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Research Article
Description of a new horned toad of Megophrys Kuhl & Van Hasselt, 1822 (Amphibia, Megophryidae) from Zhejiang Province, China
expand article infoYanqing Wu‡, Shize Li§, Wei Liu|, Bin Wang§, Jun Wu‡
‡ Nanjing Institute of Environmental Sciences, Ministry of Ecology and Environment of China, Nanjing, China
§ Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, China
| Lishui Baiyun Ecological Forest Farm, Lishui, China

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

Corresponding author: Jun Wu ( wujun@nies.org )

Academic editor: Annemarie Ohler
© 2020 Yanqing Wu, Shize Li, Wei Liu, Bin Wang, Jun Wu.
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: Wu Y, Li S, Liu W, Wang B, Wu J (2020) Description of a new horned toad of Megophrys Kuhl & Van Hasselt, 1822 (Amphibia, Megophryidae) from Zhejiang Province, China. ZooKeys 1005: 73-102. https://doi.org/10.3897/zookeys.1005.58629
ZooBank: urn:lsid:zoobank.org:pub:EF069DEA-D4AB-458A-A828-A72402F98C58
Open Access

Abstract

A new species of the Asian horned toad genus Megophrys is described from Zhejiang Province, China, based on multiple data. Molecular phylogenetic analyses based on mitochondrial DNA indicated the new species as an independent clade deeply clustered into the Megophrys clade. The new species is identified from its congeners by a combination of the following characters: body size small (SVL 28.4–32.4 mm in males); vomerine teeth absent; tongue not notched behind; tympanum distinctly visible, oval; a small horn-like tubercle present at the edge of each upper eyelid; two metacarpal tubercles distinctly visible in hand; toes without webbing; heels overlapped when thighs are positioned at right angles to the body; tibiotarsal articulation reaching the level to middle of eye when leg stretched forward; an internal single subgular vocal sac in male; in breeding male, the nuptial pads present on the dorsal base of the first two fingers.

Keywords

Molecular phylogenetic analyses, morphology, new species, taxonomy, toad

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). The generic assignment of species in the group has been controversial for decades (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; Li et al. 2020). Recent molecular phylogenetic studies proposed this group as a monophyletic group (Chen et al. 2017; Mahony et al. 2017; Li et al. 2018a; Liu et al. 2018; Liu et al. 2020; Wang et al. 2020), which was recognized as a big genus Megophrys sensu lato (Mahony et al. 2017; Li et al. 2018b; Liu et al. 2018; Liu et al. 2018; Liu et al. 2020; Lyu et al. 2020; Xu et al. 2020; Wang et al. 2020), though some studies still divided the taxa of the group into different genera and/or subgenera (Fei and Ye 2016; Chen et al. 2017; Deuti et al. 2017; Liu et al. 2018). The genus Megophrys currently contains 106 species, of which 52 species were described over the last decade (Frost 2020). A number of cryptic species were still indicated in the genus by molecular phylogenetic analyses (e.g., Chen et al. 2017; Liu et al. 2018).

Wuyi Mountain region, located in northern Fujian, southeastern Jiangxi and south Zhejiang provinces of China, is a biodiversity hotspot. In this region, four Megophrys species have been recorded, i.e., M. boettgeri (Boulenger, 1899), M. kuatunensis Pope, 1929, M. ombrophila Messenger & Dahn, 2019, and M. lishuiensis Wang, Liu & Jiang, 2017. However, many mountains in this region, especially in south Zhejiang Province, have been poorly investigated.

During field surveys in Qingyuan County, Zhejiang Province, China, we collected Megophrys specimens. Molecular phylogenetic analyses and morphological comparisons supported some of these specimens as an undescribed taxon that we describe herein as a new species.

Materials and methods

Sampling

A total of 15 specimens were sampled in this study: six adult males and one tadpole of the undescribed species and two adult males of M. boettgeri from Qingyuan County, Zhejiang Province, China, and one adult male of M. ombrophila and six adult males of M. kuatunensis from Wuyi Mountain, Fujian Province, China (Table 1; Fig. 1). The developmental stage of tadpole was identified following Gosner (1960). In the field, the toad and tadpole were euthanized using isoflurane, and the specimens were fixed in 75% ethanol. 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).

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 baishanzuensis sp. nov. CIBQY20200719001 Baishanzu National Park, Qingyuan, Zhejiang, China MW001150 MT998291
2 Megophrys baishanzuensis sp. nov. CIBQY20200719002 MW001151 MT998292
3 Megophrys baishanzuensis sp. nov. CIBQY20200719003 MW001152 MT998293
4 Megophrys baishanzuensis sp. nov. CIBQY20200719004 MW001153 MT998294
5 Megophrys baishanzuensis sp. nov. CIBQY20200719006 MW001154 MT998295
6 Megophrys baishanzuensis sp. nov. CIBQY20200726001 MW001155 MT998296
7 Megophrys baishanzuensis sp. nov. CIBQY20200726002 MW001156 MT998297
8 Megophrys kuatunensis CIBWY18082407 Wuyi Shan, Fujian, China MW001157 MT998298
9 Megophrys kuatunensis CIBWY18082408 MW001158 MT998299
10 Megophrys kuatunensis SYS a001579 KJ560376 –
11 Megophrys lini SYS a002370 Suichuan, Jiangxi, China KJ560412 –
12 Megophrys xiangnanensis SYS a002874 Yangming Shan, Hunan, China MH406713 MH406165
13 Megophrys nanlingensis SYS a001959 Nanling Nature Reserve, Guangdong, China MK524111 MK524142
14 Megophrys dongguanensis SYS a001972 Yinping Shan, Guangdong, China MK524098 MK524129
15 Megophrys nankunensis SYS a004498 Nankun Shan, Guangdong, China MK524108 MK524139
16 Megophrys cheni SYS a001427 Jinggang Shan, Jiangxi, China KJ560391 –
17 Megophrys wugongensis SYS a002610 Wugongshan Scenic Area, Jiangxi, China MK524114 MK524145
18 Megophrys ombrophila KRM18 Wuyishan, Fujian, China KX856404 –
19 Megophrys ombrophila CIBWY18082308 MW001159 MT998300
20 Megophrys obesa SYS a002272 Heishiding Nature Reserve, Guangdong, China KJ579122 –
21 Megophrys lishuiensis WYF00169 Lishui, zhejiang, China KY021418 –
22 Megophrys xianjuensis CIBXJ190505 Xianju, zhejiang, China MN563753 MN563769
23 Megophrys jinggangensis KIZ07132 Chashan Forest Farm, Jiangxi, China KX811840 KX812108
24 Megophrys boettgeri CIB20200718001 Baishanzu National Park, Qingyuan, Zhejiang, China MW001160 MT998301
25 Megophrys boettgeri CIB20200718002 Baishanzu National Park, Qingyuan, Zhejiang, China MW001161 MT998302
26 Megophrys boettgeri Tissue ID: YPXJK033 Wuyi Shan, Fujian, China KX811814 KX812104
27 Megophrys huangshanensis KIZ022004 Huang Shan, Anhui, China KX811821 KX812107
28 Megophrys liboensis GNUG:20160408003 Libo, Guizhou, China MF285262 –
29 Megophrys mufumontana SYS a006391 Mufu Shan, Hunan, China MK524105 MK524136
30 Megophrys wushanensis KIZ045469 Guangwu Shan, Sichuan, China KX811838 KX812094
31 Megophrys baolongensis KIZ019216 Baolong, Chongqing, China KX811813 KX812093
32 Megophrys tuberogranulata Tissue ID: YPX10987 Badagongshan Nature Reserve, Hunan, China KX811823 KX812095
33 Megophrys yangmingensis SYS a002877 Yangming Shan, Hunan, China MH406716 MH406168
34 Megophrys shimentaina SYS a002077 Shimentai Nature Reserve Guangdong, China MH406655 MH406092
35 Megophrys jiulianensis SYS a002107 Jiulian Shan, Jiangxi, China MK524099 MK524130
36 Megophrys shunhuangensis HNNU16SH02 Shunhuang Mountains, Hunan, China MK836037 –
37 Megophrys mirabilis SYS a002192 Huaping Nature Reserve, Guangxi, China MH406669 MH406109
38 Megophrys leishanensis CIBLS20171101001 Leigong Shan, Guizhou, China MK005310 MK005306
39 Megophrys omeimontis KIZ025765 Emei Shan, Sichuan, China KX811884 KX812136
40 Megophrys angka KIZ040591 Kiew Mae Pan nature trail, Chiang Mai, Thailand MN508052 –
41 Megophrys binchuanensis KIZ019441 Jizu Shan, Yunnan, China KX811849 KX812112
42 Megophrys palpebralespinosa KIZ011603 Pu Hu Nature Reserve, Thanh Hoa, Vietnam KX811888 KX812137
43 Megophrys spinata SYSa002227 Leigong Shan, Guizhou, China MH406676 MH406116
44 Megophrys sangzhiensis SYSa004307 Zhangjiajie, Hunan, China MH406798 MH406260
45 Megophrys binlingensis SYSa005313 Wawu Shan, Sichuan, China MH406892 MH406354
46 Megophrys wuliangshanensis KIZ046812 Huangcaoling, Yunnan, China KX811881 KX812129
47 Megophrys daweimontis KIZ048997 Dawei Shan, Yunnan, China KX811867 KX812125
48 Megophrys jingdongensis KIZ-LC0805067 Huanglianshan National Nature Reserve, Yunnan, China KX811872 KX812131
49 Megophrys fansipanensis VNMN 2018.01 Lao Cai, Sa Pa, Vietnam MH514886 –
50 Megophrys hoanglienensis VNMN 2018.02 Lao Cai, Sa Pa, Vietnam MH514889 –
51 Megophrys minor KIZ01939 Qingcheng Shan, Sichuan, China KX811896 KX812145
52 Megophrys jiangi CIBKKS20180722006 Kuankuosui Nature Reserve, Guizhou, China MN107743 MN107748
53 Megophrys chishuiensis CIBCS20190518031 Chishui Nature Reserve, Guizhou, China MN954707 MN928958
54 Megophrys brachykolos ROM 16634 Hong Kong, China KX811897 KX812150
55 Megophrys acuta SYS a001957 Heishiding Nature Reserve, Guangdong, China KJ579118 –
56 Megophrys gerti ITBCZ 1108 Nui Chua National Park, Ninh Thuan, Vietnam KX811917 KX812161
57 Megophrys elfina ZMMU ABV-00454 Bidoup Mountain, Lam Dong, Vietnam KY425379 –
58 Megophrys synoria FMNH 262778 O’Reang, Mondolkiri, Cambodia KY022198 –
59 Megophrys hansi KIZ010360 Phong Dien Nature Reserve, Thua Thien Hue, Vietnam KX811913 KX812155
60 Megophrys microstoma KIZ048799 Xiaoqiaogou Nature Reserve, Yunnan, China KX811914 KX812156
61 Megophrys pachyproctus KIZ010978 Beibeng, Xizang, China KX811908 KX812153
62 Megophrys baluensis ZMH A13125 Gunung Kinabalu National Park, Kogopan Trail, Malaysia KJ831310 –
63 Megophrys stejnegeri KU 314303 Pasonanca Natural Park, Zamboanga, Philippines KX811922 KX812052
64 Megophrys ligayae ZMMU NAP-05015 Palawan, Philippines KX811919 KX812051
65 Megophrys kobayashii UNIMAS 8148 Gunung Kinabalu National Park, Sabah, Malaysia KJ831313 –
66 Megophrys nasuta KIZ019419 Malaysia KX811921 KX812054
67 Megophrys edwardinae FMNH 273694 Bintulu, Sarawak, Malaysia KX811918 KX812050
68 Megophrys aceras KIZ025467 Khao Nan National Park, Nakhon Si Thammarat, Thailand KX811925 KX812159
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 dringi UNIMAS 8943 Gunung Mulu National Park, Sarawak, Malaysia KJ831317 –
84 Megophrys nankiangensis CIB ZYC517 Nanjiang, Sichuan, China KX811900 –
85 Megophrys wawuensis KIZ025799 Wawu Shan, Sichuan, China KX811902 KX812062
86 Megophrys gigantica SYSa003933 Wuliang shan, Yunnan, China MH406775 MH406235
87 Megophrys shapingensis KIZ014512 Liziping Nature Reserve, Sichuan, China KX811904 KX812060
88 Megophrys feae KIZ046706 Huangcaoling, Yunnan, China KX811810 KX812056
89 Megophrys chuannanensis CIB20050081 Hejiang, Sichuan, China KM504261 –
90 Megophrys carinense Tissue ID: YPX20455 Dayao Shan, Guangxi, China KX811811 KX812057
91 Megophrys popei SYS a000589 Naling Nature Reserve, Guangdong, China KM504251 –
92 Megophrys intermedia ZFMK 87596 U Bo, Phong Nha-Ke Bang NP, Vietnam HQ588950 –
93 Megophrys Montana LSUMZ 81916 Sukabumi, Java, Indonesia KX811927 KX812163
94 Megophrys lancip MZB: Amp:22233 – KY679891 –
95 Leptobrachium boringii Tissue ID: YPX37539 Emei Shan, Sichuan, China KX811930 KX812164
96 Leptobrachella oshanensis KIZ025778 Emei Shan, Sichuan, China KX811928 KX812166
Figure 1. 

Sampling localities of Megophrys baishanzuensis sp. nov. and its relatives 1 Baishanzu National Park, Qingyuan County, Zhejiang Province, China, inhabited by Megophrys baishanzuensis sp. nov. and M. boettgeri 2 Wuyi Mountain, Wuyishan City, Fujian Province, China, inhabited by M. boettgeri, M. kuatunensis, and M. ombrophila.

Molecular data and phylogenetic analyses

Six adult males and one tadpole of the undescribed species, two M. kuatunensis, one M. ombrophila, and two M. boettgeri were included in the molecular analyses (Table 1). 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 GenBank accession numbers see Table 1).

For molecular analyses, the available sequences 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 following 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 is 16S 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 (BPP) and the 50% majority-rule consensus of the post burn-in trees sampled at stationarity. Finally, mean genetic distance between Megophrys species based on uncorrected p-distance model was estimated respectively on 16S and COI genes using MEGA v. 6.06 (Tamura et al. 2013).

Morphological comparisons

Six adult males and one tadpole of the undescribed species were measured (Table 1 and Suppl. material 1). For comparisons, six adult male specimens of M. kuatunensis were also measured (Supp. material 1). The terminology and methodology followed Fei et al. (2009). Measurements were taken with a dial caliper to 0.1 mm. Twenty-two morphometric characters of adult specimens were measured:

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

FIL first finger length (distance from base to tip of finger I);

FIIL second finger length (distance from base to tip of finger II);

FIIIL third finger length (distance from base to tip of finger III);

FIVL fourth finger length (distance from base to tip of finger IV);

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);

HAL hand length (distance from tip of third digit to proximal edge of inner palmar tubercle);

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);

SNT 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).

For the single tadpole of the undescribed species, eleven morphometric characters were measured:

BH maximum body height;

BW maximum body width;

IOS interocular distance (minimum distance between eye);

MW mouth width (distance between two corners of mouth);

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

SS snout to spiraculum (distance from spiraculum to the tip of the snout);

SVL snout-vent length;

TAH tail height (maximum height between upper and lower edges of tail);

TAL tail length (distance from base of vent to the tip of tail);

TBW maximum width of tail base;

TOL total length (distance from the tip of the snout to the tip of tail).

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 the following morphometric analyses. Mann-Whitney U tests were conducted to test the significance of differences on morphometric characters between the undescribed species and M. kuatunensis. The significance level was set at 0.05. Furthermore, principal component analyses (PCA) were conducted to highlight whether the different species were separated in morphometric space.

The new species was also compared with all other Megophrys species on morphology. Comparative data were obtained for related species as described in literature (Table 2).

Table 2.
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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. awuh Mahony, Kamei, Teeling, & Biju, 2020 Mahony et al. 2020
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. boettgerii (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. dzukou Mahony, Kamei, Teeling & Biju, 2020 Mahony et al. 2020
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. 2018
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; Mahony et al. 2018
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. numhbumaeng Mahony, Kamei, Teeling, & Biju, 2020 Mahony et al. 2020
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 analyses

The advertisement calls of the undescribed species were recorded from the holotype specimen CIBQY20200726001 in the field on 26 July 2020 from Qingyuan County, Zhejiang Province, China. When registrating the male in the stream the ambient air temperature was 21.5 °C and there was air humidity of 87%. For comparisons, the advertisement calls of M. kuatunensis from Wuyi Mountain, Fujian Province, China were recorded from the specimens CIBWY18082410, CIBWY18082411 and CIBWY18082412 at an ambient air temperature of 22.0 °C and air humidity of 88% on 24 August 2018. 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 of the mitochondrial DNA dataset presented almost consistent topology, and as well, though relationships of many clades were unresolved (Fig. 2). In mitochondrial DNA trees, all samples of the undescribed species were clustered into one clade which was deeply clustered into the Megophrys clade. The species is likely sister to M. kuatunensis (bootstrap supports < 50% and BPP = 0.51) though the relationships between the two species and most other congeners were not resolved (all bootstrap supports < 50% and many BPP < 0.95).

Figure 2. 

Maximum likelihood (ML) tree of the genus Megophrys reconstructed based on 16S rRNA and COI gene sequences. Bayesian posterior probability/ML bootstrap supports were denoted beside each node. Samples 1–96 refer to Table 1.

Genetic distances based on 16S and COI genes with uncorrected p-distance model between the samples of the undescribed species were all below 0.2%. The genetic distance between the undescribed species and its closest related species M. kuatunensis were 2.1% and 8.1% on 16S and COI respectively, which was higher or at the same level with those among many pairs of sister species, for example, 1.7% and 3.8% on 16S and COI respectively between M. spinata and M. sangzhiensis (Suppl. materials 2 and 3).

In PCA for male group, the total variation of the first two principal components was 47.5%. On the two-dimensional plots of PC1 vs. PC2, the undescribed species was almost separated from M. kuatunensis (Fig. 3). The first two principal component axes could separate M. kuatunensis from the undescribed species mainly based on limb and head characteristics, namely, HDL, HDW, IND, FIL, FIIL and FL. The results of Mann-Whitney U tests indicated that in males, the undescribed species was significantly different from M. kuatunensis on UEW and TFL (p-values < 0.05; Table 3).

Table 3.
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Morphometric comparisons between the adult specimens of Megophrys baishanzuensis sp. nov. and M. kuatunensis. Units given in mm. See abbreviations for the morphological characters in Materials and methods section. P-value resulted from Mann-Whitney U test. Significant level at 0.05.

Character Megophrys baishanzuensis sp. nov. M. kuatunensis Mann-Whitney U value P-value
Male (N = 6) Male (N = 6)
Ranging Mean ± SD Ranging Mean ± SD
SVL 28.4–32.4 30.5 ± 1.8 28.4–32.4 30.5 ± 1.8 13.000 0.423
HDL 8.0–9.1 8.6 ± 0.4 8.0–9.1 8.6 ± 0.4 6.000 0.055
HDW 9.3–10.5 10.2 ± 0.4 9.3–10.5 10.2 ± 0.4 8.000 0.109
SL 3.4–4.1 3.8 ± 0.3 3.4–4.1 3.8 ± 0.3 16.000 0.749
SNT 1.5–2.6 2.0 ± 0.4 1.5–2.6 2.0 ± 0.4 18.000 1.000
IND 3.1–3.7 3.4 ± 0.3 3.1–3.7 3.48 ± 0.3 16.000 0.749
IOD 2.8–3.3 3.0 ± 0.2 2.8–3.3 3.08 ± 0.2 6.000 0.055
UEW 2.3–3.0 2.6 ± 0.2 2.3–3.0 2.6 ± 0.2 2.000 0.010
ED 3.7–4.0 3.8 ± 0.1 3.7–4.0 3.8 ± 0.1 15.000 0.631
TYD 1.5–2.1 1.8 ± 0.2 1.5–2.1 1.8 ± 0.2 16.000 0.749
LAL 13.4–14.6 14.1 ± 0.5 13.4–14.6 14.2 ± 0.5 9.000 0.150
HAL 6.6–7.9 7.1 ± 0.5 6.6–7.9 7.1 ± 0.5 6.000 0.055
LW 2.2–2.7 2.4 ± 0.2 2.2–2.7 2.4 ± 0.2 10.000 0.200
FIL 2.2–2.8 2.5 ± 0.2 2.2–2.8 2.5 ± 0.2 17.000 0.873
FIIL 2.4–3.0 2.7 ± 0.2 2.4–3.0 2.7 ± 0.2 12.000 0.200
FIIIL 4.3–5.1 4.6 ± 0.3 4.3–5.1 4.6 ± 0.3 10.000 0.200
FIVL 2.6–3.6 3.0 ± 0.4 2.6–3.6 3.0 ± 0.4 15.000 0.631
THL 12.2–13.5 12.9 ± 0.5 12.2–13.5 12.9 ± 0.5 10.000 0.200
TL 12.8–14.9 13.9 ± 0.9 12.8–14.9 13.9 ± 0.9 13.000 0.423
TW 2.7–4.2 3.3 ± 0.5 2.7–4.2 3.3 ± 0.5 13.000 0.423
TFL 17.8–20.4 19.4 ± 1.0 17.8–20.4 19.4 ± 1.0 1.000 0.006
FL 11.2–12.3 11.8 ± 0.4 11.2–12.3 11.8 ± 0.4 13.000 0.423
Figure 3. 

Plots of the first principal component (PC1) versus the second (PC2) for Megophrys baishanzuensis sp. nov. and M. kuatunensis from principal component analyses on male group.

There were two differences in sonograms and waveforms of calls between the undescribed species and M. kuatunensis (Fig. 4; Table 4). Firstly, the undescribed species had slower call repetition rate than the latter (0.79 call/s in the former vs. 1.18 call/s in the latter). Secondly, the undescribed species had lower dominant frequency (3.19–3.38 kHz in the former vs. 3.38–3.75 kHz in the latter).

Table 4.
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Comparisons of characteristics of advertisement calls of Megophrys baishanzuensis sp. nov. and M. kuatunensis.

Call character Megophrys baishanzuensis sp. nov. M. kuatunensis
CIBQY20200726001 CIBWY2018082410 CIBWY2018082412 WY2018082411
Number of call groups measured 11 30 30 20
Number of notes measured 22 30 30 40
Call duration (ms) 151.0–170.0 (162.4 ± 5.7) 131.0–163.0 (147.2 ± 7.1) 131.0–163.0 (147.2 ± 7.1) 130.0–159.0 (120.9 ± 5.9)
Call repetition rate (calls/s) 0.79 1.18 1.13 1.3
Intercall interval (ms) 682.0–1869.0 (936.8 ±349.0) 404–1548.0 (687.3 ± 206.8) 404–1548.0 (687.3 ± 206.8) 350.0–733.0 (458.4 ± 87.1)
Pulses/call 23.0–30.0 (26.0 ± 2.4) 25.0–36.0 (30.0 ± 2.3) 25.0–36.0 (30.0 ± 2.3) 32.0–40.4 (35.7 ± 2.3)
Dominant frequency (kHz) 3.19–3.38 (3.36 ± 0.06) 3.38–3.75 (3.46 ± 0.16) 3.38–3.75 (3.46 ± 0.16) 3.38–3.38 (3.38±0.01)
Pulse duration (ms) 3.0–6.0 (4.9 ± 0.6) 3.0–6.0 (4.4 ± 0.7) 3.0–6.0 (4.4 ± 0.7) 3.0–6.0 (4.5 ± 0.6)
Figure 4. 

Visualization of advertisement calls of Megophrys baishanzuensis sp. nov. and M. kuatunensis A–C waveform showing 10 seconds, waveform showing 0.2 seconds and sonogram showing 0.2 seconds of CIBQY20200726001 of Megophrys baishanzuensis sp. nov. D–F waveform showing 10 seconds, waveform showing 0.2 seconds and sonogram showing 0.2seconds of CIBWY18082410 of M. kuatunensis.

Based on the molecular phylogenetic analyses, morphological comparisons (Supp. material 4), and bioacoustics differences, the specimens from Qiangyuan County, Zhejiang Province, China represent a new species which is described as follows.

Taxonomic accounts

Megophrys baishanzuensis sp. nov.

http://zoobank.org/563EBE4E-45FF-4956-AB3B-70467B2D338E
Figs 4A, B, E, G, H, 5, 6, 7, 8; Tables 1, 2, 3, 4, Suppl. materials 1, , , 4

Holotype

CIBQY20200726001 (Figs 4A, B, E, G, H, 5), adult male, from Baishanzu National Park, Qingyuan County, Zhejiang Province, China (27.76°N, 119.18°E, ca. 1537 m a.s.l.), collected by Bin Wang on 26 July 2020.

Paratype

Five adult males collected from the same place as holotype collected by Bin Wang. CIBQY20200719001-CIBQY20200719004 collected on 19 July 2020 by Bin Wang, and CIBQY20200726002 collected by Zhonghao Luo on 26 July 2020.

Other material examined

One tadpole (CIBQY20200719005; Fig. 7) collected by Bin Wang on 19 July 2020.

Diagnosis

Megophrys baishanzuensis 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 (Fei et al. 2009).

Megophrys baishanzuensis sp. nov. could be distinguished from its congeners by a combination of the following morphological characters: body size small (SVL 28.4–32.4 mm in males); 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 distinctly visible in hand; toes without webbing; heels overlapping when thighs are positioned at right angles to the body; tibiotarsal articulation reaching the level to the middle of eye when leg stretched forward.

Description of holotype

(Figs 4A, B, E, G, H, 5). SVL 28.5 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 46.0% of head length; pupils vertical; nostril orientated laterally, closer to snout than eye; tympanum distinct, 55.8% 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 47.0% of SVL; fingers slender, relative finger lengths: I < II < IV < 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, tibia length 46.5% times of SVL; heels overlapping when thighs are positioned at right angles to the body, tibiotarsal articulation reaching the middle of 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 absent on each toes; toes without webbing but with 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 X-shaped ridge, two dorsolateral parallel ridges on either side of the X-shaped ridges; an inverted triangular brown speckle between two upper eyelidsseveral tubercles scattered on dorsal, flanks and dorsal surface of thighs and tibias; supratympanic fold distinct.

Numerous granules scattered on ventrum; pectoral and femoral glands distinct; numerous white granules on outer thighs.

Coloration of holotype in life. (Fig. 5). Dorsal brown, several pink tubercles scattered on dorsal, an inverted triangular brown speckle between the eyes; X-shaped ridges on the dorsum brown, four dark transverse bands on the dorsal surface of the thigh and shank; ventral surface of body white with brown spots; two dark brown dark bars on the flanks, throat brown; white vertical bars on lower and upper lip; ventral surface of anterior limb dark reddish purple, posterior limb orange with numerous white granules; tip of digits pale grey; inner metatarsal tubercle and two metacarpal tubercles pinkish; soles uniform dark reddish purple; pectoral glands white.

Figure 5. 

Photos of the holotype specimen CIBQY20200726001 of Megophrys baishanzuensis sp. nov. and topotype specimen of M. kuatunensis A, B, E, G, H dorsal view of body, ventral view of body, lateral view of head, ventral view of hand, and ventral view of foot of CIBQY20200726001, respectively C, D, F, I, J dorsal view of body, ventral view of body, lateral view of head, ventral view of hand, and ventral view of foot of CIBWY18082413, respectively. Red arrow points to tympanum.

Coloration of holotype in preservation. (Fig. 4A, B, E, G, H). Color of dorsal surface fades to taupe; the inverted triangular brown speckle between the eyes and brown 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.

Variation. Fig. 6. Measurements and basic statistics of adult specimens are presented in Tables 3 and Supp. material 1. All specimens were similar in morphology but some individuals different from the holotype in color pattern. In CIBQY2020200719001 the tubercles on the dorsum forming two ≻ shaped, disconnected ridges (Fig. 6A); in CIBQY2020200719004 the tubercles on the dorsum forming a big and distinct X-shaped speckle (Fig. 6B); in CIBQY2020200719003 ventral surface of body grey with brown spots (Fig. 6C); in CIBQY2020200726002 ventral surface of body and limbs brownish red (Fig. 6D).

Figure 6. 

Photos of the holotype CIBQY20200726001 of Megophrys baishanzuensis sp. nov. in life A dorsal view B ventral view C lateral view D ventral view of hand E ventral view of foot.

Tadpole description. Fig. 7. The tadpole CIBQY20200719006 (Fig. 7) was confirmed as Megophrys baishanzuensis sp. nov by molecular phylogenetic analyses. Measurements in mm. Stage 31. Body slender, body brownish black and tail pale brown, body height greater than tail height; dorsal fin arising behind the origin of the tail, the highest fin near mid-length, tapering gradually to the narrowly pointed tip; tail approximately 1.9 times as long as snout-vent length; tail height 13.6% of tail length; body width longer than body height (BW/BH1.2); eyes large, lateral, nostril near eyes; spiracle on the left side of the body and distinct; oral disk terminal, lips expanded and directed upwardly into a umbelliform oral disk; flank of body brownish black with some white spots, tail fins lightly colored, with small white and black spots. TOL 22.7; SVL 8.7; BW 3.0; BH 2.7; SL 2.0; SS 4.0; IOS 1.8; TAL 14.7; TAH 2.2; TBD 1.5; MW 1.3.

Figure 7. 

Color variation in Megophrys baishanzuensis sp. nov. in life A dorsal view of the adult male CIBQY2020200719001 B dorsal view of the adult male CIBQY2020200719004 C ventral view of the adult male CIBQY2020200719003 D ventral view of the adult male CIBQY2020200726002.

Advertisement call. Fig. 4. The call description is based on recordings of the holotype CIBQY20200726001 (Fig. 4; Table 4) from a shrub leaf near the streamlet. Call duration was 151.0–170.0 ms (mean 162.4 ± 5.7). Inter-call interval was 682.0–1869.0 ms (mean 936.8 ± 349.0). Pulse/call was 23.0–30.0 (mean 26.0 ± 2.4); pulse duration was 3.0–6.0 (mean 4.9 ± 6.0) and call repetition rate was 0.79 call/s.

Amplitude modulation within note was apparent, beginning with moderately high energy pulses, increasing to the maximum by approximately quarter, and then decreasing towards the end. The average dominant frequency was 3.36 ± 0.06 (3.19–3.38 kHz).

Secondary sexual characters. A single subgular vocal sac present in male. In breeding season, nuptial pads are present on the dorsal base of the first two fingers in males.

Comparisons

Supp. material 4. By having small body size, Megophrys baishanzuensis sp. nov. differs from M. ancrae, M. auralensis, M. awuh, M. baluensis, M. baolongensis, M. binlingensis, M. boettgeri, M. caobangensis, M. carinense, M. caudoprocta, M. chishuiensis, M. chuannanensis, M. damrei, M. daweimontis, M. dzukou, M. edwardinae, M. feae, M. flavipunctata, M. gigantica, M. glandulosa, M. hansi, M. himalayana, M. hoanglienensis, M. huangshanensis, M. insularis, M. jiangi, M. jingdongensis, M. jinggangensis, M. kalimantanensis, M. kobayashii, M. lancip, M. lekaguli, M. liboensis, M. ligayae, M. lini, M. longipes, M. major, M. mangshanensis, M. medogensis, M. megacephala, M. mirabilis, M. montana, M. monticola, M. nasuta, M. obesa, M. omeimontis, M. orientalis, M. pachyproctus, M. palpebralespinosa, M. parallela, M. parva, M. periosa, M. platyparietus, M. popei, M. sangzhiensis, M. serchhipii, M. shapingensis, M. shuichengensis, M. spinata, M. takensis M. wawuensis, and M. xiangnanensis (maximum SVL < 33.0 mm in the new species vs. minimum SVL > 34.0 mm in the latter).

By vomerine teeth absent, Megophrys baishanzuensis sp. nov. differs from M. ancrae, M. baluensis, M. carinense, M. caudoprocta, M. chuannanensis, M. damrei, M. daweimontis, M. dongguanensis, M. dzukou, 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. numhbumaeng, 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 present at the edge of each upper eyelid, Megophrys baishanzuensis 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 baishanzuensis sp. nov. differs from M. ancrae, M. baolongensis, M. binlingensis, M. boettgeri, M. carinense, M. cheni, M. chuannanensis, M. damrei, M. dringi, M. dzukou, 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. numhbumaeng, 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. notched behind in the latter).

By toes with narrow lateral fringes, Megophrys baishanzuensis 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. 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 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 without webbing, Megophrys baishanzuensis 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 baishanzuensis sp. nov. differs from M. actuta, M. brachykolos, M. dongguanensis, M. huangshanensis, M. kuatunensis, M. nankunensis, M. obesa, M. ombrophila, M. wushanensis, and M. wugongensis (vs. just meeting or not meeting in the latter).

By tibiotarsal articulation reaching to the level to the middle of eye when leg stretched forward, Megophrys baishanzuensis sp. nov. differs from M. daweimontis, M. glandulosa, M. lini, M. major, M. medogensis, M. obesa, M. sangzhiensis, and M. yangmingensis (vs. reaching the anterior corner of the eye or beyond eye or nostril and 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 baishanzuensis sp. nov. differs from M. caudoprocta, M. shapingensis, and M. shuichengensis (vs. vocal sac absent in the latter).

The congeners M. boettgeri, M. lishuiensis, M. ombrophila, and M. xianjuensis all occur in Wuyi Mountains, Fujian Province and/or Zhejiang Province, China, and probably have sympatric distribution with Megophrys baishanzuensis sp. nov. (Fei et al. 2012; Wang et al. 2017b; Messenger et al. 2019; Wang et al. 2020). The new species can be distinguished from these species by a series of morphological characters as follows. The new species differs from M. boettgeri by body size smaller (adult males with 28.4–32.4 mm vs. adult males with 34.5–37.8 mm), and in breeding male nuptial pads present on the dorsal base of the first two fingers (vs. nuptial pad only on the first finger). The new species differs from M. lishuiensis by vomerine ridges present (vs. absent), toes with narrow lateral fringe (vs. without), and tibiotarsal articulation reaching the middle of eye when leg stretched forward (vs. reaching the range from tympanum to eye). The new species differs from M. ombrophila by heels overlapping when thighs are positioned at right angles to the body (vs. not meeting), vomerine ridges present (vs. absent), and toes with narrow lateral fringe (vs. without). The new species differs from M. xianjuensis by tibiotarsal articulation reaching the middle of eye when leg stretched forward (vs. reaching the range from tympanum to eye), and toes with narrow lateral fringe (vs. without).

Figure 8. 

Photos of the tadpole CIBQY20200719006 of Megophrys baishanzuensis sp. nov. in life A dorsal view B lateral view.

Megophrys baishanzuensis sp. nov. is phylogenetically closest to M. kuatunensis. Megophrys baishanzuensis sp. nov. could be identified from M. kuatunensis distinctly by tibiotarsal articulation reaching the middle of eye when leg stretched forward (vs. reaching the range from tympanum to eye), heels overlapping when thighs are positioned at right angles to the body (vs. not meeting), tongue not notched behind (vs. notched feebly), the supratympanic fold more expanded in dorsal view and tympanum protruding (vs. concave), and having significantly lower ratios of UEW and TFL to SVL in males (all p-values < 0.05; Table 3). On call characters, the new species has slower call repetition rate (0.79 call/s in the new species vs. 1.18 call/s in M. kuatunensis), and has lower dominant frequency (3.19–3.38 kHz in the new species vs. 3.38–3.75 kHz in M. kuatunensis).

Distribution and habitat

Megophrys baishanzuensis sp. nov. is known from the type locality, Baishanzu National Park, Qingyuan County Zhejiang Province, China, at elevations between 1400–1600 m. The individuals of the new species were frequently found in the stream surrounded by evergreen broadleaved forests (Fig. 9). M. boettgeri was also found in the same stream.

Figure 9. 

Habitats of Megophrys baishanzuensis sp. nov. in the type locality, Baishanzu Naitonal Park, Qingyuan County, Zhejiang Province, China A landscape for forest B the stream under the forest inhabited by Megophrys baishanzuensis sp. nov.

Etymology

The specific name baishanzuensis refers to the distribution of this species, Baishanzu National Park, Qingyuan County, Zhejiang Province, China. We propose the common name “Baishanzu horned toad” (English) and Bai Shan Zu Jiao Chan (百山祖角蟾, Chinese).

Discussion

Although Megophrys baishanzuensis sp. nov. superficially resembles M. kuatunensis, molecular phylogenetic analyses, detailed morphological comparisons and call datas all proposed the distinct differences between them. Moreover, the breeding seasons of them are different. According to our surveys, the breeding season of M. kuatunensis is in April to May in Wuyi Mountain, Fujian Province, China. But in this season, we did not find any individual of Megophrys baishanzuensis sp. nov. in Qingyuan County, Zhejiang Province, China. And, the breeding season of the new species should be later than June because in June, we only listened to the calls of one male in the type locality (< 10 °C), and, in late July, the males of the species started to call when the temperature was just higher than 18 °C (but we did not find any female individual and egg of it). Different call characteristics and breeding ecology most probably promoted separation of the two species.

During our several and extensive surveys, we only found fewer than 15 adult males of Megophrys baishanzuensis sp. nov., only in a small stream near the top of the mountain in Baishanzu National Park, Zhejiang Province, China, and even then, we did not find any female, and only found four tadpoles of this species. Obviously, the population of the new species is very endemic and small. Fortunately, this population is in a preserved area in Baishanzu National Park. Of course, we still should make a reinforced plan to preserve this area for this toad species.

Acknowledgements

We thank Zhonghao Luo, Yang Li and Shengchao Shi for their help with collecting samples and data. This work was supported by the Biodiversity Survey Project of Lishui, Zhejiang Province and the Project supported by the Biodiversity investigation, Observation and Assessment Program (2019–2023) of Ministry of Ecology and Environment of China.

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Supplementary materials

Supplementary material 1 

Table S1

Bin Wang

Data type: morphological measurements

Explanation note: Measurements of the adult specimens of Megophrys baishanzuensis sp. nov. and M. kuatunensis. Units given in mm. See abbreviations for the morphological characters in Materials and methods section.

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 (15.88 kb)
Supplementary material 2 

Table S2

Bin Wang

Data type: genetic distance

Explanation note: Uncorrected p-distances between the Megophrys species on the 16S gene.

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 (40.18 kb)
Supplementary material 3 

Table S3

Bin Wang

Data type: genetic distance

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

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 (24.68 kb)
Supplementary material 4 

Table S4

Bin Wang

Data type: morphological comparisons

Explanation note: Diagnostic characters separating Megophrys baishanzuensis sp. nov. from other species of Megophrys.

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 (18.30 kb)
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