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Two new Leptobrachella species (Anura, Megophryidae) from the Yunnan-Guizhou Plateau, southwestern China
expand article infoJian Wang§, Zhi-Tong Lyu, Shuo Qi, Zhao-Chi Zeng§, Wen-Xiang Zhang|, Long-Shan Lu|, Ying-Yong Wang
‡ Sun Yat-sen University, Guangzhou, China
§ Shenzhen Shuanghuamu Biological Technology Co., Ltd, Shenzhen, China
| Yunnan Huanglianshan National Nature Reserve, Honghe Hani and Yi Autonomous Prefecture, China
Open Access

Abstract

Two new toad species of the genus Leptobrachella are described from the Yunnan-Guizhou Plateau of China, based on the combination of molecular and morphological data. The description of Leptobrachella aspera Wang, Lyu, Qi & Wang, sp. nov. from Huanglianshan Nature Reserve represents the thirteenth Leptobrachella species known from Yunnan Province, and the description of Leptobrachella dorsospina Wang, Lyu, Qi & Wang, sp. nov. from Yushe Forest Park represents the sixth Leptobrachella species known from Guizhou Province. These new discoveries further emphasize the extremely high diversity of the Leptobrachella toads in these regions.

Keywords

Leptobrachella aspera sp. nov., Leptobrachella dorsospina sp. nov., molecular phylogeny, morphology, taxonomy

Introduction

The generic classifications within the family Megophryidae Bonaparte, 1850 have always been controversial. For example, recent comprehensive approaches have produced different taxonomic schemes for the genus Megophrys sensu lato Kuhl and Van Hasselt 1822 (Chen et al. 2017; Mahony et al. 2017; Liu et al. 2018; Li et al. 2020b). The taxonomy of another group of megophrid toads are facing the same problem: Chen et al. (2018) presented the first well-resolved phylogenetic hypothesis for the genera Leptolalax Dubois, 1983 and Leptobrachella Bonaparte, 1850. They tended towards the most conservative “one-genus option” pending the acquisition of additional data by assigning Leptolalax as a junior synonym of Leptobrachella. Their results also rejected the hypothesis that Leptolalax consists of two subgenera as proposed by Delorme et al. (2006) and Dubois et al. (2010). In this context, the genus Leptobrachella currently contains 82 species widely distributed from southern China, west to northeastern India, through Indochina to the island of Borneo (Frost 2020). Leptobrachella is a species-rich genus of megophrid frogs, and a large number of new species have been discovered in recent years due to the application of integrative taxonomy incorporating detailed morphological, bioacoustic and molecular analyses (Rowley et al. 2016, 2017; Yang et al. 2016; Yuan et al. 2017; Eto et al. 2018; Nguyen et al. 2018; Wang et al. 2019; Chen et al. 2020; Luo et al. 2020; Qian et al. 2020).

During recent field surveys in the Yunnan-Guizhou Plateau of southwestern China, a number of megophrid specimens were collected from Yushe Forest Park in western Guizhou (Fig. 1, site 1) and Huanglianshan Nature Reserve in southern Yunnan (Fig. 1, site 2), respectively. Morphologically, all the specimens can be assigned to the genus “Leptolalax” (now a junior subjective synonym of Leptobrachella), based on the following characters: (1) small or moderate size, snout-vent length not greater than 60.0 mm, (2) rounded finger tips, the presence of an elevated inner palmar tubercle not continuous to the thumb, (3) presence of macroglands on body including supra-axillary, pectoral, femoral and ventrolateral glands, (4) vomerine teeth absent, (5) tubercles on eyelids present, and (6) anterior tip of snout with whitish vertical bar (Dubois 1983; Matsui 1997, 2006; Lathrop et al. 1998; Delorme et al. 2006; Das et al. 2010). Although their generic allocation is without doubt, some characters of these specimens do not correspond to the diagnoses of any recognized species. Subsequent molecular analysis further revealed that these specimens represent two distinct evolutionary lineages. Considering both the morphological differences and molecular divergences, these specimens are described herein as two new species.

Figure 1. 

Collection sites. Site 1-Yushe Forest Park, Shuicheng County, Guizhou Province, the type locality of Leptobrachella dorsospina sp. nov.; site 2-Huanglianshan Nature Reserve, Lyuchun County, Yunnan Province, the type locality of L. aspera sp. nov.; site 3-Xiaoqiaogou Nature Reserve, Xichou County, Yunnan Province, the type locality of L. feii; and site 4-Daweishan Nature Reserve, Pingbian County, Yunnan Province, another distribution locality of L. feii.

Materials and methods

Sampling

For the molecular analyses, a total of 80 sequences (nine muscle tissue samples was sequenced and 71 sequences obtained from GenBank) were used, including five sequences of the undescribed species from Guizhou, four sequences of the undescribed species from Yunnan, 69 sequences of 66 recognized congeners, and two out-group sequences of Oreolalax rhodostigmatus Hu & Fei, 1979 and Leptobrachium tengchongensis Yang & Huang, 2019, respectively (Table 1). Due to the presence of cryptic diversity within genus Leptobrachella, we chose sequences from type series or topotype specimens for molecular analysis if available to ensure the taxonomic identity of the species being studied.

DNA Extraction, PCR, and sequencing

DNA was extracted from muscle tissue using a DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd. The mitochondrial gene 16S ribosomal RNA gene (16S rRNA) fragment from each sample was sequenced. Fragments were amplified using the primer pairs L3975 (5'-CGCCTGTTTACCAAAAACAT-3') and H4551 (5'-CCGGTCTGAACTCAGATCACGT-3') (Simon et al. 1994). PCR amplifications were performed in a 20 μl reaction volume with the following cycling conditions: an initial denaturing step at 95 °C for five min; 35 cycles of denaturing at 95 °C for 40 s, annealing at 53 °C for 40 s and extending at 72 °C for one min; and a final extending step of 72 °C for 10 min. PCR products were purified with spin columns. The purified products were sequenced with both forward and reverse primers using BigDye Terminator Cycle Sequencing Kit according to the guidelines of the manufacturer. The products were sequenced on an ABI Prism 3730 automated DNA sequencer in Shanghai Majorbio Bio-pharm Technology Co., Ltd. All sequences have been deposited in GenBank (Table 1).

Table 1.

Collection localities, voucher data and GenBank numbers (16S rRNA) for all samples used in this study.

ID Ingroup Collection Locality Voucher No. GenBank No.
1 Leptobrachella aspera sp. nov. Huanglianshan Nature Reserve, Lyuchun, Yunnan, China SYS a007743 MW046199
2 Leptobrachella aspera sp. nov. Huanglianshan Nature Reserve, Lyuchun, Yunnan, China SYS a007744 MW046200
3 Leptobrachella aspera sp. nov. Huanglianshan Nature Reserve, Lyuchun, Yunnan, China SYS a007745 MW046201
4 Leptobrachella aspera sp. nov. Huanglianshan Nature Reserve, Lyuchun, Yunnan, China SYS a007746 MW046202
5 Leptobrachella dorsospina sp. nov. Yushe Forest Park, Shuicheng, Guizhou, China SYS a004961 MW046194
6 Leptobrachella dorsospina sp. nov. Yushe Forest Park, Shuicheng, Guizhou, China SYS a004962 MW046195
7 Leptobrachella dorsospina sp. nov. Yushe Forest Park, Shuicheng, Guizhou, China SYS a004973 MW046196
8 Leptobrachella dorsospina sp. nov. Yushe Forest Park, Shuicheng, Guizhou, China SYS a004974 MW046197
9 Leptobrachella dorsospina sp. nov. Yushe Forest Park, Shuicheng, Guizhou, China SYS a004975 MW046198
10 Leptobrachella feii Xiaoqiaogou Nature Reserve, Yunnan, China KIZ032625 MT302635
11 Leptobrachella feii Xiaoqiaogou Nature Reserve, Yunnan, China KIZ048894 MT302634
12 Leptobrachella feii Xiaoqiaogou Nature Reserve, Yunnan, China KIZ048972 MT302636
13 Leptobrachella feii Xiaoqiaogou Nature Reserve, Yunnan, China KIZ048973 MT302637
14 Leptobrachella aerea U Bo, Quang Binh, Vietnam ZFMK 86362 JN848409
15 Leptobrachella alpina Huangcaoling, Jingdong, Yunnan, China KIZ046816 MH055866
16 Leptobrachella applebyi Ngoc Linh, Kon Tum, Vietnam AMS R 173778 KR018108
17 Leptobrachella arayai Mesilau, Sabah, Malaysia BORNEENSIS 22931 AB847558
18 Leptobrachella ardens Kon Ka Kinh, Gia Lai, Vietnam AMS R 176463 KR018110
19 Leptobrachella bidoupensis Hon Giao, Lam Dong, Vietnam NCSM 77321 HQ902883
20 Leptobrachella bijie Zhaozishan Nature Reserve, Guizhou, China SYS a007320 MK414539
21 Leptobrachella botsfordi Fansipan, Lao Cai, Vietnam AMS R 176540 MH055953
22 Leptobrachella bourreti Lao Cai, Vietnam AMS R 177673 KR018124
23 Leptobrachella chishuiensis Chishui, Guizhou Province, China CIBCS20190518047 MT117053
24 Leptobrachella crocea Kon Tum, Vietnam AMS R 173740 MH055954
25 Leptobrachella dringi Gunung Mulu National Park, Sarawak, Malaysia NMBE1056372 KJ831298
26 Leptobrachella eos Long Nai, Phongsaly, Laos MNHN 2004.0274 JN848452
27 Leptobrachella firthi Ngoc Linh Nature Reserve, Kon Tum, Vietnam AMS R 176524 JQ739206
28 Leptobrachella flaviglandulosa Xiaoqiaogou Nature Reserve, Yunnan, China KIZ032626 MT302633
29 Leptobrachella fritinniens Base Camp of Mulu NP, Sarawak, Malaysia KUHE 55371 AB847557
30 Leptobrachella fuliginosa Phetchaburi, Thailand KUHE 20174 LC201987
31 Leptobrachella gracilis Camp 1 of Gunung Mulu NP, Sarawak, Malaysia NMBE1056364 KJ831300
32 Leptobrachella hamidi Bukit Lanjak, Malaysia KUHE 17545 AB969286
33 Leptobrachella heteropus Larut, Malaysia KUHE 15486 LC202005
34 Leptobrachella isos Gia Lai, Vietnam AMS R 176480 KT824769
35 Leptobrachella kajangensis Tioman, Malaysia LSUHC 4431 LC202001
36 Leptobrachella kalonensis Song Luy, Binh Thuan, Vietnam AMNH A191762 KR018115
37 Leptobrachella kecil Cameron, Malaysia KUHE 52440 LC202004
38 Leptobrachella khasiorum Meghalaya, India SDBDU 2009.329 KY022303
39 Leptobrachella laui Tai Mo Shan, Hongkong, China SYS a002057 KM014546
40 Leptobrachella liui Guadun, Mt. Wuyi, Fujian, China SYS a002479 MH605574
41 Leptobrachella macrops Phu Yen, Vietnam PYU DTD-508 MG787991
42 Leptobrachella maculosa Phuoc Binh, Ninh Thuan, Vietnam ZFMK 96600 KR018120
43 Leptobrachella mangshanensis Mangshan Nature Reserve, Hunan, China MSZTC201701 MG132196
44 Leptobrachella maoershanensis Maoershan Nature Reserve, Guangxi, China KIZ019385 KY986930
45 Leptobrachella marmorata Annah Rais, Padawan, Malaysia KUHE 53192 AB969287
46 Leptobrachella maura Kinabalu, Malaysia SP 21450 AB847559
47 Leptobrachella melanoleuca Srat Thani, Thailand KUHE 19719 LC201990
48 Leptobrachella melica Virachey, Ratanakiri, Cambodia MVZ 258197 HM133599
49 Leptobrachella minima Changdao, Thailand KUHE 23733 LC201980
50 Leptobrachella nahangensis Na Hang Nature Reserve, Tuyen Quang, Vietnam ROM 7035 MH055853
51 Leptobrachella namdongensis Thanh Hoa, Vietnam VNUF A.2017.95 MK965390
52 Leptobrachella niveimontis Daxueshan Nature Reserve, Yunnan, China KIZ015734 MT302618
53 Leptobrachella nyx Malipo, Yunnan, China ROM 35606 MH055814
54 Leptobrachella oshanensis Mt. Emei, Sichuan, China SYS a001830 KM014810
55 Leptobrachella pallida Gia Rich, Lam Dong, Vietnam UNS00510 KR018112
56 Leptobrachella pelodytoides Tam Dao, Vinh Phu, Vietnam MVZ 223642 AY236798
57 Leptobrachella petrops Tuyen Quang, Vietnam VNMN:2016 A.06 KY459998
58 Leptobrachella picta Gunung Kinabalu National Park, Sabah, Malaysia UNIMAS 8705 KJ831295
59 Leptobrachella pluvialis Sa Pa, Lao Cai, Vietnam MNHN: 1999.5675 JN848391
60 Leptobrachella puhoatensis Nghe An, Vietnam AMS R184852 KY849588
61 Leptobrachella purpura Yingjiang, Yunnan, China SYS a006531 MG520355
62 Leptobrachella purpuraventra Wujing Nature Reserve, Guizhou, China SYS a007277 MK414518
63 Leptobrachella pyrrhops Loc Bac, Lam Dong, Vietnam ZMMU ABV-00176 KP017576
64 Leptobrachella rowleyae Son Tra, Da Nang, Vietnam ITBCZ 4113 MG682549
65 Leptobrachella sabahmontana Mahua, Crocker, Malaysia BORNEENSIS 12454 AB847550
66 Leptobrachella shangsiensis Shiwandashan, Guangxi, China NHMG1401032 MK095460
67 Leptobrachella sola Terengganu, Malaysia KUHE 52342 LC202011
68 Leptobrachella suiyangensis Suiyang, Guizhou, China GZNU20180606002 MK829648
69 Leptobrachella sungi Bac Giang, Vietnam ZMMU-NAP-02269 MH055859
70 Leptobrachella tadungensis Dak Nong, Vietnam UNS00517 KR018122
71 Leptobrachella tengchongensis Tengchong, Yunnan, China SYS a004598 KU589209
72 Leptobrachella tuberosa Kon Ka Kinh National Park, Gia Lai, Vietnam ZMMU-NAP-02275 MH055959
73 Leptobrachella ventripunctata Xishuangbanna, Yunnan, China SYS a001768 KM014811
74 Leptobrachella wuhuangmontis Mt. Wuhuang, Pubei, Guangxi, China SYS a003485 MH605577
75 Leptobrachella wulingensis Tianzishan Nature Reserve, Hunan, China CSUFT 200 MT530317
76 Leptobrachella yingjiangensis Yingjiang, Yunnan, China SYS a006533 MG520350
77 Leptobrachella yunkaiensis Yunkaishan Nature Reserve, Guangdong, China SYS a004663 MH605584
78 Leptobrachella zhangyapingi Chiang Mai, Thailand KIZ07258 MH055864
79 Leptobrachium tengchongense Tengchong, Yunnan, China SYS a004603 KX066876
80 Oreolalax rhodostigmatus Da Fang, Guizhou, China CIB ZYCA746 EF397248

Phylogenetic analyses

Sequences were aligned in Clustal X 2.0 (Thompson et al. 1997) with default parameters. For GenBank sequences which lack information for part of the missing segments, we filled the blank sites with “N”. The aligned data was trimmed allowing no gap positions and default parameters in Gblocks version 0.91b (Castresana 2000). lyWe ran Jmodeltest v2.1.2 (Darriba et al. 2012) with Akaike and Bayesian information criteria on the alignment and obtained the best-fitting nucleotide substitution model of GTR + I + G. Phylogenetic analysis was using Bayesian inference (BI) in MrBayes 3.2.4 (Ronquist et al. 2012). Two independent runs with four Markov Chain Monte Carlo simulations were performed for ten million iterations and sampled every 1000 iterations. The first 25% of samples were discarded as burn-in. Convergence of the Markov Chain Monte Carlo simulations was assessed by PSRF ≤ 0.01 and ESS (effective sample size) value > 200 using Tracer 1.4 (http://tree.bio.ed.ac.uk/software/tracer/). Genetic distances among all Leptobrachella samples were calculated in MEGA 6 using the uncorrected p-distance model, with pairwise deletion of gaps and missing data.

Morphometrics

Measurements followed Fei et al. (2009) and Rowley et al. (2013), and were taken with a digital caliper to the nearest 0.1 mm. These measurements were as follows:

SVL snout-vent length (from tip of snout to vent);

HDL head length (from tip of snout to rear of jaws);

HDW head width (head width at commissure of jaws);

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 upper eyelids);

IND internasal distance (distance between nares);

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 (distance from knee to heel);

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

PL pes length (distance from tip of fourth toe to proximal edge of the inner metatarsal 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).

Sex was determined by the presence of internal vocal sac openings, and the presence of eggs in abdomen seen via external inspection.

All specimens were fixed in 10% buffered formalin and later transferred to 70% ethanol for preservation, and deposited at the Museum of Biology, Sun Yat-sen University (SYS) and Chengdu Institute of Biology, the Chinese Academy of Sciences (CIB), China; tissue samples were preserved in 95% ethanol for molecular studies.

Comparative morphological data of Leptobrachella species were obtained from examination of museum specimens (see Appendix 1) and from the references listed in Table 2. Due to the high likelihood of undiagnosed diversity within the genus (Rowley et al. 2016; Yang et al. 2016), where available, we rely on examination of topotypic material and/or original species descriptions.

Table 2.

Data sources of the 82 currently known species of the genus Leptobrachella.

ID Leptobrachella species Literature
1 L. aerea (Rowley, Stuart, Richards, Phimmachak & Sivongxay, 2010) Rowley et al. 2010c
2 L. alpina (Fei, Ye & Li, 1990) Fei et al. 2009, 2016
3 L. applebyi (Rowley & Cao, 2009) Rowley and Cao 2009
4 L. arayai (Matsui, 1997) Matsui 1997
5 L. ardens (Rowley, Tran, Le, Dau, Peloso, Nguyen, Hoang, Nguyen & Ziegler, 2016) Rowley et al. 2016
6 L. baluensis Smith, 1931 Dring 1983; Eto et al. 2016
7 L. bijie Wang, Li, Li, Chen & Wang, 2019 Wang et al. 2019
8 L. bidoupensis (Rowley, Le, Tran & Hoang, 2011) Rowley et al. 2011
9 L. bondangensis Eto, Matsui, Hamidy, Munir & Iskandar, 2018 Eto et al. 2018
10 L. botsfordi (Rowley, Dau & Nguyen, 2013) Rowley et al. 2013
11 L. bourreti (Dubois, 1983) Ohler et al. 2011
12 L. brevicrus Dring, 1983 Dring 1983; Eto et al. 2015
13 L. crocea (Rowley, Hoang, Le, Dau & Cao, 2010) Rowley et al. 2010a
14 L. chishuiensis Li, Liu, Wei & Wang, 2020 Li et al. 2020a
15 L. dringi (Dubois, 1987) Inger et al. 1995; Matsui and Dehling 2012
16 L. eos (Ohler, Wollenberg, Grosjean, Hendrix, Vences, Ziegler & Dubois, 2011) Ohler et al. 2011
17 L. feii Chen, Yuan & Che, 2020 Chen et al. 2020
18 L. firthi (Rowley, Hoang, Dau, Le & Cao, 2012) Rowley et al. 2012
19 L. fritinniens (Dehling & Matsui, 2013) Dehling and Matsui 2013
20 L. fuliginosa (Matsui, 2006) Matsui 2006
21 L. flaviglandulosa Chen, Wang & Che, 2020 Chen et al. 2020
22 L. fusca Eto, Matsui, Hamidy, Munir & Iskandar, 2018 Eto et al. 2018
23 L. gracilis (Günther, 1872) Günther 1872; Dehling 2012b
24 L. hamidi (Matsui, 1997) Matsui 1997
25 L. heteropus (Boulenger, 1900) Boulenger 1900
26 L. isos (Rowley, Stuart, Neang, Hoang, Dau, Nguyen & Emmett, 2015) Rowley et al. 2015a
27 L. itiokai Eto, Matsui & Nishikawa, 2016 Eto et al. 2016
28 L. juliandringi Eto, Matsui & Nishikawa, 2015 Eto et al. 2015
29 L. kajangensis (Grismer, Grismer & Youmans, 2004) Grismer et al. 2004
30 L. kalonensis (Rowley, Tran, Le, Dau, Peloso, Nguyen, Hoang, Nguyen & Ziegler, 2016) Rowley et al. 2016
31 L. kecil (Matsui, Belabut, Ahmad & Yong, 2009) Matsui et al. 2009
32 L. khasiorum (Das, Tron, Rangad & Hooroo, 2010) Das et al. 2010
33 L. lateralis (Anderson, 1871) Anderson 1871; Humtsoe et al. 2008
34 L. laui (Sung, Yang & Wang, 2014) Sung et al. 2014
35 L. liui (Fei & Ye, 1990) Fei et al. 2009; Sung et al. 2014
36 L. macrops (Duong, Do, Ngo, Nguyen & Poyarkov, 2018) Duong et al. 2018
37 L. maculosa (Rowley, Tran, Le, Dau, Peloso, Nguyen, Hoang, Nguyen & Ziegler, 2016) Rowley et al. 2016
38 L. mangshanensis (Hou, Zhang, Hu, Li, Shi, Chen, Mo & Wang, 2018) Hou et al. 2018
39 L. maoershanensis (Yuan, Sun, Chen, Rowley & Che, 2017) Yuan et al. 2017
40 L. marmorata (Matsui, Zainudin & Nishikawa, 2014) Matsui et al. 2014b
41 L. maura (Inger, Lakim, Biun & Yambun, 1997) Inger et al. 1997
42 L. melanoleuca (Matsui, 2006) Matsui 2006
43 L. melica (Rowley, Stuart, Neang & Emmett, 2010) Rowley et al. 2010b
44 L. minima (Taylor, 1962) Taylor 1962; Ohler et al. 2011
45 L. mjobergi Smith, 1925 Eto et al. 2015
46 L. nahangensis (Lathrop, Murphy, Orlov & Ho, 1998) Lathrop et al. 1998
47 L. natunae (Günther, 1895) Günther 1895
48 L. namdongensis Hoang, Nguyen, Luu, Nguyen & Jiang, 2019 Hoang et al. 2019
49 L. neangi Stuart & Rowley, 2020 Stuart and Rowley 2020
50 L. niveimontis Chen, Poyarkov, Yuan & Che, 2020 Chen et al. 2020
51 L. nokrekensis (Mathew & Sen, 2010) Mathew and Sen 2010
52 L. nyx (Ohler, Wollenberg, Grosjean, Hendrix, Vences, Ziegler & Dubois, 2011) Ohler et al. 2011
53 L. oshanensis (Liu, 1950) Fei et al. 2009, 2016
54 L. pallida (Rowley, Tran, Le, Dau, Peloso, Nguyen, Hoang, Nguyen & Ziegler, 2016) Rowley et al. 2016
55 L. palmata Inger & Stuebing, 1992 Inger and Stuebing 1992
56 L. parva Dring, 1983 Dring 1983
57 L. pelodytoides (Boulenger, 1893) Boulenger 1893; Ohler et al. 2011
58 L. petrops (Rowley, Dau, Hoang, Le, Cutajar & Nguyen, 2017) Rowley et al. 2017a
59 L. pictua (Malkmus, 1992) Malkmus 1992
60 L. platycephala (Dehling, 2012) Dehling 2012a
61 L. pluvialis (Ohler, Marquis, Swan & Grosjean, 2000) Ohler et al. 2000, 2011
62 L. puhoatensis (Rowley, Dau & Cao, 2017) Rowley et al. 2017b
63 L. purpura (Yang, Zeng & Wang, 2018) Yang et al. 2018
64 L. purpuraventra Wang, Li, Li, Chen & Wang, 2019 Wang et al. 2019
65 L. pyrrhops (Poyarkov, Rowley, Gogoleva, Vassilieva, Galoyan & Orlov, 2015) Poyarkov et al. 2015
66 L. rowleyae (Nguyen, Poyarkov, Le, Vo, Ninh, Duong, Murphy & Sang, 2018) Nguyen et al. 2018
67 L. sabahmontana (Matsui, Nishikawa & Yambun, 2014) Matsui et al. 2014a
68 L. serasanae Dring, 1983 Dring 1983
69 L. shangsiensis Chen, Liao, Zhou & Mo, 2019 Chen et al. 2019
70 L. sola (Matsui, 2006) Matsui 2006
71 L. suiyangensis Luo, Xiao, Gao & Zhou, 2020 Luo et al. 2020
72 L. sungi (Lathrop, Murphy, Orlov & Ho, 1998) Lathrop et al. 1998
73 L. tadungensis (Rowley, Tran, Le, Dau, Peloso, Nguyen, Hoang, Nguyen & Ziegler, 2016) Rowley et al. 2016
74 L. tamdil (Sengupta, Sailo, Lalremsanga, Das & Das, 2010) Sengupta et al. 2010
75 L. tengchongensis (Yang, Wang, Chen & Rao, 2016) Yang et al. 2016
76 L. tuberosa (Inger, Orlov & Darevsky, 1999) Inger et al. 1999
77 L. ventripunctata (Fei, Ye & Li, 1990) Fei et al. 2009, 2016
78 L. wuhuangmontis Wang, Yang & Wang, 2018 Wang et al. 2018
79 L. wulingensis Qian, Xia, Cao, Xiao & Yang, 2020 Qian et al. in publication
80 L. yingjiangensis (Yang, Zeng & Wang, 2018) Yang et al. 2018
81 L. yunkaiensis Wang, Li, Lyu & Wang, 2018 Wang et al. 2018
82 L. zhangyapingi (Jiang, Yan, Suwannapoom, Chomdej & Che, 2013) Jiang et al. 2013

Results

The BI analyses are shown in Fig. 2 with Bayesian posterior probabilities (BPP) for major nodes > 0.90. Genetic distances among all Leptobrachella samples are given in the Suppl. material 1: Table S1. Comparative morphological data of all recognized Leptobrachella species occurring north of the Kra Isthmus are listed in Table 3.

Figure 2. 

Bayesian Inference tree. The Bayesian posterior probabilities (BPP) > 0.90 were retained.

As shown by the phylogenetic result, Leptobrachella samples from Huanglianshan Nature Reserve are clustered in a distinct and robust monophyletic lineage with strong support (BPP 1.00). This lineage forms the sister taxon to L. feii occurring in Xiaoqiaogou Nature Reserve (BPP 1.00). The genetic distances between these two lineages are 3.0–3.4%, which is significantly larger than that among other recognized species (e.g., p-distance 2.6% between L. liui and L. mangshanensis). Detailed morphological examination also reveals a combination of characters that distinguish the specimens of the unnamed lineage from L. feii and other known congeners (see taxonomic comparison below). Therefore, based on the molecular and morphological differences, the population from Huanglianshan Nature Reserve is proposed as a new species, Leptobrachella aspera sp. nov.

Samples of the other unnamed lineage from Yushe Forest Park, cluster in another distinct and robust monophyletic lineage with strong support (BPP 1.00). This lineage is close to several species occurring in southwestern China, but its specific placement remains unresolved due to the insufficient support values. The smallest genetic distance between this lineage and another congener is 3.5% (vs. L. purpuraventra), which is significantly larger than that between other recognized species (e.g., p-distance 2.6% between L. liui and L. mangshanensis). Detailed morphological examination also reveals a combination of characteristics distinguishing the specimens of this lineage from all known congeners (see taxonomic comparison below). Therefore, based on the molecular and morphological differences, the population from Yushe Forest Park is proposed as a new species, Leptobrachella dorsospina sp. nov.

Table 3.

Comparisons of selected diagnostic characters for the new species described herein and congeners occurring north of the Kra Isthmus (modified from Rowley et al. 2017; Wang et al. 2019; Chen et al. 2020).

Leptobrachella species Male SVL (mm) Black spots on flanks Toe webbing Toe fringes Ventral coloration Dorsal skin texture
L. aspera sp. nov. 22.4 Present Rudimentary Narrow Creamy white with distinct dark patches on chest and abdomen Rough with dense conical granules, tubercles, and glandular folds
L. dorsospina sp. nov. 28.7–30.5 Present Rudimentary Narrow Greyish white with black spots and orange pigmentations Rough with dense conical granules, tubercles, glandular folds, and conical spines
L. aerea 25.1–28.9 Absent Rudimentary Wide Near immaculate creamy white, brown speckles on margins Finely tuberculate
L. alpina 24.0–26.4 Present Rudimentary Wide in males Creamy-white with dark spots Relatively smooth, some with small warts
L. applebyi 19.6–22.3 Present Rudimentary Absent Reddish brown with white speckles Smooth
L. ardens 21.3–24.7 Present Absent Absent Reddish brown with white speckles Smooth, finely shagreened
L. bidoupensis 18.5–25.4 Present Rudimentary Weak Reddish brown with white speckles Smooth
L. bijie 29.0–30.4 Present Rudimentary Narrow White with distinct nebulous greyish speckles on chest and ventrolateral flanks Shagreened and granular
L. botsfordi 29.1–32.6 Absent Rudimentary Narrow Reddish brown with white speckles Shagreened
L. bourreti 28.0–36.2 Present Rudimentary Weak Creamy white Relatively smooth, some with small warts
L. crocea 22.2–27.3 Absent Rudimentary Absent Bright orange Highly tuberculate
L. chishuiensis 30.8–33.4 Present Rudimentary Narrow White with distinct nebulous greyish speckles on chest and ventrolateral flanks Shagreened and granular
L. eos 33.1–34.7 Absent Rudimentary Wide Creamy white Shagreened
L. feii 21.5–22.8 Present Rudimentary Narrow Creamy white with black blotches Shagreened with small tubercles
and ridge
L. firthi 26.4–29.2 Absent Rudimentary Wide in males Creamy white Shagreened with fine tubercles
L. flaviglandulosa 23.0–27.0 Present Poorly developed Narrow Whitish with black speckles on margins Shagreened with yellowish-
brown tubercles
L. fuliginosa 28.2–30.0 Present Rudimentary Weak White with brown dusting Nearly smooth with few tubercles
L. isos 23.7–27.9 Absent Rudimentary Wide in males Creamy white with white dusting on margins Mostly smooth, females more tuberculate
L. kalonensis 25.8–30.6 Present Absent Absent Pale, speckled brown Smooth
L. khasiorum 24.5–27.3 Present Rudimentary Wide Creamy white Isolated, scattered tubercles
L. laui 24.8–26.7 Present Rudimentary Wide Creamy white with dark brown dusting on margins Round granular tubercles
L. liui 23.0–28.7 Present Rudimentary Wide Creamy white with dark brown spots on chest and margins Round granular tubercles with glandular folds
L. lateralis 26.9–28.3 Present Rudimentary Absent Creamy white Roughly granular
L. macrops 28.0–29.3 Present Rudimentary Absent Greyish violet with white speckles Roughly granular with larger tubercles
L. maculosa 24.2–26.6 Present Absent Absent Brown with few white speckles Mostly smooth
L. mangshanensis 22.2–27.8 Present Rudimentary Weak White speckles on throat and belly Nearly smooth
L. maoershanensis 25.2–30.4 Present Rudimentary Narrow Creamy white chest and belly with irregular black spots With longitudinal folds
L. melica 19.5–22.7 Present Rudimentary Absent Reddish brown with white speckles Smooth
L. minima 25.7–31.4 Present Rudimentary Absent Creamy white Smooth
L. nahangensis 40.8 Present Rudimentary Absent Creamy white with light speckles on throat and chest Smooth
L. niveimontis 22.5–23.6 Present Rudimentary Narrow Marbling with black speckles Relatively smooth with small tubercles
L. nokrekensis 26.0–33.0 Present Rudimentary Unknown Creamy white Tubercles and longitudinal folds
L. nyx 26.7–32.6 Present Rudimentary Absent Creamy white with brown margins Rounded tubercles
L. namdongensis 30.9 Present Rudimentary Absent Immaculate white, chest and belly with dark specking on outer margins Low, round tubercles, more dense
in posterior part of the back
L. neangi - Present Weak (in females) Absent (in females) Light purplish gray with dark
brown mottling on throat
Small, irregular bumps and ridges
L. oshanensis 26.6–30.7 Present Absent Absent Whitish with no markings or only small, light grey spots Smooth with few glandular ridges
L. pallida 24.5–27.7 Absent Absent Absent Reddish brown with white speckles Tuberculate
L. pelodytoides 27.5–32.3 Present Wide Narrow Whitish Small, smooth warts
L. petrops 23.6–27.6 Absent Absent Narrow Immaculate creamy white Highly tuberculate
L. pluvialis 21.3–22.3 Present Rudimentary Absent Dirty white with dark brown marbling Smooth, flattened tubercles on flanks
L. puhoatensis 24.2–28.1 Present Rudimentary Narrow Reddish brown with white dusting With longitudinal skin ridges
L. purpura 25.0–27.5 Present Rudimentary Wide Dull white with indistinct grey dusting Shagreen with small tubercles
L. purpuraventra 27.3–29.8 Present Rudimentary Narrow Grey purple with distinct nebulous greyish speckles on chest and ventrolateral flanks Shagreened with granules
L. pyrrhops 30.8–34.3 Present Rudimentary Absent Reddish brown with white speckles Slightly shagreened
L. rowleyae 23.4–25.4 Present Absent Absent Pinkish milk-white to light brown chest and belly with numerous white speckles Smooth with numerous tiny tubercles
L. suiyangensis 28.7–29.7 Present Rudimentary Narrow Yellowish creamy-white with marble texture chest and belly or with irregular light brown speckles Shagreen with small granules
L. sungi 48.3–52.7 Absent or small Wide Weak White Granular
L. tadungensis 23.3–28.2 Present Absent Absent Reddish brown with white speckles Smooth
L. tamdil 32.3 Present Wide Wide White Weakly tuberculate
L. tengchongensis 23.9–26.0 Present Rudimentary Narrow White with dark brown blotches Shagreened with small tubercles
L. tuberosa 24.4–29.5 Absent Rudimentary Absent White with small grey spots/streaks Highly tuberculate
L. ventripunctata 25.5–28.0 Present Rudimentary Absent Chest and belly with dark brown spots Longitudinal skin ridges
L. wuhuangmontis 25.6–30.0 Present Rudimentary Narrow Greyish white mixed by tiny white and black dots Rough, scattered with dense conical tubercles
L. wulingensis 22.7–30.5 Present Rudimentary Narrow Translucent creamy white, with distinct or indistinct brown speckles at margins Shagreened with sparse large warts, some with longitudinal ridges
L. yingjiangensis 25.7–27.6 Present Rudimentary Wide Creamy white with dark brown flecks on chest and margins Shagreened with small tubercles
L. yunkaiensis 25.9–29.3 Present Rudimentary Wide Belly pink with distinct or indistinct speckles Shagreened with short skin ridges and raised warts
L. zhangyapingi 45.8–52.5 Absent Rudimentary Wide Creamy-white with brown margins Mostly smooth with distinct tubercles

Taxonomic accounts

Leptobrachella aspera Wang, Lyu, Qi & Wang, sp. nov.

Fig. 3

Type material

Holotype. SYS a007743, adult male, collected by Jian Wang, Yao Li and Yu-Long Li on 31 May 2019 from Huanglianshan Nature Reserve (22.89°N, 102.29°E; ca. 1930 m a.s.l.), Lyuchun County, Yunnan Province, China.

Paratypes (N = 3). Three adult females, SYS a007744–7745, SYS a007746/CIB116080, the same collection data as the holotype.

Figure 3. 

Morphological features in life. Leptobrachella aspera sp. nov., holotype SYS a007743.

Diagnosis

(1) Small size (SVL 22.4 mm in a single adult male, 25.0–26.4 in three adult females), (2) dorsal skin rough, with dense conical granules, tubercles and glandular folds, (3) iris bicolored, amber on upper half and silver on lower half, (4) tympanum distinctly discernible, distinct black supratympanic line present, (5) absence of webbing and lateral fringes on fingers, toes with rudimentary webbing and narrow lateral fringes both in males and females, (6) longitudinal ridges under toes not interrupted at the articulations, (7) relative finger lengths I < IV < II < III, relative toe length I < II < V < III < IV, (8) heels just meeting, tibia-tarsal articulation reaches the region between middle of eye to anterior corner of eye, (9) dorsum greyish brown to yellowish brown grounding, with small light orange granules and distinct darker brown markings scattered with irregular light orange or greyish white pigmentations, (10) flanks with several enlarged dark patches with light yellowish green margin, (11) ventral surface creamy white, with distinct regular dark patches on chest and abdomen.

Comparison

From the 26 known congeners of the genus Leptobrachella occurring south of the Kra Isthmus, the presence of supra-axillary and ventrolateral glands, can easily distinguish L. aspera sp. nov. from L. arayai, L. dringi, L. fritinniens, L. gracilis, L. hamidi, L. heteropus, L. kajangensis, L. kecil, L. marmorata, L. melanoleuca, L. maura, L. picta, L. platycephala, L. sabahmontana and L. sola, all of which lack the supra-axillary and ventrolateral glands; and by the significantly larger body size, SVL 22.4 mm in a single male, L. aspera sp. nov. differs from the smaller L. baluensis (14.9–15.9 mm in males), L. brevicrus (17.1–17.8 mm in males), L. bondangensis (17.8 mm in male), L. fusca (16.3 mm in male), L. itiokai (15.2–16.7 mm in males), L. juliandringi (17.0–17.2 mm in males), L. mjobergi (15.7–19.0 mm in males), L. natunae (17.6 mm in one adult male), L. parva (15.0–16.9 mm in males), L. palmata (14.4–16.8 mm in males), and L. serasanae (16.9 mm in female).

Leptobrachella aspera sp. nov. is recovered as a sister taxon to L. feii in the phylogenetic tree (Fig. 2). However, the new species can be distinguished from L. feii by the following morphological characters: head relatively short, HDL/SVL 0.33–0.35 (vs. head relatively long, HDL/SVL 0.38–0.43); distinct regular dark patches on skin of chest and abdomen (vs. irregular black blotches scattered on skin of chest and belly); color of upper half of iris amber (vs. color of upper half of iris lighter, golden orange); ventrolateral glands forming an non-continuous line (vs. ventrolateral glands forming a continuous line); relative finger lengths I < IV < II < III (vs. relative finger lengths II < I < IV < III); tibio-tarsal articulation of adpressed limb reaching the region between middle of eye to anterior corner of eye (vs. tibio-tarsal articulation of adpressed limb reaching beyond eye).

While Leptobrachella pluvialis is distributed in the same mountain range on the Vietnamese side and possesses similar body size (Ohler et al. 2000), it can be separated from L. aspera sp. nov. by the following characters: (1) smooth dorsal skin with flattened tubercles on flanks (vs. rough dorsal skin with dense conical granules in L. aspera sp. nov.), (2) absence of webbing on toes (vs. rudimentary webbing on toes in L. aspera sp. nov.), and (3) relatively longer hindlimbs, the tibia-tarsal articulation reaching to the nostril (vs. relatively shorter hindlimbs, the tibia-tarsal articulation reaching the region between middle of eye to anterior corner of eye in L. aspera sp. nov.).

For the remaining 54 members of the genus Leptobrachella, in having SVL 22.4 mm in a single male, L. aspera sp. nov. differs from the larger L. aerea (25.1–28.9 in males), L. alpina (24.0–28.9 mm in males), L. bijie (29.0–30.4 mm in males), L. botsfordi (29.1–32.6 mm in males), L. bourreti (28.0–36.2 mm in males), L. chishuiensis (30.8–33.4 in males), L. eos (33.1–34.7 mm in males), L. firthi (26.4–29.2 mm in males), L. flaviglandulosa (23.0–27.0 mm in males), L. fuliginosa (28.2–30.0 mm in males), L. isos (23.7–27.9 mm in males), L. kalonensis (25.8–30.6 mm in males), L. khasiorum (24.5–27.3 mm in males), L. laui (24.8–26.7 mm in males), L. lateralis (26.9–28.3 mm in males), L. macrops (28.0–29.3 mm in males), L. maculosa (24.2–26.6 mm in males), L. minima (25.7–31.4 mm in males), L. nahangensis (40.8 mm in male), L. nokrekensis (26.0–33.0 mm in males), L. nyx (26.7–32.6 mm in males), L. neangi (30.9 mm in male), L. namdongensis (30.9 mm in male), L. oshanensis (26.6–30.7 mm in males), L. pallida (24.5–27.7 mm in males), L. pelodytoides (27.5–32.3 mm in males), L. petrops (23.6–27.6 mm in males), L. puhoatensis (24.2–28.1 mm in males), L. purpura (25.0–27.5 mm in males), L. purpuraventra (27.3–29.8 mm in males), L. pyrrhops (30.8–34.3 mm in males), L. rowleyae (23.4–25.4 mm in males), L. suiyangensis (28.7–29.7 mm in males), L. sungi (48.3–52.7 mm in males), L. tadungensis (23.3–28.2 mm in males), L. tamdil (32.3 mm in male), L. tengchongensis (23.9–26.0 mm in males), L. tuberosa (24.4–29.5 mm in males), L. ventripunctata (25.5–28.0 mm in males), L. wuhuangmontis (25.6–30.0 mm in males), L. yingjiangensis (25.7–27.6 mm in males), L. yunkaiensis (25.9–29.3 mm in males), and L. zhangyapingi (45.8–52.5 mm in males). By presence of black spots on flanks, the new species can be distinguished from L. crocea, versus absence of black spots on flanks; by rudimentary webbing on toes, the new species can be distinguished from L. ardens, versus absence of webbing on toes; by narrow lateral fringes on toes, the new species can be distinguished from L. applebyi, L. ardens, L. crocea, and L. melica, all having no lateral fringes on toes, and from L. liui, having wide lateral fringes on toes; by the creamy white ventral coloration and distinct regular dark patches on the chest and abdomen, the new species can be distinguished from L. applebyi, L. ardens, L. bidoupensis, and L. melica, all having reddish brown ventral coloration with white specks, from L. crocea, having bright orange ventral coloration, from L. mangshanensis, lacking dark skin patches on the chest and abdomen, from L. liui, having creamy white ventral coloration with dark brown spots on the chest and margins, and from L. niveimontis, having marbling ventral coloration with black speckling; by rough dorsal skin with dense conical granules, tubercles and glandular folds, the new species can be distinguished from L. applebyi, L. ardens, L. bidoupensis, L. mangshanensis, L. melica, and L. niveimontis, all having smooth dorsal skin.

Description of holotype

Adult male. Body size small, SVL 22.4 mm. Head length slightly larger than head width, HDW/HDL 0.99; snout slightly protruding, projecting slightly beyond margin of the lower jaw; nostril closer to snout than eye; canthus rostralis gently rounded; loreal region slightly concave; interorbital space flat, internarial distance greater than interorbital distance, IND/IOD 1.07; pineal ocellus absent; pupil vertical; snout length longer than eye diameter, SNT/EYE 1.26; tympanum distinct, rounded, and slightly concave, diameter smaller than that of the eye and larger than tympanum-eye distance, TMP/EYE 0.52 and TEY/TMP 0.44; upper margin of tympanum in contact with supratympanic ridge; distinct black supratympanic line present; vomerine teeth absent; vocal sac openings slit-like, paired, located posterolaterally on floor of mouth in close proximity to the margins of the mandible; tongue deeply notched posteriorly; supratympanic ridge distinct, extending from posterior corner of eye to supra-axillary gland.

Tips of fingers rounded, slightly swollen; relative finger lengths I < IV < II < III; nuptial pad absent; subarticular tubercles absent; large, rounded inner palmar tubercle distinctly separated from small, rounded outer palmar tubercle; webbing and lateral fringes on fingers absent. Tips of toes rounded, slightly swollen; relative toe length I < II < V < III < IV; subarticular tubercles absent; distinct longitudinal dermal ridges present under the 3rd to 5th toes, not interrupted; large, oval inner metatarsal tubercle present, outer metatarsal tubercle absent; toes webbing rudimentary; narrow lateral fringes present on all toes. Tibia 47% of snout-vent length; tibiotarsal articulation reaching to anterior corner of eye; heels slightly overlapping when thighs are appressed at right angles with respect to body.

Dorsal skin rough, with dense conical granules, tubercles and glandular folds; ventral skin smooth; sparse tiny tubercles present on surface of chest; pectoral gland and femoral gland oval; the size of pectoral glands almost equal to tips of fingers and femoral glands; femoral gland situated on posteroventral surface of thigh, closer to knee than to vent; supra-axillary glands raised. Ventrolateral glands distinctly visible, raised, forming an incomplete line.

Coloration of holotype in life

Dorsum greyish brown with small light orange granules, distinct darker brown markings scattered with irregular light orange and greyish white pigmentations. A dark brown inverted triangular pattern between the anterior corners of the eyes in connection with a dark brown W-shaped marking in the interorbital region, which is also connected to a W-shaped marking between the axillae. Tympanum dark brown. Small light orange granules present on dorsum of body and limbs; a dark brown blotch under the eye; transverse dark brown bars present on dorsal surface of limbs and digits; distinct dark brown patches with light yellowish green margin on flanks from groin to axilla; elbow and upper arms with distinct coppery orange coloration.

Ventral surface of throat, chest, and belly creamy white; presence of distinct nebulous greyish speckles present on throat, and distinct dark patches on chest and abdomen; ventral surface of limbs greyish purple, scattered with greyish white spots and small patches. Supra-axillary gland coppery orange; femoral, pectoral, and ventrolateral glands greyish white. Iris bicolored, amber on upper half and silver on lower half.

Coloration of holotype in preservative

(Fig. 4A). Dorsum of body and limbs dark brown; transverse bars on limbs become more distinct; dark brown patterns, markings and spots on the back become indistinct, orange pigmentations become dark brown, greyish white pigmentations become dark grey. Ventral surface of limbs and surface of throat light brown, surface of abdomen greyish white, nebulous speckles on throat absent, dark patches on chest, abdomen and flanks become more distinct, light yellowish green margin of patches on flanks absent. Supra-axillary, femoral, pectoral, and ventrolateral glands greyish white.

Figure 4. 

Morphological features in preserved specimens of A Leptobrachella aspera sp. nov., holotype SYS a007743 B Leptobrachella dorsospina sp. nov., holotype SYS a004974. Ellipse selected region showing the tiny spines on dorsal skin.

Variation

Measurements and body proportions are listed in Table 4. Nonsexual characters of all the female paratypes (SYS a007744, 7745, 7746) match the overall characters of the holotype except that: the dorsum is greyish brown in the holotype SYS a007743 (vs. yellowish brown in the paratypes); the size of the pectoral glands are almost equal to the tips of the fingers and the femoral glands (vs. the size of the pectoral glands are larger than the tips of fingers and the femoral glands in the paratypes); the tibia-tarsal articulation reaches forward to the anterior corner of the eye in the holotype (vs. the tibia-tarsal articulation reaches forward to the middle of the eye in the paratypes SYS a007745, 7746); the ventral skin of the thighs smooth (vs. the ventral skin of the thighs rough with dense raised tubercles in the paratypes).

Table 4.

Measurements and body proportions of Leptobrachella aspera sp. nov.

Voucher SYS a 007743 SYS a 007744 SYS a 007745 SYS a 007746
Sex Male Female Female Female
SVL 22.4 25.3 25.0 26.4
HDL 8.1 9.5 9.5 9.6
HDW 8.0 9.3 9.2 9.0
SNT 3.7 3.8 3.8 3.4
IND 2.5 2.3 2.7 2.7
IOD 2.3 2.5 2.5 2.5
EYE 2.9 3.2 3.2 3.1
TMP 1.5 1.8 1.9 1.6
TEY 0.7 1.0 1.0 0.8
ML 5.9 7.0 6.6 6.3
LAHL 11.2 13.5 12.7 12.6
PL 10.1 11.7 10.2 11.1
TIB 10.6 12.4 11.9 11.9
HLL 34.4 41.5 40.4 39.1
HDL/SVL 0.36 0.37 0.38 0.36
HDW/SVL 0.36 0.37 0.37 0.34
HDW/HDL 0.99 0.98 0.97 0.94
SNT/HDL 0.16 0.15 0.15 0.13
IND/HDW 0.31 0.25 0.29 0.30
IOD/HDW 0.29 0.27 0.27 0.28
IND/IOD 1.07 0.91 1.08 1.09
EYE/HDL 0.36 0.34 0.34 0.32
TMP/EYE 0.52 0.56 0.60 0.51
ML/SVL 0.26 0.28 0.26 0.24
LAHL/SVL 0.50 0.53 0.51 0.48
PL/SVL 0.45 0.46 0.41 0.42
TIB/SVL 0.47 0.49 0.48 0.45
HLL/SVL 1.53 1.64 1.61 1.48

Etymology

The specific epithet, aspera, is a Latin adjective which means rough, in reference to the dorsal skin texture of the new species. According to its type locality, we suggest its English common name as “Huanglianshan Leaf Litter Toad”, and the Chinese name “Huang Lian Shan Zhang Tu Chan (黄连山掌突蟾)”.

Distribution and habits

Currently, Leptobrachella aspera sp. nov. is known only from its type locality Huanglianshan Nature Reserve, near the border between China and Vietnam. The new species was found along a drainage ditch of a mountainous road. The road was surrounded by broad-leaved forest at an altitude ca. 1930 m and not close to any hillstreams. Males were not heard calling during the field survey from 31 May to 1 June 2019.

Leptobrachella dorsospina Wang, Lyu, Qi & Wang, sp. nov.

Fig. 5

Type material

Holotype. SYS a004974, adult male, collected by Zhi-Tong Lyu and Run-Lin Li on 21 June 2016 from Yushe Forest Park (26.47°N, 104.80°E; ca. 2100 m a.s.l.), Shuicheng District, Liupanshui City, Guizhou Province, China.

Paratypes (N = 6). An adult male, SYS a004977, and five adult females, SYS a004961/CIB116081, SYS a 004962, SYS a004973, 4975, 4976, collected by Zhi-Tong Lyu and Run-Lin Li on 20–21 June 2016 from the same locality as the holotype.

Figure 5. 

Morphological features in life. Leptobrachella dorsospina sp. nov., holotype SYS a004974.

Diagnosis

(1) Small size (SVL 28.7–30.5 mm in two adult males, 32.1–39.8 mm in five adult females), (2) dorsal skin rough, with dense conical granules, tubercles, glandular folds and conical spines, (3) iris bicolored, light orange on upper half and silver on lower half, (4) tympanum distinctly discernible, distinct black supratympanic line present, (5) absence of webbing and lateral fringes on fingers, toes with rudimentary webbing and narrow lateral fringes both in males and females, (6) longitudinal ridges under toes interrupted at the articulations, (7) relative finger lengths II = IV < I < III, relative toe length I < II < V < III < IV, (8) heels slightly overlapping, tibia-tarsal articulation reaches forward to the posterior corners of eyes, (9) dorsum greyish brown to dark brown grounding, with distinct darker brown markings and scattered with irregular light greyish brown pigmentations and yellowish brown spots, (10) flanks with several enlarged dark patches positioned longitudinally in two rows, (11) ventral surface greyish white with black spots and orange pigmentations.

Comparison

Compared with the 26 known congeners of the genus Leptobrachella occurring south of the Kra Isthmus, L. dorsospina sp. nov. can be easily distinguished by the presence of supra-axillary and ventrolateral glands, from L. arayai, L. dringi, L. fritinniens, L. gracilis, L. hamidi, L. heteropus, L. kajangensis, L. kecil, L. marmorata, L. melanoleuca, L. maura, L. picta, L. platycephala, L. sabahmontana and L. sola, all of which are lacking the supra-axillary and ventrolateral glands; and by the significantly larger body size, SVL 28.7–30.5 mm in two adult male, L. dorsospina sp. nov. differs from the smaller L. baluensis (14.9–15.9 mm in males), L. brevicrus (17.1–17.8 mm in males), L. bondangensis (17.8 mm in male), L. fusca (16.3 mm in male), L. itiokai (15.2–16.7 mm in males), L. juliandringi (17.0–17.2 mm in males), L. mjobergi (15.7–19.0 mm in males), L. natunae (17.6 mm in one adult male), L. parva (15.0–16.9 mm in males), L. palmata (14.4–16.8 mm in males), and L. serasanae (16.9 mm in female).

Leptobrachella dorsospina sp. nov. can be easily distinguished from Leptobrachella aspera sp. nov. by having distinctly larger body size, SVL 28.7–30.5 mm in males, 32.1–39.8 mm in females (vs. SVL 22.4 mm in male, 25.0–26.4 in females); conical spines on dorsal skin present (vs. absent); black spots on flanks in one row (vs. black spots on flanks in two rows); ventral skin greyish white with black spots and orange pigmentations (vs. ventral skin creamy white with distinct dark patches on chest and abdomen); longitudinal ridges under toes interrupted at the articulations (longitudinal ridges under toes not interrupted at the articulations).

For the remaining 56 members of the genus Leptobrachella, in having SVL 28.7–30.5 mm in two males, L. dorsospina sp. nov. differs from the larger L. eos (33.1–34.7 mm in males), L. nahangensis (40.8 mm in male), L. sungi (48.3–52.7 mm in males), L. tamdil (32.3 mm in male), and L. zhangyapingi (45.8–52.5 mm in males); and from the smaller L. alpina (24.0–26.4 mm in males), L. applebyi (19.6–22.3 mm in males), L. ardens (21.3–24.7 mm in males), L. bidoupensis (18.5–25.4 mm in males), L. crocea (22.2–27.3 mm in males), L. feii (21.5–22.8 mm in males), L. flaviglandulosa (23.0–27.0 mm in males), L. isos (23.7–27.9 mm in males), L. khasiorum (24.5–27.3 mm in males), L. laui (24.8–26.7 mm in males), L. maculosa (24.2–26.6 mm in males), L. mangshanensis (22.2–27.8 mm in males), L. melica (19.5–22.7 mm in males), L. niveimontis (22.5–23.6 mm in males), L. pallida (24.5–27.7 mm in males), L. petrops (23.6–27.6 mm in males), L. pluvialis (21.3–22.3 mm in males), L. puhoatensis (24.2–28.1 mm in males), L. purpura (25.0–27.5 mm in males), L. rowleyae (23.4–25.4 mm in males), L. tadungensis (23.3–28.2 mm in males), L. tengchongensis (23.9–26.0 mm in males), L. ventripunctata (25.5–28.0 mm in males), and L. yingjiangensis (25.7–27.6 mm in males). By having black spots on the flanks, L. dorsospina sp. nov. can be distinguished from L. aerea, L. botsfordi, L. firthi, and L. tuberosa, all of which lack black spots on the flanks. By having rough dorsal skin with conical spines, the new species can be distinguished from L. bijie, L. chishuiensis, L. liui, L. maoershanensis, L. pyrrhops, L. purpuraventra, L. suiyangensis, L. wuhuangmontis, L. wulingensis, and L. yunkaiensis (dorsal skin lacking spines); and from L. bourreti, L. fuliginosa, L. kalonensis, L. minima, L. oshanensis, and L. pelodytoides (dorsal skin smooth). By having narrow lateral fringes on the toes, the new species can be distinguished from L. lateralis, L. macrops, L. nyx, L. pyrrhops, L. namdongensis and L. neangi, all of which lack lateral fringes on the toes. The new species can be separated from the remaining L. nokrekensis by having greyish white ventral coloration with black patches and orange pigmentations (vs. creamy white), and having dense short glandular folds on the dorsal surface (vs. only a few glandular folds on the dorsal surface).

Description of holotype

Adult male. Body size rather small, SVL 30.5 mm. Head length slightly larger than head width, HDW/HDL 0.99; snout slightly protruding, projecting slightly beyond margin of the lower jaw; nostril closer to snout than eye; canthus rostralis gently rounded; loreal region slightly concave; interorbital space flat, internarial distance smaller than interorbital distance, IND/IOD 0.91; pineal ocellus absent; vertical pupil; snout length larger than eye diameter, SNT/EYE 1.29; tympanum distinct, rounded, and slightly concave, diameter smaller than that of the eye and larger than tympanum-eye distance, TMP/EYE 0.43 and TEY/TMP 0.50; upper margin of tympanum in contact with supratympanic ridge; distinct black supratympanic line present; vomerine teeth absent; vocal sac openings slit-like, paired, located posterolaterally on floor of mouth in close proximity to the margins of the mandible; tongue deeply notched posteriorly; supratympanic ridge distinct, extending from posterior corner of eye to supra-axillary gland.

Tips of fingers rounded, slightly swollen; relative finger lengths II = IV < I < III; nuptial pad absent; subarticular tubercles absent; large, rounded inner palmar tubercle distinctly separated from small, rounded outer palmar tubercle; absence of webbing and lateral fringes on fingers. Tips of toes rounded, slightly swollen; relative toe length I < II < V < III < IV; subarticular tubercles absent; distinct longitudinal dermal ridges present under the 3rd to 5th toes, interrupted; large, oval inner metatarsal tubercle present, outer metatarsal tubercle absent; toes webbing rudimentary; narrow lateral fringes present on all toes. Tibia 44% of snout-vent length; tibiotarsal articulation reaches to posterior corner of eye; heels slightly overlapping when thighs are appressed at right angles with respect to body.

Dorsal skin rough, with dense conical granules, tubercles, glandular folds and conical spines; ventral skin smooth; pectoral gland and femoral gland oval; the size of pectoral glands almost equal to tips of fingers and femoral glands; femoral gland situated on posteroventral surface of thigh, closer to knee than to vent; supra-axillary glands raised. Ventrolateral glands distinctly visible, raised, forming an incomplete line.

Coloration of holotype in life

Dorsum greyish brown with distinct darker brown markings on sides and scattered with irregular light greyish brown pigmentations and yellowish brown spots. An indistinct, darker brown inverted triangular pattern between anterior corners of the eyes, connected to an indistinct dark brown W-shaped marking between the axillae. Dense translucent spines present on dorsal skin of body and limbs. Upper 2/3 of the tympanum dark brown, lower 1/3 light orange, scattered with tiny coppery orange spots. Small greyish white and light brown granules present on the dorsum of the body and limbs; a dark brown vertical bar under the eye; transverse dark brown bars on the dorsal surface of the limbs and digits; distinct dark brown patches on the flanks, from groin to axilla; elbow and upper arms with distinct light orange coloration.

Ventral surface of throat, chest, and belly greyish white; throat with light brown speckles, chest, and abdomen with distinct dark patches; ventral surface of limbs dark grey, scattered with greyish white spots and small patches. Supra-axillary gland light orange; femoral, pectoral, and ventrolateral glands greyish white. Iris bicolored, light orange on upper half and silver on lower half.

Coloration of holotype in preservative

(Fig. 4B). Dorsum of body and limbs dark brown; transverse bars on limbs, dark brown patterns, markings, and spots on back become indistinct, light greyish brown pigmentations and yellowish spots absent. Translucent spines on dorsal skin of body and limbs become grey. Ventral surface of limbs and surface of throat light brown, surface of abdomen greyish white, dark patches on chest, abdomen and flanks become more distinct. Supra-axillary, femoral, pectoral, and ventrolateral glands greyish white.

Variations

Measurements and body proportions are listed in Table 5. All the female paratypes match the overall characters of the holotype except that: the dorsum is greyish brown in the holotype SYS a004974 (vs. dark brown in the paratypes SYS a004961, 4962), and black spots on the ventral skin are more dense and distinct in the paratypes SYS a004961, 4962.

Table 5.

Measurements, and body proportions of Leptobrachella dorsospina sp. nov.

Voucher SYS a004977 SYS a004974 SYS a004961 SYS a004962 SYS a004973 SYS a004975 SYS a004976
Sex Male Male Female Female Female Female Female
SVL 28.7 30.5 36.1 37.3 39.8 32.1 33.8
HDL 10.3 10.8 12.8 12.4 12.9 11.3 12.0
HDW 10.6 10.7 11.9 12.9 13.2 11.8 12.2
SNT 4.5 4.2 5.1 5.4 5.8 5.0 4.8
IND 3.1 3.2 3.6 3.9 4.0 3.7 3.4
IOD 3.4 2.9 3.5 3.4 3.3 3.0 2.9
EYE 3.5 3.7 3.9 3.7 4.3 4.2 3.8
TMP 1.7 1.6 2.3 2.3 2.6 2.1 2.1
TEY 1.1 0.8 1.3 1.4 1.5 1.2 1.1
ML 7.4 7.3 8.8 7.7 9.1 7.8 7.6
LAHL 14.1 14.2 17.1 16.8 17.5 16.2 15.9
PL 12.1 12.8 14.9 14.5 15.5 13.9 13.6
TIB 13.5 13.4 15.5 16.3 16.6 14.9 14.5
HLL 41.7 42.7 49.1 49.9 52.9 46.8 48.0
HDL/SVL 0.36 0.35 0.35 0.33 0.32 0.35 0.36
HDW/SVL 0.37 0.35 0.33 0.35 0.33 0.37 0.36
HDW/HDL 1.03 0.99 0.93 1.04 1.02 1.04 1.02
SNT/HDL 0.44 0.39 0.40 0.44 0.45 0.44 0.40
IND/HDW 0.29 0.30 0.30 0.30 0.30 0.31 0.28
IOD/HDW 0.32 0.27 0.29 0.26 0.25 0.25 0.24
EYE/HDL 0.34 0.34 0.30 0.30 0.33 0.37 0.32
TMP/EYE 0.49 0.43 0.59 0.62 0.60 0.50 0.55
ML/SVL 0.26 0.24 0.24 0.21 0.23 0.24 0.22
LAHL/SVL 0.49 0.47 0.47 0.45 0.44 0.50 0.47
PL/SVL 0.42 0.42 0.41 0.39 0.39 0.43 0.40
TIB/SVL 0.47 0.44 0.43 0.44 0.42 0.46 0.43
HLL/SVL 1.45 1.40 1.36 1.34 1.33 1.46 1.42

Etymology

The specific epithet, dorsospina, is in reference to the conical spines on the dorsal surface of body in the new species. According to its type locality, we suggest its English common name as “Shuicheng Leaf Litter Toad”, and the Chinese name “Shui Cheng Zhang Tu Chan (水城掌突蟾)”.

Distribution and habits

Currently, Leptobrachella dorsospina sp. nov. is known only from its type locality, Yushe Forest Park, which is near the border between Guizhou and Yunnan. The new species was found on the surface of fallen leaves by the clear-water rocky hill-stream in well-preserved montane evergreen broadleaf forest (ca. 2100 m a.s.l.). Males were not heard calling.

Discussion

In the phylogenetic tree, the Leptobrachella pelodytoides (voucher number: MVZ 223642) sample from Tam Dao, northern Vietnam is clustered together with the topotypic L. ventripunctata (voucher number: SYS a001768) sample from Xishuangbanna, Yunnan, China, with a genetic divergence of only 1.5% (Fig. 2, Suppl. material 1: Table S1), which is of a intraspecific level. In addition, the type locality of L. pelodytoides is Thao [= Thamo], Kayah State, Myanmar, which is geographically distant from northern Vietnam with a distance over 900 km. Considering the above, we recommend that the specimen MVZ 223642 be reappraised as L. ventripunctata.

Yunnan and Guizhou are both largely within the species-rich Dian freshwater zoogeographical dominion (Huang et al 2020). Spanning the Indo-Burma Hotspot and the Mountains of Southwest China Hotspot (Tordoff et al. 2012), Yunnan Province has for long been considered as one of the most biodiverse regions in China and its flora and fauna have attracted much attention. However, Guizhou Province, which also shares the Yunnan-Guizhou Plateau, remains relatively neglected; knowledge of biodiversity levels and patterns are seriously lacking. In recent years, large numbers of discoveries have been made from Guizhou, dramatically raising the number of frog species known from the region (Zhang et al. 2017; Li et al. 2018a, b, 2019a, b, 2020a; Lyu et al. 2019; Wang et al. 2019; Luo et al. 2020; Wei et al. 2020). Further comprehensive surveys are urgently needed to determine the true diversity of the amphibians of Guizhou Province.

Acknowledgements

We are grateful to Prof. Tao Thien Nguyen, Prof. Annemarie Ohler, and an anonymous reviewer for providing constructive comments that greatly improved the quality of the manuscript. We would like to thank Yu-Long Li, Run-Lin Li and Hong-Hui Chen for their help in the field work, and Yao Li for her help in the lab work.

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Appendix 1

Specimens examined

Leptobrachella alpina (n = 6): China: Yunnan Province: Jingdong County: Mt. Wuliang: CIB 24353 (holotype), CIB 24354; SYS a 003927.

Leptobrachella bijie (n = 8): China: Guizhou: Bijie City: SYS a007313–7320.

Leptobrachella laui (n = 26): China: Hong Kong: SYS a002057 (holotype), SYS a002058; China: Guangdong Province: Shenzhen City: SYSa 001505–1507, 1515–1521, 3471–3472, 5644–5645.

Leptobrachella liui (n = 18): China: Fujian Province: Mt. Wuyi: CIB 24355 (holotype), CIB 24356, SYS a001571–1578, 1595–1599, 2478–2479, 5925–5826.

Leptobrachella mangshanensis (n = 5): China: Guangdong: Nanling Nature Reserve: SYS a002827–2830, 5754.

Leptobrachella purpuraventra (n = 15): China: Guizhou: Bijie City: SYS a007277–7284, 7300–7306.

Leptobrachella tengchongensis (n = 6): China: Yunnan Province: Baoshan City: Mt. Gaoligong: SYS a004600 (holotype), 4596–4599, 4601–4602.

Leptobrachella wuhuangmontis (n = 12): China: Guangxi Province: Pubei County: Mt. Wuhuang: SYS a003500/CIB107274, SYS a000578, 0580–0581, 3485–3489, 3499, 3504–3506.

Leptobrachella yunkaiensis (n = 8): China: Guangdong Province: Maoming City: Dawuling Forest Station: SYS a004664/CIB107272, SYS a004663, 4665–4669, 4690.