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A new Megophrys Kuhl & Van Hasselt (Amphibia, Megophryidae) from southeastern China
expand article infoBin Wang, Yan-Qing Wu§, Jun-Wei Peng|, Sheng-Chao Shi, Ning-Ning Lu, Jun Wu§
‡ Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, China
§ Ministry of Ecology and Environment of China, Nanjing, China
| Xianju Biodiversity Development Company Limited, Taizhou, China
Open Access

Abstract

A new species of the genus Megophrys from Zhejiang Province, China is described. Molecular phylogenetic analyses supported the new taxon as an independent clade nested into the Megophrys clade and sister to M. lishuiensis. The new species could be distinguished from its congeners by a combination of the following morphological characteristics: (1) small size (SVL 31.0–36.3 mm in male and 41.6 mm in female); (2) vomerine ridge present and vomerine teeth absent; (3) tongue not notched behind; (4) a small horn-like tubercle at the edge of each upper eyelid; (5) tympanum distinctly visible, rounded; (6) two metacarpal tubercles in hand; (7) relative finger lengths: II < I < IV < III; (8) toes with rudimentary webbing at bases; (9) heels overlapping when thighs are positioned at right angles to the body; (10) tibiotarsal articulation reaching tympanum to eye when leg stretched forward; (11) an internal single subgular vocal sac in male; (12) in breeding male, the nuptial pads with black nuptial spines on the dorsal bases of the first and second fingers.

Keywords

Taxonomy, new species, molecular phylogenetic analysis, morphology, Zhejiang Province, China

Introduction

Megophryidae Bonaparte, 1850 (Amphibia: Anura) is a large Asian toad family which was divided into three subfamilies, i.e., Leptobrachiinae Dubois, 1983, Leptolalaginae Delorme, Dubois, Grosjean & Ohler, 2006 and Megophryinae Bonaparte, 1850 (Delorme et al. 2006). The generic and/or subgeneric classifications in Megophryinae have been under debates for a long time (Tian and Hu 1983; Dubois 1987 “1986”; Lathrop 1997; Rao and Yang 1997; Dubois and Ohler 1998; Jiang et al. 2003; Delorme et al. 2006; Frost et al. 2006; Fei and Ye 2009, 2012, 2016; Chen et al. 2017; Mahony et al. 2017; Liu et al. 2018; Munir et al. 2018) though recent phylogenetic studies clustered all recognised species of Megophryinae into one big clade (e.g., Chen et al. 2017; Mahony et al. 2017; Li et al. 2018). Mahony et al. (2017) classified all members of Megophryinae into a single genus Megophrys Kuhl & Van Hasselt, 1822 including seven subgenera (Megophrys, Xenophrys Günther, 1864, Panophrys Rao & Yang, 1997, Atympanophrys Tian & Hu, 1983, Ophryophryne Boulenger, 1908, Pelobatrachus Beddard, 1908 (1907), and Brachytarsophrys Tian & Hu, 1983) based on molecular phylogenetics and morphological comparisons. Partly based on this point, Frost (2019) placed all recognised species of Megophryinae into the genus Megophrys but with no subgeneric classifications.

The genus Megophrys is widely distributed from eastern China through the eastern and southern Himalayas, throughout mainland Indochina and the islands of the Sunda shelf in Indonesia and parts of the Philippines (Frost 2019). It currently contains 92 recognised species (Frost 2019), and noticeably, 37 species were described in the last decade (See species list of Megophrys in Frost 2019). The genus is suggested to harbour both cryptic diversity and highly localised species diversification (Chen et al. 2017; Mahony et al. 2017, 2018; Liu et al. 2018). For example, in the genus, just in China, 41 cryptic species were suggested by molecular phylogenetic analyses in Liu et al. (2018), and six of them were subsequently described (Li et al. 2018; Wang et al. 2019). To present, 48 Megophrys species have been described in China. Obviously, we still need to verify that undescribed taxa and conduct deep investigations for exploring underestimated diversity in this group especially based on detailed morphological comparisons, molecular phylogenetics and bioacoustics data.

During our field surveys in the Xianju County, Zhejiang Province, China, some Megophrys specimens were collected from the montane forests. Molecular phylogenetic analyses and morphological comparisons support that it is distinctly different from its Megophrys congeners. Therefore, we describe it herein as a new species.

Materials and methods

Specimens

Seven adult males, two adult females, and six tadpoles of the new taxon (for voucher information see Suppl. material 1: Table S1 and Suppl. material 2: Table S2) were collected from the mountain streams of Xianju County, Zhejiang Province, China (Fig. 1). The stages of tadpoles were identified following Gosner (1960). After taking photographs, they were euthanised using isoflurane, and the specimens were then fixed in 75% ethanol. Tissue samples were taken and preserved separately in 95% ethanol prior to fixation. Specimens were deposited in Chengdu Institute of Biology, Chinese Academy of Sciences (CIB, CAS).

Figure 1. 

Localities for specimens used in this study. 1, the type locality of Megophrys xianjuensis sp. nov., Xianju County, Zhejiang Province, China; 2. the common type locality of M. boettgeri (black) and M. kuatunensis (purple), Wuyi Mountain, Nanping County, Fujian Province, China; 3, the type locality of M. lishuiensis, Lishui City, Zhejiang Province, China.

Molecular data and phylogenetic analyses

Four male, two female, and three tadpole specimens of the new taxon were included in the molecular analyses (for voucher information see Table 1). For phylogenetic analyses, two topotypes of M. kuatunensis and two topotypes of M. boettgeri from Wuyi Mountain, Fujian Province, China (the common type locality of these species; Fig. 1) were also collected and sequenced (for voucher information see Table 1).

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

Species Voucher number Locality GenBank accession number
16S COI
Megophrys aceras KIZ025467 Khao Nan National Park, Nakhon Si Thammarat, Thailand KX811925 KX812159
M. acuta SYS a001957 Heishiding Nature Reserve, Guangdong Prov., China KJ579118
M. auralensis NCSM 79599 Aural, Kampong Speu, Cambodia KX811807
M. baluensis ZMH A13125 Gunung Kinabalu National Park, Kogopan Trail, Malaysia KJ831310
M. baolongensis KIZ019216 Baolong, Chongqing City, China KX811813 KX812093
M. binchuanensis KIZ019441 Mt. Jizu, Yunnan Prov., China KX811849 KX812112
M. binlingensis KIZ025807 Mt. Wawu, Sichuan Prov., China KX811852 KX812115
M. boettgeri Tissue ID: YPXJK033 Mt. Wuyi, Fujian Prov., China KX811814 KX812104
CIBWY18082307 Mt. Wuyi, Fujian Prov., China MN563762 MN563778
CIBWY18082603 Mt. Wuyi, Fujian Prov., China MN563763 MN563779
M. brachykolos ROM 16634 Hong Kong, China KX811897 KX812150
M. carinense Tissue ID: YPX20455 Dayao Shan, Guangxi Prov., China KX811811 KX812057
M. cheni SYS a001427 Jinggang Shan, Jiangxi Prov., China KJ560391
M. chuannanensis CIB20050081 Hejiang, Sichuan Prov., China KM504261
M. daweimontis KIZ048997 Dawei Shan, Yunnan Prov., China KX811867 KX812125
M. dongguanensis SYS a001972 Mt. Yinping, Guangdong Prov., China MK524098 MK524129
M. dringi UNIMAS 8943 Gunung Mulu National Park, Sarawak, Malaysia KJ831317
M. edwardinae FMNH 273694 Bintulu, Sarawak, Malaysia KX811918 KX812050
M. elfina ZMMU ABV-00454 Bidoup Mountain, Lam Dong, Vietnam KY425379
M. fansipanensis VNMN 2018.01 Lao Cai, Sa Pa, Vietnam MH514886
M. feae KIZ046706 Huangcaoling, Yunnan Prov., China KX811810 KX812056
M. flavipunctata SDBDU2009.297 East Khasi Hills dist., Meghalaya KY022307 MH647536
M. gerti ITBCZ 1108 Nui Chua National Park, Ninh Thuan, Vietnam KX811917 KX812161
M. gigantica SYSa003933 Wuliang shan, Yunnan Prov., China MH406775 MH406235
M. glandulosa KIZ048439 Husa, Yunnan Prov., China KX811762 KX812075
M. hansi KIZ010360 Phong Dien Nature Reserve, Thua Thien Hue, Vietnam KX811913 KX812155
M. himalayana SDBDU2009.75 East Siang dist., Arunachal Pradesh, India KY022311
M. hoanglienensis VNMN 2018.02 Lao Cai, Sa Pa, Vietnam MH514889
M. huangshanensis KIZ022004 Mt. Huang, Anhui Prov., China KX811821 KX812107
M. intermedia ZFMK 87596 U Bo, Phong Nha-Ke Bang NP, Vietnam HQ588950
M. jingdongensis KIZ-LC0805067 Huanglianshan, Yunnan Prov., China KX811872 KX812131
M. jinggangensis KIZ07132 Chashan Forest Farm, Jiangxi Prov., China KX811840 KX812108
M. jiulianensis SYS a002107 Mt. Jiulian, Jiangxi Prov., China MK524099 MK524130
M. kalimantanensis MZB. Amph 21482 Tanah Bumbu, Kalimantan Selatan, Borneo, Indonesia MG993554
M. kobayashii UNIMAS 8148 Gunung Kinabalu National Park, Sabah, Malaysia KJ831313
M. kuatunensis CIBWY18082407 Mt. Wuyi, Fujian Prov., China MN563764 MN563780
CIBWY18082408 Mt. Wuyi, Fujian Prov., China MN563765 MN563781
SYS a001579 Mt. Wuyi, Fujian Prov., China KJ560376
M. lancip MZB:Amp:22233 KY679891
M. leishanensis CIBLS20171101001 Mt. Leigong, Guizhou Prov., China MK005310 MK005306
M. ligayae ZMMU NAP-05015 Palawan, Philippines KX811919 KX812051
M. lini SYS a002370 Suichuan Co., Jiangxi Prov., China KJ560412
M. lishuiensis CIBWYF00169 Lishui City, Zhejiang Prov., China KY021418
CIBWYF00170 Lishui City, Zhejiang Prov., China KY113084
CIBWYF11011 Lishui City, Zhejiang Prov., China KY113085
M. major SYSa002961 Zhushihe, Yunnan Prov., China MH406728 MH406180
M. mangshanensis KIZ021786 Nanling National Forest Park, Guangdong Prov., China KX811790 KX812079
M. maosonensis KIZ016045 Xiaoqiaogou Nature Reserve, Yunnan Prov., China KX811780 KX812080
M. medogensis KIZ06621 Beibeng, Xizang Prov., China KX811767 KX812082
M. microstoma KIZ048799 Xiaoqiaogou Nature Reserve, Yunnan Prov., China KX811914 KX812156
M. minor KIZ01939 Qingcheng Shan, Sichuan Prov., China KX811896 KX812145
M. montana LSUMZ 81916 Sukabumi, Java, Indonesia KX811927 KX812163
M. monticola SDBDU 2011.1047 [h] Darjeeling dist., West Bengal, India KX894679
M. mufumontana SYS a006391 Mt. Mufu, Hunan Prov., China MK524105 MK524136
M. nankiangensis CIB ZYC517 Nanjiang, Sichuan Prov., China KX811900
M. nankunensis SYS a004498 Mt. Nankun, Guangdong Prov., China MK524108 MK524139
M. nanlingensis SYS a001959 Nanling Nature Reserve, Guangdong Prov., China MK524111 MK524142
M. nasuta KIZ019419 Malaysia KX811921 KX812054
M. obesa SYS a002272 Heishiding Nature Reserve, Guangdong Prov., China KJ579122
M. ombrophila KRM18 Mt. Wuyi, Fujian Prov., China KX856404
M. omeimontis KIZ025765 Mt. E’mei, Sichuan Prov., China KX811884 KX812136
M. oreocrypta BNHS 6046 West Garo Hills dist., Meghalaya KY022306
M. pachyproctus KIZ010978 Beibeng, Xizang Prov., China KX811908 KX812153
M. palpebralespinosa KIZ011603 Pu Hu Nature Reserve, Thanh Hoa, Vietnam KX811888 KX812137
M. parva SYSa003042 Zhushihe, Yunnan Prov., China MH406737 MH406189
M. periosa BNHS 6061 West Kameng dist., Arunachal Pradesh, India KY022309 MH647528
M. popei SYS a000589 Naling Nature Reserve, Guangdong Prov., China KM504251
M. sangzhiensis Tissue ID: YPX11006 Badagongshan Nature Reserve, Hunan Prov., China KX811856 KX812117
M. shapingensis KIZ014512 Liziping Nature Reserve, Sichuan Prov., China KX811904 KX812060
M. spinata KIZ016100 Mt. Leigong, Guizhou Prov., China KX811864 KX812119
M. stejnegeri KU 314303 Pasonanca Natural Park, Zamboanga, Philippines KX811922 KX812052
M. synoria FMNH 262778 O’Reang, Mondolkiri, Cambodia KY022198
M. tuberogranulata Tissue ID: YPX10987 Badagongshan Nature Reserve, Hunan Prov., China KX811823 KX812095
M. wawuensis KIZ025799 Wawu Shan, Sichuan Prov., China KX811902 KX812062
M. wugongensis SYS a002610 Wugongshan Scenic Area, Jiangxi Prov., China MK524114 MK524145
M. wuliangshanensis KIZ046812 Huangcaoling, Yunnan Prov., China KX811881 KX812129
M. wushanensis KIZ045469 Guangwu Mountain, Sichuan Prov., China KX811838 KX812094
M. xianjuensis sp. nov. CIBXJ190505 Xianju Co., Zhejiang Prov., China MN563753 MN563769
CIBXJ20190801 Xianju Co., Zhejiang Prov., China MN563754 MN563770
CIBXJ20190802 Xianju Co., Zhejiang Prov., China MN563755 MN563771
CIBXJ20190803 Xianju Co., Zhejiang Prov., China MN563756 MN563772
CIB20180514008 Xianju Co., Zhejiang Prov., China MN563757 MN563773
CIBXJ190503 Xianju Co., Zhejiang Prov., China MN563758 MN563774
CIBXJT19050702 Xianju Co., Zhejiang Prov., China MN563759 MN563775
CIBXJT19050703 Xianju Co., Zhejiang Prov., China MN563760 MN563776
CIBXJT19050704 Xianju Co., Zhejiang Prov., China MN563761 MN563777
M. zhangi KIZ014278 Zhangmu, Xizang Prov., China KX811765 KX812084
Leptobrachium boringii Tissue ID: YPX37539 Emei Shan, Sichuan Prov., China KX811930 KX812164
L. oshanensis KIZ025778 Emei Shan, Sichuan Prov., China KX811928 KX812166

Total DNA was extracted using a standard phenol-chloroform extraction protocol (Sambrook et al. 1989). Two fragments of the mitochondrial genes 16S rRNA and cytochrome oxidase subunit I (COI) were amplified. For 16S, the primers P7 (5’-CGCCTGTTTACCAAAAACAT-3’) and P8 (5’-CCGGTCTGAACTCAGATCACGT-3’) were used following Simon et al. (1994), and for COI, Chmf4 (5’-TYTCWACWAAYCAYAAAGAYATCGG-3’) and Chmr4 (5’-ACYTCRGGRTGRCCRAARAATCA-3’) were used following Che et al. (2012). Gene fragments were amplified under the following conditions: an initial denaturing step at 95 °C for 4 min; 36 cycles of denaturing at 95 °C for 30 sec, annealing at 52 °C (for 16S)/47 °C (for COI) for 40 sec and extending at 72 °C for 70 sec. Sequencing was conducted using an ABI3730 automated DNA sequencer in Shanghai DNA BioTechnologies Co., Ltd. (Shanghai, China). New sequences were deposited in GenBank (for GenBank accession numbers see Table 1).

For molecular analyses, the available sequence data for all related species of the genus Megophrys were downloaded from GenBank, mainly from previous studies (Chen et al. 2017; Mahony et al. 2017; Liu et al. 2018; Wang et al. 2019; for GenBank accession number see Table 1). For phylogenetic analyses, corresponding sequences of one Leptobrachella oshanensis and one Leptobrachium boringii were downloaded (for GenBank accession number see Table 1) and used as outgroups according to Chen et al. (2017).

Sequences were assembled and aligned using the Clustalw module in BioEdit v. 7.0.9.0 (Hall 1999) with default settings. Alignments were checked by eye and revised manually if necessary. 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 16S gene fragments. Non-sequenced fragments were defined as missing loci.

Phylogenetic trees were reconstructed for the concatenated data of the mitochondrial genes. Phylogenetic analyses were conducted using maximum likelihood (ML) and Bayesian Inference (BI) methods, implemented in PhyML v. 3.0 (Guindon et al. 2010) and MrBayes v. 3.12 (Ronquist and Huelsenbeck 2003), respectively. To avoid under- or over-parameterisation (McGuire et al. 2007), the best partition scheme and the best evolutionary model for each partition were chosen for the phylogenetic analyses using PARTITIONFINDER v. 1.1.1 (Robert et al. 2012). For this analysis, 16S rRNA and COI genes were defined, and Bayesian Inference Criteria (BIC) was used. As a result, the analyses suggested that the best partition scheme was 16S/COI gene, and selected GTR+I+G model as the best model for all partitions. For the ML tree, branch supports were drawn from 10,000 non-parametric bootstrap replicates. In BI analyses, two runs each with four Markov chains were run for 60 million generations with sampling every 1000 generations. The first 25% of generations were removed as the “burn-in” stage followed by calculation of Bayesian posterior probabilities and the 50% majority-rule consensus of the post burn-in trees sampled at stationarity. Finally, genetic distance was calculated with the pairwise uncorrected p-distance model between the new taxon and its congeners by 16S rRNA gene using MEGA v. 6.06 (Tamura et al. 2013).

Morphological comparisons

Nine adult specimens of the new taxon and the holotype and seven paratypes of M. lishuiensis were measured (for voucher information see Suppl. material 1: Table S1). The terminology and methods followed Fei et al. (2009). Measurements were taken with a dial calliper to 0.1 mm. In total, 22 morphometric characters of adult specimens were measured:

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

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

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

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

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

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

HAL hand length (distance from the posterior end of the inner metacarpal tubercle to the distal tip of finger III);

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

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

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

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

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

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

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

SNT distance from the tip of the snout to the naris;

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

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

THL thigh length (distance from vent to knee);

TL tibia length (distance from knee to tarsus);

TW maximal tibia width;

TYD maximal tympanum diameter;

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

For seven tadpoles of the new taxon (for voucher information see Suppl. material 2: Table S2), eleven morphometric characters were measured:

BH maximum body height;

BW maximum body width;

IOD interocular distance (minimum distance between eye);

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

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

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

SVL snout-vent length;

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

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

TBW maximum width of tail base;

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

In order to reduce the impact of allometry, a size-corrected value from the ratio of each character to SVL was calculated and then log-transformed for the following morphometric analyses. One-way analysis of variance (ANOVA) was used to test the significance of differences on morphometric characters between different sexes as well as between different species. The significance level was set at 0.05. Furthermore, to show the spatial distribution of each species on the morphometric characters, principal component analyses (PCA) were performed. These analyses were carried out in R (R Development Core Team 2008).

We compared morphological characters of the new taxon with other Megophrys species. Comparative data were obtained from the literature for 92 species of the genus (Table 2). In addition, for comparison, we examined the type and/or topotype materials for M. lishuiensis, M. boettgeri, and M. kuatunensis (for voucher numbers see Table 1 and Suppl. material 1: Table S1).

References for morphological characters for congeners of the genus Megophrys.

Species Literature obtained
M. aceras Boulenger, 1903 Taylor 1962
M. acuta Wang, Li & Jin, 2014 Li et al. 2014
M. ancrae Mahony, Teeling & Biju, 2013 Mahony et al. 2013
M. auralensis Ohler, Swan & Daltry, 2002 Ohler et al. 2002
M. baluensis (Boulenger, 1899) Boulenger 1899
M. baolongensis Ye, Fei & Xie, 2007 Ye et al. 2007
M. binchuanensis Ye & Fei, 1995 Ye and Fei 1995
M. binlingensis Jiang, Fei & Ye, 2009 Fei et al. 2009
M. boettgeri (Boulenger, 1899) Fei et al. 2012
M. brachykolos Inger & Romer, 1961 Inger and Romer 1961
M. carinense (Boulenger, 1889) Fei et al. 2009
M. caudoprocta Shen, 1994 Fei et al. 2012
M. cheni (Wang & Liu, 2014) Wang et al. 2014
M. chuannanensis (Fei, Ye & Huang, 2001) Fei et al. 2012
M. damrei Mahony, 2011 Mahony 2011
M. daweimontis Rao & Yang, 1997 Fei et al. 2012
M. dongguanensis Wang & Wang, 2019 Wang et al. 2019
M. dringi Inger, Stuebing & Tan, 1995 Inger et al. 1995
M. edwardinae Inger, 1989 Inger 1989
M. elfina Poyarkov, Duong, Orlov, Gogoleva, Vassilieva, Nguyen, Nguyen, Nguyen, Che & Mahony, 2017 Poyarkov et al. 2017
M. fansipanensis Tapley, Cutajar, Mahony, Nguyen, Dau, Luong, Le, Nguyen, Nguyen, Portway, Luong & Rowley, 2018 Tapley et al. 2018
M. feae Boulenger, 1887 Fei et al. 2009
M. feii Yang, Wang & Wang, 2018 Yang et al. 2018
M. flavipunctata Mahony, Kamei, Teeling & Biju, 2018 Mahony et al. 2018
M. gerti (Ohler, 2003) Ohler 2003
M. gigantica Liu, Hu & Yang, 1960 Fei et al. 2012
M. glandulosa Fei, Ye & Huang, 1990 Fei et al. 2012
M. hansi (Ohler, 2003) Ohler 2003
M. himalayana Mahony, Kamei, Teeling & Biju, 2018 Mahony et al. 2018
M. hoanglienensis Tapley, Cutajar, Mahony, Nguyen, Dau, Luong, Le, Nguyen, Nguyen, Portway, Luong & Rowley, 2018 Tapley et al. 2018
M. huangshanensis Fei & Ye, 2005 Fei et al. 2012
M. insularis (Wang, Liu, Lyu, Zeng & Wang, 2017) Wang et al. 2017a
M. intermedia Smith, 1921 Rao and Yang 1997
M. jingdongensis Fei & Ye, 1983 Fei et al. 2012
M. jinggangensis (Wang, 2012) Wang et al. 2012
M. jiulianensis Wang, Zeng, Lyu & Wang, 2019 Wang et al. 2019
M. kalimantanensis Munir, Hamidy, Matsui, Iskandar, Sidik & Shimada, 2019 Munir et al. 2019
M. kobayashii Malkmus & Matsui, 1997 Malkmus and Matsui 1997
M. koui Mahony, Foley, Biju & Teeling, 2017 Mahony et al. 2017
M. kuatunensis Pope, 1929 Fei et al. 2012
M. lancip Munir, Hamidy, Farajallah & Smith, 2018 Munir et al. 2018
M. leishanensis Li, Xu, Liu, Jiang, Wei & Wang, 2018 Li et al. 2018
M. lekaguli Stuart, Chuaynkern, Chan-ard & Inger, 2006 Stuart et al. 2006
M. liboensis (Zhang, Li, Xiao, Li, Pan, Wang, Zhang & Zhou, 2017) Zhang et al. 2017
M. ligayae Taylor, 1920 Taylor 1920
M. lini (Wang & Yang, 2014) Wang et al. 2014
M. lishuiensis (Wang, Liu & Jiang, 2017) Wang et al. 2017b
M. longipes Boulenger, 1886 Taylor 1962
M. major Boulenger, 1908 Mahony et al. 2018
M. mangshanensis Fei & Ye, 1990 Fei et al. 2012
M. maosonensis Bourret, 1937 Bourret 1937
M. medogensis Fei, Ye & Huang, 1983 Fei et al. 2012
M. megacephala Mahony, Sengupta, Kamei & Biju, 2011 Mahony et al. 2011
M. microstoma (Boulenger, 1903) Fei et al. 2012
M. minor Stejneger, 1926 Fei et al. 2012
M. montana Kuhl & Van Hasselt, 1822 Kuhl & Van Hasselt 1822
M. monticola (Günther, 1864) Mahony et al. 2018
M. mufumontana Wang, Lyu & Wang, 2019 Wang et al. 2019
M. nankiangensis Liu & Hu, 1966 Fei et al. 2012
M. nankunensis Wang, Zeng &. Wang, 2019 Wang et al. 2019
M. nanlingensis Lyu, Wang, Liu & Wang, 2019 Wang et al. 2019
M. nasuta (Schlegel, 1858) Taylor 1962
M. obesa Wang, Li & Zhao, 2014 Wang et al. 2014
M. ombrophila Messenger & Dahn, 2019 Munir et al. 2019
M. omeimontis Liu, 1950 Fei et al. 2009
M. oreocrypta Mahony, Kamei, Teeling & Biju, 2018 Mahony et al. 2018
M. oropedion Mahony, Teeling & Biju, 2013 Mahony et al. 2013
M. pachyproctus Huang, 1981 Fei et al. 2009
M. palpebralespinosa Bourret, 1937 Fei et al. 2012
M. parallela Inger & Iskandar, 2005 Inger and Iskandar 2005
M. parva (Boulenger, 1893) Fei et al. 2009
M. periosa Mahony, Kamei, Teeling & Biju, 2018 Mahony et al. 2018
M. popei (Zhao, Yang, Chen, Chen & Wang, 2014) Zhao et al. 2014
M. robusta Boulenger, 1908 Mahony et al. 2018
M. rubrimera Tapley, Cutajar, Mahony, Chung, Dau, Nguyen, Luong & Rowley, 2017 Tapley et al. 2017
M. sangzhiensis Jiang, Ye & Fei, 2008 Jiang et al. 2008
M. serchhipii (Mathew & Sen, 2007) Mathew and Sen 2007
M. shapingensis Liu, 1950 Fei et al. 2009
M. shuichengensis Tian & Sun, 1995 Fei et al. 2009
M. shunhuangensis Wang, Deng, Liu, Wu & Liu, 2019 Wang et al. 2019
M. spinata Liu & Hu, 1973 Fei et al. 2009
M. stejnegeri Taylor, 1920 Taylor 1920
M. synoria (Stuart, Sok & Neang, 2006) Stuart et al. 2006
M. takensis Mahony, 2011 Mahony 2011
M. tuberogranulata Shen, Mo & Li, 2010 Fei et al. 2012
M. vegrandis Mahony, Teeling, Biju, 2013 Mahony et al. 2013
M. wawuensis Fei, Jiang & Zheng, 2001 Fei et al. 2012
M. wugongensis Wang, Lyu & Wang, 2019 Wang et al. 2019
M. wuliangshanensis Ye & Fei, 1995 Fei et al. 2012
M. wushanensis Ye & Fei, 1995 Fei et al. 2012
M. zhangi Ye & Fei, 1992 Fei et al. 2012
M. zunhebotoensis (Mathew & Sen, 2007) Mathew and Sen 2007

Bioacoustics notes

The advertisement calls of the new taxon from Xianju County, Zhejiang Province, China were recorded in the field. SONY PCM-D50 digital sound recorder was used to record within 20 cm of the calling individuals. The sound files in wave format were resampled at 48 kHz with sampling depth 24 bits. The sonograms and waveforms were generated by WaveSurfer software (Sjöander and Beskow 2000) from which all parameters and characters were measured. Ambient temperature was taken by a digital hygrothermograph.

Results

Phylogenetic analyses

Aligned sequence matrix of 16S+COI contained 1104 bps. ML and BI trees of the mitochondrial DNA dataset presented almost consistent topology, though relationships of some lineages were unresolved (Fig. 2). All individuals of the new taxon were clustered into one clade which was independently nested into the Megophrys clade and sister to the M. lishuiensis clade. Genetic distances on 16S gene with uncorrected p-distance model between specimens of the new taxon were less than 0.7% (ranging from 0.0% to 0.6%), being lower than most interspecific genetic distances in the genus Megophrys (0.4%–19.3%; Suppl. material 3: Table S3). Genetic distance between the new taxon and its closely-related species M. lishuiensis is 2.8%, higher than that between many substantial species, for example, M. wushanensis vs. M. baolongensis (2.1%), M. baolongensis vs. M. tuberogranulata (1.5%), M. sangzhiensis vs. M. binlingensis (1.7%), and M. omeimontis vs. M. jingdongensis (2.1%).

Figure 2. 

Maximum Likelihood tree of the genus Megophrys reconstructed based on the 16S rRNA and COI gene sequences. ML bootstrap support/Bayesian posterior probability was denoted beside each node. Samples 1-89 refer to Table 1.

Morphometric analyses

The results of one-way ANOVA showed that in the new taxon, the male group was significantly different from the female group on SVL and the ratio of ED and IND to SVL (p-value < 0.05). Therefore, morphometric analyses between the new taxon and M. lishuiensis were conducted separately for male and female. In PCA, the total variation of the first two principal components was 56.2% in male and 74.9% in female respectively. In both male and female, the new taxon could be distinctly separated from its phylogenetically sister species M. lishuiensis on the two-dimensional plots of PC1 vs. PC2 (Fig. 3). The results of one-way ANOVA indicated that the new taxon was significantly different from M. lishuiensis on many morphometric characters (all p-values < 0.05; Table 3). More detailed descriptions of results from morphological comparisons between the new taxon and its congeners were presented in the following sections for describing the new species.

Figure 3. 

Plots of the first principal component (PC1) versus the second (PC2) for Megophrys xianjuensis sp. nov. and M. lishuiensis from principal component analyses A male B female.

Morphometric comparisons between the adult specimens of Megophrys xianjuensis sp. nov. and M. lishuiensis. Units are in mm. See abbreviations for the morphological characters in Materials and methods section.

Character Megophrys xianjuensis sp. nov. (MX) M. lishuiensis (ML) P-value from ANOVA
Males (N = 7) Female (N = 2) Males (N = 6) Female (N = 2)
Range Mean ± SD Range Mean ± SD Range Mean ± SD Range Mean ± SD Male vs. female in MX MX vs. ML in male MX vs. ML in female
SVL 31.0–36.3 33.1 ± 1.8 32.8–41.6 37.2 ± 6.2 30.5–34.7 32.5 ± 1.6 37.6–40.4 39.0 ± 1.9 0.119 0.633 0.728
HDL 9.4–10.5 9.9 ± 0.4 10.1–10.8 10.4 ± 0.5 9.4–10.3 9.8 ± 0.4 9.9–11.4 10.6 ± 1.1 0.365 0.916 0.705
HDW 10.6–11.5 11.1 ± 0.3 10.7–12.9 11.8 ± 1.5 10.6–11.6 10.9 ± 0.3 12.1–12.1 12.1 ± 0.02 0.361 0.813 0.561
SL 3.8–4.7 4.2 ± 0.3 3.6–5.2 4.4 ± 1.1 4.1–4.5 4.3 ± 0.2 4.3–4.4 4.3 ± 0.1 0.318 0.569 0.510
SNT 1.9–2.5 2.2 ± 0.2 1.8–2.5 2.1 ± 0.5 2.0–2.5 2.3 ± 0.2 2.4–2.7 2.6 ± 0.2 0.165 0.177 0.177
IND 3.2–3.7 3.4 ± 0.2 3.2–3.6 3.4 ± 0.3 3.1–4.0 3.5 ± 0.4 4.2–4.2 4.2 ± 0.01 0.045* 0.470 0.121
IOD 2.3–3.3 2.8 ± 0.4 2.8–2.9 2.8 ± 0.1 2.4–2.8 2.5 ± 0.1 2.8–3.1 2.9 ± 0.2 0.345 0.140 0.959
UEW 2.3–2.9 2.7 ± 0.2 2.7–3.4 3.1 ± 0.5 2.6–3.6 3.2 ± 0.3 3.7–3.7 3.7 ± 0.02 0.807 0.004** 0.047*
ED 3.3–4.0 3.6 ± 0.3 2.9–4.0 3.4 ± 0.8 3.8–4.6 4.1 ± 0.2 4.1–4.6 4.3 ± 0.3 0.002** 0.002** 0.046*
TYD 1.8–2.2 2.0 ± 0.1 1.7–2.7 2.2 ± 0.6 1.9–2.5 2.1 ± 0.2 2.2–2.3 2.3 ± 0.1 0.571 0.044* 0.962
LAL 14.2–17.2 15.3 ± 1.0 13.7–19.2 16.5 ± 3.9 14.6–15.7 15.2 ± 0.4 17.5–17.6 17.5 ± 0.04 0.279 0.792 0.767
HAL 7.6–9.2 8.2 ± 0.6 7.8–10.0 8.9 ± 1.5 8.0–8.8 8.4 ± 0.3 8.7–9.3 9.0 ± 0.4 0.489 0.220 0.021*
LW 2.1–2.7 2.5 ± 0.2 2.0–2.6 2.3 ± 0.4 2.4–3.1 2.8 ± 0.2 2.4–2.4 2.4 ± 0.01 0.062 0.042* 0.879
FIL 2.5–3.1 2.9 ± 0.2 2.8–3.9 3.4 ± 0.78 2.4–3.0 2.7 ± 0.2 2.8–3.6 3.2 ± 0.5 0.488 0.644 0.602
FIIL 2.7–3.5 3.0 ± 0.3 3.0–4.0 3.5 ± 0.78 2.5–3.3 2.9 ± 0.3 3.0–3.2 3.1 ± 0.2 0.675 0.975 0.151
FIIIL 4.4–6.2 4.9 ± 0.6 4.8–5.9 5.4 ± 0.8 4.9–5.7 5.2 ± 0.2 5.3–5.8 5.6 ± 0.3 0.622 0.111 0.949
FIVL 2.9–4.1 3.3 ± 0.2 3.0–4.4 3.6 ± 1.0 3.2–3.9 3.5 ± 0.2 3.2–3.6 3.4 ± 0.3 0.726 0.110 0.471
THL 13.8–16.0 14. ± 0.8 13.1–17.6 15.3 ± 3.2 13.7–14.7 14.2 ± 0.4 15.7–16.3 16. 0 ± 0.4 0.177 0.646 0.996
TL 14.5–16.1 15.0 ± 0.5 13.0–17.8 15.4 ± 3.4 14.3–15.5 15.0 ± 0.3 16.4–16.7 16.5 ± 0.2 0.019* 0.558 0.628
TW 2.8–4.4 3.4 ± 0.6 2.9–3.3 3.1 ± 0.3 3.9–4.4 4.1 ± 0.2 4.0–4.7 4.3 ± 0.5 0.124 0.004** 0.050
TFL 18.0–21.5 19.5 ± 1.14 19.3–23.5 21.4 ± 3.0 18.6–20.4 19.7 ± 0.7 21.0–22.4 21.7 ± 1.0 0.483 0.444 0.697
FL 11.2–14.8 12.9 ± 1.1 12.4–15.6 14.0 ± 2.2 12.3–13.5 12.840 ± 0.5 13.7–14.8 14.3 ± 0.8 0.562 0.946 0.734

Taxonomic accounts

Megophrys xianjuensis sp. nov.

Figures 1, 2, 3, 4, 5, 6, 7, 8, 9

Type material

Holotype. CIBXJ190503 (Fig. 4A, B, E, G, I), adult male, from Shenxianju scenic area, Danzhu Township, Xianju County, Zhejiang Province, China (28.677483N, 120.594888E, 350 m a. s. l.), collected by Bin Wang on 7 May 2019.

Figure 4. 

Holotype CIBXJ190503 of Megophrys xianjuensis sp. nov. and holotype CIBWYF00164 of M. lishuiensis A, B dorsal view and ventral of Megophrys xianjuensis sp. nov., respectively C, D dorsal view and ventral of M. lishuiensis, respectively E oral of Megophrys xianjuensis sp. nov. showing vomerine ridges (red arrows) F oral of M. lishuiensis showing no vomerine ridge G ventral view of hand of Megophrys xianjuensis sp. nov. H ventral view of hand of M. lishuiensis I ventral view of foot of Megophrys xianjuensis sp. nov. J ventral view of foot of M. lishuiensis.

Paratype. One adult male CIB20180514007 and one adult female CIB20180514008 collected from the type locality of the new species by Bin Wang on 14 May 2018; one adult male CIBXJ190501 and one adult female CIBXJ190505 collected from the type locality by Bin Wang on 7 May 2019; five adult males CIBXJ20190801, CIBXJ20190802, CIBXJ20190803, CIBXJ20190804 and CIBXJ20190805 collected from the type locality by Bin Wang on 28 August 2019.

Diagnosis

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

The new species could be identified from its congeners by a combination of the following morphological characters: (1) small size (SVL 31.0–36.3 mm in males and 41.6 mm in female); (2) vomerine ridge present and vomerine teeth absent; (3) tongue not notched behind; (4) a small horn-like tubercle at the edge of each upper eyelid; (5) tympanum distinctly visible, rounded; (6) two metacarpal tubercles in hand; (7) relative finger lengths: II < I < IV < III; (8) toes with rudimentary webbing at bases; (9) heels overlapping when thighs are positioned at right angles to the body; (10) tibiotarsal articulation reaching tympanum to eye when leg stretched forward; (11) an internal single subgular vocal sac in male; (12) in breeding male, the nuptial pads with black nuptial spines on the dorsal bases of the first and second fingers.

Description of holotype

SVL 34.4 mm; head wider than long (HDW/HDL ratio 1.2); snout obtusely pointed, protruding well beyond the margin of the lower jaw in ventral view; loreal region vertical and concave; canthus rostralis well developed; top of head flat on in dorsal view; an small horn-like tubercle at the edge of the upper eyelid; eye large and convex, eye diameter 40.4% of head length; pupils vertical; nostril orientated laterally, closer to snout than eye; tympanum distinct, TYP/EYE ratio 0.56; vomerine ridges present and vomerine teeth absent; margin of tongue smooth, not notched behind (Fig. 4A, B, E).

Forelimbs slender, the length of lower arm and hand 44.9% of SVL; fingers slender, relative finger lengths: II < I < IV < III; tips of digits globular, without lateral fringes; subarticular tubercle distinct at the base of each fingers; two metacarpal tubercles, prominent, oval-shaped, the inner one bigger than the outer (Fig. 4G).

Hindlimbs slender, heels overlapping when thighs are positioned at right angles to the body, tibiotarsal articulation reaching tympanum to eye when leg stretched forward; tibia length slightly longer than thigh length; relative toe lengths I < II < V < III < IV; tips of toes round, slightly dilated; subarticular tubercle absent; toes with rudimentary webbing at bases; lateral fringe narrow; inner metatarsal tubercle oval-shaped; outer metatarsal tubercle absent (Fig. 4I).

Dorsal rough, with numerous granules; several large warts scattered on flanks; an small horn-like tubercle at the edge of each upper eyelid; a dark brown inverted triangular pattern between anterior corner of eyes, tubercles on the dorsum forming a weak X-shaped ridge and two discontinuous dorsolateral parallel ridges on either side of the X-shaped ridge; several tubercles on the flanks and dorsal surface of thighs and tibias, and limbs barred with dark brown forming four transverse rows; supratympanic fold distinct (Fig. 4A).

Ventral surface with numerous white granules; chest gland distinct and round, closer to the axilla than to midventral line; femoral gland on rear of thigh; posterior end of the body protrudes distinct and appears as an arc-shaped swelling, upper the anal region (Fig. 4B).

Colouration of holotype in life

An inverted triangular brown speckle between the eyes; an X-shaped ridges on the dorsum of body, four transverse bands on the dorsal surface of the hindlimb; several dark brown and white vertical bars on the lower and upper lip; the venter purple and the colour of throat is deeper than belly, flank and middle of throat with black brown spots, numerous white granules on the ventral surface and limbs; palms and soles purple and the metacarpal tubercles are orange, tip of digits greyish white; pectoral and femoral glands white (Fig. 5).

Figure 5. 

Photos of the holotype CIBXJ190503 of Megophrys xianjuensis sp. nov. in life A dorsal view B ventral view C ventral view of hand D ventral view of foot E dorsolateral view showing the single external subgular vocal sac (red arrow).

Preserved holotype colouration

Dorsal surface fade to olive; the inverted triangular brown speckle between the eyes, X-shaped ridges on dorsum and transverse bands on limbs and digits and brown spots on flank and middle throat are more distinct; ventral surface greyish white; creamy-white substitutes the orange in metacarpal tubercles; the posterior of ventral surface of body, inner of thigh and upper of tibia creamy-white (Fig. 4).

Variation

In some adult individuals a brown Y-shaped marking on the dorsum of head and disconnected with a X-shaped marking on back (Fig. 6A); an inverted triangular brown speckle between two upper eyelids with Y-shaped marking on back of trunk, the colouration of dorsum is brown with brick-red and the caudal vertebra is pointed in the adult female (Fig. 6B); in some adult individuals the metacarpal tubercles in palm is grey-white but the tip of fingers is orange and the black spots in flank is smaller (Fig. 6C); in some adult individuals the white granules in ventral surface are more intensive (Fig. 6D).

Figure 6. 

Colour variation in Megophrys xianjuensis sp. nov. in life A, B dorsolateral view of a male (Voucher: CIBXJ190501) and a female (Voucher: CIBXJ190505), respectively C, D ventral view of CIBXJ190501 and CIBXJ190505, respectively.

Advertisement calls

Ten advertisement calls from two individuals of the new species were recorded in the Xianju County, Zhejiang Province, China between 21:00–23:00 on 7 May 2019. The call description is based on recordings of the holotype CIBXJ090503 (Fig. 7) from the stone near the streamlet, and the ambient air temperature was 24.5 °C. Each call consists of 22–62 (mean 49.33 ± 14.50, N = 6) notes. Call duration was 4.90–14.22 second (mean10.95 ± 3.42, N = 6). Call interval was 4.12–7.64 second (mean 6.23 ± 1.56, N = 5). Each note had a duration of 0.06– 0.90 second (mean 0.10 ± 0.05, N = 290) and the intervals between notes 0.07–0.88 second (mean 0.13 ± 0.05, N = 284). Amplitude modulation within note was apparent, beginning with lower energy pulses, increasing slightly to a maximum by approximately mid note, and then decreasing towards the end of each note. The average dominant frequency was 6400 ± 465.79 (5520–6840 Hz, N = 6).

Figure 7. 

Advertisement call of the holotype CIBXJ190503 of Megophrys xianjuensis sp. nov. A waveform showing one note B waveform showing three notes of one advertisement call C sonogram showing one note D sonogram showing three notes of one call.

Secondary sexual characteristics

Adult females with SVL 41.6 mm, larger than adult males with 31.0–36.3 mm. Adult males have a single subgular vocal sac (Fig. 5E). In breeding males, the brownish red nuptial pads on the dorsal bases of the first finger and second fingers with black nuptial spines under microscope.

Tadpole description

The tadpole was confirmed as Megophrys xianjuensis sp. nov. by molecular phylogenetic analyses. The following tadpole description is based on the specimen CIBXJT19050704 at Stage 31 (Fig. 8). Body slender, body and tail yellow-brown in life and fade to light brown and creamy-white of body and tail respectively in preserved specimens; two dotted lines on flank of dorsal from mouth to fin; tail height greater than body height; dorsal fin arising, behind the origin of the tail, height near mid-length, tapering gradually to narrow, tip pointed; tail approximately 1.9 times as long as snout-vent length; tail height 19.9% of tail length; body width slightly longer than body height (BW/BH = 1.1); tail fins lightly coloured, tail muscles with small black spots; eyes large, lateral, nostril near eyes; spiracle on the left side of the body and distinct; oral disk terminal, lips expanded and directed upwardly into a umbelliform oral disk; transverse width of expanded funnel 18.1% of snout-vent length.

Figure 8. 

Tadpole specimen CIBXJT19050704 of Megophrys xianjuensis sp. nov. A dorsal view B lateral view C ventral view. Arrows indicate two dotted lines on flank of dorsum from mouth to fin.

Morphological comparisons

By having small size body, Megophrys xianjuensis sp. nov. differs from M. aceras, M. auralensis, M. binlingensis, M. carinense, M. caudoprocta, M. chuannanensis, M. damrei, M. edwardinae (in female), M. feae, M. flavipunctata, M. gigantica, M. glandulosa, M. himalayana, M. intermedia, M. jingdongensis, M. kobayashii, M. kalimantanensis, M. lekaguli, M. liboensis, M. ligayae, M. longipes, M. major, M. mangshanensis, M. maosonensis, M. medogensis, M. omeimontis, M. oreocrypta, M. periosa, M. popei, M. robusta, M. spinata, M. sangzhiensis, M. shapingensis and M. shuichengensis and M. takensis (maximum SVL < 42.0 mm in the new species vs. minimum SVL > 45 mm in the latter).

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

By having a small horn-like tubercle at the edge of each upper eyelid, Megophrys xianjuensis sp. nov. differs from M. binchuanensis, M. binlingensis, M. damrei, M. gigantica, M. minor, M. nasuta, M. nankiangensis, M. oropedion, M. pachyproctus, M. spinata, M. stejnegeri, M. takensis, M. wuliangshanensis, M. wushanensis, M. zhangi, and M. zunhebotoensis (vs. tubercle lacking in the latter) and differs from M. carinense, M. feae, M. gerti, M. hansi, M. intermedia, M. kalimantanensis, M. koui, M. liboensis, M. microstoma, M. palpebralespinosa, M. popei, M. shuichengensis, and M. synoria (vs. having a prominent and elongated tubercle at the edge of each upper eyelid in the latter).

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

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

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

By tibiotarsal articulation reaching forward to the region between tympanum and eye when hindlimb is stretched along the side of the body, Megophrys xianjuensis sp. nov. differs from M. baolongensis, M. nankiangensis, M. pachyproctus, M. serchhipii, M. shuichengensis, and M. tuberogranulata (vs. reaching posterior corner of the eye in the latter); differs from M. daweimontis, M. glandulosa, M. lini, M. major, M. medogensis, M. obesa, and M. sangzhiensis (vs. reaching the anterior corner of the eye or beyond eye or nostril and tip of snout in the latter); differs from M. leishanensis (vs. reaching middle part of eye); differs from M. mufumontana (vs. reaching tympanum in males and to the eye in females).

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

By having nuptial pads and nuptial spines on the dorsal base of the first and second fingers in breeding male, Megophrys xianjuensis sp. nov. differs from M. acuta, M. feii, M. shapingensis, and M. shuichengensis (vs. nuptial pads and nuptial spines lacking in the latter); differs from M. boettgeri, and M. elfina (vs. nuptial pads and nuptial spines only on the first finger in the latter).

Megophrys boettgeri and M. kuatunensis were suggested to be distributed in Zhejiang Province, China and might be sympatric with Megophrys xianjuensis sp. nov. (Fei et al. 2016; Wang et al. 2017b). The new species can be distinguished from these species by a series of morphological characters as follows. The new species vs. M. boettgeri: vomerine ridges present vs. vomerine ridges absent, tibiotarsal articulation reaching forward to the region between tympanum and eye when hindlimb is stretched along the side of the body vs. tibiotarsal articulation reaching forward to eye, having nuptial pads and nuptial spines on the dorsal base of the first and second fingers in breeding male vs. nuptial pads and nuptial spines only on the first finger, heels overlapping when thighs are positioned at right angles to the body vs. heels just meeting, light round patches on the shoulder absent vs. present. The new species vs. M. kuatunensis: heels overlapping when thighs are positioned at right angles to the body vs. heels not meeting, toes with rudimentary webbing at bases vs. toes without webbing.

Molecular phylogenetic analyses revealed that the new species was genetically closer to M. lishuiensis. The new species can be distinguished from M. lishuiensis by a series of morphological characters as follows. Vomerine ridges present vs. vomerine ridges absent; heels overlapping when thighs are positioned at right angles to the body vs. heels just meeting or not meeting; light round patches on the shoulder absent vs. present; having significantly lower ratios of UEW, ED, TYD, LW and TW to SVL in males; and having significantly higher ratios of UEW, ED and HAL to SVL in females (all p-values < 0.05; Table 3).

Distribution and habitats

Megophrys xianjuensis sp. nov. is known from the type locality, Xianju County, Zhejiang Province, China at elevations between 320–480 m a.s.l. This new species is frequently found on stones in the streams in the subtropical montane forests (Fig. 5E; Fig. 9). Six sympatric amphibian species, i.e., Amolops wuyiensis, Odorrana tianmuensis, O. graminea, O. tormota, Limnonectes fujianensis, and Quasipaa spinosa, were found.

Figure 9. 

Habitats of Megophrys xianjuensis sp. nov. in the type locality, Xianju County, Zhejiang Province, China A landscape showing mountain forests B a mountain stream where toads of the new species occur.

Etymology

The specific epithet xianjuensis refers to Xianju County, Zhejiang Province, China, where the type locality of the species is located. We propose the common name “Xianju horned toad” in English and Xian ju Jiao Chan in Chinese.

Discussion

Although Megophrys xianjuensis sp. nov. superficially resembles M. lishuiensis, our integrative comparisons with morphological and molecular data can clearly identify the new species from the latter. This indicates that conserved morphology could hamper species delineation, requiring the incorporation of detailed morphological, genetic, and bioacoustic data to recognise cryptic species. The discovery of Megophrys xianjuensis sp. nov. brings the total number of species in the genus to 93, with 49 of them recorded in China (Fei et al. 2016; Frost 2019). Yet, there remain still dozens of undescribed species just in China (Chen et al. 2017; Liu et al. 2018). As noted, the new species was a newly-found clade which has not been reported in previous phylogenetic works although it belongs to the subgenus Panophrys according to our phylogenetic frameworks and previous classifications (Mahony et al. 2017; Liu et al. 2018).

According to records in Fei et al. (2016) and Wang et al. (2017b), M. boettgeri is widely distributed in Zhejiang Province, China. However, with two-years’ field surveys and detailed comparisons with many Megophrys specimens from Xianju County, Zhejiang Province, we have not found M. boettgeri in this region. Similarly, in this county, we have also not found M. kuatunensis and M. lishuiensis even though they were reported to be distributed in western and southern parts of Zhejiang Province, China (Fei et al. 2016; Wang et al. 2017b); therefore, we suggest that probably in Xianju County, Zhejiang Province, China, there is no M. boettgeri, M. kuatunensis or M. lishuiensis. Considering the high underestimated species level and localised diversification in Megophrys (Chen et al. 2017; Liu et al. 2018; Wang et al. 2019), wider and in-depth surveys should be conducted in the eastern part of Zhejiang Province and adjacent areas for detecting distributional range of the new species and for finding cryptic species.

Many Megophrys species have narrow distributions fitting the “micro-endemism” model of Liu et al. (2018) and Wang et al. (2019). Similarly, according to our surveys, Megophrys xianjuensis sp. nov. is probably only distributed in a narrow range in Xianju County and/or adjacent regions. Hence, this species is likely to be threatened as scenic sites in Xianju County harbouring its habitats (Fig. 9) are increasingly impacted by tourists and considerable developments in tourist infrastructure easily leading to habitat loss. Additionally, an increased frequency of extreme weather events in recent years also influenced the species; for example, the super typhoon “Lekima” in August 2019 causing heavy rain seriously broke many channels and forests where the species occurs in Xianju County. It is urgent to conduct more surveys to understand the population status of the new species and to develop appropriate protection strategies.

Acknowledgements

We are grateful to the editors and reviewers for their working on the manuscript. Collections in field were permitted by Administration of Xianju National Park (No. AXJNP2018020801). This study was approved by the animal ethical committee of Chengdu Institute of Biology, Chinese Academy of Sciences, and animal experiments were carried out following the institutional guidelines (No. 2018CIBAEC0345). The work was supported by Demonstration Project on Biodiversity Conservation and Development of Xianju County Project Financed by AFD, Project supported by the biodiversity investigation, observation and assessment program (2019–2023) of Ministry of Ecology and Environment of China, and the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDPB0202).

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