﻿A new Asian leaf litter toad of the genus Leptobrachella (Amphibia, Anura, Megophryidae) from central south China

﻿Abstract A new species of the Asian leaf litter toad genus Leptobrachella from central south China is described. Molecular phylogenetic analyses, based on mitochondrial 16S rRNA and nuclear RAG1 gene sequences indicated the new species as an independent clade in the genus. The new species could be distinguished from its congeners by a combination of the following characters: body of medium size (SVL 29.2–34.2 mm in 15 adult males and 34.4–43.1 mm in seven adult females); distinct black spots present on flanks; toes rudimentary webbed, with wide lateral fringes; ventral belly white with distinct nebulous brown speckling on ventrolateral flanks; skin on dorsum shagreened with fine tiny granules or short ridges; iris copper above, silver below; heels overlapped when thighs are positioned at right angles to the body; tibia-tarsal articulation reaches the middle eye; dorsal surface of tadpole semi-transparent light brown, spots on tail absent, keratodont row formula I: 3+3/2+2: I; call series basically consist of repeated long calls, at dominant frequency (5093 ± 412 Hz).


Introduction
The Asian leaf litter toads of the genus Leptobrachella Smith, 1925 (Anura, Megophryidae) are widely distributed from southern China west to north-eastern India and Myanmar, through mainland Indochina to peninsular Malaysia and the island of Borneo Frost 2022). The toads inhabit the forest floor in montane evergreen forest. The taxa in the group had been classified into different genera, i.e. Paramegophrys Liu, 1964, Carpophrys Sichuan Biological Research Institute, 1977, Leptolalax Dubois, 1980, Lalax Delorme, Dubois, Grosjean & Ohler, 2006and Lalos Dubois, Grosjean, Ohler, Adler & Zhao, 2010. Based on large-scale molecular phylogenetic analyses on this group, Chen et al. (2018) suggested that the above genera were synonymised with Leptobrachella. Currently, the genus Leptobrachella contains 95 species, of which, as noted, 34 species were described in the last five years (Frost 2022). The species diversity in the genus was indicated to be much underestimated and many cryptic species have not been described until now .
In recent years, we carried out a series of biodiversity surveys in Hunan and Guizhou Provinces, China and collected some specimens of Leptobrachella. Morphological comparisons, molecular phylogenetic analyses and bioacoustic comparisons consistently indicated these specimens as an undescribed species. We describe it herein as a new species.

Specimens
A total of 22 specimens of the new species (Suppl. material 1) were collected from Tongdao and Suining County, Hunan Province and Congjiang County, Guizhou Province, China ( Fig. 1). After taking photographs, they were euthanised using isoflurane and then the specimens were fixed in 10% buffered formalin. Tissue samples were taken and preserved separately in 95% ethanol prior to fixation. Specimens were deposited in Chengdu Institute of Biology, the Chinese Academy of Sciences (CIB, CAS), China.

Molecular phylogenetic analyses
A total of 15 samples of the new species were used for 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), based on previous studies Shi et al. 2021). Corresponding sequences of one Leptobrachium huashen Fei & Ye, 2005 and one Megophrys glandulosa (Fei, Ye & Huang, 1990) were downloaded and used as outgroups.
Sequences were assembled and aligned using the Clustalw module in BioEdit v. 7.0.9.0 (Hall 1999) with default settings. Alignments were checked by eye and revised manually, if necessary. For the phylogenetic analyses, we constructed two sequence matrices for reconstructing the phylogenetic trees, i.e. mitochondrial 16S and RAG1 gene datasets. 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 16S data and GTR + R for the RAG1 data. For the ML tree, branch supports were drawn from 10,000 non-parametric bootstrap replicates. In BI analyses, 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. To detect the haplotype relationships and genetic isolation between the undescribed species and its related species on nuclear DNA, a haplotype network, based on RAG1 gene sequences, was constructed using the maximum parsimony method in TCS v.1.21 (Clement et al. 2000). 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 22 specimens of the new taxon were measured. The terminology and methods followed Fei and Ye (2005), Mahony et al. (2011 and Shi et al. (2021). Measurements were made with a dial caliper to the nearest 0.1 mm (Watters et al. 2016) with digital calipers. Fourteen morphometric characters of adult specimens were measured:

ED
eye diameter (distance from the anterior corner to the posterior corner of the eye); 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 hind-limb 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); 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); TL tibia length (distance from knee to tarsus); TYD maximal tympanum diameter; UEW upper eyelid width (greatest width of the upper eyelid margins measured perpendicular to the anterior-posterior axis).
One tadpole specimen of the undescribed species was measured. Nineteen morphometric characters were measured for the tadpole: The new taxon was also compared with all other congeners of Leptobrachella, based on morphological characters. Comparative morphological data were obtained from literature ( Table 2).

Bioacoustics analyses
The advertisement calls of the new taxon were recorded from specimens CIB SSC1757, CIB SSC1754, CIB SSC1760, CIB LB20220311001, CIB LB20220311002 and CIB ZNY2022012. The advertisement call of the toad was recorded in the stream at ambient air temperature of 11.2-15.0 °C. Sony PCM-D50, Philips VTR 6900 digital sound recorder, Huawei Mate 30E Pro smart phone were 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. Terminology of advertisement call analyses and description followed Köhler et al. (2017) and Wang et al. (2019). Call recordings were analysed and visualised by Raven Pro 1.5 software (Cornell Laboratory of Ornithology, Ithaca, NY, USA) (window size 256 points, fast-Fourier transform, Hanning windows). Ambient temperature was taken by a digital hygrothermograph.

Results
The aligned sequence matrix of 16S and RAG1 gene contained 498 bps and 888 bps, respectively. ML and BI analyses, based on the 16S gene matrix, resulted in essentially identical topologies (Fig. 2). All 15 samples of the undescribed species were clustered into one clade being deeply clustered into the Leptobrachella clade and seemly being sister to a clade comprising of L. graminicola and L. yeae ( Fig. 2A). ML and BI analyses, based on the RAG1 gene matrix, also resulted in essentially identical topologies (Fig. 2B). In the RAG1 tree, eleven samples of the undescribed species were clustered together into an independent clade which was distantly divergent from other congeners. Four haplotypes were found for eleven samples of the undescribed species in the  Table 1 C the haplotype network constructed, based on RAG1 gene sequences. RAG1 gene and there was no common haplotype between the undescribed species and its related species (Fig. 2C). The genetic distance of the p-distance model on the 16S gene within the specimens of the undescribed species ranged from 0.0% to 0.4%, being much lower than the interspecific genetic distance (all higher than 1.8%; Suppl. material 2). The smallest pairwise genetic divergence between the undescribed species and its congeners is 2.2% (vs. L. bourreti), being higher than or at the same level with that between some pairs of related species, such as L. bijie and L. jinshaensis (2.0%), L. purpuraventra and and L. jinshaensis (2.1%).
The undescribed species could be identified from its congeners in a series of morphological and bioacoustics characters. For the detailed demonstration, based on morphological and bioacoustics comparisons, see the following section describing the new species.
Molecular phylogenetic analyses, morphological comparisons and bioacoustics analyses indicated that the specimens from Tongdao and Suining County, Hunan Province and Congjiang County, Guizhou Province, China represent an undescribed species which is described as follows. Diagnosis. Leptobrachella dong sp. nov. is assigned to the genus Leptobrachella, based on molecular data and the following morphological characters: medium size, rounded finger tips, the presence of an elevated inner palmar tubercle not continuous to the thumb, presence of macroglands on body (including supra-axillary, pectoral and femoral glands), vomerine teeth absent, tubercles on eyelids and anterior tip of snout with vertical white bar (Dubois 1983;Fei et al. 2009). Leptobrachella dong sp. nov. could be distinguished from its congeners by a combination of the following characters: (1) body of medium size (SVL 29.2-32.0 mm in 15 adult males and 37.4-43.1 mm in seven adult females); (2) distinct black spots present on flanks; toes rudimentary webbed, with wide lateral fringes; (3) ventral belly white with distinct nebulous brown speckling on ventrolateral flanks; (4) skin on dorsum shagreened with fine tiny granules or short ridges; (5) heels overlapped when thighs are positioned at right angles to the body; (6) tibia-tarsal articulation reaches the middle eye; (7) dorsal surface of tadpole semi-transparent light brown, spots on tail absent, keratodont row formula I: 3+3/2+2: I; (8) calls with two types, at dominant frequency (5.1 ± 0.4 kHz).
Description of holotype. Adult male. SVL in 32.0 mm. Head width almost equal with head length slightly (HDW / HDL 1.03); snout rounded in both ventral view and lateral view, projecting slightly beyond margin of the lower jaw; nostril closer to snout than eye; loreal region oblique; canthus rostralis indistinct; eyes large (ED / HDL 0.40), eye diameter slightly longer than snout length (ED / SL 1.07), eyes notably protuberant in both dorsal and lateral views, pupil vertical; tympanum distinct, rounded, tympanum diameter smaller than eye (TYD / ED 0.38), upper margin of tympanum in contact with supratympanic ridge; vomerine teeth absent; tongue notched behind; supratympanic ridge distinct, extending from posterior corner of eye to supra-axillary gland.
Fore-limb relatively long (LAL / SVL 0.46), fingers long and slender (ML / SVL 0.25), webbing absent, lateral fringes on fingers narrow; relative finger lengths II < I < IV < III; tips of fingers rounded and slightly swollen; subarticular tubercles absent on fingers, inner metacarpal tubercle large and rounded, separated from the smaller, round outer metacarpal; supra-axillary glands oval.
Hind-limb relatively long (HLL / SVL 1.53), heels overlapping when the tibias perpendicular to the body axis; tibio-tarsal articulation of adpressed limb reaching middle of eye, tibia length about half of snout-vent length (TL / SVL 0.49); relative toe length: I < II < V < III < IV; toe tips rounded and slightly swollen; rudimentary webbing present between all five toes; wide lateral fringes present on all toes; dermal ridges under fourth toes interrupted; subarticular tubercles distinct under the base of II, III and IV toe; inner metatarsal tubercle oval and distinct, outer metatarsal tubercle absent (Fig. 3C).  Dorsal skin relatively smooth with small tubercles and short folds; supra-axillary gland distinct and yellowish; pectoral gland small and indistinct; round femoral glands present and protuberant on rear of thigh, closer to knee than to vent; femoral adipose glands distinct, attached to inner side of skin on posterior ventral surface of thigh; ventral skin smooth; ventrolateral glands forming a distinct white line on flanks.

Colouration of holotype in life.
In life, dorsal surface of head and trunk yellowish-brown, with distinct olive reverse-triangle dark markings between eyes connecting to a dark W-shaped marking between axillae that are fringed with greyish-white colour; elbow to upper arm distinctly yellowish-orange in colour on the dorsum; four transverse black bars present on dorsal surface of thighs and three on dorsal surface of lower arm; one dark blotch between nostril and eyes on loreal region and a dark blotch under the eye; supratympanic ridge reddish and a large black marking under supratympanic ridge; distinct dark blotches on flanks from groin to axilla, longitudinally in two rows; ventral surfaces light coloured; throat and ventral arms pinkish with cream speckling on margins; chest and belly cream white, on the lateral belly with dense brown speckling; ventral hind-limbs pinkish with sparse white glands; upper iris copper, lower iris silver.
Preserved holotype colouration. Dorsum of body and limbs fading to brown copper; transverse bars on limbs becoming more distinct. Ventral surface of body and limbs fading to cream white. Supra-axillary, femoral and pectoral glands fading to cream yellow.
Variations. Measurements and basic statistics of adult specimens are presented in Suppl. materials 1, 3, respectively. Females larger than males (29.2-34.2 mm in 15 adult males and 34.4-43.1 mm in seven adult females) and, in CIB LB20220311002, dark blotch between nostril and eyes on loreal region absent (Fig. 5A); in CIBSCC1754, a patch on the outside of the reverse-triangle dark markings between eyes (Fig. 5B); in CIB LB20220305005, no longitudinal stripes along dorsolateral body (Fig. 5C); in CIB LB20220306008, the colouration of head and anterior dorsum is darker than the posterior (Fig. 5D).
Bioacoustics. (Fig. 6; Suppl. material 3). Calls recorded at temperatures 11.2 to 15.0 °C. Descriptions based on six sequenced adults (Suppl. material 3) and 157 calls were measured. Dominant frequency of all type of calls is 4.4-5.6 kHz (5.1 ± 0.4) kHz, call duration is 203.783 ± 161.7 ms, call interval is 1238.3 ± 2034.5 ms, call repetition rate is 1.61 ± 0.9 (calls/s). Additionally, the calls were of two types. The first type (type A) consists of repeated short notes (Fig. 6A, B), and the second type (type B) consists of two repeated short notes and with longer call duration (524.2 ± 64.2 ms) and shorter call interval (148.8 ± 72.8 ms) than type A (Fig. 6C, D). Amplitude of type A was largest at first pulse, drastic reducing in the following pulses; amplitude of second note of type A about half of the first note; amplitude of type B with highest pulse at the beginning of each note and decreasing towards to the end.
Comparisons. Compared with the 26 known congeners occurring south of the Isthmus of Kra, Leptobrachella dong sp. nov. could be distinguished from them by several characters: by having supra-axillary and ventrolateral glands, the new species differs from L. arayai, L. dringi, L. fritinniens, L. gracilis, L. hamidi, L. heteropus, L. kajangensis, L. kecil, L. marmorata, L. maura, L. melanoleuca, L. picta, L. platycephala, L. sabahmontana, and L. sola (vs. absent in the latter); by having rounded fingertips and moderate body size (29.2-34.2 mm in 15 adult males and 34.4-43.1 mm in seven adult females), the new species differs from the following species with pointed      Fig. 3) differs from all other congeners occurring north of the Isthmus of Kra for which comparable acoustic data are available consisting of uniform and continuous calls with four to five pulses in each note. Of the congeners in the region with known calls, the new species can be separated from L. purpurus, L. tuberosus, L. puhoatensis and L. yingjiangensis by not having an invariably single-note call with irregular intervals. In addition, the dominant frequency of 4.4-5.6 kHz (at 11.2-15 °C) further distinguishes the call of Leptobrachella dong sp. nov. from that of the higher frequency calls of L. aereus , L. yingjiangensis (5.7-5.9 kHz at 19 °C), L. isos  and L. ventripunctatus (6.1-6.4    and L. tuberosus . In mitochondrial DNA trees, Leptobrachella dong sp. nov. was clustered as an independent clade and sister to a clade comprising of L. graminicola and L. yeae. The new species differs from L. graminicola by the following characters: body size larger with SVL 29.2-34.2 mm in adult males and 34.4-43.1 mm in adult females (vs. 23.1-24.6 mm in adult males and 28.6-32.9 mm in adult females); black spots on flanks present (vs. absent); ventral surface white with distinct nebulous brown speckling on ventrolateral flanks (vs. white with brown spots); dorsal surface shagreened with fine tubercles (vs. smooth, with many tubercles); and tibiotarsal articulation reaching to middle of eye (vs. anterior edge of eye). The new species differs from L. yeae by having wide fringes on toes (vs. narrow); dorsal surface shagreened with fine tubercles (vs. relatively smooth with fine tiny granules or short ridges); and males with a pair of subgular internal vocal sacs (vs. internal single subgular vocal sac).
Secondary sexual characteristics. Adult males with a pair of subgular vocal sacs (Fig. 8B), femoral adipose glands present on posterior surface of thigh and tiny transparent spines on chest during breeding season. Nuptial pads and spines absent on males.  Ecology notes. Leptobrachella dong sp. nov. has been found in three localities: Tongdao County and Suining County, Hunan Province and Congjiang County, Guizhou Province, China. Elevations recorded range from 620 m to 1200 m. Population from the Tongdao County inhabited a torrent stream covered by evergreen shrubs and the new species always found on the stones (Fig. 8A, B). Population from Congjiang County inhabited slow-flowing streams surrounded by evergreen broadleaf forest (Fig. 8C). Populations from Suining County, Hunan Province inhabited broad mountain stream surrounded by evergreen broadleaf forest (Fig. 8D). Tadpoles could be found at daytime and night. Gravid females were found by the streams in the type locality (2 April 2017) and Suining County (15 March 2022).
Etymology. This specific name "dong" refers to the Dong people, as the new species distributed in the concentrated area of Dong people. We suggest its English common name "Dong leaf litter toads" and Chinese name "Dong Zhang Tu Chan (侗掌突蟾)".

Discussion
South-western China was proposed as a biodiversity hotspot (Myers et al. 2000). In the past five years, 34 new species of the genus Leptobrachella have been discovered (Frost 2022), while the species of Leptobrachella have low vagility and an exclusive association with montane forests and their populations are often highly structured and underestimation of species diversity occurs in the genus, which suggests a high degree of localised diversification and micro-endemism (Fei et al. 2012;Chen et al. 2018). Therefore, a lot of cryptic species were proposed by molecular analyses in areas where surveys are weak .
This new species was found in three localities and the largest geographical distance between the localities is over 200 km. However, in this study, phylogenetic analyses, based on mitochondrial DNA, suggested the three populations as the same species and different from its congeners on a series of morphological characters. This perhaps indicated that the species have a widespread distribution. Further surveys are needed to evaluate the population status of the species. This work was supported by the Projects from the National Natural Science Foundation of China (Nos. 32270498, 31960099, 32260136, and 32070426