Confirmation of Leptobrachellaventripunctata (Fei, Ye, and Li, 1990), based on molecular and morphological evidence in Thailand

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 Leptobrachellaventripunctata (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.


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. , Suwannapoom et al. 2017. Currently, Thailand is known to be home to 194 amphibians, while at least 29 species are considered as being endemic ). 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). According to , 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 , Frost 2021. There is no evidence of records for L. ventripunctata in Thailand . 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 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.

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.

Molecular analysis
Genomic DNA was extracted from the liver tissue sample using standard phenolchloroform 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   . Apart from the phylogenetic tree-based methods, we also calculated the degree of row pairwise sequence divergence using uncorrected pdistances 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)

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%).
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).
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.
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: I4).
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 reddishbrown 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.  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.  (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.

Discussion
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. 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.