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Research Article
A new species of Leptobrachella (Anura, Megophryidae) from Guizhou Province, China
expand article infoTao Luo‡, Ning Xiao§, Kai Gao|, Jiang Zhou‡
‡ Guizhou Normal University, Guiyang, China
§ Guiyang Nursing Vocational College, Guiyang, China
| Beijing Normal University, Beijing, China

Corresponding author: Jiang Zhou ( zhoujiang@ioz.ac.cn )

Academic editor: Angelica Crottini
© 2020 Tao Luo, Ning Xiao, Kai Gao, Jiang Zhou.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Luo T, Xiao N, Gao K, Zhou J (2020) A new species of Leptobrachella (Anura, Megophryidae) from Guizhou Province, China. ZooKeys 923: 115-140. https://doi.org/10.3897/zookeys.923.47172
ZooBank: urn:lsid:zoobank.org:pub:104B0C71-0826-4C50-938B-FD78C4D403A6
Open Access

Abstract

This study describes a new species of the genus Leptobrachella, Leptobrachella suiyangensis sp. nov. from the Huoqiuba Nature Reserve, Suiyang County, Guizhou Province, China, based on morphological data and phylogenetic analyses (16S rRNA mtDNA). The new species can be distinguished from other congeners by the molecular divergence and by a combination of morphological characters, including body size, dorsal and ventral patterns, dorsal skin texture, size of the pectoral and femoral glands, degree of webbing and fringing on the toes and fingers, dorsum coloration, and iris coloration in life. Currently, the genus Leptobrachella contains 75 species, 21 of which are found in China, including seven species reported from Guizhou Province. The uncorrected sequence divergence percentage between Leptobrachella suiyangensis sp. nov. and all homologous DNA sequences available for the 16S rRNA gene was found to be >4.7%. The new record of the species and its relationships with others in the same genus imply that species distribution, habitat variation, environmental adaptation, and diversity of the genus Leptobrachella in southwest China need to be further investigated.

Keywords

Leptobrachella suiyangensis sp. nov., mitochondrial DNA, morphology, Southwest China

Introduction

The genus Leptolalax Dubois, 1983 in the family Megophryidae Bonaparte, 1850 is regarded to be closely associated with the genus Leptobrachella Smith, 1925 and has been assigned as a synonym of the genus Leptobrachella based on a large-scale molecular analysis (Chen et al. 2018). The genus Leptobrachella is now considered to contain 74 species. The genus is widely distributed from southwestern China to northeastern India and Myanmar (Fei et al. 2012; Frost 2019), extending to mainland Indochina, peninsular Malaysia, and the islands of Borneo (Rowley et al. 2016, 2017a; Yang et al. 2016; Yuan et al. 2017; Wang et al. 2018; Nguyen et al. 2018). Currently, 20 species of this genus are known from China. They are: Leptobrachella alpina (Fei, Ye & Li, 1990) and L. bourreti (Dubois, 1983) from Yunnan and Guangxi; L. eos (Ohler, Wollenberg, Grosjean, Hendrix, Vences, Ziegler & Dubois, 2011) and L. nyx (Ohler, Wollenberg, Grosjean, Hendrix, Vences, Ziegler & Dubois, 2011) from Yunnan; L. laui (Sung, Yang & Wang, 2014) and L. yunkaiensis Wang, Li, Lyu & Wang, 2018 from southern Guangdong, including Hong Kong; L. liui (Fei & Ye, 1990) from Fujian, Jiangxi, Guangdong, Guangxi, Hunan, and Guizhou; L. oshanensis (Liu, 1950) from Gansu, Sichuan, Chongqing, Guizhou, and Hubei; L. purpuraventra Wang, Li, Li, Chen & Wang, 2019 and L. bijie Wang, Li, Li, Chen & Wang, 2019 from Guizhou; L. purpurus (Yang, Zeng & Wang, 2018), L. pelodytoides (Boulenger, 1893), L. tengchongensis (Yang, Wang, Chen & Rao, 2016) and L. yingjiangensis (Yang, Zeng & Wang, 2018) from Yunnan; L. ventripunctata (Fei, Ye & Li, 1990) from Guizhou and Yunnan; L. mangshanensis (Hou, Zhang, Hu, Li, Shi, Chen, Mo & Wang, 2018) from southern Hunan, and L. sungi (Lathrop, Murphy, Orlov & Ho, 1998), L. maoershanensis (Yuan, Sun, Chen, Rowley & Che, 2017), L. shangsiensis Chen, Liao, Zhou & Mo, 2019, and L. wuhuangmontis Wang, Yang & Wang, 2018 from Guangxi (Sung et al. 2014; Yang et al. 2016, 2018; Yuan et al. 2017; Wang et al. 2018, 2019; Hou et al. 2018; Chen et al. 2018, 2019; Wang et al. 2019; AmphibiaChina 2019).

During a field survey in June 2018 in a montane evergreen forest, Suiyang County, Guizhou Province (Fig. 1), we collected three different species of the family Megophryidae co-occurring in this small-fragmented forest. The specimens could be morphologically separated from one another. Subsequent studies based on morphological and molecular data indicated that two of the three could be classified as Megophrys minor Stejneger and M. spinata Liu and Hu, while the third population, differing significantly from the other two, was further analyzed via morphological characters. Subsequent 16S rRNA sequences from these specimens revealed that the collection represented distinct evolving lineages and belong to the genus Lepobrachella. Combining morphological characters, acoustic data, and molecular divergence, we described the specimens as a new species.

Materials and methods

Sampling

Eight specimens collected from the aforementioned area (Fig. 1) were euthanized with chlorobutanol solution and fixed in 10% formalin for 24 h, and then stored in 75% ethanol. Liver and muscular tissues were taken before fixing and preserved in 95% alcohol at -20 °C. All of the specimens are kept at the College of Life Sciences, Guizhou Normal University (GZNU), Guiyang City, Guizhou Province, China.

Figure 1. 

Collection locality (red circle) of Leptobrachella suiyangensis sp. nov. from Suiyang County, Guizhou province, China used in this study.

DNA Extraction, PCR and sequencing

DNA samples were extracted from muscular tissues with a DNA extraction kit (Tiangen Biotech (Beijing) Co. Ltd). The mitochondrial gene and 16S ribosomal RNA gene (16S rRNA) were sequenced (951bp). The fragmented genes were amplified with 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 1 min, followed by 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 a BigDye Terminator Cycle Sequencing Kit according to the guidelines of the manufacturer. The products were sequenced on an ABI Prism 3730 automated DNA sequencer at Shanghai Majorbio Bio-pharm Technology Co. Ltd. All sequences have been deposited in GenBank (Table 1). For molecular analyses, a total of 77 sequences (74 sequences downloaded from GenBank and three our new sequences) from 55 species of the genus Leptobrachella were used, including one undescribed species from C hina, that is, the populations from Huoqiuba Nature Reserve, Suiyang County, and Guizhou Province. Three species which sequences downloaded from GenBank are used as outgroups (Leptobrachium huashen Fei & Ye, 2005, Leptobrachium cf. chapaense (Bourret, 1937) and Megophrys major Boulenger, 1908 (Chen et al. 2018; Wang et al. 2019; Table 1).

Table 1.
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Localities and voucher data for all specimens used in this study.

ID Species Locality Voucher no. GenBank no.
1 Leptobrachella suiyangensis sp.nov. Suiyang County, Guizhou, China GZNU20180606002 MK829648
2 Leptobrachella suiyangensis sp.nov. Suiyang County, Guizhou, China GZNU20180606005 MK829649
3 Leptobrachella suiyangensis sp.nov. Suiyang County, Guizhou, China GZNU20180606006 MK829650
4 Leptobrachella aerea Vilabuly, Savannakhet, Laos NCSM 76038 MH055809
5 Leptobrachella aerea Phong Nha-Ke Bang, Quang Binh, Vietnam RH60165 JN848437
6 Leptobrachella alpina Huangcaoling, Yunnan, China KIZ046816 MH055866
7 Leptobrachella applebyi Song Thanh Nature Reserve, Quang Nam, Vietnam AMS R171704 HM133598
8 Leptobrachella baluensis Tambunan, Sabah, Borneo, Malaysia SP 21604 LC056792
9 Leptobrachella bidoupensis Bidoup, Lam Dong, Vietnam NCSM 77321 HQ902883
10 Leptobrachella bijie Zhaozishan Nature Reserve, Bijie City, Guizhou, China SYS a007313/CIB110002 MK414532
11 Leptobrachella bijie Zhaozishan Nature Reserve, Bijie City, Guizhou, China SYS a007314 MK414533
12 Leptobrachella botsfordi Fansipan, Lao Cai, Vietnam AMS R 176540 MH055952
13 Leptobrachella bourreti Sapa, Lao Cai, Vietnam 1999.566 KR827860
14 Leptobrachella brevicrus Gunung Mulu National Park, Sarawak, Malaysia UNIMAS 8957 KJ831303
15 Leptobrachella dringi Gunung Mulu, Malaysia KUHE:55610 AB847553
16 Leptobrachella eos Boun Tay, Phongsaly, Laos NCSM 80551 MH055887
17 Leptobrachella eos Zhushihe, Yunnan, China SYS a003959 MH055888
18 Leptobrachella firthi Ngoc Linh Nature Reserve, Kon Tum, Vietnam AMS: R 176506 JQ739207
19 Leptobrachella fritinniens Gunung Mulu, Malaysia KUHE55371 AB847557
20 Leptobrachella gracilis Gunung Mulu, Malaysia KUHE55624 AB847560
21 Leptobrachella hamidi Bukit Lanjan, Selangor, Malaysia KUHE17545 AB969286
22 Leptobrachella heteropus Larut, Perak, Malaysia KUHE15487 AB530453
23 Leptobrachella isos Gia Lai, Vietnam AMS R 176469 KT824767
24 Leptobrachella itiokai Mulu NP, Sarawak, Borneo, Malaysia KUHE 55845 LC137802
25 Leptobrachella juliandringi Mulu NP, Sarawak, Borneo, Malaysia KUHE 55333 LC056780
26 Leptobrachella kajangensis Tioman, Malaysia LSUHC 4431 LC202001
27 Leptobrachella kecil Cameron, Malaysia KUHE 52440 LC202004
28 Leptobrachella khasiorum Khasi Hills, Meghalaya, India SDBDU 2009.329 KY022303
29 Leptobrachella liui Wuyi Shan, Fujian, China SYS a001597 KM014547
30 Leptobrachella liui Wuyi Shan, Fujian, China ZYCA907 MH055908
31 Leptobrachella laui Shenzhen, Guangdong, China SYS a002450 MH055904
32 Leptobrachella laui Shenzhen, Guangdong, China SYS a001515 KM014545
33 Leptobrachella macrops Phu Yen, Vietnam ZMMU-A5823 MG787993
34 Leptobrachella mangshanensis Mangshan, Hunan, China MSZTC201701 MG132196
35 Leptobrachella mangshanensis Mangshan, Hunan, China MSZTC201702 MG132197
36 Leptobrachella maoershanensis Mao’er Shan, Guangxi, China KIZ07614 MH055927
37 Leptobrachella maoershanensis Mao’er Shan, Guangxi, China KIZ027236 MH055928
38 Leptobrachella marmorata Borneo, Malaysia KUHE53227 AB969289
39 Leptobrachella maura Borneo, Malaysia SP21450 AB847559
40 Leptobrachella melanoleucus Surat Thani, Thailand KUHE:23845 LC201999
41 Leptobrachella melica Cambodia, Ratanakiri MVZ258198 HM133600
42 Leptobrachella minimus Doi Chiang Dao, Chiangmai, Thailand THNHM07418 JN848402
43 Leptobrachella minimus Doi Suthep, Thailand KUHE:19201 LC201981
44 Leptobrachella mjobergi Gading NP, Sarawak, Borneo, Malaysia KUHE:47872 LC056787
45 Leptobrachella nahangensis Na Hang Nature Reserve, Tuyen Quang, Vietnam ROM 7035 MH055853
46 Leptobrachella nahangensis Na Hang, Tuyen Quang, Vietnam ZMMU-NAP-02259 MH055854
47 Leptobrachella nyx Ha Giang, Vietnam ROM 36692 MH055816
48 Leptobrachella oshanensis Emei Shan, Sichuan, China KIZ025776 MH055895
49 Leptobrachella oshanensis Emei Shan, Sichuan, China Tissue ID: YPX37492 MH055896
50 Leptobrachella pallida Vietnam: Lam Dong UNS00511 KU530190
51 Leptobrachella parva Mulu National Park, Sarawak, Malaysia KUHE:55308 LC056791
52 Leptobrachella petrops Cham Chu Nature Reserve, Tuyen Quang, Vietnam VNMN:2016 A.06 KY459998
53 Leptobrachella picta Borneo, Malaysia UNIMAS 8705 KJ831295
54 Leptobrachella pluvialis Fansipan, Lao Cai, Vietnam ROM 30685 MH055843
55 Leptobrachella pluvialis Sapa, Lao Cai, Vietnam ZMMU-A-5222-02262 MH055844
56 Leptobrachella puhoatensis Pu Hu, Thanh Hoa, Vietnam VNMN:2016 A.23 KY849587
57 Leptobrachella purpura Yingjiang, Yunnan Province, China SYS a006530 MG520354
58 Leptobrachella purpura Yingjiang, Yunnan Province, China SYS a006531 MG520355
59 Leptobrachella purpuraventra Wujing Nature Reserve, Bijie City, Guizhou, China SYS a007081 MK414517
60 Leptobrachella purpuraventra Wujing Nature Reserve, Bijie City, Guizhou, China SYS a007277/CIB110003 MK414518
61 Leptobrachella pyrrhops Lam Dong, Vietnam ZMMU A-5208 KP017575
62 Leptobrachella sabahmontana Borneo, Malaysia BORNEENSIS 12632 AB847551
63 Leptobrachella shangsiensis Guangxi, China NHMG1401032 MK095460
64 Leptobrachella shangsiensis Guangxi, China NHMG1401033 MK095461
65 Leptobrachella solus Hala-Bala, Thailand KUHE:23261 LC202007
66 Leptobrachella solus Tam Dao, Vinh Phuc, Vietnam ROM 20236 MH055858
67 Leptobrachella tengchongensis Gaoligong Shan, Yunnan, China SYS a004598 KU589209
68 Leptobrachella tengchongensis Gaoligong Shan, Yunnan, China SYS a003766 MH055897
69 Leptobrachella ventripunctatus Zhushihe, Yunnan, China SYS a004536 MH055831
70 Leptobrachella wuhuangmontis Mt. Wuhuang, Pubei County, Guangxi, China SYS a003485 MH605577
71 Leptobrachella wuhuangmontis Mt. Wuhuang, Pubei County, Guangxi, China SYS a003486 MH605578
72 Leptobrachella yingjiangensis Yingjiang, Yunnan, China SYS a006533 MG520350
73 Leptobrachella yingjiangensis Yingjiang, Yunnan, China SYS a006532 MG520351
74 Leptobrachella yunkaiensis Dawuling Forest Station, Maoming City, Guangdong, China SYS a004663 MH605584
75 Leptobrachella yunkaiensis Dawuling Forest Station, Maoming City, Guangdong, China SYS a004664 / CIB107272 MH605585
76 Leptobrachella zhangyapingi Chiang Mai, Thailand KIZ07258 MH055864
77 Leptobrachella zhangyapingi Pang Num Poo, Chiang Mai Province,Thailand JK-2013 JX069979
78 Leptobrachium huashen Yunnan, China KIZ049025 KX811931
79 Leptobrachium cf. chapaense Sapa, Lao Cai, Vietnam AMS R 171623 KR018126
80 Megophrys major Kon Tum, Vietnam AMS R 173870 KY476333

Phylogenetic analyses

All sequences were aligned by MUSCLE v. 3.6 with the default settings (Edgar 2004). Trimming with the gaps partially deleted was performed in MEGA 7.0 (Kumar et al. 2016), while within high variable regions, all gaps were removed.

Phylogenetic trees were constructed with both Maximum Likelihood (ML) and Bayesian Inference (BI). The ML was conducted in IQ-TREE (Nguyen et al. 2015) with 2000 ultrafast bootstrapping (Hoang et al. 2018) and was performed until a correlation coefficient of at least 0.99 was reached. The BI was performed in MrBayes v. 3.2.1 (Ronquist et al. 2012), and the best-fit model was obtained by the Akaike Information Criterion (AIC) computed with PartitionFinder 2 (Lanfear et al. 2016), resulting in the best-fitting nucleotide substitution models of GTR + I + G with for BI and ML analysis. Two independent processes were conducted for 10 million generations, sampling every 1000, with four independent chains and a burn-in of 25%. Convergence was assessed referring to the criteria of all parameters having reached stationarity and having obtained satisfactory effective sample sizes (>200) using Tracer v. 1.6. (Rambaut et al. 2014). Nodes in the trees were considered well supported when Bayesian posterior probabilities (BPP) were ≥0.95 and ML ultrafast bootstrap values (UFB) was ≥95 % (Chen et al. 2018; Hoang et al. 2018). Uncorrected p-distances based on 16S rRNA were calculated in MEGA v. 7.0 (Kumar et al. 2016).

Morphological and morphometric analyses

Morphometric data were taken from eight of most well-preserved adult specimens. Measurements were recorded to the nearest 0.1 mm (Watters et al. 2016) with digital calipers following the methods of Fei et al. (2009) and Rowley et al. (2013). 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 the anterior corner of the eye)

EYE eye diameter (diameter of the exposed portion of the eyeballs)

IOD interorbital distance (minimum distance between upper eyelids)

IND internasal distance (distance between nares)

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

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

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

TMP tympanum diameter (horizontal diameter of tympanum)

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

TIB tibia length (distance from knee to heel)

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

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

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

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

Sex was determined by direct observation of calls in life, the presence of internal vocal sac openings, and the presence of eggs in the abdomen through external inspection. Comparative morphological data of Leptobrachella species were obtained 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.

Table 2.
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Obtained references of 74 known congeners of the genus Leptobrachella, respectively.

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

Results

Phylogenetic trees from Maximum likelihood (ML) and Bayesian inference (BI) were constructed based on DNA sequences of the mitochondrial 16S rRNA gene with a length of 500 bp. The trees present identical topologies (Fig. 2) with the clustered population of Leptobrachella from Huoqiuba Nature Reserve, in which L. alpina + L. purpurus and the population of Leptobrachella from Huoqiuba Nature Reserve show relatively high node supporting values (0.68 in BI and 71% in ML) and exhibit a separate evolving lineage. The smallest pairwise genetic divergence between the population from Suiyang County and all other species of the genus Leptobrachella is 4.71%. This indicates that there is substantial genetic divergence between the species in Leptobrachella and the specimens from Suiyang County, indicating that this new population can be regarded to be a separate lineage and is valid to be described as a new species as below.

Figure 2. 

Bayesian inference tree derived from partial DNA sequences of the mitochondrial 16S r RNA gene. Numbers before slashes indicate Bayesian posterior probabilities (displayed >0.60 values), and numbers after slashes are ultrafast bootstrap support for maximum likelihood (2000 replicates) analyses (>60 retained). The symbol “–” represents value below 0.60/60. The scale bar represents 0.05 nucleotide substitutions per site.

Taxonomic account

Leptobrachella suiyangensis sp. nov.

http://zoobank.org/75EDCF88-0293-40E9-83FE-47785145864C
Table 3, Figs 3, 4

Type material

Holotype. GZNU20180606007, adult male, collected by Tao Luo (TL hereafter) on 7 June 2018 from the Huoqiuba Nature Reserve (28.4805°N, 107.0764°E, 1501 m. a.s.l.; Fig. 1), Suiyang County, Guizhou Province, China.

Paratypes. Five adult males (GZNU20180606002, GZNU20180606005, GZNU20180606006, GZNU20180606008), and three adult females (GZNU20180606001, GZNU20180606003, GZNU20180606004). They were collected from the holotype locality on 6 June 2018.

Etymology

The specific epithet “suiyangensis” refers to the name of the holotype locality, Taibai Town in Suiyang County of Guizohu Province, China. We suggest as its English name “Suiyang Leaf-litter Toad,” and its Chinese name as “Sui Yang Zhang Tu Chan (绥阳掌突蟾)”.

Table 3.
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Measurements (in mm) of the type series of Leptobrachella suiyangensis sp.nov. (H = holotype, P = paratype,M= male, F= female, another abbreviations defined in text).

Specimen Type status Sex SVL HDL HDW SNT EYE IOD IND UEW NEL NSL TMP TEY TIB HND LAHL HLL FOT
GZNU20180606007 H M 28.7 9.9 9.3 4.1 2.4 2.9 2.8 1.6 2.8 1.1 2.1 1.1 13.1 7.2 13.4 43.3 12.5
GZNU20180606008 P M 29.2 10.5 9.8 4.6 2.8 2.8 2.9 2.1 2.3 1.7 1.2 1.9 13.4 7.0 13.2 43.4 12.9
GZNU20180606002 P M 29.7 12.1 10.1 5.0 3.9 3.2 3.7 3.1 2.3 1.3 2.3 1.4 13.8 7.1 13.3 44.4 12.3
GZNU20180606005 P M 29.0 11.8 10.3 4.5 3.3 3.4 3.1 2.0 2.7 1.1 1.9 1.6 13.5 6.5 13.4 41.8 12.9
GZNU20180606006 P M 29.2 11.4 10.4 4.0 3.8 3.2 3.2 2.6 2.6 2.2 1.8 1.3 13.6 7.4 13.3 42.8 12.6
GZNU20180606001 P F 32.0 12.6 10.7 4.7 3.7 3.5 3.5 3.0 2.4 1.3 2.6 1.7 15.2 7.1 13.4 44.7 13.9
GZNU20180606003 P F 30.5 10.3 10.9 4.7 3.7 3.1 3.1 2.2 2.3 1.5 3.5 1.4 15.2 7.4 13.8 45.3 16.6
GZNU20180606004 P F 33.5 13.1 12.1 4.9 3.6 3.1 3.6 2.8 3.1 1.8 3.8 1.7 17.4 8.1 16.7 53.8 14.4

Diagnosis

The specimens were assigned to the genus Leptobrachella on the basis of the following characters: (1) small body size; (2) having an elevated inner metacarpal tubercle; (3) having macro-glands on body (including supra-axillary, femoral and ventrolateral glands); (4) lacking vomerine teeth; (5) having small tubercles on eyelids; (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). Leptobrachella suiyangensis sp. nov. can be distinguished from its congeners by referring to the following characters: (1) small body size (SVL 28.7–29.7 mm in males, 30.5–33.5 mm in females); (2) dorsal skin shagreened, with some of the granules forming longitudinal short skin ridges; (3) tympanum distinctly discernible, slightly concave, with a deep, black, supratympanic line; (4) ventrolateral glands are distinct, forming a dotted line; (5) dorsal surface shagreened and granular, lacking enlarged tubercles or warts, with some of the granules forming short longitudinal folds; (6) flanks with several distinct and large dark blotches; (7) ventral surface of throat grey-white, and surface of chest and belly yellowish creamy-white with marbled texture or with irregular light brown speckling; (8) supra-axillary, femoral, pectoral and ventrolateral glands are distinctly visible; (9) absence of webbing and lateral fringes on fingers, and toes feature rudimentary webbing and a weak lateral fringes; (10) relatively short hindlimbs (TIB/SVL ratio in males 0.46–0.47); (11) longitudinal ridges under the toes are interrupted at the articulations; (12) relative finger lengths I <II < IV < III, relative toe lengths I < II < V < III < IV; (13) dorsum greyish-brown, with small light-orange granules and distinct darker brown markings scattered with irregular light-orange pigmentation, and bicolored iris, coppery orange on the upper half and silver grey on the lower half.

Description of the holotype. GZNU20180606007 (adult male), small body size (SVL 28.7 mm); the head length is slightly larger than the head width (HDL/HDW ratio 1.06); the snout is slightly protruding, projecting beyond the margin of the lower jaw; the nostril is between the snout and the eye (NSL/NEL ratio 0.39); the canthus rostralis is gently rounded; the loreal region is slightly concave; the interorbital space is flat; larger (IOD 2.9 mm) than the upper eyelid (1.6 mm in width), and the internarial distance is 2.8 mm; with vertical pupil; snout length is slightly larger than eye diameter (SNT/EYE ratio 1.71); tympanum is distinct and rounded, its diameter (TMP 2.1 mm) is smaller than that of the eye diameter (EYE 2.4 mm) and longer than the tympanum-eye distance (TMP/TEY ratio 1.91); deep black supratympanic line is present; weakly black supratympanic line exists (Fig. 3C); tympanic rim is distinctly elevated relative to the skin of the temporal region; supratympanic ridge is distinct, extending from the eye to the supra-axillary gland; a few indistinct tubercles present on supratympanic ridge; absent vomerine teeth; vocal sac openings is slit-like, located posterior-laterally on the floor of the mouth close to the margins of the mandible; long and wide tongue, with a small shallow notch at the posterior tip.

Figure 3. 

Holotype of Leptobrachella suiyangensis sp. nov. (GZNU20180606007) in life. A Dorsal view B Dorsolateral view C Ventral view D Right eye shown iris coloration E Volar view of the left hand F Plantar view of the left foot.

The tips of the fingers are rounded, slightly swollen; relative finger lengths are presented as: I <II < IV < III; nuptial pad is absent; absent subarticular tubercles (Fig. 3F); a large, round inner palmar tubercle is distinctly separated from a small, round outer palmar tubercle; finger webbing and dermal fringes absent. Toe tips are similar to those of the fingers; the relative toe length is presented as: I <II <V<III <IV; absent subarticular tubercles; distinct dermal ridges present under the 3rd to 5th toes; pronounced larger, oval inner metatarsal tubercle, outer metatarsal tubercle is absent; rudimentary toe webbing; weak lateral fringes present on all toes. Tibia is slightly shorter than half of the snout-vent length (TIB/SVL ratio 0.46); tibiotarsal articulation reaches to the anterior eye; heels meet each other when thighs are appressed at right angles referring to the body.

Dorsal skin is shagreened and scattered with fine and rounded granules, some of the granules forming short longitudinal folds; ventral skin smooth; large pectoral gland, elongated oval, 1.5 mm in length; small femoral gland, rounded, 0.7 mm in diameter, situated on the posteroventral surface of the thigh, closer to tibiotarsal articulation than to the vent; risen supra-axillary gland, 1.3 mm in diameter; ventrolateral gland is distinct as small white dots forming an incomplete line (Fig. 3D).

Measurements of holotype (in mm). Holotype: SVL 28.7, HDL 9.9, HDW 9.3, SNT 4.1, EYE 2.4, IOD 2.9, INT 2.8, UEW 1.6, NEL 2.8, NSL 1.1, TMP 2.1, TEY 1.1, TIB 13.1, HND 7.2, LAHL 13.4, HLL 43.3, FOT 12.5.

Coloration of holotype in life. Dorsal skin purple-brown; brown-purplish with dark-brown marks between the eyes and the scapular region, which are scattered with some deep yellow-orange granules more concentrated on the upper eyelid (Fig. 3C). A dark brown ϒ-pattern exists between eyes, linked with dark brown W-shaped marks between axillae. Tympanum is light brown-grey; black-brown tubercles present on dorsum of the body and the limb; those on dorsal side are much more distinct and dense; anterior upper lip features distinct blackish-brown patches; transverse dark-brown bars exist on dorsal surface of the limbs two or three (elbow and upper arms are an exception); indistinct black or brown blotches present on the flanks from groin to axilla; elbow and upper arms have no dark bars but with distinct dark-orange coloration; fingers and toes show indistinct brown blotches; a black spot is present on the loreal region; lower edge of the upper drum ridge is prominently black; ventral surface of the throat is grey-white, and surface of chest and belly is yellowish creamy-white, ventral part with distinct or indistinct light brown speckling mixed with marble texture; ventral surface of the thighs is dark grey and scattered with small light white spots. Supra-axillary gland milky yellow; iris is bicolored, coppery orange on the upper half and silver grey on the lower half.

Figure 4. 

Holotype of Leptobrachella suiyangensis sp. nov. (GZNU20180606007) in preservative. A Dorsal view B Ventral views C Lateral views.

Coloration of holotype in preservative

In preservation, there are dark brown marks on the dorsum and flanks; dorsum of the body and hindlimbs are dark brown, while dorsum of the forelimbs is yellowish brown; transverse bars on the limbs become more distinct, and dark-brown patterns, marks and spots on the back are indistinct; ventral surface of the body is yellowish brown with brown marbling on the sides and chest; orange supra-axillary, femoral, pectoral and ventrolateral glands fade to greyish white.

Variations

Measurements of the type series are shown in Table 4. Females (mean of SVL (32.0 ± 1.5 mm, n = 3) have larger body size than males (mean of SVL 29.2 ± 0.4 mm, n = 5) (Table 4). In life (Fig. 5), all paratypes match overall characters of the holotype, except the surface of the belly that is scattered with brown speckling in the holotype (that for females is more distinct; GZNU20180606001). Under the condition of preservation, however, some specimens become slightly darker brown compared to the holotype.

Figure 5. 

Paratypes of Leptobrachella suiyangensis sp. nov. in life. A GZNU20180606005, adult male (A), (B) GZNU20180606002, adult male C GZNU20180606003, adult female.

Distribution and habitats

Currently, Leptobrachella suiyangensis sp. nov. is known only from its holotype locality, Huoqiuba Nature Reserve, Suiyang County, Guizhou Province, China (Fig. 1). The specimens were collected in a stream (ca 1.5 m in width and ca 10 cm in depth) and from nearby well-preserved bamboo forests (1501 m a.s.l.). During June, males were calling from under bamboo leaves; others perch on or under rocks by the side of the stream.

Table 4.
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Measurements (in mm), and body proportions of Leptobrachella suiyangensis sp.nov. from Suiyang County, Guizhou Province, China.

Measurements Males Range (mean ± SD), n = 5 Females Range (mean ± SD), n = 3
SVL 28.7–29.7 (29.2 ± 0.4) 30.5–33.5 (32.0 ± 1.5)
HDL 9.9–12.1 (11.1 ± 0.9) 10.3–13.1 (12.0 ± 1.5)
HDW 9.3–10.4 (10.0 ± 0.4) 10.7–12.1 (11.2 ± 0.8)
SNT 4.0–5.0 (4.4 ± 0.4) 4.7–4.9 (4.8 ± 0.1)
EYE 2.4–3.9 (3.2 ± 0.6) 3.6–3.7 (3.7 ± 0.1)
IOD 2.8–3.4 (3.1 ± 0.2) 3.1–3.5 (3.2 ± 0.2)
INT 2.8–3.7 (3.1 ± 0.4) 3.1–3.6 (3.4 ± 0.3)
UEW 1.6–3.1 (2.3 ± 0.6) 2.2–3.0 (2.7 ± 0.4)
NEL 2.3–2.8 (2.5 ± 0.2) 2.3–3.1 (2.6 ± 0.4)
NSL 1.1–2.2 (1.5 ± 0.5) 1.3–1.8(1.5 ± 0.3)
TMP 1.2–2.3 (1.9 ± 0.4) 2.6–3.8 (3.3 ± 0.6)
TEY 1.1–1.9 (1.5 ± 0.3) 1.4–1.7 (1.6 ± 0.2)
TIB 13.1–13.8 (13.5 ± 0.3) 15.2–17.4 (15.9 ± 1.3)
HND 6.5–7.4 (7.0 ± 0.3) 7.1–8.1 (7.5 ± 0.5)
LAHL 13.2–13.4 (13.3 ± 0.1) 13.4–16.7 (14.6 ± 1.8)
HLL 41.8–44.4 (43.1 ± 0.9) 44.7–53.8 (47.9 ± 5.1)
FOT 12.3–12.9 (12.6 ± 0.3) 13.9–16.6 (15.0 ± 1.4)
HDL/HDW 1.06–1.20 (1.12 ± 0.06) 0.90–1.20 (1.07 ± 0.15)
HDL/SVL 0.34–0.41 (0.38 ± 0.03) 0.30–0.40 (0.37 ± 0.06)
SNT/HDL 0.35–0.44 (0.40 ± 0.03) 0.40–0.50 (0.43 ± 0.06)
SNT/EYE 1.05–1.71 (1.41 ± 0.27) 1.30–1.40 (1.33 ± 0.06)
EYE/TMP 1.14–2.33 (1.80 ± 0.45) 1.00–1.40 (1.17 ± 0.21)
EYE/SNT 0.59–0.95 (0.73 ± 0.15) 0.70–0.80 (0.77 ± 0.06)
TMP/EYE 0.43–0.88 (0.59 ± 0.18) 0.70–1.10 (0.93 ± 0.21)
TIB/SVL 0.46–0.47 (0.46 ± 0.01) 0.50
LAHL/SVL 0.45–0.47 (0.46 ± 0.01) 0.40–0.50 (0.47 ± 0.06)
HLL/SVL 1.44–1.51 (1.48 ± 0.03) 1.40–1.60 (1.50 ± 0.10)
TIB/HLL 0.30–0.32 (0.31 ± 0.01) 0.30

Comparisons

Leptobrachella suiyangensis sp. nov. differs from all other species of Leptobrachella based on morphological and molecular evidence. Phylogenetically, L. suiyangensis sp. nov., L. alpina and L. purpurus form a clade. Genetically, among this clade, the smallest genetic distance, at 5.49%, is between L. suiyangensis sp. nov. and L. alpina, and the largest genetic distance is 6.27% (L. suiyangensis sp. nov. and L. purpurus). Morphologically, the new species can be distinguished from L. alpina by having a larger body size of males (28.7–29.7 mm vs 24.0–26.4 mm); having narrower lateral fringes on the toes of the male (vs wide in males); dorsum purple-brown to dark purple-brown or grey-purple ground colour; ventral yellowish creamy-white with marbled texture on the chest and belly or with irregular light-brown speckling (vs almost uniformly gray-brown on dorsal part, ventral nearly immaculately creamy white, brown specking on margins); ventrolateral glands are characterized by small white dots forming an incomplete line (vs small white dots forming a complete line longitudinally); shoulder-gland is orange-yellow (vs white, around gland); head length greater than head width, HDL/HDW ratio 1.12 (vs head length equal to head width, HDL/HDW ratio 1.00). The new species can be distinguished from L. purpurus by body size of males (28.7–29.7 mm vs 25.0–27.5 mm); having narrow lateral fringes on the toes of males (vs wide in males); dorsum purple-brown to dark purple-brown or grey-purple ground color, ventral yellowish creamy-white with marbled texture on the chest and belly or with irregular light-brown speckling (vs dorsum coloration purplish brown, ventral side dull white with an indistinct grey dusting); throat immaculate gray (vs throat immaculate pinkish; almost dark orange-yellow on the upper arm (vs upper arms with distinct coppery orange coloration); dark bars on dorsal surface of tibia and tarsus very narrow, especially those on dorsal skin of tarsus (vs relatively broader dark bars on dorsal surface of tibia and tarsus); tibiotarsal articulation reaches to the anterior eye (vs tibiotarsal articulation reaches to posterior corner of the eye); relative length of fingers I <II < IV < III (vs I = II = IV < III).

Compared with the 26 known congeners in the genus Leptobrachella found south of the Isthmus of Kra, referring to the presence or absence of supra-axillary and ventrolateral glands, L. suiyangensis sp. nov. can be easily distinguished from L. arayai, L. dringi, L. fritinniens, L. gracilis, L. hamidi, L. heteropus, L. kajangensis, L. kecil, L. marmorata, L. maura, L. melanoleuca, L. picta, L. platycephala, L. sabahmontana and L. sola, all of which are lack of supra-axillary and ventrolateral glands (Dubois et al. 2010; Dehling and Matsui 2013; Matsui et al. 2014). As for the comparison referring to the body size, the new species shows a significantly larger body size (SVL, 28.7–29.7 mm in males) than 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), 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), L. serasanae (16.9 mm in one adult male), and Leptobrachella sp. 3 “baluensis” (15.0–16.0 mm in males).

From the remaining 48 known congeners in the genus Leptobrachella found north of the Isthmus of Kra (Table 5) with SVL 28.7–29.7 mm in males and SVL 30.5–33.5 mm in females, L. suiyangensis sp. nov. can be distinguished from the larger L. bourreti (42.0–45.0 mm in females), L. eos (33.1–34.7 mm in males and 40.7 in one female), L. lateralis (36.6 mm in females), L. nahangensis (40.8 mm in one male), L. nyx (37.0–41.0 mm in females), L. pyrrhops (30.8–34.3 mm in males), L. sungi (48.3–52.7 mm in males and 56.7–58.9 mm in females), L. tamdil (32.3 mm in males) 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 and 21.7–26.4 mm in females), L. ardens (21.3–24.7 mm in males and 24.5 mm in female), L. bidoupensis (18.5–25.4 mm in males and 29.2–29.4 mm in females), L. khasiorum (24.5–27.3 mm in males), L. laui (24.8–26.7 mm in males), L. maculosa (24.2–26.6 in males and 27.0 mm in one female), L. melica (19.5–22.7 mm in males), L. maoershanensis (29.1 mm in one female), L. petrops (23.6–27.6 mm in males), L. pluvialis (21.3–22.3 mm in males), L. purpurus (25.0–27.5 mm in males), L. rowleyae (23.1–28.1 mm in males and 27.0–27.8 mm in females), L. ventripunctata (25.5–28.0 mm in males), L. tengchongensis (23.9–26.0 mm in males and 28.8–28.9 mm in females) and L. yingjiangensis (25.7–27.6 mm in males).

Table 5.
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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; Hou et al. 2018; Wang et al. 2018).

ID Species Males SVL (mm) Black spots on flanks Toes webbing Fringes on toes Ventral coloration Dorsal skin texture
1 L. suiyangensis sp. nov. 28.7–29.7 Yes Rudimentary Narrow Yellowish creamy-white with marble texture chest and belly or with irregular light brown speckling Shagreen with small granules
2 L. aerea 25.1–28.9 No Rudimentary Wide Near immaculate creamy white, brown specking on margins Finely tuberculate
3 L. alpinus 24.0–26.4 Yes Rudimentary Wide in males Creamy-white with dark spots Relatively smooth, some with small warts
4 L. applebyi 19.6–22.3 Yes Rudimentary No Reddish brown with white speckling Smooth
5 L. ardens 21.3–24.7 Yes No No Reddish brown with white speckling Smooth- finely shagreened
6 L. bidoupensis 18.5–25.4 Yes Rudimentary Weak Reddish brown with white speckling Smooth
7 L. botsfordi 29.1–32.6 No Rudimentary Narrow Reddish brown with white speckling Shagreened
8 L. bourreti 28.0–36.2 Yes Rudimentary Weak Creamy white Relatively smooth, some with small warts
9 L. crocea 22.2–27.3 No Rudimentary No Bright orange Highly tuberculate
10 L. eos 33.1–34.7 No Rudimentary Wide Creamy white Shagreened
11 L. firthi 26.4–29.2 No Rudimentary Wide in males Creamy white Shagreened with fine tubercles
12 L. fuliginosa 28.2–30.0 Yes Rudimentary Weak White with brown dusting Nearly smooth, few tubercles
13 L. isos 23.7–27.9 No Rudimentary Wide in males Creamy white with white dusting on margins Mostly smooth, females more tuberculate
14 L. kalonensis 25.8–30.6 Yes No No Pale, speckled brown Smooth
15 L. khasiorum 24.5–27.3 Yes Rudimentary Wide Creamy white Isolated, scattered tubercles
16 L. lateralis 26.9–28.3 Yes Rudimentary No Creamy white Roughly granular
17 L. laui 24.8–26.7 Yes Rudimentary Wide Creamy white with dark brown dusting on margins Round granular tubercles
18 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
19 L. macrops 28.0–29.3 Yes Rudimentary No Greyish-violet with white speckling Roughly granular with larger tubercles
20 L. maculosa 24.2–26.6 Yes No No Brown, less white speckling Dorsum mostly smooth with numerous tiny tubercles
21 L. mangshanensis 22.2–27.8 Yes Rudimentary Weak White speckles on throat and belly Nearly smooth
22 L. maoershanensis 25.2–30.4 Yes Rudimentary Narrow Creamy white chest and belly with irregular black spots Longitudinal folds
23 L. melica 19.5–22.7 Yes Rudimentary No Reddish brown with white speckling Smooth
24 L. minima 25.7–31.4 Yes Rudimentary No Creamy white Smooth
25 L. nahangensis 40.8 Yes Rudimentary No Creamy white with light specking on throat and chest Smooth
26 L. nokrekensis 26.0–33.0 Yes Rudimentary unknown Creamy white Tubercles and longitudinal folds
27 L. nyx 26.7–32.6 Yes Rudimentary No Creamy white with white with brown margins Rounded tubercles
28 L. oshanensis 26.6–30.7 Yes No No Whitish with no markings or only small, light grey spots Smooth with few glandular ridges
29 L. pallida 24.5–27.7 No No No Reddish brown with white speckling Tuberculate
30 L. pelodytoides 27.5–32.3 Yes Wide Narrow Whitish Small, smooth warts
31 L. petrops 23.6–27.6 No No Narrow Immaculate creamy white Highly tuberculate
32 L. pluvialis 21.3–22.3 Yes Rudimentary No Dirty white with dark brown marbling Smooth, flattened tubercles on flanks
33 L. puhoatensis 24.2–28.1 Yes Rudimentary Narrow Reddish brown with white dusting Longitudinal skin ridges
34 L. purpurus 25.0–27.5 Yes Rudimentary Wide Dull white with indistinct grey dusting Shagreen with small tubercles
35 L. pyrrhops 30.8–34.3 Yes Rudimentary No Reddish brown with white speckling Slightly shagreened
36 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
37 L. sungi 48.3–52.7 No or small Wide Weak White Granular
38 L. shangsiensis 24.9–29.4 Yes Narrow Narrow Yellowish creamy-white with marble texture Smooth
39 L. tadungensis 23.3–28.2 Yes No No Reddish brown with white speckling Smooth
40 L. tamdil 32.3 Yes Wide Wide White Weakly tuberculate
41 L. tengchongensis 23.9–26.0 Yes Rudimentary Narrow White with dark brown blotches Shagreened with small tubercles
42 L. tuberosa 24.4–29.5 No Rudimentary No White with small grey spots/streaks Highly tuberculate
43 L. ventripunctata 25.5–28.0 Yes Rudimentary No Chest and belly with dark brown spots Longitudinal skin ridges
44 L. wuhuangmontis 25.6–30.0 Yes Rudimentary Narrow Greyish white mixed by tiny white and black dots Rough, scattered with dense conical tubercles
45 L. yingjiangensis 25.7–27.6 Yes Rudimentary Wide Creamy white with dark brown flecks on chest and margins Shagreened with small tubercles
46 L. yunkaiensis 25.9–29.3 Yes Rudimentary Wide Belly pink with distinct or indistinct speckling Shagreened with short skin ridges and raised warts
47 L. zhangyapingi 45.8–52.5 No Rudimentary Wide Creamy-white with white with brown margins Mostly smooth with distinct tubercles
48 L. bijie 29.0–30.4 Yes Rudimentary Narrow White with distinct nebulous greyish speckling on chest and ventrolateral flanks Shagreened and granular
49 L. purpuraventra 27.3–29.8 Yes Rudimentary Narrow Grey purple with distinct nebulous greyish speckling on chest and ventrolateral flanks Shagreened and granular

In having irregular, light-brown speckling on the flanks, the new species differs from L. aerea, L. botsfordi, L. crocea, L. firthi, L. isos, L. pallida, L. petrops and L. tuberosa, all of which lack distinct irregular, light-brown speckling on the flanks. By having rudimentary webbing on the toes, the new species differs from L. kalonensis, L. oshanensis, L. pallida, L. petrops, and L. tadungensis, all of which lack webbing on the toes; and from L. pelodytoides, which has wide webbing on the toes. By having narrow lateral fringes on toes, the new species differs from L. ardens, L. eos, L. firthi, L. isos, L. khasiorum, L. laui, L. liui, L. purpurus, L. tamdil, L. yingjiangensis and L. yunkaiensis, all of which have wide lateral fringes on the toes; from L. bidoupensis, L. bourreti, L. fuliginosa and L. mangshanensis, all of which have weak lateral fringes on the toes; and from L. crocea, L. kalonensis, L. lateralis, L. macrops, L. minima, L. nyx, L. oshanensis, L. pallida, L. pyrrhops, L. tadungensis, L. tuberosa, and L. ventripunctata, all of which lack lateral fringes on the toes. By having dorsal surface shagreened with small granules, and in lacking enlarge tubercles or warts, the new species differs from L. applebyi, L. bidoupensis, L. kalonensis, L. melica, L. minima, L. nahangensis, L. shangsiensis and L. tadungensis, all of which have the dorsum smooth, and L. alpina (dorsum smooth, some with small warts), L. fuliginosa (dorsum smooth with fine tubercles), L. laui (dorsum with round granular tubercle, lacking skin ridges), L. liui (dorsum with round tubercles), L. macrops (dorsum roughly granular with large tubercles), L. maoershanensis (dorsum smooth with small warts), L. nokrekensis (dorsum tubercles and longitudinal folds), L. pelodytoides (dorsum with small, smooth warts), L. puhoatensis (dorsum longitudinal skin ridges), L. tuberosa (dorsum highly tuberculate), L. yunkaiensis (dorsum with raised warts), L. wuhuangmontis (dorsum rough with conical tubercles), and L. bijie and L. purpuraventra (dorsum shagreened and granular). By the yellowish creamy-white with marbled chest and belly or with irregular light-brown speckling, the new species differs from L. alpinus, L. applebyi, L. ardens, L. bidoupensis, L. botsfordi and L. pyrrhops (ventral reddish brown with white speckling), L. aerea (ventral nearly immaculate creamy-white with brown specking on margins), L. bijie (ventral white with distinct nebulous greyish speckling on chest and ventrolateral flanks), L. crocea (ventral bright orange), L. khasiorum, L. nokrekensis and L. yingjiangensis (ventral creamy white), L. macrops (ventral greyish-violet with white speckling), L. puhoatensis (ventral reddish-brown with white dusting), L. purpurus (ventral dull white with indistinct grey dusting), L. purpuraventra (ventral grey-purple with distinct nebulous greyish speckling on the chest and ventrolateral flanks), L. tuberosa (ventral white with small grey spots and streaks), L. ventripunctata (chest and belly with large dark brown spots), L. wuhuangmontis (ventral greyish white), and L. yunkaiensis (belly pink with speckling). A comparative morphological data (selection) of Leptobrachella suiyangensis sp. nov. and 48 recognized Leptobrachella species occurring north of the Isthmus of Kra are listed in Table 5.

Discussion

Phylogenetic analyses based on mitochondrial DNA and nuclear DNA all suggested that the new species belongs to Leptobrachella but is separate from its congeners. Genetic distance of the 16S rRNA gene between the new species and its closely related species (L. bijie, L. purpuraventra, L. alpina and L. purpurus) was 4.71–6.27%, within the expected range of interspecific divergences in amphibians (Fouquet et al. 2007), and this genetic distance is much higher than between many sister species, of which, most species have been completely recognized as valid species. For example, in Leptobrachella, the p-distance = 2.35% between L. purpurus and L. alpina. Finally, a series of morphological characters were found to be different between the new species and its congenerson. All in all, multiple pieces of evidence support the validity of the new species.

The new species described in this study increases the number of species of Leptobrachella to 75, with 21 recorded from China (Fei et al. 2012; Sung et al. 2014; Yang et al. 2016, 2018; Yuan et al. 2017; Hou et al. 2018; Wang et al. 2018, 2019; Chen et al. 2018, 2019; Frost 2019). Before the description of the new species herein, only 12 species were recorded from southwest China. This highlights the underestimation of the species diversity of the genus Leptobrachella. Additional field surveys are required to understand the true diversity of amphibians in this genus, which will be useful for conservation strategies.

Studies on the taxonomy and phylogeny of the genus Leptobrachella were difficult to perform because of the morphological conservativeness of the species; in the field, many species appear to be very similar morphologically, and there exist sympatric species. This likely hinders our understanding of these cryptic species (Ohler et al. 2010; Sung et al. 2014; Yang et al. 2016, 2018; Yuan et al. 2017; Hou et al. 2018; Wang et al. 2018, 2019; Chen et al. 2019). The high species diversity and the degree of endemism indicated that the speciation pattern and sympatry mechanism of species in the genus Leptobrachella also need additional investigation.

Currently, to our knowledge, L. suiyangensis sp. nov. is restricted to rocky streams in bamboo forests. However, the type locality of L. suiyangensis sp. nov. has faced habitat loss and human disturbance, such as artificial grazing and herb collection, which could possibly threaten this species. Leptobrachella suiyangensis sp. nov. is range-restricted to Kuankuoshui National Nature Reserve, which borders the nearby Huoqiuba Nature Reserve and is in the eastern Ta-lou Mountains. These areas feature subtropical evergreen broad-leaved forest and evergreen deciduous broad-leaved mixed forest. Thus, it is likely that other populations of L. suiyangensis sp. nov. may be discovered in the Kuankuoshui Nature Reserve in the near future.

Acknowledgements

This project was supported by the key project of science-technology of basic condition platform granted by The Ministry of Science and Technology of the People’s Republic of China (Grant No. 2005DKA21402); The National Top Discipline Construction Project of Guizhou Province; Geography in Guizhou Normal University (85 2017 Qianjiao Keyan Fa); and The Project of Science and Technology Program of Guizhou Province, “Study of biological and ecological vales for Fanjingshan World Natural Heritage Nomination of Tongren City (3052 2015 Qiankehe SY)”. We thank Bo Song and Hongtao Cui for their help in sample collection. We thank Professor Ruliang Pan and LetPub (http://www.letpub.com) for its linguistic assistance during the preparation of this manuscript.

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