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

Abstract A new species of the Asian leaf litter toad genus Leptobrachella from Guizhou Province, China is described based on molecular phylogenetic analyses, morphological comparisons, and bioacoustics data. Phylogenetic analyses based on the mitochondrial 16S rRNA gene sequences supported the new species as an independent clade nested into the Leptobrachella clade and sister to L. bijie. The new species could be distinguished from its congeners by a combination of the following characters: small body size (SVL 30.8–33.4 mm in seven adult males, and 34.2 mm in one adult female); dorsal skin shagreened, some of the granules forming longitudinal short skin ridges; tympanum distinctly discernible, slightly concave; internasal distance longer than interorbital distance; supra-axillary, femoral, pectoral and ventrolateral glands distinctly visible; absence of webbing and lateral fringes on fingers; toes with rudimentary webbing and shallow lateral fringes, relative finger lengths II < IV < I < III; heels overlapped when thighs are positioned at right angles to the body; and tibia-tarsal articulation reaches the tympanum.

. Location of the type locality of Leptobrachella chishuiensis sp. nov., Chishui National Nature Reserve, Chishui City, Guizhou Province, China. fixed in 10% buffered formalin. Before fixing, muscle tissue was taken and preserved separately in 95% ethanol. Specimens were deposited in Chengdu Institute of Biology, Chinese Academy of Sciences (CIB, CAS). Molecular phylogenetic analyses. All eight specimens of the new taxon were included in the 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) mainly based on previous studies Wang et al. 2019;Luo et al. 2020). Corresponding sequences of Leptobrachium tengchongensis, one Leptobrachium huashen, and one Megophrys major were also downloaded from GenBank, and used as outgroups according to previous phylogenetic works Wang et al. 2019;Luo et al. 2020).
Total DNA was extracted using a standard phenol-chloroform extraction protocol (Sambrook et al. 1989). The mitochondrial 16S rRNA gene (16S) sequences were amplified, and the primers P7 (5'-CGCCTGTTTACCAAAAACAT-3') and P8 (5'-CCG-GTCTGAACTCAGATCACGT-3') were used following Simon et al. (1994). Gene fragments were amplified under the following conditions: an initial denaturing step at 95 °C for 4 min; 36 cycles of denaturing at 95 °C for 30 s, annealing at 51 °C for 30 s and extending at 72 °C for 70 s. Sequencing was conducted using an ABI3730 automated DNA sequencer in Shanghai DNA BioTechnologies Co., Ltd. (Shanghai, China). New sequences were deposited in GenBank (for GenBank accession numbers see Table 1).
Sequences were assembled and aligned using the Clustalw module in BioEdit v. 7.0.9.0 (Hall 1999) with default settings. 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 data used in ML and BI analyses. For the ML analysis, branch supports were drawn from 10,000 nonparametric bootstrap replicates. In BI analysis, 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. 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 eight adult specimens (Table 2)

ED
eye diameter (distance from the anterior corner to the posterior corner of the eye); FIL first finger length (distance from base to tip of finger I); FIIL second finger length (distance from base to tip of finger II); FIIIL third finger length (distance from base to tip of finger III); FIVL fourth finger length (distance from base to tip of finger IV); 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 hindlimb 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); LW lower arm width (maximum width of the lower arm); 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); SVL snout-vent length (distance from the tip of the snout to the posterior edge of the vent); TYD maximal tympanum diameter; TEY tympanum-eye distance (distance from anterior edge of tympanum to posterior corner of eye); TFL length of foot and tarsus (distance from the tibiotarsal articulation to the distal end of the toe IV); THL thigh length (distance from vent to knee); TL tibia length (distance from knee to tarsus); TW maximal tibia width; UEW upper eyelid width (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 character to SVL was calculated and then was log-transformed for the following morphometric analyses. Mann-Whitney U tests were conducted to test the significance of differences on morphometric characters between the undescribed species, L. bijie and L. purpuraventra. The significance level was set at 0.05. Furthermore, principal component analyses (PCA) were conducted to highlight whether the different species were separated in morphometric space. Due to only the measurements SVL, HDL, HDW, SL, IND, IOD, ED, TYD, TEY, LAL, ML, TL, HLL, and FL of male L. bijie and L. purpuraventra being available from Wang et al. (2019), the morphometric analyses were conducted only based on these 14 morphometric characters for male group.
The new taxon was also compared with all other congeners of Leptobrachella based on morphological characters. Comparative morphological data were obtained from literatures (Table 3).
Bioacoustics analyses. The advertisement calls of the new taxon were recorded from the holotype specimen CIBCS20190518047 in the field on 18 May 2019 in Chishui National Nature Reserve, Chishui City, Guizhou Province, China. The advertisement call of the new species was recorded in the stream at ambient air temperature of 20 °C and air humidity of 87%. SONY PCM-D50 digital sound recorder was 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. Calls were recoded and examined as described by Wijayathilaka and Meegaskumbura (2016). Call recordings were visualized and edited with SoundRuler v. 0.9.6.0 (Gridi-Papp 2003-2007 and Raven Pro v. 1.5 software (Cornell Laboratory of Ornithology, Ithaca, NY, USA). Ambient temperature of the type locality was taken by a digital hygrothermograph.

results
Aligned sequence matrix of 16S contained 537 bps. ML and BI analyses based on the 16S matrix resulted in essentially identical topologies (Fig. 2). All six samples of the new taxon were clustered into one monophyletic group (node supports in ML and BI: 94 and 0.95) nested into Leptobrachella, and was a sister taxon to L. bijie (node supports in ML and BI: 92 and 1.00). The genetic distance between the new taxon and its closest relatives L. bijie was 2.1%, at the same level with that between L. alpina and L. purpurus (2.1%; Suppl. material 1: Table S1).  In PCA for male group, the total variation of the first two principal components was 64.6%. In males on the two-dimensional plots of PC1 vs. PC2, the undescribed species could be distinctly separated from L. bijie and L. purpuraventra (Fig. 3). The results of Mann-Whitney U tests indicated that in males, the new taxon was significantly different from L. bijie and L. purpuraventra on many morphometric characters (all p-values < 0.05; Table 4).
There were many differences in sonograms and waveforms of calls between the new species L. bijie, and L. purpuraventra. Firstly, a call contains 1-4 notes in the new species and only contains two notes of each call in L. bijie and L. purpuraventra. Secondly, the dominant frequency of the new species is higher than L. bijie and L. purpuraventra.
Based on the molecular, morphological, and bioacoustics differences, the specimens from Chishui City, Guizhou Province, China represent a new species which is described as follows.    Diagnosis. Leptobrachella chishuiensis sp. nov. is assigned to the genus Leptobrachella based on molecular phylogenetic analyses and the following morphological characters: small body size; having an elevated inner metacarpal tubercle; having macro-glands on body (including supra-axillary, femoral andventrolateral glands); lacking vomerine teeth; having small tubercles on eyelids; anterior tip of snout with whitish vertical bar (Dubois 1983;Lathrop et al. 1998;Delorme et al. 2006;Das et al. 2010;Luo et al. 2020).
Leptobrachella chishuiensis sp. nov. could be distinguished from its congeners by a combination of the following characters: (1) small body size  mm in seven adult males, and 34.2 mm in one adult female); (2) dorsal skin shagreened, some of the granules forming longitudinal short skin ridges; (3) tympanum distinctly discernible, slightly concave; (4) internasal distance longer than interorbital distance; (5) supra-axillary, femoral, pectoral and ventrolateral glands distinctly visible; (6) absence of webbing and lateral fringes on fingers; (7) toes with rudimentary webbing and shallow lateral fringes; (8) relative finger lengths II < IV < I < III; (9) heels overlapped when thighs are positioned at right angles to the body; and (10) tibia-tarsal articulation reaches the tympanum.
Description of holotype. Measurements in mm. Adult male (CIBCS20190518047). SVL 32.4. Head length slightly longer than head width (HDL/HDW ratio 1.04); snout slightly protruding, projecting slightly beyond margin of the lower jaw; nostril closer to snout than eye; canthus rostralis gently rounded; loreal region slightly concave; interorbital space flat, internarial distance longer than interorbital distance (IND/IOD ratio 1.23); pineal ocellus absent; vertical pupil; snout length larger than eye diameter; tympanum distinct, rounded, and slightly concave, diameter smaller than that of the eye (TMP/ED ratio 0.57); upper margin of tympanum in contact with supratympanic ridge; distinct black supratympanic line present; vomerine teeth absent; tongue notched behind; supratympanic ridge distinct, extending from posterior corner of eye to supra-axillary gland.
Hindlimbs slender, tibia 49% of snout-vent length; heels overlapped when thighs are positioned at right angles to the body, tibiotarsal articulation reaching tympanum when leg stretched forward; tibia length slightly longer than thigh length; relative toe lengths I < II < V < III < IV; tips of toes rounded, slightly dilated; subarticular tubercle small, distinct at the base of each toes; toes without webbing; narrow lateral fringes present on all toes; inner metatarsal tubercle present, large, oval, outer metatarsal tubercle absent; dorsal surface shagreened and granular, some of the granules forming short longitudinal folds on the flank of dorsal; ventral skin smooth; dense tiny granules present on surface of chest and ventral surface of thigh and tibia; pectoral gland and femoral gland oval, distinctly visible. Ventrolateral gland distinctly visible and forming an incomplete line.
Colouration of holotype in life. Dorsum brown, with small, distinct darker brown markings and spots and scattered with irregular light orange pigmentation. A dark brown inverted triangular pattern between anterior corner of eyes. Tympanum brown, a dark brown bar above tympanum, and a dark brown bar under the eye; transverse dark brown bars on dorsal surface of limbs; distinct dark brown blotches on flanks from groin to axilla, longitudinally in two rows; elbow and upper arms with dark bars and distinct coppery orange coloration; fingers and toes with distinct dark bars. Ventral surface of throat grey purple, chest and belly white, presence of distinct nebulous greyish speckling on flanks; ventral surface of limbs grey purple. Supra-axillary gland, femoral, pectoral and ventrolateral glands white (Fig. 5).
Preserved holotype colouration. Dorsum of body and limbs fade to dark brown; transverse bars on limbs become more distinct ventral surface of body and limbs fade to greyish white. Supra-axillary, femoral, pectoral and ventrolateral glands fade to greyish white (Fig. 4).
Variations. Morphological measurements were showed in Table 2. All specimens were similar in morphology but some individuals different from the holotype in color pattern. In some adult males, a dark brown inverted triangular pattern between anterior corner of eyes, in connected to the dark brown W-shaped marking on interorbital region (Fig. 6A); in adult female, the color of dorsum is blacker (Fig. 6B) and some patchiness on the chest and the flank of belly (Fig. 6C); in some adult males, the throat and bell creamy and white patchiness sparse on the ventral surface of limbs (Fig. 6D); in some specimens, the tibiotarsal articulation reaching tympanum to eye when leg stretched forward.
Secondary sexual characteristics. Adult males with a large subgular vocal sac, and nupital pads and spines absent.
By the finger II < I, Leptobrachella chishuiensis sp. nov. differs from L. tamdil (vs. II > I in the latter).
By head length slightly longer than wide, Leptobrachella chishuiensis sp. nov. differs from L. namdongensis (vs. head wider than long in the latter).
Six Leptobrachella species were reported to be distributed in Guizhou Province, China, they are: L. liui, L. oshanensis, L. purpuraventra, L. bijie, L. ventripunctata, and L. suiyangensis (Fei et al. 2012;Li et al. 2016;Wang et al. 2019;Luo et al. 2020). We make a comparative note between them and the new species as follows. Leptobrachella chishuiensis sp. nov. differs from L. liui by having shallow lateral fringes on toes (vs. wide lateral fringes on the toes in the latter), dorsal surface shagreened with small granules, lacking enlarge tubercles or warts (vs. dorsum with round tubercles in the latter); from L. oshanensis by having rudimentary webbing on the toes (vs. lack webbing on the toes in the latter), having shallow lateral fringes on toes (vs. lacking lateral fringes on the toes in the latter), from L. suiyangensis by heels overlapping when thighs are positioned at right angles to the body (vs. just meeting in the latter), tibia-tarsal articulation reaches tympanum or tympanum to eye (vs. reaches to the anterior corner of eye in the latter); from L. ventripunctata by bigger body size .0 mm in males in the latter), chest and belly without large dark brown spots (vs. with large dark brown spots in the latter).
Leptobrachella chishuiensis sp. nov. is genetically closer to L. bijie and L. purpuraventra. The new species differs from L. bijie by the following characters: larger body size  mm in males in the latter), internasal distance longer than interorbital distance (vs. equal to interorbital distance in the latter), heels overlapping (vs. just meeting in the latter), tibia-tarsal articulation reaches the tympanum or tympanum to eye (vs. reaching the region between middle of eye to anterior corner of eye in the latter), one call contains 1-4 notes (vs. 2 notes in each call in the latter), having shorter call interval (60 ± 21, N = 31 in the new species vs. 101.9 ± 6.4, N = 33 in the latter), having significantly higher value of SVL in males, and having significantly higher value of HDL, HDW, SL, IND, IOD, TEY, TL and FL to SVL in males (all P-values < 0.05; Table 5). Leptobrachella chishuiensis sp. nov. differs from L. purpuraventra by larger body size  mm in seven adult males vs.  mm in eleven adult males in the latter), tibia-tarsal articulation reaches the tympanum or tympanum to eye (vs. reaching the middle of eye in the latter), the call contains 1-4 notes (vs. 2 notes in each call in the latter), having longer call duration (200 ± 67, N = 32 vs. 192.2 ± 13.0 as the longest call duration in L. purpuraventra), shorter call interval (60 ± 21, N = 31 vs. 90.8 ± 5.6, N = 20 as the shortest call interval in L. bijie), having significantly higher value of SVL in males, and having significantly higher value of SVL, HDL,HDW, SL, IOD, ED, TYD, LAL, TL and FL to SVL in males (all P-values < 0.05; Table 5).
Etymology. This specific name chishuiensis refers to the distribution of this species, Chishui City, Guizhou Province, China. We propose the common English name "Chishui leaf litter toads" (English) and its Chinese as "Chi Shui Zhang Tu Chan (赤水掌突蟾)".

Discussion
The Asian leaf litter toads of Leptobrachella have low vagility and are in exclusive association with montane forests, and their populations are often highly structured. Underestimation of species diversity occurs in the genus, which suggests a high degree of localized diversification and micro-endemism (Fei et al. 2012;Chen et al. 2018). Many cryptic species were proposed by molecular analyses in areas where surveys are weak ), but in Guizhou Province the investigation into the genus was poor although this area was likely to be an important transition zone for many clades or lineages . Additionally, in Guizhou Province, many new amphib-ian species has been described in recent years (Zhang et al. 2017;Li et al. 2018a, b;Li et al. 2019a, b;Lyu et al. 2019;Wang et al. 2019;Wei et al. 2020), including two species of Leptobrachella, indicating the underestimated species diversity of amphibians in this region. To date, in Guizhou Province, seven Leptobrachella species were recorded, i.e., Leptobrachella chishuiensis sp. nov., L. liui, L. oshanensis L. purpuraventra, L. bijie, L. ventripunctata, and L. suiyangensis (Fei et al. 2012;Li et al. 2016;Wang et al. 2019;Luo et al. 2020). It is expected that in this area, the species diversity of Leptobrachella may be underestimated, and more investigation should be conducted for detecting richness of the toad species.
The new species is found along clear water rocky streams from Chishui County, Guizhou Province, China, and little is known about the population status of the new species. Thus, further research on the true distribution, population size and trends, and conservation actions are required.