A new species of the genus Leptobrachella (Anura, Megophryidae) from northwestern Guangdong Province, China

The genus Leptobrachella is a species-rich genus of megophrid frog. Rapid discovery of many new species within this genus emphasizes the importance of regional research. In this study, we describe a new species of Leptobrachella , Leptobrachella verrucosa sp. nov. , from northwestern Guangdong Province, China, based on a combination of molecular and morphological data. A key in-cluding congeners from the same province, namely L. laui , L. liui , L. mangshanensis , L. shimentaina , and L. yunkaiensis , is provided.


Introduction
Frogs of the genus Leptobrachella Smith, 1925, are forest dwellers that are widely distributed in China, Myanmar, northeastern India, the Indochina region, Borneo and Natuna Island (AmphibiaWeb 2022;Frost 2022). Leptobrachella represents one of the most species-rich groups of frogs with 93 recognized species. However, the morphological conservativeness of this group makes it hard to study their taxonomy, hindering our understanding of the true diversity of this group . Combining detailed morphological examination and molecular phylogeny, During recent field surveys in mountainous areas of northwestern Guangdong which border with Hunan Province and Guangxi Zhuang Autonomous Region (Fig. 1), we collected five adult male specimens of Leptobrachella. Preliminary morphological examination indicated that they can be distinguished from recognized congeners by a series of discrete features. Subsequent molecular analysis further revealed that these specimens represent a separate evolutionary lineage. Considering both the morphological differences and their genetic allocation and divergences, these specimens are described herein as a new species. A key for congeners that occur in Guangdong Province is also provided.

Phylogenetic analyses
In total, 108 samples of 80 Leptobrachella species plus 2 outgroup samples were used in this study, encompassing five newly sequenced individuals and others downloaded from GenBank. Detailed information for all samples is given in Suppl. material 1: Table S1. All newly collected specimens were euthanized with MS 222 and fixed in 10% buffered formalin and later transferred to 70% ethanol for preservation, and deposited at the Guangdong Polytechnic of Environmental Protection Engineering (GEP), Foshan City, Guangdong, China; muscle tissue samples were preserved in 95% ethanol for molecular studies.
The 16S ribosomal RNA mitochondrial gene (16S rRNA) fragment of 528 base pairs was sequenced for new samples; DNA extraction, PCR, and sequencing follow . Sequences were aligned with Clustal X 2.0 (Thompson et al. 1997) with default parameters. We ran Jmodeltest v2.1.2 (Darriba et al. 2012; with Akaike and Bayesian information criteria) on our alignment and obtained the bestfitting nucleotide substitution model of GTR + I + G (General Time Reversible model, with variable sites modelled according to the Gamma distribution, and a portion of the sites invariant). Phylogenetic analysis was conducted using Bayesian inference (BI) in MrBayes 3.2.4 (Ronquist et al. 2012) and maximum likelihood (ML) in maximum likelihood (ML) in Randomized Axelerated Maximum Likelihood (RAxML, Stamatakis 2006) with RAxML GUI 1.3 (Silvestro and Michalak 2012). For the ML analysis, an optimal tree was obtained and branch supports were evaluated with 1000 rapid bootstrapping replicates. For the BI analysis, two independent runs with four Markov Chain Monte Carlo simulations were performed for ten million iterations and sampled every 1000 iterations. The first 25% of the samples were discarded as burn-in, leaving 7500 samples in the final summary. Convergence of the Markov Chain Monte Carlo simulations was assessed by PSRF < 0.01 and ESS (effective sample size) value > 200 using Tracer 1.4 (http://tree.bio.ed.ac.uk/software/ tracer/). Nodes having ML bootstrap values (BS) ≥ 70 and BI posterior probabilities (BPP) ≥ 0.90 were considered well supported. Genetic distances among all Leptobrachella samples were calculated in MEGA 6 using the uncorrected p-distance model. Following Fei et al. (2009) and Rowley et al. (2013), measurements were taken with digital calipers from preserved specimens (Neiko 01407A Stainless Steel 6-Inch Digital Calipers) to the nearest 0.1 mm. Measurements include: snout-vent length (SVL) from the tip of the snout to posterior margin of the vent; head length (HDL) from the tip of the snout to the articulation of the jaw; head width (HDW) at the commissure of the jaws; snout length (SNT) from the tip of the snout to the anterior corner of the eye; eye diameter (EYE) from the anterior corner of the eye to posterior corner of the eye; internasal distance (IND); interorbital distance (IOD); horizontal diameter of tympanum (TMP); tympanum-eye distance (TEY) from the anterior edge of tympanum to posterior corner of the eye; tibial length (TIB) from the outer surface of the flexed knee to the heel; manus length (ML) from the tip of the third finger to the proximal edge of inner palmar tubercle; pes length (PE) from the tip of the fourth toe to the proximal edge of the inner metatarsal tubercle; length of lower arm and hand (LAHL) from the tip of the third finger to elbow; and hindlimb length (HLL) from the tip of the fourth toe to vent.

Morphological examination
Sex was determined by direct observation of calling in life. Comparative morphological data for other congeneric species of Leptobrachella were obtained from museum specimens (Appendix 1) and from the literature (Table 1).

Results
The ML and Bayesian approach results identical topologies, and all Leptobrachella samples form a monophyletic clade strongly-supported in both phylogenetic trees (Fig. 2). The Leptobrachella samples (ID 1-5, Suppl. material 1: Table S1) from Lianshan Bijiashan Nature Reserve form a monophyletic clade which is strongly supported (BS = 100; BPP = 1.00). This clade represents an independent lineage with strong divergence and it is grouped with the clade composed of L. liui and L. mangshanensis. However, the relationship of these three lineages is not well resolved.
Although we did not use genetic distances to diagnose the new species (which we define on the basis of discrete character differences), we note that the Leptobrachella lineage from Lianshan Bijiashan Nature Reserve differs from its closely-related congeners by 2.4% (with L. liui) and 2.6% (with L. mangshanensis) in the mean p-distance of 16S RNA ribosomal mitochondrial gene (Suppl. material 2: Table S2), which approximates the level of genetic divergence observed in uncontroversial species of Leptobrachella:  Table S1. Etymology. The specific epithet "verrucosa" is an adjective meaning "verrucous", referring to the tuberculate dorsal skin texture of the new species. Diagnosis.
Description of holotype. Adult male. Body size small, SVL 23.2 mm. Head length slightly larger than head width, HDW/HDL 0.95; snout slightly protruding, projecting slightly beyond margin of lower jaw; nostril closer to snout than eye; canthus rostralis gently rounded; loreal region slightly concave; interorbital space flat, internarial distance slightly larger than interorbital distance, IND/IOD 1.05; pineal ocellus absent; pupil vertical; snout slightly longer than eye diameter, SNT/EYE 1.03; tympanum distinct, rounded, diameter smaller than that of eye and larger than tympanum-eye distance, TMP/ EYE 0.54, TEY/TMP 0.46; upper margin of tympanum in contact with supratympanic ridge; vomerine teeth absent; a single vocal sac; vocal sac openings slit-like, paired, located posterolaterally on floor of mouth, close to margins of mandible; tongue deeply notched distally; supratympanic ridge distinct, extending from posterior corner of eye to supra-axillary gland.
Tips of fingers rounded, slightly swollen; relative finger lengths I = II = IV < III; nuptial pad absent; subarticular tubercles absent; inner palmar tubercle large, rounded, distinctly separated from outer palmar tubercle; outer palmar tubercle small, rounded; fingers lacking interdigital webbing and lateral fringes. Tips of toes rounded, slightly swollen; relative toe length I < II < V < III < IV; longitudinal ridges under toes continuous; inner metatarsal tubercle large, oval; outer metatarsal tubercle absent; interdigital webbing between toes rudimentary; narrow lateral fringes present on all toes. Tibia 49% of snout-vent length; tibiotarsal articulation reaches to anterior corner of eye; heels not meeting when thighs are appressed at right angles with respect to the body.
Dorsal surface shagreened with numerous conical tubercles, lacking spines, enlarged warts or skin ridges; ventral skin smooth; pectoral and femoral glands oval; both larger in diameter than tip of fingers; femoral glands larger in diameter than tip of toes; femoral gland situated on posteroventral surface of thigh, closer to knee than to vent; supra-axillary gland raised. Ventrolateral gland distinctly visible, forming a longitudinal uncontinuous series.

Coloration of holotype in life.
Dorsal background color grayish brown, with distinct dark brown markings, and rounded spots, and scattered with irregular light orange pigmentation. A dark brown inverted triangular marking in interorbital region, connecting to dark brown W-shaped marking on occipital region. Tympanum dark brown, lower margin grayish white. Supratympanic line black. A pair of dark brown vertical bars present under eyes; transverse dark brown bars on dorsal surfaces of distal limbs and digits; distinct dark brown blotches on flanks; surfaces of elbows and upper arms coppery orange, with dark markings and spots.
Surface of throat, chest, belly and underside of forelimbs creamy white, with distinct grayish white and dark brown spots; under side of hindlimbs grayish purple with numerous white spots. Supra-axillary gland coppery orange, pectoral glands grayish white, femoral glands coppery orange, anterior half of ventrolateral glands coppery and posterior half grayish white. Iris bicolored, upper half coppery orange, lower half grayish brown.
Coloration of holotype in preservative. Dorsal background color dark brown scattered with irregular gray   pigmentations. All markings, bars and spots become indistinct. Tympanum dark brown. Ventral surface grayish white; grayish white spots absent, dark brown spots become more distinct. Supra-axillary, pectoral, and ventrolateral glands greyish white.
Distribution, ecology and conservation. Leptobrachella verrucosa sp. nov. is currently only known from its type locality ( Fig. 1; ca. 500-600 m a.s.l.) which is located at the boundary of Guangxi Zhuang Autonomous Region, and Guangdong and Hunan provinces. The new species inhabits clear-water rocky streams in primary forests surrounded by broadleaved forest in granite landforms. Males were observed calling while perched on rocks or leaves of dwarf shrubs near flowing seeps. Data of larvae and females remain unknown. The breeding season lasts from April to June according to our long-term observation. We recommend the new species be listed as LC (Least Concerned) in the IUCN categorization as the forest is well protected and the distribution of the species is potentially wider than currently known, i.e. the adjacent Hunan Province and Guangxi Zhuang Autonomous Region.
Comparisons. In the phylogenetic tree (Fig. 2), Leptobrachella verrucosa sp. nov. is most closely related to the clade composed of L. liui and L. mangshanensis, therefore, the latter two are selected for detailed comparison ( Table 3). The new species differs from L. liui by its narrow fringes along lateral surfaces of toes (vs. wide), continuous longitudinal ridges under toes (vs. interrupted), dorsal surface with numerous conical tubercles and lacking skin ridges (vs. dorsal surface with rounded warts and sparse short skin ridges), heels not meeting when adpressed (vs. heels meeting). The new species differs from L. mangshanensis by its shagreened dorsal surface with numerous conical tubercles, lacking spines, enlarged warts or skin ridges (vs. dorsal surface almost smooth with tiny transparent spines, small tubercles and sparse short skin ridges), ventral surface of thighs lacking conical spines (vs. ventral surface bearing conical spines), tibial-tarsal articulation reaching anterior corner of eye (vs. reaching tip of snout, well beyond anterior corner of eye).
Leptobrachella verrucosa sp. nov. can be distinguished from L. yunkaiensis and L. shimentaina by having no fringes along lateral surfaces of fingers (vs. lateral fringes present in L. yunkaiensis and males of L. shimentaina); narrow fringes along lateral surfaces of toes (vs. lateral fringes wide in L. yunkaiensis and males of L. shimentaina); shagreened dorsal surface with numerous conical tubercles, lacking spines, enlarged warts or skin ridges (vs. dorsal surface with skin ridges in L. shimentaina, dorsal surface with short skin ridges and raised warts in L. yunkaiensis); heels not meeting when adpressed (vs. heels slightly overlapping in L. shimentaina, heels meeting in L. yunkaiensis). Leptobrachella verrucosa sp. nov. can be distinguished from L. bashaensis and L. maoershanensis by having shagreened dorsal surface with numerous conical tubercles, lacking spines, enlarged warts or skin ridges (vs. dorsum shagreened with small tubercles in L. bashaensis, dorsum with skin ridges in L. maoershanensis); tibialtarsal articulation reaching anterior corner of eye (vs. reaching tip of snout, well beyond anterior corner of eye in L. bashaensis and L. maoershanensis). Leptobrachella verrucosa sp. nov. can be distinguished from L. laui by having no lateral fringes along lateral surfaces of fingers (vs. moderate lateral fringes present), narrow lateral fringes along lateral surfaces of toes (vs. lateral fringes wide). Leptobrachella verrucosa sp. nov. differs from L. flaviglandulosa by having continuous longitudinal ridges under toes (vs. interrupted), heels not meeting when adpressed (vs. heels meeting).

Discussion
Leptobrachella pelodytoides (Boulenger, 1893) was described from Thao [= Thamo], Kayah State, Myanmar. Pope (1931) first reported L. pelodytoides in China based on a series of specimens from Fujian Province. The identification was revised by Fei and Ye (1992) and it was recognized as a distinct species, L. liui. Fei et al. (2009) further extended the known distribution of L. liui to Guangxi Zhuang Autonomous Region, Fujian, Zhejiang, Jiangxi, Hunan, and Guizhou Provinces, and two localities bordering Guangdong, i.e. Hunan Province (Yizhang county) and Hong Kong. Later, Li et al. (2011) recognized Leptobrachella populations from Hong Kong and Guangdong Province (Xinyi City, Fengkai County, Shenzhen City) as L. pelodytoides (Boulenger, 1893). However, all these proposals, excluding the record from Fengkai County, have since been revised: (1) the Leptobrachella population from Hong Kong and adjacent Shenzhen City of Guangdong Province should actually be L. laui (Sung et al. 2014), neither L. pelodytoides nor L. liui; (2) the population recorded as L. liui from Hunan Province was revised as L. mangshanensis (Hou et al. 2018), which was confirmed to occur in northern Guangdong Province by Wang et al. (2022); (3) the population recorded as L. pelodytoides from Xinyi City was revised as L. yunkaiensis by Wang et al. (2018); (4) Wang et al. (2022) further described a new species from northern Guangdong Province, L. shimentaina, which was indicated by Chen et al. (2018) as a putatively undescribed species "Leptobrachella sp. 6" (refer to Suppl. material 1: Table S1 and Fig. 1 for detailed distribution information). The taxonomic status of the population from Fengkai County, western Guangdong remains unresolved due to the lack of morphological and molecular data and the distribution of L. pelodytoides in China has been indirectly ruled out (Ohler, 2011;Chen et al. 2017). Thus, we propose to remove L. pelodytoides from the herpetofauna of Guangdong Province. Most importantly, the historic records of L. liui and L. pelodytoides from other areas require reevaluation.
Below, we provide a key for all species within the genus Leptobrachella occurring in Guangdong Province, which includes L. laui, L. liui, L. mangshanensis, L. shimentaina, L. yunkaiensis, and L. verrucosa.