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Multiple data revealed two new species of the Asian horned toad Megophrys Kuhl & Van Hasselt, 1822 (Anura, Megophryidae) from the eastern corner of the Himalayas
expand article infoShengchao Shi§|, Meihua Zhang, Feng Xie, Jianping Jiang, Wulin Liu, Li Ding, Li Luan#, Bin Wang
‡ Chinese Academy of Sciences, Chengdu, China
§ Sichuan University, Chengdu, China
| University of Chinese Academy of Sciences, Beijing, China
¶ Forestry Survey and Design Research Institute of the Tibet Autonomous Region, Lhasa, China
# Chengdu Survey and Design Research Institute of China Electric Power Construction Group Co., Ltd., Chengdu, China
Open Access

Abstract

Multiple disciplines can help to discover cryptic species and resolve taxonomic confusions. The Asian horned toad genus Megophrys sensu lato as a diverse group was proposed to contain dozens of cryptic species. Based on molecular phylogenetics, morphology, osteology, and bioacoustics data, the species profiles of Megophrys toads in the eastern corner of Himalayas in Medog County, Tibet Autonomous Region, China was investigated. The results indicated that this small area harbored at least four Megophrys species, i.e., M. medogensis, M. pachyproctus, Megophrys zhoui sp. nov., and Megophrys yeae sp. nov., the latter two being described in this study. Additionally, the mitochondrial DNA trees nested the low-middle-elevation and high-elevation groups of M. medogensis into a monophyletic group, being in discordance with the paraphyletic relationship between them revealed in the nuclear DNA trees. The findings highlighted the underestimated biodiversity in Himalayas, and further indicated that the Megophrys toads here have been probably experienced complicated evolutionary history, for example, introgression between clades or incomplete lineage sorting and niche divergences in microhabitats. Anyway, it is urgent for us to explore the problems because these toads are suffering from increasing threats from human activities and climatic changes.

Keywords

Multiple data, taxonomy, molecular phylogenetic analyses, morphology, introgression

Introduction

Species are the basic units of biodiversity, and species taxonomy is central to biodiversity explorations, further contributing to evolutionary biology, conservation biology and other categories of biological studies (Queiroz 2007; Condon et al. 2008; Wheeler et al. 2012). Increasing numbers of studies have advocated integrative taxonomy mainly because the findings from different disciplines would improve rigor (Dayrat 2005; Pante et al. 2014; Gómez Daglio and Dawson 2019). Integrative taxonomy has strongly promoted the discovery of cryptic species either in the understudied taxa (Larsen 2001; Bickford et al. 2007; Burns et al. 2008; Yoder et al. 2005) or in well-studied biomes (Rissler and Apodaca 2007; Stockman and Bond 2007). It could also resolve the taxonomic confusions like through demonstration of conspecificity of described species (Petrusek et al. 2008; Seifert 2009). And finally, multiple disciplines may further bring out clues for understanding the evolutionary processes of species for example in cases of disagreement among disciplines (DeSalle and Giddings 1986; Degnan and Rosenberg 2009; Thielsch et al. 2017).

The Asian horned toad Megophrys sensu lato Kuhl and Van Hasselt, 1822 (Anura, Megophryidae Bonaparte, 1850) widely inhabit mountain forests in the tropical and subtropical regions of Asia, ranging from India to south-central China and south to the Sundas and the Philippines (Frost 2020). The generic classifications of the group have been controversial for a long time (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. 2016; Mahony et al. 2017; Liu et al. 2018; Frost 2020). Most recent phylogenetic studies, however, clustered all members of the group into a monophyletic group (Chen et al. 2016; Mahony et al. 2017; Liu et al. 2018; Li et al. 2018; Liu et al. 2020; Wang et al. 2020), which was defined as one big genus Megophrys sensu lato by Mahony et al. (2017). The genus currently contains 95 species, of which, noticeably, 39 species were discovered in this decade (Frost 2020; Liu et al. 2020; Wang et al. 2020). What’s more, molecular phylogenetic studies still put forward dozens of cryptic species in the group (Chen et al. 2016; Liu et al. 2018). Misleading taxonomic judgements without precise and adequate comparisons and insufficient field work often hinder the discovery of cryptic diversity in the group (Mahony et al. 2018; Liu et al. 2018). Hence, comprehensive examinations with multiple data (e.g., molecular phylogenetic, morphological, and bioacoustics data) are needed for describing new taxon and furtherly recognizing underestimated species diversity in this diverse group.

Himalaya Mountains holds high level of biodiversity, and with increasingly deep surveys, species diversity in this region was indicated to be much underestimated. For example, just in Medog County, Tibet Autonomous Region, China in the eastern corner of Himalayas, several new frog or toad species has been found in recent years (e.g., Jiang et al. 2012; Jiang et al. 2016a, b, c). In Medog County, two Megophrys species has been recorded, i.e., M. pachyproctus Huang, 1981 and M. medogensis Fei, Ye and Huang 1983. Nevertheless, for ca. four decades, there have been only incomplete morphological reports (e.g., Fei et al. 2009; Fei and Ye 2016) or separate molecular data for them (Chen et al. 2016; Liu et al. 2018) but no detailed evaluation on taxonomic profiles of their populations especially using multiple disciplines. According to the hypothesis “lots of cryptic species in Megophrys” (Chen et al. 2016; Liu et al. 2018), it is expected that the toad populations in this high-profile biodiversity hotspot may contain cryptic species.

In recent years, we conducted a series of field surveys in Medog County, Tibet Autonomous Region, China, and collected a series of specimens of Megophrys sensu lato. Based on molecular phylogenetic, morphological, osteological and bioacoustics data, we will explore the species composition of the Asian horned toad Megophrys in Medog County, Tibet Autonomous Region, China in the eastern corner of Himalayas. Our multiple-data comparisons proposed that the specimens contained two undescribed species. Herein we describe them as two new species.

Materials and methods

Sampling

A total of 50 Megophrys specimens was collected from nine sites in Medog County, Tibet Autonomous Region, China (Fig. 1; for voucher numbers see Table 1, Suppl. material 1: Tables S1, S2). The specimens were identified as four species, i.e., M. medogensis, M. cf. pachyproctus, and the two undescribed species (Megophrys zhoui sp. nov. and Megophrys yeae sp. nov.) based on morphology. Megophrys cf. pachyproctus was defined because the specimens were collected from the type locality of M. pachyproctus (a stream in Gelin village, Medog County), and they are morphologically similar to the holotype of M. pachyproctus although with some morphological differences. For caution, we regarded M. pachyproctus and M. cf. pachyproctus as two groups in the following analyses and descriptions. In addition, for comparison, we also divided M. medogensis specimens into two groups, i.e., high-elevation group (above ca. 2100 m a.s.l.) and low-middle-elevation group (500–1600 m). The high-elevation group contained five tadpoles collected from 80k and Gedang village, and the low-middle-elevation group contained five adult males, six adult females, and four tadpoles from the urban area of Medog town, Bari village, Beibeng village, Gelin village and Didong village (Fig. 1; Tables 1, Suppl. material 1: Tables S1, S2). Sex and maturity of each toad were determined by direct observation of advertisement calls or inspection of vocal sac openings and gonads. The tadpoles were identified based on their phylogenetic positions after representatives of the population with almost identical morphology were sequenced.

Table 1.

Sampling information and GenBank accession numbers of samples used in the molecular analyses.

ID Species Voucher number Locality 12S 16S COI RAG1 CXCR-4
1 M. medogensis low-middle elevation CIB022017061502 Gelin, Medog, Tibet, China / MN963236 MN964296 MN984365 /
2 CIB022017060801 Beibeng, Medog, Tibet, China / MN963245 MN964287 MN984356 /
3 CIB022017060904 Didong, Medog, Tibet, China / MN963244 MN964288 MN984357 /
4 CIB022017061808 Bari, Medog, Tibet, China / MN963225 MN964306 MN984376 /
5 CIB022017061810 Bari, Medog, Tibet, China / MN963223 MN964308 MN984378 /
6 CIB022017061801 Bari, Medog, Tibet, China / MN963230 MN964301 MN984371 /
7 CIB022017061604 Beibeng, Medog, Tibet, China / MN963232 MN964299 MN984369 /
8 CIB022017061601 Beibeng, Medog, Tibet, China / MN963235 MN964297 MN984366 /
9 CIB022017061602 Beibeng, Medog, Tibet, China / MN963234 MN964298 MN984367 /
10 CIB022017061603 Beibeng, Medog, Tibet, China / MN963233 / MN984368 /
11 CIB022017061501 Gelin, Medog, Tibet, China / MN963237 MN964295 MN984364 /
12 CIB022017061404DD Didong, Medog, Tibet, China / MN963240 MN964292 MN984361 /
13 CIB022017061406MT Suburb Medog, Tibet, China / MN963239 MN964293 MN984362 /
14 CIBMT1710104 Bari, Medog, Tibet, China / MN963212 MN964317 MN984385 /
15 CIBMT1710101 Yadong, vicinity of suburb Medog, Tibet, China / MN963213 MN964316 MN984384 /
16 KIZ06621 Beibeng, Tibet, China / KX811767 KX812082 KX812197 /
17 M. medogensis unknown elevation SYSa002932 Motuo, Tibet, China MH406458 MH406725 MH406177 MH404950 /
18 M. glandulosa SYSa003795 Jingdong County, Yunnan, China MH406493 MH406760 MH406219 MH404995 /
19 M. medogensis high elevation CIB022017062002 80K, Medog, Tibet, China / MN963219 MN964310 / /
20 CIB022017062003 80K, Medog, Tibet, China / MN963218 MN964311 / /
21 CIBMT1710106 Gutang (Gedang), Medog, Tibet, China / MN963211 MN964318 MN984386 /
22 CIBMT1710107 Gutang (Gedang), Medog, Tibet, China / MN963210 MN964319 MN984387 /
23 CIBMT1710112 80K, Medog, Tibet, China MN963176 MN963209 MN964320 MN984388 /
24 M. medogensis unknown elevation SYSa002934 Motuo, Tibet, China MH406459 MH406726 MH406178 MH404952 /
25 M. mangshanensis KIZ021786 Nanling National Forest Park, Guangdong, China / KX811790 KX812079 KX812194 /
26 M. maosonensis ROM 16679 Tam Dao, Vinh Phuc, Vietnam / KX811784 KX812081 KX812196 /
27 M. periosa BNHS 6055 [SDBDU 2009.793] 28°12'33.96"N, 94°59'10.02"E MH647522 MH647522 MH647529 MH647553 MH647537
28 M. himalayana BNHS 6050 [SDBDU 2009.1227] 27°4'56.52"N, 92°34'50.22"E MH647526 MH647526 / MH647554 MH647538
29 M. flavipunctata SDBDU 2009.297 East Khasi Hills dist, Meghalaya, India KY022307 KY022307 MH647536 KY022352 KY022330
30 M. robusta SDBDU 2011.1057 Darjeeling, West Bengal, India KY022314 KY022314 / KY022365 KY022343
31 M. oreocrypta SDBDU 2009.1104 West Garo Hills dist, Meghalaya, India KY022306 KY022306 / KY022351 KY022329
32 M. major SDBDU 2007.229 Kohima dist, Nagaland, India MH647514 MH647514 / MH647550 MH647540
33 M. zhangi KIZ014278 Zhangmu, Tibet, China / KX811765 KX812084 KX812200 /
34 M. monticola middle elevation SDBDU 2011.420 Darjeeling dist, West Bengal, India MH647510 MH647510 / KY022359 KY022337
35 M. monticola high elevation SDBDU 2011.1047 Darjeeling dist, West Bengal, India KY022312 KY022312 / KY022358 KY022336
36 ZSI11401 Kabi, North district, Sikkim, India / KX894667 / / /
37 M. lekaguli FMNH 265955 Pang Si Da, Sa Kaeo, Thailand KY022214 KY022214 / KY022241 KY022177
38 M. auralensis NCSM 79599 Aural, Kampong Speu, Cambodia / KX811807 / / /
39 M. takensis FMNH 261711 Khlong Lan National Park, Kampaeng, Thailand KY022215 KY022215 / KY022246 KY022183
40 M. cf. parva KIZ048507 Tongbiguan Nature Reserve, Yunnan, China / KX811796 KX812071 KX812180 /
41 M. zunhebotoensis RGK 0041 Nagaland, India KY022322 KY022322 / KY022367 KY022345
42 M. serchhipii SDBDU 2009.612 Tripura, India KY022323 KY022323 / KY022366 KY022344
43 M. ancrae SDBDU 2009.727 27°29.833'N 96°23.467'E KY022318 KY022318 / KY022350 KY022328
44 M. oropedion SDBDU 2009.299 Mawphlang, Mawphlang Sacred Forest, East Khasi Hills, Meghalaya, India KY022317 KY022317 / KY022360 KY022338
45 M. megacephala ZSI A 11213 East Khasi Hills, northern Meghalaya, India KY022315 KY022315 / KY022357 KY022335
46 M. aceras LSUHC 7038 Tremengor Forest, Perak, Peninsular Malaysia, Malaysia / GQ995534 / / /
47 M. longipes IABHU 21101 Genting highland, Malaysia / AB530656 / / /
48 Megophrys yeae sp. nov. CIB201706MT01 Didong, Medog, Tibet, China MN963172 MN963217 MN964312 MN984380 /
49 CIB201706MT02 Beibeng, Medog, Tibet, China MN963173 MN963216 MN964313 MN984381 /
50 CIB201706MT03 Suburb of Medog, Tibet, China MN963174 MN963215 MN964314 MN984382 /
51 CIB022017061102 Didong, Medog, Tibet, China MN963162 MN963243 MN964289 MN984358 /
52 CIB022017061407b Beibeng, Medog, Tibet, China MN963165 MN963238 MN964294 MN984363 MN984402
53 CIB022017061804 Bari, Medog, Tibet, China MN963167 MN963229 MN964302 MN984372 MN984403
54 CIB022017061809 Bari, Medog, Tibet, China MN963171 MN963224 MN964307 MN984377 /
55 CIB022017061811 Bari, Medog, Tibet, China / MN963222 MN964309 MN984379 /
56 CIB022017061103 Didong, Medog, Tibet, China MN963163 MN963242 MN964290 MN984359 /
57 CIB022017061104 Didong, Medog, Tibet, China MN963164 MN963241 MN964291 MN984360 /
58 CIB022017061606 Beibeng, Medog, Tibet, China MN963166 MN963231 MN964300 MN984370 /
59 CIBMT171064 Yadong, vicinity of suburb Medog, Tibet, China MN963187 MN963198 / MN984399 /
60 CIBMT171065 Yarang, Medog, Tibet, China MN963188 MN963197 / MN984400 /
61 CIBMT171066 Yarang, Medog, Tibet, China MN963189 MN963196 / MN984401 /
62 KIZ010978 Beibeng, Tibet, China / KX811908 KX812153 KX812265 /
63 KIZ011175 Beibeng, Tibet, China / KX811909 KX812154 KX812266 /
64 M. vegrandis SDBDU 2009.1272 /ZSI A 11605 27°06.067'N 92°31.642'E KY022305 KY022305 / KY022349 KY022327
65 Megophrys cf. pachyproctus CIBMT171053 Renqinbeng, Medog, Tibet, China MN963178 MN963207 MN964322 MN984390 /
66 CIBMT171060 Renqinbeng, Medog, Tibet, China MN963185 MN963200 MN964329 MN984397 /
67 CIBMT171062 Renqinbeng, Medog, Tibet, China MN963186 MN963199 MN964330 MN984398 /
68 CIB022017061813 Bari, Medog, Tibet, China / MN963220 / / /
69 CIBMT171054 Renqinbeng, Medog, Tibet, China MN963179 MN963206 MN964323 MN984391 /
70 CIBMT171052 Renqinbeng, Medog, Tibet, China MN963177 MN963208 MN964321 MN984389 /
71 CIB201706MT04 Bari, Medog, Tibet, China MN963175 MN963214 MN964315 MN984383 /
72 CIB022017061805 Bari, Medog, Tibet, China MN963168 MN963228 MN964303 MN984373 MN984404
73 CIB022017061806 Bari, Medog, Tibet, China MN963169 MN963227 MN964304 MN984374 MN984405
74 CIB022017061807 Bari, Medog, Tibet, China MN963170 MN963226 MN964305 MN984375 MN984406
75 CIB022017061812 Bari, Medog, Tibet, China / MN963221 / / /
76 Megophrys cf. pachyproctus CIBMT171055 Renqinbeng, Medog, Tibet, China MN963180 MN963205 MN964324 MN984392 /
77 CIBMT171056 Renqinbeng, Medog, Tibet, China MN963181 MN963204 MN964325 MN984393 /
78 CIBMT171057 Renqinbeng, Medog, Tibet, China MN963182 MN963203 MN964326 MN984394 /
79 CIBMT171058 Renqinbeng, Medog, Tibet, China MN963183 MN963202 MN964327 MN984395 /
80 CIBMT171059 Renqinbeng, Medog, Tibet, China MN963184 MN963201 MN964328 MN984396 /
81 M. xianjuensis CIBXJ190503 Xianju, Zhejiang, China / MN563758 MN563774 / /
82 M. lishuiensis WYF00169 Lishui, Zhejiang, China / KY021418 / / /
83 M. shunhuangensis HNNU16SH02 Shunhuang Mountains, Hunan, China MK836034 MK836037 / / /
84 M. brachykolos SYSa002258 Hong Kong, China MF667851 KJ560403 MH406120 MH404888 /
85 M. kuatunensis SYSa003449 Guadun, Fujian, China MF667850 MF667881 MH406206 MH404982 /
86 M. dongguanensis SYSa001971/ CIB110006 Mt. Yinping, Dongguan City, Guangdong, China / MK524097 MK524128 / /
87 M. nankunensis SYSa004498 Mt. Nankun, Huizhou City,Guangdong, China / MK524108 MK524139 / /
88 M. wugongensis SYSa002610 Wugongshan Scenic Area, Anfu County, Jiangxi, China / MK524114 MK524145 / /
89 M. ombrophila NJFU2015201 KRM15 Mt. Wuyi, Fujian, China KX856422 KX856401 / / /
90 M. obesa SYSa002271 Heishiding, Guangdong, China MH406410 KJ579121 MH406123 MH404891 /
91 M. lini SYSa002381 Mt. Jinggang, Jiangxi, China MF667842 MF667874 MH406135 MH404903 /
92 M. nanlingensis SYSa001959 Nanling Nature Reserve, Shaoguan City, Guangdong, China / MK524111 MK524142 / /
93 M. cheni SYSa002126 Taoyuandong, Hunan, China MH406389 MH406659 MH406096 MH404864 /
94 M. insularis SYSa002169 Nan’ao Island, Guangdong, China MH406393 MH406663 MH406103 MH404871 /
95 M. jinggangensis SYSa004824 Mt. Sifang, Hunan, China MH406590 MH406857 MH406319 MH405100 /
96 M. caudoprocta SYSa004281 Zhangjiajie, Hunan, China MH406528 MH406795 MH406257 MH405036 /
97 M. tuberogranulatus SYSa004310 Zhangjiajie, Hunan, China MH406534 MH406801 MH406263 MH405042 /
98 M. wushanensis SYSa003008 Mt. Wu, Hubei, China MH406465 MH406732 MH406184 MH404959 /
99 M. leishanensis SYSa002213 Mt. Leigong, Guizhou, China MH406403 MH406673 MH406113 MH404881 /
100 M. acuta SYSa002276 Heishiding, Guangdong, China MH406413 KJ579124 MH406126 MH404894 /
101 M. boettgeri SYSa004149 Mt. Wuyi, Fujian, China MF667847 MF667878 MH406247 MH405026 /
102 M. huangshanensis SYSa002703 Huangshan, Anhui, China MF667854 MF667883 MH406161 MH404929 /
103 M. liboensis GNUG20150813001 Libo Country, Guizhou, China MF285242 MF285253 / / /
104 M. jiulianensis SYSa002107 Mt. Jiulian, Ganzhou City, Jiangxi, China / MK524099 MK524130 / /
105 M. mufumontana SYSa006390 CIB110012 Mt. Mufu, Pingjiang County, Hunan, China / MK524104 MK524135 / /
106 M. baolongensis KIZ019216 Baolong, Chongqing, China / KX811813 KX812093 KX812202 /
107 M. sangzhiensis SYSa004306 Zhangjiajie, Hunan, China MH406530 MH406797 MH406259 MH405038 /
108 M. spinata SYSa002226 Mt. Leigong, Guizhou, China MH406405 MH406675 MH406115 MH404883 /
109 M. binlingensis SYSa005313 Wawu Shan, Sichuan, China MH406625 MH406892 MH406354 MH405137 /
110 M. wuliangshanensis SYSa003924 Mt. Wuliang, Yunnan, China MH406504 MH406771 MH406230 MH405007 /
111 M. jingdongensis SYSa003928 Mt. Wuliang, Yunnan, China MH406506 MH406773 MH406232 MH405009 /
112 M. daweimontis KIZ048997 Dawei Shan, Yunnan, China / KX811867 KX812125 KX812248 /
113 M. omeimontis KIZ025765 Emei Shan, Sichuan, China / KX811884 KX812136 KX812223 /
114 M. binchuanensis KIZ019441 Jizu Shan, Yunnan, China / KX811849 KX812112 KX812219 /
115 M. rubrimera AMS R177676 Sa Pa, Lao Cai, Vietnam / MF536419 / / /
116 M. jiangi CIBKKS20180722006 Kuankuosui Nature Reserve, Guizhou, China / MN107743 MN107748 / /
117 M. minor SYSa003209 Dujiangyan, Sichuan, China MF667825 MF667862 MH406194 MH404969 /
118 M. hansi AMCC 144729 Thua Tien Hue, A Luoi District, A Roang Commune, Viet Nam KY022204 KY022204 / KY022229 KY022165
119 M. microstoma KU KUH 311601 Shiwan Dashang Nature Reserve, Guangxi, China KY022200 KY022200 / KY022234 KY022170
120 M. gerti AMCC 106456 Quang Nam, Tra My Dist., Tra Don Commune, Viet Nam KY022201 KY022201 / KY022231 KY022167
121 M. synoria FMNH 262778 Mondolkiri, Cambodia KY022198 KY022198 / KY022235 KY022171
122 M. elfina ZMMU NAP-02658 Chu Pan Fan Mt, Chu Yang Sin N.P., Dak Lak Prov., Vietnam KY425389 KY425389 / / /
123 M. palpebralespinosa FMNH 258098 Phou Dendin National Biodiversity Conservation Area, Phongsaly, Laos KY022209 KY022209 / KY022238 KY022174
124 M. intermedia FMNH 258093 Xe Kong, Kaleum District, Xe Sap National Biodiversity Conservation Area, Laos KY022196 KY022196 / KY022221 KY022157
125 M. carinense CAS 243791 Khotama Camp, Yephyu, Dawei, Tanintharyi, Myanmar KY022197 KY022197 / KY022219 KY022155
126 M. lancip MZB:Amp:22233 Ngarip, Ulubelu, Lampung, Sumatra, Indonesia / KY679891 / / /
127 M. montana LSUMZ 81916 Sukabumi, Java, Indonesia / KX811927 KX812163 KX812281 /
128 M. chuannanensis SYSa004926 Hejiang County, Sichuan, China MH406635 MH406901 MH406364 MH405147 /
129 M. feae SYSa003912 Jingdong County, Yunnan, China MH406633 MH406899 MH406362 MH405145 MH450011
130 M. popei SYSa001864 Taoyuandong, Hunan, China MH406632 KM504256 MH406361 MH405144 /
131 M. gigantica SYSa003883 Ailao Shan, Yunnan, China MH406499 MH406766 MH406225 MH405001 MH450010
132 M. wawuensis SYSa005311 Wawu Shan, Sichuan, China MH406624 MH406891 MH406353 MH405136 /
133 M. nankiangensis CIB ZYC517 Nanjiang, Sichuan, China / KX811900 / / /
134 M. shapingensis KIZ014512 Liziping Nature Reserve, Sichuan, China / KX811904 KX812060 KX812274 /
135 M. dringi UNIMAS 8948 Gunung Mulu, Sarawak, Malaysia / KJ831316 / / /
136 M. nasuta MBH 5357 Bengkulu, Sumatra, Indonesia KY022185 KY022185 / KY022225 KY022161
137 M. kalimantanensis FMNH 236525 Crocker Range National Park, Tenom Dist, Sabah, Borneo, Malaysia DQ283342 DQ283342 / / /
138 M. kobayashii UNIMAS 8148 Gunung Kinabalu National Park, Sabah, Malaysia / KJ831313 / / /
139 M. baluensis voucher not preserved Kinabalu, Borneo DQ642146 DQ642121 / / /
140 M. stejnegeri KU 314303 Pasonanca Natural Park, Zamboanga City, Philippines / KX811922 KX812052 KX812172 /
141 M. edwardinae FMNH 273694 Bintulu, Sarawak, Malaysia / KX811918 KX812050 KX812168 /
142 M. ligaya ZMMU NAP-05015 Palawan, Philippines / KX811919 KX812051 KX812169 /
143 Leptolalax alpinus SYSa003927 Jingdong County, Yunnan, China MH406639 MH406905 MH406368 MH405151 /
144 Leptobrachium cf. rakhinensis SDBDU 2009.49 Trishna Wildlife Sanctuary, South Dist, Tripura state, India KY022304 KY022304 / KY022347 KY022325
Figure 1. 

Distributional localities for specimens of the Megophrys species used in this study in Medog County, Tibet Autonomous Region, China. 1 80k 2 Gedang village 3 vicinity of Medog urban area 4 Bari village 5 vicinity of Renqingbeng Temple 6 Beibeng village 7 Gelin village 8 Didong village 9 Yarang village. Species were denoted as different color.

In the field, after taking photographs, the toads and tadpoles were euthanized using isoflurane, and then the specimens were fixed in 75% ethanol. Tissue samples were taken and preserved separately in 95% ethanol prior to fixation. Specimens collected in this work were deposited in Chengdu Institute of Biology, Chinese Academy of Sciences (CIB, CAS). The Animal Care and Use Committee of Chengdu Institute of Biology, CAS provided full approval for this research (Number: CIB2016012301). Field work was approved by the Management Office of the Administration of Yarlung Zangbo Grand Canyon National Nature Reserve (YLZB000342).

Molecular phylogenetic analyses

Total genomic DNA was extracted from each specimen collected in this study using QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany), following manufacturer instructions. Three mitochondrial genes (12S rRNA, 16S rRNA, and COI) and two nuclear protein-coding genes (RAG1 and CXCR-4) were amplified and sequenced. Primer sequences were retrieved from literatures for 12S (Sumida et al. 2000), 16S (Simon et al. 1994), COI (Che et al. 2011), RAG1 (Mauro et al. 2004; Fu et al. 2007), and CXCR-4 (Biju and Bossuyt 2003) genes. PCR amplifications for mitochondrial genes were performed in a 30 μl volume reaction with the following conditions: an initial denaturing step at 95 °C for 4 min; 36 cycles of denaturing at 95 °C for 40 s, annealing at 55 °C (for 12S and 16S)/52 °C (for COI) for 40 s and extending at 72 °C for 70 s, and a final extending step of 72 °C for 10 min. Amplifications of nuclear genes were according to Mahony et al. (2017). PCR products were sequenced with both forward and reverse primers same as used in PCR. Sequencing was conducted using an ABI3730 automated DNA sequencer in Sangon Biotechnologies Co., Ltd. (Shanghai, China). New sequences were uploaded to GenBank (see Table 1).

For phylogenetic comparisons, corresponding sequences of Megophrys species were downloaded from GenBank especially for their holotypes and/or topotypes for which comparable sequences were available (Table 1). Corresponding sequences of one Leptobrachium rakhinensis and one Leptobrachella khasiorum (Table 1) were also downloaded and used as outgroups according to previous studies (Mahony et al. 2017; Chen et al. 2016).

Sequences were assembled and aligned using BioEdit v. 7.0.9.0 (Hall 1999) with default settings, and were further revised manually if necessary. To avoid bias in alignments, GBLOCKS v. 0.91.b (Castresana 2000) with default settings was used to extract regions of defined sequence conservation from the length-variable 12S and 16S fragments. The protein-coding gene (COI, RAG1, and CXCR-4) sequences were translated to amino acid sequences in MEGA v. 7.0 (Kumar et al. 2016), adjusted for open reading frames, and checked to ensure absence of premature stop codons. No-sequenced fragments were treated as missing data. At last, for phylogenetic analyses, two datasets were obtained, i.e., three-mitochondrial genes concatenated dataset of 12S+16S+COI and two-nuclear genes concatenated dataset of RAG1+CXCR-4.

Phylogenetic analyses were conducted on each dataset using maximum likelihood (ML) and Bayesian Inference (BI) methods, implemented in PhyML v. 3.0 (Guindon et al. 2010) and MrBayes v. 3.2 (Ronquist et al. 2012), respectively. For the phylogenetic analyses, each gene was regarded as one partition, and the best evolutionary model for each partition were chosen under the Bayesian Inference Criteria (BIC) using jModelTest v. 2.1.3 (Darriba 2012). The analyses selected GTR + I + G model for each mitochondrial gene, and HKY + I for each nuclear gene. For the ML tree, branch supports were drawn from 10000 non-parametric bootstrap replicates. In BI analyses, the parameters for each partition were unlinked, and branch lengths were allowed to vary proportionately across partitions. Two runs each with four Markov chains were simultaneously run for 80 million generations with sampling every 1000 generations. The first 25% of trees were removed as the “burn-in” stage followed by calculations of Bayesian posterior probabilities at stationarity, and the 50% majority-rule consensus of the post burn-in trees sampled. Finally, genetic distance between species with uncorrected p-distance model on the 16S gene was estimated using MEGA.

Morphological analyses

In total, 38 adult specimens of four species (the two undescribed species, M. medogensis, and M. cf. pachyproctus) were measured (Suppl. material 1: Table S1). The terminology and methods followed Mahony (2011). Measurements were taken with a dial caliper to the nearest 0.1 mm. Twenty-two characters of adult specimens were measured:

EL eye length (horizontal distance between the anterior and posterior borders of orbit);

EN eye-nostril length (distance from front of eye to the center of nostril);

FAL forearm length (distance from elbow to wrist);

FIIIW finger III width (largest width of tip of finger III);

FIVW finger IV width (largest width of tip of finger IV);

FOL foot length (distance from the proximal end of the inner metatarsal tubercle to the tip of the fourth digit);

HAL hand length (distance from wrist to tip of third digit);

HL head length (distance from the rear of the mandible to the tip of the snout);

HLL hindlimb length;

HW head width (distance between the posterior angles of jaw);

IBE internal back of eyes (the shortest distance between the posterior borders of the orbits);

IFE internal front of eyes (shortest distance between the anterior borders of orbits);

IMT ength of the inner metatarsal tubercle;

IN internarial distance (shortest distance between two nostrils);

IUE inter upper eyelid width (shortest distance between upper eyelids);

SHL shank length (distance from knee to ankle);

SL snout length (distance from tip of snout to anterior border of the orbit);

SN nostril-snout length (distance from center of the nostril to tip of the snout);

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

TFOL tarsal-foot length (distance from heel to the tip of the fourth digit);

TL thigh length (distance from cloaca to knee);

TYD largest tympanum diameter;

TYE tympanum-eye distance (distance from the anterior border of the tympanum to the posterior orbital border);

UEW maximum upper eyelid width.

Thirteen tadpoles of four groups (i.e., Megophrys yeae sp. nov., M. cf. pachyproctus, and two elevation groups of M. medogensis) were measured (Suppl. material 1: Table S2). The stages of tadpoles were identified following Gosner (1960). Seventeen morphometric characters of tadpoles were measured:

BH maximum body height;

BL body length (distance from tip of snout to trunk-tail junction);

BW maximum body width;

ED maximum eye diameter;

IND internasal distance (distance between center of two naris);

LF maximum height of lower tail fin;

NE naris-eye distance (distance from center of naris to anterior corner of eye);

ODW oral disc width (largest width of oral disc);

PP interpupilar distance;

RN rostro-narial distance (distance from tip of snout to center of naris);

SS snout-spiracle distance (distance from tip of snout to opening of spiracle);

SU snout-upper fin distance (distance from snout to beginning of upper tail fin);

TAL tail length (distance between posterior side of opening of cloaca to tip of tail);

TMH maximum tail muscle width;

TMW maximum tail muscle height;

TOL total length;

UF maximum height of upper tail fin.

For morphometric comparisons, the corresponding morphometric data of the holotype and two topotypes of M. vegrandis were retrieved from Mahony et al. (2013), and that of the allotype and one paratype of M. pachyproctus from Huang and Fei (1981). To reduce the impact of allometry, the correct value from the ratio of each measurement to SVL was calculated and then log-transformed for the following morphometric analyses. Mann-Whitney U test was used to test the significance of difference on each character between different species in each gender group. In the analyses for male group, 13 characters of 28 individuals of five species (Megophrys yeae sp. nov., M. cf. pachyproctus, M. pachyproctus, M. medogensis, and M. vegrandis) were included, and for female, 26 characters of 13 individuals of four species (Megophrys yeae sp. nov., Megophrys zhoui sp. nov., M. cf. pachyproctus, and M. medogensis) were included. The significance level was set at 0.05. The analyses were carried out in R (R Development Core Team 2008).

The two undescribed species were compared with each other as well with other congeners of Megophrys sensu lato on morphology. Comparative morphological data were obtained from literatures (Table 2). In addition, the holotype of M. pachyproctus and topotypes of M. medogensis were also examined for comparisons (Suppl. material 1: Tables S1, S2).

Table 2.

References utilized for morphological characters of congeners of the genus Megophrys.

No. Species Literature obtained
1 Megophrys aceras Boulenger, 1903 Bourret 1942; Munir et al. 2018
2 Megophrys acuta Wang, Li, and Jin, 2014 Li et al. 2014
3 Megophrys ancrae Mahony, Teeling, and Biju, 2013 Mahony et al. 2013
4 Megophrys angka Wu, Suwannapoom, Poyarkov, Chen, Pawangkhanant, Xu, Jin, Murphy, and Che, 2019 Wu et al. 2019
5 Megophrys auralensis Ohler, Swan, and Daltry, 2002 Ohler et al. 2002
6 Megophrys baluensis Boulenger, 1899 Boulenger 1899, 1908
7 Megophrys baolongensis Ye, Fei, and Xie, 2007 Ye et al. 2007; Fei and Ye 2016
8 Megophrys binchuanensis Ye and Fei, 1995 Ye et al. 1995; Fei and Ye 2016
9 Megophrys binlingensis Jiang, Fei, and Ye, 2009 Fei et al. 2009
10 Megophrys boettgeri Boulenger, 1899 Boulenger 1908; Fei et al. 2009
11 Megophrys brachykolos Inger and Romer, 1961 Inger et al. 1961; Fei et al. 2009; Li et al. 2014
12 Megophrys carinense Boulenger, 1889 Boulenger 1908; Bourret 1942
13 Megophrys caudoprocta Shen, 1994 Shen 1994; Shen et al. 2013
14 Megophrys cheni Wang and Liu, 2014 Wang et al. 2014
15 Megophrys chuannanensis Fei, Ye, and Huang, 2001 Fei and Ye 2001; Fei et al. 2009
16 Megophrys damrei Mahony, 2011 Mahony 2011
17 Megophrys daweimontis Rao and Yang, 1997 Rao and Yang 1997; Fei and Ye 2016
18 Megophrys dongguanensis Wang and Wang, 2019 Wang et al. 2019
19 Megophrys dringi Inger, Stuebing, and Tan, 1995 Inger et al. 1995; Oberhummer et al. 2014
20 Megophrys edwardinae Inger, 1989 Inger et al. 1989
21 Megophrys elfina Poyarkov, Duong, Orlov, Gogoleva, Vassilieva, Nguyen, Nguyen, Nguyen, Che, and Mahony, 2017 Poyarkov et al. 2017
22 Megophrys fansipanensis Tapley, Cutajar, Mahony, Nguyen, Dau, Luong, Le, Nguyen, Nguyen, Portway, Luong, and Rowley, 2018 Tapley et al. 2018
23 Megophrys feae Boulenger, 1887 Boulenger 1908; Fei et al. 2009
24 Megophrys feii Yang, Wang, and Wang, 2018 Yang et al. 2018
25 Megophrys flavipunctata Mahony, Kamei, Teeling, and Biju, 2018 Mahony et al. 2018
26 Megophrys gerti Ohler, 2003 Poyarkov et al. 2017
27 Megophrys gigantica Liu, Hu, and Yang, 1960 Liu et al. 1960; Fei et al. 2009
28 Megophrys glandulosa Fei, Ye, and Huang, 1990 Fei et al. 1990; Fei et al. 2009; Fei and Ye 2016
29 Megophrys hansi Ohler, 2003 Ohler 2003
30 Megophrys himalayana Mahony, Kamei, Teeling, and Biju, 2018 Mahony et al. 2018
31 Megophrys hoanglienensis Tapley, Cutajar, Mahony, Nguyen, Dau, Luong, Le, Nguyen, Nguyen, Portway, Luong, and Rowley, 2018 Tapley et al. 2018
32 Megophrys huangshanensis Fei and Ye, 2005 Fei and Ye 2005, 2016; Fei et al. 2009
33 Megophrys insularis Wang, Liu, Lyu, Zeng, and Wang, 2017 Wang et al. 2017a
34 Megophrys intermedia Smith, 1921 Smith 1921
35 Megophrys Jiangi Liu, Li, Wei, Xu, Cheng, Wang and Wu, 2020 Liu et al. 2020
36 Megophrys jingdongensis Fei and Ye, 1983 Fei at al. 1983, 2009; Fei and Ye 2016
37 Megophrys jinggangensis Wang, 2012 Wang et al. 2012
38 Megophrys jiulianensis Wang, Zeng, Lyu, and Wang, 2019 Wang et al. 2019
39 Megophrys kalimantanensis Munir, Hamidy, Matsui, Iskandar, Sidik, and Shimada, 2019 Munir et al. 2019
40 Megophrys kobayashii Malkmus and Matsui, 1997 Malkmus and Matsui 1997
41 Megophrys koui Mahony, Foley, Biju, and Teeling, 2017 Yang 1991
42 Megophrys kuatunensis Pope, 1929 Pope 1929; Fei et al. 2009; Tapley et al. 2017
43 Megophrys lancip Munir, Hamidy, Farajallah, and Smith, 2018 Munir et al. 2018
44 Megophrys leishanensis Li, Xu, Liu, Jiang, Wei, and Wang, 2019 “2018” Li et al. 2018a
45 Megophrys lekaguli Stuart, Chuaynkern, Chan-ard, and Inger, 2006 Stuart et al. 2006a
46 Megophrys liboensis Zhang, Li, Xiao, Li, Pan, Wang, Zhang, and Zhou, 2017 Zhang et al. 2017
47 Megophrys ligayae Taylor, 1920 Taylor 1920
48 Megophrys lini Wang and Yang, 2014 Wang et al. 2014
49 Megophrys lishuiensis Wang, Liu and Jiang, 2017 Wang et al. 2017b
50 Megophrys longipes Boulenger, 1886 Boulenger 1908; Bourret 1942
51 Megophrys major Boulenger, 1908 Boulenger 1908
52 Megophrys mangshanensis Fei and Ye, 1990 Fei et al. 1990; Fei and Ye 2016
53 Megophrys maosonensis Bourret, 1937 Bourret 1942
54 Megophrys medogensis Fei, Ye, and Huang, 1983 Fei at al. 1983, 2009; Fei and Ye 2016; This paper
55 Megophrys megacephala Mahony, Sengupta, Kamei, and Biju, 2011 Mahony et al. 2011
56 Megophrys microstoma Boulenger, 1903 Fei et al. 2009
57 Megophrys minor Stejneger, 1926 Stejneger 1926; Li et al. 2014; Fei and Ye 2016
58 Megophrys montana Kuhl and Van Hasselt, 1822 Munir et al. 2018
59 Megophrys monticola Günther, 1864 Mahony et al. 2018
60 Megophrys mufumontana J. Wang, Lyu, and Y.Y. Wang, 2019 Wang et al. 2019
61 Megophrys nankiangensis Liu and Hu, 1966 Fei et al. 2009
62 Megophrys nankunensis Wang, Zeng, and. Wang, 2019 Wang et al. 2019
63 Megophrys nanlingensis Lyu, J. Wang, Liu, and Y.Y. Wang, 2019 Wang et al. 2019
64 Megophrys nasuta Schlegel, 1858 Mahony et al. 2018
65 Megophrys obesa Wang, Li, and Zhao, 2014 Li et al. 2014
66 Megophrys ombrophila Messenger and Dahn, 2019 Messenger et al. 2019
67 Megophrys omeimontis Liu, 1950 Fei et al. 2009; Fei and Ye 2016
68 Megophrys oreocrypta Mahony, Kamei, Teeling, and Biju, 2018 Mahony et al. 2018
69 Megophrys oropedion Mahony, Teeling, and Biju, 2013 Mahony et al. 2013
70 Megophrys pachyproctus Huang, 1981 Huang and Fei 1981 Huang et al. 1998; This paper
71 Megophrys palpebralespinosa Bourret, 1937 Bourret 1942
72 Megophrys parallela Inger and Iskandar, 2005 Inger and Iskandar 2005
73 Megophrys parva Boulenger, 1893 Boulenger 1908; Deuti et al. 2017
74 Megophrys periosa Mahony, Kamei, Teeling, and Biju, 2018 Mahony et al. 2018
75 Megophrys popei Zhao, Yang, Chen, Chen, and Wang, 2014 Zhao et al. 2014
76 Megophrys robusta Boulenger, 1908 Boulenger 1908; Mahony et al. 2018
77 Megophrys rubrimera Tapley, Cutajar, Mahony, Chung, Dau, Nguyen, Luong, and Rowley, 2017 Tapley et al. 2017
78 Megophrys sangzhiensis Jiang, Ye, and Fei, 2008 Jiang et al. 2008
79 Megophrys serchhipii Mathew and Sen, 2007 Mathew and Sen 2007
80 Megophrys shapingensis Liu, 1950 Liu et al. 1950; Fei et al. 2009
81 Megophrys shuichengensis Tian and Sun, 1995 Tian and Sun 1995; Fei et al. 2009
82 Megophrys shunhuangensis Wang, Deng, Liu, Wu, and Liu, 2019 Wang et al. 2019a
83 Megophrys spinata Liu and Hu, 1973 Fei et al. 2009; Fei and Ye 2016
84 Megophrys stejnegeri Taylor, 1920 Taylor 1920
85 Megophrys synoria Stuart, Sok, and Neang, 2006 Stuart et al. 2006b
86 Megophrys takensis Mahony, 2011 Mahony 2011
87 Megophrys tuberogranulata Shen, Mo and Li, 2010 Mo et al. 2010
88 Megophrys vegrandis Mahony, Teeling, Biju, 2013 Mahony et al. 2013
89 Megophrys wawuensis Fei, Jiang, and Zheng, 2001 Fei et al. 2009
90 Megophrys wugongensis J. Wang, Lyu, and Y.Y. Wang, 2019 Wang et al. 2019
91 Megophrys wuliangshanensis Ye and Fei, 1995 Fei et al. 2009; Fei and Ye 2016
92 Megophrys wushanensis Ye and Fei, 1995 Fei et al. 2009; Fei and Ye 2016
93 Megophrys xianjuensis Wang, Wu, Peng, Shi, Lu and Wu, 2020 Wang et al. 2020
94 Megophrys zhangi Ye and Fei, 1992 Ye and Fei 1992; Fei et al. 2009; Fei and Ye 2016
95 Megophrys zunhebotoensis Mathew and Sen, 2007 Mathew and Sen 2007

Bioacoustics

We recorded advertisement calls of three species: six males (CIB022017061804, CIB022017061101–CIB022017061103, CIBMT171064, and one unvouchered individual) of Megophrys yeae sp. nov., three males (CIB022017061805–CIB022017061807) of M. cf. pachyproctus, and three unvouchered males of M. medogensis (Suppl. material 1: Table S3). Each calling individual was recorded at a distance between 0.5–1.0 m using a Philip VTR6900 digital voice recorder with a build-in microphone with sampling rate 96 kHz. Temperature was recorded using HTC-1 hygrothermograph. All callings were recorded between a relatively concentrated temperature range of 17–25 °C. Calls were analyzed using Raven Pro© v.1.5 beta software (http://www.birds.cornell.edu/raven) with fast-Fourier transform (FFT) of 512 points, 50% overlap, and 188 Hz grid-spacing using Hanning windows. Sonograms and spectrograms were presented in figures using Praat (Boersma 2001) after de-noised using Audition 3. Terminology of advertisement call analyses and description followed Köhler et al. (2017). Call duration (ms), intercall interval (ms), number of calls per call group, call repetition rate (calls/s), number of pulses per call, and dominant frequency (kHz) were applied in measurement. To compare acoustic characteristics between the species, one-way ANOVA was conducted with LSD post hoc.

Skull scanning. The holotype CIB201706MT02 of Megophrys yeae sp. nov., holotype CIBMT171053 of Megophrys zhoui sp. nov., and the adult male CIB022017061805 of M. cf. pachyproctus were scanned. For comparisons, the holotype NWIPB 770650 of M. pachyproctus and the adult male topotype CIB022017061406 of M. medogensis were also scanned. In the high-resolution X-ray scanner (Quantum GX micro-CT Imaging System, PerkinElmer®), the specimens were scanned along the coronal axis at an image resolution of 1024× 1024 pixels. Segmentation and three-dimensional reconstruction of the CT images were made using VG57 Studio Max 2.2 (Volume Graphics, Heidelberg, Germany). Terminology of skull description follows Fei and Yei (2016).

Results

Phylogenetic analyses

Aligned sequence matrix of mitochondrial DNA and nuclear DNA contained 2890 bp and 2058 bp, respectively. ML and BI analyses based on the mitochondrial DNA matrix resulted in essentially consistent topologies (Fig. 2A), and all analyses on nuclear DNA matrix also obtained generally consistent topologies (Fig. 2B), though some relationships were not r|solved in these trees.

Figure 2. 

Phylogenetic trees respectively based on the mitochondrial DNA and nuclear DNA. A Maximum Likelihood (ML) tree based on the mitochondrial DNA B ML tree based on the nuclear DNA. ML bootstrap support/Bayesian posterior probability was denoted beside node. Samples 1–144 refer to Suppl. material 1: Table S1.

All samples of Megophrys sensu lato were strongly clustered into a clade in all trees. In all trees, each of the two new species was well supported as an independent clade, and all of them were then clustered into a big clade also containing M. cf. pachyproctus and M. vegrandis. In all trees, in this clade, M. cf. pachyproctus was indicated to be at the basal position. In mitochondrial DNA trees, the relationships of other three species were supported as (Megophrys zhoui sp. nov. (M. vegrandis, Megophrys yeae sp. nov.)), but in nuclear DNA trees, as (M. vegrandis (Megophrys yeae sp. nov., Megophrys zhoui sp. nov.)). This clade with the four species was phylogenetically far from the clade containing all samples of M. medogensis in all trees. As note, in nuclear DNA trees, M. medogensis was resolved as a monophyletic group because the high-elevation and low-middle-elevation groups of M. medogensis were nested into one clade, but in mitochondrial DNA trees, the low-middle-elevation group of M. medogensis was clustered as a clade sister to M. robusta, being paraphyly with the clade in comprising of the high-elevation group of M. medogensis.

Genetic distance among samples of each new species is below 0.4%, much lower than the interspecific distance of Megophrys (mean: 10.5%; range: 0.8%–26.1%; Suppl. material 1: Table S4). Genetic distance between Megophrys zhoui sp. nov. and other congeners was at least 4.0% (Megophrys zhoui sp. nov. vs. M. vegrandis), and that between Megophrys yeae sp. nov. and other congeners was at least 5.4% (Megophrys yeae sp. nov. vs. M. vegrandis). As note, genetic distance between the low-middle-elevation and high-elevation groups of M. medogensis was 5.0% on 16S gene. These values were much higher than interspecific genetic distance between many pairs of Megophrys species (Suppl. material 1: Table S4).

Morphological analyses

On many morphometric characters, the two new species were significantly different from each other as well from M. vegrandis, M. medogensis, and M. pachyproctus (Table 3). In male, ten characters were significantly different at least between one pair of species, i.e., SVL, HL, SL, EL, UEW, TYD, FAL, HAL, SHL, and FOL (all P-values < 0.05; Table 3); and in female, 14 characters were significant different at least between one pair of species, i.e., SVL, HW, HL, SL, IFE, IBE, TYD, TYE, FAL, HAL, FIL, FIIIL, FIIIW, and FIVW (all P-values < 0.05; Table 3).

Table 3.

Morphometric comparisons between the Megophrys species from the eastern corner of Himalayas. P-value is resulted from Mann-Whitney U test on each character between species. Significant level at 0.05 (* P-value < 0.05). Abbreviation for species name: MCP, M. cf. pachyproctus; MZ, Megophrys zhoui sp. nov.; MY, Megophrys yeae sp. nov.; MP, M. pachyproctus; MM, M. medogensis; and MV, M. vegrandis. See abbreviations for the morphological characters in Materials and methods section.

Sex Character MCP vs. MY MCP vs. MZ MCP vs. MM MCP vs. MP MCP vs. MV MZ vs. MY MZ vs. MM MY vs. MP MY vs. MM MP vs. MV MM vs. MV
Female SVL 0.133 0.133 0.016* / / 0.333 0.095 / 0.095 / /
HW 0.133 0.267 0.016* / / 0.333 0.095 / 0.095 / /
HL 1.000 0.267 0.032* / / 1.000 0.571 / 0.190 / /
SL 0.533 0.267 0.032* / / 0.333 0.095 / 1.000 / /
SN 1.000 0.267 1.000 / / 0.333 0.190 / 0.857 / /
EN 0.800 1.000 0.286 / / 1.000 0.570 / 1.000 / /
IN 1.000 0.267 0.286 / / 1.000 1.000 / 0.857 / /
EL 0.133 1.000 1.000 / / 0.667 1.000 / 0.095 / /
IUE 0.533 0.533 0.111 / / 1.000 1.000 / 1.000 / /
UEW 0.533 0.533 0.730 / / 1.000 1.000 / 1.000 / /
IFE 0.800 0.267 0.111 / / 1.000 0.950 / 0.571 / /
IBE 0.133 0.133 1.000 / / 0.667 0.190 / 0.190 / /
TYD 0.533 0.133 0.032* / / 0.333 0.095 / 0.095 / /
TYE 0.800 0.133 0.016* / / 0.667 0.095 / 0.095 / /
FAL 0.133 0.800 0.286 / / 0.333 0.381 / 0.095 / /
HAL 0.133 1.000 0.016* / / 0.333 0.095 / 0.095 / /
FIL 1.000 0.133 0.016* / / 0.333 0.095 / 0.095 / /
FIIL 0.133 0.533 0.016* / / 0.333 0.095 / 0.381 / /
FIIIL 0.133 0.267 0.286 / / 0.333 1.000 / 0.095 / /
FIVL 1.000 0.533 0.730 / / 0.333 0.381 / 0.381 / /
TL 0.133 0.533 0.413 / / 0.333 0.571 / 1.000 / /
SHL 0.533 0.800 0.730 / / 0.333 0.857 / 0.857 / /
TFOL 1.000 0.133 0.730 / / 0.333 0.857 / 1.000 / /
FOL 1.000 0.267 1.000 / / 0.333 0.571 / 1.000 / /
FIIIW 0.133 0.133 0.556 / / 0.333 0.095 / 0.095 / /
FIVW 0.133 0.133 0.730 / / 0.333 0.095 / 0.095 / /
Male SVL 0.001* / 0.476 0.533 0.029* / / / 0.005* 0.643 0.038*
HW 0.446 / 1.000 0.533 0.486 / / / 0.180 0.286 0.067
HL 0.599 / 0.038* 1.000 0.057 / / / 0.005* 0.143 0.171
SL 0.521 / 0.610 0.533 0.200 / / / 0.125 0.286 0.067
IN 0.262 / 0.257 1.000 0.686 / / / 1.000 1.000 0.352
EL 0.262 / 0.380 0.533 0.886 / / / 0.000* 0.710 0.670
UEW 0.133 / 0.190 1.000 0.029* / / / 0.180 0.710 0.010*
TYD 0.262 / 0.380 0.533 0.343 / / / 0.018* 1.000 0.010*
FAL 0.002* / 0.010* 1.000 0.029* / / / 0.000* 0.710 0.010*
HAL 0.133 / 0.010* 0.800 0.029* / / / 0.000* 0.710 0.010*
SHL 0.684 / 0.010* 0.533 0.343 / / / 0.102 0.710 0.914
TFOL 0.212 / 0.171 1.000 0.343 / / / 0.964 0.643 0.171
FOL 0.020* / 1.000 0.533 0.886 / / / 0.007* 1.000 0.762

On morphology, the two new species could be identified from each other as well as from their congeners by a series of characters (for morphological differences between the five groups of Megophrys species from Medog County see Suppl. material 1: Table S5; Fig. 3). Detailed comparisons on morphological characters between the new species and other congeners were demonstrated in detail in the sections for describing the new species.

Figure 3. 

Photos of specimens of Megophrys species in Medog. A–E dorsal views of adult male holotype NWIPB770650 of M. pachyproctus, adult male topotype CIB022017061406 of M. medogensis, adult male CIB022017061805 of M. cf. pachyproctus, adult male holotype CIBMT171053 of Megophrys zhoui sp. nov., and adult male holotype CIB201706MT02 of Megophrys yeae sp. nov., respectively F–J ventral views of the specimens, respectively K–O lateral view of head of the specimens, respectively P–T ventral view of hand of the specimens, respectively U–Y ventral view of foot of the specimens, respectively. Scale bar for body view equal to 10 mm, and for partial view 5 mm.

Bioacoustics comparisons. The advertisement calls of Megophrys yeae sp. nov., M. cf. pachyproctus, and M. medogensis were obviously different (Fig. 4; Tables 4; Suppl. material 1: Table S3). Megophrys yeae sp. nov. vocalizes continuous fast short calls in high-frequency, M. medogensis vocalizes sparse relatively deep calls in moderate speed, and as for M. cf. pachyproctus, the calls are moderate in frequency and repetition rate, but distinctly longer (call duration 491–889 ms) than the former two species (Fig. 4; Tables 4; Suppl. material 1: Table S3).

Table 4.

Comparisons of advertisement calls between three Megophrys species in Medog. P-value is resulted from Mann-Whitney U test on each character between species. Significant level at 0.05 (* P-value < 0.05). Abbreviation for species names: MCP, M. cf. pachyproctus; MY, Megophrys yeae sp. nov.; and MM, M. medogensis.

Call character MCP MY MM P-value
Mean ± SD (range) Mean ± SD (range) Mean ± SD (range) MH vs. MY MH vs. MM MY vs. MM
Number of individuals 6 3 3 / / /
Total number of calls analyzed 3.2 ± 2.6 (1–8) 5.0 ± 2.6 (2–7) 5.3 ± 3.5 (2–9) / / /
Call repetition rate (calls/s) 3.0 ± 0.7 (1.9–4.1) 0.9 ± 0.2 (0.7–1.1) 1.2 ± 0.9 (0.6–2.2) 0.024* 1 0.048*
Calls/call group 68.9 ± 46.7 (10.3–109.3) 10.8 ± 3.3 (7.1–13.3) 4.3 ± 1.6 (2.8–6.0) 0.229 0.1 0.057
Call duration (ms) 139 ± 39 (99–212) 746 ± 221 (491–889) 176 ± 61 (121–241) 0.024* 0.1 0.381
Intercall interval (ms) 218 ± 81 (146–370) 580 ± 122 (493–720) 205 ± 514 (153–254) 0.024* 0.1 0.905
Pulses/call 9.2 ± 0.6 (8.5–9.9) 42.1 ± 2.0 (40.6–44.4) 17.1 0.024* 0.5 0.286
Dominant frequency (kHz) 4.7 ± 0.3 (4.4–5.2) 3.2 ± 0.1 (3.2–3.3) 2.5 ± 0.1 (2.4–2.6) 0.024* 0.1 0.024*
Temperature (°C) 17–25 17–21 19–20 / / /
Figure 4. 

Visualization of advertisement calls of three Megophrys species from Medog. A–C visualizations of 60 seconds waveform of relative amplitude over time for M. medogensis (one unvouchered individual recorded in the vicinity of Medog urban area), M. cf. pachyproctus (CIB022017061807), and Megophrys yeae sp. nov. (paratype CIB022017061804), respectively D–F visualizations of 20 seconds waveform of relative amplitude over time G–I visualizations of two seconds waveform of relative amplitude for the species, respectively J–L visualizations of two seconds waveform of spectrogram for the species, respectively.

Skull comparisons. Skulls of the four toad species in Medog were different on many aspects (Fig. 5; Suppl. material 1: Table S5). In general, the skulls of these five species are weakly ossified except for M. medogensis. Skulls of them differ from each other on the following characters: premaxillary and maxillary teeth, nasal bones contact with sphenethmoid or not, texture and shape of sphenethmoid, the shape of frontoparietal, opening of anterior fontanelle and sagittal suture, front part of anterior process parasphenoid, relatively position of exoccipitals with the line connecting conjunctions of quadratojugal and mandible, and columella auris (Fig. 5; Suppl. material 1: Table S5).

Figure 5. 

Skull of Megophrys species in Medog. A–E dorsal views of adult male holotype NWIPB770650 of M. pachyproctus, adult male topotype CIB022017061406 of M. medogensis, adult male CIB022017061805 of M. cf. pachyproctus, adult male holotype CIBMT171053 of Megophrys zhoui sp. nov., and adult male holotype CIB201706MT02 of Megophrys yeae sp. nov., respectively F–J ventral views of the specimens, respectively. Key to skull: 1 premaxillary; 2 maxillary; 3 nasal; 4 sphenethmoid; 5 anterior fontanelle; 6 frontoparietal; 7 sagittal suture; 8 pterygoid; 9 squamosal; 10 quadratojugal; 11 prootic; 12 exoccipital; 13 vomerine ridge; 14 mandible; 15 anterior process of parasphenoid; 16 columella auris. Scale bar equal to 5 mm.

Taxonomic accounts

Megophrys pachyproctus Huang, 1981

Figs 3A, F, K, P, U, 5A, F, Suppl. material 2: Fig. S5C; Tables 1, Suppl. material 1: Tables S1, S2, S5

Megophrys pachyproctus Huang, 1981* in Huang & Fei, 1981: 211–212. Holotype: NWIPB 770650, by original designation. Type locality: Gelin, Medog (29°11'N, 95°10'E), Xizang, China; altitude 1530 m, China.

Specimens examined

Holotype: adult male NWIPB 770650.

Description of holotype

(Fig. 3A, F, K, P, U; Suppl. material 1: Table S1). Measurements in mm. Adult male. Body moderate, SVL 35.7; a large swollen arc-shaped protuberance present on vent beyond cloaca and visible on both dorsal and lateral view, its length 6.8 (measured dorsally), width 4.7, and thickness 2.7.

Head wider than long (HW/HL 1.13); snout blunt in dorsal view, obtusely protruding beyond mandible in lateral view; rostral appendage absent; canthus rostralis well developed, loreal region concave; dorsal surface of snout slightly concave; nostril oval, slightly closer to snout than eye (EN/SN 1.04); eyes lager than twice tympanum (EL/TYD 2.24); eye-tympanum distance smaller than tympanum diameter (TYE/TYD 0.86); tympanum oval, obliquely orientated, upper 1/3 concealed with supratympanic ridge; interorbital space flat, wider than upper eyelids (UEW/IUE 0.89); pineal ocellus not visible; vomerine ridges well developed, acutely angled, enlarged at ends where bearing several vomerine teeth; maxillary teeth present; tongue notched posteriorly, medial lingual process absent.

Forearm moderately long and wide; fingers long and thin, without webbing and lateral fringes; subarticular tubercles absent; inner and outer metacarpal tubercles small and oval, weakly connected at lower half; finger relative lengths I < II < IV < III; base of finger I strong, larger than base of finger II; tips of fingers slightly swollen and rounded (FIIIW 0.8), without pads.

Hindlimbs relatively thin and long; thighs ca. equal length of shanks and feet; toes long and thin, relative lengths I < II < V < III < IV; tips of toes rounded; toes rudimentary webbed; lateral fringes narrow; continuous dermal ridges present under toes; outer metatarsal, and subarticular tubercles absent; inner metatarsal tubercle distinct, rounded, separate from base of toe I at a distance nearly twice its diameter; tips of toes rounded.

Dorsal surface of head and body relativity rough, densely scattered with small granules; temporal region and upper corner of mandible scattered small granules; tympanum border slightly raised; upper eyelid without pointed edge; supratympanic ridges extend from posterior upper eyelid border to region above forearm insertions, not curving above tympanum, rear part thicker than front; flanks densely covered with small granules and scattered several larger tubercles; two longitudinal ridges on dorsolateral body distinct, nearly parallel, extending from above shoulder to nearby groin; parietoscapular-sacral ridges forming a “> <” configuration, composed by rows of small tubercles, dorsal surface of forearm thighs and shanks with several rows of small tubercles transversely arranged; dorsal upper arm and other dorsal surfaces of hindlimbs covered with dense small granules; ventral surface of body and limbs smooth; pectoral glands small (diameter 0.8) and rounded, close to axilla on chest; femoral glands small (diameter 1.0) and rounded, closer to outer edge of knee than to cloaca.

Coloration of holotype in preservative

(Fig. 3A, F, K, P, U). Dorsal and lateral surface of body, dorsal surface of head mostly tan; a brown triangle present between eyes, little lighter in center, anterior corners reach to near out edge of upper eyelids; indistinct “X”-shaped markings on dorsum, with small tubercles in center; darker brown stripe along with dorsolateral ridges; tubercles on flanks white, edged with dark patches; lateral surface of head tan with brown stripes radiating from orbit to upper mandible and upper eyelid; iris dark brown; a brown stripe extending from posterior corner of orbit under former half supratympanic ridges to behind tympanum, a clear thin dark stripe under edges of supratympanic ridges after tympanum, no long white stripe present on upper lip; dorsal and lateral surface of limbs mostly tan, two broad brown transverse bands on forearms, and four thin indistinct transverse bands on dorsal thighs and shanks; dorsal tarsal pale gray with three indistinct transverse bands, outer three fingers with tan blotches; gular region and chest dusty tan with a short longitudinal brown stripe in middle of throat; two light patches on edges of jaw corresponding anterior corners of eyes and front edge of brown stripe at posterior end of jaw; and one brown stripe extending from posterior end of jaws to base of forearms on both sides; abdomen dusty tan, with a dozen darker patches on middle and upper abdomen, large longitudinal dark patches present on ventral lateral abdomen hardly present; ventral surface of forelimbs and hindlimbs dusty tan mottled with light patches; pectoral and femoral glands light tan.

Coloration of holotype in life

According to Huang and Fei (1981): dorsum brown or dark brown; two to four dark colored transverse bands present on forearms; and four to five dark colored transverse bands present on thighs and shanks; places around cloaca, groin, and anterior, posterior, ventral thigh orange; tips of fingers and toes light red; ventral surfaces of tarsi, metatarsus, and toes grayish brown or black-brown; lateral and ventral surface with lots of grayish black spots; a longitudinal short grayish black stripe present on middle throat; granules on dorsal surface of body and limbs light red.

Skull

(Fig. 5A, F). Description based on scan of the holotype. Skull weakly ossified, width 1.12× of length; maxillary overlapping with the quadratojugal; premaxillary and maxillary teeth strong, most tooth closely positioned with others, 9/9 teeth present on left/right premaxillary, no teeth present on mandible; vomerine ridge well developed, two vomerine teeth present on enlarged posterior end of each vomerine ridge; nasal process of premaxilla protruding beyond skull; nasal bones separated from each other, inner edge mostly contact with sphenethmoid; sphenethmoid relatively smooth with few small pits on both dorsal and ventral surface, the middle one third of front edge not contacting nasal bones and truncate, separated from premaxilla; frontoparietal divided by a distinctly opening sagittal suture, sagittal suture slightly wider posteriorly; anterior fontanelle small, only slightly wider than sagittal suture; front and rear part of frontoparietal almost equally wide; posterior edge of exoccipitals posterior to the line connecting conjunctions of quadratojugal and mandible; pterygoid moderate; anterior process of squamosal slender and sharp, tip closer to the junction of pterygoid and quadratojugal than its base, posterior process present; prootic relatively smooth, separated from exoccipitals; anterior process of parasphenoid in shape of fusiform, anterior part not raised above sphenethmoid, conjunction of parasphenoid anterior process meet with sphenethmoid moderate, width ca. three quarters of the constriction near its base; columella auris short.

Variations

See for morphometric variation within the three types (two adult males and one adult female) in Suppl. material 1: Table S1. According to the photo of dorsal view of the only adult male paratype NPIB 770651 presented by Fei and Yei (2016), the adult male paratype resemble the holotype in general, also has a distinct swollen arc-shaped protuberance present on end of body beyond cloaca, but different in color on dorsal body darker, and not having distinguishable “X”-shaped markings on dorsum. The adult female allotype NPIB 770652 do not have a distinct projection on vent, and the coloration on dorsal and ventral surface of body lighter than males.

Secondary sexual characters

Male with gray nuptial pad on inner side of the first finger, spines on nuptial pad dense and small; single subgular vocal sac; vocal sac opening small, slit like; a distinct fatty swollen rounded projection present on the end of body beyond cloaca.

Distribution and natural history

According to Huang and Fei (1981), this species was first collected at elevation 1530 m in the type locality, Gelin, Medog, Xizang, China; two adult males were found on shrubs emitting continuous calls sounds like “gazhi...gazhi...gazhi...” to the human ear; the female was found on the road near a brook.

Comparisons

Megophrys pachyproctus differs from all other known congeners except M. koui and M. caudoprocta by having a distinct protuberance above vent, and further differs from the latter two species in protuberance above vent being swollen and arc-shaped (vs. not). For comparisons with subsequent undescribed species covered in this paper, refer to relevant morphological comparison sections for those species.

Remarks

Megophrys pachyproctus was originally described by Huang and Fei (1981) with description and figures of the holotype, measurements of types, secondary sexual characters, and brief natural history. And then were translated into English and measurements of snout length, internasal space, interorbital space, eyelid width, diameter of eye, tympanum, and tibia width were supplemented by Huang et al. (1998). Fei et al. (2009) provided illustration of the holotype. Fei et al. (2010) and Fei and Ye (2016) provided colored drawings of the holotype and the paratype (NPIB) 770651, and colored photos of dorsal and lateral views of one living topotype from Medog (photographs by Ke Jiang). The topotype possess expanded fingertips with small disk and two large longitudinal dark patches on ventral lateral abdomen (while the holotype of M. pachyproctus does not have these character), and not having a distinct swollen arc-shaped protuberance present above vent (while the holotype of M. pachyproctus possess). Li et al. (2010) provided similar photos of a living specimen and measurements of seven specimens from Maniwong and Yarang, Medog, under the name M. pachyproctus. The body length of these specimens ranges from 26.1 mm to 27.9 mm, and Li et al. (2010) cited the body length of the male types of M. pachyproctus as 25.3 mm to 36.2 mm, which should be 35.3 mm to 36.2 mm (Huang and Fei 1981). These specimens with small body size, expanded fingertips with small disk, and two large longitudinal dark patches on ventral lateral abdomen turn out to be mostly similar with Megophrys yeae sp. nov. (see description of Megophrys yeae sp. nov.). We suggest reexamination of these specimens should be taken. Saikia and Sinha (2018) reported M. pachyproctus from Southern Xizang (27.547681°N, 93.897555°E, 1855 m), provided description, measurements (body length 37.8), and a photo of dorsal view of the single male voucher specimen V/A/NERC/1352. But the photo does not present an arc-shaped swelling above vent (Saikia and Sinha 2018: fig. 3A) as the holotype. We suggest further examination should be made to confirm the identification of the specimen. This species was also reported new range in Lao Cai and Ha Tinh Province, Vietnam (Orlov et al. 2002; Nguyen et al. 2005). Since Megophrys inhibits astonishing cryptic species biodiversity, and species like M. pachyproctus thought to be widespread from Southwest China to Vietnam confirmed to be another species (Chen et al. 2016; Tapley et al. 2017; Liu et al. 2018), records from Vietnam where more than 1000 km from type locality should be questioned and specimens should be reexamined.

Megophrys medogensis Fei et al., 1983

Figs 3B, G, L, Q, V, 4A, D, G, J, 5B, G, 6A, B, E, F, I, J, M, N, 10A–C, E, F, Suppl. material 2: Figs S1, S5A–H; Tables 1, 2, 3, 4, Suppl. material 1: Table S1-S5

Megophrys omeimontis medogensis Fei, Ye and Huang (1983)**: 49–52.

Specimens examined

Five adult females and six adult males from Medog (Suppl. material 1: Table S1).

Holotype description

Refer to Fei et al. (1983) for holotype description, Mahony et al. (2018) for picture of holotype CIB 73II0015, Fei et al. (2009) and Fei and Ye (2016) for description of coloration and picture of topotypes.

Skull. (Fig. 5B, G). Description based on sequenced adult male topotype CIB022017061406. Skull well ossified, width 1.21× length; maxillary overlapping with the quadratojugal; premaxillary and maxillary teeth well developed, and closely positioned with others, 11/13 teeth present on left/right of premaxillary; vomerine ridge robust; few vomerine teeth strong, present on posterior end of vomerine ridge; nasal process of premaxilla protruding beyond skull; nasal bones separated, posterior one third of inner edge contact with sphenethmoid; frontoparietal distinctly wider in front than rear; sphenethmoid relatively smooth with few small pits on dorsal and ventral surface, the middle half of front edge not contacting nasal bones and protruding forward, separated from premaxilla; frontoparietal not divided, sagittal suture occlusive; anterior fontanelle occlusive; front part of frontoparietal distinctly wider than rear; posterior edge of exoccipitals anterior to the line connecting conjunctions of quadratojugal and mandible; pterygoid robust; anterior process of squamosal slender, tip much closer to the junction of pterygoid and quadratojugal than its base, posterior process present; prootic relatively smooth, separated from exoccipitals; anterior process of parasphenoid in shape of fusiform, the front part raise above sphenethmoid from ventral view, the conjunction with sphenethmoid with width equals the constriction near base of anterior process of parasphenoid; columella slender and long.

Secondary sexual characters

Adult female generally with larger body size. Average body length females 79.7 mm (n = 5, 75.7–85.5 mm), male 65.3 mm (n = 6, 63.1–68.7 mm). Males with brown nuptial pads on fingers I and II, spines on nuptial pad dense; single subgular vocal sac.

Tadpole

(Fig. 6A, B, E, F, I, J, M, N; Suppl. material 1: Table S2). For low-middle-elevation tadpoles of M. medogensis, description was based on tadpole CIBMT20170621 (stage 35) which shared the same pond of sequenced tadpole CIBMT022017061808 in Bari village. They are similar on morphology. For coloration at stage 26, description based on sequenced specimen CIBMT1710101 from Yadong village. Measurements in mm. For stage 35, body 13.3, elongated; head slightly narrower than trunk, oral disk large, funnel like, 1.2× body width; three rows of short oval submarginal papillae on lower lip; middle of lower lip protruding forward, with five rounded papillae longitudinal arranged from the tip middle lower lip to oral cavity; corner of mouth with six papillae arranged in a transverse row on both sides; three transverse rows of short oval papillae on upper lip; keratodonts absent; nares closer to eyes than tip of snout (RN/NE 1.6); eyes round, positioned dorsolaterally; internarial distance (IND 3.0) 61% of the interpupilar distance (PP 4.9); spiracle mostly in left side of body, in right-handed helix from ventral view; spiracular tube not protruding beyond body wall, positioned 60% of the distance between tip of the snout and trunk-tail junction, and below the horizontal mid trunk line; tail accounts 69% of total length; dorsal fin arise above trunk-tail junction, 35% of maximum body height; ventral fin connected to the trunk, with lesser height, 27% of maximum body height; anal siphon opens medially; maximum tail muscle height 72% of maximum body height, maximum tail muscle strong, width 53% of maximum body width; 12 small curves present on both lateral side of tail muscle. For stage 26, dorsal fin arises behind trunk-tail junction. For stage 43, clear “X” and “l_l” skin ridges have present on dorsum, limbs are well developed. For high-elevation tadpoles of M. medogensis, description mostly based on sequenced tadpole CIBMT171001 (at stage 27), coloration based on sequenced tadpoles CIBMT1710106 and CIBMT1710112, collected from Gedang, Medog, Tibet Autonomous Region, China (29.463916°N, 95.769507°E, 2142 m). Body 9.5, elongated; head slightly narrower than trunk; oral disk moderate, funnel like, positioned anterior-dorsal, width equal with body width; 5 transverse rows of short oval papillae on upper lip; keratodonts absent; nares much closer to eyes than tip of snout (RN/NE 2.2); eyes round, positioned dorsolaterally; internarial distance (IND1.9) 61% of the interpupil distance (PP 3.2); spiracle barely visible from ventral view; the spiracular tube not protruding beyond body wall, positioned 63% of the distance between tip of the snout and trunk-tail junction, and below the horizontal mid trunk line; tail accounts 72% of total length; dorsal fin arise above anal siphon opens, 40% of maximum body height; ventral fin connected to the trunk, with lesser height than dorsal fin, 37% of maximum body height; anal siphon opens medially; tail muscle relatively weak, maximum height 72% of maximum body height, width only 44% of maximum body width; eleven small curves present on both lateral side of tail muscle.

Figure 6. 

Tadpole specimens of four groups of three Megophrys species from Medog. A–D dorsal views of the low-middle-elevation tadpole CIBMT20170621 of M. medogensis (Goser stage 35), the high-elevation tadpole CIBMT171001 of M. medogensis (stage 27), tadpole CIBMT20170611 of M. cf. pachyproctus (stage 25), tadpole CIBMT170604 of Megophrys yeae sp. nov. (stage 35), respectively E–H lateral views of the tadpoles, respectively IL ventral views of the tadpoles, respectively. M–P dorsal views of head of the tadpoles, respectively. Scale bar for body view equal to 10 mm, and for head view 2 mm.

Coloration of tadpoles. Low-middle-elevation tadpoles. In preservation (based on CIBMT20170621; Fig. 6A, B, E, F): dorsal body brown; lips semitransparent, papillae on lips brown; dorsal tail light brown, marbled with deep brown markings; lateral side of tail densely covered with tiny brown pigment spots, also mottled with small light colored patches, and scattered with deep brown pigments piles; fins semitransparent; ventral surface of body semitransparent, sparsely covered with brown pigments. In live: dorsal and lateral body generally in light yellow-brown; lateral side of tail mottled with light colored patches; ventral body without white patches; iris brown.

High elevation tadpoles (Fig. 6I, J, M, N). In preservation: dorsal body and tail with brown pigmentation; lateral body brown, skin coloration lighter below spiracular tube, with clear white patches; lateral tail muscle brown mottled with dense tiny white dots; upper and lower fin semitransparent brown, lower fin colored lighter; no dark patches on lateral and dorsal tail; ventral body semitransparent white, stained with light brown pigments; lips semitransparent white, papillae brown. When alive, dorsal body and tail basically deep brown, mottled with copper pigmentation, especially dense on body; lateral body brown, with cream-white patches near abdomen; lateral tail brown, scattered with tiny white pigment spots, no dark brown patches on tail; ventral surface of body semitransparent brown, covered with small white pigments; iris brown.

Bioacoustics

(Fig. 4A, D, G, J; Suppl. material 1: Tables 4; Suppl. material 2: Fig. S3). A total of 16 call groups and 62 calls were analyzed. Average dominant frequency of calls low, 2.5 kHz (2.3–3.0 kHz); call repetition rate moderate, average 1.2 calls per seconds; call interval short, average 153–254 ms; call groups with average 10.8 calls; call duration long (average 746 ms), and with lots of notes (average 42.1). To the human ear, the call sound like “ga ga ga...”.

Distribution and natural history

The species is currently known with certainty from the type locality in Medog County, and its distribution elevation was recorded between 680–2200 m (Fei et al. 1983, 2012, Fei and Ye, 2016; this study). This species was recorded in or near small mountain streams of tropical rain forests, sit on rocks, leaf litter, and sometimes bare soil. Calls heard between 11 June to 5 August (this study; Fei et al. 2019). Four in five females recorded during 11 June to 18 June were gravid. Males start calling before dusk under dense vegetation. Normally, two or more males call in small groups along stream banks, spacing themselves ca. 3–5 meters from each other. Sequenced tadpoles in metamorphosis were recorded on 18 June, in small mountain stream pond at 1560 m. Tadpoles of two other species of Megophrys share the same ponds. See description in following. Breeding season is supposed to including early June and may last to early August.

Comparison

Refer to Mahony et al. (2018) for comparison with other species of M. major group. M. medogensis differs from M. pachyproctus by much larger body size (SVL 57.2–68.7 in 21 males vs. 35.3–35.7 in two males in the latter), absence of large protuberance above vent (vs. present in the latter), skin relatively smooth (vs. rough in the latter), frontoparietal distinctly wider in front than rear (vs. almost equally wide in the latter), sagittal suture occlusive (vs. distinctly open in the latter), and columella auris long (vs. short in the latter). For comparisons with species studied in this paper, refer to relevant morphological comparison sections for those species.

Megophrys cf. pachyproctus

Figs 3C, H, M, R, W, 4B, E, H, K, 5C, H, 6C, G, K, O, 7, 10C, D, H, I, Suppl. material 2: Figs S2, S5B, S5F; Tables 1, 3, 4, Suppl. material 1: Tables S1, S2, S3, S5

Megophrys cf. pachyproctus Huang, 1981***

Specimens examined

Four adult males, CIB022017061805 (Figs 3C, H, M, R, W, 5C, H, 7), CIB201706MT04, CIB022016061806, CIB022017061807, collected from Bari village, Medog County, Tibet Autonomous Region, China (29.32947°N, 95.36016°E, 1780 m) by SC Shi and L Ding, on 18 June 2017. One adult male (CIBMT171056), four adult females in gravidity (CIBMT171052, CIBMT171057, CIBMT171058, CIBMT171054), and one male toadlet (CIBMT171059) were collected from vicinity of Renqingbeng Temple (29.304832°N, 95.361682°E, 2003 m) by SC Shi on 26 October 2017.

Description of the representative (referred) specimen

Adult male, CIB022017061805 (Figs 3C, H, M, R, W, 5C, H, 7). Measurements in mm. Body stout, relatively small size (SVL 34.8); protuberance beyond cloaca small, barely visible from ventral view, not swollen.

Head moderately large, wider than long (HW 12.3, HL 11.0, IFE 6.5, IBE 10.4); snout rounded in dorsal view, slightly projecting in profile, protruding beyond lower jaw; rostral appendage absent (SL 4.6); canthus rostralis blunt; loreal region concave, dorsal surface of snout slightly concave; nostril oval, nearly in the middle of distance from snout to eye (SN 2.2, EN 2.3), distance between nostrils almost equal to distance between upper eyelids (IN 3.9, IUE 3.8); tympanum smaller than half of eyes (EL 4.5, TYD 1.8); eye-tympanum distance subequal to tympanum diameter (TYE 1.7); tympanum irregular rounded, upper 1/3 conceal with supratympanic ridge; interorbital space flat, larger than upper eyelid (UEW 3.2); pineal ocellus not visible; vomerine ridges distinct, orientation of two ridges acutely angled, enlarged at ends where bearing several vomerine teeth; maxillary teeth present; tongue notched posteriorly, medial lingual process absent.

Forearm moderately long and wide, similar size of upper arms, shorter than hand (FAL 7.5, HAL 9.6); fingers long and thin, with rudimentary webbing; narrow lateral fringes present on finger III, indistinct on other fingers; subarticular tubercles absent; inner and outer metacarpal tubercles mostly fused, large, with the size of base of finger I; finger length formula I < II < IV < III; base of finger I strong, larger than base of finger II; tips of fingers slightly swollen, without pads (FIIIW 1.1).

Hindlimbs thin and long; tibio-tarsal articulation reaches middle eye; thighs shorter than shanks but longer than feet (TL 16.5, SHL 17.2, FOL 15.2, TFOL 24.0); toes long and thin, relative lengths I < II < V < III < IV, rudimentary webbed, with narrow lateral fringes, tips rounded, dermal ridges continuously present on ventral surface; subarticular tubercles absent; outer metatarsal tubercle tiny and rounded; inner metatarsal tubercle distinct (IMT 1.6), nearly oval, partially fused with toe I.

Skin. Dorsal surface of head and body rough, densely scattered with small granules; temporal region and upper corner of mandible with rough granules; tympanum ring slightly raised; several small granules on edges of upper eyelids; supratympanic ridges extend from posterior upper eyelids to above forearm insertions, curving above tympanum, rear part thicker than the front; skin on flanks smoother than skin on dorsum, with several large warts and lesser granules; dorsolateral ridges distinct, irregularly stretch from above shoulder to near groin; a transverse skin ridge between upper eyelids; a near “V”-shaped skin ridge between shoulders, connected with the right dorsolateral ridge by a short skin ridge, a tubercle present near the end of “V”-shaped skin ridge; two oblique skin ridges connected with dorsolateral ridges at posterior; dorsal surface of upper arm covered with small granules in three rows from shoulder to elbow; small granules on dorsum of lower arm, hand, and hindlimbs, four transverse rows of granules on thighs and shanks; ventral surface of body and limbs smooth; pectoral glands small and rounded, with the size of first fingertip, close to axilla on chest; femoral glands small, closer to outer edge of knee than to cloaca.

Coloration in preservative

(Fig. 3C, H, M, R, W). Dorsal surface of head gray; dorsal surface of body pale gray; a darker gray triangle bet between eyes, little lighter in center, anterior corners reach to near out edge of upper eyelids; area around dorsal skin ridges darker, no clear “X”-shaped markings on dorsum; tubercles on flanks white and edged with dark patches on one side; lateral surface of head mottled with pale gray and grayish white; a dark stripe extend from behind upper eyelid to behind corners of the mouth, thicker in the middle, and covers tympanum entirely; no long white stripe present on upper lip; two dark strips from eyes to upper lips, two short dark bands on upper lips before eyes; iris dark covered with silver pigments radiated from pupil; dorsal and lateral surface of limbs pale gray with darker transverse bands, one or two broad dark brown transverse bands on forearms, and four indistinct transverse bands on dorsal thighs and shanks, dorsal tarsal pale gray with three indistinct transverse bands, dorsal surface of fingers and toes also covered with several darker transverse bands; gular and chest dusty white; edge of lower mandible white with five brown patches, the pair corresponding to places between nasals and eyes are largest; a short longitudinal light brown stripe present in middle of throat, two pairs of faint brown patches beside the short longitudinal light brown stripe; a brown stripes extending from posterior end of jaws to base of forearms on both sides; skin around pectoral glands faint brown, three medium size faint brown patches present on dusty white upper abdomen, lower abdomen cream-white reticulated with dusty brown pigments, no large longitudinal dark patches present on ventral lateral abdomen; ventral surface of forelimbs and hindlimbs dusty brown, mottled with several irregular brown patches; pectoral and femoral glands cream-white; nuptial pad grayish black.

Coloration in life

(Fig. 7). Markings as described in preservative; flanks, lateral sides of head, dorsal surface of head, body, and limbs light brown in general with orange-red granules; tympanum and stripes under eyes brown; several cream-white dots present on flanks; two dark brown transverse bands present on forearms; four dark brown transverse bands present on thighs, shanks, and tarsi; ventral surface of head, and abdomen grayish white basically, two large longitudinal brown present on lateral sides of abdomen; chest, ventral surface of hand, thigh, and feet flesh colored; skins around cloaca, on groin, and anterior, posterior and ventral thigh without orange patches; iris dark brown reticulated with dense golden pigments, pupil edged with diamond-shaped golden ring; nuptial pad gray.

Figure 7. 

The adult male CIB022017061805 of M. cf. pachyproctus in life. A dorsolateral view of body B ventral view of body C dorsolateral view of head D ventral view of hand E ventral view of foot.

Skull

(Fig. 5C, H). Skull weakly ossified, width 1.15× of length; maxillary overlapping with the quadratojugal; premaxillary and maxillary teeth weakly developed, most tooth separated from others by a distinct gap, 10/10 teeth present on left/right premaxillary, teeth absent on mandible; vomerine ridge moderate, few vomerine teeth weak, present on enlarged posterior end of vomerine ridge; nasal process of premaxilla protruding beyond skull; nasal bones separated from each other, half connected with sphenethmoid; sphenethmoid rough with curves and pits on dorsal and ventral surface, middle one third of front edge free from nasal bones, and protruding forward, separated from premaxilla; frontoparietal divided by a distinctly open sagittal suture , slightly wider posteriorly; anterior fontanelle small, slightly wider sagittal suture; front and rear part of frontoparietal almost equally wide; posterior edge of exoccipitals posterior to the line connecting conjunctions of quadratojugal and mandible; pterygoid moderate; anterior process of squamosal slender and sharp, tip closer to the junction of pterygoid and quadratojugal than its base, posterior process present; front part of prootic smooth, rear part rough, separated from exoccipitals; anterior process of parasphenoid in shape of fusiform, anterior part not raised above sphenethmoid, conjunction of parasphenoid anterior process meet with sphenethmoid narrow, width ca. half the constriction near its base; columella auris short.

Variation

(Suppl. material 2: Fig. S2). The topotypes have the following differences: tympanum diameter/eye diameter ratio varies from 0.40 to 0.60 for adults, 0.36 for the juvenile CIBMT171059; skin ridges between dorsolateral ridges on dorsum vary in configuration, i.e., “> <” (CIB022016061806) and “>” (CIB022017061807); and specimen CIB022017061807 with orange-red patches on groin, and flanks stained with orange-red. The three skulls similar in morphology (e.g., premaxillary and maxillary teeth weak; the nasal bones half connected with sphenethmoid; the sphenethmoid rough and protruding forward; tip of anterior process of parasphenoid narrow), but possessing following variation: vomerine teeth only present on left vomerine ridge of CIBMT171056; sagittal suture of CIBMT171056 partially occlusive near the center.

Secondary sexual characters

Adult female with larger body size, average 1.17× of males. Male with gray nuptial pad on inner first finger, spines on nuptial pad dense and small; single subgular vocal sac; vocal sac opening small, slit like; lineae musculinae absent.

Bioacoustics

(Fig. 4B, E, H, K; Tables 4; Suppl. material 1: Table S3). A total of 15 call groups and 82 calls were analyzed. Average dominant frequency of calls moderate, 3.2 kHz (3.2–3.3 kHz); call repetition rate moderate, average 0.9 calls per seconds; call interval long, average 493–720 ms; call groups with average 10.8 calls; call duration long (average 746 ms), and with lots of notes (average 42.1). To human ears, sound like pebbles hitting ground continuously.

Tadpole

(Fig. 6C, G, K, O; Suppl. material 1: Table S2). Description based on measurements and observation of tadpole specimen CIBMT20170611 at stage 25. Measurements in mm. Body 6.1, elongated; head slightly narrower than trunk; oral disk large, funnel like, positioned anterior-dorsal, width 1.6× of body width; 4 rows of oval submarginal papillae on middle lower lip, 3 rows of oval submarginal papillae on both sides of upper lips; all these papillae range towards oral cavity; keratodonts absent; nares much closer to eyes than tip of snout (RN/NE 3.8); eyes round, positioned dorsolaterally; internarial distance (IND1.4) 85% of the interpupil distance (PP 1.63); spiracle mostly in left side of body, in right-handed helix from ventral view, the spiracular tube not protruding beyond body wall, positioned 63% of the distance between tip of the snout and trunk-tail junction, and below the horizontal mid trunk line; tail accounts 68% of total length; dorsal fin arise above trunk-tail junction, 38% of maximum body height; ventral fin connected to the trunk, with lesser height than dorsal fin, 31% of maximum body height; anal siphon opens medially; maximum tail muscle height 72% of maximum body height, maximum tail muscle width 53% of maximum body width; eleven small curves present on both lateral side of tail muscle.

When alive, dorsal body and tail basically with yellow-brown pigmentation; two golden spots in size of eyes present on dorsolateral mid body. In preservation, dorsal body, and most part of lateral tail with brown pigmentation; ventral body and tail fin semitransparent; lateral body and tail with pigmentation, but lower fin and ventral body barely pigmented.

Comparison

By having relative smaller body size (males 33.6–36.6, n = 5; females 40.6–42.8, n = 4; measurements in mm), Megophrys cf. pachyproctus differs from M. medogensis (males 57.2–68.7, n = 21), M. caudoprocta (males 70.8–81.3, n = 4); M. hoanglienensis (males 37.4–47.6, n = 11), M. jingdongensis (males 53.0–56.5, n = 3), M. liboensis (males 61.6–62.9, n = 4), M. omeimontis (males 56.0–59.5, n = 10), M. aceras (males 55.8–62.4, n = 6); M. ancrae (males 39.1–45.3, n = 8), M. damrei (male 57.1, n = 1), M. flavipunctata (males 56.9–68.4, n = 4), M. glandulosa (males 76.3–81.0, n = 10), M. himalayana (males 68.0–73.5, n = 6), M. lekaguli (males 55.6–66.6, n = 8), M. major (males 71.6–87.5, n = 12), M. mangshanensis (male 62.5, n = 1), M. maosonensis (male 77, n = 1), M. megacephala (males 45.9–53.4, n = 12), M. monticola (males 38.4–49.5, n = 17), M. periosa (males 71.3–93.8, n = 12), M. robusta (males 73.5–83.1, n = 6), M. longipes (male 47, n = 1; female 65, n = 1), M. oreocrypta (female 94.9, n = 1), M. serchhipii (male 37.1, n = 1), and M. takensis (males 47.3–53.0, n = 3).

By having relative larger body size (males 33.6–36.6, n = 5; females 40.6–42.8, n = 4; measurements in mm), Megophrys cf. pachyproctus differs from M. zunhebotoensis (male 30.0, n = 1; female 39.0, n = 1), M. rubrimera (males 26.7–30.5, n = 8), and M. angka (males 31.2–32.1, n = 2).

By tympanum present distinctly, Megophrys cf. pachyproctus differs from M. gigantica, M. nankiangensis, and M. shapingensis (vs. absent or concealed in the latter).

By vomerine ridge and teeth present, Megophrys cf. pachyproctus differs from M. wawuensis (vs. absent in the latter).

By maxillary teeth present, Megophrys cf. pachyproctus differs from M. elfina, M. gerti, M. hansi, M. koui, M. microstoma, and M. synoria (vs. absent in the latter).

By hind limbs long and head not wide and flat, Megophrys cf. pachyproctus differs from M. carinense, M. chuannanensis, M. feae, M. intermedia, and M. popei (vs. hind limbs short and head flat wide in the latter).

By lacking a single, wide and flat palpebral projection on the edge of the upper eyelid, Megophrys cf. pachyproctus differs from M. lancip, M. montana, M. parallela, M. baluensis, M. edwardinae, M. kobayashii, M. ligayae, M. nasuta, and M. kalimantanensis (vs. present in the latter).

By lacking rostral appendage, Megophrys cf. pachyproctus differs from M. stejnegeri (vs. having less rostral appendage in the latter).

By lacking a distinct horn-like tubercle at edge of upper eyelid, Megophrys cf. pachyproctus differs from M. dringi (vs. present in the latter).

By vomerine teeth present, Megophrys cf. pachyproctus differ from M. vegrandis, M. baolongensis, M. binchuanensis, M. binlingensis, M. boettgeri, M. brachykolos, M. cheni, M. kuatunensis, M. lini, M. lishuiensis, M. minor, M. obesa, M. palpebralespinosa, M. sangzhiensis, M. shuichengensis, M. spinata, M. tuberogranulata, M. wuliangshanensis, M. wushanensis, M. ombrophila, M. leishanensis, M. wugongensis, M. mufumontana, M. feii, M. auralensis, and M. huangshanensis, M. angka, M. shunhuangensis, M. jiangi, and M. xianjuensis (vs. absent in the latter).

By relatively finger lengths I < II < IV < III and nuptial pads present only on finger I, Megophrys cf. pachyproctus differs from M. nanlingensis (vs. relatively finger lengths II < I < IV < III, nuptial pads and nuptial spines invisible in males during breeding season in the latter).

By toes with rudimentary webbing, Megophrys cf. pachyproctus differs from M. serchhipii (vs. at least one fourth webbed in the latter).

By toes with narrow lateral fringes, Megophrys cf. pachyproctus differs from M. binchuanensis, M. cheni, M. jingdongensis, M. lini, M. rubrimera, M. shuichengensis, M. spinata, M. feii, M. vegrandis, and M. glandulosa (vs. wide in the latter).

By dorsal skin rough but without spines, Megophrys cf. pachyproctus differs from the following species: M. vegrandis (vs. smooth); M. medogensis (vs. smooth with small granules); M. daweimontis (vs. smooth); M. fansipanensis (vs. smooth with small granules); M. oropedion (vs. smooth with small granules); M. parva (vs. smooth); M. zhangi (vs. smooth); and M. jiulianensis (vs. dorsal skin rough with spines).

By snout rounded in dorsal view and nuptial pad only present only on finger I, Megophrys cf. pachyproctus differs from M. dongguanensis (vs. snout pointed, nuptial pads present on the first two fingers in the latter).

Megophrys cf. pachyproctus further differs from M. medogensis by the following characters: nuptial pads only present on finger I in males (vs. on the first two fingers in the latter); dorsal skin rough (vs. relatively smooth in the latter); vomerine ridge moderate, vomerine teeth weak (vs. both strong in the latter).

By having following characters of skull, Megophrys cf. pachyproctus differs from M. medogensis: skull weakly ossified, opening of anterior fontanelle present, sagittal suture distinctly open (vs. skull well ossified, opening of anterior fontanelle and sagittal suture occlusive in the latter); frontoparietal front equals rear (vs. distinctly wider in the latter); sphenethmoid rough with curves and pits, middle front edge protruding (vs. relatively smooth with few pits, truncate in the latter); exoccipitals posterior to the line connecting conjunctions of quadratojugal and mandible (vs. anterior in the latter); and columella auris short (vs. long in the latter).

By having following characters of bioacoustics, Megophrys cf. pachyproctus differs from M. medogensis (Tables 3, Suppl. material 1: Table S5): call duration significantly much longer (491–889 ms vs. 121–241 ms; P < 0.001); dominant frequency significantly higher (3.2–3.3kHz vs. 2.3–3.0 kHz; P < 0.01); and call intervals significantly longer (493–720 ms vs. 153–254 ms; P < 0.001).

Megophrys cf. pachyproctus very resemble M. pachyproctus on morphology, but differs from the latter in the following characters: protuberance beyond cloaca small, barely visible from ventral view, not swollen (vs. protuberance present on vent beyond cloaca large, swollen, arc-shaped, can be seen on both dorsal and lateral view in the latter); inner metatarsal tubercle distinct partially fused with toe I (vs. inner metatarsal tubercle separate from base of toe I at a distance nearly twice its diameter in the latter). Megophrys cf. pachyproctus further differs from M. pachyproctus by having the following characters on skull morphology: premaxillary and maxillary teeth weak, separated from others by gaps (vs. strong, closely positioned with others in the latter); inner edge of nasal bones half contact with sphenethmoid (vs. mostly in the latter); sphenethmoid rough with curves and pits, middle front edge protruding (vs. relatively smooth with few pits, truncate in the latter); and conjunction of parasphenoid anterior process meet with sphenethmoid narrow, width ca. half the constriction near its base (vs. moderate, ca. three quarters in the latter).

Distribution and natural history

This group is currently known at elevation from 1560 m to 2003 m in Medog County, Tibet Autonomous Region, China. It inhabits mountain streams of subtropical forests. During June, males call on branches and leaves of bushes near mountain stream with a distance at least three meters from others, where covered with dense broad leaf forests (Figs 10C, D, H, I, Suppl. material 2: Fig. S5B, S5F). Females collected during October were gravid with well-developed eggs, and also found on leaves of floor vegetation like Elatostema species and ferns near small mountain streams. Distribution elevation overlap with M. medogensis at 1560 m, where a small stream pond was found to have tadpoles of three Megophrys species on 18 June, including M. medogensis (at stage 42), Megophrys cf. pachyproctus (at stages 26–27), and Megophrys yeae sp. nov. (at stages 28–35). Theloderma sp. and Amolops medogensis Li and Rao, 2005 were recorded at the same habitat.

Megophrys zhoui sp. nov.

Figs 3D, I, N, S, X, 5D, I, 8, 10D, J, Suppl. material 2: Figs S3, S5B; Tables 1, 3, Suppl. material 1: Tables S1, S5

Holotype

(Figs 3D, I, N, S, X, 8). Adult male CIBMT171053, collected from vicinity of Renqingbeng Temple, Medog County, Tibet Autonomous Region, China (29.304832°N, 95.361682°E, 2003 m) by SC Shi on 26 October 2017.

Paratypes

(Suppl. material 2: Fig. S3). Two adult gravid females CIBMT171060 and CIBMT171062, collected along with the holotype.

Etymology

The specific name is in honor of Professor Zhou Kai-Ya, for his contribution to Chinese amphibian research.

Suggested vernacular name

Zhou’s horned toad (English), Zhou Shi Jiao Chan (周氏角蟾, Chinese).

Diagnoses

Megophrys zhoui sp. nov. is assigned to the genus Megophrys sensu lato based upon molecular phylogenetic analyses and the following morphological characters: canthus rostralis well-developed; supratympanic fold distinct; axillary glands small and tit-like, on sides of the breast; head length more than 25% of body size; upper jaw protruding beyond the margin of the lower jaw; no skin fold on back of head; maxillary teeth present; tympanum distinct; hind legs long and thin.

Megophrys zhoui sp. nov. is distinguished from its congeners by a combination of following characters: body small (male 23.0, n = 1; females 23.5–23.9, n = 2); vomerine ridge weak, vomerine teeth absent; tympanum present, moderate; base of finger I in similar size with finger II, relative finger lengths I < II < IV < III, fingertips not expanded into small pads; toes with narrow lateral fringes or absent; inner metatarsal tubercle long oval, positioned on base of toe I; dorsal skin relatively smooth; protuberance beyond cloaca indistinct, barely visible from ventral view, not swollen; skull weakly ossified, premaxillary and maxillary teeth weak; skull wider slightly than long; nasal bones not contact with sphenethmoid.

Holotype description

(Figs 3D, I, N, S, X, 8). Measurements in mm. Adult male, with well-developed testes; body slender, extremely small (SVL 23.0); protuberance beyond cloaca small, not visible from ventral view, not swollen.

Head moderate, longer than wide (HW 7.8, HL 8.3, IFE 4.5, IBE 7.2); snout near rounded in dorsal view, slightly protruding beyond lower jaw; rostral appendage absent (SL 3.6); canthus rostralis blunt; loreal region slightly concave, dorsal surface of snout slightly concave; nostril oval, closer to eye than tip of snout (SN 1.8, EN 1.4); distance between nostrils approximate distance between upper eyelids (IN 3.0, IUE 2.7); eyes twice size of tympanum (EL 2.7, TYD 1.3); pupils diamond, inferior angle slightly concave; eye-tympanum distance subequal with tympanum diameter (TYE 1.1); tympanum rounded, upper 1/3 conceal with supratympanic ridge; interorbital space flat, wider than upper eyelids (UEW 2.3); pineal ocellus not visible; two arcuate vomerine ridges present, orientation of two ridges acutely angled, not enlarged at posterior ends, shortest distance between two ridges equal to length of vomerine ridges; vomerine teeth absent; maxillary teeth present; tongue weakly notched behind, medial lingual process absent.

Forearm slender, not wider than upper arms, shorter than hand (FAL 5.2, HAL 7.1); fingers thin, without rudimentary webbing; subarticular tubercles absent; inner and outer metacarpal tubercles indistinct; base of finger I equal wide with base of finger II; finger relative length I < II < IV < III; tips of fingers slightly swollen, without pads (FIIIW 0.5).

Hindlimbs thin and long, tibio-tarsal articulation reaches middle eye; thighs shorter than shanks but longer than feet (TL 11.5, SHL 12.5, FOL10.9, TFOL 16.7); toes slender, relative length I < II < V < III < IV, rudimentary webbed, without lateral fringes, tips slightly swollen, no dermal ridges on ventral surface; subarticular tubercles absent; outer metatarsal tubercle absent; inner metatarsal tubercle long oval (IMT 1.1), positioned on base of toe I.

Dorsal surface of head and body basically smooth, with skin ridges formed by small disconnected granules; lateral surface of head smooth, tympanum ring not raised; two small granules on out edges of upper eyelid; supratympanic ridges nearly straight, extend from behind upper eyelids to above forearm insertions, rear part not thicker than the front; flanks smoother than dorsum, with several small tubercles one or two × size of nostril; skin on head scattered with tiny granules, some lager granules form a triangle between eyes; a “Y”-shaped skin ridges present between shoulders, but posterior part connected the middle of a “W”-shaped skin ridge on dorsum; several larger granules on rear dorsum behind the “W”; dorsal surface of arm smooth, scattered with tiny granules; dorsal hand and feet smooth; dorsal thighs and shanks smooth, with several larger granules; ventral surface of body and limbs smooth; pectoral glands tiny, barely visible, close to axilla on chest; pectoral glands small and rounded, slightly larger than fingertips; closer to outer edge of knee than to cloaca.

Coloration of holotype in preservative

(Fig. 3D, I, N, S, X). Dorsal surface of body and limbs covered with dense gray pigments; larger granules on body and limbs light colored; a brown triangle present between eyes on head; markings on dorsum, and larger granules on dorsal thighs and shanks with brown fringes around; one broad brown transverse bands present on finger II, III and IV; two narrow transverse short bands present on lower arms; one or two faint brown transverse bands on dorsal toes. Lateral side of head pale gray mostly; skin on upper jaw between nostril and below eyes colored lighter; eyes dark with silver dense fiber around pupils and radiate on iris; supratympanic ridge light colored; chest, ventral surface of head, arms and shanks and feet covered with dense smoky gray pigments; abdomen ivory stained with smoky pigments, and scattered with several dark dots; several small ivory patches present on ventral margin of mandible; a darker brown patches with light colored inner edges extend from posterior end of jaws to ventral surface of upper arms on both sides; ventral surface hand mostly with smoky gray pigments, but base of finger I and II ivory; a brown stripe present on ventrolateral body; ventral surface of thighs smoky gray; tips of digits light colored; pectoral and femoral glands ivory.

Coloration of holotype in life

(Fig. 8). Dorsal body and limbs orange-brown, granules on body orange-red; markings on dorsal body as described above; lateral head basically brown; supratympanic ridge orange; temporal region under supratympanic ridge dark brown; upper lips and canthus rostralis stained with orange; dark patches present on upper lips under eyes; iris orange-red, brighter around pupils. Flanks with several larger orange dots, ventrolateral trunk with white pigments and larger white dots. Throat, chest, arms orange-brown, mottled with dense white pigments; chin stained with orange, several small white patches present on lower lips; brown patches from posterior end of jaws to ventral surface of upper arms edged with white at inner side; upper abdomen orange-brown, stained with several faint orange dots; lower abdomen white, scattered with several clean orange dots; both lateral sides of abdomen with broad brown strips; ventral surface of thighs and shanks flesh brown, with several white tiny granules around cloaca; ventral hand with dense gray-brown pigments, base of finger I and II fleshy; inner and outer metacarpal tubercle, and tips of fingers light orange; ventral feet brown; inner metatarsal tubercle, tips of toes light orange.

Figure 8. 

The holotype adult male CIBMT171053 of Megophrys zhoui sp. nov. in life. A dorsolateral view of body B ventral view of body C dorsolateral view of head D ventral view of hand E ventral view of foot.

Skull

(Fig. 5D, J). Skull weakly ossified, width equal to length; maxillary overlapping with the quadratojugal; premaxillary and maxillary teeth weak, barely visible; teeth absent on mandible; vomerine ridge weak, vomerine teeth absent; nasal process of premaxilla protruding beyond skull; nasal bones separated from each other, completely disconnected with sphenethmoid; sphenethmoid relatively smooth with several small pits on dorsal surface and ventral surface, the front edge of sphenethmoid rounded and protruding forward, separated from premaxilla; frontoparietal partially divided by a narrowly opening sagittal suture; anterior fontanelle almost occlusive; front and rear part of frontoparietal almost equally wide; posterior edge of exoccipitals posterior to the line connecting conjunctions of quadratojugal and mandible; pterygoid moderate; anterior process of squamosal slender and sharp, tip in the middle of the distance from the base to the junction of pterygoid and quadratojugal, posterior process present; prootic relatively smooth, separated from exoccipitals; anterior process of parasphenoid in shape of narrow trapezoid, anterior part not raised above sphenethmoid, conjunction of anterior process of parasphenoid with width ca. two thirds of the constriction near the base; columella auris short.

Variation

(Suppl. material 2: Fig. S3). Paratypes resemble the holotype in general but with following differences: for CIBMT171060, narrow fringes present on toes, tympanum slightly larger than half eyes, orange granules on dorsum form an “X”-shaped skin ridge and a pair of nearly parallel ridges on dorsolateral trunk, and ventral surface less brown and more orange; for CIBMT171060, a broad “X” pattern present on dorsum, and parallel skin ridges on dorsolateral trunk do not make contact with skin ridges between the left parallel ridge.

Secondary sexual characters

Male with single subgular vocal sac; nuptial pad not observed in October; lineae musculinae absent.

Distribution and natural history

The species is currently only discovered from type locality Renqingbeng Temple area at elevation 2003 m in Medog County, Tibet Autonomous Region, China, inhabits small streams in subtropical forests (Fig. 10D, J; Suppl. material 1: Table S5B). All three individuals were found on short herds or ferns beside small mountain stream at a distance shorter than 0.5 m to the ground (Fig. 10J). Females were found to be gravid during October, but eggs not well developed. Advertisement calls not head in October. Several gravid females of Megophrys cf. pachyproctus were collected from the same small stream with types of Megophrys zhoui sp. nov. on the same night. Theloderma sp. and Amolops nyingchiensis Jiang, Wang, Xie, Jiang, and Che, 2016 were recorded at the same habitat.

Comparison

By body relatively smaller (male 23.0, n = 1; females 23.5–23.9, n = 2 measurements in mm), Megophrys zhoui sp. nov. differs from M. pachyproctus (males 35.3–35.7, n = 2; female 35.8, n = 1), Megophrys cf. pachyproctus (males 33.6–36.6, n = 5; females 40.6–42.8, n = 4), M. medogensis (males 57.2–68.7, n = 21), M. acuta (males 27.1–33.0, n = 10), M. baolongensis (males 41.8–45.0, n = 5), M. binchuanensis (males 32.0–36.0, n = 4), M. binlingensis (males 45.1–51.0, n = 3), M. boettgeri (males 34.5–37.8, n = 20), M. brachykolos (males 33.7–39.3, n = 5), M. caudoprocta (males 70.8–81.3, n = 4), M. cheni (males 26.2–29.5, n = 15), M. daweimontis (males 34–37, n = 18), M. fansipanensis (males 30.9–44.3, n = 13), M. hoanglienensis (males 37.4–47.6, n = 11), M. insularis (males 36.8–41.2, n = 5), M. jingdongensis (males 53.0–56.5, n = 3), M. jinggangensis (males 35.1–36.7, n = 2), M. kuatunensis (males 26.2–31.4, n = 18), M. liboensis (males 61.6–62.9, n = 4), M. lini (males 34.1–39.7, n = 20), M. lishuiensis (males 30.7–34.7, n = 13), M. minor (males 34.5–41.2, n = 4), M. obesa (male 35.6, n = 1; females 37.5–41.2, n = 6), M. omeimontis (males 56.0–59.5, n = 10), M. palpebralespinosa (male 36, n = 1; female 41, n = 1), M. rubrimera (males 26.7–30.5 n = 8), M. sangzhiensis (male 54.7, n = 1), M. shuichengensis (males 102.0–118.3, n = 7), M. spinata (males 47.2–54.4, n = 18), M. tuberogranulata (males 33.2–39.6, n = 9), M. wuliangshanensis (males 27.3–31.6, n = 10), M. wushanensis (males 30.4–35.5, n = 10), M. ombrophila (males 27.4–34.5, n = 5), M. leishanensis (males 32.1–42.3, n = 10), M. dongguanensis (males 30.2–39.3, n = 9), M. nankunensis (males 29.9–34.9, n = 11), M. jiulianensis (males 30.4–33.9, n = 9), M. nanlingensis (males 30.5–37.3, n = 10), M. wugongensis (males 31.0–34.1, n = 4), M. mufumontana (males 30.1–30.8, n = 2), M. feii (males 24.5–25.1, n = 4; female 28.2–28.9, n = 2), M. vegrandis (males 27.5–30.6, n = 4), M. aceras (males 55.8–62.4, n = 6); M. ancrae (males 39.1–45.3, n = 8), M. auralensis (males 76.7, n = 1), M. damrei (male 57.1, n = 1; female 69.1, n = 1), M. flavipunctata (males 56.9–68.4, n = 4), M. glandulosa (males 76.3–81.0, n = 10), M. himalayana (males 68.0–73.5, n = 6), M. huangshanensis (males 36.0–41.6, n = 4), M. katabhako (males 35.4–37.0, n = 3), M. lekaguli (males 55.6–66.6, n = 8), M. longipes (male 47, n = 1; female 65, n = 1), M. major (males 71.6–87.5, n = 12), M. mangshanensis (male 62.5, n = 1; female 73.0, n = 1), M. maosonensis (male 77, n = 1; female 94, n = 1), M. megacephala (males 45.9–53.4, n = 12), M. monticola (males 38.4–49.5, n = 17), M. periosa (males 71.3–93.8, n = 12), M. robusta (males 73.5–83.1, n = 6), M. longipes (male 47, n = 1; female 65, n = 1), M. oreocrypta (female 94.9, n = 1), M. oropedion (males 32.8–39.2, n = 7), M. parva (males 35.6–50.6, n = 5), M. periosa (males 71.3–93.8, n = 12), M. robusta (males 73.5–83.1, n = 6), M. sanu (males 39.0–46.7, n = 5), M. serchhipii (male 37.1, n = 1), M. takensis (males 47.3–53.0, n = 3), M. zhangi (males 32.5–37.2, n = 3), M. zunhebotoensis (male 30.0, n = 1; female 39.0, n = 1), M. angka (males31.2–32.1, n = 2), M. shunhuangensis (males 30.3–33.7, n = 10), M. jiangi (males 34.4–39.2, n = 9), and M. xianjuensis (males 31.0–36.3, n = 7).

By tympanum distinct moderate, larger than half eye diameter, Megophrys zhoui sp. nov. differs from M. gigantica, M. nankiangensis, M. shapingensis, and M. wawuensis (vs. tympanum absent, concealed or very small in the latter).

By maxillary teeth present, Megophrys zhoui sp. nov. differs from M. elfina, M. gerti, M. hansi, M. koui, M. microstoma, and M. synoria (vs. absent in the latter).

By hind limbs long and head not wide and flat, Megophrys zhoui sp. nov. differs from M. carinense, M. chuannanensis, M. feae, M. intermedia, and M. popei (vs. head wide flat and hind limbs short in the latter).

By lacking a single, wide and flat palpebral projection on the edge of the upper eyelid, Megophrys zhoui sp. nov. differs from M. lancip, M. montana, M. parallela, M. baluensis, M. edwardinae, M. kobayashii, M. ligayae, M. nasuta, and M. kalimantanensis (vs. present in the latter).

By lacking rostral appendage, Megophrys zhoui sp. nov. differs from M. stejnegeri (vs. having less rostral appendage in the latter).

By lacking a distinct horn-like tubercle at edge of upper eyelid, Megophrys zhoui sp. nov. differs from M. dringi (vs. present in the latter).

By vomerine ridge weak, Megophrys zhoui sp. nov. differs from M. pachyproctus, M. medogensis, and Megophrys cf. pachyproctus (vs. vomerine ridge stronger in the latter); differs from M. vegrandis, M. baolongensis, M. binchuanensis, M. boettgeri, M. kuatunensis, M. lishuiensis, M. wuliangshanensis, M. wushanensis, M. ombrophila, M. leishanensis, M. feii, M. huangshanensis, M. shunhuangensis, and M. jiangi (vs. absent in the latter).

By vomerine teeth absent, Megophrys zhoui sp. nov. differs from Megophrys cf. pachyproctus, M. pachyproctus, M. medogensis, M. caudoprocta, M. daweimontis, M. fansipanensis, M. hoanglienensis, M. insularis, M. jingdongensis, M. jinggangensis, M. liboensis, M. omeimontis, M. rubrimera, M. dongguanensis, M. nankunensis, M. jiulianensis, M. nanlingensis, M. aceras, M. ancrae, M. damrei, M. flavipunctata, M. glandulosa, M. himalayana, M. katabhako, M. lekaguli, M. longipes, M. major, M. mangshanensis, M. maosonensis, M. megacephala, M. monticola, M. oreocrypta, M. oropedion, M. parva, M. periosa, M. serchhipii, M. takensis, M. zhangi, and M. zunhebotoensis (vs. present in the latter).

By toes with narrow lateral fringes or absent, Megophrys zhoui sp. nov. differs from M. binchuanensis, M. cheni, M. jingdongensis, M. lini, M. rubrimera, M. shuichengensis, M. spinata, M. feii, M. vegrandis, and M. glandulosa (vs. wide in the latter).

By dorsal skin relatively smooth, Megophrys zhoui sp. nov. differs from M. pachyproctus, Megophrys cf. pachyproctus, M. insularis, M. jinggangensis, M. tuberogranulata, M. wuliangshanensis, M. leishanensis, M. dongguanensis, M. jiulianensis, M. nanlingensis, M. wugongensis, M. mufumontana, and M. feii (vs. rough in the latter).

By tympanum moderate (TYD/EL 0.40–0.60, n = 9), Megophrys zhoui sp. nov. differs from species with large tympanum: M. brachykolos (0.70–0.75, n = 7); M. jinggangensis (0.73–0.88, n = 5), and M. takensis (0.71–0.77, n = 4).

By fingertips not expanded into small pads, Megophrys zhoui sp. nov. differs from M. vegrandis, M. ancrae, and M. feii (vs. fingertips with small pads in the latter).

By the following characters, Megophrys zhoui sp. nov. differs from M. pachyproctus: protuberance beyond cloaca small, not visible from ventral view, not swollen (vs. protuberance present on vent beyond cloaca large, swollen, arc-shaped, visible on both dorsal and lateral view in the latter); and inner metatarsal tubercle long oval, positioned on base of toe I (vs. inner metatarsal tubercle rounded, separate from base of toe I at a distance nearly twice its diameter in the latter).

By having following differences on skull morphology, Megophrys zhoui sp. nov. differs from M. pachyproctus: premaxillary and maxillary teeth weak, barely visible or separated from others by gaps (vs. strong, closely positioned with others in the latter); nasal bones not contact with sphenethmoid (vs. mostly in the latter); and middle front edge of sphenethmoid protruding (vs. truncate in the latter).

By base of finger I in similar size with finger II, relative finger lengths I < II < IV < III, Megophrys zhoui sp. nov. differs from M. medogensis (vs. base of finger I distinctly larger than finger II, relative finger lengths II < I < IV < III in the latter).

By having following differences on skull, Megophrys zhoui sp. nov. differs from M. medogensis: skull weakly ossified, opening of anterior fontanelle present, sagittal suture narrowly or wide open (vs. skull well ossified, opening of anterior fontanelle and sagittal suture occlusive in the latter); premaxillary and maxillary teeth weak, barely visible or separated from others by gaps (vs. strong, closely positioned with others in the latter); frontoparietal front equals rear (vs. distinctly wider in the latter); exoccipitals posterior to the line connecting conjunctions of quadratojugal and mandible (vs. anterior); and columella auris short (vs. long in the latter).

By base of finger I similar in size with finger II, nasal bones not in contact with sphenethmoid, and texture of sphenethmoid relatively smooth with several small pits, Megophrys zhoui sp. nov. differs from Megophrys cf. pachyproctus (vs. base of finger I larger than the base of finger II, nasal bones mostly contact with sphenethmoid, and sphenethmoid rough with curves and pits in the latter).

Megophrys yeae sp. nov.

Figs 3E, J, O, T, Y, 4C, F, I, L, 5E, J, 6D, H, L, P, 9, 10A, C, G, Suppl. material 2: Figs S4, S5A, S5B, S5E, S5F; Tables 1, 2, 3, 4, Suppl. material 1: Tables S1-S3, S5

Holotype

(Figs 3E, J, O, T, Y, 4C, F, I, L, 9). CIB201706MT02, adult male, collected in Beibeng village, Medog County, Tibet Autonomous Region, China (29.24292°N, 95.18561°E, 870 m), at 1:40 h on 15 June 2017 by SC Shi and L Ding.

Paratypes

Thirteen specimens (eleven males and two females) from Medog County, Tibet Autonomous Region, China. Four adult males (CIB201706MT01, CIB022017061102, CIB022017061103, and CIB022017061104) collected in Didong village (29.22508°N, 95.12463°E, 670 m) on 11 June 2017 by SC Shi and L Ding. One adult female (CIB201706MT03) collected on 13 June 2017 in Medog urban neighborhood (29.32213°N, 95.31324°E, 907 m) by SC Shi and L Ding. One adult female (CIBMTXC-201701-043) and one adult male (CIBMTXC-201701-044) collected on 28 May 2017 in Medog City neighborhood by F Xie and DW Yang. Two adult males (CIB022017061606 and CIB022017061407) collected in the same location of holotype by SC Shi and L Ding. One male (CIB022017061804) collected in Bari village (29.32947°N, 95.36016°E, 1780 m) at 21:01 18 June 2017 by S.C. Shi. Two adult males (CIBMT171065 and CIBMT171066) collected on 10 and 24 October 2017 in Yarang village (29.29485°N, 95.28126°E, 795 m) by F Xie and DW Yang. One adult male (CIBMT171064) collected at 23:54, 25 October 2017 in Yadong village in the vicinity of Medog city suburb (29.32654°N, 95. 34397°E, 1073 m) by SC Shi and B Wang.

Etymology

The specific name yeae is in honor of Professor Ye Chang-Yuan, for her contribution to Chinese amphibian research and inspiration for younger generations of Chinese herpetologists.

Suggested vernacular name

Ye’s horned toad (English), Ye Shi Jiao Chan (叶氏角蟾, Chinese).

Diagnoses

Megophrys yeae sp. nov. is assigned to the genus Megophrys sensu lato based on molecular phylogenetic analyses and the following morphological characters: canthus rostralis well-developed; a tiny horn’-like tubercle at edge of upper eyelid present; supratympanic fold distinct; axillary glands small and tit-like, on sides of the breast; oral disc of tadpoles funnel-like; mouth of tadpoles lacking transverse rows of teeth; head length more than 25% of body size; upper jaw protruding beyond the margin of the lower jaw; no skin fold on back of head; maxillary teeth present; tympanum distinct; hind legs long and thin.

Megophrys yeae sp. nov. is distinguished from its congeners by a combination of following characters: body relatively small (males 23.8–29.1 mm, n = 12; females 27.9–31.3 mm, n = 2); vomerine ridge weak, vomerine teeth absent; base of first finger weak, size equal to the base of second finger, tips of fingers II-IV flat, expand to small pad; foot of males shorter (FOL 10.8–12.6 mm, n = 12); dorsal skin being relatively smooth; protuberance beyond cloaca small, not visible from ventral view, not swollen; nuptial pad absent; skull weakly ossified, wider than long; premaxillary and maxillary teeth weak, separated from others by gaps; texture of sphenethmoid smooth, without curves and pits; anterior fontanelle opening large, sagittal suture occlusive; advertisement call short and fast (duration 99–212 ms, repetition rate 1.9–4.1 call/s, intervals, n = 6), and dominant frequency high (4.4–5.2 kHz, n = 6).

Description of holotype

(Figs 3E, J, O, T, Y, 4C, F, I, L, 9). Measurements in mm. Adult male. Body small and slender (SVL 27.5); protuberance beyond cloaca small, not visible from ventral view, not swollen.

Head moderate, wider slightly than long (HW 9.8, HL 9.0, IFE 5.1, IBE 8.7); snout rounded in dorsal view, slightly projecting in profile, protruding beyond lower jaw, rostral appendage absent (SL 3.6); loreal region vertical and concave; canthus rostralis blunt; dorsal surface of snout slight concave; nostrils oval, nearly in the middle of distance from snout to eye(SN 1.9, EN 2.0); distance between nostrils (IN 3.2) almost equal with the shortest distance between upper eyelids (IUE 3.1); tympanum small, rounded, diameter (TYD 1.6) less than half of eye length (EL 3.8 mm), upper one third of tympanum anulus merge with supratympanic fold (Figure 5C); eye-tympanum distance (TYE 1.6) equal to tympanum diameter; pupil near oval, with a gap at lower edge; visible pineal ocellus absent; vomerine ridges weak, interval longer than its length, vomerine teeth absent; tongue feebly notched behind, hardly visible, with no medial lingual process.

Forearm long and slim, forearm length (FAL 7.0) 25% of body length, slightly shorter than hand (HAL 8.3), not enlarged relative to the upper arm; relative finger lengths I < II < IV < III; base of first finger weak, size equal to the base of second finger; tips of finger I rounded, slightly swollen, tips of fingers II-IV flat and expanded, forming small oval pads (FIIIW 1.2, FIVW 1.3), pads without grooves and distinctively larger than terminal phalanges; fingers rudimentary webbed, with ventral callous ridges and narrow lateral fringes; subarticular and supernumerary tubercles absent, palmar tubercles indistinct.

Hindlimbs long and thin, tibio-tarsal articulation reaches area between nostril and eye; heels meet when thighs are positioned at right angles to the body, shank (SHL 14.1) slightly longer than thigh (TL 12.3) and feet (FOL 12.5, TFOL 19.7); toes thin, rudimentary webbed, with ventral callous ridges and narrow fringes; relatively toes lengths I < II < V < III < IV; tips of toes flat and slightly dilated, without grooves, slightly larger than terminal phalanges; inner metatarsal tubercle weak (IMT 1.9) and elliptical, outer metatarsal tubercle, subarticular and supernumerary tubercles absent.

Dorsal body and head relatively smooth, with tiny tubercles scattered on dorsal part of body and limbs; tiny tubercles on most of dorsum form a large “W” skin ridge from behind supratympanic fold curve to ca. one third distance left of groin, a “V” between shoulders ahead of “W”, and a triangle between eyes; edges of snout, eyelids, especially supratympanic fold and flanks scattered with larger tubercles; supratympanic fold thin, extend from rear of eyelid, curves down above tympanum to shoulder; small tubercles on dorsal thigh and shank arranged in several transversal rows. Ventral surface of body smooth; a granular line present on ventrolateral side of belly, interrupted on left side; several small glandular tubercles present around cloaca; pectoral glands small, as large as tips of finger II, raised slightly, close to axilla; single femoral gland on ventral thigh small and slightly raised, closer to knee than cloaca.

Coloration of holotype in life

(Fig. 9). Dorsal head and body light brown, tiny tubercles scattered on dorsum and head orange-red; skin ridges edged with faint brown, forming a barely visible “X” pattern on dorsum and triangular on head; skin ridges on thigh edged with narrow black-brown; supratympanic fold orange-red; one short black vertical bar on each side of upper lips beneath the eyes; tympanum pale gray; a black streak under supratympanic fold; tubercles on flanks edged with small dark blotches; two thin ambiguous transverse dark band on dorsal forearms; fingers II-IV with transverse dark band on dorsal surface; dorsal surface of fingers and toes colored with orange-red; flanks light brown; throat pale dusty gray; chest mottled gray stained with light purple between axillary glands; two pale gray streaks from lower place of joins of jaws extend to half of ventral upper arm; abdomen ivory, mottled gray on upper part, several small dark dots scattered rear; a large dark streaks present on both lateral sides of abdomen, from behind axilla to near groin, bordering the creamy white ventrolateral granular line on belly; groin not colored with red; ventral surfaces of thighs light purple mottled with tiny smoky white pigments; small glandular tubercles around cloaca and ivory; ventral surface of shanks and arms with large dark patches; palm and ventral surfaces of foot purplish gray; tips of digits orange edges; pectoral glands and femoral glands creamy white; iris orange-bronze.

Figure 9. 

The holotype adult male CIB201706MT02 of Megophrys yeae sp. nov. in life. A dorsolateral view of body B ventral view of body C dorsolateral view of head D ventral view of hand E ventral view of foot.

Coloration of holotype in preservative

(Fig. 3E, J, O, T, Y). Dorsal body gray, triangle on head and “X” pattern on dorsum barely visible; tubercles on dorsum, dorsal surface of head and limbs light gray; tympanum brown, a black streak under supratympanic fold; vertical bar beneath the eyes pale gray; tubercles on flanks edged with small black blotches; tubercles rows on dorsal thigh and shank with dark edges more or less; dorsal surface of forelimbs and hindlimbs dark gray with several ambiguous transverse dark bands; fingers II, III and IV with transverse dark band on dorsal surface; tips of digits light colored; throat, chest, and upper abdomen dusty gray, lower part of abdomen off-white; two dark streaks from lower place of joins of jaws extend to half of ventral upper arm; several small dark patches scattered on lower abdomen; two large dark streaks on lateral sides of abdomen; ventral surface of lower arm whitish, with a pale gray patches connected to hand; palm and ventral surface of foot dusty gray, with light colored fingertips; ventral surface of thigh dusty gray, femoral glands and glandular tubercles around cloaca white; ventral surface of shank dusty gray with several large pale gray patches; iris mottled copper.

Skull

(Fig. 5E, J). Skull rather small and weakly ossified, width 1.16× of length; maxillary overlapping with the quadratojugal; premaxillary and maxillary teeth moderately developed, independent with each other, 10/7 teeth present on left/right premaxillary, teeth absent on mandible; vomerine ridge weak, vomerine teeth absent; nasal process of premaxilla protruding beyond skull; nasal bones separated, posterior edges completely contact with sphenethmoid; sphenethmoid smooth on both dorsal and ventral surface, without curves and pits, the front edge of sphenethmoid concave, in contact with premaxillary; frontoparietal not divided, sagittal suture occlusive; anterior fontanelle opening large, triangular, width approximately the same as nasal bones; the front and rear part of frontoparietal almost equally wide; posterior edge of exoccipitals posterior to the line connecting conjunctions of quadratojugal and mandible; pterygoid moderate; anterior process of squamosal slender and sharp, tip closer to the junction of pterygoid and quadratojugal than its base, posterior process present; prootic relatively smooth, separated from exoccipitals; anterior process of parasphenoid in shape of narrow trapezoid, anterior part not raised above sphenethmoid, conjunction of anterior process of parasphenoid with width approximately the same as the constriction near the base; columella auris short.

Variation

(Suppl. material 2: Fig. S4; Suppl. material 1: Table S1). Two female specimens with no lateral fringes on toes. A distinct brown “X” marking on dorsum and a clear triangular on head present on CIB022017061407, CIBMT171064. Abdomens of CIB201706MT01, CIB022017061103 with dark patches on both sides instead of two large streaks. Overall coloration of CIB022017061102, CIB022017061103, CIB022017061103, CIB201706MT01 lighter, without visible “X” marking on dorsum. The “W” skin ridges on dorsum of CIBMT171065 and CIBMT171066 shattered into short disconnected bars. The tympanum of CIBMT171065 not merged with supratympanic fold. The ventrolateral line varies among individuals, in some (e.g., CIB201706MT01) it is interrupted and short, in others (e.g., CIBMT171066) it is simply formed by two separated granules.

Secondary sexual characters

An internal single subgular vocal sac present in male. Vocal openings present at rear of part the mouth. Calling males without nuptial pad on finger.

Advertisement call

(Fig. 4C, F, I, L; Tables 4; Suppl. material 1: Table S3). A total of 19 call groups and 176 calls were analyzed. Megophrys yeae sp. nov. has a high dominant frequency (average 4.7 kHz, range 4.4–5.2 kHz). Calls frequent, average calls per seconds 3.0, vary from 1.9 to 4.1; average intercall interval 218 ms, vary from 649 ms to 119 ms when ambient temperatures vary from 17 °C to 25 °C. The number of calls in each call group average 68.9, range from 5 to 187. Calls short, duration average 139 ms, range from 89 ms to 246 ms. Pules per call average 9.2, vary from 7 to 12. To human ears, sound like cricket.

Tadpole

(Fig. 6D, H, L, P; Suppl. material 1: Table S2). Stages 28–35. Body length range from 10.2–11.4 mm, elongated and slender; oral disk funnel like; positioned anterior-dorsal, large, width average 1.4 (1.1–1.5) × of maximum body width, with five nearly parallel rows of oval submarginal papillae on middle lower lip, three rows of oval submarginal papillae on both sides of upper lips, both submarginal papillae rows on upper and lower lips rows pointing towards oral cavity, smaller outer sides; nares oval and are closer to the eye than to the snout (RN/NE average 2.2, 1.8–2.6); internarial distance average 69% (64–78%) of the interorbital distance; eyes positioned dorsolaterally, the pupils rounded; spiracle in right-handed helix from ventral view, spiracular tube not protruding beyond body wall, positioned 53% (47–57%) of the distance between tip of the snout and trunk-tail junction, and opens laterally; the tail makes up average 69% (67–72%) of the total body length; dorsal fin arise behind trunk-tail junction, average 35% (30–41%) of maximum body height; the basal tail width average 60% (48–65%) of the maximal trunk width; keratodonts absent.

Coloration of tadpoles in life: dorsal body brown with dense copper pigments; dorsal tail brown, scattered with copper pigments; lateral tail above lower fin mottled with copper patches; ventral surface of body, and lower fin semi-transparent; iris light brown. Coloration in preservative: dorsal body brown; dorsal tail light brown scattered with brown patches; lateral sides of body brown; lateral tail semitransparent brown, muscle scattered with a lot of distinct brown patches; fins semitransparent stained with little brown, no pigments on lower fin except latter 1/3; ventral body semitransparent white, with tiny gray pigments scattered on throat and chest; ventral tail off-white; lips semitransparent white, papillae brown.

Distribution and natural history

This species is currently known from five localities in Medog County, Tibet Autonomous Region, China (Fig. 1). All calling males recorded on June and October were found on herb leaves near or upon small stream in tropical forest (Fig. 10A, C, G; Suppl. material 2: Fig. S5A, B, E, F). Eggs in adult female (CIB201706MT03) are in two different development stage: pure yellow eggs with diameter of 1.1 mm, and semitransparent eggs with size half or less of the former. A total of 45 larger yellow eggs were counted, smaller semitransparent eggs more than 70. Thus, breeding season is suggested including June to October, and this species may lay eggs more than once during one season. The new species was recorded at elevation between 670 m to 1780 m. On 18 June 2017, four males of Megophrys pachyproctus (CIB201706MT04–CIB022016061806, CIB022017061807) were calling in the same stream where one male (CIB022017061804) of the new species was calling together at nearest distance ca. 3 meters in Bari Village (29.32947°N, 95.36016°E, 1780 m). From its habitat, other amphibians like Megophrys medogensis, Megophrys cf. pachyproctus, Odorrana zhaoi Li, Lu, and Rao, 2008, Amolops medogensis, and Huangixalus translineatus Wu, 1977 were also recorded.

Figure 10. 

Microhabitats of Megophrys toads in the field in Medog. A stream at elevation 850 m in Didong village, harboring the low-middle-elevation M. medogensis and Megophrys yeae sp. nov. B a stream at 1530 m in Gelin village, hosting the low-middle-elevation M. medogensis and M. pachyproctus C a stream at 1780 m in Bari village, harboring low-middle-elevation M. medogensis, M. cf. pachyproctus and Megophrys yeae sp. nov. D a stream at 2003 m in the vicinity of Renqingbeng Temple, hosting M. cf. pachyproctus and Megophrys zhoui sp. nov. E a stream at 2142 m in Gedang village, hosting M. medogensis F one adult male of low-middle-elevation M. medogensis calling on a dead leaf on the tropical forest ground nearby a stream in Didong village G the adult male paratype CIB022017061102 of Megophrys yeae sp. nov. calling on a leaf of dense bushes under tropical forest, ca. 0.5 m above a stream in Didong village H the adult male CIB022017061806 of M. cf. pachyproctus calling on a branch of dead bush, ca. 0.5 m above ground under subtropical forest in Bari village I the gravid female CIBMT171054 of M. cf. pachyproctus precariously climbing up onto a stem of herb, ca. 0.3 m above a tiny stream under subtropical forest in Renqingbeng J the adult male holotype CIBMT171053 of Megophrys zhoui sp. nov. sitting on a split of a fern leaf in a small stream under subtropical forest in the vicinity of Renqingbeng Temple.

Comparison

By body relatively smaller (males 23.8–29.1, n = 12; females 27.9–31.3, n = 2; measurements in mm), Megophrys yeae sp. nov. differs from M. pachyproctus (males 35.3–35.7, n = 2; female 35.8, n = 1), Megophrys cf. pachyproctus (males 33.6–36.6, n = 5; females 40.6–42.8, n = 4), M. medogensis (males 57.2–68.7, n = 21), M. baolongensis (males 41.8–45.0, n = 5), M. binchuanensis (males 32.0–36.0, n = 4), M. binlingensis (males 45.1–51.0, n = 3), M. boettgeri (males 34.5–37.8, n = 20), M. brachykolos (males 33.7–39.3, n = 5), M. caudoprocta (males 70.8–81.3, n = 4), M. daweimontis (males 34–37, n = 18), M. fansipanensis (males 30.9–44.3, n = 13), M. hoanglienensis (males 37.4–47.6, n = 11), M. insularis (males 36.8–41.2, n = 5), M. jingdongensis (males 53.0–56.5, n = 3), M. jinggangensis (males 35.1–36.7, n = 2), M. liboensis (males 61.6–62.9, n = 4), M. lini (males 34.1–39.7, n = 20), M. lishuiensis (males 30.7–34.7, n = 13), M. minor (males 34.5–41.2, n = 4), M. obesa (male 35.6, n = 1; females 37.5–41.2, n = 6), M. omeimontis (males 56.0–59.5, n = 10), M. palpebralespinosa (male 36, n = 1; female 41, n = 1), M. sangzhiensis (male 54.7, n = 1), M. shuichengensis (males 102.0–118.3, n = 7), M. spinata (males 47.2–54.4, n = 18), M. tuberogranulata (males 33.2–39.6, n = 9), M. wushanensis (males 30.4–35.5, n = 10), M. leishanensis (males 32.1–42.3, n = 10), M. dongguanensis (males 30.2–39.3, n = 9), M. nankunensis (males 29.9–34.9, n = 11), M. jiulianensis (males 30.4–33.9, n = 9), M. nanlingensis (males 30.5–37.3, n=10), M. wugongensis (males 31.0–34.1, n = 4), M. mufumontana (males 30.1–30.8, n = 2), M. aceras (males 55.8–62.4, n = 6); M. ancrae (males 39.1–45.3, n = 8), M. auralensis (males 76.7, n = 1), M. damrei (male 57.1, n = 1; female 69.1, n = 1), M. flavipunctata (males 56.9–68.4, n = 4), M. glandulosa (males 76.3–81.0, n = 10), M. himalayana (males 68.0–73.5, n = 6), M. huangshanensis (males 36.0–41.6, n = 4), M. katabhako (males 35.4–37.0, n = 3), M. lekaguli (males 55.6–66.6, n = 8), M. longipes (male 47, n = 1; female 65, n = 1), M. major (males 71.6–87.5, n = 12), M. mangshanensis (male 62.5, n = 1; female 73.0, n = 1), M. maosonensis (male 77, n = 1; female 94, n = 1), M. megacephala (males 45.9–53.4, n = 12), M. monticola (males 38.4–49.5, n = 17), M. periosa (males 71.3–93.8, n = 12), M. robusta (males 73.5–83.1, n = 6), M. longipes (male 47, n = 1; female 65, n = 1), M. oreocrypta (female 94.9, n = 1), M. oropedion (males 32.8–39.2, n = 7), M. parva (males 35.6–50.6, n = 5), M. periosa (males 71.3–93.8, n = 12), M. robusta (males 73.5–83.1, n = 6), M. sanu (males 39.0–46.7, n = 5), M. serchhipii (male 37.1, n = 1), M. takensis (males 47.3–53.0, n = 3), M. zhangi (males 32.5–37.2, n = 3), M. zunhebotoensis (male 30.0, n = 1; female 39.0, n = 1), M. angka (males 31.2–32.1, n = 2), M. shunhuangensis (males 30.3–33.7, n = 10), M. jiangi (males 34.4–39.2, n = 9), and M. xianjuensis (males 31.0–36.3, n = 7).

By tympanum distinct moderate, Megophrys yeae sp. nov. differs from M. gigantica, M. nankiangensis, M. shapingensis, and M. wawuensis (vs. absent, concealed or very small in the latter).

By maxillary teeth present, Megophrys yeae sp. nov. differs from M. elfina, M. gerti, M. hansi, M. koui, M. microstoma, and M. synoria (vs. absent in the latter).

By hind limbs long and head not wide and flat, Megophrys yeae sp. nov. differs from M. carinense, M. chuannanensis, M. feae, M. intermedia, and M. popei (vs. head wide flat and hind limbs short in the latter).

By lacking a single, wide and flat palpebral projection on the edge of the upper eyelid, Megophrys yeae sp. nov. differs from M. lancip, M. montana, M. parallela, M. baluensis, M. edwardinae, M. kobayashii, M. ligayae, M. nasuta, and M. kalimantanensis (vs. present in the latter).

By lacking rostral appendage, Megophrys yeae sp. nov. differs from M. stejnegeri (vs. having less rostral appendage in the latter).

By lacking a distinct horn-like tubercle at edge of upper eyelid, Megophrys yeae sp. nov. differs from M. dringi (vs. present in the latter).

By vomerine ridge weak, Megophrys yeae sp. nov. differs from M. pachyproctus, M. medogensis, and Megophrys cf. pachyproctus (vs. stronger in the latter); differs from M. vegrandis, M. baolongensis, M. binchuanensis, M. boettgeri, M. kuatunensis, M. lishuiensis, M. wuliangshanensis, M. wushanensis, M. ombrophila, M. leishanensis, M. feii, M. huangshanensis, M. shunhuangensis, M. jiangi, and M. xianjuensis (vs. absent in the latter).

By vomerine teeth absent, Megophrys yeae sp. nov. differs from Megophrys cf. pachyproctus, M. pachyproctus, M. medogensis, M. caudoprocta, M. daweimontis, M. fansipanensis, M. hoanglienensis, M. insularis, M. jingdongensis, M. jinggangensis, M. liboensis, M. omeimontis, M. rubrimera, M. dongguanensis, M. nankunensis, M. jiulianensis, M. nanlingensis, M. aceras, M. ancrae, M. damrei, M. flavipunctata, M. glandulosa, M. himalayana, M. katabhako, M. lekaguli, M. longipes, M. major, M. mangshanensis, M. maosonensis, M. megacephala, M. monticola, M. oreocrypta, M. oropedion, M. parva, M. periosa, M. serchhipii, M. takensis, M. zhangi, and M. zunhebotoensis (vs. present in the latter).

By tips of fingers II-IV flat, expand to small pad, Megophrys yeae sp. nov. differs from Megophrys cf. pachyproctus, Megophrys zhoui sp. nov., M. pachyproctus, M. acuta, M. binlingensis, M. brachykolos, M. cheni, M. lini, M. minor, M. obesa, M. palpebralespinosa, M. sangzhiensis, M. shuichengensis, M. spinata, M. tuberogranulata, M. wugongensis, M. mufumontana, M. auralensis, and M. robusta (vs. expanded pads on fingertips absent in the latter).

By foot of males shorter (FOL 10.8–12.6 mm, n = 12), tympanum relatively smaller (males TD/EL 0.36–0.46, n = 12), and toes with narrow lateral fringes, Megophrys yeae sp. nov. further differs from M. vegrandis (vs. FOL 13.2–13.8 mm, n = 4, P < 0.001; TYD/EL 0.44–0.56, n = 4, P < 0.03; and fringes on toes wide in the latter).

By dorsal skin being relatively smooth, Megophrys yeae sp. nov. differs from M. feii (vs. dorsal skin rough in the latter).

Megophrys yeae sp. nov. differs from M. medogensis by the following characters: nuptial pad absent (vs. present in the latter); and base of first finger weak, size equal to the base of second finger, relative finger lengths I < II < IV < III (vs. base of finger I strong, larger than base of finger II, relative finger lengths II < I < IV < III in the latter).

By having following differences on skull morphology, Megophrys yeae sp. nov. differs from M. medogensis: skull weakly ossified, opening of anterior fontanelle large (vs. skull well ossified, opening of anterior fontanelle occlusive in the latter); premaxillary and maxillary teeth weak, separated from others by gaps (vs. strong, closely positioned with others in the latter); texture of sphenethmoid smooth, without curves and pits (vs. relatively smooth, with few pits in the latter); frontoparietal front equals rear (vs. distinctly wider in the latter); exoccipitals posterior to the line connecting conjunctions of quadratojugal and mandible (vs. anterior); and columella auris short (vs. long in the latter).

By having following differences on bioacoustics, Megophrys yeae sp. nov. differs from M. medogensis: dominant frequency significantly higher (4.4–5.2 kHz vs. 2.3–3.0 kHz in the latter; P < 0.001); call significantly faster (repetition rate average 3.0, vary from 1.9 to 4.1 vs. average 1.2 vary from 0.6 to 2.2 in the latter); and call intervals significantly longer (493–720 ms vs. 153–254 ms in the latter; P < 0.001).

By having the following characters, Megophrys yeae sp. nov. differs from M. pachyproctus: lacking a swollen protruding beyond cloaca (vs. present in the latter); nuptial pad absent (vs. present in the latter); and base of first finger weak, size equal to the base of second finger (vs. base of finger I strong, larger than base of finger II in the latter).

By having the following characters on skull morphology, Megophrys yeae sp. nov. differs from M. pachyproctus: premaxillary and maxillary teeth weak, separated from others by gaps (vs. strong, closely positioned with others in the latter); texture of sphenethmoid smooth, without curves and pits (vs. relatively smooth, with few pits in the latter); anterior fontanelle opening large (vs. occlusive in the latter); and sagittal suture occlusive (vs. distinctly open in the latter).

By having the following characters, Megophrys yeae sp. nov. differs from Megophrys cf. pachyproctus: nuptial pad absent (vs. present on finger I in the latter); and base of first finger weak, size equal to the base of second finger (vs. strong, larger than base of finger II in the latter).

By having following characters on skull morphology, Megophrys yeae sp. nov. differs from Megophrys cf. pachyproctus: texture of sphenethmoid smooth, without curves and pits (vs. rough, with curves and pits in the latter); anterior fontanelle opening large (vs. small, width equals sagittal suture in the latter); and sagittal suture occlusive (vs. distinctly open in the latter).

By having the following acoustical characters, Megophrys yeae sp. nov. differs from Megophrys cf. pachyproctus: call significantly shorter (99–212 ms, n = 6 vs. 491–889 ms, n = 3 in the latter; P < 0.001); dominant frequency much higher (4.4–5.2 kHz, n = 6 vs. 3.2–3.3 kHz, n = 3 in the latter; P < 0.001); call intervals significantly shorter (146–370 ms, n = 6, vs. 493–720 ms, n = 3 in the latter; P < 0.001); and calls significantly faster (call repetition rate1.9–4.1 call/s, n = 6, vs. 0.7–1.1call/s, n = 3 in the latter; P < 0.01).

By having following characters on skull morphology, Megophrys yeae sp. nov. differs from Megophrys zhoui sp. nov.: texture of sphenethmoid smooth, without curves and pits (vs. relatively smooth, with several small pits in the latter); and sagittal suture occlusive (vs. narrowly or wide open in the latter).

Discussion

Similar to our surveys, only relatively few herpetologists have conducted field work in the eastern corner of Himalayas, mainly in Medog County, China (e.g., Huang and Fei 1981; Fei et al. 1983; Li et al. 2010; Jiang et al. 2012; Jiang et al. 2016a, b, and c). Several factors probably hindered the discoveries of the three new Megophrys species described here. First, in this region, M. medogensis, especially its tadpoles, are almost sympatric with all other related species’ tadpoles at extensive elevations even in the microhabitats, probably arousing the judgement of “one population with one species”. Moreover, the related species were superficially similar morphologically, easily misleading the identifications if made without detailed examination, especially for the first identification in the field. Of course, the third was insufficient expeditions. Chen et al. (2016) recognized two specimens KIZ010978 and KIZ011175 from Medog County as M. pachyproctus without reporting their morphological information. But in our phylogenetic trees, these two specimens were deeply nested into the Megophrys yeae sp. nov. clade (Fig. 2). Additionally, our results suggested that Megophrys yeae sp. nov. differs distinctly from M. pachyproctus on morphology (Figs 3, 5; Tables 1, 2). Hence, we propose that the two specimens were misidentified in this literature and that they should be classified as Megophrys yeae sp. nov. Similarly, Liu et al. (2018) treated one sample SYSa002934 from Medog County as M. pachyproctus. Our analyses, however, nested this sample into the M. medogensis clade (Fig. 2). Megophrys pachyproctus and M. medogensis should be classified as different species groups based on their morphology: the much larger body size of M. medogensis in the large-body-size clade (M. major complex proposed in Mahony et al. 2018), also indicating that the two species should be phylogenetically distinct. In any case, all these specimens should be reexamined.

By the protruding vent, M. pachyproctus differs from almost all species of Megophrys except M. caudoprocta and M. koui. The protruding vent of M. caudoprocta includes an elongated urostyle that slightly exceed ischium (Shen et al. 2013: fig. 1). However, the protruding vent of M. pachyproctus is a swelling and the urostyle does not exceed the vent. Furthermore, according to Yang and Rao (2008), the specimens of Megophrys (Ophryophyne) from the type locality (Zhushihe, Mengla, Yunnan Province, China) of M. koui vary in the presence of protruding vent while they share other morphological characters (identical skin ridge patterns and horn on outer edge of upper eyelid). Furthermore, M. pachyproctus was described based only on two males and one female. All these observations increase the uncertainty of whether the swelling protrusion can be used as a diagnostic character of M. pachyproctus. Our specimens M. cf. pachyproctus from Renqinbeng and Bari differ from the holotype of M. pachyproctus from Gelin mostly in the following characters: protuberance beyond cloaca small, barely visible from ventral view, not swollen (vs. protuberance present on vent beyond cloaca large, swollen, arc-shaped, visible on both dorsal and lateral view in the latter); and inner metatarsal tubercle distinct partially fused with toe I (vs. separate from base of toe I at a distance nearly twice its diameter in the latter). But M. cf. pachyproctus is similar to M. pachyproctus on many other morphological characters (e.g., body measurements, skin texture and skin ridges, and most characters on skull; Suppl. material 1: Table S5). For the moment, only one specimen (the holotype) of M. pachyproctus was examined, and there is no available molecular evidence from samples from Gelin; therefore, it is not prudent to erect a new name while there are still enigmas. Thus, we temporally treat these specimens from Renqingbeng and Bari as M. cf. pachyproctus. Further sampling at Gelin would help to resolve this problem in the future.

In this work, we classified samples of M. medogensis as low-middle-elevation group (682–1560 m) and high-elevation group (> 2100 m), because these samples phylogenetically clustered into two lineages based on mitochondrial DNA dataset but formed a single lineage when based on nuclear DNA dataset. The discordance indicates introgression between these two groups. The tadpoles of high-elevation group are morphologically different from the low-middle-elevation group: body coloration deep brown with copper pigmentation vs. body yellow-brown without copper pigmentation; tail muscle weaker (TMW/BW 44%) than the latter (TMW/BW 53–57%); lateral tail without dark patches vs. present. The morphological comparisons between adults of the two groups were not applicable in this work because no adults of the high-elevation groups were collected. The scenario of phylogenetical discordance between different gene datasets was also found in M. monticola (Mahony et al. 2018). The mechanism of two discovered cases of introgression from southeastern Himalayan is fascinating for further study. Note that the phylogenetically sister species of Megophrys in this region (i.e., Megophrys yeae sp. nov., Megophrys zhoui sp. nov., and M. vegrandis being genetically closer; Fig. 2) are distributed in different sites or altitudes, i.e., Megophrys zhoui sp. nov. just lives above 2000 m near the Renqingbeng Temple in Medog County, Megophrys yeae sp. nov. has a larger range but in some other sites at elevations between 500–1800 m (Figs 1, 10, and Suppl. material 2: Fig. S5) in Medog, and M. vegrandis has been just found at 1110 m in a southwestern locality away from the type localities of the first two relatives (Mahony et al. 2013). This case fits the “micro-endemism” model (Liu et al. 2018; Wang et al. 2019) for separating closely related species. On the other hand, the more “phylogenetically distant” species are often sympatric in microhabitat, such as tadpoles of Megophrys cf. pachyproctus and Megophrys yeae sp. nov. in the same pond, and M. medogensis with Megophrys cf. pachyproctus, Megophrys zhoui sp. nov., and Megophrys yeae sp. nov. in the same stream, indicating the “sympatric but phylogenetically distant” model. These biogeographical patterns have often been found in Megophrys (Mahony et al. 2018; Liu et al. 2018; Wang et al. 2019), indicating a complicated picture of biogeographical history of this taxonomically diverse toad group.

Separations of the horned toad species in Medog are also likely reflected on their different behaviors. Although being sympatric even in the same stream at elevations between 1500–1800 m in Bari village (Fig. 1), M. medogensis prefers tropical and subtropical forest floor (Fig. 10F), while Megophrys yeae sp. nov. is typically found calling on the leaves of tall dense plants (Fig. 10G), and Megophrys cf. pachyproctus calls on the branches of bushes (Fig. 10H). It is interesting that Megophrys cf. pachyproctus possess unique long calls, making a distinctly contrast with Megophrys yeae sp. nov. in the same stream which emit short calls (Fig. 4B, C; Table 3). The distinct calling patterns especially in the two “standing-upper” species probably prevent their calls overlapping in the upper space. This kind of “so-small-microhabitat” niche divergences may be also related with phenotype differences between them. The “floor” toad M. medogensis presents bigger body size, while the two “standing-on-plants or leaves” species have a pale body. Probably for further isolations, Megophrys cf. pachyproctus with relatively moderate body size prefers relative harder branches, vines, or stem of plants (Fig. 10H, I), while the sympatric species Megophrys yeae sp. nov. and Megophrys zhoui sp. nov. often stand on soft leaves and/or grass by their lighter body (Fig. 10G, J), even developing finger pads for climbing like tree frogs (Table 2; Fei et al. 2009). It is fascinating on exploring how their behaviors with corresponding morphological characteristics have been evolved to fitting corresponding environments.

The discoveries of the new species indicate a much-underestimated biodiversity in the Himalayan Mountains. Yet, the amphibians in the region are suffering from obvious threats in their habitats, for example, the ongoing construction of roads, towns, and houses, the use of pesticide chemicals for farming, and increasing activities of tourists. And, we also still have a poor understanding of the influences of local and/or global climatic changes. Undoubtedly, it is urgent to investigate their population status for the conservation of these extraordinary toads.

Acknowledgements

We thank Dengwei Yang, Yin Qi, Bo Cai, Guocheng Shu, and Yong Huang for their assistance in the field. We are grateful to the Administration of Yarlung Zangbo Grand Canyon National Nature Reserve for issuing relevant permits for our field work. We are grateful to the editor Nathalie Yonow who provided many suggestions on language and descriptions. This research is supported by the Second Tibetan Plateau Scientific Expedition and Research Program (STEP) (Grant Nos.: 2019QZKK05010503 and 2019QZKK0501), Project supported by the biodiversity investigation, observation and assessment program (2019–2023) of Ministry of Ecology and Environment of China (2019HJ2096001006), and China Biodiversity Observation Networks (Sino BON–Amphibian & Reptile).

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1 For detailed comparisons, we re-described M. pachyproctus mainly based on the holotype NWIPB 770650 and added our skull information for it.
2 For detailed comparisons, we supplied morphology description of skull and tadpole for M. medogensis mainly based on the specimens collected in this study and added bioacoustics data for this species.
3 For detailed comparisons between these all related species, we independently described specimens of M. cf. pachyproctus in detail.