Description of a new Asian Leaf Litter Toad of the genus Leptobrachella Smith, 1925 (Anura, Megophryidae) from southern Guizhou Province, China

Abstract Background The Asian leaf litter toads of the genus Leptobrachella Smith, 1925 (Anura, Megophryidae) inhabit the forest floor and rocky streams in hilly evergreen forests and are widely distributed from southern China, west to north-eastern India and Myanmar, through mainland Indochina to Peninsular Malaysia and the Island of Borneo. New information A new species of the Asian leaf litter toad genus Leptobrachella from Guizhou Province, China is described. Molecular phylogenetic analyses, based on mitochondrial 16S rRNA and COI genes and nuclear RAG1 gene sequences indicated that the new species is genetically divergent from its congeners. The new species could be distinguished from its congeners by a combination of the following characters: (1) body of medium size in males (SVL 31.9 – 32.9 mm); (2) distinct black spots present on flanks; (3) toes rudimentarily webbed, with wide lateral fringes; (4) skin on dorsum shagreened with fine tiny granules and short ridges; (5) heels overlapped when thighs are positioned at right angles to the body; (6) tibia-tarsal articulation reaching interior corner of the eye. A new species of the Asian leaf litter toad genus Leptobrachella from Guizhou Province, China is described. Molecular phylogenetic analyses, based on mitochondrial 16S rRNA and COI genes and nuclear RAG1 gene sequences indicated that the new species is genetically divergent from its congeners. The new species could be distinguished from its congeners by a combination of the following characters: (1) body of medium size in males (SVL 31.9 – 32.9 mm); (2) distinct black spots present on flanks; (3) toes rudimentarily webbed, with wide lateral fringes; (4) skin on dorsum shagreened with fine tiny granules and short ridges; (5) heels overlapped when thighs are positioned at right angles to the body; (6) tibia-tarsal articulation reaching interior corner of the eye.


Introduction
The Asian leaf litter toads of the genus Leptobrachella Smith, 1925 (Anura, Megophryidae) inhabit the forest floor and rocky streams in hilly evergreen forests and 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 2023).The species 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. Chen et al. (2018) suggested that the above genera were synonymised with Leptobrachella, based on large-scale molecular phylogenetic analyses.Currently, the genus contains 102 species, of which there are 40 species described in the last five years (Frost 2023).The species diversity in the genus was indicated to be much underestimated and many cryptic species have not been described till now (Chen et al. 2018), especially in some species group which were formerly recorded as widespread species, such as L. oshanensis complex ( Nguyen et al. 2021, Shi et al. 2021) and more fieldwork and investigations should focus on them.
From 2022 to 2023, four specimens of Leptobrachella were collected from Dushan County, Guizhou Province, China.Morphologically, these specimens most closely resemble L. dong, but differ from L. dong and all other Leptobrachella from China and adjoining countries.To distinguish these specimens, we conducted phylogenetic analyses, based on mitochondrial DNA, nuclear DNA and morphological comparisons.All of the analyses consistently indicated that the specimens from Dushan County are a new taxon.Herein, we describe this taxon as a new species.

Specimens
Four specimens of the undescribed species (Table 1 and Suppl.material 1) were collected from Dushan County, Guizhou Province, China (Fig. 1).After taking photographs, they were euthanized 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 Moutai Institute (MT).
For phylogenetic analyses, we downloaded the corresponding gene sequences for all related species in the genus Leptobrachella from GenBank, based on previous studies (Chen et al. 2018, Shi et al. 2021).Corresponding sequences of one Leptobrachium huashen Fei and Ye (2005) and one Megophrys glandulosa (Fei et al. 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.Phylogenetic analyses of mitochondrial DNA, the datasets of 16S and COI gene sequences were prepared using Maximum Likelihood (ML) and Bayesian Inference (BI) methods, implemented in PhyML v. 3.0 (Guindon et al. 2010) andMrBayes v. 3.12 (Ronquist andHuelsenbeck 2003), respectively.The best-fit nucleotide substitution models for the sequence super-matrices were selected in PartitionFinder 2.1.1 (Lanfear et al. 2012 ) using the Bayesian Information Criterion (BIC).As a result, the analysis suggested that the best model for 16S was GTR + R and, for the COI gene, it was TN93 + G + I.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 the uncorrected p-distance model, was estimated on 16S and COI genes using MEGA v. 6.06 (Tamura et al. 2013), respectively.

Morphological comparisons
Morphologically, these specimens most closely resemble L. dong.To explore the morphological differences between the new taxon and L. dong, four specimens of the new taxon and 15 specimens of L. dong containing four specimens from Guizhou Province and 11 specimens from Hunan Province were measured.The terminology and methods followed Fei et al. (2005)and Liu et al. (2023).Measurements were made to the nearest 0.1 mm (Watters et al. 2016) with digital calipers.Fourteen morphometric characters of adult specimens were measured: eye diameter (ED, distance from the anterior corner to the posterior corner of the eye); foot length (FL, distance from tarsus to the tip of the fourth toe); head length (HDL, distance from the tip of the snout to the articulation of jaw); head width (HDW, greatest width between the left and right articulations of jaw); hind-limb length (HLL, distance from tip of fourth toe to vent); internasal distance (IND, minimum distance between the inner margins of the external nares); interorbital distance (IOD, minimum distance between the inner edges of the upper eyelids); length of lower arm and hand (LAL, distance from the elbow to the distal end of the Finger IV); snout length (SL, distance from the tip of the snout to the anterior corner of the eye); snout-vent lengt (SVL, distance from the tip of the snout to the vent); maximal tympanum diameter (TD); tibia length (TL, distance from knee to tarsus); tibia width (TW, the widest length of the tibia); upper eyelid width (UEW, 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 measurement to SVL was calculated and then log-transformed for the following morphometric analyses.One-way analysis of variance (ANOVA) was used to test the significance of differences on morphometric characters between the undescribed species and L. dong from different populations in males.The significance level was set at 0.05.To show the spatial distribution of different species on the morphometric characters, principal component analyses (PCA) were performed.All statistical analyses were performed using SPSS 21.0 (SPSS, Inc., Chicago, IL, USA).
The undescribed taxon was also compared with all other congeners of Leptobrachella, based on morphological characters.Comparative morphological data were obtained from literature (Table 2).

Diagnosis
Leptobrachella dushanensis 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 two 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 dushanensis sp.nov.could be distinguished from its congeners by a combination of the following characters: body of medium size in males (SVL 31.9 -32.9 mm); distinct black spots present on flanks; toes rudimentarily webbed, with wide lateral fringes; skin on dorsum shagreened with fine tiny granules and short ridges; heels overlapped when thighs are positioned at right angles to the body; tibia-tarsal articulation reaching interior corner of the eye.
Description of holotype: Adult male.SVL in 33.2 mm.Head length slightly wider than head width (HDL/HDW = 1.04); snout sharply rounded in dorsal view, projecting slightly beyond margin of the lower jaw; nostril closer to snout than eye; loreal region oblique; canthus rostralis indistinct; eyes large and convex (ED/HDL = 0.37), slightly shorter than snout length (ED/SL = 0.93), pupil vertical; tympanum distinct, rounded, tympanum diameter smaller than eye (TD/ED = 0.43), 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 = 0.45), fingers long and slender (ML/SVL = 0.25), without webbing, 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 = 1.56), heels overlapping when the tibiae perpendicular to the body axis; tibio-tarsal articulation of adpressed limb reaching interior corner of the eye, tibia length about half of snout-vent length (TL/SVL = 0.49); relative toe length: I < II < III < V < IV; toe tips rounded and slightly swollen; rudimentary webbing present between all five toes; wide lateral fringes present on all toes; subarticular tubercles indistinct on the base of toe; inner metatarsal tubercle oval and distinct, outer metatarsal tubercle absent.
Skin on dorsum shagreened with fine tiny granules and short ridges; supra-axillary glands long, oval, close to the armpit; pectoral gland 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 distinctly visible and raised, forming an incomplete line.

Colouration of holotype in life:
In life, dorsal surface of head and trunk earth brown, with a distinct reverse-triangle taupe markings between eyes connecting to a taupe Wshaped marking between axillae that are fringed with greyish-white colour; very distinct, light brown markings between the nostrils; bicoloured iris, with the upper 1/3 of the iris being copper-orange and the lower 2/3 a light silvery-grey and a dark blotch under the eye; sparse, small, light brown granules and small dark brown patches present on the dorsum of the limbs; elbow to upper arm distinctly greyish-brown in colour on the dorsum; three transverse black bars present on dorsal surface of lower arm; distinct dark blotches on flanks from groin to axilla, longitudinally in two rows; ventral surfaces light coloured, throat and ventral arms pinkish, chest and belly cream-white and flanks of ventral with several granules and brown spots; ventral hind-limbs pinkish with sparse white glands (Fig. 2).
Preserved holotype colouration: Dorsum of body and limbs fade to brown; transverse bars on limbs become more distinct.Ventral surface of body and limbs fade to creamwhite.Supra-axillary, femoral and pectoral glands fade to cream-yellow (Fig. 3).
Leptobrachella dushanensis sp.nov.could also be identified from 76 known Leptobrachella species occurring north of the Isthmus of Kra by some characters (Suppl.material 2).

Etymology
This specifc name "Dushan" refers to the distribution of this species in Dushan County, Guizhou Province, China.We suggest its English common name "Dushan leaf litter toad" and Chinese name "Dushan Zhang Tu Chan (独山掌突蟾)".

Distribution
Leptobrachella dushanensis sp.nov.was only found in Dushan County, Guizhou Province, China.Elevations recorded range from 1000 m to 1200 m.

Ecology
Leptobrachella dushanensis sp.nov.was found under stones in fast-flowing mountain streams surrounded by evergreen broadleaf forest (Fig. 5) and we did not find eggs, nor tadpoles or females.Based on our surveys, we speculate that the breeding season is probably in late March.

Analysis Phylogenetic analyses
Aligned sequence matrix of 16S, COI and RAG1 genes contained 519, 615 and 831 bps respectively.ML and BI analyses, based on 16S and COI, resulted in essentially identical topologies.All samples of the undescribed species were clustered into one clade being deeply clustered into the Leptobrachella clade and being sister to L. dong (Fig. 6A and B).
Only one haplotype was found for all samples of the undescribed species in RAG1 gene and there was no common haplotype between the undescribed species and its related species (Fig. 6C).The smallest pairwise genetic divergence between the undescribed species and its close congener L. dong from Guizhou population and Hunan population were both 1.6% on 16S (Suppl.material 3) and, on COI, were 7.3% and 7.4%, respectively (Suppl.material 4), which was higher than the divergence between the populations of L. dong (0.3% on 16S and 1.6% on COI) and also higher or at the same level with those amongst many pairs of congeners, such as L. bijie and L. jinyunensis (1.6% on 16S), L. bijie and L. jinyunensis was 4.4%, L. bijie and L. chishuiensis was 4.1%, L. chishuiensis and L. jinshaensis 5.8%, L. jinshaensis and L. purpuraventra was 4.0% on COI (Suppl.materials 3, 4).

Morphological analyses
The results of one-way ANOVA showed that males of the undescribed species differed significantly from L. dong from both Guizhou and Hunan populations, based on several morphometric characters.From Guizhou population, the undescribed species was larger in SVL, HDW and HLL and shorter in SL, IND, ED and TYD; from Hunan population, the undescribed species was larger in HDL and shorter in HDW, SL, IND, IOD, ED and LAL (all p-values < 0.05; Table 3).In PCA for males, the total variation of the first two principal components was 57.52% and, on the two-dimensional plots of PC1 vs. PC2, the undescribed species could be separated from the specimens of L. dong both from Guizhou and Hunan populations (Fig. 7).
More detailed descriptions of results from morphological comparisons between the undescribed species and its congeners were presented in the Taxon Treatment section for describing the new species.
As a result, molecular and morphological results supported that our specimens from Guizhou Province, China were a new taxon.

Discussion
The Asian leaf litter toads 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).In recent years, the approach of integrative taxonomy has made substantial progress with this species-rich group.However, only a short mitochondrial fragment of 16S gene was sequenced for many species in this genus and the phylogenetic relationships between most species in the genus have not been resolved (Ohler et al. 2011, Rowley et al. 2017b, Chen et al. 2018, In this phylogenetic tree, Bayesian posterior probabilities (BPP) from BI analyses/bootstrap supports (BS) from ML analyses are listed beside the nodes.The symbol "-" represents a value below 0.50/50.For information of samples 1-126, refer to Table 1.Chen et al. 2023, Liu et al. 2023 etc.).This is most probably due to the insufficient phylogenetic information content in this kind of gene fragment (Chan et al. 2022).
In this study, two mitochondrial genes and one nuclear gene were amplified.On the 16S gene, the genetic distance between Leptobrachella dushanensis sp.nov.and its closelyrelated species L. dong was 1.6% which is at the same level with L. bijie and L. jinyunensis but on COI gene, the genetic distance between them is 7.3%, much larger than that between other solid species (Suppl.materials 3, 4).Additionally, the haplotype network constructed, based on RAG1 gene sequences, also showed that all samples of Leptobrachella dushanensis sp.nov.shared one haplotype and there was no common haplotype between Leptobrachella dushanensis sp.nov.and its related species.This observation indicates a clear genetic differentiation between Leptobrachella dushanensis sp.nov.and its relatives, supporting its recognition as a distinct species.The lack of shared haplotypes further implies limited gene flow and potential geographic isolation between Leptobrachella dushanensis sp.nov.and its related species.The evidence for the interspecific divergence was further confirmed by the significant morphological differences.Accordingly, the results of molecular and morphological comparisons indicated the validation of these species.

Figure 2 .
Figure 2. Photos of the holotype CIB DS20220409002 Leptobrachella dushanensis sp.nov. in life.A dorsolateral view; B ventral view; C dorsal view; D ventral view of hand E ventral view of foot.

Figure 3 .
Figure 3.The holotype specimen MT DS20220409002 Leptobrachella dushanensis sp.nov.A dorsal view; B ventral view; C ventral view of hand; D ventral view of foot.

Figure 4 .
Figure 4. Colour variation in Leptobrachella dushanensis sp.nov.A dorsal view of the male specimen MT DS20230310001; B ventral view of the male specimen MT DS20230310001; C dorsal view of the male specimen MT DS20230310002; D ventral view of the female specimen MT DS20230310002.

Figure 5 .
Figure 5. Habitats of Leptobrachella dushanensis sp.nov.A landscape of the type locality Dushan County, Guizhou Province, China; B a mountain stream in the type locality.

Figure 6 .
Figure 6.Phylogenetic trees of the genus Leptobrachella and a haplotype network constructed, based on RAG1 gene sequences.A Bayesian inference (BI) tree, based on mitochondrial COI gene; B Bayesian Inference (BI) tree, based on mitochondrial 16S gene; C the haplotype network constructed, based on RAG1 gene sequences.In this phylogenetic tree, Bayesian posterior probabilities (BPP) from BI analyses/bootstrap supports (BS) from ML analyses are listed beside the nodes.The symbol "-" represents a value below 0.50/50.For information of samples 1-126, refer to Table1.

Figure 7 .
Figure 7. Plots of principal component analyses of Leptobrachella dushanensis sp.nov.and L. dong in males.PC1, the first principal component; PC2, the second principal component.Different species were denoted as different shapes, the red triangle represents Leptobrachella dushanensis sp.nov., black square represents L. dong from Guizhou Province and blue square represents L. dong from Hunan Province.
Description of a new Asian Leaf Litter Toad of the genus Leptobrachella ... Description of a new Asian Leaf Litter Toad of the genus Leptobrachella ... Description of a new Asian Leaf Litter Toad of the genus Leptobrachella ...

Table 2 .
References for morphological characters for congeners of the genus Leptobrachella.Description of a new Asian Leaf Litter Toad of the genus Leptobrachella ...

Table 3 .
Measurements of Leptobrachella dushanensis sp.nov and L. dong from Guizhou and Hunan populations.Units in mm.In addition, the results of one-way ANOVA with p-values for morphometric comparisons between Leptobrachella dushanensis sp.nov.and L. dong from Guizhou and Hunan populations (* p < 0.05, ** p < 0.01) are given.See abbreviations for characters in the Materials and Methods section.
, L. yeae and L. yunyangensis (vs.fringes on toes narrow or absent in the latter)..shangsiensisand L. tadungensis, all of which have the dorsum smooth and L. bourreti (dorsum smooth with small warts), L. fuliginosa (dorsum smooth with fine tubercles), L. liui (dorsum with round tubercles), L. macrops (dorsum roughly granular with large tubercles), L. maoershanensis (dorsum shagreened with tubercles), L. minima (dorsum smooth), L. neangi (dorsum with small, irregular bumps and ridges), L. nyx (dorsum with round tubercles), L. nokrekensis (dorsum tubercles and longitudinal folds), L. pelodytoides (dorsum with small, smooth warts), L. tamdil (dorsum weakly tuberculate, with low, oval tubercles), L. tuberosa (dorsum highly tuberculate), L. yunkaiensis (dorsum with raised warts) and L. wuhuangmontis (dorsum rough with conical tubercles).In mitochondrial DNA trees, Leptobrachella dushanensis sp.nov.and L. dong clustered into one clade, being sisters.The new species differs from L. dong by the following characters: head length slightly wider than head width (vs.head width slightly wider than head length); males with internal single subgular vocal sac (vs. a pair of subgular internal vocal sacs); tibiotarsal articulation reaching to anterior edge of eye (vs.reaching to middle of eye).Adult males with internal single subgular vocal sac, 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.