Biodiversity Data Journal : Taxonomy & Inventories
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Taxonomy & Inventories
Description of a new Asian Leaf Litter Toad of the genus Leptobrachella Smith, 1925 (Anura, Megophryidae) from southern Guizhou Province, China
expand article infoShize Li, Wei Li§, Yanlin Cheng, Jing Liu, Gang Wei|, Bin Wang§
‡ Moutai Institute, Guizhou, China
§ Chengdu Institute of Biology, the Chinese Academy of Sciences, Chengdu, China
| Guiyang College, Guizhou, China
Open Access

Abstract

Background

The Asian leaf litter toads of the genus Leptobrachella Smith, 1925 (Anura, Megophryidae) inhabit the forest floor and rocky streams in hilly evergreen forests and are widely distributed from southern China, west to north-eastern India and Myanmar, through mainland Indochina to Peninsular Malaysia and the Island of Borneo.

New information

A new species of the Asian leaf litter toad genus Leptobrachella from Guizhou Province, China is described. Molecular phylogenetic analyses, based on mitochondrial 16S rRNA and COI genes and nuclear RAG1 gene sequences indicated that the new species is genetically divergent from its congeners. The new species could be distinguished from its congeners by a combination of the following characters: (1) body of medium size in males (SVL 31.9 – 32.9 mm); (2) distinct black spots present on flanks; (3) toes rudimentarily webbed, with wide lateral fringes; (4) skin on dorsum shagreened with fine tiny granules and short ridges; (5) heels overlapped when thighs are positioned at right angles to the body; (6) tibia-tarsal articulation reaching interior corner of the eye.

A new species of the Asian leaf litter toad genus Leptobrachella from Guizhou Province, China is described. Molecular phylogenetic analyses, based on mitochondrial 16S rRNA and COI genes and nuclear RAG1 gene sequences indicated that the new species is genetically divergent from its congeners. The new species could be distinguished from its congeners by a combination of the following characters: (1) body of medium size in males (SVL 31.9 – 32.9 mm); (2) distinct black spots present on flanks; (3) toes rudimentarily webbed, with wide lateral fringes; (4) skin on dorsum shagreened with fine tiny granules and short ridges; (5) heels overlapped when thighs are positioned at right angles to the body; (6) tibia-tarsal articulation reaching interior corner of the eye.

Keywords

Taxonomy, molecular phylogenetic analyses, morphology, new species

Introduction

The Asian leaf litter toads of the genus Leptobrachella Smith, 1925 (Anura, Megophryidae) inhabit the forest floor and rocky streams in hilly evergreen forests and are widely distributed from southern China, west to north-eastern India and Myanmar, through mainland Indochina to Peninsular Malaysia and the Island of Borneo (Frost 2023). The species in the group had been classified into different genera, i.e. Paramegophrys Liu, 1964, Carpophrys Sichuan Biological Research Institute, 1977, Leptolalax Dubois, 1980, Lalax Delorme, Dubois, Grosjean & Ohler, 2006 and Lalos Dubois, Grosjean, Ohler, Adler & Zhao, 2010. Chen et al. (2018) suggested that the above genera were synonymised with Leptobrachella, based on large-scale molecular phylogenetic analyses. Currently, the genus contains 102 species, of which there are 40 species described in the last five years (Frost 2023). The species diversity in the genus was indicated to be much underestimated and many cryptic species have not been described till now (Chen et al. 2018), especially in some species group which were formerly recorded as widespread species, such as L. oshanensis complex (Nguyen et al. 2021, Shi et al. 2021) and more fieldwork and investigations should focus on them.

From 2022 to 2023, four specimens of Leptobrachella were collected from Dushan County, Guizhou Province, China. Morphologically, these specimens most closely resemble L. dong, but differ from L. dong and all other Leptobrachella from China and adjoining countries. To distinguish these specimens, we conducted phylogenetic analyses, based on mitochondrial DNA, nuclear DNA and morphological comparisons. All of the analyses consistently indicated that the specimens from Dushan County are a new taxon. Herein, we describe this taxon as a new species.

Materials and methods

Specimens

Four specimens of the undescribed species (Table 1 and Suppl. material 1) were collected from Dushan County, Guizhou Province, China (Fig. 1). After taking photographs, they were euthanized using isoflurane and then the specimens were fixed in 10% buffered formalin. Tissue samples were taken and preserved separately in 95% ethanol prior to fixation. Specimens were deposited in Moutai Institute (MT).

Table 1.

Information for samples used in molecular phylogenetic analyses in this study (/ = not available)

ID Species Locality Voucher number GenBank accession number
16S COI RAG1
1 Leptobrachella dushanensis sp. nov. Dushan County, Guizhou Province, China CIB DS20220409002 PP061389 PP061403 PP067958
2 Leptobrachella dushanensis sp. nov. Dushan County, Guizhou Province, China CIB DS20230310002 PP061391 PP061405 /
3 Leptobrachella dushanensis sp. nov. Dushan County, Guizhou Province, China CIB DS20230310001 PP061390 PP061404 /
4 Leptobrachella dushanensis sp. nov. Dushan County, Guizhou Province, China CIB DS20220409001 PP061388 PP061402 PP067957
5 L. graminicola Mount Pu Ta Leng, Lao Cai, Vietnam VNMN 010905 MZ224648 / /
6 L. graminicola Mount Pu Ta Leng, Lao Cai, Vietnam VNMN 010908 MZ224653 / /
7 L. graminicola Mount Pu Ta Leng, Lao Cai, Vietnam VNMN 010909 MZ224649 / /
8 L. graminicola Mount Pu Ta Leng, Lao Cai, Vietnam TOLO2022.518 PP061392 PP061406 /
9 L. graminicola Mount Pu Ta Leng, Lao Cai, Vietnam VNMN 010904 MZ224651 / /
10 L. graminicola Mount Pu Ta Leng, Lao Cai, Vietnam VNMN 010912 MZ224647 / /
11 L. graminicola Mount Pu Ta Leng, Lao Cai, Vietnam VNMN 010910 MZ224655 / /
12 L. yeae Changshouqiao, Mount Emei, Sichuan, China CIBEM1867 MT957005 / MT975978
13 L. yeae Linggongli, Mount Emei, Sichuan, China CIBEMLGL19052104 MT957006 / MT975979
14 L. yeae Mount Emei, Sichuan, China KIZ025778 KX811928 KX812166 /
15 L. dong Congjiang County, Guizhou Province, China CIB LB20220305005 OP764533 PP061396 /
16 L. dong Congjiang County, Guizhou Province, China CIB LB20220311002 OP764535 PP061398 OP776441
17 L. dong Congjiang County, Guizhou Province, China CIB LB20220306008 OP764534 PP061397 OP776439
18 L. dong Suining County, Hunan Province, China CIB ZNY2022001 OP764538 PP061399 OP776442
19 L. dong Suining County, Hunan Province, China CIB ZNY2022002 OP764539 PP061400 OP776443
20 L. dong Tongdao County, Hunan Province, China CIB WB2020277 OP764531 PP061401 OP776448
21 L. bourreti Sapa, Lao Cai, Vietnam 1999.566 KR827860 / /
22 L. bourreti Sapa, Lao Cai Province, Vietnam AMS R 177673 KR018124 / /
23 L. bourreti Ky Quan San, Lao Cai, Vietnam AMS R.188515 MZ208835 / /
24 L. bourreti Mount Pu Ta Leng, Lao Cai, Vietnam VNMN 010916 MZ209167 / /
25 L. bourreti Bat Xat District, Lao Cai, Vietnam VNMN 010073 PP061393 PP061407 /
26 L. bourreti Bat Xat District, Lao Cai, Vietnam ZMMU-A5636-02280 MH055872 / /
27 L. jinyunensis Mt.Jinyun, Beibei District, Chongqing, China CIB 119058 OQ024797 OQ024396 OQ031199
28 L. jinyunensis Mt.Jinyun, Beibei District, Chongqing, China CIB 119061 OQ024800 OQ024399 OQ031202
29 L. chishuiensis Chishui National Nature Reserve, Chishui, Guizhou, China CIBCS20190518047 MT117055 OQ024422 OQ031225
30 L. chishuiensis Chishui National Nature Reserve, Chishui, Guizhou, China CIBCS20190518049 MT117053 OQ024424 OQ031227
31 L. bijie Mt. Baima, Qixingguan, Bijie, Guizhou, China CIB 119068 OQ024807 OQ024406 OQ031208
32 L. bijie Mt. Baima, Qixingguan, Bijie, Guizhou, China CIB 119069 OQ024808 OQ024407 OQ031209
33 L. jinshaensis Lengshuihe Nature Reserve, Jinsha County, Guizhou, China CIBJS20200516005 OQ024816 OQ024417 OQ031220
34 L. jinshaensis Lengshuihe Nature Reserve, Jinsha County, Guizhou, China CIBJS20200516002 OQ024815 OQ024419 OQ031222
35 L. suiyangensis Huoqiuba Nature Reserve, Suiyang County, Guizhou, China GZNU20180606005 MK829649 / OL800396
36 L. suiyangensis Huoqiuba Nature Reserve, Suiyang County, Guizhou, China GZNU20180606006 MK829650 / OL800397
37 L. purpuraventra Mt. Baima, Qixingguan, Bijie, Guizhou, China CIB 119072 OQ024811 OQ024412 OQ031215
38 L. purpuraventra Mt. Baima, Qixingguan, Bijie, Guizhou, China CIB 119073 OQ024812 OQ024413 OQ031216
39 L. niveimontis Daxueshan Nature Reserve, Yunnan Province, China KIZ015734 MT302618 / OL800394
40 L. yunyangensis Qiyaoshan Nature Reserve, Yunyang County, Chongqing, China GZNU20210622001 OL800364 / OL800393
41 L. eos Long Nai Khao, Phongsali, Laos MNHN: 2004.0276 KR827862 KR087758 /
42 L. eos Boun Tay, Phongsaly, Laos NCSM 80551 MH055887 / /
43 L. purpura Yingjiang, Yunnan Province, China SYS a006530 MG520354 / /
44 L. alpina Caiyanghe, Yunnan Province, China KIZ049024 MH055867 / /
45 L. wulingensis Tianquanshan Forest Park, Zhangjiajie, Hunan Province, China CSUFT 177 MT530315 / /
46 L. dorsospina Yushe Forest Park, Shuicheng County, Guizhou Province, China SYS a004961 MW046194 / /
47 L. oshanensis Baoguosi, Mount Emei, Sichuan Province, China CIBEMS20190421BGS1 MT957023 / /
48 L. oshanensis Shengshuige, Mount Emei, Sichuan, China CIBEMS20190422SSG1-4 MT957025 / /
49 L. oshanensis Heilongjiang, Mount Emei, Sichuan, China CIBEMS20190422HLJ1-4 MT957027 / /
50 L. oshanensis Shengshuige, Mount Emei, Sichuan, China CIBEMS20190421SSG1-10 MT957031 / /
51 L. oshanensis Heilongjiang, Mount Emei, Sichuan, China CIBEMS20190422HLJ1-2 MT957033 / /
52 L. korifi Doi Inthanon,Thailand KUHE 19134 LC741033 / /
53 L. sinorensis Mae Hong Son,Thailand KUHE 19809 LC741034 / /
54 L. murphyi Doi Inthanon, Chiang Mai, Thailand KIZ034039 MZ710519 / /
55 L. tengchongensis Gaoligong Shan, Yunnan Province, China SYS a004598 KU589209 / /
56 L. khasiorum Khasi Hills, Meghalaya, India SDBDU 2009.329 KY022303 / /
57 L. tamdil Mizoram, India MZMU2224 MW665130 / /
58 L. yingjiangensis Yingjiang County, Yunnan Province, China SYS a006533 MG520350 / /
59 L. puhoatensis Pu Hu, Thanh Hoa, Vietnam VNMN:2016 A.23 KY849587 / /
60 L. petrops Ba Vi National Park, Ha Tay, Vietnam ROM 13483 MH055901 / /
61 L. namdongensis Thanh Hoa Province, Vietnam VNUF A.2017.37 MK965389 / /
62 L. liui Wuyi Shan City, Fujian Province, China SYS a001597 KM014547 / /
63 L. mangshanensis Mangshan, Hunan Province, China MSZTC201701 MG132196 / /
64 L. verrucosa Lianshan Bijiashan Nature Reserve, Guangdong, China GEP a059 OP279589 / /
65 L. shimentaina Shimentai Nature Reserve, Guangdong, China SYS a004712 MH055926 / /
66 L. flaviglandulosa Xiaoqiaogou Nature Reserve, Yunnan Province, China KIZ016072 MH055934 / /
67 L. yunkaiensis Dawuling Forest Station, Maoming City, Guangdong Province, China SYS a004663 MH605584 / /
68 L. maoershanensis Mao’er Shan, Guangxi Province, China KIZ07614 MH055927 / /
69 L. bashaensis Basha Nature Reserve, Congjiang County, Guizhou Province, China GIB196403 MW136294 / /
70 L. laui Shenzhen City, Guangdong Province, China SYS a002450 MH055904 / /
71 L. firthi Quang Nam Province, Vietnam AMS R 171714 JQ739203 / /
72 L. isos Gia Lai, Vietnam AMS R 176469 KT824767 / /
73 L. sungi Tam Dao, Vinh Phuc, Vietnam ROM 20236 MH055858 / /
74 L. zhangyapingi Chiang Mai, Thailand KIZ07258 MH055864 / /
75 L. pelodytoides Tam Dao, Vinh Phu, Vietnam ROM18282 EF397244 / /
76 L. ventripunctata Wenlong, Yunnan Province, China KIZ013621 MH055824 / /
77 L. feii Xiaoqiaogou Nature Reserve, Yunnan Province, China KIZ048894 MT302634 / /
78 L. aspera Huanglianshan Nature Reserve, Lyuchun, Yunnan, China SYS a007743 MW046199 / /
79 L. aerea Vilabuly, Savannakhet, Laos NCSM 76038 MH055809 / /
80 L. minima Doi Phu Fa, Nan, Thailand KIZ024317 MH055852 / /
81 L. nyx Ha Giang, Vietnam ROM 36692 MH055816 / /
82 L. wuhuangmontis Pubei County, Guangxi Province, China SYS a003485 MH605577 / /
83 L. shiwandashanensis Fangcheng City, Guangxi Province, China NNU202103146 MZ326691 / /
84 L. wumingensis Wuming County, Guangxi, China NNU 00283 OM935575 / /
85 L. pluvialis Fansipan, Lao Cai, Vietnam ROM 30685 MH055843 / /
86 L. shangsiensis Guangxi Province, China NHMG1401032 MK095460 / /
87 L. nahangensis Na Hang Nature Reserve, Tuyen Quang, Vietnam ROM 7035 MH055853 / /
88 L. damingshanensis Guangxi Province., China NNU202103281 MZ145229 / /
89 L. kalonensis Binh Thuan Province, Vietnam AMNH A191762 KR018115 / /
90 L. bidoupensis Bidoup-Nui Ba National Park, Lam Dong, Vietnam ZMMU-A-4797-01454 MH055945 / /
91 L. pallida Lam Dong, Vietnam UNS00511 KU530190 / /
92 L. tadungensis Dak Nong Province, Vietnam UNS00515 KR018121 / /
93 L. maculosa Ninh Thuan Province, Vietnam AMS R 177660 KR018119 / /
94 L. pyrrhops Loc Bac, Lam Dong, Vietnam ZMMU-A-4873-00158 MH055950 / /
95 L. macrops Phu Yen, Vietnam ZMMU-A5823 MG787993 / /
96 L. rowleyae Da Nang City, Vietnam ITBCZ2783 MG682552 / /
97 L. applebyi Phong Dien Nature Reserve, Thua Thien-Hue, Vietnam KIZ010701 MH055947 / /
98 L. melica Cambodia, Ratanakiri MVZ258198 HM133600 / /
99 L. ardens Kon Ka Kinh National Park, Gia Lai, Vietnam ZMMU-NAP-06099 MH055949 / /
100 L. crocea Thua Thien-Hue, Vietnam ZMMU-NAP-02274 MH055955 / /
101 L. tuberosa Kon Ka Kinh National Park, Gia Lai, Vietnam ZMMU-NAP-02275 MH055959 / /
102 L. botsfordi Fansipan, Lao Cai, Vietnam AMS R 176540 MH055952 / /
103 L. fuliginosa Phetchaburi, Thailand KUHE 20197 LC201988 / /
104 L. melanoleuca Kapoe, Ranong, Thailand KIZ018031 MH055967 / /
105 L. neangi Veal Veng District, Pursat Province, Cambodia CBC 1624 MT644613 / /
106 L. brevicrus Gunung Mulu National Park, Sarawak, Malaysia UNIMAS 8957 KJ831303 / /
107 L. itiokai Mulu National Park, Sarawak, Malaysia KUHE:55897 LC137805 / /
108 L. parva Mulu National Park, Sarawak, Malaysia KUHE:55308 LC056791 / /
109 L. baluensis Tambunan, Sabah, Borneo, Malaysia SP 21604 LC056792 / /
110 L. mjobergi Gading NP, Sarawak, Borneo, Malaysia KUHE:47872 LC056787 / /
111 L. juliandringi Mulu NP, Sarawak, Borneo, Malaysia KUHE 55333 LC056780 / /
112 L. sabahmontana Borneo, Malaysia BORNEENSIS 12632 AB847551 / /
113 L. dringi Gunung Mulu, Malaysia KUHE:55610 AB847553 / /
114 L. picta Borneo, Malaysia UNIMAS 8705 KJ831295 / /
115 L. fritinniens Danum Valley Field Center, Sabah, Malaysia FMNH 244800 MH055971 / /
116 L. arayai Liwagu, Kinabalu, Malaysia BORNEEISIS 22931 AB847558 / /
117 L. hamidi Bukit Lanjan, Selangor, Malaysia KUHE17545 AB969286 / /
118 L. marmorata Borneo, Malaysia KUHE53227 AB969289 / /
119 L. gracilis Bukit Kana, Sarawak, Malaysia FMNH 273682 MH055972 / /
120 L. maura Borneo, Malaysia SP21450 AB847559 / /
121 L. heteropus Larut, Perak, Malaysia KUHE15487 AB530453 / /
122 L. sola Gunung Stong, Kelantan, Malaysia KU RMB20973 MH055973 / /
123 L. kecil Cameron, Malaysia KUHE 52440 LC202004 / /
124 L. kajangensis Tioman, Malaysia LSUHC 4431 LC202001 / /
125 Megophrys glandulosa Yunnan Province, China KIZ048439 KX811762 KX812165 /
126 Leptobrachium huashen Yunnan Province, China KIZ049025 KX811931 KX812075 /
Figure 1.  

Sampling localities of Leptobrachella dushanensis sp. nov., L. bourreti, L. dong and L. graminicola. 1 Dushan County, the type locality of Leptobrachella dushanensis sp. nov.; 2-4 represent the sampling localities of L. dong; 2 Congjiang County, Guizhou Province; 3 Tongdao County, Hunan Province; 4 Suining County, Hunan Province, China; 5 represent the sampling locality of L. bourreti from Bat Xat District, Lao Cai, Vietnam; 6 the sampling locality of L. graminicola from Mount Pu Ta Leng, Lao Cai, Vietnam.

Molecular phylogenetic analyses

Total DNA was extracted using a standard phenol-chloroform extraction protocol (Sambrook et al. 1989). The sequences of mitochondrial 16S rRNA (16S) and cytochrome oxidase subunit I (COI) genes and nuclear gene recombination activating gene 1 (RAG1) were amplified. For 16S, the primers P7 (5’-CGCCTGTTTACCAAAAACAT-3’) and P8 (5’-CCGGTCTGAACTCAGATCACGT-3’) were used following Simon et al. (1994), for COI, Chmf4 (5’-TYTCWACWAAYCAYAAAGAYATCGG-3’) and Chmr4 (5’-ACYTCRGGRTGRCCRAARAATCA-3’) were used following Che et al. (2012) and for RAG1, RAG1-F (AGCTGCAGYCARTACCAYAARATGTA) and RAG1-R (GCAAAGTTTCCGTTCATTCTCAT) were used following Mauro et al. (2004). The PCR amplification procedures are as follows: an initial denaturing step at 95°C for 4 min; 36 cycles of denaturing at 95°C for 30 s, annealing at 51°C (for 16S)/47°C (for COI)/57°C (for RAG1) for 30 s and extending at 72°C for 70 s. The fragments were sequenced on an ABI Prism 3730 automated DNA sequencer at Chengdu TSING KE Biological Technology Co. Ltd (Chengdu, China). New sequences were deposited in GenBank (for GenBank accession numbers, see Table 1).

For phylogenetic analyses, we downloaded the corresponding gene sequences for all related species in the genus Leptobrachella from GenBank, based on previous studies (Chen et al. 2018, Shi et al. 2021). Corresponding sequences of one Leptobrachium huashen Fei and Ye (2005) and one Megophrys glandulosa (Fei et al. 1990) were downloaded and used as outgroups.

Sequences were assembled and aligned using the Clustalw module in BioEdit v. 7.0.9.0 (Hall 1999) with default settings. Alignments were checked by eye and revised manually, if necessary. Phylogenetic analyses of mitochondrial DNA, the datasets of 16S and COI gene sequences were prepared using Maximum Likelihood (ML) and Bayesian Inference (BI) methods, implemented in PhyML v. 3.0 (Guindon et al. 2010) and MrBayes v. 3.12 (Ronquist and Huelsenbeck 2003), respectively. The best-fit nucleotide substitution models for the sequence super-matrices were selected in PartitionFinder 2.1.1 (Lanfear et al. 2012) using the Bayesian Information Criterion (BIC). As a result, the analysis suggested that the best model for 16S was GTR + R and, for the COI gene, it was TN93 + G + I. For the ML tree, branch supports were drawn from 10,000 non-parametric bootstrap replicates. In BI analyses, the parameters for each partition were unlinked and branch lengths were allowed to vary proportionately across partitions. Two runs each with four Markov chains were simultaneously run for 60 million generations with sampling every 1,000 generations. The first 25% trees were removed as the “burn-in” stage followed by calculations of Bayesian posterior probabilities and the 50% majority-rule consensus of the post burn-in trees sampled at stationarity. To detect the haplotype relationships and genetic isolation between the undescribed species and its related species on nuclear DNA, a haplotype network, based on RAG1 gene sequences, was constructed using the maximum parsimony method in TCS v.1.21 (Clement et al. 2000). Finally, genetic distance between Leptobrachella species, based on the uncorrected p-distance model, was estimated on 16S and COI genes using MEGA v. 6.06 (Tamura et al. 2013), respectively.

Morphological comparisons

Morphologically, these specimens most closely resemble L. dong. To explore the morphological differences between the new taxon and L. dong, four specimens of the new taxon and 15 specimens of L. dong containing four specimens from Guizhou Province and 11 specimens from Hunan Province were measured. The terminology and methods followed Fei et al. (2005)and Liu et al. (2023). Measurements were made to the nearest 0.1 mm (Watters et al. 2016) with digital calipers. Fourteen morphometric characters of adult specimens were measured: eye diameter (ED, distance from the anterior corner to the posterior corner of the eye); foot length (FL, distance from tarsus to the tip of the fourth toe); head length (HDL, distance from the tip of the snout to the articulation of jaw); head width (HDW, greatest width between the left and right articulations of jaw); hind-limb length (HLL, distance from tip of fourth toe to vent); internasal distance (IND, minimum distance between the inner margins of the external nares); interorbital distance (IOD, minimum distance between the inner edges of the upper eyelids); length of lower arm and hand (LAL, distance from the elbow to the distal end of the Finger IV); snout length (SL, distance from the tip of the snout to the anterior corner of the eye); snout-vent lengt (SVL, distance from the tip of the snout to the vent); maximal tympanum diameter (TD); tibia length (TL, distance from knee to tarsus); tibia width (TW, the widest length of the tibia); upper eyelid width (UEW, greatest width of the upper eyelid margins measured perpendicular to the anterior-posterior axis).

In order to reduce the impact of allometry, the correct value from the ratio of each measurement to SVL was calculated and then log-transformed for the following morphometric analyses. One-way analysis of variance (ANOVA) was used to test the significance of differences on morphometric characters between the undescribed species and L. dong from different populations in males. The significance level was set at 0.05. To show the spatial distribution of different species on the morphometric characters, principal component analyses (PCA) were performed. All statistical analyses were performed using SPSS 21.0 (SPSS, Inc., Chicago, IL, USA).

The undescribed taxon was also compared with all other congeners of Leptobrachella, based on morphological characters. Comparative morphological data were obtained from literature (Table 2).

Table 2.

References for morphological characters for congeners of the genus Leptobrachella.

ID Species References
1 L. aerea (Rowley, Stuart, Richards, Phimmachak & Sivongxay, 2010) Rowley et al. 2010a
2 L. alpina (Fei, Ye & Li, 1990) Fei et al. 1990
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. aspera Wang, Lyu, Qi & Wang, 2020 Wang et al. 2020
7 L. baluensis Smith, 1931 Smith 1931
8 L. bashaensis Lyu, Dai, Wei, He, Yuan, Shi, Zhou, Ran, Kuang, Guo, Wei & Yuan, 2020 Lyu et al. 2020
9 L. bidoupensis (Rowley, Le, Tran & Hoang, 2011) Rowley et al. 2011
10 L. bijie Wang, Li, Li, Chen & Wang, 2019 Wang et al. 2019
11 L. bondangensis Eto, Matsui, Hamidy, Munir & Isk &ar, 2018 Eto et al. 2018
12 L. botsfordi (Rowley, Dau & Nguyen, 2013) Rowley et al. 2013
13 L. bourreti (Dubois, 1983) Dubois 1983
14 L. brevicrus Dring, 1983 Dring 1983, Eto et al. 2015
15 L. chishuiensis Li, Liu, Wei & Wang, 2020 Li et al. 2020
16 L. crocea (Rowley, Hoang, Le, Dau & Cao, 2010) Rowley et al. 2010b
17 L. damingshanensis Chen, Yu, Cheng, Meng, Wei, Zhou, Lu, 2021 Chen et al. 2021b
18 L. dorsospina Wang, Lyu, Qi & Wang, 2020 Wang et al. 2020
19 L. dringi (Dubois, 1987) Dubois 1987
20 L. dong Liu, Shi, Li, Zhang, Xiang, Wei, and Wang, 2023 Liu et al. 2023
21 L. eos (Ohler, Wollenberg, Grosjean, Hendrix, Vences, Ziegler & Dubois, 2011) Ohler et al. 2011
22 L. feii Chen, Yuan & Che, 2020 Chen et al. 2020
23 L. firthi (Rowley, Hoang, Dau, Le & Cao, 2012) Rowley et al. 2012
24 L. flaviglandulosa Chen, Wang & Che, 2020 Chen et al. 2020
25 L. fritinniens (Dehling & Matsui, 2013) Dehling and Matsui 2013
26 L. fuliginosa (Matsui, 2006) Matsui 2006
27 L. fusca Eto, Matsui, Hamidy, Munir & Isk &ar, 2018 Eto et al. 2018
28 L. gracilis (Günther, 1872) Dehling 2012a
29 L. graminicola Nguyen, Tapley, Nguyen, Luong & Rowley, 2021 Nguyen et al. 2021
30 L. hamidi (Matsui, 1997) Matsui 1997
31 L. heteropus (Boulenger, 1900) Boulenger 1900
32 L. isos (Rowley, Stuart, Neang, Hoang, Dau, Nguyen & Emmett, 2015) Rowley et al. 2015
33 L. itiokai Eto, Matsui & Nishikawa, 2016 Eto et al. 2016
34 L. jinshaensis Cheng, Shi, Li, Liu, Li & Wang, 2021 Cheng et al. 2021
35 L. jinyunensis Shi, Shen, Wang, Jiang & Wang, 2023 Shi et al. 2023
36 L. juliandringi Eto, Matsui & Nishikawa, 2015 Eto et al. 2015
37 L. kajangensis (Grismer, Grismer & Youmans, 2004) Grismer et al. 2004
38 L. kalonensis (Rowley, Tran, Le, Dau, Peloso, Nguyen, Hoang, Nguyen & Ziegler, 2016) Rowley et al. 2016
39 L. kecil (Matsui, Belabut, Ahmad & Yong, 2009) Matsui et al. 2009
40 L. khasiorum (Das, Tron, Rangad & Hooroo, 2010) Das et al. 2010
41 L. korifi Matsui, Panha & Eto, 2023 Matsui et al. 2023
42 L. lateralis (Anderson, 1871) Anderson 1871, Humtsoe et al. 2008
43 L. laui (Sung, Yang & Wang, 2014) Sung et al. 2014
44 L. liui (Fei & Ye, 1990) Fei et al. 1990, Sung et al. 2014
45 L. macrops (Duong, Do, Ngo, Nguyen & Poyarkov, 2018) Duong et al. 2018
46 L. maculosa (Rowley, Tran, Le, Dau, Peloso, Nguyen, Hoang, Nguyen & Ziegler, 2016) Rowley et al. 2016
47 L. mangshanensis (Hou, Zhang, Hu, Li, Shi, Chen, Mo & Wang, 2018) Hou et al. 2018
48 L. maoershanensis (Yuan, Sun, Chen, Rowley & Che, 2017) Yuan et al. 2017
49 L. marmorata (Matsui, Zainudin & Nishikawa, 2014) Matsui et al. 2014b
50 L. maura (Inger, Lakim, Biun & Yambun, 1997) Inger et al. 1997
51 L. melanoleuca (Matsui, 2006) Matsui 2006
52 L. melica (Rowley, Stuart, Neang & Emmett, 2010) Rowley et al. 2010a
53 L. minima (Taylor, 1962) Ohler et al. 2011, Taylor 1962
54 L. mjobergi Smith, 1925 Smith 1925
55 L. murphyi Chen, Suwannapoom, Wu, Poyarkov, Xu, Pawangkhanant & Che, 2021 Chen et al. 2021
56 L. nahangensis (Lathrop, Murphy, Orlov & Ho, 1998) Lathrop et al. 1998
57 L. namdongensis Hoang, Nguyen, Luu, Nguyen & Jiang, 2019 Hoang et al. 2019
58 L. natunae (Günther, 1895) Günther 1895
59 L. neangi Stuart & Rowley, 2020 Stuart and Rowley 2020
60 L. niveimontis Chen, Poyarkov, Yuan & Che, 2020 Chen et al. 2020
61 L. nokrekensis (Mathew & Sen, 2010) Mathew and Sen 2010
62 L. nyx (Ohler, Wollenberg, Grosjean, Hendrix, Vences, Ziegler & Dubois, 2011) Ohler et al. 2011
63 L. oshanensis (Liu, 1950) Liu 1950, Shi et al. 2023
64 L. pallida (Rowley, Tran, Le, Dau, Peloso, Nguyen, Hoang, Nguyen & Rowley et al. 2016
65 L. palmata Inger & Stuebing, 1992 Inger and Stuebing 1992
66 L. parva Dring, 1983 Dring 1983
67 L. pelodytoides (Boulenger, 1893) Boulenger 1893, Ohler et al. 2011
68 L. petrops (Rowley, Dau, Hoang, Le, Cutajar & Nguyen, 2017) Rowley et al. 2017b
69

L. phiadenensis Luong, Hoang, Pham, Ziegler, and Nguyen, 2023

Luong et al. 2023
70

L. phiaoacensisLuong, Hoang, Pham, Ziegler, and Nguyen, 2023

Luong et al. 2023
71 L. picta (Malkmus, 1992) Malkmus 1992
72 L. pingbianensis (Rao, Hui, Zhu & Ma, 2022 "2020") Zhu and Rao 2020
73 L. platycephala (Dehling, 2012) Dehling 2012b
74 L. pluvialis (Ohler, Marquis, Swan & Grosjean, 2000) Ohler et al. 2000
75 L. puhoatensis (Rowley, Dau & Cao, 2017) Rowley et al. 2017a
76 L. purpuraventra Wang, Li, Li, Chen & Wang, 2019 Wang et al. 2019
77 L. purpurus (Yang, Zeng & Wang, 2018) Yang et al. 2018
78 L. pyrrhops (Poyarkov, Rowley, Gogoleva, Vassilieva, Galoyan & Orlov, 2015) Poyarkov et al. 2015
79 L. rowleyae (Nguyen, Poyarkov, Le, Vo, Ninh, Duong, Murphy & Sang, 2018) Nguyen et al. 2018
80 L. sabahmontana (Matsui, Nishikawa & Yambun, 2014) Matsui et al. 2014a
81 L. serasanae Dring, 1983 Dring 1983
82 L. shangsiensis Chen, Liao, Zhou & Mo, 2019 Chen et al. 2019
83 L. shiwanshanensis Chen, Peng, Pan, Liao, Liu & Huang, 2021 Chen et al. 2021a
84 L. shimentaina Wang, Lyu & Wang, 2022 Wang et al. 2022
85 L. sinorensis Matsui, Panha & Eto, 2023 Matsui et al. 2023
86 L. sola (Matsui, 2006) Matsui 2006
87 L. suiyangensis Luo, Xiao, Gao & Zhou, 2020 Luo et al. 2020
88 L. sungi (Lathrop, Murphy, Orlov & Ho, 1998) Lathrop et al. 1998
89 L. tadungensis (Rowley, Tran, Le, Dau, Peloso, Nguyen, Hoang, Nguyen & Ziegler, 2016) Rowley et al. 2016
90 L. tamdil (Sengupta, Sailo, Lalremsanga, Das & Das, 2010) Sengupta et al. 2010
91 L. tengchongensis (Yang, Wang, Chen & Rao, 2016) Yang et al. 2018
92 L. tuberosa (Inger, Orlov & Darevsky, 1999) Inger et al. 1999
93 L. ventripunctata (Fei, Ye & Li, 1990) Fei et al. 1990
94 L. verrucosa Wang, Zeng, Lin & Li, 2022 Lin et al. 2022
95 L. wuhuangmontis Wang, Yang & Wang, 2018 Wang et al. 2018
96 L. wulingensis Qian, Xiao, Cao, Xiao & Yang, 2020 Qian et al. 2020
97 L. wumingensis Chen, Peng, Li, and Yu, 2023 Chen et al. 2023
98 L. yeae Shi, Hou, Song, Jiang & Wang, 2021 Shi et al. 2021
99 L. yingjiangensis (Yang, Zeng & Wang, 2018) Yang et al. 2018
100 L. yunyangensis Luo, Deng & Zhou, 2022 Luo et al. 2022
101 L. yunkaiensis Wang, Li, Lyu & Wang, 2018 Wang et al. 2018
102 L. zhangyapingi (Jiang, Yan, Suwannapoom, Chomdej & Che, 2013) Jiang et al. 2013

Data resources

All the sequences in this study were retrieved from GenBank and the accession numbers of the newly-determined sequences in this study are shown in Table 1.

Taxon treatment

Leptobrachella dushanensis Li, Li, Cheng, Liu, Wei, Wang sp. nov.

Materials   Download as CSV 
Holotype:
  1. scientificName:
    Leptobrachella dushanensis
    ; kingdom:
    Animalia
    ; phylum:
    Chordata
    ; class:
    Amphibia
    ; order:
    Anura
    ; family:
    Megophryidae
    ; genus:
    Leptobrachella
    ; country:
    China
    ; stateProvince:
    Guizhou
    ; county:
    Dushan
    ; decimalLatitude:
    25.961719
    ; decimalLongitude:
    107.649194
    ; eventDate:
    2022-04-09
    ; sex:
    male
    ; lifeStage:
    adult
    ; recordedBy:
    Jing Liu
    ; identifiedBy:
    Shize Li
    ; institutionID:
    MT DS20220409002
    ; collectionID:
    DS20220409002
    ; occurrenceID:
    B8B9E666-2D12-5015-8398-05CB6F244FCD
Paratype:
  1. scientificName:
    Leptobrachella dushanensis
    ; kingdom:
    Animalia
    ; phylum:
    Chordata
    ; class:
    Amphibia
    ; order:
    Anura
    ; family:
    Megophryidae
    ; genus:
    Leptobrachella
    ; country:
    China
    ; stateProvince:
    Guizhou
    ; county:
    Dushan
    ; decimalLatitude:
    25.961719
    ; decimalLongitude:
    107.649194
    ; sex:
    3 males
    ; lifeStage:
    adult
    ; recordedBy:
    Jing Liu and Wei Li
    ; identifiedBy:
    Shize Li
    ; type:
    Event
    ; institutionID:
    MT DS20220409001, MT DS20230310001 and MT DS20230310002
    ; collectionID:
    DS20220409001, DS20230310001 and DS20230310002
    ; occurrenceID:
    83945396-6D45-5955-875D-54024A94F66A

Diagnosis

Leptobrachella dushanensis sp. nov. is assigned to the genus Leptobrachella, based on molecular data and the following morphological characters: medium size, rounded finger tips, the presence of two elevated inner palmar tubercle not continuous to the thumb, presence of macroglands on body (including supra-axillary, pectoral and femoral glands), vomerine teeth absent, tubercles on eyelids and anterior tip of snout with vertical white bar (Dubois 1983; Fei et al. 2009).

Leptobrachella dushanensis sp. nov. could be distinguished from its congeners by a combination of the following characters: body of medium size in males (SVL 31.9 – 32.9 mm); distinct black spots present on flanks; toes rudimentarily webbed, with wide lateral fringes; skin on dorsum shagreened with fine tiny granules and short ridges; heels overlapped when thighs are positioned at right angles to the body; tibia-tarsal articulation reaching interior corner of the eye.

Description of holotype: 

Adult male. SVL in 33.2 mm. Head length slightly wider than head width (HDL/HDW = 1.04); snout sharply rounded in dorsal view, projecting slightly beyond margin of the lower jaw; nostril closer to snout than eye; loreal region oblique; canthus rostralis indistinct; eyes large and convex (ED/HDL = 0.37), slightly shorter than snout length (ED/SL = 0.93), pupil vertical; tympanum distinct, rounded, tympanum diameter smaller than eye (TD/ED = 0.43), upper margin of tympanum in contact with supratympanic ridge; vomerine teeth absent; tongue notched behind; supratympanic ridge distinct, extending from posterior corner of eye to supra-axillary gland.

Fore-limb relatively long (LAL / SVL = 0.45), fingers long and slender (ML/SVL = 0.25), without webbing, lateral fringes on fingers narrow; relative finger lengths II < I < IV < III; tips of fingers rounded and slightly swollen; subarticular tubercles absent on fingers, inner metacarpal tubercle large and rounded, separated from the smaller, round outer metacarpal; supra-axillary glands oval.

Hind-limb relatively long (HLL/SVL = 1.56), heels overlapping when the tibiae perpendicular to the body axis; tibio-tarsal articulation of adpressed limb reaching interior corner of the eye, tibia length about half of snout-vent length (TL/SVL = 0.49); relative toe length: I < II < III < V < IV; toe tips rounded and slightly swollen; rudimentary webbing present between all five toes; wide lateral fringes present on all toes; subarticular tubercles indistinct on the base of toe; inner metatarsal tubercle oval and distinct, outer metatarsal tubercle absent.

Skin on dorsum shagreened with fine tiny granules and short ridges; supra-axillary glands long, oval, close to the armpit; pectoral gland indistinct; round femoral glands present and protuberant on rear of thigh, closer to knee than to vent; femoral adipose glands distinct, attached to inner side of skin on posterior ventral surface of thigh; ventral skin smooth; ventrolateral glands distinctly visible and raised, forming an incomplete line.

Colouration of holotype in life: 

In life, dorsal surface of head and trunk earth brown, with a distinct reverse-triangle taupe markings between eyes connecting to a taupe W-shaped marking between axillae that are fringed with greyish-white colour; very distinct, light brown markings between the nostrils; bicoloured iris, with the upper 1/3 of the iris being copper-orange and the lower 2/3 a light silvery-grey and a dark blotch under the eye; sparse, small, light brown granules and small dark brown patches present on the dorsum of the limbs; elbow to upper arm distinctly greyish-brown in colour on the dorsum; three transverse black bars present on dorsal surface of lower arm; distinct dark blotches on flanks from groin to axilla, longitudinally in two rows; ventral surfaces light coloured, throat and ventral arms pinkish, chest and belly cream-white and flanks of ventral with several granules and brown spots; ventral hind-limbs pinkish with sparse white glands (Fig. 2).

Figure 2.  

Photos of the holotype CIB DS20220409002 Leptobrachella dushanensis sp. nov. in life. A dorsolateral view; B ventral view; C dorsal view; D ventral view of hand E ventral view of foot.

Preserved holotype colouration: 

Dorsum of body and limbs fade to brown; transverse bars on limbs become more distinct. Ventral surface of body and limbs fade to cream-white. Supra-axillary, femoral and pectoral glands fade to cream-yellow (Fig. 3).

Figure 3.  

The holotype specimen MT DS20220409002 Leptobrachella dushanensis sp. nov. A dorsal view; B ventral view; C ventral view of hand; D ventral view of foot.

Variations: 

Measurements of adult specimens are presented in Suppl. material 1 and Table 3, respectively. In MT DS20230310001, dorsal surface of head and trunk reddish-brown (Fig. 4A) and femoral adipose glands more obvious, ventral skin of thigh smooth (Fig. 4B); in MT DS20230310002, the black bars on dorsum more obvious (Fig. 4C) and glands on ventral surface of hind-limbs more dense (Fig. 4D).

Table 3.

Measurements of Leptobrachella dushanensis sp. nov and L. dong from Guizhou and Hunan populations. Units in mm. In addition, the results of one-way ANOVA with p-values for morphometric comparisons between Leptobrachella dushanensis sp. nov. and L. dong from Guizhou and Hunan populations (* p < 0.05, ** p < 0.01) are given. See abbreviations for characters in the Materials and Methods section.

Character Leptobrachella dushanensis sp.nov. (n = 4) L. dong (n = 4) Guizhou L. dong (n = 11) Hunan p-value
Ranging Mean ± SD Ranging Mean ± SD Ranging Mean ± SD Guizhou Hunan
SVL 31.9 – 32.9 32.3 ± 0.4 29.5 – 31.2 30.4 ± 0.86 29.2 – 34.2 31.1 ± 1.4 0.006** 0.169
HDL 10.5 – 10.8 10.6 ± 0.1 9.6 – 10.8 10.1 ± 0.5 10.0 – 11.7 10.7 ± 0.5 0.694 0.025*
HDW 10.0 – 10.4 10.1 ± 0.2 9.8 – 10.9 10.5 ± 0.5 10.5 – 12.2 11.2 ± 0.6 0.001** 0.000**
SL 4.2 – 4.4 4.2 ± 0.1 4.2 – 4.9 4.6 ± 0.3 4.0 – 5.0 4.4 ± 0.3 0.027* 0.044*
IND 2.6 – 2.7 2.6 ± 0.1 3.5 – 4.4 3.8 ± 0.4 2.8 – 3.6 3.1 ± 0.3 0.000** 0.000**
IOD 2.8 – 3.1 2.9 ± 0.1 2.3 – 3.2 2.8 ± 0.4 2.8 – 3.9 3.4 ± 0.4 0.788 0.001**
UEW 2.9 – 3.1 3.0 ± 0.1 2.8 – 3.4 3.1 ± 0.3 2.6 – 3.1 2.9 ± 0.2 0.094 0.946
ED 3.8 – 4.0 3.9 ± 0.1 3.9 – 4.4 4.1 ± 0.2 3.6 – 4.4 4.2 ± 0.2 0.039* 0.006**
TYD 1.6 – 1.8 1.7 ± 0.1 1.9 – 2.2 2.1 ± 0.1 1.5 – 1.7 1.6 ± 0.1 0.001** 0.705
LAL 14.4 – 14.5 14.5 ± 0.1 13.5 – 14.2 13.9 ± 0.3 14.3 – 15.5 14.8 ± 0.4 0.127 0.011*
ML 8.0 – 8.5 8.1 ± 0.2 7.3 – 8.3 7.9 ± 0.4 7.2 – 8.6 7.9 ± 0.4 0.216 0.750
TL 15.9 – 16.0 15.9 ± 0.1 14.5 – 14.7 14.6 ± 0.1 14.5 – 16.4 15.5 ± 0.6 0.079 0.749
FL 14.8 – 15.0 14.9 ± 0.1 13.5 – 15 14.2 ± 0.6 13.5 – 15.5 15.0 ± 0.6 0.625 0.408
HLL 50.0 – 50.5 50.3 ± 0.3 41.3 – 44.2 43.3 ± 1.3 46.2 – 51.6 49.1 ± 1.6 0.018* 0.564
Figure 4.  

Colour variation in Leptobrachella dushanensis sp. nov. A dorsal view of the male specimen MT DS20230310001; B ventral view of the male specimen MT DS20230310001; C dorsal view of the male specimen MT DS20230310002; D ventral view of the female specimen MT DS20230310002.

Comparisons: 

Compared with the 26 known congeners occurring south of the Isthmus of Kra, Leptobrachella dushanensis sp. nov. could be distinguished from them by several characters. By having supra-axillary and ventrolateral glands, the new species differs 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 (vs. absent in the latter); by having rounded fingertips and moderate body size (31.9 – 32.9 mm in four adult males), the new species differs from the following species with pointed fingertips and smaller body size: L. baluensis (14.9–15.9 mm in males), L. bondangensis (17.8 mm in male), L. brevicrus (17.1–17.8 mm in males), L. fusca (16.3 mm in male), L. itiokai (15.2–16.7 mm in males), L. juliandringi (17.0–17.2 mm in males), L. mjobergi (15.7–19.0 mm in males), L. natunae (17.6 mm in one adult male), L. palmata (14.4–16.8 mm in males), L. parva (15.0–16.9 mm in males) and L. serasanae (16.9 mm in female).

Leptobrachella dushanensis sp. nov. could also be identified from 76 known Leptobrachella species occurring north of the Isthmus of Kra by some characters (Suppl. material 2).

By having medium size of body (SVL 31.9–32.9 mm in males), Leptobrachella dushanensis sp. nov. differs from the the smaller in males L. aerea (25.1–28.9 mm), L. alpina (24.0–26.4 mm), L. applebyi (19.6–22.3 mm), L. ardens (21.3–24.7 mm), L. aspera (22.4 mm), L. bashaensis (22.9–25.6 mm), L. bidoupensis (23.6–24.6), L. bijie (29.0–30.4), L. crocea (22.2–27.3 mm), L. dorsospina (28.7–30.5 mm), L. feii (21.5–22.8 mm), L. firthi (26.4–29.2 mm), L. flaviglandulosa (23.0–27.0 mm), L. fuliginosa (28.2–30.0 mm), L. graminicola (23.1–24.6 mm) , L. isos (23.7–27.9 mm), L. khasiorum (24.5–27.3 mm), L. lateralis (26.9–28.3 mm), L. laui (24.8–26.7 mm), L. liui (24.8–26.7 mm), L. macrops (28.0–29.3 mm), L. maculosa (24.2–26.6 mm), L. mangshanensis (22.22–27.76 mm), L. maura (26.1 mm), L. melica (19.5–22.8 mm), L. murphyi (23.2–24.9 mm), L. niveimontis (22.5–23.6 mm), L. pallida (24.5–27.7 mm, L. petrops (23.6–27.6 mm), L. pingbianensis (28 mm), L. pluvialis (21.3–22.3 mm), L. puhoatensis (24.2–28.1 mm), L. purpuraventra (27.3–29.8 mm), L. purpurus (25.0–27.5 mm), L. rowleyae (23.4–25.4 mm), L. shangsiensis (24.9–29.4 mm), L. shiwandashanensis (26.8–29.7 mm), L. shimentaina (26.4–28.9 mm), L. sinorensis (26.6–27.1 mm), L. suiyangensis (28.7–29.7 mm), L. tadungensis (23.3–28.2 mm), L. tengchongensis (23.9–26.0 mm), L. tuberosa (24.4–29.5 mm), L. ventripunctata (23.7–27.7 mm), L. verrucosa (23.2–25.9 mm), L. wuhuangmontis (25.6–30.0 mm), L. wumingensis (26.0–26.7 mm), L. yingjiangensis (25.7–27.6 mm) and L. yunkaiensis (25.9–29.3 mm); differs from the larger in males L. nahangensis (40.8 mm), L. platycephala (35.1 mm), L. sungi (48.3–52.7 mm in males) and L. zhangyapingi (45.8–52.5 mm).

By having black spots on flanks, Leptobrachella dushanensis sp. nov. differs from L. aerea, L. botsfordi, L. crocea, L. eos, L. firthi, L. isos, L. pallida, L. petrops, L. tuberosa and L. zhangyapingi (vs. lacking distinct black spots on the flanks in the latter).

By having rudimentary webbing, Leptobrachella dushanensis sp. nov. differs from L. ardens, L. jinshaensis, L. kalonensis, L. maculosa, L. oshanensis, L. pallida, L. petrops, L. rowleyae, L. shiwandashanensis and L. tadungensis (vs. absent webbing in the latter).

By having wide fringes on toes, Leptobrachella dushanensis sp. nov. differs from L. applebyi, L. ardens, L. aspera, L. bashaensis, L. bidoupensis, L. bijie, L. botsfordi, L. bourreti, L. chishuiensis, L. crocea, L. damingshanensis, L. dorsospina, L. feii, L. flaviglandulosa, L. fuliginosa, L. jinshaensis, L. kalonensis, L. korifi, L. lateralis, L. macrops, L. maculosa, L. mangshanensis, L. melica, L. minima, L. nahangensis, L. namdongensis, L. niveimontis, L. nyx, L. oshanensis, L. pallida, L. pelodytoides, L. petrops, L. phiaoacensis, L. phiadenensis,

L. pluvialis, L. puhoatensis, L. purpuraventra, L. pyrrhops, L. rowleyae, L. shangsiensis, L. sinorensis, L. shiwandashanensis, L. sungi, L. tengchongensis, L. tuberosa, L. ventripunctata, L. wuhuangmontis, L. wulingensis, L. wumingensis, L. yeae and L. yunyangensis (vs. fringes on toes narrow or absent in the latter).

By having dorsal surface shagreened with fine tubercles, Leptobrachella dushanensis sp. nov. 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. bourreti (dorsum smooth with small warts), L. fuliginosa (dorsum smooth with fine tubercles), L. liui (dorsum with round tubercles), L. macrops (dorsum roughly granular with large tubercles), L. maoershanensis (dorsum shagreened with tubercles), L. minima (dorsum smooth), L. neangi (dorsum with small, irregular bumps and ridges), L. nyx (dorsum with round tubercles), L. nokrekensis (dorsum tubercles and longitudinal folds), L. pelodytoides (dorsum with small, smooth warts), L. tamdil (dorsum weakly tuberculate, with low, oval tubercles), L. tuberosa (dorsum highly tuberculate), L. yunkaiensis (dorsum with raised warts) and L. wuhuangmontis (dorsum rough with conical tubercles).

In mitochondrial DNA trees, Leptobrachella dushanensis sp. nov. and L. dong clustered into one clade, being sisters. The new species differs from L. dong by the following characters: head length slightly wider than head width (vs. head width slightly wider than head length); males with internal single subgular vocal sac (vs. a pair of subgular internal vocal sacs); tibiotarsal articulation reaching to anterior edge of eye (vs. reaching to middle of eye).

Secondary sexual characteristics: 

Adult males with internal single subgular vocal sac, femoral adipose glands present on posterior surface of thigh and tiny transparent spines on chest during breeding season. Nuptial pads and spines absent on males.

Etymology

This specifc name “Dushan” refers to the distribution of this species in Dushan County, Guizhou Province, China. We suggest its English common name “Dushan leaf litter toad” and Chinese name “Dushan Zhang Tu Chan (独山掌突蟾)”.

Distribution

Leptobrachella dushanensis sp. nov. was only found in Dushan County, Guizhou Province, China. Elevations recorded range from 1000 m to 1200 m.

Ecology

Leptobrachella dushanensis sp. nov. was found under stones in fast-flowing mountain streams surrounded by evergreen broadleaf forest (Fig. 5) and we did not find eggs, nor tadpoles or females. Based on our surveys, we speculate that the breeding season is probably in late March.

Figure 5.  

Habitats of Leptobrachella dushanensis sp. nov. A landscape of the type locality Dushan County, Guizhou Province, China; B a mountain stream in the type locality.

Analysis

Phylogenetic analyses

Aligned sequence matrix of 16S, COI and RAG1 genes contained 519, 615 and 831 bps respectively. ML and BI analyses, based on 16S and COI, resulted in essentially identical topologies. All samples of the undescribed species were clustered into one clade being deeply clustered into the Leptobrachella clade and being sister to L. dong (Fig. 6A and B). Only one haplotype was found for all samples of the undescribed species in RAG1 gene and there was no common haplotype between the undescribed species and its related species (Fig. 6C). The smallest pairwise genetic divergence between the undescribed species and its close congener L. dong from Guizhou population and Hunan population were both 1.6% on 16S (Suppl. material 3) and, on COI, were 7.3% and 7.4%, respectively (Suppl. material 4), which was higher than the divergence between the populations of L. dong (0.3% on 16S and 1.6% on COI) and also higher or at the same level with those amongst many pairs of congeners, such as L. bijie and L. jinyunensis (1.6% on 16S), L. bijie and L. jinyunensis was 4.4%, L. bijie and L. chishuiensis was 4.1%, L. chishuiensis and L. jinshaensis 5.8%, L. jinshaensis and L. purpuraventra was 4.0% on COI (Suppl. materials 3, 4).

Figure 6.  

Phylogenetic trees of the genus Leptobrachella and a haplotype network constructed, based on RAG1 gene sequences. A Bayesian inference (BI) tree, based on mitochondrial COI gene; B Bayesian Inference (BI) tree, based on mitochondrial 16S gene; C the haplotype network constructed, based on RAG1 gene sequences. In this phylogenetic tree, Bayesian posterior probabilities (BPP) from BI analyses/bootstrap supports (BS) from ML analyses are listed beside the nodes. The symbol “-” represents a value below 0.50/50. For information of samples 1–126, refer to Table 1.

Morphological analyses

The results of one-way ANOVA showed that males of the undescribed species differed significantly from L. dong from both Guizhou and Hunan populations, based on several morphometric characters. From Guizhou population, the undescribed species was larger in SVL, HDW and HLL and shorter in SL, IND, ED and TYD; from Hunan population, the undescribed species was larger in HDL and shorter in HDW, SL, IND, IOD, ED and LAL (all p-values < 0.05; Table 3). In PCA for males, the total variation of the first two principal components was 57.52% and, on the two-dimensional plots of PC1 vs. PC2, the undescribed species could be separated from the specimens of L. dong both from Guizhou and Hunan populations (Fig. 7).

Figure 7.  

Plots of principal component analyses of Leptobrachella dushanensis sp. nov. and L. dong in males. PC1, the first principal component; PC2, the second principal component. Different species were denoted as different shapes, the red triangle represents Leptobrachella dushanensis sp. nov., black square represents L. dong from Guizhou Province and blue square represents L. dong from Hunan Province.

More detailed descriptions of results from morphological comparisons between the undescribed species and its congeners were presented in the Taxon Treatment section for describing the new species.

As a result, molecular and morphological results supported that our specimens from Guizhou Province, China were a new taxon.

Discussion

The Asian leaf litter toads of Leptobrachella have low vagility and an exclusive association with montane forests and their populations are often highly structured and underestimation of species diversity occurs in the genus, which suggests a high degree of localised diversification and micro-endemism (Fei et al. 2012, Chen et al. 2018). In recent years, the approach of integrative taxonomy has made substantial progress with this species-rich group. However, only a short mitochondrial fragment of 16S gene was sequenced for many species in this genus and the phylogenetic relationships between most species in the genus have not been resolved (Ohler et al. 2011, Rowley et al. 2017b, Chen et al. 2018, Chen et al. 2023, Liu et al. 2023 etc.). This is most probably due to the insufficient phylogenetic information content in this kind of gene fragment (Chan et al. 2022).

In this study, two mitochondrial genes and one nuclear gene were amplified. On the 16S gene, the genetic distance between Leptobrachella dushanensis sp. nov. and its closely-related species L. dong was 1.6% which is at the same level with L. bijie and L. jinyunensis, but on COI gene, the genetic distance between them is 7.3%, much larger than that between other solid species (Suppl. materials 3, 4). Additionally, the haplotype network constructed, based on RAG1 gene sequences, also showed that all samples of Leptobrachella dushanensis sp. nov. shared one haplotype and there was no common haplotype between Leptobrachella dushanensis sp. nov. and its related species. This observation indicates a clear genetic differentiation between Leptobrachella dushanensis sp. nov. and its relatives, supporting its recognition as a distinct species. The lack of shared haplotypes further implies limited gene flow and potential geographic isolation between Leptobrachella dushanensis sp. nov. and its related species. The evidence for the interspecific divergence was further confirmed by the significant morphological differences. Accordingly, the results of molecular and morphological comparisons indicated the validation of these species.

References

Supplementary materials

Suppl. material 1: Measurements 
Authors:  Shize Li, Wei Li, Yanlin Cheng, Jing Liu, Gang Wei, Bin Wang
Data type:  morphological
Brief description: 

Measurements of adult specimen of Leptobrachella dushanensis sp. nov. and L. dong. Units in mm. See abbreviations for characters in the Materials and Methods section.

Suppl. material 2: Diagnosis characters on morphology 
Authors:  Shize Li, Wei Li, Yanlin Cheng, Jing Liu, Gang Wei, Bin Wang
Data type:  morphological
Brief description: 

Diagnosis characters on morphology of Leptobrachella dong sp. nov. from other congeners.

Suppl. material 3: Uncorrected p-distance on the 16S rRNA gene 
Authors:  Shize Li, Wei Li, Yanlin Cheng, Jing Liu, Gang Wei, Bin Wang
Data type:  phylogenetic
Brief description: 

Uncorrected p-distance between Leptobrachella species on the 16S rRNA gene. Mean value of genetic distance is given in the lower half of the table.

Suppl. material 4: Uncorrected p-distance on the COI 
Authors:  Shize Li, Wei Li, Yanlin Cheng, Jing Liu, Gang Wei, Bin Wang
Data type:  phylogenetic
Brief description: 

Uncorrected p-distance between Leptobrachella species on the COI. Mean value of genetic distance is given in the lower half of the table.

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