A new species of the genus Leptobrachella Smith, 1925 (Anura, Megophryidae) from Guizhou, China

Abstract Asian leaf-litter toads of the genus Leptobrachella represent charismatic anuran diversification with 80 species, of which 25 are from China. Recent new discoveries suggest that the diversity of this genus is underestimated. Here, we describe a new species of Leptobrachella, Leptobrachella bashaensissp. nov. from the Basha Nature Reserve, Congjiang County, Guizhou Province, China. The new species is distinguished from its congeners by the following suite of morphological traits: small body size (SVL 22.9–25.6 mm in six adult males and 27.1 mm in one adult female); head longer than wide; dorsal skin slightly shagreened with small tubercles; creamy-white chest and belly with irregular black spots; distinct ventrolateral glands forming a white line; finger webbing and fringes absent; toe webbing rudimentary and lateral fringes narrow; iris bicolored with bright orange in upper half and silver in lower half; dorsal surface of tadpole head dark brown with small, brown, irregular spot, air sac-shaped bulges on both sides of body. The new species differs from all known congeners by an uncorrected p-distance of >5.3% of the 16S rRNA gene fragment examined, and the phylogenetic analysis clusters the new species with L. maoershanensis and L. laui. At present, the new species is only known from a small range of montane evergreen secondary forests in Basha Nature Reserve approximately 900 m elevation. Its natural history and conservation status are discussed.

were euthanized using chlorobutanol solution, fixed in 10% formalin for 24 hours, then stored in 75% ethanol. Liver or muscle tissues were taken from the specimens before fixing, and preserved in 95% alcohol at -20 °C. These newly collected specimens were subsequently deposited in the Museum of Biology of the Guizhou Institute of Biology, Guizhou Academy of Sciences (GIB, GAS), Guiyang, China.

Morphology and morphometrics
All measurements were obtained to the nearest 0.1 mm (Watters et al. 2016) with digital calipers following the methods of Fei et al. (2009). The measurements of seven adult individuals were as follows: SVL snout-vent length (distance from the tip of the snout to the posterior edge of the vent); 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); SNT snout length (from tip of snout to anterior corner of eye); EYE eye diameter (diameter of exposed portion of eyeball); IOD interorbital distance (minimum distance between the inner edges of the upper eyelids); IND internasal distance (minimum distance between the inner margins of the external nares); UEW upper eyelid width (measured as the greatest width of the upper eyelid); NEL nostril-eyelid length (distance from nostril to eyelid); NSL nostril-snout length (distance from nostril to snout); TMP tympanum diameter (horizontal diameter of tympanum); TEY tympanum-eye distance (distance from anterior edge of tympanum to posterior corner of eye); TIB tibia length (tibia length with hindlimb flexed); ML manus length (distance from tip of third digit to proximal edge of inner palmar tubercle); LAHL length of lower arm and hand (distance from tip of the third finger to elbow); HLL hindlimb length (distance from tip of fourth toe to vent); FOT foot length (from proximal edge of inner metatarsal tubercle to tip of fourth toe).
Morphometrics of a single tadpole followed Ohler et al. (2011), a total of nine morphometric characters of tadpole were measured:

BH
maximum body height; BL body length (distance from base of vent to the tip of snout); BW maximum body width; ED maximum eye diameter (diameter of exposed portion of eyeball); TH maximum tail height (maximum height between upper and lower edges of tail); SS snout to spiraculum (distance from the tip of the snout to the opening of the spiracle); TMW maximum tail muscle width; TL tail length (distance from base of vent to the tip of tail); TOL total length (distance from the tip of snout to the tip of tail).

DNA sequencing and analysis of sequences
Standard phenol-chloroform extraction with ethanol precipitation protocol was used to extract genomic DNA from tissues (Sambrook et al.1989). We amplified a 565~567 bp fragment 16S rRNA using polymerase chain reactions (PCRs), using the primers, and cycling conditions given by Kocher et al. (1989). Amplified PCR products were purified with enzymatic purification using exonuclease 1 and shrimp alkaline phosphatase that was incubated at 37 °C for 15 min followed by 80 °C to inactivate the enzymes. Purified PCR products were used as templates for Big Dye (Applied Biosystems, Foster City, CA) cycle sequencing. We conducted all sequencing on an ABI 3100 automated sequencers (Applied Biosystems) at Suoqin, Beijing. Considering that the morphological characters of the newly collected specimens are closest to those of L. maoershanensis, 19 sequences representing 16 recognized species of the seven clades belonging to the genus Leptobrachella from southern China (A1-A7; Chen et al. 2018) were retrieved from GenBank and included in the following phylogenetic analyses (Table 1). Megophrys major Boulenger, 1908 was selected as the outgroup based on the current phylogenetic hypothesis of Leptobrachella . All the sequences were aligned using MUSCLE v. 3.6 with the default settings (Edgar 2004).
Phylogenetic relationships were inferred using both maximum likelihood (ML) and Bayesian inference (BI). ML analysis was performed with RAx ML Black Box (Stamatakis et al. 2008) under the GTR + G model (1000 replicates). BI was performed using MrBayes v. 3.2.1 (Ronquist et al. 2012). The best-fit model was determined using the Akaike Information Criterion (AIC) computed with jModel Test 2 (Darriba et al.2012). Two independent runs were conducted for 30 million generations, sampling every 1000, with four independent chains and a burn-in of 25%. Convergence was assessed by checking stationary distribution and effective sample sizes (>200) using Tracer v. 1.6. (Rambaut et al.2014). We used MEGA v. 5.2 (Tamura et al. 2011) to calculate the interspecific mean uncorrected pairwise distances between the samples from Basha and its most closely related species based on the phylogenetic results. Besides, intraspecific distance was calculated among the samples from Basha.

Results
A total of 565~567 base pairs (bp) of the 16S gene were obtained in the final alignment. The two sequences belonging to the newly acquired specimens (GenBank accession numbers MW136294 and MW136295) shared a same haplotype. The alignment contained 242 variable sites and 186 parsimony informative sites. The observed interspecific uncorrected p-distances between the new population from Basha and all species from clade A based on the study of Chen et al. (2018) varied from 5.3% (L. maoershanensis) to 15.7% (L. firthi) ( Table 2).
Both ML and BI (Fig. 2) analyses yielded near-identical results except for the poorly supported nodes (BS < 75% or BPP < 0.95). The relationships of the most clades (A1-A7) of Leptobrachella were not resolved, which is similar to previous studies (Yang et al. 2016;Yuan et al. 2017) due to limited information of 16S. The samples from Basha, Guizhou, formed a monophyletic group (Clade A4, Fig. 2) with L. liui, L. laui, L. mangshanensis, L. maoershanensis, and L. yunkaiensis with high support in both analyses, but the relationships among these species were not resolved. The samples from Basha formed a lineage that clustered with L. maoershanensis, but with low support (BPP < 95% and BS < 70%). In combination with morphological differences (see below), we conclude that the newly collected Basha specimens represent a distinct species, and we describe it herein.

Taxonomic account
Leptobrachella bashaensis sp. nov. http://zoobank.org/6257CDCB-B124-4649-BE10-9A4874C67F20 Figs 3-7; Tables 1-3 Type material. Holotype. GIB196403, adult male, from Basha Nature Reserve, Congjiang, Guizhou Province, China (25.8534°N, 108.7356°E, elevation 900 m. a.s.l.;  Diagnosis. The new species is assigned to the genus Leptobrachella on the basis of the following characters: small size, rounded fingertips, presence of an elevated inner palmar tubercle not continuous to the thumb, presence of supra-axillary, femoral and ventrolateral glands, vomerine teeth absent, tubercles on eyelids, and pale vertical bar present on anterior tip of snout (Dubois , 1983Ohler et al. 2011;Rowley and Cao 2009;Rowley et al. 2013). Leptobrachella bashaensis sp. nov. is distinguished from its congeners by a combination of the following morphological characters: (1) small size (SVL 22.9-25.6 mm in six adult males and 27.1 mm in one adult female), (2) head longer than wide, (3) externally distinct tympanum, (4) dorsal skin slightly shagreened with small tubercles and irregular brown stripes, (5) distinct dark spots on the flank, (6) creamy-white chest and off-white belly with irregular black spots, (7) grey-pinkish to dark brownish-violet ventral skin of limbs with numerous whitish speckles, (8) distinct ventrolateral glands, forming a distinct white line, (9) finger webbing and fringes absent, (10) toe webbing rudimentary and lateral fringes narrow, (11) longitudinal ridges under toes and not interrupted at the articulations, (12) a distinctly bicolored iris, typically bright orange in upper half, fading to silver in lower half.
Description of the holotype. Adult male. SVL 24.0 mm; head slightly longer than wide (HDL/HDW = 1.10), rectangular in dorsal view (Fig. 3A, B); snout rounded in both ventral view and lateral view, protruding slightly beyond lower jaw (Fig. 3D); nostril oval, located closer to tip of snout than to eye; loreal region oblique; Table 2. Pairwise genetic divergence between Leptobrachella species of monophyletic clades A based on uncorrected p-distance at a 16S rRNA fragment.    (Fig. 3C); vomerine teeth absent; vocal sac openings slit-like, located posterolateral on floor of mouth; tongue long and moderately wide, with a shallow notch at posterior tip; supratympanic ridge distinct, running from eye towards axillary with raised tubercles. Forelimb long and slender, fingers long and slender, without webbing and lateral fringes; relative length of fingers II<I<IV<III; tips of fingers rounded and slightly swollen; nuptial pad absent; subarticular tubercles absent in fingers; inner metacarpal tubercle large and rounded, separated from laterally compressed and much smaller outer metacarpal tubercle (Fig. 4A, B). Hindlimb moderately long; tibia half of snout-vent length (TIB/SVL = 0.50); tibiotarsal articulation of adpressed limb reaching snout, well beyond anterior margin of eye, but not beyond snout tip; relative toe lengths I<II<V<III<IV; toe tips rounded and thickened; greatly reduced basal webbing present between all five toes; narrow lateral fringes present only on II and III toes (Fig. 4C, D); subarticular tubercles hardly discernible under toes II and III; dorsal skin slightly rough with small tubercles and irregular pustules; ventral skin smooth; oval supra-axillary gland present at forelimb base on ventral surface of axillary region (Fig. 3F); oval femoral glands distinct on posteroventral surface of thigh, closer to knee than to vent; ventrolateral glands forming a distinct line on flanks.
Coloration of holotype in life. Dorsal surface of head and trunk brown with small, dark brown, irregularly shaped markings; large reverse-triangle dark brown marking between eyes, connected to the W-shaped mark between axillae (Fig. 3); supratympanic ridge present; elbow to upper arm reddish in color; fine, distinct reddish tubercles scattered on upper eyelids, dorsal surfaces of head, body, and limbs. Ventral surface of throat and belly off-white, with faint spots; chest creamy white, with irregular black spots (Fig. 3G, H); ventral surfaces of lip and limbs covered with irregular white speckles; ventral surface of thighs grey-brown with white spots. Supra-axillary gland, ventrolateral glands, and femoral glands white. Iris bicolored: bright orange in upper half, silver in lower half, with black reticulations throughout. Fine, faint transverse dark brown bars on dorsal surface of fingers and toes, lower arms, tarsus, thighs, and tibia (Fig. 4).
Coloration of holotype in preservation. In preservative (75% ethanol), dorsal surface dark grey-brown, and ventral and lateral white glands not evident (Fig. 5); ventral surface of throat, chest, belly, and interior portions of arms fade to creamy white, ventral surface of thighs dark brown with white spots; bars and blotches on dorsum and limbs are dark brown and less apparent. The color of the tympanum fades to brown.
Tadpoles. Brown, narrow; BH 4.4 mm; BL 14.1 mm; BW 6.2; ED 2.0 mm; TH 3.8; SS 4.9 mm; TMW 3.4 mm; TL 29.3 mm; TOL 43.8 mm. TAL/TOL = 0.67, with a I:3+3/1+1:II labial tooth row formula (Fig. 6). In life, dorsal surface of head dark brown with small, brown, irregularly shaped spot; air sac-shaped bulges on both sides of the body; upper lip and lower lip nearly round shape. Tadpoles were collected in the field from a stream surveyed on 16 October 2017 in Basha Nature Reserve by Lyu. Etymology. The specific epithet, "bashaensis"/Basha Zhang Tu Chan (岜沙掌突蟾 in Chinese), refers to the location where the specimens were collected, Basha Nature Reserve of Guizhou Province, China.
Morphological variation. Variation in size and body proportions of the type series are shown in Table 3. Representative photographs of paratypes in life are shown in Fig. 7. In life, the dorsal surface of the head and trunk varies from olive-brown to reddish; the ventral surface of the lower jaw among these individuals varies from cream-yellow, pink, to grey; the ventral surface of chest and belly is grey-pink to white. There is variation among individuals in the shape of tubercles, pustules, black ventrolateral blotches, and dark stripes, bars on the dorsum or dorsal surface of limbs. The W-shaped marking between axillae is distinct in all individuals.
Natural history. All specimens were collected at night in small streams in Basha Nature Reserve approximately 900 m elevation (Fig. 8). Calling males were found along the streams, perching on large rocks, in rocky crevices, or under dead wood. Insect-like calls could be heard in June. The breeding season of this species is likely to occur from June to July, as females collected during these months were gravid, and males were heard calling only from June to the beginning of July. During both surveys, the number of males observed was much greater than females (males:females = 12:1).
Comparisons. Leptobrachella bashaensis sp. nov. differs from all known Leptobrachella species distributed north of the Isthmus of Kra by a combination of male body size,    externally distinct tympanum, and black spots on the flank, plus ventral coloration, degree of webbing and fringing on the toes, iris coloration, and dorsal skin texture. Care was taken to differentiate Leptobrachella bashaensis sp. nov. from the five most morphologically and molecularly similar species, L. maoershanensis, L. laui, L. mangshanensis, L. yunkaiensis, and L. liui from China.
Leptobrachella bashaensis sp. nov. differs from its sister taxon L. maoershanensis by the following: smaller body size (SVL 22.9-25.6 mm in males, 27.1 mm in female vs 25.2-30.4 mm in males, 28.6 mm in female); bicolored iris with bright orange in upper half, fading to silver in lower half (vs typically bright orange-red in upper half, fading to silver in lower half ); vertical and distinct narrow pupil (vs vertical and wide pupil); narrow lateral fringes present only on II and III toes (vs narrow lateral fringes present on all toes).
Leptobrachella bashaensis sp. nov. differs from L. laui in having slightly a smaller body size (SVL 22.9-25.6 mm in males, 27.1 mm in female vs 24.8-26.7 mm in males, 28.1 mm in female); bicolored iris with bright orange in upper half, fading to silver in lower half (vs uniformly coppery orange with fine black reticulations throughout); vertical and distinct narrow pupil (vs vertical and wide pupil); narrow lateral fringes present only on II and III toes (vs wide lateral fringes present on all toes); distinct black spots on the flank (vs indistinct black spots on the flank).
Leptobrachella bashaensis sp. nov. differs from L. mangshanensis by having slightly smaller body size (SVL 22.9-25.6 mm in males, 27.1 mm in female vs 22.2-27.8 mm in males, 30.2 mm in female); bicolored iris with bright orange in upper half, fading to silver in lower half (vs bright orange in upper half, greyish cream in lower half ); narrow lateral fringes present only on II and III toes (vs weak lateral fringes on toes).
Leptobrachella bashaensis sp. nov. differs from L. yunkaiensis by having smaller body size (SVL 22.9-25.6 mm in males, 27.1 mm in female vs 25.9-29.3 mm in males, 34.0-35.3 mm in females); bicolored iris with bright orange in upper half, fading to silver in lower half (vs coppery orange in upper half and silver in lower half ); narrow lateral fringes present only on II and III toes (vs wide lateral fringes present on all toes); distinct black spots on the flank (vs indistinct black spots on the flank).
Leptobrachella bashaensis sp. nov. differs from L. liui in having slightly smaller body size (SVL 22.9-25.6 mm in males, 27.1 mm in female vs 23.0-28.7 mm in males, 23.1-28.1 mm in females); vertical and distinctly narrow pupil (vs vertical and wide pupil); narrow lateral fringes present only on II and III toes (vs wide lateral fringes present on all toes). Chen et al. (2018) suggested that Leptobrachella may contain hidden species diversity based on multiple nuclear DNA markers assessed using larger sample sizes, combined with morphological, nuclear gene, and bioacoustic data. With the development of DNA barcoding technology and extensive field work, more and more cryptic species in this genus have been reported. The number of Leptobrachella species recorded in China is now up to 25, including the new species. The discovery of this species reaffirms that the diversity within the genus is underestimated, with many species yet to be discovered.

Discussion
Guizhou Province is located in the typical Karst landform area of southern China with an abundance of species diversity. So far, eight species of the genus Leptobrachella (L. liui, L. oshanensis, L. pelodytoides, L. ventripunctatus, L. bijie, L. purpuraventra, L. chishuiensis, and L. suiyangensis) were recorded in Guizhou province, while L. bashaensis sp. nov. has not been included in previous studies, which indicates that there may potentially be more new species to be found by further field research (Wu et al. 1987;Wang et al. 2019).
We observed a restricted distribution of the species in Basha Natural Reserve. We believe that L. bashaensis sp. nov. has a specific niche associated with small streams. The following threats were observed: reduced forest coverage, soil erosion, habitat destruction, and local people using the tadpole of L. bashaensis sp. nov. for food. Hence, greater protection is required for this endemic species inhabiting this highdiversity region.