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Morphology and molecular genetics reveal two new Leptobrachella species in southern China (Anura, Megophryidae)
expand article infoJian Wang, Jianhuan Yang§, Yao Li, Zhitong Lyu, Zhaochi Zeng, Zuyao Liu, Youhua Ye|, Yingyong Wang
‡ Sun Yat-sen University, Guangzhou, China
§ Kadoorie Farm and Botanic Garden, Hong Kong, Hong Kong
| Zhongkai University of Agriculture and Engineering, Guangzhuo, China
Open Access

Abstract

Based on morphological and phylogenetic analyses (16S rRNA mtDNA), two new species of the genus Leptobrachella are described from southern China, namely L. yunkaiensis Wang, Li, Lyu & Wang, sp. n. from Dawuling Forest Station of Guangdong Province and L. wuhuangmontis Wang, Yang & Wang, sp. n. from Mt. Wuhuang of Guangxi Province. To date, the genus Leptobrachella contains 68 species, among which 13 species are known from China. The descriptions of the two new species further emphasize that the species diversity of the genus Leptobrachella from China is still highly underestimated and requires further investigations.

Keywords

China, Leptobrachella yunkaiensis sp. n., L. wuhuangmontis sp. n., morphology, phylogenetic, species diversity

Introduction

The genus Leptolalax Dubois, 1983 within the family Megophryidae Bonaparte, 1850 was currently found to be non-monophyletic with Leptobrachella Smith, 1925, and was assigned as a junior synonym of Leptobrachella based on a large-scale molecular analysis (Chen et al. 2018).Their results also rejected the hypothesis that Leptolalax consists of two subgenera as proposed by Delorme et al. (2006) and Dubois et al. (2010). At present, the genus Leptobrachella contains sixty-six species, widely distributed from southern China west to northeastern India and Myanmar, through mainland Indochina to peninsular Malaysia and the island of Borneo (Frost 2017; Nguyen et al. 2018; Rowley et al. 2016, 2017; Yang et al. 2016; Yuan et al. 2017). They are commonly known as Asian leaf litter frogs. Currently, eleven species of this genus are known from China, i.e., L. alpinus from Yunnan and Guangxi provinces, L. laui from southern Guangdong including Hong Kong, L. liui from Fujian, Jiangxi, Guangdong, Guangxi, Hunan and Guizhou provinces, L. oshanensis from Gansu, Sichuan, Chongqing, Guizhou and Hubei provinces, L. cf. pelodytoides (which may represents a undescribed taxon), L. purpura, L. tengchongensis, L. ventripuntatus, and L. yingjiangensis from Yunnan Province, and L. sungi and L. maoershanensis from Guangxi Province (Sung et al. 2014; Yang et al. 2016; Yuan et al. 2017; Yang et al. 2018).

During field surveys in southern China from 2009 to 2016, a number of specimens were collected from Dawuling Forest Station of Guangdong Province and Mt. Wuhuang of Guangxi Province, respectively (Figure 1), that can all be morphologically assigned to the genus Leptobrachella, based on the following characters: (1) comparatively small size, snout-vent length no overlap 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). Subsequent molecular studies on 16S rRNA mtDNA sequences revealed that this collection represents two different undescribed species which can be distinguished from each other and from all other recognized congeners by a combination of morphological characters and molecular divergences; they are described herein as two new species.

Figure 1. 

Collection localities of the two new Leptobrachella species: 1 the type locality of Leptobrachella yunkaiensis sp. n., Dawuling Forest Station in Guangdong Province 2 the type locality of L. wuhuangmontis sp. n., Mt. Wuhuang in Guangxi Province.

Materials and methods

Sampling. For molecular analyses, a total of 65 samples (19 muscle tissues and 46 sequences downloaded from Genbank) from 29 species of the genus Leptobrachella were sequenced, in addition to two undescribed species from China, i.e., the population from Dawuling Forest Station of Guangdong Province and Mt. Wuhuang of Guangxi Province. Additionally, four sequences were downloaded from GenBank as the outgroups (see Table 1; Pelobates syriacus, Pelobates varaldii, Leptobrachium cf. chapaense and Megophrys major).

Localities and voucher data for all specimens used in this study.

ID Species Locality Voucher no. GenBank No. 16S rRNA
1 Leptobrachella yunkaiensis sp. n. China: Dawuling Forest Station, Maoming City, Guangdong SYS a004663 MH605584
2 Leptobrachella yunkaiensis sp. n. China: Dawuling Forest Station, Maoming City, Guangdong SYS a004664 / CIB107272 MH605585
3 Leptobrachella yunkaiensis sp. n. China: Dawuling Forest Station, Maoming City, Guangdong SYS a004665 MH605586
4 Leptobrachella yunkaiensis sp. n. China: Dawuling Forest Station, Maoming City, Guangdong SYS a004666 MH605587
5 Leptobrachella yunkaiensis sp. n. China: Dawuling Forest Station, Maoming City, Guangdong SYS a004667 MH605588
6 Leptobrachella yunkaiensis sp. n. China: Dawuling Forest Station, Maoming City, Guangdong SYS a004668 MH605589
7 Leptobrachella yunkaiensis sp. n. China: Dawuling Forest Station, Maoming City, Guangdong SYS a004669 MH605590
8 Leptobrachella yunkaiensis sp. n. China: Dawuling Forest Station, Maoming City, Guangdong SYS a004690 MH605591
9 Leptobrachella wuhuangmontis sp. n. China: Mt. Wuhuang, Pubei County, Guangxi SYS a003485 MH605577
10 Leptobrachella wuhuangmontis sp. n. China: Mt. Wuhuang, Pubei County, Guangxi SYS a003486 MH605578
11 Leptobrachella wuhuangmontis sp. n. China: Mt. Wuhuang, Pubei County, Guangxi SYS a003487 MH605579
12 Leptobrachella wuhuangmontis sp. n. China: Mt. Wuhuang, Pubei County, Guangxi SYS a003499 MH605580
13 Leptobrachella wuhuangmontis sp. n. China: Mt. Wuhuang, Pubei County, Guangxi SYS a003500 / CIB107274 MH605581
14 Leptobrachella wuhuangmontis sp. n. China: Mt. Wuhuang, Pubei County, Guangxi SYS a003504 MH605582
15 Leptobrachella aerea Vietnam: Quang Binh RH60165 JN848437
16 Leptobrachella applebyi Vietnam: Kon Tum AMS R 173778 KR018108
17 Leptobrachella applebyi Vietnam: Kon Tum AMS R 173635 KU530189
18 Leptobrachella bidoupensis Vietnam: Lam Dong AMS R 173133 HQ902880
19 Leptobrachella bidoupensis Vietnam: Lam Dong NCSM 77321 HQ902883
20 Leptobrachella bourreti Vietnam: Lao Cai AMS R 177673 KR018124
21 Leptobrachella eos Laos: Phongsaly MNHN : 2004.0278 JN848450
22 Leptobrachella firthi Vietnam: Kon Tum AMS R 176524 JQ739206
23 Leptobrachella fritinniens Malaysia: Borneo KUHE55371 AB847557
24 Leptobrachella gracilis Malaysia: Borneo KUHE 55624 AB847560
25 Leptobrachella hamidi Malaysia: Borneo KUHE 17545 AB969286
26 Leptobrachella heteropus Malaysia: Peninsula KUHE 15487 AB530453
27 Leptobrachella isos Vietnam: Gia Lai VNMN A 2015.4 / AMS R 176480 KT824769
28 Leptobrachella laui China: Tai Mo Shan, Hong Kong SYS a002057 KM014546
29 Leptobrachella laui China: San zhoutian, Shenzhen SYSa002450 MH055904
30 Leptobrachella laui China: Mt. Wutong, Shenzhen SYS a003477 MH605576
31 Leptobrachella liui China: Mt. Wuyi, Fujian SYS a002478 MH605573
32 Leptobrachella liui China: Mt. Wuyi, Fujian SYS a002479 MH605574
33 Leptobrachella liui China: Mt. Wuyi, Fujian SYS a001597 KM014547
34 Leptobrachella liui China: Mt. Tongbo, Jiangxi SYS a001702 KM014548
35 Leptobrachella liui China: Mt. Daiyun, Fujian SYS a001736 KM014550
36 Leptobrachella liui China: Dongkeng Town, Jingning County, Zhejiang SYSa002732 MH605575
37 Leptobrachella liui China: Dongkeng Town, Jingning County, Zhejiang SYSa002733 MH055909
38 Leptobrachella marmorata Malaysia: Borneo KUHE 53227 AB969289
39 Leptobrachella maura Malaysia: Borneo SP 21450 AB847559
40 Leptobrachella maoershanensis China: Maoershan, Guangxi KIZ 019386 KY986931
41 Leptobrachella melica Cambodia: Ratanakiri MVZ 258198 HM133600
42 Leptobrachella minima Thailand: Chiangmai / JN848369
43 Leptobrachella nyx Vietnam: Ha Giang AMNH A 163810 DQ283381
44 Leptobrachella oshanensis China: Sichuan SYS a001830 KM014810
45 Leptobrachella pallida Vietnam: Lam Dong UNS 00511 KU530190
46 Leptobrachella picta Malaysia: Borneo UNIMAS 8705 KJ831295
47 Leptobrachella pluvialis Vietnam: Lao Cai MNHN:1999.5675 JN848391
48 Leptobrachella pyrrhops Vietnam: Lam Dong ZMMU A-5208 KP017575
49 Leptobrachella pyrrhops Vietnam: Lam Dong ZMMU A-4873 (ABV-00213) KP017576
50 Leptobrachella sabahmontana Malaysia: Borneo BORNEENSIS 12632 AB847551
51 Leptobrachella rowleyae Vietnam: Da Nang City, Son Tra ITBCZ 4113 MG682549
52 Leptobrachella rowleyae Vietnam: Da Nang City, Son Tra ITBCZ 4114 MG682550
53 Leptobrachella rowleyae Vietnam: Da Nang City, Son Tra ITBCZ 2790 MG682551
54 Leptobrachella rowleyae Vietnam: Da Nang City, Son Tra ITBCZ 2783 MG682552
55 Leptobrachella tengchongensis China: Tengchong County, Yunnan SYS a004596 KU589208
56 Leptobrachella tengchongensis China: Tengchong County, Yunnan SYS a004598 KU589209
57 Leptobrachella tengchongensis China: Tengchong County, Yunnan SYS a004600 KU589210
58 Leptobrachella ventripunctata Laos: Phongsaly MNHN 2005.0116 JN848410
59 Leptobrachella ventripunctata China: Zhushihe, Xishuangbanna, Yunnan SYS a001768 KM014811
60 Leptobrachella ventripunctata China: Zhushihe, Xishuangbanna, Yunnan SYS a003957 MH605583
61 Leptobrachella zhangyapingi Thailand: Chiang Mai KJ-2013 JX069979
62 Leptobrachium cf. chapaense Vietnam: Lao Cai AMS R 171623 KR018126
63 Pelobates syriacus / MVZ 234658 AY236807
64 Pelobates varaldii / / AY236808
65 Megophrys major Vietnam: Kon Tum AMS R 173870 KY476333

All specimens were previous to fixation 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.

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) from each sample was sequenced. Fragments of the genes were amplified using primer pairs L3975 (5’-CGCCTGTTTACCAAAAACAT-3’) and H4551 (5’-CCGGTCTGAACTCAGATCACGT-3’) for 16S rRNA (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).

Phylogenetic analyses. Sequence alignments were first conducted using Clustal X 2.0 (Thompson et al. 1997), with default parameters and the alignment being checked and manually revised, if necessary. Tested in Jmodeltest v2.1.2 (Darriba et al. 2012) with Akaike and Akaike information criteria, the best-fitting nucleotide substitution models are GTR + I + G. Phylogenetic trees were analyzed using maximum likelihood (ML) implemented in RaxmlGUI 1.3 (Silvestro and Michalak 2012), and Bayesian inference (BI) using MrBayes 3.2.4 (Ronquist et al. 2012). For ML analysis, the maximum likelihood tree inferred from 1000 replicates was used to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 60% of bootstrap replicates were collapsed. For BI analysis, two independent runs with four Markov Chain Monte Carlo simulations were performed for ten million iterations and sampled every 1000th iteration. The first 25% of samples were discarded as burn-in. Convergence of the markov Chain monte carlo simulations was assessed with PSRF ≤ 0.01 and ESS (effective sample size) value > 200 using Tracer v.1.4 (http://tree.bio.ed.ac.uk/software/tracer/). We also calculated pairwise sequence divergence based on uncorrected p-distance using MEGA 6.06 (Tamura et al. 2013).

Morphometrics. Measurements followed Fei et al. (2009) and Rowley et al. (2013), and were taken with digital calipers 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 of 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);

INDY 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);

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

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

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

Sex was determined by direct observation of calls in life, the presence of internal vocal sac openings, and the presence of eggs in abdomen through external inspection. 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 relied on examination of topotypic material and/or original species descriptions.

Obtained references of 66 known congeners of the genus Leptobrachella, respectively.

ID Leptobrachella species Literature obtained
1 L. aereus (Rowley, Stuart, Richards, Phimmachak & Sivongxay, 2010) Rowley et al. 2010c
2 L. alpinus (Fei, Ye & Li, 1990) Fei et al. 2009
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. bidoupensis (Rowley, Le, Tran & Hoang, 2011) Rowley et al. 2011
8 L. botsfordi (Rowley, Dau, & Nguyen, 2013) Rowley et al. 2013
9 L. bourreti (Dubois, 1983) Ohler et al. 2011
10 L. brevicrus Dring, 1983 Dring 1983; Eto et al. 2015
11 L. crocea (Rowley, Hoang, Le, Dau & Cao, 2010) Rowley et al. 2010a
12 L. dringi (Dubois, 1987) Inger et al. 1995; Matsui and Dehling 2012
13 L. eos (Ohler, Wollenberg, Grosjean, Hendrix, Vences, Ziegler & Dubois, 2011) Ohler et al. 2011
14 L. firthi (Rowley, Hoang, Dau, Le & Cao, 2012) Rowley et al. 2012
15 L. fritinniens (Dehling & Matsui, 2013) Dehling and Matsui 2013
16 L. fuliginosa (Matsui, 2006) Matsui 2006
17 L. gracilis (Günther, 1872) Günther 1872; Dehling 2012b
18 L. hamidi (Matsui, 1997) Matsui 1997
19 L. heteropus (Boulenger, 1900) Boulenger 1900
20 L. isos (Rowley, Stuart, Neang, Hoang, Dau, Nguyen & Emmett, 2015) Rowley et al. 2015a
21 L. itiokai Eto, Matsui & Nishikawa, 2016 Eto et al. 2016
22 L. juliandringi Eto, Matsui & Nishikawa, 2015 Eto et al. 2015
23 L. kajangensis (Grismer, Grismer & Youmans, 2004) Grismer et al. 2004
24 L. kalonensis (Rowley, Tran, Le, Dau, Peloso, Nguyen, Hoang, Nguyen & Ziegler, 2016) Rowley et al. 2016
25 L. kecil (Matsui, Belabut, Ahmad & Yong, 2009) Matsui et al. 2009
26 L. khasiorum (Das, Tron, Rangad & Hooroo, 2010) Das et al. 2010
27 L. lateralis (Anderson, 1871) Anderson 1871; Humtsoe et al. 2008
28 L. laui (Sung, Yang & Wang, 2014) Sung et al. 2014
29 L. liui (Fei & Ye, 1990) Fei et al. 2009; Sung et al. 2014
30 L. macrops (Duong, Do, Ngo, Nguyen & Poyarkov, 2018) Duong et al. 2018
31 L. maculosa (Rowley, Tran, Le, Dau, Peloso, Nguyen, Hoang, Nguyen & Ziegler, 2016) Rowley et al. 2016
32 L. maoershanensis (Yuan, Sun, Chen, Rowley & Che, 2017) Yuan et al. 2017
33 L. marmorata (Matsui, Zainudin & Nishikawa, 2014) Matsui et al. 2014b
34 L. maura (Inger, Lakim, Biun & Yambun, 1997) Inger et al. 1997
35 L. melanoleuca (Matsui, 2006) Matsui 2006
36 L. melica (Rowley, Stuart, Neang & Emmett, 2010) Rowley et al. 2010b
37 L. minima (Taylor, 1962) Taylor 1962; Ohler et al. 2011
38 L. mjobergi Smith, 1925 Eto et al. 2015
39 L. nahangensis (Lathrop, Murphy, Orlov & Ho, 1998) Lathrop et al. 1998
40 L. natunae (Günther, 1895) Günther 1895
41 L. nokrekensis (Mathew & Sen, 2010) Mathew and Sen 2010
42 L. nyx (Ohler, Wollenberg, Grosjean, Hendrix, Vences, Ziegler & Dubois, 2011) Ohler et al. 2011
43 L. oshanensis (Liu, 1950) Fei et al. 2009
44 L. pallida (Rowley, Tran, Le, Dau, Peloso, Nguyen, Hoang, Nguyen & Ziegler, 2016) Rowley et al. 2016
45 L. palmata Inger & Stuebing, 1992 Inger and Stuebing 1992
46 L. parva Dring, 1983 Dring 1983
47 L. pelodytoides (Boulenger, 1893) Boulenger 1893; Ohler et al. 2011
48 L. petrops (Rowley, Dau, Hoang, Le, Cutajar & Nguyen, 2017) Rowley et al. 2017
49 L. pictua (Malkmus, 1992) Malkmus 1992
50 L. platycephala (Dehling, 2012) Dehling 2012a
51 L. pluvialis (Ohler, Marquis, Swan & Grosjean, 2000) Ohler et al. 2000, 2011
52 L. puhoatensis (Rowley, Dau & Cao, 2017) Rowley et al. 2016
53 L. purpura (Yang, Zeng & Wang, 2018) Yang et al. 2018
54 L. pyrrhops (Poyarkov, Rowley, Gogoleva, Vassilieva, Galoyan & Orlov, 2015) Poyarkov et al. 2015
55 L. rowleyae (Nguyen, Poyarkov, Le, Vo, Ninh, Duong, Murphy & Sang, 2018) Nguyen et al. 2018
56 L. sabahmontana (Matsui, Nishikawa & Yambun, 2014) Matsui et al. 2014a
57 L. serasanae Dring, 1983 Dring 1983
58 L. sola (Matsui, 2006) Matsui 2006
59 L. sungi (Lathrop, Murphy, Orlov & Ho, 1998) Lathrop et al. 1998
60 L. tadungensis (Rowley, Tran, Le, Dau, Peloso, Nguyen, Hoang, Nguyen & Ziegler, 2016) Rowley et al. 2016
61 L. tamdil (Sengupta, Sailo, Lalremsanga, Das & Das, 2010) Sengupta et al. 2010
62 L. tengchongensis (Yang, Wang, Chen & Rao, 2016) Yang et al. 2016
63 L. tuberosa (Inger, Orlov & Darevsky, 1999) Inger et al. 1999
64 L. ventripunctata (Fei, Ye & Li, 1990) Fei et al. 2009
65 L. yingjiangensis (Yang, Zeng & Wang) Yang et al. 2018
66 L. zhangyapingi (Jiang, Yan, Suwannapoom, Chomdej & Che, 2013) Jiang et al. 2013

Results

Bayesian inference (BI) and Maximum likelihood (ML) phylogenetic tree were constructed based on DNA sequences of the mitochondrial 16S gene with a total length of 476 bp. The two analyses resulted in essentially identical topologies (Figure 2) with clustered the population of Leptobrachella from Dawuling Forest Station with L. laui, L. liui, and L. maoershanensis with very high node supporting values (1.00 in BI and 91% in ML) and represented a separately evolving lineage. Besides, the population from Mt. Wuhuang was a distinct separately evolving lineage with high node supporting values (1.00/100% in BI and ML). The smallest pairwise genetic divergences between the population from Dawuling Forest Station and all other species of the genus Leptobrachella for which comparable sequences were included was 6.0–6.7% (with L. liui), and between population from Mt. Wuhuang and all other species was 7.4% (with L. aerea) (Table 3). These values were significantly larger than observed pairwise genetic distances between recognized species (p-distance = 2.6%, between L. bourreti and L. oshanensis). Given that the two populations both can be morphologically distinguished with each other, and from all known congeners, we herein describe these specimens as two new species, respectively.

Figure 2. 

Bayesian inference tree derived from partial DNA sequences of the mitochondrial 16S r RNA gene. Numbers before slashes indicate Bayesian posterior probabilities (>60 retained) and numbers after slashes are bootstrap support for maximum likelihood (1000 replicates) analyses (>60 retained). The symbol “-” represents bootstrap value below 0.60/60%.

Uncorrected p-distances among L. species and outgroups based on 16S rRNA fragment (To be continued).

NO. Species 1–8 9–14 15 16–17 18–19 20 21 22 23 24 25 26 27 28–30 31–37 38 39 40
1–8 Leptobrachella yunkaiensis sp. n. 0–0.3
9–14 Leptobrachella wuhuangmontis sp. n. 11.1–12.3 0–0.3
15 Leptobrachella aerea 10.7–11.5 7.4 0
16–17 Leptobrachella applebyi 15.4–15.9 13.8–14.2 14.5 0
18–19 Leptobrachella bidoupensis 15.6–16.0 13.4–13.5 15.4 9.6 0
20 Leptobrachella bourreti 8.1–8.9 10.3–10.7 10.3 14.3 17.2 0
21 Leptobrachella eos 8.1–8.9 11.1–11.5 11.4 14.7 16.0 3.9 0
22 Leptobrachella firthi 14.1–14.6 13.3–13.7 12.2 16.8 18.3 12.6 13.8 0
23 Leptobrachella fritinniens 18.2–18.6 15.9–16.3 15.1 17.7 14.5 17.3 17.0 17.6 0
24 Leptobrachella gracilis 20.3–20.8 19.9–20.4 18.1 16.4 18.7 19.5 20.8 22.2 13.0 0
25 Leptobrachella hamidi 17.9–18.3 15.4–15.8 15.3 12.7 15.6 16.2 14.3 17.9 9.3 10.7 0
26 Leptobrachella heteropus 20.1–21.0 16.6–17.7 17.5 15.5 17.4 20.5 21.4 22.4 19.6 20.8 17.3 0
27 Leptobrachella isos 12.7–13.1 11.8–12.2 12.1 14.3 13.9 10.4 12.3 12.1 17.3 20.1 14.6 19.6 0
28–30 Leptobrachella laui 6.3–6.7 12.5–12.9 10.7 16.1 17.9 8.8 8.8 13.4 18.0 19.4 15.8 20.8 13.3 0
31–37 Leptobrachella liui 6.0–6.7 9.6 8.9 14.6 14.3 8.1 8.1 12.6 17.3 22.4 16.2 19.2 12.2 5.6 0
38 Leptobrachella marmorata 16.7–16.8 13.9–14.3 14.5 10.9 15.7 14.7 14.3 15.6 9.6 11.4 4.2 18.2 14.7 16.2 15.1 0
39 Leptobrachella maura 17.5–17.9 15.8 14.5 12.6 15.5 15.8 15.8 18.0 11.1 11.5 8.8 18.6 14.7 17.4 17.0 8.4 0
40 Leptobrachella maoershanensis 6.7–7.1 10.0 18.5 15.1 14.8 9.9 9.9 16.6 18.5 21.2 16.3 19.2 13.4 6.7 5.7 16.3 17.5 0
41 Leptobrachella melica 16.6–17.0 13.0 12.6 5.6 9.1 14.3 15.4 17.1 16.1 15.2 12.3 16.0 15.3 16.5 15.7 12.4 13.1 15.8
42 Leptobrachella minima 11.1–11.9 10.8–11.2 6.3 15.0 16.0 11.1 11.9 12.8 17.8 20.0 16.6 18.6 13.3 8.9 8.2 15.8 16.6 9.2
43 Leptobrachella nyx 9.3–10.0 7.7–8.1 4.9 13.7 14.3 9.2 10.0 10.8 15.6 19.8 15.0 16.6 12.2 8.9 7.1 14.3 15.4 8.9
44 Leptobrachella oshanensis 8.5–9.2 11.1–11.5 10.7 15.1 18.1 2.6 5.0 12.6 17.3 19.5 16.7 22.2 11.2 8.1 8.5 16.3 16.3 11.1
45 Leptobrachella pallida 16.0–16.5 14.7–15.1 15.8 10.4 5.3 17.6 15.6 18.8 14.4 16.9 14.8 19.0 16.6 16.2 15.2 14.5 14.7 15.6
46 Leptobrachella picta 18.4–18.9 16.9–17.4 15.8 14.5 15.7 17.1 17.2 15.9 6.0 11.9 10.3 19.1 17.6 17.1 16.4 8.9 10.7 17.6
47 Leptobrachella pluvialis 8.2–8.6 8.2–8.5 6.4 13.9 14.8 10.3 11.1 13.5 16.6 19.2 16.7 16.0 14.2 8.9 7.9 15.2 16.7 6.8
48–49 Leptobrachella pyrrhops 14.3–15.5 13.1–14.0 13.5–13.9 12.3–12.7 9.0–9.3 16.7–17.1 16.0–16.5 17.2–17.6 15.8–16.2 17.4–17.8 16.1–16.5 17.0–17.4 14.3–14.7 15.5–15.9 15.2–15.6 15.8–16.2 17.2–17.6 14.4–14.8
50 Leptobrachella sabahmontana 17.9–18.4 15.4–15.9 15.4 12.9 15.0 15.8 16.0 15.9 7.0 12.7 10.0 21.3 16.3 17.0 16.3 8.5 8.9 17.9
51–53 Leptobrachella tengchongensis 11.1–11.9 12.2–12.6 8.5 15.3 15.8 8.1 7.8 11.2 16.1 21.2 14.2 19.1 9.3 8.1 8.5 14.7 15.4 10.3
54–56 Leptobrachella ventripunctata 11.5–12.7 8.5–10.0 6.7–7.7 16.3–16.6 17.5–18.0 11.1–12.3 11.9–13.1 11.1–11.5 15.6–16.5 20.4–21.2 14.7–15.1 18.5 11.5–11.8 10.4–11.6 9.0–10.1 14.0 15.0–15.8 10.0–10.4
57 Leptobrachella zhangyapingi 12.5–12.9 13.3 10.6 15.4 16.2 11.0 10.3 13.5 18.9 22.4 18.3 20.5 12.0 10.3 10.6 16.8 16.8 12.1
58–61 Leptobrachella rowleyae 16.3–16.7 13.5–14.0 14.3 7.8 10.7 15.1 16.3 17.3 16.8 19.1 14.1 17.8 16.3 15.8 15.1 13.4 14.8 15.1
62 Leptobrachium cf. chapaense 25.3–25.7 23.8–24.3 25.5 23.7 27.5 28.4 28.8 29.3 27.4 27.0 24.8 25.7 25.6 26.8 25.6 23.7 22.2 25.2
63 Pelobates syriacus 26.6–27.6 24.4–24.9 26.2 23.2 25.0 26.7 27.2 27.2 22.3 22.6 24.0 26.0 29.4 26.1 25.8 22.4 22.6 26.8
64 Pelobates varaldii 27.0–28.0 25.1–25.6 25.3 23.1 25.4 25.7 25.4 27.5 22.4 23.9 24.1 27.4 28.9 25.7 25.4 21.6 24.3 27.2
65 Megophrys major 28.8–29.9 26.4–27.0 27.6 25.6 28.8 25.8 26.2 30.9 28.1 25.9 25.1 27.3 27.4 29.4 27.6 25.2 23.1 28.2

Continued.

NO. Species 41 42 43 44 45 46 47 48–49 50 51–53 54–56 57 58–61 62 63 64 65
1–8 Leptobrachella yunkaiensis sp. n.
9–14 Leptobrachella wuhuangmontis sp. n.
15 Leptobrachella aerea
16–17 Leptobrachella applebyi
18–19 Leptobrachella bidoupensis
20 Leptobrachella bourreti
21 Leptobrachella eos
22 Leptobrachella firthi
23 Leptobrachella fritinniens
24 Leptobrachella gracilis
25 Leptobrachella hamidi
26 Leptobrachella heteropus
27 Leptobrachella isos
28–30 Leptobrachella laui
31–37 Leptobrachella liui
38 Leptobrachella marmorata
39 Leptobrachella maura
40 Leptobrachella maoershanensis
41 Leptobrachella melica 0
42 Leptobrachella minima 14.6 0
43 Leptobrachella nyx 11.8 5.7 0
44 Leptobrachella oshanensis 15.1 10.4 8.9 0
45 Leptobrachella pallida 11.1 14.7 15.2 16.4 0
46 Leptobrachella picta 14.4 16.5 14.7 17.6 15.3 0
47 Leptobrachella pluvialis 14.2 7.8 6.4 11.1 14.8 15.7 0
48–49 Leptobrachella pyrrhops 12.3–12.7 15.6–16.0 14.4–14.8 16.0–16.4 8.3–8.6 16.4–16.8 14.3–14.8 0.3
50 Leptobrachella sabahmontana 12.9 15.7 15.1 15.9 13.3 5.0 16.9 15.0–15.4 00
51–53 Leptobrachella tengchongensis 14.5 7.8 8.2 8.2 16.7 16.0 10.3 16.8–17.2 16.4 0
54–56 Leptobrachella ventripunctata 15.5–15.9 6.8–7.9 5.0–6.0 11.1–12.3 17.6–18.1 15.5–16.4 8.2–8.6 15.2–15.6 14.8–15.6 9.7–10.8 0–0.1
57 Leptobrachella zhangyapingi 17.0 11.4 9.5 11.1 17.5 18.1 10.3 16.7–17.1 18.5 9.2 11.1–11.5 0
58–61 Leptobrachella rowleyae 6.3 14.7 12.7 15.9 10.8 15.5 13.5 11.2–11.6 13.9 15.0 16.1–16.5 17.1 0
62 Leptobrachium cf. chapaense 26.1 25.5 25.7 27.5 26.2 25.9 25.1 23.8–24.3 27.5 26.3 24.4–24.8 29.1 25.9 0
63 Pelobates syriacus 23.5 26.4 24.9 26.2 25.6 21.1 27.9 24.3–24.7 20.8 28.2 25.4–26.9 28.6 24.7 21.3 0
64 Pelobates varaldii 23.5 27.4 24.0 25.7 25.8 21.2 27.8 25.5–26.0 21.6 28.1 24.5–25.9 27.2 25.0 23.1 3.6 0
65 Megophrys major 27.1 30.5 28.0 26.6 30.3 27.6 28.2 29.0–29.5 27.2 26.0 26.2–27.7 31.3 29.2 27.9 24.3 22.4 0

Systematics

Leptobrachella yunkaiensis Wang, Li, Lyu & Wang, sp. n.

Figures 3, 4C1–C3

Holotype

SYS a004665, adult male, collected on 15 April 2016 by Jian Wang (JW hereafter), Zhao-Chi Zeng (ZCZ hereafter), Ying-Yong Wang (YYW hereafter), Zu-Yao Liu (ZYL hereafter), Hai-Long He (HLH hereafter) and Zhi-Tong Lyu (ZTL hereafter) from Dawuling Forest Station (DWL hereafter) (22°16'32.9"N, 111°11'42.87"E; 1600 m a.s.l.), Maoming City, Guangdong Province, China.

Paratypes

Collectors and locality data of paratypes were the same as holotype: adult males, SYS a004664 / CIB107272, SYS a004666–4669 and an adult female SYS a004663, collected on 15 April 2016, the other adult female, SYS a004690, collected on 16 April 2017.

Diagnosis

(1) small size (SVL 25.9–29.3 mm in males, 34.0–35.3 mm in females), (2) dorsal skin shagreened with short skin ridges and raised warts, (3) iris bicolored, coppery orange on upper half and silver on lower half, (4) tympanum distinctly discernible, slightly concave, weakly black supratympanic line present, (5) dorsal surface yellowish-brown grounding, with distinct darker brown markings and rounded spots and scattered with irregular orange patches, (6) flanks with several dark blotches, (7) surface of belly pinkish, with distinct or indistinct light dark brown speckling, (8) supra-axillary, femoral, pectoral and ventrolateral glands distinctly visible, (9) absence of webbing and presence of distinct lateral fringes on fingers, toes with rudimentary webbing and wide lateral fringes, (10) longitudinal ridges under toes not interrupted at the articulations, and (11) dense conical spines present on lateral and ventral surface of tarsus, surface of tibia-tarsal, inner-side surface of shank and surface around cloacal region.

Figure 3. 

General aspect in life: A–D SYS a004665, the male holotype of Leptobrachella yunkaiensis sp. n. E SYS a004667, the male paratype F SYS a004690, the female paratype.

Comparisons

Comparative morphological data of Leptobrachella yunkaiensis sp. n. with 66 recognized Leptobrachella species were obtained from examination of museum specimens (see Appendix 1) and from the references listed in Table 2. All comparative data were shown in Tables 4, 5.

Compared with the 24 known congeners of the genus Leptobrachella occurring south of the Isthmus of Kra, by the presence of supra-axillary and ventrolateral glands, L. yunkaiensis sp. n. can be easily distinguished 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 lacking supra-axillary and ventrolateral glands; and by the significantly larger body size, SVL 25.9–29.3 mm in males, 34.0–35.3 mm in females, L. yunkaiensis sp. n. differs from the smaller L. baluensis (14.9–15.9 mm in males), L. brevicrus (17.1–17.8 mm in males), L. itiokai (15.2–16.7 mm in males), L. juliandringi (17.0–17.2 mm in males and 18.9–19.1 mm in females), L. mjobergi (15.7–19.0 mm in males), L. natunae (17.6 mm in male), L. parva (15.0–16.9 mm in males and 17.8 mm in female), L. palmata (14.4–16.8 mm in males), L. serasanae (16.9 mm in female) and Dring’s (1983) Leptobrachella sp. 3 “baluensis” (15.0–16.0 mm in males).

Leptobrachella yunkaiensis sp. n. is most similar to L. laui and L. liui, but it can be distinguished by the larger body sized, SVL 34.0–35.3 mm in females (vs. SVL 28.1 mm in a single female of L. laui; SVL 23.0–28.0 mm in females of L. liui), presence of short skin ridge and raised warts on dorsum (vs. absent in L. laui), black supratympanic line weak (vs. black supratympanic line distinct in L. liui), longitudinal ridges under toes not interrupted at the articulations (vs. interrupted in L. liui) (Figure 4), belly pinkish with distinct or indistinct speckling (vs. belly creamy white with dark brown dusting on margins in L. laui; belly creamy white with dark brown spots on chest and margins in L. liui).

Figure 4. 

Specimens in preservative: A1–A3 SYS a002957, the holotype of Leptobrachella laui B1–B3 SYS a005925, the topotype of L. liui C1–C3 SYS a004665, the holotype of L. yunkaiensis sp. n..

From the remaining 40 known congeners (Table 5), with SVL 25.9–29.3 mm in six males, SVL 34.0–35.3 mm in two females in Leptobrachella yunkaiensis sp. n., it can be distinguished from the larger L. eos (males 33.1–34.7 mm, female 40.7 mm), L. nahangensis (male 40.8 mm), L. pyrrhops (males 30.8–34.3 mm), L. sungi (males 48.3–52.7 mm, females 56.7–58.9 mm) and L. zhangyapingi (males 45.8–52.5 mm), and the smaller L. applebyi (males 19.6–22.3 mm, females21.7–25.9 mm), L. melica (males 19.5–22.7 mm), and L. pluvialis (males 21.3–22.3 mm). By having wide fringes on toes, the new species differs from L. applebyi, L. ardens, L. crocea, L. kalonensis, L. lateralis, L. maculosa, L. macrops, L. melica, L. minima, L. nahangensis, L. nyx, L. oshanensis, L. pallida, L. pluvialis, L. pyrrhops, L. rowleyae, L. tadungensis, L. tuberosa, and L. ventripunctata, all of which have no lateral fringes on toes; L. bidoupensis, L. bourreti, L. fuliginosa, and L. sungi, all of which have weak lateral fringes on toes; L. botsfordi, L. maoershanensis, L. pelodytoides, L. petrops, L. puhoatensis, and L. tengchongensis, all of which have narrow lateral fringes on toes; L. alpinus, L. firthi, and L. isos, all of which have wide lateral fringes only in males. With rudimentary webbing on toes, the new species differs from L. ardens, L. kalonensis, L. maculosa, L. oshanensis, L. pallida, L. petrops, L. rowleyae, and L. tadungensis, all of which have no webbing on toes; L. pelodytoides, L. sungi, and L. tamdil, all of which have wide webbing on toes. By having black spots on flanks, the new species differs from L. aerea, L. botsfordi, L. eos, L. firthi, L. isos, L. pallida, L. petrops, L. tuberosa, and L. zhangyapingi, all of which have no black spots on flanks. With belly pink with distinct or indistinct speckling, the new species differs from L. bourreti, L. eos, L. firthi, L. khasiorum, L. lateralis, L. minima, L. nahangensis, and L. nokrekensis, all of which have creamy white belly without patterns; from L. macrops, which have greyish-violet with white speckling; from L. purpura, which have dull white belly with indistinct grey dusting; and from L. yingjiangensis, which have creamy white belly with dark brown flecks on chest and margins. By dorsal skin shagreened with short skin ridges and raised warts, the new species differs from L. purpura, L. yingjiangensis and L. tengchongensis, all of which have shagreened dorsal skin with small tubercles, and from L. macrops, which have no skin ridges dorsally.

Measurements (minimum–maximum (mean ± SD); in mm), and body proportions of Leptobrachella yunkaiensis sp. n. from Dawuling Forest Station.

Measurements Males (n = 6) Females (n = 2)
SVL 25.9–29.3 (27.6 ± 1.4) 34.0–35.3 (34.7 ± 0.9)
HDL 9.3–10.3 (9.9 ± 0.4) 12.2–12.6 (12.4 ± 0.2)
HDW 9.0–10.0 (9.7 ± 0.4) 12.0–12.2 (12.1 ± 0.1)
SNT 3.6–3.8 (3.7 ± 0.1) 4.4–4.7 (4.6 ± 0.2)
EYE 3.4–3.7 (3.6 ± 0.1) 3.8–3.9 (3.9 ± 0.1)
IOD 2.7–2.9 (2.8 ± 0.1) 3.0–3.2 (3.1 ± 0.1)
IND 2.5–2.8 (2.7 ± 0.1) 2.9–3.0 (3.0 ± 0.1)
TMP 1.5–1.7 (1.6 ± 0.1) 2.0
TEY 0.7–0.8 (0.8 ± 0.1) 1.0
TIB 12.2–12.8 (12.5 ± 0.2) 15.0–15.2 (15.1 ± 0.2)
ML 5.8–7.3 (6.9 ± 0.6) 7.4–7.8 (7.6 ± 0.2)
PL 10.8–12.4 (11.9 ± 0.6) 12.7–12.9 (12.8 ± 0.1)
LAHL 12.0–12.6 (12.3 ± 0.2) 14.7–15.0 (14.8 ± 0.2)
HLL 37.0–40.3 (38.7 ± 1.2) 47.0–49.5 (48.3 ± 1.8)
HDL/HDW 1.01–1.03 (1.02 ± 0.01) 1.02–1.03 (1.02 ± 0.01)
HDL/SVL 0.34–0.39 (0.36 ± 0.02) 0.36
SNT/HDL 0.36–0.41 (0.38 ± 0.02) 0.37
SNT/ED 1.03–1.06 (1.05 ± 0.02) 1.16–1.21 (1.18 ± 0.03)
EYE/TMP 2.12–2.40 (2.25 ± 0.13) 1.90–1.95 (1.93 ± 0.04)
TMP/EYE 0.42–0.47 (0.45 ± 0.03) 0.51–0.53 (0.52 ± 0.01)
TEY/TMP 0.47–0.53 (0.48 ± 0.03) 0.50
TIB/SVL 0.43–0.48 (0.45 ± 0.02) 0.43–0.44 (0.44 ± 0.01)
LAHL/SVL 0.43–0.47 (0.45 ± 0.02) 0.42–0.43 (0.43 ± 0.01)
HLL/SVL 1.33–1.51 (1.41 ± 0.06) 1.38–1.40 (1.39 ± 0.01)
TIB/HLL 0.31–0.33 (0.32 ± 0.01) 0.31–0.32 (0.32 ± 0.01)

Selected diagnostic characters for species described herein and species in the genus Leptobrachella occurring north of the Isthmus of Kra (modified from Rowley et al. 2017; Yuan et al. 2017).

Species Male SVL (mm) Black spots on flanks Toes webbing Fringes on toes Ventral coloration Dorsal skin texture
L. yunkaiensis sp. n. 25.9–29.3 Yes Rudimentary Wide Belly pink with distinct or indistinct speckling Shagreened with short skin ridges and raised warts
L. wuhuangmontis sp. n. 25.6–30.0 Yes Rudimentary Narrow Greyish white mixed by tiny white and black dots Rough, scattered with dense conical tubercles
L. aerea 25.1–28.9 No Rudimentary Wide Near immaculate creamy white, brown specking on margins Finely tuberculate
L. alpinus 24.0–26.4 Yes Rudimentary Wide in males Creamy-white with dark spots Relatively smooth, some with small warts
L. applebyi 19.6–22.3 Yes Rudimentary No Reddish brown with white speckling Smooth
L. ardens 21.3–24.7 Yes No No Reddish brown with white speckling Smooth- finely shagreened
L. bidoupensis 18.5–25.4 Yes Rudimentary Weak Reddish brown with white speckling Smooth
L. botsfordi 29.1–32.6 No Rudimentary Narrow Reddish brown with white speckling Shagreened
L. bourreti 28.0–36.2 Yes Rudimentary Weak Creamy white Relatively smooth, some with small warts
L. crocea 22.2–27.3 No Rudimentary No Bright orange Highly tuberculate
L. eos 33.1–34.7 No Rudimentary Wide Creamy white Shagreened
L. firthi 26.4–29.2 No Rudimentary Wide in males Creamy white Shagreened with fine tubercles
L. fuliginosa 28.2–30.0 Yes Rudimentary Weak White with brown dusting Nearly smooth, few tubercles
L. isos 23.7–27.9 No Rudimentary Wide in males Creamy white with white dusting on margins Mostly smooth, females more tuberculate
L. kalonensis 25.8–30.6 Yes No No Pale, speckled brown Smooth
L. khasiorum 24.5–27.3 Yes Rudimentary Wide Creamy white Isolated, scattered tubercles
L. lateralis 26.9–28.3 Yes Rudimentary No Creamy white Roughly granular
L. laui 24.8–26.7 Yes Rudimentary Wide Creamy white with dark brown dusting on margins Round granular tubercles
L. liui 23.0-28.7 Yes Rudimentary Wide Creamy white with dark brown spots on chest and margins Round granular tubercles with glandular folds
L. macrops 28.0–29.3 Yes Rudimentary No Greyish-violet with white speckling Roughly granular with larger tubercles
L. maculosa 24.2–26.6 Yes No No Brown, less white speckling Mostly smooth
L. maoershanensis 25.2–30.4 Yes Rudimentary Narrow Creamy white chest and belly with irregular black spots Longitudinal folds
L. melica 19.5–22.7 Yes Rudimentary No Reddish brown with white speckling Smooth
L. minima 25.7–31.4 Yes Rudimentary No Creamy white Smooth
L. nahangensis 40.8 Yes Rudimentary No Creamy white with light specking on throat and chest Smooth
L. nokrekensis 26.0–33.0 Yes Rudimentary unknown Creamy white Tubercles and longitudinal folds
L. nyx 26.7–32.6 Yes Rudimentary No Creamy white with white with brown margins Rounded tubercles
L. oshanensis 26.6–30.7 Yes No No Whitish with no markings or only small, light grey spots Smooth with few glandular ridges
L. pallida 24.5–27.7 No No No Reddish brown with white speckling Tuberculate
L. pelodytoides 27.5–32.3 Yes Wide Narrow Whitish Small, smooth warts
L. petrops 23.6–27.6 No No Narrow Immaculate creamy white Highly tuberculate
L. pluvialis 21.3–22.3 Yes Rudimentary No Dirty white with dark brown marbling Smooth, flattened tubercles on flanks
L. puhoatensis 24.2–28.1 Yes Rudimentary Narrow Reddish brown with white dusting Longitudinal skin ridges
L. purpura 25.0–27.5 Yes Rudimentary Wide Dull white with indistinct grey dusting Shagreen with small tubercles
L. pyrrhops 30.8–34.3 Yes Rudimentary No Reddish brown with white speckling Slightly shagreened
L. rowleyae 23.4–25.4 Yes No No Pinkish milk-white to light brown chest and belly with numerous white speckles Smooth with numerous tiny tubercles
L. sungi 48.3–52.7 No or small Wide Weak White Granular
L. tadungensis 23.3–28.2 Yes No No Reddish brown with white speckling Smooth
L. tamdil 32.3 Yes Wide Wide White Weakly tuberculate
L. tengchongensis 23.9–26.0 Yes Rudimentary Narrow White with dark brown blotches Shagreened with small tubercles
L. tuberosa 24.4–29.5 No Rudimentary No White with small grey spots/streaks Highly tuberculate
L. ventripunctata 25.5–28.0 Yes Rudimentary No Chest and belly with dark brown spots Longitudinal skin ridges
L. yingjiangensis 25.7–27.6 Yes Rudimentary Wide Creamy white with dark brown flecks on chest and margins Shagreened with small tubercles
L. zhangyapingi 45.8–52.5 No Rudimentary Wide Creamy-white with white with brown margins Mostly smooth with distinct tubercles

Description of holotype

Adult male. Body size small, SVL in 28.7 mm. Head length slightly larger than head width, HDL/HDW 1.03; snout slightly protruding, projecting slightly beyond margin of the lower jaw; nostril equidistance between snout and eye; canthus rostralis gently rounded; loreal region slightly concave; interorbital space flat, larger internarial distance; pineal ocellus absent; vertical pupil; snout length slightly larger than eye diameter, SNT/EYE 1.03; tympanum distinct, rounded, and slightly concave, diameter smaller than that of the eye and larger than tympanum-eye distance, TMP/EYE 0.46 and TEY/TMP 0.47; weakly black supratympanic line present; vomerine teeth absent; vocal sac openings slit-like, located posterolaterally on floor of mouth in close proximity to the margins of the mandible; tongue deeply notched behind; supratympanic ridge distinct, extending from posterior corner of eye to supra-axillary gland; tubercles present on supratympanic ridge.

Tips of fingers rounded, slightly swollen; relative finger lengths I = II = IV < III; nuptial pad absent; subarticular tubercles absent; a large, rounded inner palmar tubercle distinctly separated from small, round outer palmar tubercle; absence of webbing and presence of distinct lateral fringes on fingers. Tips of toes like fingers; relative toe length I < II < V < III < IV; subarticular tubercles absent; distinct dermal ridges present under the 3rd to 5th toes; large, oval inner metatarsal tubercle present, outer metatarsal tubercle absent; toes webbing rudimentary; wide lateral fringes present on all toes. Tibia 43% of snout-vent length; tibiotarsal articulation reaches to middle of eye; heels just meeting each other when thighs are appressed at right angles with respect to body.

Skin on dorsum shagreened and scattered with fine, round tubercles; short skin ridges and raised warts on dorsum surface present; ventral skin smooth; pectoral gland and femoral gland large, oval; pectoral glands greater than tips of fingers and femoral glands; femoral gland situated on posteroventral surface of thigh, closer to knee than to vent; supra-axillary gland raised. Ventrolateral gland distinctly visible, forming an incomplete line. Dense conical spines on lateral and ventral surface of tarsus, surface of tibia-tarsal, inner-side surface of shank and surface around cloacal region present.

Measurements of holotype

(in mm). SVL 28.7, HDL 10.3, HDW 10.0, SNT 3.8, EYE 3.7, IOD 2.9, IND 2.8, TMP 1.7, TEY 0.8, TIB 12.4, ML 7.2, PL 12.1, LAHL 12.3, HLL 38.3.

Coloration of holotype in life

Dorsal surface orange-brown with distinct dark brown blotches edged distinct light orange pigmentation. A dark brown triangular pattern between eyes, connected to the dark brown W-shaped marking between axillae. Tympanum black. Orange-brown tubercles present on dorsum of body and limb, those on flanks much distinct and dense; anterior upper lip with distinct blackish brown patches; transverse dark brown bars on dorsal surface of limbs; indistinct dark brown blotches on flanks from groin to axilla; elbow and upper arms without dark bars but with distinct coppery orange coloration; fingers and toes with indistinct dark brown blotches.

Surface of throat creamy white and scattered with small whitish dots; belly pinkish and scattered with small brown speckling; ventral surface of thighs pinkish and scattered with small light orange-brown spots. Supra-axillary coppery orange; femoral, pectoral and ventrolateral glands whitish orange. Iris bicolored, coppery orange on upper half and silver on lower half.

Coloration of holotype in preservative

Dorsum of body and hindlimbs dark brown while dorsum of forelimbs yellowish brown; transverse bars on limbs become more distinct, dark brown patterns, markings and spots on back become indistinct. Ventral surface of body yellowish brown, with brown marbling on sides and chest. Orange supra-axillary, femoral, pectoral and ventrolateral glands fade to greyish white (Figure 4C1–C3).

Sexual dimorphism

Females with a larger body size than males, SVL 34.0–35.3 mm (34.7 ± 0.9) (vs. SVL 25.9–29.3 mm (27.6 ± 1.4) in males); presence of a single vocal sac in males (vs. absent in females); dense conical spines on lateral and ventral surface of tarsus, surface of tibia-tarsal, inner-side surface of shank, surface of thighs and surface around cloacal region distinct in males, and barely visible in females.

Variations

All paratypes match the overall characters of the holotype except that: the heels just meeting each other when thighs are appressed at right angles with respect to body, tibiotarsal articulation reaches to middle of eye in holotype SYS a004665 (vs. tibiotarsal articulation reaches to anterior corner of eye in SYS a004666, reaches the posterior corner of eye in SYS a004669). Surface of belly scattered with distinctly dark brown speckling in holotype (vs. such speckling indistinct in female paratypes SYS a004663, 4690. Tympanum black in the holotype (vs. tympanum black grounding with orange speckling in SYS a004667–4668). Distinct black spots present on dorsum in the female paraype SYS a004690 (Figure 3).

Etymology

The specific epithet, yunkaiensis, is in reference to the type locality, DWL of Guangdong, China located in the Yunkai Mountains Range. For the common name, we suggest “Yunkai Mountain’s Leaf Litter Toad”, and Chinese name “Yun Kai Zhang Tu Chan (云开掌突蟾)”.

Distribution and habits

Currently, Leptobrachella yunkaiensis sp. n. is known only from its type locality DWL of Guangdong Province (Figure 1). The new species was found along a clear-water rocky stream (ca. 2–3 m in width and ca. 20–30 cm in depth) and small nearby seeps in well-preserved montane evergreen broadleaf forest (1600 m a.s.l.) (Figure 5). During April and June, males were found calling mainly hidden under leaf litter, and some were found calling perching on the rocks or under rocks by the side of the stream. Females collected on April bear pure white oocytes.

Figure 5. 

The habitat of Leptobrachella yunkaiensis sp. n. in Dawuling Forest Station of Guangdong Province.

Leptobrachella wuhuangmontis Wang, Yang & Wang, sp. n.

Figures 6, 7

Holotype

SYS a003486, adult male, collected on 29 March 2015 by JW, ZTL, YYW and ZYL from Mt. Wuhuang (MWH hereafter) (22°08'30.77"N, 109°24'43.90"E; 500 m a.s.l.), Pubei County, Qinzhou City, Guangxi Province, China.

Paratypes

Adult males SYS a000578, 581 and an adult female SYS a000580, collected on 28 April 2009 by Jian-Huan Yang (JHY hereafter) and Run-Lin Li (RLL hereafter), adult males SYS a003487–3489, 3505–3506, SYS a003500 / CIB107274 and adult females SYS a003485, 3499, 3504,, collected from 29–30 March 2015 by JW, ZTL, YYW and ZYL, all from the same locality as the holotype.

Diagnosis

(1) small size (SVL 25.6–30.0 mm in males, 33.0–36.0 mm in females), (2) dorsal surface rough with skin ridges and dense conical tubercles, (3) iris bicolored, coppery yellow on upper half and silver on lower half, (4) tympanum distinctly discernible, slightly concave, dark brown, distinct black supratympanic line present, (5) dorsal surface greyish purple background with dark brown markings and scattered with orange-yellow blotches and white speckling, (6) distinct dark blotches on flanks, (7) ventral surface greyish white mixed by tiny white and black dots, (8) lateral fringes on fingers absent, (9) toes with narrow lateral fringes and rudimentary webbing, (10) longitudinal ridges under toes not interrupted at the articulations, and (11) dense conical spines on lateral and ventral surface of tarsus, dorsal surface of tibia-tarsal and surface of inner-side shank and surface around cloacal region.

Figure 6. 

General aspect in life of SYS a003486 (A–D), the male holotype of Leptobrachella wuhuangmontis sp. n. and the female paratype SYS a003499 (E, F).

Figure 7. 

The holotype of Leptobrachella wuhuangmontis sp. n., SYS a003486 in preservative.

Comparisons

Comparative morphological data of Leptobrachella wuhuangmontis sp. n. with the 66 recognized Leptobrachella species were obtained from examination of museum specimens (see Appendix 1) and from the references listed in Table 2. All comparative data were shown in Tables 4, 5, 6.

Compared with the 24 known congeners of the genus Leptobrachella occurring south of the Isthmus of Kra, by the presence of supra-axillary and ventrolateral glands, L. wuhuangmontis sp. n. can be easily distinguished 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 lacking supra-axillary and ventrolateral glands; and by the significantly larger body size, SVL 25.6–30.0 mm in males, 33.0–36.0 mm in females, L. wuhuangmontis sp. n. differs from the smaller L. baluensis (14.9–15.9 mm in males), L. brevicrus (17.1–17.8 mm in males), L. itiokai (15.2–16.7 mm in males), L. juliandringi (17.0–17.2 mm in males and 18.9–19.1 mm in females), L. mjobergi (15.7–19.0 mm in males), L. natunae (17.6 mm in male), L. parva (15.0–16.9 mm in males and 17.8 mm in female), L. palmata (14.4–16.8 mm in males), L. serasanae (16.9 mm in female) and Dring’s (1983) Leptobrachella sp. 3 “baluensis” (15.0–16.0 mm in males).

Leptobrachella wuhuangmontis sp. n. significantly differs from L. yunkaiensns sp. n. by a large genetic divergence (p=10.2–11.1%), lateral fringes on toes narrow (vs. wide), black supratympanic line distinct (vs. weak), dorsal surface of body rough and scattered with dense conical tubercles (vs. shagreened with short skin ridges and raised warts), belly greyish white mixed by tiny white and black dots (vs. belly pink with distinct or indistinct speckling).

From the rest 42 known congeners (Table 5), with SVL 25.6–30.0 mm in nine males and 33.0–36.0 mm in four females, Leptobrachella wuhuangmontis sp. n. differs from the larger L. bourreti (females 42.0–45.0 mm), L. eos (males 33.1–34.7 mm, female 40.7 mm), L. lateralis (female 36.6 mm), L. nahangensis (male 40.8 mm), L. nyx (females 37.0–41.0 mm), L. sungi (males 48.3–52.7 mm, females 56.7–58.9 mm), L. tamdil (male 32.3 mm) and L. zhangyapingi (males 45.8–52.5 mm); and from the smaller L. aerea (females 28.8–28.9 mm), L. ardens (female 24.5 mm), L. alpinus (females 32.1–32.5 mm in), L. applebyi (males 19.6–20.8 mm, female 21.7 mm), L. bidoupensis (males 18.5–25.4 mm), L. botsfordi (females 30.0–31.8 mm), L. kalonensis (females 28.9–30.6 mm), L. laui (female 28.1 mm), L. liui (females 23.0–28.0 mm), L. maculosa (female 27.0 mm), L. maoershanensis (female 29.1 mm), L. melica (males 19.5–22.7 mm), L. oshanensis (female 31.6 mm), L. pluvialis (males 21.0–22.0 mm), L. puhoatensis (females 27.3–31.5 mm), L. rowleyae (females 27.0–27.8 mm), L. tadungensis (female 32.1 mm), and L. tengchongensis (females 28.9–28.9 mm). Having head longer than wide in the new species (vs. head wider than long in L. bourreti, L. khasiorum, L. lateralis and L. sungi, and head width equal to or wider than long in L. nokrekensis). By having narrow fringes on toes, the new species differs from L. applebyi, L. ardens, L. crocea, L. kalonensis, L. lateralis, L. maculosa, L. macrops, L. melica, L. minima, L. nahangensis, L. nyx, L. oshanensis, L. pallida, L. pluvialis, L. pyrrhops, L. rowleyae, L. tadungensis, L. tuberosa, and L. ventripunctata, all of which have no lateral fringes on toes; L. bidoupensis, L. bourreti, L. fuliginosa, and L. sungi, all of which have weak lateral fringes on toes; L. alpinus, L. firthi and L. isos, all of which have wide lateral fringes only in males; L. aerea, L. eos, L. khasiorum, L. laui, L. liui, L. purpura, L. tamdil, L. yingjiangensis, and L. zhangyaping, all of which have wide lateral fringes both in males and females. By having rudimentary webbing on toes, the new species differs from L. ardens, L. kalonensis, L. maculosa, L. oshanensis, L. pallida, L. petrops, L. rowleyae, and L. tadungensis, all of which have no webbing on toes; L. pelodytoides, L. sungi, and L. tamdil, all of which have wide webbing on toes. By having black spots on flanks, the new species differs from L. aerea, L. botsfordi, L. eos, L. firthi, L. isos, L. pallida, L. petrops, L. tuberosa, and L. zhangyapingi, all of which have no black spots on flanks. By having rough dorsal skin with skin ridges and dense conical tubercles, the new species differs from L. applebyi, L. bidoupensis, L. kalonensis, L. melica, L. minima, L. nahangensis, and L. tadumgensis, all of which have smooth dorsal skin, and from L. purpura, L. tengchongensis, and L. yingjiangensis, all of which have shagreened dorsal skin with small tubercles.

Measurements (minimum–maximum (mean ± SD); in mm), and body proportions of Leptobrachella wuhuangmontis sp. n. from Mt. Wuhuang.

Measurements Males (n = 9) Females (n = 4)
SVL 25.6–30.0 (28.5 ±1.5) 33.0–36.0 (34.9 ± 1.4)
HDL 10.5–11.5 (10.9 ± 0.4) 12.4–12.6 (12.5 ± 0.1)
HDW 10.0–11.2 (10.5 ± 0.4) 12.1–12.3 (12.2 ± 0.1)
SNT 3.6–4.4 (4.1 ± 0. 2) 4.6–4.7 (4.6 ± 0.1)
EYE 3.5–4.4 (4.0 ± 0.3) 4.5–4.6 (4.6 ± 0.1)
IOD 2.8–3.0 (2.9 ± 0.1) 3.1–3.3 (3.2 ± 0.1)
IND 2.9–3.2 (3.1 ± 0.1) 3.2–3.4 (3.3 ± 0.1))
TMP 2.1–2.6 (2.4 ± 0.1) 2.6–2.8 (2.7 ± 0.1)
TEY 0.7–0.9 (0.8 ± 0.1) 0.8–0.9 (0.9 ± 0.1)
TIB 12.5–13.6 (13.3 ± 0.3) 15.0–16.3 (15.7 ± 0.5)
ML 7.0–8.0 (7.6 ± 0.3) 8.0–9.2 (8.5 ± 0.5)
PL 11.7–13.0 (12.5 ± 0.5) 13.9–14.8 (14.4 ± 0.4)
LAHL 14.2–16.0 (14.9 ± 0.6) 15.8–17.0 (16.4 ± 0.5)
HLL 38.8–44.9 (42.8 ± 1.9) 47.5–54.0 (51.2 ± 2.9)
HDL/HDW 1.03–1.06 (1.04 ± 0.01) 1.02–1.03 (1.03 ± 0.01)
HDL/SVL 0.36–0.41 (0.38 ± 0.02) 0.35–0.38 (0.36 ± 0.01)
SNT/HDL 0.34–0.40 (0.38 ± 0.02) 0.37 (0.37 ± 0)
SNT/ED 1.00–1.08 (1.03 ± 0.03) 1.00–1.02 (1.01 ± 0.01)
EYE/TMP 1.56–1.79 (1.68 ± 0.06) 1.64–1.73 (1.69 ± 0.04)
TMP/EYE 0.58–0.64 (0.60 ± 0.02) 0.58–0.61 (0.59 ± 0.01)
TEY/TMP 0.28–0.38 (0.33 ± 0.04) 0.30–0.33 (0.31 ± 0.02)
TIB/SVL 0.45–0.50 (0.47 ± 0.02) 0.44–0.47 (0.45 ± 0.01)
LAHL/SVL 0.50–0.55 (0.52 ± 0.02) 0.46–0.49 (0.47 ± 0.02)
HLL/SVL 1.45–1.54 (1.50 ± 0.03) 1.41–1.52 (1.47 ± 0.05)
TIB/HLL 0.29–0.33 (0.31 ± 0.01) 0.29–0.32 (0.31 ± 0.01)

Description of holotype

Adult male. Body size small, SVL in 30.0 mm. Head slightly longer than wide, HDL/HDW 1.04; snout rounded in dorsal view, nostril rounded, closer to tip of snout than to eye; canthus rostralis distinct; lores slightly concave; eye large, diameter equal to snout length, in 4.3 mm; tympanum distinct, rounded and slightly concave, its diameter significantly shorter than eye, TMP/EYE 0.56; distinct black supratympanic line present; vomerine teeth absent; vocal sac opening slit-like; tongue deeply notched behind; supratympanic ridge distinct, running from eye towards supra-axillary gland with raised tubercles.

Tips of fingers rounded, slightly swollen; relative finger lengths I = II < IV < III; nuptial pad absent; subarticular tubercles absent; a large, rounded inner palmar tubercle distinctly separated from small, round outer palmar tubercle; finger webbing absent and lateral fringes absent. Tips of toes like fingers; relative toe length I < II < V < III < IV; subarticular tubercles absent; dermal ridges undeveloped but present under the 3rd to 5th toes; large, oval inner metatarsal tubercle present, outer metatarsal tubercle absent; toes webbing rudimentary; narrow lateral fringes present on all toes. Tibia 45% of snout-vent length; tibiotarsal articulation reaches to middle of eye; heels just meeting each other when thighs are appressed at right angles with respect to body.

Skin on dorsum body and limbs rough with skin ridges and dense conical tubercles, ventral skin smooth; pectoral gland and femoral gland large, oval, slightly elevated; femoral gland situated on posterovertral surface of thigh, closer to knee than to vent; supra-axillary gland raised. Ventrolateral gland distinct, forming an incomplete line. Dense conical spines present on surface of lateral and ventral tarsus, surface of tibia-tarsal, inner-side surface of shank and surface around cloacal region.

Measurements of holotype

(in mm). SVL 30.0, HDL 10.9, HDW 10.5, SNT 4.3, EYE 4.3, IOD 2.9, IND 3.0, TMP 2.4, TEY 0.8, TIB 13.5, ML 7.8, PL 13.0, LAHL 15.4, HLL 44.9.

Coloration of holotype in life

Dorsal surface greyish purple with distinct dark brown markings and scattered with yellow blotches; distinct small white speckling present on edges of dark markings. A distinct dark brown triangle pattern between eyes, connected to the incomplete W-shaped dark brown marking between axillae. Tubercles on dorsum of body and limbs brown, those on lower flanks somewhat whitish; anterior upper lip with distinct blackish brown patches; transverse dark brown bars on dorsal surface of limbs; distinct dark brown blotches on flanks from groin to axilla; elbow and upper arms coppery orange and with distinct dark bars; fingers and toes with distinct dark brown blotches.

Ventral surface greyish-white mixed with tiny white and black dots. Supra-axillary, femoral, and ventrolateral glands white, pectoral gland greyish white as the color of ventral surface. Iris bicolored, coppery yellow on upper half and silver on lower half.

Coloration of holotype in preservative

Dorsal of body dark with greyish white dots on flanks, while dorsal of limbs dark brown, transverse bars on dorsal of forelimbs become more distinct, and indistinct on dorsal of hindlimbs, dark brown patterns, markings and spots on back become indistinct. Ventral surface light yellow with brown speckling. Supra-axillary, femoral, ventrolateral and pectoral glands light yellow (Figure 7).

Sexual dimorphism

Females with a larger body size than males, SVL 33.0–36.0 mm (34.9 ± 1.4) (vs. SVL 25.6–30.0 mm (28.5 ±1.5) in males); presence of a single vocal sac in males (vs. absent in females); dense conical spines on lateral and ventral surface of tarsus, surface of tibia-tarsal, inner-side surface of shank and surface around cloacal region distinct in males (vs. barely visible in females); pectoral gland and femoral gland large, oval, slightly elevated in males (vs. indistinct in females).

Variations

All paratypes match the overall characters of the holotype except that: tibiotarsal articulation reaches to posterior corner of eye in female paratypes SYS a003499, 3504 and reaches to anterior corner of eye in male paratypes SYS a003487 and SYSa 003500 / CIB 107274; pectoral gland large, oval, slightly elevated in all individuals in life, and become indistinct in preservation. Yellow blotches and white speckling present on dorsum in the holotype (vs. indistinct in the female paratype SYS a003499). Elbow and upper arms coppery orange and with distinct dark bars in the holotype (vs. elbow and upper arms light orange, dark bars indistinct in the male paratypes SYS a003488, 3505 and the female paratype SYS a003499) (Figure 6).

Etymology

The specific epithet, wuhuangmontis, is in reference to the type locality, Mt. Wuhuang of Guangxi Province, China. For the common name, we suggest “Mt. Wuhuang’s Leaf Litter Toad”, and for the Chinese name “Wu Huang Shan Zhang Tu Chan (五皇山掌突蟾)”.

Distribution and habits

Currently, Leptobrachella wuhuangmontis sp. n. is only known from its type locality MWH from Guangxi Province of China (Figure 1). The new species was found along a clear-water rocky streams and small steep rocky streams in well-preserved montane evergreen broadleaf forest (500 m a.s.l.) (Figure 8). During field surveys in March, males were found calling exposed on the rocks or hiding in the rock seams; gravid female collected on March and April bear pure white oocytes.

Figure 8. 

The habitat of Leptobrachella wuhuangmontis sp. n. in Mt. Wuhuang of Guangxi Province.

Discussion

Studies of the taxonomy and phylogeny of Leptobrachella are difficult to perform because of the morphological conservativeness and very similar characters (for example, the coloration and the texture of skin) in different environments, which may cause misidentifications (Ohler et al. 2010; Sung et al. 2014). With the evidence of both morphological and phylogenetic analyses, 15 cryptic species of the genus Leptobrachella have been discovered and described since 2010 (Frost 2017; Rowley et al. 2016, 2017; Yang et al. 2016; Yuan et al. 2017). With the description of L. yunkaiensis sp. n. and L. wuhuangmontis sp. n. based on an taxonomical approach, the number of the genus Leptobrachella herein is increased to 68, indicating the underestimated diversity.

During our examination, it was observed that the dense tiny conical spines present on the surface of the lateral and ventral aspects of the tarsus, surface of tibia-tarsal, the inner surface of the shank and surface around cloacal region (distinct in males and barely visible in females) in the two new Leptobrachella species described in this study are also present in examined specimens of L. alpinus, L. laui, L. liui, and L. tengchongensis as well as in other cryptic taxa (Wang et al. unpublished data). Thus, this neglected morphological character may be common among congeners of the genus Leptobrachella, and further morphological studies are needed to study this in more detail.

Mt. Wuhuang of Guangxi Province in southern China is known for the extraordinarily high biodiversity, with some new national records discovered in recent years, for example, the national records of Opisthotropis maculosa and Sphenomorphus tonkinensis from Mt. Wuhuang were recorded (Wang et al. 2013; Yang et al. 2011). Except for the new species (Leptobrachella yunkaiensis sp. n.) described in this study, several new species of amphibians and reptiles have been discovered from Dawuling Forest Station during field surveys in the last two years (Wang et al. unpublished data; Wang et al. 2018; Lyu et al. 2018), which suggests a high herpetofaunal biodiversity of Dawuling Forest Station localized in western Guangdong Province, China. Recently, these areas have been subjected to tourism development; thus, conservation strategies and measures for references and enforcements are urgently needed.

Acknowledgments

We would like to thank Jian Zhao for providing the photo of the habitat of Leptobrachella yunkaiensis sp. n., thank Canrong Lin, Chaoyu Lin, Hailong He, Honghui Chen, Jiahe Li, Runlin Li, Siyu Zhang and Youyu Li for their help in the field work, and we are grateful to Yun Li and Chenghui Xu for their help and suggestions on the figures and with specimen photography. This work was supported by the Biodiversity Conservation Program of Ministry of Environmental Protection of P. R. China to Ying-Yong Wang.

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Appendix 1

Specimens examined

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

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