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Research Article
A new species of the genus Leptobrachella Smith, 1925 (Anura, Megophryidae) from Guizhou, China
expand article infoJing-Cai Lyu‡, Liang-Liang Dai‡, Ping-Fan Wei§, Yan-Hong He§, Zhi-Yong Yuan§, Wen-Li Shi|, Sheng-lun Zhou, Si-yu Ran, Zhong-Fan Kuang‡, Xuan Guo‡, Gang Wei#, Guo Yuan‡
‡ Guizhou Institute of Biology, Guizhou Academy of Sciences, Guiyang, China
§ Southwest Forestry University, Kunming, China
| Forestry Bureau of Congjiang County, Guizhou, China
¶ Mt. Foding National Nature Reserve Bureau, Shiqian, China
# Guiyang College, Guiyang, China

Corresponding author: Gang Wei ( wg198553@126.com )

Corresponding author: Guo Yuan ( 875932286@qq.com )

Academic editor: Anthony Herrel
© 2020 Jing-Cai Lyu, Liang-Liang Dai, Ping-Fan Wei, Yan-Hong He, Zhi-Yong Yuan, Wen-Li Shi, Sheng-lun Zhou, Si-yu Ran, Zhong-Fan Kuang, Xuan Guo, Gang Wei, Guo Yuan.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Lyu J-C, Dai L-L, Wei P-F, He Y-H, Yuan Z-Y, Shi W-L, Zhou S-l, Ran S-y, Kuang Z-F, Guo X, Wei G, Yuan G (2020) A new species of the genus Leptobrachella Smith, 1925 (Anura, Megophryidae) from Guizhou, China. ZooKeys 1008: 139-157. https://doi.org/10.3897/zookeys.1008.56412
ZooBank: urn:lsid:zoobank.org:pub:0D4C047F-1B79-4FE5-9ED4-D93C62BCF6A0
Open Access

Abstract

Asian leaf-litter toads of the genus Leptobrachella represent charismatic anuran diversification with 80 species, of which 25 are from China. Recent new discoveries suggest that the diversity of this genus is underestimated. Here, we describe a new species of Leptobrachella, Leptobrachella bashaensis sp. nov. from the Basha Nature Reserve, Congjiang County, Guizhou Province, China. The new species is distinguished from its congeners by the following suite of morphological traits: small body size (SVL 22.9–25.6 mm in six adult males and 27.1 mm in one adult female); head longer than wide; dorsal skin slightly shagreened with small tubercles; creamy-white chest and belly with irregular black spots; distinct ventrolateral glands forming a white line; finger webbing and fringes absent; toe webbing rudimentary and lateral fringes narrow; iris bicolored with bright orange in upper half and silver in lower half; dorsal surface of tadpole head dark brown with small, brown, irregular spot, air sac-shaped bulges on both sides of body. The new species differs from all known congeners by an uncorrected p-distance of >5.3% of the 16S rRNA gene fragment examined, and the phylogenetic analysis clusters the new species with L. maoershanensis and L. laui. At present, the new species is only known from a small range of montane evergreen secondary forests in Basha Nature Reserve approximately 900 m elevation. Its natural history and conservation status are discussed.

Keywords

Integrated taxonomy, morphology, tadpole, vocalization

Introduction

At present, the megophryid genus Leptobrachella Smith, 1925 comprises 80 nominal species that are widely distributed from southwestern China to northeastern India, Southeast Asia and Myanmar, through mainland Indochina to peninsular Malaysia and the island of Borneo (Chen et al. 2018; Frost 2020). With the development of DNA barcoding technology and extensive fieldwork, more and more cryptic species in this genus have been reported. From 2016 to 2020, a total of 28 species were described (Eto et al. 2016, 2018; Rowley et al. 2016, 2017a, b; Yang et al. 2016, 2018; Yuan et al. 2017; Duong et al. 2018; Hou et al. 2018; Nguyen et al. 2018; Wang et al. 2018, 2019; Chen et al. 2019, 2020; Hoang et al. 2019; Li et al. 2020; Luo et al. 2020). The discovery of these species indicates that the species diversity of the genus is underestimated, and there still may be a large number of undiscovered cryptic species.

In China, the genus Leptobrachella is currently known to comprise 25 species, including L. alpinus, L. bourreti, L. eos, L. laui, L. liui, L. mangshanensis, L. maoershanensis, L. nyx, L. oshanensis, L. pelodytoides, L. purpura, L. sungi, L. tengchongensis, L. ventripunctatus, L. wuhuangmontis, L. yingjiangensis, L. yunkaiensis, L. shangsiensis, L. bijie, L. purpuraventra, L. chishuiensis, L. feii, L. flaviglandulosa, L. niveimontis, and L. suiyangensis, occurs widely in Yunnan, Guangxi and Guizhou Province (Chen et al. 2018; Wang et al. 2018; Yang et al. 2018; Chen et al. 2019; Wang et al. 2019; Amphibian China 2020; Li et al. 2020; Luo et al. 2020). More than half of the species of this genus were described in last three years, with more potential new species suggested by previous studies (Chen et al. 2018).

From 2017 to 2019, we collected a series of specimens during the field surveys in Basha Nature Reserve, Guizhou Province, Southern China. These specimens were assigned to genus Leptobrachella based on a combination of the following characteristics: small body size, rounded fingertips, presence of an elevated inner palmar tubercle not continuous to the thumb, absence of vomerine teeth, and vertical bars on the anterior tip of the snout. However, distinct morphological and genetic differences were found between the specimens and all recognized species. Subsequent molecular analyses confirmed that these specimens represent an as yet unknown lineage within the Leptobrachella. Therefore, we describe these specimens as a new species here.

Materials and methods

Sampling

A total of seven specimens were collected during fieldwork in October 2017 and March to June 2019 within Basha Nature Reserve, Guizhou Province (Fig. 1). All specimens were euthanized using chlorobutanol solution, fixed in 10% formalin for 24 hours, then stored in 75% ethanol. Liver or muscle tissues were taken from the specimens before fixing, and preserved in 95% alcohol at -20 °C. These newly collected specimens were subsequently deposited in the Museum of Biology of the Guizhou Institute of Biology, Guizhou Academy of Sciences (GIB, GAS), Guiyang, China.

Figure 1. 

Collection locality (Basha, red star) of Leptobrachella bashaensis sp. nov. specimens from Basha, Guizhou, China used in this study.

Morphology and morphometrics

All measurements were obtained to the nearest 0.1 mm (Watters et al. 2016) with digital calipers following the methods of Fei et al. (2009). The measurements of seven adult individuals were as follows:

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

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

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

SNT snout length (from tip of snout to anterior corner of eye);

EYE eye diameter (diameter of exposed portion of eyeball);

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

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

UEW upper eyelid width (measured as the greatest width of the upper eyelid);

NEL nostril-eyelid length (distance from nostril to eyelid);

NSL nostril-snout length (distance from nostril to snout);

TMP tympanum diameter (horizontal diameter of tympanum);

TEY tympanum-eye distance (distance from anterior edge of tympanum to posterior corner of eye);

TIB tibia length (tibia length with hindlimb flexed);

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

LAHL length of lower arm and hand (distance from tip of the third finger to elbow);

HLL hindlimb length (distance from tip of fourth toe to vent);

FOT foot length (from proximal edge of inner metatarsal tubercle to tip of fourth toe).

Morphometrics of a single tadpole followed Ohler et al. (2011), a total of nine morphometric characters of tadpole were measured:

BH maximum body height;

BL body length (distance from base of vent to the tip of snout);

BW maximum body width;

ED maximum eye diameter (diameter of exposed portion of eyeball);

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

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

TMW maximum tail muscle width;

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

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

DNA sequencing and analysis of sequences

Standard phenol-chloroform extraction with ethanol precipitation protocol was used to extract genomic DNA from tissues (Sambrook et al.1989). We amplified a 565~567 bp fragment 16S rRNA using polymerase chain reactions (PCRs), using the primers, and cycling conditions given by Kocher et al. (1989). Amplified PCR products were purified with enzymatic purification using exonuclease 1 and shrimp alkaline phosphatase that was incubated at 37 °C for 15 min followed by 80 °C to inactivate the enzymes. Purified PCR products were used as templates for Big Dye (Applied Biosystems, Foster City, CA) cycle sequencing. We conducted all sequencing on an ABI 3100 automated sequencers (Applied Biosystems) at Suoqin, Beijing. Considering that the morphological characters of the newly collected specimens are closest to those of L. maoershanensis, 19 sequences representing 16 recognized species of the seven clades belonging to the genus Leptobrachella from southern China (A1–A7; Chen et al. 2018) were retrieved from GenBank and included in the following phylogenetic analyses (Table 1). Megophrys major Boulenger, 1908 was selected as the outgroup based on the current phylogenetic hypothesis of Leptobrachella (Chen et al. 2018). All the sequences were aligned using MUSCLE v. 3.6 with the default settings (Edgar 2004).

Table 1.
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List of voucher specimens and GenBank accession numbers for all DNA sequences of the mitochondrial 16S rRNA gene fragments of Leptobrachella included in this study.

ID Species Locality Voucher no. GenBank no.
1 L. aereus Vilabuly, Savannakhet, Laos NCSM 76038 MH055809
2 L. aereus Savannakhet, Laos SAM R64242 KR018123
3 L. alpinus Huangcaoling, Yunnan, China KIZ046816 MH055866
4 L. bijie Bijie City, Guizhou, China SYS a007313 MK414532
5 L. bijie Bijie City, Guizhou, China SYS a007314 MK414533
6 L. bourreti Sapa,Lao Cai, Vietnam 1999.5660 KR827860
7 L. bourreti Bat Xat, Lao Cai, Vietnam ZMMU-A-5636-02280 MH055872
8 L. chishuiensis Chishui City,Guizhou Province, China CIBCS20190518047 MT117053
9 L. chishuiensis Chishui City,Guizhou Province, China CIBCS20190518042 MT117054
10 L. eos Boun Tay, Phongsaly, Laos NCSM 80551 MH055887
11 L. eos Boun Tay, Phongsaly, Laos MNHN:2004.0277 JN848448
12 L. feii Wenshan Prefecture, Yunnan Province, China KIZ032625 MT302635
13 L. feii Honghe, Yunnan Province, Chin KIZ048973 MT302637
14 L. firthi Ngoc Linh Nature Reserve, Kon Tum, Vietnam AMS: R 176506 JQ739207
15 L. flaviglandulosa Xiaoqiaogou Nature Reserve, Yunnan, China KIZ016064 MT302624
16 L. flaviglandulosa Xiaoqiaogou Nature Reserve, Yunnan, China KIZ032626 MT302633
17 L. isos Gia Lai, Vietnam AMS: R 176469 KT824767
18 L. khasiorum Khasi Hills, Meghalaya, India SDBDU 2009.329 KY022303
19 L. laui San zhoutian, Shenzhen, China SYS a002540 MH055904
20 L. laui Mt. Wutong, Shenzhen, China SYS a003477 MH605576
21 L. liui Mt. Wuyi, Fujian, China SYS a002478 MH605573
22 L. liui Mt. Huanggang, Jiangxi, China SYS a001620 KM014549
23 L. mangshanensis Mangshan, Hunan, China MSZTC201702 MG132197
24 L. mangshanensis Mangshan, Hunan, China MSZTC201703 MG132198
25 L. maoershanensis Maoershan, Guangxi, China KIZ019386 KY986931
26 L. maoershanensis Mao’er Shan, Guangxi, China KIZ07614 MH055927
27 L. maoershanensis Mao’er Shan, Guangxi, China KIZ027236 MH055928
28 L. bashaensis sp. nov. Basha Nature Reserve, Guizhou, China GIB196403 MW136294
29 L. bashaensis sp. nov. Basha Nature Reserve, Guizhou, China GIB196404 MW136295
30 L. minimus Doi Chiang Dao, Chiangmai, Thailand THNHM07418 JN848402
31 L. minimus Doi Suthep, Thailand KUHE:19201 LC201981
32 L. nahangensis Na Hang Nature Reserve, Tuyen Quang, Vietnam ROM 7035 MH055853
33 L. nahangensis Na Hang, Tuyen Quang, Vietnam ZMMU-NAP-02259 MH055854
34 L. namdongensis Thanh Hoa Provincen, Vietnam VNUF A.2017.37 MK965389
35 L. niveimongtis Daxueshan Nature Reserve, Yunnan, China KIZ028276 MT302620
36 L. niveimongtis Daxueshan Nature Reserve, Yunnan, China KIZ028277 MT302621
37 L. nyx Mount Tay Conn Linh, Ha Giang, Vietnam AMNH A163810 DQ283381
38 L. nyx Ha Giang, Ha Giang, Vietnam ROM 36692 MH055816
39 L. oshanensis Emei Shan, Sichuan, China KIZ025776 MH055895
40 L. oshanensis Emei Shan, Sichuan, China YPX37492 MH055896
41 L. petrops Cham Chu Nature Reserve, Tuyen Quang, Vietnam VNMN:2016 A.06 KY459998
42 L. pluvialis Fansipan, Lao Cai, Vietnam ROM 30685 MH055843
43 L. puhoatensis Pu Hu Nature Reserve, Thanh Hoa, Vietnam VNMN:2016 A.23 KY849587
44 L. puhoatensis Pu Hu Nature Reserve, Thanh Hoa, Vietnam AMS:R184852 KY849588
45 L. purpuraventra Bijie City, Guizhou, China SYS a007081 MK414517
46 L. purpuraventra Bijie City, Guizhou, China SYS a007277 MK414518
47 L. shangsiensis Guangxi Prov.,China NHMG1401032 MK095460
48 L. shangsiensis Guangxi Prov.,China NHMG1401033 MK095461
49 L. suiyangensis Suiyang County,Guizhou, China GZNU20180606002 MK829648
50 L. suiyangensis Suiyang County,Guizhou, China GZNU20180606006 MK829649
51 L. sungi Tam Dao, Vinh Phuc, Vietnam ROM 20236 MH055858
52 L. tengchongensis Gaoligong Shan, Yunnan, China SYS a004598 KU589209
53 L. tengchongensis Gaoligong Shan, Yunnan, China SYS a003766 MH055897
54 L. ventripunctatus Zhushihe, Yunnan, China SYS a004536 MH055831
55 L. yunkaiensis Dawuling Forest Station, Maoming City, Guangdong, China SYS a004663 MH605584
56 L. yunkaiensis Dawuling Forest Station, Maoming City, Guangdong, China SYS a004664 MH605585
57 L. zhangyapingi Chiang Mai, Thailand KIZ07258 MH055864
58 Megophrys major Kon Tum, Vietnam AMS R173870 KY476333

Phylogenetic relationships were inferred using both maximum likelihood (ML) and Bayesian inference (BI). ML analysis was performed with RAx ML Black Box (Stamatakis et al. 2008) under the GTR + G model (1000 replicates). BI was performed using MrBayes v. 3.2.1 (Ronquist et al. 2012). The best-fit model was determined using the Akaike Information Criterion (AIC) computed with jModel Test 2 (Darriba et al.2012). Two independent runs were conducted for 30 million generations, sampling every 1000, with four independent chains and a burn-in of 25%. Convergence was assessed by checking stationary distribution and effective sample sizes (>200) using Tracer v. 1.6. (Rambaut et al.2014). We used MEGA v. 5.2 (Tamura et al. 2011) to calculate the interspecific mean uncorrected pairwise distances between the samples from Basha and its most closely related species based on the phylogenetic results. Besides, intraspecific distance was calculated among the samples from Basha.

Results

A total of 565~567 base pairs (bp) of the 16S gene were obtained in the final alignment. The two sequences belonging to the newly acquired specimens (GenBank accession numbers MW136294 and MW136295) shared a same haplotype. The alignment contained 242 variable sites and 186 parsimony informative sites. The observed interspecific uncorrected p-distances between the new population from Basha and all species from clade A based on the study of Chen et al. (2018) varied from 5.3% (L. maoershanensis) to 15.7% (L. firthi) (Table 2).

Table 2.
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Pairwise genetic divergence between Leptobrachella species of monophyletic clades A based on uncorrected p-distance at a 16S rRNA fragment.

Species 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
1 L. bashaensis sp. nov. –
2 L. maoershanensis 0.053 –
3 L. mangshanensis 0.073 0.064 –
4 L. liui 0.089 0.070 0.026 –
5 L. laui 0.068 0.081 0.081 0.085 –
6 L. yunkaiensis 0.082 0.086 0.078 0.082 0.082 –
7 L. flaviglandulosa 0.061 0.074 0.066 0.061 0.077 0.062 –
8 L. niveimontis 0.112 0.117 0.131 0.108 0.116 0.113 0.087 –
9 L. chishuiensis 0.103 0.121 0.130 0.112 0.120 0.121 0.087 0.049 –
10 L. namdongensis 0.107 0.119 0.116 0.112 0.099 0.130 0.104 0.131 0.116 –
11 L. suiyangensis 0.120 0.130 0.148 0.139 0.138 0.130 0.112 0.057 0.053 0.143 –
12 L. bijie 0.116 0.134 0.135 0.117 0.121 0.134 0.095 0.053 0.015 0.125 0.057 –
13 L. purpuraventra 0.103 0.104 0.130 0.113 0.129 0.121 0.095 0.054 0.034 0.117 0.049 0.045 –
14 L. shangsiensis 0.107 0.107 0.126 0.122 0.129 0.125 0.095 0.140 0.148 0.131 0.166 0.157 0.139 –
15 L. feii 0.086 0.090 0.130 0.124 0.120 0.135 0.091 0.131 0.131 0.130 0.135 0.140 0.135 0.091 –
16 L. tengchongensis 0.098 0.103 0.108 0.095 0.086 0.125 0.099 0.103 0.111 0.070 0.119 0.119 0.102 0.116 0.121 –
17 L. oshanensis 0.105 0.104 0.113 0.096 0.127 0.126 0.091 0.062 0.077 0.132 0.086 0.082 0.057 0.131 0.133 0.108 –
18 L. eos 0.107 0.107 0.125 0.108 0.115 0.107 0.082 0.042 0.069 0.121 0.073 0.073 0.053 0.134 0.139 0.094 0.061 –
19 L. bourreti 0.060 0.067 0.080 0.062 0.075 0.073 0.047 0.025 0.032 0.081 0.040 0.043 0.022 0.077 0.069 0.063 0.043 0.025 –
20 L. alpinus 0.095 0.117 0.122 0.116 0.107 0.113 0.074 0.053 0.042 0.126 0.049 0.057 0.049 0.140 0.109 0.107 0.074 0.057 0.030 –
21 L. zhangypingi 0.115 0.132 0.121 0.125 0.124 0.127 0.086 0.148 0.138 0.130 0.150 0.147 0.141 0.133 0.125 0.111 0.139 0.128 0.090 0.120 –
22 L. sungi 0.111 0.116 0.134 0.121 0.133 0.121 0.112 0.140 0.138 0.104 0.139 0.143 0.130 0.116 0.112 0.091 0.139 0.134 0.075 0.139 0.081 –
23 L. nahangensis 0.098 0.103 0.121 0.099 0.115 0.116 0.082 0.117 0.108 0.096 0.126 0.121 0.108 0.073 0.086 0.104 0.113 0.108 0.043 0.104 0.120 0.099 –
24 L. pluvialis 0.087 0.078 0.105 0.095 0.115 0.098 0.074 0.130 0.130 0.131 0.130 0.148 0.122 0.073 0.082 0.126 0.122 0.130 0.069 0.117 0.112 0.099 0.069 –
25 L. ventripunctatus 0.112 0.108 0.127 0.104 0.124 0.128 0.091 0.144 0.148 0.118 0.176 0.158 0.149 0.082 0.082 0.113 0.141 0.144 0.077 0.144 0.112 0.103 0.061 0.066 –
26 L. nyx 0.090 0.095 0.109 0.091 0.107 0.121 0.078 0.099 0.112 0.109 0.139 0.121 0.121 0.078 0.065 0.104 0.122 0.116 0.045 0.108 0.120 0.095 0.053 0.074 0.062 –
27 L. aereus 0.086 0.086 0.117 0.099 0.119 0.134 0.082 0.126 0.113 0.113 0.139 0.122 0.122 0.082 0.049 0.116 0.129 0.134 0.065 0.108 0.124 0.098 0.061 0.074 0.068 0.046 –
28 L. miniums 0.079 0.079 0.105 0.101 0.113 0.100 0.075 0.119 0.118 0.096 0.127 0.136 0.109 0.063 0.075 0.105 0.110 0.105 0.050 0.110 0.103 0.079 0.055 0.059 0.063 0.062 0.071 –
29 L. puhoatensis 0.111 0.121 0.126 0.121 0.116 0.148 0.130 0.153 0.129 0.062 0.157 0.147 0.129 0.141 0.148 0.087 0.142 0.152 0.092 0.143 0.153 0.114 0.105 0.131 0.136 0.127 0.144 0.111 –
30 L. petrops 0.129 0.124 0.117 0.109 0.125 0.140 0.118 0.150 0.143 0.086 0.143 0.153 0.134 0.154 0.139 0.082 0.149 0.134 0.094 0.139 0.152 0.126 0.118 0.134 0.150 0.123 0.140 0.115 0.082 –
31 L. khasiorum 0.156 0.139 0.148 0.126 0.143 0.138 0.144 0.167 0.166 0.135 0.186 0.185 0.176 0.182 0.191 0.125 0.167 0.152 0.124 0.176 0.175 0.157 0.136 0.154 0.168 0.159 0.176 0.114 0.135 0.122 –
32 L. isos 0.134 0.139 0.154 0.134 0.155 0.153 0.121 0.144 0.167 0.135 0.158 0.177 0.158 0.149 0.148 0.131 0.141 0.149 0.098 0.149 0.153 0.149 0.112 0.153 0.129 0.134 0.145 0.132 0.151 0.170 0.193 –
33 L. firthi 0.157 0.172 0.153 0.130 0.143 0.169 0.130 0.143 0.152 0.141 0.162 0.166 0.162 0.153 0.153 0.131 0.149 0.162 0.097 0.152 0.153 0.140 0.107 0.144 0.117 0.117 0.137 0.133 0.146 0.156 0.156 0.136 –

Both ML and BI (Fig. 2) analyses yielded near-identical results except for the poorly supported nodes (BS < 75% or BPP < 0.95). The relationships of the most clades (A1–A7) of Leptobrachella were not resolved, which is similar to previous studies (Yang et al. 2016; Yuan et al. 2017) due to limited information of 16S. The samples from Basha, Guizhou, formed a monophyletic group (Clade A4, Fig. 2) with L. liui, L. laui, L. mangshanensis, L. maoershanensis, and L. yunkaiensis with high support in both analyses, but the relationships among these species were not resolved. The samples from Basha formed a lineage that clustered with L. maoershanensis, but with low support (BPP < 95% and BS < 70%). In combination with morphological differences (see below), we conclude that the newly collected Basha specimens represent a distinct species, and we describe it herein.

Figure 2. 

Bayesian Inference of Leptobrachella inferred from a 567 bp fragment of the 16S rRNA gene. Node support is indicated on branches as Bayesian posterior probabilities (BPP) (displayed values > 0.95) and Maximum Likelihood support (displayed values > 70); “-” represents low support values. The scale bar represents 0.07 nucleotide substitutions per site.

Taxonomic account

Leptobrachella bashaensis sp. nov.

http://zoobank.org/6257CDCB-B124-4649-BE10-9A4874C67F20
Figs 3, 4, 5, 6, 7; Tables 1, 2, 3

Type material

Holotype. GIB196403, adult male, from Basha Nature Reserve, Congjiang, Guizhou Province, China (25.8534°N, 108.7356°E, elevation 900 m. a.s.l.; Fig. 1), collected by Jingcai Lyu and Liangliang Dai on 4 June 2019. Paratypes. GIB196401–02, GIB196404, GIB196406–07 (five adult males) collected from the holotype locality by Jingcai Lyu on 4 June 2019.

Diagnosis

The new species is assigned to the genus Leptobrachella on the basis of the following characters: small size, rounded fingertips, presence of an elevated inner palmar tubercle not continuous to the thumb, presence of supra-axillary, femoral and ventrolateral glands, vomerine teeth absent, tubercles on eyelids, and pale vertical bar present on anterior tip of snout (Dubois 1980, 1983; Ohler et al. 2011; Rowley and Cao 2009; Rowley et al. 2013). Leptobrachella bashaensis sp. nov. is distinguished from its congeners by a combination of the following morphological characters: (1) small size (SVL 22.9–25.6 mm in six adult males and 27.1 mm in one adult female), (2) head longer than wide, (3) externally distinct tympanum, (4) dorsal skin slightly shagreened with small tubercles and irregular brown stripes, (5) distinct dark spots on the flank, (6) creamy-white chest and off-white belly with irregular black spots, (7) grey-pinkish to dark brownish-violet ventral skin of limbs with numerous whitish speckles, (8) distinct ventrolateral glands, forming a distinct white line, (9) finger webbing and fringes absent, (10) toe webbing rudimentary and lateral fringes narrow, (11) longitudinal ridges under toes and not interrupted at the articulations, (12) a distinctly bicolored iris, typically bright orange in upper half, fading to silver in lower half.

Description of the holotype

Adult male. SVL 24.0 mm; head slightly longer than wide (HDL/HDW = 1.10), rectangular in dorsal view (Fig. 3A, B); snout rounded in both ventral view and lateral view, protruding slightly beyond lower jaw (Fig. 3D); nostril oval, located closer to tip of snout than to eye; loreal region oblique; canthus rostralis distinct; eye large (EYE/HDL = 0.35), diameter slightly smaller than snout length (EYE/SNT = 0.88), notably protuberant in both dorsal and lateral view; pupil vertical; tympanum distinct, rounded, diameter smaller than that of eye (TMP/EYE = 0.60) (Fig. 3C); vomerine teeth absent; vocal sac openings slit-like, located posterolateral on floor of mouth; tongue long and moderately wide, with a shallow notch at posterior tip; supratympanic ridge distinct, running from eye towards axillary with raised tubercles. Forelimb long and slender, fingers long and slender, without webbing and lateral fringes; relative length of fingers II<I<IV<III; tips of fingers rounded and slightly swollen; nuptial pad absent; subarticular tubercles absent in fingers; inner metacarpal tubercle large and rounded, separated from laterally compressed and much smaller outer metacarpal tubercle (Fig. 4A, B). Hindlimb moderately long; tibia half of snout-vent length (TIB/SVL = 0.50); tibiotarsal articulation of adpressed limb reaching snout, well beyond anterior margin of eye, but not beyond snout tip; relative toe lengths I<II<V<III<IV; toe tips rounded and thickened; greatly reduced basal webbing present between all five toes; narrow lateral fringes present only on II and III toes (Fig. 4C, D); subarticular tubercles hardly discernible under toes II and III; dorsal skin slightly rough with small tubercles and irregular pustules; ventral skin smooth; oval supra-axillary gland present at forelimb base on ventral surface of axillary region (Fig. 3F); oval femoral glands distinct on posteroventral surface of thigh, closer to knee than to vent; ventrolateral glands forming a distinct line on flanks.

Figure 3. 

Holotype of Leptobrachella bashaensis sp. nov. (GIB196403) in life A frontolateral view B lateral view C iris coloration D W-shaped marking E dorsal view of thighs F posterioventral view of thighs G ventral view H throat view.

Figure 4. 

Holotype of Leptobrachella bashaensis sp. nov. (GIB196403) in life A, B ventral and dorsal view of the hand C, D dorsal and ventral views of the foot.

Coloration of holotype in life

Dorsal surface of head and trunk brown with small, dark brown, irregularly shaped markings; large reverse-triangle dark brown marking between eyes, connected to the W-shaped mark between axillae (Fig. 3); supratympanic ridge present; elbow to upper arm reddish in color; fine, distinct reddish tubercles scattered on upper eyelids, dorsal surfaces of head, body, and limbs. Ventral surface of throat and belly off-white, with faint spots; chest creamy white, with irregular black spots (Fig. 3G, H); ventral surfaces of lip and limbs covered with irregular white speckles; ventral surface of thighs grey-brown with white spots. Supra-axillary gland, ventrolateral glands, and femoral glands white. Iris bicolored: bright orange in upper half, silver in lower half, with black reticulations throughout. Fine, faint transverse dark brown bars on dorsal surface of fingers and toes, lower arms, tarsus, thighs, and tibia (Fig. 4).

Coloration of holotype in preservation

In preservative (75% ethanol), dorsal surface dark grey-brown, and ventral and lateral white glands not evident (Fig. 5); ventral surface of throat, chest, belly, and interior portions of arms fade to creamy white, ventral surface of thighs dark brown with white spots; bars and blotches on dorsum and limbs are dark brown and less apparent. The color of the tympanum fades to brown.

Figure 5. 

Holotype of Leptobrachella bashaensis sp. nov. (GIB196403) in preservation A dorsal view B ventral view C volar view of hand D plantar view of foot.

Tadpoles

Brown, narrow; BH 4.4 mm; BL 14.1 mm; BW 6.2; ED 2.0 mm; TH 3.8; SS 4.9 mm; TMW 3.4 mm; TL 29.3 mm; TOL 43.8 mm. TAL/TOL = 0.67, with a I:3+3/1+1:II labial tooth row formula (Fig. 6). In life, dorsal surface of head dark brown with small, brown, irregularly shaped spot; air sac-shaped bulges on both sides of the body; upper lip and lower lip nearly round shape. Tadpoles were collected in the field from a stream surveyed on 16 October 2017 in Basha Nature Reserve by Lyu.

Figure 6. 

Tadpole of Leptobrachella bashaensis sp. nov. in preservative A dorsal view B ventral view C dorsal view D labial tooth.

Etymology

The specific epithet, “bashaensis”/Basha Zhang Tu Chan (岜沙掌突蟾 in Chinese), refers to the location where the specimens were collected, Basha Nature Reserve of Guizhou Province, China.

Morphological variation

Variation in size and body proportions of the type series are shown in Table 3. Representative photographs of paratypes in life are shown in Fig. 7. In life, the dorsal surface of the head and trunk varies from olive-brown to reddish; the ventral surface of the lower jaw among these individuals varies from cream-yellow, pink, to grey; the ventral surface of chest and belly is grey-pink to white. There is variation among individuals in the shape of tubercles, pustules, black ventrolateral blotches, and dark stripes, bars on the dorsum or dorsal surface of limbs. The W-shaped marking between axillae is distinct in all individuals.

Figure 7. 

Paratypes of Leptobrachella bashaensis sp. nov. in life (GIB196404).

Table 3.
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Morphometric measurements (in mm) of Leptobrachella bashaensis sp. nov. Abbreviations are defined in the text.

Voucher number GIB196401 GIB196402 GIB196403 GIB196404 GIB196406 GIB196407 GIB196405
Sex Male Male Male Male Male Male Female
SVL 22.9 23.0 24.0 24.6 25.6 24.2 27.1
HDL 8.4 8.3 9.4 8.7 9.4 9.1 9.6
HDW 8.0 7.5 8.7 8.1 8.2 8.0 8.2
SNT 3.3 3.4 3.5 3.5 4.0 3.8 3.8
IND 2.5 2.4 2.4 2.7 2.7 2.5 2.9
IOD 3.2 3.5 3.3 3.3 3.3 3.2 3.3
UEW 2.3 2.1 2.2 2.2 2.3 2.5 2.3
EYE 2.8 2.7 2.9 3.1 3.5 3.3 3.6
NEL 1.5 1.6 1.8 2.0 1.9 1.8 2.0
NSL 1.9 1.8 1.7 1.7 1.9 1.6 1.9
TEY 0.7 0.9 1.1 0.9 1.0 1.0 1.1
ML 6.2 5.6 6.1 6.0 6.0 6.3 6.3
TMP 2.1 1.6 2.2 1.9 1.9 1.7 1.9
LAHL 11.7 10.4 10.8 10.9 11.5 11.6 11.9
HLL 37.4 37.6 40.7 40.3 38.8 39.7 41.3
TIB 12.1 11.4 12.6 12.3 12.1 13.2 12.9
FOT 11.0 10.9 11.2 11.3 10.6 11.6 11.8

Natural history

All specimens were collected at night in small streams in Basha Nature Reserve approximately 900 m elevation (Fig. 8). Calling males were found along the streams, perching on large rocks, in rocky crevices, or under dead wood. Insect-like calls could be heard in June. The breeding season of this species is likely to occur from June to July, as females collected during these months were gravid, and males were heard calling only from June to the beginning of July. During both surveys, the number of males observed was much greater than females (males:females = 12:1).

Figure 8. 

Habitat at the type locality of Leptobrachella bashaensis sp. nov., Basha Nature Reserve, Congjiang, Guizhou province, China.

Comparisons

Leptobrachella bashaensis sp. nov. differs from all known Leptobrachella species distributed north of the Isthmus of Kra by a combination of male body size, externally distinct tympanum, and black spots on the flank, plus ventral coloration, degree of webbing and fringing on the toes, iris coloration, and dorsal skin texture.

Care was taken to differentiate Leptobrachella bashaensis sp. nov. from the five most morphologically and molecularly similar species, L. maoershanensis, L. laui, L. mangshanensis, L. yunkaiensis, and L. liui from China.

Leptobrachella bashaensis sp. nov. differs from its sister taxon L. maoershanensis by the following: smaller body size (SVL 22.9–25.6 mm in males, 27.1 mm in female vs 25.2–30.4 mm in males, 28.6 mm in female); bicolored iris with bright orange in upper half, fading to silver in lower half (vs typically bright orange-red in upper half, fading to silver in lower half); vertical and distinct narrow pupil (vs vertical and wide pupil); narrow lateral fringes present only on II and III toes (vs narrow lateral fringes present on all toes).

Leptobrachella bashaensis sp. nov. differs from L. laui in having slightly a smaller body size (SVL 22.9–25.6 mm in males, 27.1 mm in female vs 24.8–26.7 mm in males, 28.1 mm in female); bicolored iris with bright orange in upper half, fading to silver in lower half (vs uniformly coppery orange with fine black reticulations throughout); vertical and distinct narrow pupil (vs vertical and wide pupil); narrow lateral fringes present only on II and III toes (vs wide lateral fringes present on all toes); distinct black spots on the flank (vs indistinct black spots on the flank).

Leptobrachella bashaensis sp. nov. differs from L. mangshanensis by having slightly smaller body size (SVL 22.9–25.6 mm in males, 27.1 mm in female vs 22.2–27.8 mm in males, 30.2 mm in female); bicolored iris with bright orange in upper half, fading to silver in lower half (vs bright orange in upper half, greyish cream in lower half); narrow lateral fringes present only on II and III toes (vs weak lateral fringes on toes).

Leptobrachella bashaensis sp. nov. differs from L. yunkaiensis by having smaller body size (SVL 22.9–25.6 mm in males, 27.1 mm in female vs 25.9–29.3 mm in males, 34.0–35.3 mm in females); bicolored iris with bright orange in upper half, fading to silver in lower half (vs coppery orange in upper half and silver in lower half); narrow lateral fringes present only on II and III toes (vs wide lateral fringes present on all toes); distinct black spots on the flank (vs indistinct black spots on the flank).

Leptobrachella bashaensis sp. nov. differs from L. liui in having slightly smaller body size (SVL 22.9–25.6 mm in males, 27.1 mm in female vs 23.0–28.7 mm in males, 23.1–28.1 mm in females); vertical and distinctly narrow pupil (vs vertical and wide pupil); narrow lateral fringes present only on II and III toes (vs wide lateral fringes present on all toes).

Discussion

Chen et al. (2018) suggested that Leptobrachella may contain hidden species diversity based on multiple nuclear DNA markers assessed using larger sample sizes, combined with morphological, nuclear gene, and bioacoustic data. With the development of DNA barcoding technology and extensive field work, more and more cryptic species in this genus have been reported. The number of Leptobrachella species recorded in China is now up to 25, including the new species. The discovery of this species reaffirms that the diversity within the genus is underestimated, with many species yet to be discovered.

Guizhou Province is located in the typical Karst landform area of southern China with an abundance of species diversity. So far, eight species of the genus Leptobrachella (L. liui, L. oshanensis, L. pelodytoides, L. ventripunctatus, L. bijie, L. purpuraventra, L. chishuiensis, and L. suiyangensis) were recorded in Guizhou province, while L. bashaensis sp. nov. has not been included in previous studies, which indicates that there may potentially be more new species to be found by further field research (Wu et al. 1987; Wang et al. 2019).

We observed a restricted distribution of the species in Basha Natural Reserve. We believe that L. bashaensis sp. nov. has a specific niche associated with small streams. The following threats were observed: reduced forest coverage, soil erosion, habitat destruction, and local people using the tadpole of L. bashaensis sp. nov. for food. Hence, greater protection is required for this endemic species inhabiting this high-diversity region.

Acknowledgements

This study was supported by the Forestry Bureau of Congjiang County, Guizhou Province. We thank Gui-Wang Gao and Lao-Bin Zhang from the Forestry Bureau of Congjiang County for support. This work was supported by Guizhou Provincial Science and Technology Projects (2020)4Y029#.

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