15.5 18.3 6.8 3.0 3.5 5.7 5.3 5.4 5.3 20.4 6.6 76.9 24.5 25.0 7.0 34.5 24.5 3.4 7.8 14.0
M. spinata CIBLS20190801002 male 56.2 14.9 18.4 6.0 3.0 3.4 5.8 4.2 5.1 6.1 24.2 5.5 93.7 27.4 29.9 6.1 40.8 28.6 2.7 9.1 14.0
M. spinata CIBLS20190801004 male 53.5 14.5 19.1 7.1 2.8 4.1 6.0 5.0 5.7 5.0 24.1 5.9 99.0 29.8 30.4 8.0 43.1 28.1 2.8 9.6 14.4
M. spinata CIBLS20190801001 male 54.8 14.6 18.6 6.7 2.8 3.9 6.0 4.8 5.8 4.5 24.3 6.1 87.7 27.6 28.9 7.0 39.7 26.4 2.8 9.2 14.2
M. spinata CIBLS20190801003 male 51.2 14.3 18.8 6.6 2.9 3.6 6.1 5.2 6.0 5.1 25.1 6.5 93.0 26.1 29.6 7.7 41.6 29.6 2.5 9.1 14.0
M. spinata CIBLS20160610008 male 53.8 15.8 18.4 5.7 2.7 3.8 6.3 5.3 5.5 4.5 24.0 7.4 85.7 26.9 29.0 6.0 39.2 27.9 2.9 8.5 14.3
M. sangzhiensis CIBSZ2012062005 male 59.8 17.8 20.6 7.2 3.1 4.1 7.3 4.8 7.1 5.7 26.6 6.6 105.0 31.6 32.6 7.8 46.1 29.4 3.1 10.4 16.1
M. sangzhiensis CIBSZ2012062008 male 58.8 17.8 21.5 7.6 3.0 4.4 7.3 4.7 7.4 6.1 26.8 6.2 97.3 30.3 31.6 7.8 42.9 26.7 3.4 10.9 17.3
M. sangzhiensis CIBSZ2012062006 male 59.5 16.1 21.0 8.2 3.5 4.6 7.7 5.0 6.7 6.1 26.6 6.3 99.8 27.9 32.3 7.3 43.8 29.2 3.4 10.1 17.3
M. sangzhiensis CIBSZ2012062019 male 57.4 18.0 20.9 7.3 3.5 3.7 6.8 5.0 6.4 5.1 26.2 6.1 99.5 30.2 32.6 8.1 43.2 29.9 3.7 10.4 17.3
M. sangzhiensis CIBSZ2012062007 male 56.1 16.1 20.0 6.6 3.9 4.1 6.9 5.8 6.1 5.7 28.2 6.7 100.0 28.0 32.0 7.5 44.9 29.4 3.4 9.4 16.2

In order to reduce the impact of allometry, the correct value from the ratio of each character to SVL was calculated, and then was log-transformed for subsequent morphometric analyses. One-way analysis of variance (ANOVA) was used to test the significance of differences on morphometric characters between different species. The significance level was set at 0.05. To show the spatial distribution of different species on the morphometric characters, principal component analyses (PCA) were performed. These analyses were carried out in the R (R Development Core Team 2008). The new species was also compared with all other Megophrys species on morphology. Comparative data were obtained from related species as described in literature (Table 3).

Table 3.
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References for morphological characters for congeners of the genus Megophrys.

Species Literature
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, & 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. 2018b
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, 2020 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, 2020 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. 2010
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, 2020 Lyu et al. 2020
M. yangmingensis Lyu, Zeng & Wang, 2020 Lyu et al. 2020
M. zhangi Ye & Fei, 1992 Ye and Fei 2012
M. zunhebotoensis (Mathew & Sen, 2007) Mathew and Sen 2007

Bioacoustics analyses

The advertisement calls of the undescribed species were recorded from the holotype specimen CIBTZ20190608017 in a stream at ambient air temperature of 20.5 °C and air humidity of 87 % in the field on 8 June 2019 in Huanglian Nature Reserve, Tongzi County, Guizhou Province, China. The advertisement calls of M. sangzhiensis were recorded from the specimen CIBSZ2012062005 in a stream at ambient air temperature of 18.5 °C and air humidity of 85 % in the field on 20 June 2012 in Sangzhi County, Hunan Province, China. The advertisement calls of M. spinata were recorded from the specimen CIBLS20190801001 in a stream at ambient air temperature of 19.0 °C and air humidity of 85 % in the field on 1 August 2019 in Leishan County, Guizhou Province, China. SONY PCM-D50 digital sound recorder was used to record within 20 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 had almost consistent topology though relationships of some lineages were unresolved (Fig. 2). In trees, the undescribed species was clustered as an independent clade and sister to a clade in comprising of M. sangzhiensis and M. spinata (Fig. 2).

Figure 2. 

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

Genetic distances on COI gene with uncorrected p-distance model between all samples of the undescribed species were below 0.2%. The genetic distance between the undescribed species and its closest related species M. sangzhiensis was 4.3 % on COI gene, which was higher or at the same level with those among many pairs of congeners, for example, 3.6 % between M. spinata and M. sangzhiensis, 1.8% between M. huangshanensis and M. boettgeri, and 4.3 % between M. maosonensis and M. mangshanensis (Suppl. material 1: Table S1).

Morphological comparisons

In PCA for males, the total variation of the first two principal components was 63.2 %. In males on the two-dimensional plots of PC1 vs. PC2, the undescribed species could be distinctly separated from M. sangzhiensis and M. spinata (Fig. 3). The results of one-way ANOVA indicated that in males, the undescribed species was significantly different from M. sangzhiensis and M. spinata on many morphometric characters (all p-values < 0.05; Table 4). More detailed descriptions of results from morphological comparisons between the undescribed species and its congeners were presented in the following sections for describing the new species.

Figure 3. 

Plots of the first principal component (PCA1) versus the second (PCA2) for Megophrys qianbeiensis sp. nov., M. sangzhiensis, and M. spinata from principal component analyses.

Table 4.
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Morphometric comparisons between Megophrys qianbeiensis sp. nov., M. sangzhiensis, and M. spinata. Units given in mm. Abbreviations for the species name: MQ, Megophrys qianbeiensis sp. nov.; MSZ, M. sangzhiensis; MSP, M. spinata. See abbreviations for morphometric characters in Materials and methods section.

Megophrys qianbeiensis sp. nov. M. sangzhiensis M. spinata p-value from ANOVA in male
males (N = 6) males (N = 5) males (N = 5)
Range Mean ± SD Range Mean ± SD Range Mean ± SD MQ vs. MSZ MQ vs. MSP
SVL 49.3–58.2 54.3 ± 3.09 56.1–59.8 58.3 ± 1.56 51.2–56.2 53.9 ± 1.84 0.029 0.851
HDL 14.6–17.0 15.6 ± 0.93 16.1–18.0 17.2 ± 0.96 14.3–15.8 14.8 ± 0.59 0.027 0.124
HDW 18.3–21.0 19.9 ± 1.08 20.0–21.5 20.8 ± 0.55 18.4–19.1 18.7 ± 0.30 0.123 0.037
SL 6.7–7.2 6.9 ± 0.17 6.6–8.2 7.4 ± 0.58 5.7–7.1 6.4 ± 0.56 0.067 0.085
TYD 3.2–4.3 3.5 ± 0.39 3.1–3.7 3.4 ± 0.21 2.5–2.9 2.7 ± 0.15 0.639 0.001
IFE 7.8–10.9 9.7 ± 1.13 9.4–10.9 10.2 ± 0.54 8.5–9.6 9.1 ± 0.39 0.340 0.348
IAE 14.0–16.4 15.5 ± 0.86 16.1–17.3 16.8 ± 0.62 14.0–14.4 14.2 ± 0.18 0.019 0.009
NED 2.4–3.4 2.9 ± 0.36 3.0–3.9 3.4 ± 0.36 2.7–3.0 2.8 ± 0.11 0.060 0.618
NSD 3.5–4.3 3.8 ± 0.29 3.7–4.6 4.2 ± 0.34 3.4–4.1 3.8 ± 0.27 0.101 0.683
IND 5.7–7.5 6.7 ± 0.62 6.8–7.7 7.2 ± 0.34 5.8–6.3 6.0 ± 0.18 0.150 0.056
IOD 3.7–5.3 4.5 ± 0.59 4.7–5.8 5.1 ± 0.45 4.2–5.3 4.9 ± 0.44 0.117 0.257
ED 5.4–6.9 6.3 ± 0.58 6.1–7.4 6.7 ± 0.51 5.1–6.0 5.6 ± 0.34 0.280 0.041
UEW 5.1–6.2 5.6 ± 0.43 5.1–6.1 5.7 ± 0.42 4.5–6.1 5.0 ± 0.65 0.484 0.126
LAL 20.4–25.4 24.0 ± 1.84 26.2–28.2 26.9 ± 0.78 24.0–25.1 24.3 ± 0.44 0.014 0.654
LW 5.7–7.4 6.6 ± 0.55 6.1–6.7 6.4 ± 0.24 5.5–7.4 6.3 ± 0.72 0.394 0.364
HLL 76.9–93.7 87.8 ± 6.07 97.3–105.0 100.3 ± 2.84 85.7–99.0 91.8 ± 5.27 0.003 0.280
THL 24.1–28.4 26.6 ± 1.82 27.9–31.6 29.6 ± 1.59 26.1–29.8 27.6 ± 1.38 0.019 0.343
TL 25.0–32.4 29.2 ± 2.63 31.6–32.6 32.2 ± 0.42 28.9–30.4 29.6 ± 0.63 0.038 0.713
TW 7.0–8.7 8.0 ± 0.58 7.3–8.1 7.7 ± 0.32 6.0–8.0 7.0 ± 0.91 0.404 0.053
TFL 34.5–44.0 40.4 ± 3.58 42.9–46.1 44.2 ± 1.32 39.2–43.1 40.9 ± 1.55 0.062 0.761
FL 24.5–29.4 27.5 ± 1.95 26.7–29.9 28.9 ± 1.28 26.4–29.6 28.1 ± 1.16 0.201 0.531

Bioacoustics comparisons

There were many differences in sonograms and waveforms of calls between the undescribed species, M. sangzhiensis, and M. spinata (Fig. 4; Table 5). Firstly, in the note interval, the undescribed species were shorter than those of both M. sangzhiensis and M. spinata. Secondly, the dominant frequency of call in the undescribed species was lower than both of M. sangzhiensis and M. spinata. Thirdly, the amplitude of the undescribed species beginning with moderately high energy pulses, increasing slightly to a maximum by approximately mid note, and then decreasing towards the end of each note, in M. sangzhiensis beginning with maximum energy pulses and then decreasing towards the end of note, and in M. spinata beginning with lower energy pulses, then increasing to the maximum by approximately one-four note and then decreasing to the mid note then increasing to the second highest energy pulses and then decreasing towards the end of note.

Figure 4. 

Visualization of advertisement calls of Megophrys qianbeiensis sp. nov., M. sangzhiensis, and M. spinata A1 waveform showing one note of Megophrys qianbeiensis sp. nov. A2 sonogram showing one note of Megophrys qianbeiensis sp. nov. A3 waveform showing 25 notes of one call of Megophrys qianbeiensis sp. nov. A4 sonogram showing 25 notes of one call of Megophrys qianbeiensis sp. nov. B1 waveform showing one note of M. sangzhiensis B2 sonogram showing one note of M. sangzhiensis B3 waveform showing 38 notes of one call of M. sangzhiensis. B4 sonogram showing 38 notes of one call of M. sangzhiensis C1 waveform showing one note of M. spinata C2 sonogram showing one note of M. spinata C3 waveform showing 20 notes of one call of M. spinata C4 sonogram showing 20 notes of one call of M. spinata.

Table 5.
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Comparisons of characteristics of advertisement calls of Megophrys qianbeiensis sp. nov., M. sangzhiensis, and M. spinata.

Call character Megophrys qianbeiensis sp. nov. M. sangzhiensis M. spinata
Range Mean ± SD Range Mean ± SD Range Mean ± SD
Number of notes in a call 14–26 22.5 ± 4.4 38 / 7–28 17 ± 7.92
Call duration (ms) 2832–5621 4413 ± 972 8152 / 1500–6623 3905 ± 2010
Call interval (ms) 6812–14387 10878 ± 2701 / / 592–5770 2708 ± 1863.33
Note duration (ms) 129– 211 167 ± 0.02 107–155 120.3 ± 8.73 107–123 114 ± 3.79
Note interval (ms) 34–94 57 ± 0.01 72–132 95.6 ± 13.89 113–232 147 ± 33.12
Dominant frequency (Hz) 2250–3000 2469 ± 197.47 10380–13200 11795 ± 670.58 4260–4589 4416 ± 130.04
Temperature (°C) 20.5 18.5 19.0

Taxonomic accounts

Megophrys qianbeiensis sp. nov.

http://zoobank.org/C6C89A51-8178-4C7B-A100-80C0D2D42AD3
Figures 4A1–C1, 5, 6A1–A6, 7; Tables 1, 2, 4, 5, Suppl. material 2: Table S2

Type material

. Holotype. CIBTZ20190608017 (Figs 5, 6), adult male, from Huanglian Nature Reserve, Tongzi County, Guizhou Province, China (28.498056°N, 107.046944°E, ca. 1500 m a.s.l.), collected by Shi-Ze Li 8 June 2019.

Paratype. Four adult males from the same place as holotype, and one from Kuankuoshui National Nature Reserve (28.21835°N, 107.166388°E, ca.1520 m a.s.l.) collected by Shi-Ze Li. CIBKKS20180722001 collected 22 July 2018 from Kuankuoshui National Nature Reserve and CIBTZ20160715003 collected 15 July 2016, CIBTZ20190608015, CIBTZ20190608016 and CIBTZ20190608018 collected 8 June 2019 from Kuankuoshui National Nature Reserve.

Figure 5. 

Photos of the holotype CIBTZ20190608017 of Megophrys qianbeiensis 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.

Diagnosis

Megophrys qianbeiensis 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 qianbeiensis sp. nov. could be distinguished from its congeners by a combination of the following morphological characters: body size moderate (SVL 49.3–58.2 mm in males); vomerine ridges present distinctly, vomerine teeth present; tongue feebly notched behind; tympanum distinctly visible, oval; two metacarpal tubercles in hand; toes with one-third webbing and wide lateral fringes; heels overlapped when thighs are positioned at right angles to the body; tibiotarsal articulation reaching the level between tympanum and eye when leg stretched forward; an internal single subgular vocal sac present in male; in breeding male, the nuptial pads with large and sparse black nuptial spines present on the dorsal bases of the first two fingers.

Figure 6. 

Photos of specimens of Megophrys qianbeiensis sp. nov., M. sangzhiensis and M. spinata A1–A6 dorsal view, ventral view, dorsal view of hand, ventral view of hand, ventral view of foot and view of oral cavity of the holotype specimen CIBTZ20190608017 of Megophrys qianbeiensis sp. nov. B1–B6 dorsal view, ventral view, dorsal view of hand, ventral view of hand, ventral view of foot and view of oral cavity of CIBSZ2012062005 of M. sangzhiensis C1–C6 dorsal view, ventral view, dorsal view of hand, ventral view of hand, ventral view of foot and view of oral cavity of CIBLS20190801001 of M. spinata. Arrow point to vomerine ridge.

Description of holotype

(Figs 5, 6). SVL 56.3 mm; head width larger than head length (HDW/HDL ratio ca. 1.3); 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 44.5 % of head length; pupils vertical; nostril orientated laterally, closer to snout than eye; tympanum distinct, TYP/EYE ratio 0.49; vomerine ridges present distinctly as V-shape, vomerine teeth present; margin of tongue smooth, feebly notched behind.

Forelimbs slender, the length of lower arm and hand 42.6 % of SVL; fingers burly, relative finger lengths: II < I < 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, 1.54 times of SVL; heels overlapping when thighs are positioned at right angles to the body, tibiotarsal articulation reaching tympanum to 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 the base of each toes ; toes with one-third webbing and relative wide lateral fringe; inner metatarsal tubercle oval-shaped; outer metatarsal tubercle absent.

Dorsal skin rough, with numerous granules with black spins; several large warts scattered on flanks; tubercles on the dorsum forming a weak V-shaped ridge; two discontinuous dorsolateral parallel ridges on either side of the V-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 with numerous white granules; glands on chest indistinct; femoral glands on rear of thighs, numerous white granules on outer thighs; posterior end of the body distinctly protruding and forming an arc-shaped swelling above the anal region.

Coloration of holotype in life

(Fig. 5). An inverted triangular brown speckle between the eyes; V-shaped ridges on the dorsum with brown speckle, on transverse bands on the dorsal surface of the thigh and shank; several dark brown and white vertical bars on the lower and upper lip; belly whitish grey with dark brown marbling; ventral surface of posterior limb orange with numerous granules; palms, soles and tip of digits uniform purple grey; femoral glands white.

Coloration of holotype in preservation

(Fig. 6). Color of dorsal surface fades to brownness; the inverted triangular brown speckle between the eyes and V-shaped ridges on dorsum indistinct; 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.

Variation

In CIBTZ20160715003 the dorsolateral parallel ridges are short, just a little bit above the shoulder (Fig. 7A); in CIBTZ20190608015 the X-shaped marking on back of trunk consists of a ridge with brown spots (Fig. 7B) and the posterior belly are orange with black spots on the flank belly (Fig. 7C); in CIBKKS20180722001 the belly is grey brown with some white spots (Fig. 7D).

Figure 7. 

Color variation in Megophrys qianbeiensis sp. nov. A dorsolateral view of the specimen CIBTZ20160715003 B dorsolateral view of the specimen CIBTZ20190608015 C ventral view of the male specimen CIBTZ20190608015 D ventral view of the specimen CIBKKS20180722001.

Advertisement call

The call description is based on recordings of the holotype CIBTZ20190608017 (Fig. 4) from the shrub leaf near the streamlet, and the ambient air temperature was 20.5 °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).

Secondary sexual characters

Adult males have a single subgular vocal sac. In breeding males, brownish red nuptial pads are present on the dorsal bases of the first two fingers with big and sparse black nuptial spines (Fig. 5A).

Comparisons

By having moderate body size (minimum SVL > 49.8 mm in males), Megophrys qianbeiensis sp. nov. differs from M. aceras, M. acuta, M. angka, M. ancrae, M. baluensis, M. baolongensis, M. binchuanensis, M. binlingensis, M. boettgeri, M. brachykolos, M. caobangensis, M. cheni, M. daweimontis, M. dongguanensis, M. dringi, M. edwardinae, M. elfina, M. fansipanensis, M. feii, M. gerti, M. hansi, M. hoanglienensis, M. huangshanensis, M. insularis, M. jiangi, M. jinggangensis, M. jiulianensis, M. kuatunensis, M. lancip, M. leishanensis, M. lini, M. lishuiensis, M. longipes, M. major, M. microstoma, M. minor, M. monticola, M. mufumontana, M. nankunensis, M. nanlingensis, M. obesa, M. ombrophila, M. oropedion, M. pachyproctus, M. palpebralespinosa, M. parallela, M. parva, M. rubrimera, M. serchhipii, M. shimentaina, M. shunhuangensis, M. tuberogranulata, M. vegrandis, M. wawuensis, M. wugongensis, M. wuliangshanensis, M. wushanensis, M. xianjuensis, M. zhangi, M. zunhebotoensis, M. xiangnanensis, and M. yangmingensis (vs. minimum SVL < 48.0 mm).

By having moderate body size (minimum SVL < 59.0 mm in males), Megophrys qianbeiensis sp. nov. differs from M. auralensis, M. carinense, M. caudoprocta, M. caudoprocta, M. chuannanensis, M. feae, M. gigantica, M. glandulosa, M. himalayana, M. kalimantanensis, M. kobayashii, M. ligayae, M. mangshanensis, M. orientalis, M. periosa, M. platyparietus, M. popei, M. shapingensis, and M. shuichengensis (vs. minimum SVL > 60.0 mm).

By having vomerine teeth, Megophrys qianbeiensis sp. nov. differs from M. aceras, M. acuta, M. angka, M. auralensis, M. baolongensis, M. binchuanensis, M. binlingensis, M. boettgeri, M. brachykolos, M. caobangensis, M. cheni, M. chishuiensis, M. dringi, M. jiangi , M. leishanensis, M. lini , M. lishuiensis, M. major , M. microstoma, M. minor,. M. mirabilis, M. mufumontana, M. nankiangensis, M. obesa, M. ombrophila, M. shapingensis, M. shuichengensis, M. shunhuangensis, M. tuberogranulata, M. vegrandis, M. wawuensis, M. wugongensis, M. wuliangshanensis, M. wushanensis, M. xianjuensis, M. xiangnanensis, and M. yangmingensis (vs. absent).

By the absence of horn-like tubercle at the edge of each upper eyelid, Megophrys qianbeiensis sp. nov. differs from M. aceras, M. acuta, M. angka, M. ancrae, M. auralensis, M. baluensis, M. baolongensis, M. boettgeri, M. brachykolos, M. caobangensis, M. carinense, M. caudoprocta, M. cheni, M. chishuiensis, M. chuannanensis, M. daweimontis, M. dongguanensis, M. dringi, M. edwardinae, M. elfina, M. fansipanensis, M. feae, M. feii, M. flavipunctata, M. gerti, M. glandulosa, M. hansi, M. himalayana, M. hoanglienensis, M. huangshanensis, M. insularis, M. intermedia, M. jiangi, M. jingdongensis, M. jinggangensis, M. jiulianensis, M. kalimantanensis, M. koui, M. kuatunensis, M. lancip, M. leishanensis, M. lekaguli, M. liboensis, M. ligayae, M. lini, M. lishuiensis, M. longipes, M. mangshanensis, M. medogensis, M. microstoma, M. mirabilis, M. montana, M. mufumontana, M. nankunensis, M. nanlingensis, M. nasuta, M. obesa, M. ombrophila, M. omeimontis, M. oreocrypta, M. orientalis, M. palpebralespinosa, M. parallela, M. parva, M. periosa, M. platyparietus, M. popei, M. rubrimera, M. shimentaina, M. shuichengensis, M. shunhuangensis, M. stejnegeri, M. synoria, M. vegrandis, M. wugongensis, M. xianjuensis, M. xiangnanensis, and M. yangmingensis (vs. present).

With the tongue feebly notched behind, Megophrys qianbeiensis sp. nov. differs from M. aceras, M. acuta, M. angka, M. auralensis, M. brachykolos, M. caobangensis, M. caudoprocta, M. dongguanensis, M. elfina, M. hansi, M. jiangi, M. jinggangensis, M. lancip, M. leishanensis, M. lekaguli, M. lini, M. lishuiensis, M. megacephala, M. mufumontana, M. nankunensis, M. obesa, M. ombrophila, M. orientalis, M. palpebralespinosa, M. parallela, M. parva, M. shunhuangensis, M. takensis, M. wushanensis, and M. xianjuensis (vs. tongue not notched behind in the latter), and differs from M. cheni, M. damrei, M. dringi, M. flavipunctata, M. gigantica, and M. popei (vs. tongue notched behind).

By having lateral wide fringes on toes, Megophrys qianbeiensis sp. nov. differs from M. angka, M. baolongensis, M. brachykolos, M. caobangensis, 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. leishanensis, 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).

By toes with one-third webs, Megophrys qianbeiensis sp. nov. differs from M. aceras, M. acuta, M. angka, M. auralensis, M. baluensis, M. baolongensis, M. binchuanensis, M. binlingensis, M. boettgeri, M. brachykolos, M. caobangensis, M. caudoprocta, M. cheni, M. chuannanensis, M. damrei, M. daweimontis, M. dongguanensis, M. dringi, M. elfina, M. fansipanensis, M. feae, M. feii, M. flavipunctata, M. gerti, M. gigantica, M. glandulosa, M. hansi, M. hoanglienensis, M. huangshanensis, M. insularis, M. jiangi, M. jinggangensis, M. jiulianensis, M. kalimantanensis, M. koui, M. kuatunensis, M. lancip, M. leishanensis, M. lekaguli, M. liboensis, M. lini, M. lishuiensis, M. longipes, M. major, M. mangshanensis, M. medogensis, M. medogensis, M. megacephala, M. microstoma, M. minor, M. mufumontana, M. nankiangensis, M. nankunensis, M. nanlingensis, M. obesa, M. ombrophila, M. omeimontis, M. oropedion, M. pachyproctus, M. parva, M. periosa, M. robusta, M. rubrimera, M. serchhipii, M. shunhuangensis, M. takensis, M. tuberogranulata, M. vegrandis, M. wawuensis, M. wugongensis, M. wuliangshanensis, M. wushanensis, M. xianjuensis, and M. zhangi (vs. with rudimentary or without webs).

By heels overlapping when thighs are positioned at right angles to the body, Megophrys qianbeiensis 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).

By the tibiotarsal articulation reaching to the level between tympanum and eye when leg stretched forward, Megophrys qianbeiensis sp. nov. differs from M. daweimontis, M. glandulosa, M. lini, M. major, M. medogensis, and M. obesa (vs. reaching the anterior corner of the eye or beyond eye or nostril and tip of snout).

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

The congeners M. carinense and M. jiangi have sympatric distribution with Megophrys qianbeiensis sp. nov. (Fei et al. 2012). The new species can be distinguished from these species by a series of morphological characters as follows. The new species differs from M. carinense in the smaller body size in the new species (adult males with 49.3–58.2 mm vs. adult males with 92–123 mm in the latter), a horn-like tubercle at the edge of each upper eyelid absent (vs. prominent in the latter), the tongue feebly notched behind (vs. notched behind in the latter). The new species differs from M. jiangi by a larger body size (49.3–58.2 mm in males in the new species vs. 34.4–39.2 mm in the latter), a horn-like tubercle at the edge of each upper eyelid absent (vs. present in the latter), the tongue feebly notched behind (vs. notched behind in the latter), presence of wide lateral fringes on the toes (vs. lacking in the latter), and toes with one-third webbing (vs. rudimentary webbing in the latter).

Megophrys qianbeiensis sp. nov. is phylogenetically closest to M. sangzhiensis and M. spinata. The new species differs from M. sangzhiensis by the following characters: horn-like tubercle absent at the edge of each upper eyelid (vs. present in the latter), toes with one-third webs (vs. with rudimentary webbing in the latter), vomerine ridges present distinctly as V-shape and vomerine teeth present (vs. vomerine ridges separated and weak, vomerine teeth absent in the latter), tibiotarsal articulation reaching to the level between tympanum and eye when leg stretched forward (vs. reaching the anterior corner of eye in the latter), spines on nuptial pads on the first two fingers larger and sparser (vs. finer and thicker in the latter), and having significantly higher ratios of HDL, LAL, HLL, TL, and IAE to SVL. On bioacoustics, the new species differs from M. sangzhiensis in the following characters: lower dominant frequency (2250–3000 Hz in the new species vs. 10380 – 13200 Hz in the latter), the amplitude beginning with moderately high energy pulses, increasing slightly to a maximum by approximately mid note, and then decreasing towards the end of each note (vs. beginning with maximum energy pulses and then decreasing towards the end of note in the latter).

The new species differs from M. spinata by the following characters: tibiotarsal articulation reaching the level between tympanum to eye when leg stretched forward (vs. reaching the anterior corner of eye in the latter), present distinctly as V-shape and vomerine teeth present (vs. vomerine ridges separated and weak, vomerine teeth absent in the latter), spines on nuptial pads on the first two fingers little weaker (vs. spines larger in the latter), and having significantly higher ratios of HDW, ED, LAL, TYD and IAE to SVL. On bioacoustics, the new species differs from M. spinata in the following characters: lower dominant frequency (2250–3000 Hz in the new species vs. 4260–4589 Hz in the latter), the amplitude beginning with moderately high energy pulses, increasing slightly to a maximum by approximately mid note, and then decreasing towards the end of each note (vs. beginning with lower energy pulses, then increasing to the maximum by approximately one-four note and then decreasing to the mid note then increasing to the second highest energy pulses and then decreasing towards the end of note in the latter).

Distribution and habitats

Megophrys qianbeiensis sp. nov. is known from Huanglian Nature Reserve, Tongzi County and Kuankuoshui National Nature Reserve, Suiyang County, Guizhou Province, China at elevations between 1400–1600 m. The individuals of the new species were frequently found on stone in the streams surrounded by evergreen broadleaved forests (Fig. 8), and three sympatric amphibian species were found, i.e., Megophrys jiangi, Odorrana margaratae (Liu, 1950), and Zhangixalus omeimontis (Stejneger, 1924).

Figure 8. 

Habitats of Megophrys qianbeiensis sp. nov. in the type locality, Huanglian Nature Reserve, Tongzi 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 CIBTZ20190608017standing on the stone).

Etymology

The specific epithet qianbeiensis refers to northern part of Guizhou, also called “黔”, the province where the type locality of the species belongs to. We propose the common English name “Qianbei horned toad” and Chinese name “Qian Bei Jiao Chan (黔北角蟾)”.

Discussion

The new species Megophrys qianbeiensis sp. nov. resembles M. spinata and M. sangzhiensis, and detailed comparisons with different data are important for recognizing them. Our molecular phylogenetic data on mitochondrial DNA and morphological comparisons both separated the new species from the two closely related species. Megophrys spinata were reported to be distributed widely through the provinces of Sichuan, Guizhou, Hunan, Chongqing, Yunnan, and Guangxi (Fei et al. 2012), but detailed investigations with multiple data suggested that several populations of the species should contain cryptic species (including Megophrys qianbeiensis sp. nov. and M. sangzhiensis). In recent years, many new species of the genus Megophrys have been gradually described, of which a large part of number was found in China (Frost 2020). To date, among the 106 species of Megophrys, 56 species were discovered in China. Even so, many cryptic species still need to be described just in southern China (Chen et al. 2017; Liu et al. 2018).

South-western China has long been proposed as biodiversity hotspot (Myers et al. 2000). Guizhou Province is an important part of southwest China, especially with the particular environments of karst rocky desertifcation, and knowledge of biodiversity levels and/or patterns are still seriously lacking. Recently, a series of new amphibian species were described from this 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), indicating that species diversity of amphibians in this region is highly underestimated. It is urgent for herpetologists to conduct comprehensive and in-depth surveys to explore the level of amphibian species diversity in this region under accelerating climate changes. Obviously, more work should be conducted on detecting the differentiation of the populations and further describe the cryptic species in this region.

Acknowledgements

We are grateful to the editors and reviewers for their working on the manuscript. This work was supported by National Natural Sciences Foundation of China (NSFC31960099), Key project for bio-diversity conservation, Ministry of Ecology and Environment of People’s Republic of China. (Code: 2018-02-06-M2019-49/50), 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).

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Supplementary materials

Supplementary material 1 

Table S1

Haijun Su, Shengchao Shi, Yanqing Wu, Guangrong Li, Xiaogang Yao, Bin Wang, Shize Li

Data type: molecular data

Explanation note: Uncorrected p-distances between the Megophrys species based on COI gene sequences

Download file (24.66 kb)
Supplementary material 2 

Table S2

Bin Wang

Data type: morphological data

Explanation note: Diagnostic characters separating the new species described in this study from other species of Megophrys

Download file (37.70 kb)
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