Research Article
Print
Research Article
A new species of the genus Leptobrachella Smith 1925 (Amphibia, Anura, Megophryidae) from Guangxi, China
expand article infoWei-Cai Chen, Wan-Xiao Peng, Peng Li, Gui-Dong Yu§
‡ Nanning Normal University, Nanning, China
§ Guangxi Forest Inventory and Planning Institute, Nanning, China
Open Access

Abstract

A new species of Leptobrachella, L. wumingensis sp. nov., was described from the Damingshan National Nature Reserve, Wuming District, Nanning City, Guangxi, China based on morphological, molecular and bioacoustic data. Phylogenetic analysis of 16S mtDNA fragments revealed that the new species is closely related to L. damingshanensis. Uncorrected p-distances between the new species and all homologous DNA sequences available for the 16S gene of Leptobrachella are greater than 7.1%. Morphologically, L. wumingensis sp. nov. differs from its congeners in several ways, including a medium body size (SVL 26.0–26.7 mm in males, 30.6–34.8 mm in females), lack of toe webbing and lateral fringes, shagreened and granular dorsal surface, pale brown dorsum with darker brown markings, iris bicolored, with the upper half copper and fading to silver in the lower half, and the presence of small irregular black spots and tangerine tubercles on the flanks. Furthermore, we found the new species to have two types of advertisement calls and relatively high dominant frequencies, making it distinct from its congeners.

Key words

Bioacoustics, molecular analyses, morphological characters, sympatric species, taxonomy

Introduction

The genus Leptobrachella Smith, 1925 is distributed in northeastern India, Southeast Asia, Vietnam, Thailand, Myanmar, Malaya, Borneo and Natuna Island (Frost 2023). The species diversity of Leptobrachella has been drastically underestimated due to its small body size, conserved morphology and high degree of sympatry (Chen et al. 2018; Chen et al. 2021a, 2021b). However, by utilizing morphological, molecular and bioacoustic data, numerous new Leptobrachella species have been described in recent decades (Frost 2023). The genus now comprises 99 species, of which 37 were described in the last five years (Frost 2023). In China, there are 39 Leptobrachella species, and 27 of them were described in the past five years, mostly occurring in southern and western China (AmphibiaChina 2023), indicating an underestimation of the diversity of Leptobrachella.

Between 2019 and 2023, we conducted surveys at the Damingshan National Nature Reserve (DMS), Wuming District, Nanning City, Guangxi, China. We collected nine specimens (hereafter DMS specimens) of Leptobrachella that significantly differed from the sympatric species, L. damingshanensis Chen, Yu, Cheng, Meng, Wei, Zhou & Lu, 2021 and other congeners in morphological characters, including body size and color pattern as well as molecular data. The uncorrected p-distances between 16S mtDNA fragments of DMS specimens and all homologous sequences of Leptobrachella available in GenBank were greater than 7.1%. Additionally, the bioacoustic data of the DMS specimens distinguished them from L. damingshanensis and other advertisement calls available in the genus Leptobrachella. Given the unique morphological characters, the relatively high degree of mtDNA divergence, and the distinct bioacoustics data, we describe the DMS specimens as a new species of Leptobrachella.

Material and methods

Nine Leptobrachella specimens were collected from the Damingshan National Nature Reserve, Wuming District, Nanning City, Guangxi, China (Fig. 1). After fixing the specimens in 10% formalin, they were stored in 75% ethanol and deposited at Nanning Normal University (NNU). Muscle tissue was taken before fixing and stored in 100% ethanol for DNA isolation. We measured all specimens to the nearest 0.1 mm using a digital caliper, following Fei et al. (2009) and Rowley et al. (2016), which included the snout-vent length (SVL) , head length from tip of snout to rear of jaws (HL) , head width at commissure of jaws (HW) , snout length from tip of snout to the anterior eye (SNT) , diameter of the exposed portion of eye (ED) , interorbital distance (IOD) , internarial distance (IN) , horizontal diameter of tympanum (TD) , distance from anterior edge of the tympanum to posterior corner of eye (TED) , manus length from tip of third finger to base of inner palmar tubercle (ML) , forelimb length from elbow to the tip of third finger (FLL) , thigh length from vent to knee (THL) , tibia length with flexed hindlimb (TIB) , pes length from tip of fourth toe to base of inner metatarsal tubercle (PL) , and maximum diameter of femoral gland (FEM). Sex was determined by the calls of males or gonadal inspection. Comparative morphological data were collected from the literature (Suppl. material 1: table S1) and from the collected specimens (Suppl. material 1: table S2).

Figure 1. 

Type locality of L. wumingensis sp. nov. (A) and its habitat (B).

Genomic DNA was isolated from muscle tissue using Tiangen Biotech Co. Ltd. tissue extraction kits (Beijing, China). The primer pairs 16Sar_L 5’-CGCCTGTTTAC CAAAAACAT-3’ and 16Sbr_H 5’-CCGGTCTGAACTCAGATCACGT-3’ (Palumbi et al. 1991) were used to amplify and sequence mitochondrial 16S rRNA gene fragments (~530 bp). The polymerase chain reaction (PCR) amplification was carried out in a 20 μl reaction volume involving the following steps: an initial denaturation step at 95 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 35 s, annealing at 58 °C for 40 s, and extension at 72 °C for 40 s, and a final extension step at 72 °C for 10 min. The fragments were sequenced using an ABI Prism 3730 automated DNA sequencer (Applied Biosystems, USA), and the newly obtained sequences were submitted to GenBank (accession numbers OM935575OM935578, OR194551OR194553).

Phylogenetic relationships were inferred using 16S mtDNA fragments through maximum likelihood (ML) and Bayesian inference (BI) analyses. Table 1 provides detailed information on the sequences used in the molecular analyses, which were aligned using the Clustalw module in MEGA v. 7 (Kumar et al. 2016) with default settings. The best-fit models of evolution (GTR+I+G) were determined using MrModeltest v. 2.3 (Nylander 2004) with Akaike and Bayesian information criteria. ML analyses were conducted on the CIPRES science gateway, estimating the proportion of invariable sites from the data and performing 1000 bootstrap pseudo replicates (https://www.phylo.org/portal2) (Miller et al. 2010). BI was performed using MrBayes v. 3.2 (Ronquist et al. 2012), with two independent runs and four Markov Chain Monte Carlo simulations for 30 million iterations. Trees were sampled every 1000 generations, with the first 25% of trees discarded as burn-in. Node values were considered well supported if ML bootstrap supports (BS) were ≥ 70% and Bayesian posterior probabilities (PP) were ≥ 0.95. Outgroups used were Leptobrachium huashen and Xenophrys glandulosa following Chen et al. (2018). Uncorrected p-distances were calculated based on 16S gene fragments using Mega v. 7 with default settings.

Table 1.

DNA sequences used in this study. ‘*’ represents type locality.

ID Species Locality Voucher no. GenBank No. 16S rRNA
1 L. wumingensis sp. nov. Wuming County, Guangxi, China* NNU 00283 OM935575
2 L. wumingensis sp. nov. Wuming County, Guangxi, China* NNU 00284 OM935576
3 L. wumingensis sp. nov. Wuming County, Guangxi, China* NNU 00285 OM935577
4 L. wumingensis sp. nov. Wuming County, Guangxi, China* NNU 00286 OM935578
5 L. wumingensis sp. nov. Wuming County, Guangxi, China* NNU 01058 OR194551
6 L. wumingensis sp. nov. Wuming County, Guangxi, China* NNU 01059 OR194552
7 L. wumingensis sp. nov. Wuming County, Guangxi, China* NNU 01086 OR194553
8 L. aerea Quang Binh, Vietnam ZFMK 86362 JN848409
9 L. alpina Caiyanghe, Yunnan, China KIZ 049024 MH055867
10 L. applebyi Phong Dien Nature Reserve, Thua Thien-Hue, Vietnam KIZ 010701 MH055947
11 L. arayai Borneo, Malaysia* AE 100/S9 DQ642119
12 L. ardens Kon Ka Kinh National Park, Gia Lai, Vietnam* ZMMU-NAP-06099 MH055949
13 L. aspera Huanglianshan Nature Reserve, Lyuchun, Yunnan, China* SYS a007743 MW046199
14 L. baluensis Sabah, Borneo, Malaysia* SP 21604 LC056792
15 L. bashaensis Basha Nature Reserve, Guizhou, China* GIB 196404 MW136295
16 L. bidoupensis Bidoup-Nui Ba National Park, Lam Dong, Vietnam* ZMMU-A-4797-01454 MH055945
17 L. bijie Bijie City, Guizhou, China* SYS a007313 MK414532
18 L. botsfordi Lao Cai, Vietnam* AMS R 176540 MH055952
19 L. bourreti Mao’er Shan, Guangxi, China KIZ 019389 MH055869
20 L. brevicrus Sarawak, Borneo, Malaysia* ZMH A09365 KJ831302
21 L. chishuiensis Guizhou, China* CIB CS20190518047 MT117053
22 L. crocea Thua Thien-Hue, Vietnam ZMMU-NAP-02274 MH055955
23 L. damingshanensis Wuming County, Guangxi, China* NNU 202103281 MZ145229
24 L. damingshanensis Wuming County, Guangxi, China* NNU 202103282 MZ145230
25 L. damingshanensis Wuming County, Guangxi, China* NNU 202103283 MZ145231
26 L. dong Tongdao County, Hunan Province, China* CIB SSC1758 OP764529
27 L. dorsospina Yushe Forest Park, Shuicheng, Guizhou, China* SYS a004961 MW046194
28 L. dringi Borneo, Malaysia* KUHE:55610 AB847553
29 L. eos Phongsaly, Laos* MNHN 2004.0274 JN848452
30 L. feii Yunnan, China* KIZ048894 MT302634
31 L. firthi Kon Tum, Vietnam* AMS: R 176524 JQ739206
32 L. flaviglandulosa Xiaoqiaogou Nature Reserve, Yunnan, China* KIZ 016072 MH055934
33 L. fritinniens Danum Valley Field Center, Sabah, Malaysia FMNH 244800 MH055971
34 L. fuliginosa Phetchaburi, Thailand KUHE:20197 LC201988
35 L. gracilis Bukit Kana, Sarawak, Malaysia FMNH 273682 MH055972
36 L. graminicola Mount Pu Ta Leng, Lao Cai, Vietnam* VNMN 010909 MZ224649
37 L. hamidi Borneo, Malaysia* KUHE 17545 AB969286
38 L. heteropus Peninsular, Malaysia KUHE 15487 AB530453
39 L. isos Gia Lai, Vietnam* AMS R 176480 KT824769
40 L. itiokai Gunung Mulu National Park, Sarawak, Malaysia* KUHE:55897 LC137805
41 L. jinshaensis Lengshuihe Nature Reserve, Jinsha County, Guizhou, China* CIB JS20200516001 MT814014
42 L. juliandringi Sarawak, Borneo, Malaysia* KUHE 17557 LC056784
43 L. kajangensis Tioman, Malaysia* LSUHC:4439 LC202002
44 L. kalonensis Binh Thuan, Vietnam* IEBR A.2014.15 KR018114
45 L. kecil Cameron, Malaysia* KUHE:52439 LC202003
46 L. khasiorum Meghalaya, India* SDBDU 2009.329 KY022303
47 L. korifi Doi Inthanon, Thailand* KUHE 19134 LC741033
48 L. laui Wutongshan, Shenzhen city, China* SYS a001507 KM014544
49 L. liui Wuyi Shan, Fujian, China* ZYCA907 MH055908
50 L. macrops Dak Lak, Vietnam* AMS R177663 KR018118
51 L. maculosa Ninh Thuan, Vietnam* AMS: R 177660 KR018119
52 L. mangshanensis Manghan, Hunan, China* MSZTC201703 MG132198
53 L. maoershanensis Mao’er Shan, Guangxi, China KIZ 07614 MH055927
54 L. marmorata Borneo, Malaysia* KUHE 53227 AB969289
55 L. maura Borneo, Malaysia SP 21450 AB847559
56 L. melanoleuca Kapoe, Ranong, Thailand KIZ 018031 MH055967
57 L. melica Ratanakiri, Cambodia* MVZ 258198 HM133600
58 L. minima Doi Phu Fa, Nan, Thailand KIZ 024317 MH055852
59 L. mjobergi Sarawak, Borneo, Malaysia* KUHE 47872 LC056787
60 L. murphyi Doi Inthanon, Chiang Mai, Thailand* KIZ 031199 MZ710523
61 L. nahangensis Tuyen Quang, Vietnam* ROM 7035 MH055853
62 L. namdongensis Thanh Hoa, Vietnam* VNUF A.2017.95 MK965390
63 L. neangi Veal Veng District, Pursat, Cambodia* CBC 1609 MT644612
64 L. niveimontis Yongde County, Yunnan, China* KIZ 028276 MT302620
65 L. nyx Ha GiangProv., Vietnam* AMNH A 163810 DQ283381
66 L. oshanensis Emei Shan, Sichuan, China* Tissue ID: YPX37492 MH055896
67 L. pallida Lam Dong, Vietnam* UNS 00510 KR018112
68 L. parva Mulu National Park, Sarawak, Malaysia* KUHE:55308 LC056791
69 L. pelodytoides NA TZ 819 AF285192
70 L. petrops Ba Vi National Park, Ha Tay, Vietnam ROM 13483 MH055901
71 L. picta Borneo, Malaysia UNIMAS 8705 KJ831295
72 L. pluvialis Lao Cai, Vietnam* MNHN:1999.5675 JN848391
73 L. puhoatensis Nghe An, Vietnam* VNMN 2016 A.22 KY849586
74 L. purpurus Yunnan, China* SYS a006530 MG520354
75 L. purpuraventra Guizhou, China* SYS a007281 MK414517
76 L. pyrrhops Loc Bac, Lam Dong, Vietnam* ZMMU-A-4873-00158 MH055950
77 L. rowleyae Da Nang City, Vietnam* ITBCZ2783 MG682552
78 L. sabahmontanus Borneo, Malaysia* BORNEENSIS 12632 AB847551
79 L. shangsiensis Shangsi County, Guangxi, China* NHMG1401032 MK095460
80 L. shimentaina Shimentai Nature Reserve, Guangdong, China* SYS a004712 MH055926
81 L. shiwandashanensis Fangcheng City, Guangxi, China* NNU202103146 MZ326691
82 L. sinorensis Mae Hong Son, Thailand* KUHE 19809 LC741034
83 L. sola Gunung Stong, Kelantan, Malaysia KU RMB20973 MH055973
84 L. suiyangensis Guizhou, China* GZNU 20180606005 MK829649
85 L. sungi Vinh Phuc, Vietnam* ROM 20236 MH055858
86 L. tadungensis Dak Nong, Vietnam* UNS 00515 KR018121
87 L. tengchongensis Yunnan, China* SYS a004598 KU589209
88 L. tuberosa Kon Ka Kinh National Park, Gia Lai, Vietnam* ZMMU-NAP-02275 MH055959
89 L. ventripunctata Wenlong, Yunnan, China KIZ 013621 MH055824
90 L. wuhuangmontis Pubei County, Guangxi, China* SYS a003486 MH605578
91 L. wulingensis Hunan, China* CSUFT194 MT530316
92 L. yeae Mount Emei, Sichuan, China* CIBEMS20190422HLJ1-6 MT957019
93 L. yingjiangensis Yunnan, China* SYS a006532 MG520351
94 L. yunkaiensis Guangdong, China* SYS a004663 MH605584
95 L. yunyangensis Yunyang County, Chongqing, China* GZNU 20210629001 OL800366
96 L. zhangyapingi Chiang Mai, Thailand* KIZ 07258 MH055864
97 Leptobrachium huashen Yunnan, China KIZ 049025 KX811931
98 Xenophrys glandulosa Yunnan, China KIZ 048439 KX811762

Two male advertisement calls were recorded using a SONY ICD-TX50 recorder at an ambient temperature of approximately 21 °C. Acoustic analysis was performed using Raven Pro 1.6 (Cornell Laboratory of Ornithology, Ithaca, NY, USA) following the method outlined by Köhler et al. (2017). The protocol involved calculating audio-spectrograms with a fast-Fourier transform (FFT) of 512 points, 50% overlap, and 172 Hz grid-spacing, using Hanning windows. Refer to Suppl. material 1: table S3 for comparison data on bioacoustics.

Results

Molecular analyses

The molecular analyses conducted using BI and ML methods produced similar topologies, as depicted in Fig. 2. However, the phylogenetic relationships of Leptobrachella species showed weak internal node support and remained unresolved (Fig. 2). The phylogenetic trees indicated that the DMS specimens are closely related to L. damingshanensis, L. nahangensis and L. nyx. Furthermore, the genetic divergence of the 16S gene fragment between the DMS specimens and all other available homologous sequences of Leptobrachella species was found to be greater than 7.1% for the analyzed fragment (Suppl. material 1: table S4).

Figure 2. 

BI trees based on the 16S mtDNA fragments. Node support is indicated on branches as Bayesian posterior probabilities (upper half; >0.95 = grey, 1 = black) and maximum likelihood support (lower half; >70%<90% = grey, >90% = black).

Bioacoustics

The calls of two individuals were recorded in the field (NNU 01058 and NNU 01086). Two types of advertisement calls (Type A and Type B; Fig. 3) were detected. The call durations of Type A ranged from 0.2454 s to 0.4306 s, with an average of 0.3407 ± 0.0423 s, while the call intervals of Type A ranged from 0.1784 s to 0.3756 s, with an average of 0.2364 ± 0.0333 s. For Type B, the call durations ranged from 0.0416 s to 0.0903 s, with an average of 0.0605 ± 0.0094 s, and the call intervals ranged from 0.3700 s to 1.7740 s, with an average of 0.8932 ± 0.3810 s. The dominant frequencies were observed to be between 6.0–7.5 kHz (at 21.0 °C). The DMS specimens exhibited two types of calling models and relatively high dominant frequencies, which distinguishes them from the known advertisement callings in the genus Leptobrachella (Suppl. material 1: table S3).

Figure 3. 

Advertisement calls spectrograms of L. wumingensis sp. nov.

Morphological characters

The DMS specimens can be readily differentiated from other species in the same phylogenetic clade. For instance, adult males of L. damingshanensis have a significantly larger body size (SVL 33.6–34.4 mm) and possess a pair of distinct tangerine glands on the proximal thigh area, as well as rudimentary webbing and narrow lateral fringes on their toes. In contrast, the DMS specimens have indistinct tangerine glands on the proximal thigh area, and no toe webbing or lateral fringes.

Taken together, molecular data, acoustic analyses and morphological characters support the conclusion that the DMS specimens represent a distinct and previously unrecognized species of Leptobrachella, which is described below.

Taxonomic account

Leptobrachella wumingensis sp. nov.

Figs 4, 5

Type materials

Holotype. NNU 01058, adult male, collected at the Damingshan National Nature Reserve, Wuming District, Nanning City, Guangxi, China (23.507°N, 108.395°E; elevation 1214 m), collected by Wei-Cai Chen on 12 April 2023. Paratypes. NNU 201907009, NNU 01086, two adult males, collected at the same locality as the holotype; NNU 201907009 collected by Gui-Dong Yu on 23 May 2019, NNU 01086 collected by Wei-Cai Chen on 14 April 2023; NNU 00283–6, four adult females collected at the same locality as the holotype on 16 June 2021 by Wei-Cai Chen; NNU 01059–60, two adult females collected at the same locality as the holotype on 12 April 2023 by Wei-Cai Chen.

Etymology

The specific name ‘wumingensis’ is derived from the type locality, Wuming District, Nanning City, Guangxi, China. The proposed common name in English is Wuming Leaf Litter Toad, and in Chinese, it is called Wu Ming Zhang Tu Chan (武鸣掌突蟾).

Diagnosis

Leptobrachella wumingensis sp. nov. is classified under Leptobrachella based on specific morphological features, including its relatively small body size, presence of an inner metacarpal tubercle, macro-glands on the supra-axillary and femoral glands, lack of vomerine teeth, and a whitish vertical bar on the anterior tip of the snout, according to previous studies (Dubois 1983; Lathrop et al. 1998; Delorme et al. 2006; Matsui 2006). Leptobrachella wumingensis sp. nov. can be differentiated from other species in its genus by a combination of the following characters: (1) medium size (SVL 26.0–26.7 mm in males, 30.6–34.8 mm in females); (2) absence of toe webbing and lateral fringes; (3) shagreened and granular dorsal surface; (4) pale brown dorsum with darker brown markings; (5) iris bicolored, with the upper half copper and fading to silver in the lower half; (6) presence of small irregular black spots and tangerine tubercles on the flanks; and (7) two types of advertisement callings and high dominant frequencies.

Description of holotype

Head length almost equal to width (HW/HL = 1.02); snout bluntly rounded in profile and dorsal view, projecting slightly over lower jaw; nostril oval-shaped, closer to tip of snout than eye; canthus rostralis distinct; loreal region distinctly sloping, slightly concave; pupil vertical; eye diameter less than snout length (ED/SNT = 0.96); tympanum distinct and rounded, diameter about 49% that of eye; vomerine teeth absent; tongue with a deep notch at posterior tip; supratympanic fold distinctly raised from corner of eye to the posterior of tympanum (Fig. 4A–D).

Figure 4. 

Morphological characters of L. wumingensis sp. nov. (NNU 01058) A dorsal view B ventral view C, D lateral view E ventral view of hand F ventral view of foot.

Tips of fingers slightly swollen; relative finger lengths I < II < IV < III; subarticular tubercles absent; prominent inner palmar tubercle, small outer palmar tubercle; finger webbing and dermal fringes absent; nuptial pad on fingers absent. Tips of toes rounded, slightly swollen; relative toe lengths I < II < V = III < IV; subarticular tubercles absent, replaced by dermal ridges; prominent and elongated inner metatarsal tubercle; outer metatarsal tubercle absent; toe webbing and lateral fringes absent. Tibia 48% of SVL; tibiotarsal articulation reaching middle of eye; heels meeting when hindlimbs flexed at right angles with respect to body (Fig. 4E, F).

Dorsal surface shagreened and granular; upper eyelid with small tubercles; ventral surface without tubercles; flanks with sparse tubercles; pectoral glands elongated, approximately 2.1 mm in diameter; small femoral glands oval, approximately 0.6 mm in diameter, closer to knee than to vent; supra-axillary glands oval, approximately 0.8 mm in diameter; ventrolateral glandular line discrete; ventral surface of thigh with some tubercles (Fig. 4A–D).

Color in life

Dorsum light brown with brown markings, an inverted triangle marking between eyes, an irregular brown marking on scapular region, a brown ‘Λ’ marking on rear of dorsum; tympanum pale brown; supratympanic fold black from the posterior corner of eye to the anterior of supra-axillary glands; two wide black bars on upper lip; five irregular black spots and several small light tangerine tubercles on flanks; transverse brown bars on dorsal surface of hindlimbs; upper arms light orange; belly with tiny creamy white spots; throat creamy white with tiny light brown spots; lower jaw with creamy white tubercles; ventral surfaces of limbs with sparse creamy white tubercles; pectoral and femoral glands creamy white, supra-axillary glands light orange; pupil black; iris bicolored, upper half copper, fading to silver in lower half (Fig. 4A–D).

Color in preservative

The dorsum and limb surfaces are faded to uniform light brown. Irregular black spots on flanks and bars on limbs are darkish brown. The throat, chest, and belly are creamy white, and pectoral, femoral, supra-axillary, and ventrolateral glands are also creamy white.

Variation

The measurements of the type series are shown in Table 2. The holotype and paratypes exhibit similar color patterns. Females are larger than males in terms of body size. The number of black spots on the flanks varies, ranging from five to eight (Fig. 5A, B). Some individuals have more tubercles on their flanks and their dorsum are rougher compared to the holotype (Fig. 5C).

Table 2.

Measurements of voucher specimens of L. wumingensis sp. nov. (mm). Abbreviations defined in text.

NNU 01058 NNU 01086 NNU 201907009 NNU 00283 NNU 00284 NNU 00285 NNU 00286 NNU 01059 NNU 01060
Sex Male Male Male Female Female Female Female Female Female
SVL 26.7 26.6 26.0 31.2 31.5 30.6 31.9 34.8 31.6
HL 8.7 8.7 8.9 10.2 10.0 10.2 10.8 10.4 11.2
HW 8.9 9.0 9.3 10.5 10.6 10.8 10.8 11.0 11.2
SNT 3.4 3.8 3.7 4.3 4.2 4.1 4.0 4.5 4.3
ED 3.2 3.6 3.4 4.3 4.8 4.6 4.1 3.7 3.6
IOD 3.0 2.7 2.8 3.1 3.3 3.6 3.3 3.4 2.9
IN 2.7 2.6 2.2 3.4 3.5 3.6 3.4 3.2 3.0
TD 1.6 1.8 1.8 2.4 2.4 1.9 2.1 2.1 2.0
TED 0.9 1.0 0.8 1.3 1.3 1.1 1.4 1.1 0.9
ML 6.2 5.8 6.1 7.3 7.3 7.6 7.4 7.3 7.2
FLL 11.5 11.9 11.4 14.3 14.5 14.4 14.4 13.8 14.4
THL 12.3 11.7 12.9 14.9 16.3 15.4 15.6 14.1 14.0
TIB 12.7 12.8 12.8 14.4 15.9 15.4 15.9 14.9 15.2
PL 11.5 11.4 11.4 13.4 14.4 14.0 14.2 13.6 13.7
FEM 0.6 0.7 0.7 1.5 1.5 1.4 1.2 1.1 1.0
Figure 5. 

A dorsolateral view of NNU 01086 B dorsolateral view of NNU 01059 C dorsolateral view of NNU 00285 D NNU 201907009 in habitat (photo taken in situ) E NNU 00283 in habitat (photo taken in situ) F eggs (NNU 01059).

Ecology and distribution

Leptobrachella wumingensis sp. nov. inhabits evergreen forests in the DMS at elevations of 1000–1200 m. All specimens were discovered over 10 m away from rocky streams (Fig. 5D, E). The species’ advertisement calls were heard on the rocks in mid-April, and creamy white eggs were found on females’ abdomens in April (Fig. 5F). We dissected four females collected in June and found no eggs in their abdomens. We speculate that the breeding period of the new species is between April and May. Since 2019, we have surveyed over ten rocky streams similar to the type locality but failed to find more sites where the new species occurs. The population of the new species is very rare; we have visited the type locality more than ten times since 2019, but only encountered nine adults. Although both L. wumingensis sp. nov. and L. damingshanensis occur in the DMS, the two species do not inhabit the same streams, and the closest site where both species are found is 10 km away. Currently, L. wumingensis sp. nov. is only known to occur in the DMS (Fig. 1).

Comparisons

To begin with, L. wumingensis sp. nov. is distinguished from Leptobrachella species found south of the Isthmus of Kra, Malay Peninsula by the presence of supra-axillary and ventrolateral glands (vs absent in the latter). Furthermore, Leptobrachella wumingensis sp. nov. and L. damingshanensis are sympatric. Leptobrachella wumingensis sp. nov. can be differentiated from L. damingshanensis by several characters, including smaller body size in males (SVL 26.0–26.7 mm vs 33.6–34.4 mm); indistinct orange glands on the proximal thigh area (vs a pair of distinct tangerine glands on the proximal thigh area); pale brown dorsal skin with brown markings and a shagreened and granular dorsal surface (vs rough dorsal skin with sparse tangerine tubercles and some short longitudinal ridges); the absence of toe webbing and lateral fringes on toes (vs rudimentary toes webbing and narrow lateral fringes on toes); distinct transverse dark brown bars on the dorsal surface of hindlimbs (vs indistinct transverse dark brown bars on the dorsal surface of hindlimbs); and different bioacoustics, including two types of calling models and dominant frequency at 6.0–7.5 kHz (vs a single calling model and dominant frequency at 4.6–5.2 kHz) (Fig. 4, Suppl. material 1: table S3).

According to the phylogenetic analysis, L. wumingensis sp. nov., L. damingshanensis, L. nahangensis, and L. nyx constitute a monophyletic group (Fig. 2). In terms of morphology, L. wumingensis sp. nov. can be differentiated from L. nahangensis by its notably smaller body size in males (SVL 26.0–26.7 mm vs SVL 40.8 mm); lack of toe webbing (vs rudimentary toes webbing); pale brown dorsum with brown markings, a triangle-shaped marking between the eyes, an irregular brown marking on the scapular region, and a brown ‘Λ’ marking on the posterior dorsum (vs dorsum covered with irregular, diffuse dark gray and black spots); and iris that is bicolored, with the upper half copper and the lower half fading to silver (vs uniformly gold iris). Similarly, L. wumingensis sp. nov. differs from L. nyx in lacking toe webbing (vs rudimentary toes webbing); pale brown dorsum with brown markings, a triangle-shaped marking between eyes, an irregular brown marking on the scapular region, and a brown ‘Λ’ marking on the posterior of dorsum (vs dorsum being greyish brown with dark regularly set rounded spots); and flanks with small, irregular black spots and tangerine tubercles (vs flanks with poorly distinct spots).

In comparison to other recognized Leptobrachella species from north of the Isthmus of Kra, L. wumingensis sp. nov. is distinguishable from smaller species such as L. applebyi (19.6–22.3 mm in males), L. ardens (21.3–24.7 mm in males), L. aspera (22.4 mm in male), L. feii (21.5–22.8 mm in males), L. korifi (22.7 mm in female), L. melica (19.5–22.7 mm in males), L. murphyi (23.2–24.9 mm in males), L. niveimontis (22.5–23.6 mm in males) and L. pluvialis (21.3–22.3 mm in males) due to its larger size (26.0–26.7 mm in males). Additionally, L. wumingensis sp. nov. is distinct from the notably larger L. sungi (48.3–52.7 mm in males) and L. zhangyapingi (45.8–52.5 mm in males).

Leptobrachella wumingensis sp. nov. can be distinguished from L. aerea, L. alpina, L. dong, L. eos, L. firthi, L. graminicola, L. isos, L. khasiorum, L. laui, L. liui, L. murphyi, L. purpurus, L. shimentaina, L. tamdil, L. yingjiangensis, L. yunkaiensis and L. zhangyapingi by the absence of lateral fringes on its toes (vs. wide lateral fringes); from L. aspera, L. bashaensis, L. bidoupensis, L. bijie, L. botsfordi, L. bourreti, L. chishuiensis, L. damingshanensis, L. dorsospina, L. feii, L. flaviglandulosa, L. fuliginosa, L. jinshaensis, L. jinyunensis, L. korifi, L. mangshanensis, L. maoershanensis, L. niveimontis, L. pelodytoides, L. petrops, L. puhoatensis, L. purpuraventra, L. shangsiensis, L. sinorensis, L. suiyangensis, L. sungi, L. tengchongensis, L. ventripunctata, L. verrucosa, L. wuhuangmontis, L. wulingensis, L. yeae, and L. yunyangensis by the absence of lateral fringes on its toes (vs. narrow lateral fringes). Leptobrachella wumingensis sp. nov. is also differentiated from L. aerea, L. botsfordi, L. crocea, L. graminicola, L. eos, L. firthi, L. isos, L. pallida, L. petrops, and L. tuberosa by the presence of black spots on its flanks (vs. absent). Leptobrachella wumingensis sp. nov. differs from L. applebyi, L. bidoupensis, L. kalonensis, L. melica, L. minima, L. nahangensis, and L. tadungensis by the presence of shagreened and granular dorsal surface (vs smooth).

Lastly, L. wumingensis sp. nov. differs from other Leptobrachella species in terms of acoustic features such as relatively high dominant frequencies and two distinct types of callings (Fig. 3, Suppl. material 1: table S3).

Discussion

Based on morphological characters, molecular data and bioacoustics, we have identified the DMS specimens as a new species. The validation of this assignment is supported by significant genetic divergence (>7.1%), high dominant frequencies, complicated calling styles and substantial morphological characters. The DMS reserve is located in the central region of Guangxi, and previous fieldwork by many investigation teams did not discover any Leptobrachella species until we reported the first one in 2021 (Chen et al. 2021b). We have conducted annual amphibian surveys in the reserve since 2019 and have found that the population of L. wumingensis sp. nov. is rare, with only nine individuals found along with L. damingshanensis. Further field surveys are required to determine their distributions for conservation purposes.

According to previous research and our new data (AmphibiaChina 2023; Frost 2023), at least ten Leptobrachella species occur in Guangxi, including L. alpina, L. bourreti, L. damingshanensis, L. liui, L. maoershanensis, L. shangsiensis, L. shiwandashanensis, L. ventripunctata, L. wuhuangmontis and L. wumingensis sp. nov. Six of these species have been described in the past five years. The weak dispersal abilities and forest-dependent niches of Leptobrachella species may contribute to underestimating their diversity and distribution in Guangxi. Thus, more surveys are needed to understand the true species diversity and distribution of Leptobrachella in the region.

Acknowledgments

The authors are grateful to the Damingshan National Nature Reserve staff (Li Lin, Chang-Gui Cen, and De-Qi Zhang) for assistance during fieldwork.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This work was supported by the National Natural Science Foundation of China (32060116) and Guangxi Natural Science Foundation, China (2020GXNSFDA 238022).

Author contributions

CWC designed the study and wrote the manuscript. PWX, LP and YGD took part in fieldwork. CWC and LP performed the molecular experiments and analyzed the data. PWX drew the figures.

Author ORCIDs

Wei-Cai Chen https://orcid.org/0000-0002-2398-4079

Wan-Xiao Peng https://orcid.org/0000-0001-5635-9061

Peng Li https://orcid.org/0000-0001-8311-0544

Gui-Dong Yu https://orcid.org/0009-0008-1928-6594

Data availability

All of the data that support the findings of this study are available in the main text or Supplementary Information.

References

  • AmphibiaChina (2023) The database of Chinese amphibians. Electronic Database accessible at http://www.amphibiachina.org/. Kunming Institute of Zoology (CAS), Kunming, Yunnan, China.
  • Chen JM, Poyarkov Jr NJ, Suwannapoom C, Lathrop A, Wu YH, Zhou WW, Yuan ZY, Jin JQ, Chen HM, Liu HQ, Nguyen TQ, Nguyen SN, Duong TV, Eto K, Nishikawa K, Matsui M, Orlov NL, Stuart BL, Brown RM, Rowley JJL, Murphy RW, Wang YY, Che J (2018) Largescale phylogenetic analyses provide insights into unrecognized diversity and historical biogeography of Asian leaf-litter frogs, genus Leptolalax (Anura: Megophryidae). Molecular Phylogenetics and Evolution 124: 162–171. https://doi.org/10.1016/j.ympev.2018.02.020
  • Chen WC, Peng WX, Pan WH, Liao NY, Liu YJ, Huang Y (2021a) A new species of Leptobrachella Smith 1925 (Anura: Megophryidae) from southern Guangxi, China. Zootaxa 5020(3): 581–596. https://doi.org/10.11646/zootaxa.5020.3.8
  • Delorme M, Dubois A, Grosjean S, Ohler A (2006) Une nouvelle ergotaxinomie des Megophryidae (Amphibia, Anura). Alytes 24: 6–21.
  • Dubois A (1983) Note préliminaire sur le genre Leptolalax Dubois, 1980 (Amphibiens, Anoures), avec diagnose d’une espèce nouvelle du Vietnam. Alytes 2: 147–153. https://doi.org/10.5962/p.297429
  • Fei L, Hu SQ, Ye CY, Huang YZ (2009) Fauna Sinica. Amphibia. Vol. 2. Anura. Science Press, Beijing. [In Chinese]
  • Köhler J, Jansen M, Rodríguez A, Kok PJR, Toledo LF, Emmrich M, Glaw F, Haddad CFB, Rödel MO, Vences M (2017) The use of bioacoustics in anuran taxonomy: Theory, terminology, methods and recommendations for best practice. Zootaxa 4251(1): 1–124. https://doi.org/10.11646/zootaxa.4251.1.1
  • Kumar S, Stecher G, Tamura K (2016) MEGA 7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Molecular Biology and Evolution 33(7): 1870–1874. https://doi.org/10.1093/molbev/msw054
  • Lathrop A, Murphy RW, Orlov N, Ho CT (1998) Two new species of Leptolalax (Anura: Megophryidae) from northern Vietnam. Amphibia-Reptilia 19(3): 253–267. https://doi.org/10.1163/156853898X00160
  • Miller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: Proceedings of the Gateway Computing Environments Workshop (GCE), 14 Nov. 2010, New Orleans, LA, 1–8. https://doi.org/10.1109/GCE.2010.5676129
  • Nylander JAA (2004) MrModeltest v 2: Program distributed by the author. Evolutionary Biology Centre, Uppsala University.
  • Palumbi SR, Martin R, Romano S, McMillan WO, Stice L, Grabowski G (1991) The Simple Fool’s Guide To PCR, v2.0. Special publication of the University of Hawaii Department of Zoology and Kewalo Marine Laboratory.
  • Ronquist F, Teslenko M, Van Der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) Mrbayes 3.2: Efficient bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61(3): 539–542. https://doi.org/10.1093/sysbio/sys029
  • Rowley JJL, Tran DTA, Le DTT, Dau VQ, Peloso PLV, Nguyen TQ, Hoang HD, Nguyen TT, Ziegler T (2016) Five new, microendemic Asian Leaf-litter Frogs (Leptolalax) from the southern Annamite mountains, Vietnam. Zootaxa 4085(1): 63–102. https://doi.org/10.11646/zootaxa.4085.1.3
  • Smith MA (1925) Contributions to the herpetology of Borneo. Sarawak Museum Journal 3: 15–34.

Supplementary material

Supplementary material 1 

Supplementary information

Wei-Cai Chen, Wan-Xiao Peng, Peng Li, Gui-Dong Yu

Data type: morphological, phylogenetic

Explanation note: table S1: Literature for morphological characters for species of the genus Leptobrachella; table S2: Comparative material examined; table S3: Dominant frequency of advertisement calls of species available in the genus Leptobrachella; table S4: Uncorrected p-distance in Leptobrachella species based on 16S mtDNA fragments.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (86.90 kb)
login to comment