We report the first country record of Leptobrachella eos (Ohler, Wollenberg, Grosjean, Hendrix, Vences, Ziegler & Dubois, 2011) from Thailand, based on one specimen collected from Chom poo Phuka nature trail, Bo Kluea District. Morphologically, the specimen displayed good agreement with the original descriptions provided for L. eos. Phylogenetically, the specimens clustered according to the sequences of type locality of L. eos. Notably, our discovery increases the number of Leptobrachella species known to occur in Thailand to nine.

Amphibia, Leptobrachella eos, new record, Thailand

Thailand is an important component of the Indo-Burma biodiversity hotspot and its northern region is located in the same zoogeographic region where the faunas of China, Indochina, India and Himalaya converge (Myers et al. 2000). The amphibian fauna in Thailand is rich in terms of species count and endemism (Frost 2021). In recent years, several new and new record species of amphibians have been described (e.g. Matsui et al. 2018; Wu et al. 2019; Suwannapoom et al. 2021). These results suggest that the rich amphibian diversity in the region remains underestimated.

The genus Leptobrachella was originally described by Smith (1925) and Leptobrachella mjöbergi Smith from Sarawak (Malaysia) was designated as the type species. Dubois (1980) erected a new genus Leptolalax with the type species Leptobrachium gracile Günther. However, Chen et al. (2018) suggested that Leptolalax was a junior synonym of the genus Leptobrachella based on a large-scale molecular analysis. Currently, frogs of the genus Leptobrachella are renowned for their extraordinary diversity in small-sized, forest-floor specialists that inhabit montane evergreen forest throughout Oriental Zoogeographic Region (Chen et al. 2018; Frost 2021). This genus is comprised of 89 currently recognized species (Frost 2021). Among these, more than 70% of recognized species were described in the last two decades (Rowley et al. 2016; Yuan et al. 2017; Chen et al. 2020; Nguyen et al. 2021), which is attributed to intensified survey efforts and use of more integrative taxonomic approaches.

Rosy Litter Frog L. eos was recently described by Ohler et al. (2011) from Long Nai, Phongsaly Province, Laos. The species has since been reported from Dien Bien, Thanh Hoa, and Son La Provinces, northwestern Vietnam and in adjacent Yunnan, China (Pham et al. 2014; Chen et al. 2018; Nguyen et al. 2020).

During fieldwork in 2018, we collected a specimen in Nan Province of northern Thailand that can morphologically be assigned to the genus Leptobrachella. Subsequent studies based on morphological and molecular data indicate that the specimen should be classified as L. eos. Herein, we have reported on one Asian leaf-litter frogs, namely L. eos, for the first time from Thailand.

Field surveys were conducted in Chom poo Phuka nature trail, Nan Province, Bo Kluea District, Thailand (Fig. 1). The specimen was collected and euthanized with ethyl acetate and then fixed in 75% ethanol for storage after taking photographs. Liver tissue sample was preserved in absolute ethyl alcohol for molecular analysis. The specimen and tissue sample were 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. eos (red star) in Laos.

Total genomic DNA was extracted from tissue sample using a standard phenol-chloroform extraction protocol (Sambrook et al. 1989). The mitochondrial gene 16S ribosomal RNA gene (16S rRNA) was amplified and sequenced using the primer pairs 16S rRNA-F (CGCCTGTTTAYCAAAAACAT) and 16S rRNAR (CCGGTYTGAACTCAGATCAYGT) (Kocher et al. 1989). PCR amplification was performed in a 25 μl reaction volume with the following cycling conditions: an initial denaturing step at 95 °C for 5 min; 35 cycles of denaturing at 95 °C for 40 s, annealing at 55 °C for 40 s and extending at 72 °C for 1 min, followed by a final extension step of 72 °C for 10 min. The amplified PCR product was purified using Qiagen PCR purification kit and sequences were obtained from an ABI 3100 automated sequencer. The sequence was deposited in GenBank under accession number (GenBank accession number is available in Table 1).

The newly obtained nucleotide sequence was first assembled and edited using AutoSeqMan (Sun 2018). New sequences incorporated with homologous data downloaded from GenBank were aligned using MUSCLE 3.8 with default settings (Edgar 2004), and then visually checked by eye for accuracy and trimmed to minimize missing characters in MEGA6 (Tamura et al. 2013). Leptobrachium boringii and Xenophrys glandulosa were selected as the outgroups (Chen et al. 2018).

Phylogenetic reconstructions using Bayesian inference (BI) and maximum likelihood (ML) were executed in the CIPRES web server (Miller et al. 2010). ML analysis was conducted with the rapid bootstrapping algorithm using the program RAxML v8.00 (Stamatakis 2014). Nodal support for ML was assessed with 1000 rapid bootstrap replicates (BS). The best-fit substitution model was selected under the Bayesian Information Criterion by the program jModeltest 2.1.4 (Darriba et al. 2012). The best fit substitution model for the 16S dataset was GTR + I + G. For BI analysis, two independent runs were initiated each with four simultaneous Markov Chain Monte Carlo (MCMC) chains for 10 million generations and sampled every 1000 generations. The first 25% generations were discarded as burn-in, and the last remaining trees were used to create a 50% majority-rule consensus tree and estimate Bayesian posterior probabilities (BPP). Pairwise divergences (uncorrected p-distance) between species on 16S dataset were calculated using MEGA6 (Tamura et al. 2013).

Measurements were taken using a digital caliper to the nearest 0.1 mm (Table 1). 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 center 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) Hindlimb 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).

Aligned sequence matrix of 16S gene contained 508 bp, among which, 195 sites were variable and 148 were parsimony-informative (include outgroups). Phylogenetic trees from ML and BI present identical topologies and the node supports are very high except for some internal nodes (Fig. 2). The results indicated that the monophyly of the genus Leptobrachella was strongly supported, and in agreement with results of Chen et al. (2018). The phylogenetic analysis suggested that this specimen was nested in the genus Leptobrachella and formed a monophyletic clade with L. eos obtained from China, Laos, and Vietnam.

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.

Genetic distance on 16S between the specimen of the L. eos collected from Nan province and L. eos from Laos, Vietnam, and China was 3.5%, lower than interspecific genetic distances of other species, varying from 3.9% (between L. bourreti) to 12.8% (between L. minima, L. zhangyapingi, and L. ventripunctata) (Table 2).

Morphologically, the specimen from Nan province shows a similar appearance to the original description of L. eos. Therefore, we considered AUP 00377 to belong to L. eos.

Taxonomic account

 Leptobrachella eos (Ohler, Wollenberg, Grosjean, Hendrix, Vences, Ziegler & Dubois, 2011)

Specimen examined

Adult female (AUP 00377) collected on 05 October 2018 by the Chatmongkon Suwannapoom and Parinya Pawangkhanant from Chom poo Phuka nature trail, Nan Province, Bo Kluea District, Thailand (19.0181°N, 100.9731°E, 1300 m elevation).

Morphological description

(measurements in mm; provided in Table 3). Morphological characters of the specimen from Thailand agreed well with the original description of Ohler et al. (2011). Adult female with SVL 34.2 mm; head length (HL 13.6 mm, 39.7% of SVL) slightly longer than width (HW 12.1 mm, 35.5% of SVL); snout slightly protruding, its length (SL 5.3 mm, 15.5% of SVL) longer than horizontal diameter of eye (EL 4.4 mm, 12.8% of SVL); canthus rostralis rounded, loreal region concave; interorbital space flat, larger (IOD 4.0 mm, 11.6% of SVL) than width of upper eyelid (UEW 2.8 mm, 8.2% of SVL) and internarial distance (IN 3.6 mm, 10.6% of SVL); snout longer than eye diameter (SL/ED 120.5%); tympanum distinct (TD 4.0 mm), rounded, about half eye diameter (ED 4.4 mm); vomerine teeth absent; supratympanic fold distinct; pupil vertical (Fig. 3).

Table 3.

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Measurement (in mm) of and proportions of the Leptobrachella eos (see Materials and methods section for list of abbreviations).

Characters	AUP-00377	Ratio (-/SVL)
SEX	F	-
SVL	34.2	-
HL	13.6	39.7%
SL	5.3	15.5%
ED	4.4	12.8%
N-EL	3.2	9.4%
HW	12.1	35.5%
IND	3.6	10.6%
IOD	4.0	11.6%
UEW	2.8	8.2%
FLL	22.0	64.3%
LAL	16.2	47.3%
HAL	10.1	29.6%
1FL	4.8	13.9%
IPTL	1.5	4.4%
OPTL	1.6	4.6%
3FDD	0.8	2.2%
HLL	53.3	155.8%
TL	16.4	47.9%
FL	23.5	68.6%
IMTL	2.0	5.7%
1TOEL	4.5	13.0%
4TDD	0.8	2.4%
TD	4.0	11.7%
OMTL	2.2	6.3%

Figure 3. 

Dorsolateral view and lateral view of head of L. eos (photos taken by Parinya Pawangkhanant).

Forelimbs slender; lower arm length (LAL 22.0 mm, 47.3% of SVL) shorter than hand length (HAL 10.1 mm, 29.6% of SVL); relative finger lengths: I<II<IV<III; tips of all fingers slightly enlarged; no webbing between fingers; subarticular tubercles distinct, big; two metacarpal tubercles, inner metacarpal tubercle (IPTL 1.5 mm, 4.4% of SVL) almost equal to outer metacarpal tubercle relatively (OPTL 1.6 mm, 4.6% of SVL).

Hindlimbs long, tibia (TL 16.4 mm) about half SVL and shorter and foot (FL 23.5 mm); relative length of toes: I<II<III<V<IV; tibiotarsal articulation reaching the nostril when the leg is stretched forward; heels overlapping when thighs are positioned at right angles to the body; tips of toes rounded and not swollen; rudimentary webbing between toes; subarticular tubercles distinct, rounded; inner metatarsal tubercle distinct and oval (IMTL 2.0 mm, 5.7% of SVL), outer metatarsal tubercle distinct (OMTL 2.2 mm, 6.3% of SVL).

Dorsal skin relatively smooth, with small tubercles; side of head and dorsum shagreened; tiny warts scattered on flanks; supratympanic fold prominent, running from posterior corner of eye towards axilla; dorsal parts of limbs: forelimbs shagreened; thigh and shank with glandular warts; tarsus smooth; femoral glands and pectoral gland distinct, oval; axillary glands indistinct; ventrolateral glands forming continuous white line on flanks.

In life, dorsal surface brown, with reddish-brown W-shaped marking on scapular region; distinct reverse-triangle black marking between eyes; tympanic region brown gray; dorsal surfaces of elbow to upper arm with distinctive reddish-brown coloration; transverse dark-brown bars present on dorsal surface of the limbs; iris distinctly bicolored, bright orange-red in upper half and silvery-white in lower half (Fig. 3).

In preservation. Dorsum of the body and hindlimbs light brownish gray; transverse bars on the limbs distinct, and dark-brown patterns; marks and spots on the back are indistinct; ventral surface of the body is yellowish brown with brown marbling on the sides and chest; axillary glands, femoral, pectoral and ventrolateral glands fade to grayish white.

Ecological notes

Leptobrachella eos was found along a rocky stream in Montane Forest, with dense vegetation of Wild Banana (Musa acuminata) and Bamboo (Cephalostachyum sp.) (Fig. 4). The male was found calling mainly hidden under leaf litter.

Figure 4. 

Habitat at collection site of L. eos in Chiang Rai Province, Thailand (photos taken by Parinya Pawangkhanant)

Distribution

Leptobrachella eos is currently known in Phongsaly, Bolikhamxay, Oudomxai, and Xiasomboun Provinces, Laos; Dien Bien, Thanh Hoa, and Son La Provinces, northwestern Vietnam; Yunnan province, China and Nan province, Thailand.

Currently, seven Leptobrachella species including L. fuliginosa, L. melanoleuca, L. minima, L. pelodytoides, L. sola, L. ventripunctata, L. murphyi, and L. zhangyapingi are recorded in Thailand (Chen et al. 2021; Frost 2021; Wu et al. 2021). The present study supports that this specimen from Chom poo Phuka nature trail, Nan Province, Thailand belongs to L. eos, representing the first record of this species in Thailand. The discovery of L. eos in this study increases the total number of known species in Thailand from 125 (Khonsue and Thirakhupt 2001) to 195 (Frost 2021), along with the known number of Leptobrachella species from eight to nine (Frost 2021). In addition, our results further confirm that amphibian diversity in Thailand has been underestimated. Therefore, investigation and research need to be strengthened in the future.

Although phylogenetic analyses based on 16S suggested that the L. eos is monophyletic it contains three genetically independent clades, and in agreement with results of Chen et al. (2018). In addition, intraspecific genetic distance of this species was over 3.0% , a value that was earlier proposed as a good indicator for candidate new species in frogs (Fouquet et al. 2007). But it is also common to the intraspecific genetic distance over 3% in amphibians and reptiles, such as L. minima (Chen et al. 2018) and Amolops spinapectoralis (Wu et al. 2020). Some taxa also exhibit very small interspecific genetic divergence, such as Amolops mantzorum group (Wu et al. 2020) and the genus Nidirana (Lyu et al. 2020). Therefore, there is no clear threshold for genetic distance to distinguish intraspecific and interspecific, but merely a reference.

There are two main reasons for high genetic differentiation within species. The first is the possible existence of cryptic species, and the second is geographical genetic variation in a low-dispersal group. To investigate these possibilities, further studies employing more comprehensive sampling, integrative taxonomy methods, and nuclear genetic data will be necessary to investigate the species diversity within L. eos.

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), the Animal Branch of the Germplasm Bank of Wild Species, CAS (Large Research Infrastructure Funding) to J.C. and Thailand science research and innovation fund and the University of Phayao (Grant No. FF65-RIM010) to C.S., Southeast Asia Biodiversity Research Institute, CAS, Specimen was collected under approval from the Institute of Animal for Scientific Purposes Development (IAD), which issued fieldwork permission (No. 610104022). This research study was also granted permission by the Department of National Parks, Wildlife and Plant Conservation (DNP).