Biodiversity Data Journal : Taxonomic Paper
PDF
Taxonomic Paper
Confirmation of Leptobrachella ventripunctata (Fei, Ye, and Li, 1990), based on molecular and morphological evidence in Thailand
expand article infoYun-He Wu, Parinya Pawangkhanant§, Jin-Min Chen|, Wei Gao, Chatmongkon Suwannapoom§, Jing Che
‡ State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China
§ Division of Fishery, School of Agriculture and Natural Resources, University of Phayao, Phayao, Thailand
| Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological Resources, College of Life Sciences, Anhui Normal University, Anhui, China
Open Access

Abstract

Background

Thailand is considered a global biodiversity hotspot that is known to harbour a striking diversity of endemic species. However, several research studies have determined that the level of amphibian diversity in the country has been significantly underestimated. The megophryid genus Leptobrachella Smith, 1925 is currently known to include 89 species that are primarily distributed throughout southern China and Southeast Asia; however, only seven species have been found in Thailand.

New information

Based on an integrative approach encompassing genetic and morphological analyses, we have concluded that the population identified from Chiang Rai Province of Thailand is conspecific with Leptobrachella ventripunctata (Fei, Ye, and Li, 1990). Importantly, this is the first confirmation record of this species, based on molecular and morphological evidence in Thailand. The discovery of this species reaffirms that the diversity within the genus has been underestimated with many species yet to be discovered. In addition, the findings of our study further highlight the lack of existing knowledge on amphibian taxonomy and an underestimation of the biodiversity that exists along these national border areas.

Keywords

Megophryidae, national new record, Chiang Rai Province, 16S rRNA

Introduction

As a result of the increasing amounts of attention and effort devoted to herpetological research studies in Thailand over the past decade, the present knowledge of Thailand’s amphibian fauna has grown rapidly with newly-described taxa and the addition of new records from the country (e.g. Poyarkov et al. 2018, Suwannapoom et al. 2017, Suwannapoom et al. 2016, Wu et al. 2019, Yan et al. 2016). Currently, Thailand is known to be home to 194 amphibians, while at least 29 species are considered as being endemic (Poyarkov et al. 2021). However, many regions of the country, particularly along the northern borders of Thailand, have not yet been surveyed in full detail. Perhaps the most notable of these regions would be the tropical region along the Thailand-Myanmar border in Doi Tung, Chiang Rai Province.

The frog genus Leptobrachella Smith, 1925 is a key component of the Southeast Asian herpetofauna, which currently includes 89 species mainly being distributed throughout southern China, north-eastern India, Indochina, Malaya, Borneo and Natuna Island (Chen et al. 2018, Frost 2021). Frogs of this genus are small in size and inhabiting forest floors and rocky streams in hilly evergreen forests. Furthermore, frogs of this genus are often difficult to find and identify down to the species level due to their inconspicuous and morphological conservatism. However, recent increases in survey efforts, along with the use of updated molecular and acoustic data, have revealed an underestimation of the taxonomic diversity of the genus. These new efforts have established descriptions of many new species, while also yielding some newly-recorded species (e.g. Chen et al. 2018, Stuart and Rowley 2020, Yuan et al. 2017). According to Poyarkov et al. (2021), nine species of the genus Leptobrachella are distributed in Thailand, which include L. fuliginosa, L. heteropus, L. melanoleuca, L. minima, L. pelodytoides, L. sola, L. bourreti, L. ventripunctata and L. zhangyapingi. However, records of L. bourreti for Thailand seem to be based on misidentifications and need to be verified by further studies (Poyarkov et al. 2021, Frost 2021). There is no evidence of records for L. ventripunctata in Thailand (Poyarkov et al. 2021). Thus, only seven species of this genus are currently undisputed in Thailand.

The species Leptobrachella ventripunctata was originally described from Mengla County, Yunnan, China (Fei et al. 1990). Recently, relevant research studies have observed new distributions of this species. For example, Luong et al. (2019) reported distribution of this species in Dien Bien Province, Vietnam. In addition, Chen et al. (2018) recorded this species in Caiyanghe, Yunnan, China, as well as localities in Tuyen Quang, Thanh Hoa, Cao Bang, Lao Cai and Phu Tho Provinces in Vietnam. To date, this species is known to be distributed widely throughout southern Yunnan in China, Phongsaly in Laos and in the Son La, Vinh Phuc, Thanh Hoa, Tuyen Quang, Cao Bang and Dien Bien Provinces of northern Vietnam (Frost 2021).

During recent field surveys, conducted in Chiang Rai Province of northern Thailand in 2017, we collected one specimen that we have assigned to the genus Leptobrachella, based on morphological characteristics. Subsequent detailed morphological comparisons and phylogenetic analyses indicate that the newly-identified species in Chiang Rai Province should be assigned to L. ventripunctata. Therefore, we have further confirmed the presence of L. ventripunctata in Thailand.

Materials and methods

Sampling

Field work was conducted in the environs of Doi Tung, Chiang Rai Province, Thailand (20°19'36.1"N, 99°49'35.0"E, 650 m a.s.l.) and one specimen was collected by Chatmongkon Suwannapoom on 16 July 2017 (Fig. 1). The specimen was photographed in situ. The specimen was euthanised using benzocaine, then liver tissue was extracted, which was stored in 95% ethanol. The voucher specimen was fixed with 10% formalin and later stored in 70% ethanol. The voucher specimen and tissue sample were then deposited in the herpetological collections of the School of Agriculture and Natural Resources, University of Phayao (AUP), Phayao, Thailand.

Figure 1.  

Map showing the new record in Thailand (red circle) and the type locality of L. ventripunctata (red star) in China.

Molecular analysis

Genomic DNA was extracted from the liver tissue sample using standard phenol-chloroform protocols (Sambrook et al. 1989). A partial fragment of the mitochondrial gene 16S rRNA (16S) was amplified and sequenced using the following primers: 16SAR (5'-CGCCTGTTTAYCAAAAACAT-3'; Kocher et al. 1989) and 16SBR (5'-CCGGTYTGAACTCAGATCAYGT-3'; Kocher et al. 1989). Amplification was performed in a 25 µl volume reaction according to the following procedure: initial denaturation at 95°C for 5 min, 35 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1 min and a final extension at 72°C for 10 min. PCR products were purified using a Gel Extraction Mini Kit (A T G C, Bangkok, Thailand). All sequencing was conducted on an ABI PRISM 3730 automated sequencer (Applied Biosystems, Foster City, CA, USA). The new sequence was first assembled and edited using AutoSeqMan (Sun 2018).

To study the existing phylogenetic relationships amongst Leptobrachella, phylogenetic trees were reconstructed, based on the partial mitochondrial 16S rRNA gene. Homologous sequences of the related species in the genus Leptobrachella and those of the outgroups Megophrys glandulosa (KIZ048439) and Leptobrachium huashen (KIZ049025) (Chen et al. 2018) were downloaded from GenBank (Table 1). All sequences were aligned using MUSCLE 3.6 (Edgar 2004), visually checked for accuracy and then trimmed to minimise missing characters in MEGA v.6.0.6 (Tamura et al. 2013).

Table 1.

Localities, voucher ID and GenBank numbers for all samples used in this study.

ID

Species

Voucher ID

Locality

16S rRNA

Reference

Ingroup

1

Leptobrachella bourreti

KIZ01599

Jinxiu, Guangxi, China

MH055870

Chen et al. 2018

2

Leptobrachella bourreti

KIZ048947

Dawei Shan, Yunnan, China

MH055871

Chen et al. 2018

3

Leptobrachella eos

ZMMU-NAP-02278

Vientiane, Laos

MH055880

Chen et al. 2018

4

Leptobrachella tengchongensis

SYS a004598

Gaoligong Shan, Yunnan, China

KU589209

Yang et al. 2016

5

Leptobrachella tengchongensis

SYS a003766

Gaoligong Shan, Yunnan, China

MH055897

Chen et al. 2018

6

Leptobrachella puhoatensis

IEBR ADPH049

Pu Hu Nature Reserve, Thanh Hoa, Vietnam

MH055898

Chen et al. 2018

7

Leptobrachella liui

SYS a004035

Wugong Shan, Jiangxi, China

MH055916

Chen et al. 2018

8

Leptobrachella laui

SYS a002450

Shenzhen, Guangdong, China

MH055904

Chen et al. 2018

9

Leptobrachella maoershanensis

KIZ07614

Mao’er Shan, Guangxi, China

MH055927

Chen et al. 2018

10

Leptobrachella minima

NCSM 79224

Louangphrabang, Luang Prabang, Laos

MH055845

Chen et al. 2018

11

Leptobrachella aereus

NCSM 76038

Vilabuly, Savannakhet, Laos

MH055809

Chen et al. 2018

12

Leptobrachella ventripunctata

ROM 35750

Pu'er, Yunnan, China

MH055828

Chen et al. 2018

13

Leptobrachella ventripunctata

ROM 28378

Sapa, Lao Cai, Vietnam

MH055829

Chen et al. 2018

14

Leptobrachella ventripunctata

KIZ020421

Jinuo Shan, Yunnan, China

MH055825

Chen et al. 2018

15

Leptobrachella ventripunctata

KIZ014379

Caiyanghe, Yunnan, China

MH055826

Chen et al. 2018

16

Leptobrachella ventripunctata

KIZ020428

Maandi, Yunnan, China

MH055827

Chen et al. 2018

17

Leptobrachella ventripunctata

IEBR ADPH050

Pu Hu Nature Reserve, Thanh Hoa, Vietnam

MH055819

Chen et al. 2018

18

Leptobrachella ventripunctata

NCSM 77715

Viengthong, Houaphanh, Laos

MH055820

Chen et al. 2018

19

Leptobrachella ventripunctata

NCSM 80550

Boun Tay, Phongsaly, Laos

MH055821

Chen et al. 2018

20

Leptobrachella ventripunctata

NCSM 80783

Kham, Xiangkhouang, Laos

MH055822

Chen et al. 2018

21

Leptobrachella ventripunctata

Tissue ID: YPX32065

Huanglianshan National Nature Reserve, Yunnan, China

MH055823

Chen et al. 2018

22

Leptobrachella ventripunctata

KIZ013621

Wenlong, Yunnan, China

MH055824

Chen et al. 2018

23

Leptobrachella ventripunctata

ZMMU-NAP-05008

Muong Nhe Nature Reserve, Dien Bien, Vietnam

MH055830

Chen et al. 2018

24

Leptobrachella ventripunctata

ZMMU-A-5225

Xuan Son National Park, Phu Tho, Vietnam

MH055835

Chen et al. 2018

25

Leptobrachella ventripunctata

ZMMU-NAP-02272

Hoa Binh, Vietnam

MH055836

Chen et al. 2018

26

Leptobrachella ventripunctata

ZMMU-NAP-05061

Kim Son, Nghe An, Vietnam

MH055837

Chen et al. 2018

27

Leptobrachella ventripunctata

ZMMU-NAP-02277

Thuong Xuan, Thanh Hoa, Vietnam

MH055838

Chen et al. 2018

28

Leptobrachella ventripunctata

ROM 27051

Quang Thanh Village, Cao Bang, Vietnam

MH055832

Chen et al. 2018

29

Leptobrachella ventripunctata

ZMMU-NAP-02270

Bac Giang, Vietnam

MH055833

Chen et al. 2018

30

Leptobrachella ventripunctata

ZMMU-A-5604-06331

Vinh Phuc, Vietnam

MH055834

Chen et al. 2018

31

Leptobrachella ventripunctata

SYS a004536

Zhushihe, Yunnan, China

MH055831

Chen et al. 2018

32

Leptobrachella ventripunctata

ROM 6945

Na Hang Nature Reserve, Tuyen Quang, Vietnam

MH055839

Chen et al. 2018

33

Leptobrachella ventripunctata

AUP-00326

Doi Tung, Chiang Rai, Thailand

OK430887

This study

34

Leptobrachella nyx

AMNH A163810

Mount Tay Conn Linh, Ha Giang, Vietnam

DQ283381

Frost 2021

35

Leptobrachella nyx

ROM 35606

Malipo, Yunnan, China

MH055814

Chen et al. 2018

36

Leptobrachella pluvialis

ROM 30685

Fansipan, Lao Cai, Vietnam

MH055843

Chen et al. 2018

37

Leptobrachella nahangensis

ROM 7035

Na Hang Nature Reserve, Tuyen Quang, Vietnam

MH055853

Chen et al. 2018

38

Leptobrachella melanoleuca

KIZ018031

Kapoe, Ranong, Thailand

MH055967

Chen et al. 2018

39

Leptobrachella dringi

KUHE:55610

Gunung Mulu, Malaysia

AB847553

Matsui et al. 2014

40

Leptobrachella heteropus

KUHE 15487

Larut, Perak, Malaysia

AB530453

Matsui et al. 2014

41

Leptobrachella kajangensis

LSUHC:4431

Tioman, Malaysia

LC202001

Matsui et al. 2017

Outgroups

42

Megophrys glandulosa

KIZ048439

Yunnan, China

KX811762

Chen et al. 2016

43

Leptobrachium huashen

KIZ049025

Yunnan, China

KX811931

Chen et al. 2016

Phylogenetic reconstructions using Bayesian Inference (BI) and Maximum Likelihood (ML) were executed in the CIPRES web server (Miller et al. 2010). Data were tested in JMODELTEST 2.1.7 (Darriba et al. 2012) using Bayesian Information Criteria to provide the best-fitting nucleotide substitution models (BIC; Posada 2008). For BI analyses, two separate runs were performed with four Markov chains using the GTR+I+G model. Each run was conducted for 10 million generations, while every 100 generations were sampled with a burn-in value of 25%. Convergence was assessed by the average standard deviation of split frequencies (below 0.01) and ESS values (over 200) in TRACER 1.5 (Rambaut and Drummond 2009). ML analysis was performed using RAxML with 1,000 bootstrap replications using the rapid bootstrap feature under the GTR+G model (random seed value of 12,345) (Stamatakis 2014). Apart from the phylogenetic tree-based methods, we also calculated the degree of row pairwise sequence divergence using uncorrected p-distances and complete deletion implemented in MEGA v.6.0.6 (Tamura et al. 2013).

Morphology

Measurements were taken using a digital caliper to the nearest 0.1 mm. Abbreviations are presented following the method employed by Matsui (1984) for 25 morphological characteristics: (1) Snout-vent length (SVL); (2) Head length (HL); (3) Head width (HW); (4) Snout length (SL); (5) Distance from the centre of the nostril to the tip of the snout (SN); (6) Nostril-eye distance (N-EL); (7) Eye diameter (ED); (8) Tympanum diameter (TD); (9) Internarial distance (IND); (10) Interorbital distance (IOD); (11) Upper eyelid width (UEW); (12) Forelimb length (FLL); (13) Lower arm length (LAL); (14) Hand length (HAL); (15) First finger length (1FL); (16) Third finger disc diameter (3FDD); (17) Outer palmar tubercle length (OPTL); (18) Inner palmar tubercle length (IPTL); (19) Tibia length (TL); (20) Foot length (FL); (21) Hind-limb length (HLL); (22) Fourth toe disc diameter (4TDD); (23) Inner metatarsal tubercle length (IMTL); (24) Outer metatarsal tubercle length (OMTL) and (25) First toe length (1TOEL).

Data resources

Molecular Phylogeny

The final aligned dataset of 16S nucleotide sequences contained 41 individuals with 510 bp. Amongst the 510 sites, 324 were established as conserved sites and 184 were considered variable sites, of which 127 were found to be potentially parsimony-informative sites (excluding outgroups). The Bayesian Inference (BI) and Maximum Likelihood (ML) phylogenetic trees yielded essentially identical topologies. This was true, except for the poorly-supported nodes, which have been integrated in Fig. 2. The phylogenetic analysis suggested that the newly-collected singular specimen from Chiang Rai was nested in the genus Leptobrachella and formed a monophyletic clade with L. ventripunctata obtained from China, Laos and Vietnam with strong support (Bayesian posterior probabilities (PP) = 1.00; ML bootstrap support (BS) = 100%).

Figure 2.  

Phylogram of Leptobrachella resulting from the analyses of one fragment of the mitochondrial 16S gene. Nodal support values with Bayesian posterior probabilities (BPP) >= 95%/bootstrap support (BS) >= 70 are shown near the node. A “–” denotes Bayesian posterior probabilities (BPP) < 95% and bootstrap support (BS) < 70. Node values with Bayesian posterior probabilities (BPP) < 95%/bootstrap support (BS) < 70 are not shown.

Interspecific genetic divergene (uncorrected p-distance) between the new sample obtained from Chiang Rai, Thailand and the other species of Leptobrachella varied from 4.9% (versus L. bourreti) to 17.9% (versus L. heteropus) (Fig. 3, Suppl. material 1, Suppl. material 2). The genetic divergence between the individual specimen of Leptobrachella, collected from Chiang Rai and the L. ventripunctata specimens collected from Vietnam, Laos and China, was found to be very small (0–0.4%, Suppl. material 2).

Figure 3.  

Heatmap of pairwise uncorrected p-distances of the 16S rRNA gene of Leptobrachella species included in phylogenetic analyses. The colours red to blue indicate high to low divergences.

Morphologically, the specimen from Chiang Rai shows a similar appearance with original description of L. ventripunctata: pupil vertical; iris distinctly bicoloured; fingers with lateral dermal fringes, absent for toes; belly creamy white with many scattered brown spots. Therefore, we determined that AUP-00326 belonged to L. ventripunctata.

Taxon treatment

Leptobrachella ventripunctata (Fei, Ye & Li, 1990)

Material    Download as CSV 
  1. scientificName:
    Leptobrachella ventripunctata
    ; class:
    Amphibia
    ; order:
    Anura
    ; family:
    Megophryidae
    ; genus:
    Leptobrachella
    ; specificEpithet:
    ventripunctata
    ; country:
    Thailand
    ; countryCode:
    TL
    ; stateProvince:
    Chiang Rai
    ; locality:
    Doi Tung
    ; verbatimElevation:
    650 m
    ; verbatimLatitude:
    20°19'36.1"N
    ; verbatimLongitude:
    99°49'35.0"E
    ; individualCount:
    1
    ; sex:
    male
    ; lifeStage:
    adult
    ; catalogNumber:
    AUP-00326
    ; basisOfRecord:
    preserved specimen

Description

Morphological descriptions of the specimen obtained from Thailand (measurements shown in Suppl. material 3) are as follows: adult male with SVL 28.9 mm; head length (HL 13.1 mm, 45.3% of SVL) longer than width (HW 11.6 mm, 40.1% of SVL); snout slightly pointed, projecting beyond lower jaw; nostrils slightly closer to eyes than tip of snout (N-EL 2.5 mm, 8.7% of SVL; SN 2.7 mm, 9.3% of SVL); canthus rostralis distinct; loreal region concave; snout (SL 5.3 mm, 18.3% of SVL) longer than eye diameter (EL 4.6 mm, 15.9% of SVL); width of upper eyelid (UEW 4.5 mm) larger than interorbital distance (IOD 3.5 mm) and internasal distance (IND 3.1 mm); tympanum distinct, rounded, length (TD 2.4 mm, 8.3% of SVL) nearly half the diameter of the eye; vomerine teeth absent; pupil vertical; tongue pyriform, deeply notched posteriorly; supratympanic fold distinct, running from posterior corner of eye towards axilla (Fig. 4).

Figure 4.  

Male of L. ventripunctata (AUP-00326) in life. A. Dorsolateral view B. ventral view C. lateral view of the head. Photo by P. Pawangkhanant.

Forelimbs slender; relative finger lengths: I<II<IV<III; tips of all fingers rounded and slightly swollen; fingers with lateral dermal fringes; no webbing between fingers; subarticular tubercles distinct, large, near the palm smaller; two palmar tubercles, inner palmar tubercle large, rounded (IPTL 1.6 mm, 5.5% of SVL), outer palmar tubercle relatively small (OPTL 1.0 mm, 3.5% of SVL) (Fig. 4).

Hind-limbs long, foot slightly shorter than tibia, tibia length (TL) 56.1% of SVL, foot length (FL) 51.6% of SVL; tibial-tarsal articulation beyond the tip of the eye when the hind-limb is adpressed along the side of the body; heels overlapping when the flexed legs are held at right angles to the body axis; relative toe length: I<II<III<IV<V; tips of toes rounded and slightly swollen; rudimentary webbing between toes; subarticular tubercles distinct, rounded, inner metatarsal tubercle distinct and oval (IMTL 0.9 mm, 3.1% of SVL), outer metatarsal tubercle distinct (1.0 mm, 3.5% of SVL) (Fig. 4).

Dorsal surfaces of head, body, thigh, tibia and flank of body appear relatively rough with numerous granular spots; ventral surfaces smooth; pectoral gland and femoral gland distinct, oval; pectoral glands larger than femoral glands; supra-axillary gland raised. Ventrolateral gland distinctly visible, forming discontinuous cream-white lines on flanks (Fig. 4).

Color in life: 

Dorsal surface appears reddish brown with small reddish tubercles; a dark inverted triangular marking in the interorbital region, a "W"-shaped marking appears between axillae; flanks scattered with some distinct moderate black blotches; upper lips with three black vertical bars; loreal and tympanic region with distinct black markings; supratympanic ridge appears reddish and lower margin of supratympanic fold appears black; elbow to upper arm and tibio-tarsal articulation distinct and reddish-brown in colour on the dorsum; dorsal surface of lower arms, legs, fingers and toes with dark brown crossbars; throat pale with some dusting; belly creamy white with many scattered brown spots; ventrolateral glands, pectoral glands and femoral glands appear white or yellowish-white; ventral surface of limbs appear grey brown with dark brown and white speckling or dots; iris distinctly bicoloured, typically bright orange-red on upper half and silvery-white on lower half (Fig. 4).

Distribution

This species was previously only known to be indigenous to extreme southern Yunnan, China; Phongsaly, Xiangkhouang and Houaphanh Provinces in Laos; as well as Son La, Vinh Phuc, Thanh Hoa, Tuyen Quang, Cao Bang, Nghe An, Bac Giang, Phu Tho, Hoa Binh, Lao Cai and Dien Bien Provinces in Vietnam (Chen et al. 2018, Frost 2021). Importantly, this first specific record of L. ventripunctata in Thailand substantially expands the known area of distribution of this species.

Ecology

This species lives mainly in the litter on both sides of streams. An individual specimen was observed at night sitting on the branch of a shrub that was about 1-2 m off the ground in an evergreen forest surrounded by trees near a stream with nearby herbaceous plants (Fig. 5). This species has been associated with the sympatric distribution of Micryletta inornata and Limnonectes taylori.

Figure 5.  

Habitat at collection site of L. ventripunctata in Chiang Rai Province, Thailand.

Discussion

Poyarkov et al. (2021) mentioned the distribution of L. ventripunctata in the extreme north of Thailand, but did not provide any evidence, such as the specific locality, voucher specimens or DNA sequences. This is the first record of the species with morphological data and molecular evidence.

Thailand represents an important component of the Indo-Burma biodiversity hot-spot. Its northern region lies at a biogeographic crossroads where the fauna of China, Indochina, India and Himalaya converge (Myers et al. 2000). Recently, along with an increasing intensity of field investigations and the application of new techniques, biodiversity studies in this region have expanded. Consequently, many new species and records have been described and reported (e.g. Lorphengsy et al. 2021, Wu et al. 2019). The confirmation of L. ventripunctata in this study increases the total number of known amphibian species in Thailand from 125 (Khonsue and Thirakhupt 2001) to 194, along with the known number of Leptobrachella species from seven to eight. The following species of Leptobrachella are known to be from Thailand: L. ventripunctata, L. pelodytoides, L. fuliginosa, L. heteropus, L. melanoleuca, L. minima, L. sola and L. zhangyaping. The discovery of this species reaffirms that, to date, the diversity within the genus has been underestimated with many species yet to be discovered in this country.

Furthermore, our study further highlights a lack of existing knowledge on amphibian taxonomy and identifies an underestimation of the potential biodiversity along these national border areas. In recent years, many new recorded species have been found in border areas, such as Nidirana chapaensis (Yuan et al. 2019), Gracixalus quangi (Lorphengsy et al. 2021) and Theloderma pyaukkya (Du et al. 2020), while a number of studies have called for enhanced surveys of species diversity in these border areas (Chen et al. 2020, Chen et al. 2018, Wu et al. 2020, Yuan et al. 2019). The findings of our research further support this view. The newly-recorded species we reported was found along the border of Thailand and Myanmar, which is very close to Laos. Currently, distributions of this species have been recorded in Laos and Thailand, whereas they have not been definitively reported in Myanmar (Chen et al. 2018, Frost 2021). Considering that it is located in the same zoogeographic region comprised of continuous forests habitats, we suspect that this species is also present in Myanmar. In the future, it will be important to strengthen levels of international cooperation in order to further clarify the full range of this species.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (NSFC 31900323, 32100371) to J.M.C and Y.H.W., the International Partnership Program of CAS (152453KYSB20170033), Southeast Asia Biodiversity Research Institute, CAS (Y4Y4ZK111B01: 2017CASSEABRIQG002) and the Animal Branch of the Germplasm Bank of Wild Species, CAS (Large Research Infrastructure Funding) to J.C.; the Unit of Excellent 2022 on Biodiversity and Natural Resources Management, University of Phayao (FF65-UoE003) and the Thailand Research Fund: DBG6180001 to C.S. We thank Mali Naiduangchan for their assistance and support in fieldwork. We thank reviewers and editor for their helpful comments and constructive suggestions. Specimen was collected under approval from the Institute of Animal for Scientific Purposes Development (IAD), which issued fieldwork permission (No. 610104022).

References

Supplementary materials

Suppl. material 1: Mean pairwise uncorrected p-distance 
Authors:  Wu et al.
Data type:  Table
Brief description: 

The mean pairwise uncorrected p-distance (%) of 16S rRNA gene amongst the species of Leptobrachella. Sample ID corresponds to those in Table 1

Suppl. material 2: Average uncorrected p-distances 
Authors:  Wu et al.
Data type:  Table
Brief description: 

Average uncorrected p-distances amongst those calculated from 16S rRNA gene sequences

Suppl. material 3: Measurement and proportions 
Authors:  Wu et al.
Data type:  Table
Brief description: 

Measurement (in mm) of and proportions of Leptobrachella ventripunctata.