A new species of the Asian leaf litter toad genus Leptobrachella from Guizhou Province, China is described based on molecular phylogenetic analyses, morphological comparisons, and bioacoustics data. Phylogenetic analyses based on the mitochondrial 16S rRNA gene sequences supported the new species as an independent clade nested into the Leptobrachella clade and sister to L. bijie. The new species could be distinguished from its congeners by a combination of the following characters: small body size (SVL 30.8–33.4 mm in seven adult males, and 34.2 mm in one adult female); dorsal skin shagreened, some of the granules forming longitudinal short skin ridges; tympanum distinctly discernible, slightly concave; internasal distance longer than interorbital distance; supra-axillary, femoral, pectoral and ventrolateral glands distinctly visible; absence of webbing and lateral fringes on fingers; toes with rudimentary webbing and shallow lateral fringes, relative finger lengths II < IV < I < III; heels overlapped when thighs are positioned at right angles to the body; and tibia-tarsal articulation reaches the tympanum.

Guizhou, molecular phylogenetic analyses, morphology, new species, taxonomy

The Asian leaf litter toads of the genus Leptobrachella Smith, 1925 (Anura, Megophryidae) are widely distributed from southern China west to northeastern India and Myanmar, through mainland Indochina to peninsular Malaysia and the island of Borneo (Frost 2020). Many species in this genus had been ever classified into Leptolalax Dubois, 1983 (e.g., Fei et al. 2009, 2012), and Chen et al. (2018) placed Leptolalax as a junior synonym of Leptobrachella based on large-scale molecular analyses. Currently, the genus Leptobrachella contains 76 species, of which44 species have been described in the past ten years (Frost 2020). Currently, 21 species of the genus Leptobrachella are known from China: 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, L. bijie Wang, Li, Li, Chen & Wang, 2019, and L. suiyangensis Luo, Xiao, Gao & Zhou, 2020 from Guizhou; L. purpura (Yang, Zeng & Wang, 2018), L. pelodytoides (Boulenger, 1893), L. tengchongense (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; Li et al. 2016; Yang et al. 2016, 2018; Yuan et al. 2017; Chen et al. 2018, 2019; Hou et al. 2018; Wang et al. 2018, 2019; Wang et al. 2019; Luo et al. 2020). Even more, a series of cryptic species in the genus were still proposed in Chen et al. (2018).

In recent years, we carried out a series of biodiversity surveys in Chishui City, Guizhou Province, China, and collected some specimens of the genus Leptobrachella. Molecular phylogenetic analyses, morphological comparisons, and bioacoustics comparisons consistently indicated these specimens as an undescribed species of Leptobrachella. Hence, we describe it herein as a new species.

Specimens. Seven adult males and one adult female of the undescribed species were collected from the mountain streams in Chishui National Nature Reserve, Chishui City, Guizhou Province, China (for voucher information see Table 1; Fig. 1). After taking photographs, they were euthanized using isoflurane, and then the specimens were fixed in 10% buffered formalin. Before fixing, muscle tissue was taken and preserved separately in 95% ethanol. Specimens were deposited in Chengdu Institute of Biology, Chinese Academy of Sciences (CIB, CAS).

Figure 1. 

Location of the type locality of Leptobrachella chishuiensis sp. nov., Chishui National Nature Reserve, Chishui City, Guizhou Province, China.

Molecular phylogenetic analyses. All eight specimens of the new taxon were included in the molecular analyses (Table 1). For phylogenetic analyses, the corresponding gene sequences for all those related species for which comparable sequences were available were also downloaded from GenBank (Table 1) mainly based on previous studies (Chen et al. 2018; Wang et al. 2019; Luo et al. 2020). Corresponding sequences of Leptobrachium tengchongense, one Leptobrachium huashen, and one Megophrys major were also downloaded from GenBank, and used as outgroups according to previous phylogenetic works (Chen et al. 2018; Wang et al. 2019; Luo et al. 2020).

Total DNA was extracted using a standard phenol-chloroform extraction protocol (Sambrook et al. 1989). The mitochondrial 16S rRNA gene (16S) sequences were amplified, and the primers P7 (5’-CGCCTGTTTACCAAAAACAT-3’) and P8 (5’-CCGGTCTGAACTCAGATCACGT-3’) were used following Simon et al. (1994). Gene fragments were amplified under the following conditions: 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 30 s and extending at 72 °C for 70 s. Sequencing was conducted using an ABI3730 automated DNA sequencer in Shanghai DNA BioTechnologies Co., Ltd. (Shanghai, China). New sequences were deposited in GenBank (for GenBank accession numbers see Table 1).

Sequences were assembled and aligned using the Clustalw module in BioEdit v. 7.0.9.0 (Hall 1999) with default settings. Phylogenetic analyses were conducted 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. We ran JMODELTEST v. 2.1.2 (Darriba et al. 2012) with Akaike and Bayesian information criteria on the alignment, resulting in the best-fitting nucleotide substitution models of GTR + I + G for the data used in ML and BI analyses. For the ML analysis, branch supports were drawn from 10,000 nonparametric bootstrap replicates. In BI analysis, 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. Finally, genetic distance between Leptobrachella species based on uncorrected p-distance model was estimated on 16S gene using MEGA v. 6.06 (Tamura et al. 2013).

Morphological comparisons. All eight adult specimens (Table 2) of the new taxon were measured. The terminology and methods followed Fei et al. (2005), Mahony et al. (2011), and Wang et al. (2019). Measurements were made with a dial caliper to the nearest 0.1 mm (Watters et al. 2016) with digital calipers. Corresponding measurements of L. bijie and L. purpuraventra were retrieved from Wang et al. (2019). Twenty-three morphometric characters of adult specimens were measured:

ED eye diameter (distance from the anterior corner to the posterior corner of the eye);

FIL first finger length (distance from base to tip of finger I);

FIIL second finger length (distance from base to tip of finger II);

FIIIL third finger length (distance from base to tip of finger III);

FIVL fourth finger length (distance from base to tip of finger IV);

FL foot length (distance from tarsus to the tip of the fourth toe);

HDL head length (distance from the tip of the snout to the articulation of jaw);

HDW head width (greatest width between the left and right articulations of jaw);

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

IND internasal distance (minimum distance between the inner margins of the external nares);

IOD interorbital distance (minimum distance between the inner edges of the upper eyelids);

LAL length of lower arm and hand (distance from the elbow to the distal end of the Finger IV);

LW lower arm width (maximum width of the lower arm);

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

SL snout length (distance from the tip of the snout to the anterior corner of the eye);

SVL snout-vent length (distance from the tip of the snout to the posterior edge of the vent);

TYD maximal tympanum diameter;

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

TFL length of foot and tarsus (distance from the tibiotarsal articulation to the distal end of the toe IV);

THL thigh length (distance from vent to knee);

TL tibia length (distance from knee to tarsus);

TW maximal tibia width;

UEW upper eyelid width (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 character to SVL was calculated and then was log-transformed for the following morphometric analyses. Mann-Whitney U tests were conducted to test the significance of differences on morphometric characters between the undescribed species, L. bijie and L. purpuraventra. The significance level was set at 0.05. Furthermore, principal component analyses (PCA) were conducted to highlight whether the different species were separated in morphometric space. Due to only the measurements SVL, HDL, HDW, SL, IND, IOD, ED, TYD, TEY, LAL, ML, TL, HLL, and FL of male L. bijie and L. purpuraventra being available from Wang et al. (2019), the morphometric analyses were conducted only based on these 14 morphometric characters for male group.

The new taxon was also compared with all other congeners of Leptobrachella based on morphological characters. Comparative morphological data were obtained from literatures (Table 3).

Bioacoustics analyses. The advertisement calls of the new taxon were recorded from the holotype specimen CIBCS20190518047 in the field on 18 May 2019 in Chishui National Nature Reserve, Chishui City, Guizhou Province, China. The advertisement call of the new species was recorded in the stream at ambient air temperature of 20 °C and air humidity of 87%. SONY PCM-D50 digital sound recorder was used to record within 20 cm of the calling individual. The sound files in wave format were resampled at 48 kHz with sampling depth 24 bits. Calls were recoded and examined as described by Wijayathilaka and Meegaskumbura (2016). Call recordings were visualized and edited with SoundRuler v. 0.9.6.0 (Gridi-Papp 2003–2007) and Raven Pro v. 1.5 software (Cornell Laboratory of Ornithology, Ithaca, NY, USA). Ambient temperature of the type locality was taken by a digital hygrothermograph.

Aligned sequence matrix of 16S contained 537 bps. ML and BI analyses based on the 16S matrix resulted in essentially identical topologies (Fig. 2). All six samples of the new taxon were clustered into one monophyletic group (node supports in ML and BI: 94 and 0.95) nested into Leptobrachella, and was a sister taxon to L. bijie (node supports in ML and BI: 92 and 1.00). The genetic distance between the new taxon and its closest relatives L. bijie was 2.1%, at the same level with that between L. alpina and L. purpura (2.1%; Suppl. material 1: Table S1).

Figure 2. 

Maximum Likelihood (ML) tree based on the mitochondrial 16S rRNA sequences. Bootstrap supports from ML analyses/Bayesian posterior probabilities from Bayesian Inference (BI) analyses are labelled beside nodes. Information of samples 1–83 refer to Table 1.

In PCA for male group, the total variation of the first two principal components was 64.6%. In males on the two-dimensional plots of PC1 vs. PC2, the undescribed species could be distinctly separated from L. bijie and L. purpuraventra (Fig. 3). The results of Mann-Whitney U tests indicated that in males, the new taxon was significantly different from L. bijie and L. purpuraventra on many morphometric characters (all p-values < 0.05; Table 4).

Figure 3. 

Plots of the first principal component (PC1) versus the second (PC2) for Leptobrachella chishuiensis sp. nov., L. bijie, and L. purpuraventra in males from a principal component analysis.

There were many differences in sonograms and waveforms of calls between the new species L. bijie, and L. purpuraventra. Firstly, a call contains 1–4 notes in the new species and only contains two notes of each call in L. bijie and L. purpuraventra. Secondly, the dominant frequency of the new species is higher than L. bijie and L. purpuraventra.

Based on the molecular, morphological, and bioacoustics differences, the specimens from Chishui City, Guizhou Province, China represent a new species which is described as follows.

The Asian leaf litter toads of Leptobrachella have low vagility and are in exclusive association with montane forests, and their populations are often highly structured. Underestimation of species diversity occurs in the genus, which suggests a high degree of localized diversification and micro-endemism (Fei et al. 2012; Chen et al. 2018). Many cryptic species were proposed by molecular analyses in areas where surveys are weak (Chen et al. 2018), but in Guizhou Province the investigation into the genus was poor although this area was likely to be an important transition zone for many clades or lineages (Chen et al. 2018). Additionally, in Guizhou Province, many new amphibian species has been described in recent years (Zhang et al. 2017; Li et al. 2018a, b; Li et al. 2019a, b; Lyu et al. 2019; Wang et al. 2019; Wei et al. 2020), including two species of Leptobrachella, indicating the underestimated species diversity of amphibians in this region. To date, in Guizhou Province, seven Leptobrachella species were recorded, i.e., Leptobrachella chishuiensis sp. nov., L. liui, L. oshanensis L. purpuraventra, L. bijie, L. ventripunctata, and L. suiyangensis (Fei et al. 2012; Li et al. 2016; Wang et al. 2019; Luo et al. 2020). It is expected that in this area, the species diversity of Leptobrachella may be underestimated, and more investigation should be conducted for detecting richness of the toad species.

The new species is found along clear water rocky streams from Chishui County, Guizhou Province, China, and little is known about the population status of the new species. Thus, further research on the true distribution, population size and trends, and conservation actions are required.

We would like to thank Jian Wang for supplying measurements of some species. This work was supported by Project supported by the Biodiversity investigation, Observation and Assessment Program (2019–2023) of Ministry of Ecology and Environment of China, National Natural Science Foundation of China (No. 31960099), Basic research project of science and technology department of Guizhou Province (No. [2020] 1Y083), Science and technology support project of science and technology department of Guizhou Provincial (No. [2020] 4Y029), Guizhou Provincial Department of Education Youth Science and Technology Talents Growth Project (Nos. KY [2018] 455 and KY [2018] 468). Forestry Science and Technology Research Project of Guizhou Forestry Department (No. [2020] 13).