Morphological and molecular evidence for a new species of the genus Leptobrachella (Anura, Megophryidae) from Gaoligong Mountain Range, Yunnan, China

Yun-He Wu; Zhong-Bin Yu; Shen-Pin Yang; Zheng-Pan Duan; An-Ru Zuo; Ding-Can Zhang; Felista Kasyoka Kilunda; Robert W. Murphy; Jing Che

doi:10.3897/zse.101.135560

Abstract

The Gaoligong Mountain Range in Yunnan Province, China, is characterized by its large variation in elevation and topography, together with its wide latitudinal range, resulting in extremely high levels of biodiversity. Studies show that the amphibian diversity of the Gaoligong Mountain Range is largely underestimated, especially in the south. During herpetological surveys in 2023 and 2024, three specimens of Leptobrachella were collected from the mountain at Tongbiguan Provincial Nature Reserve. Subsequent morphological comparisons and a phylogenetic reconstruction revealed that these specimens belonged to a previously unknown and morphologically distinct lineage of Leptobrachella, which we formally describe. Our discovery brings the number of species in the genus to 108, 44 of which occur in China, and seven on Gaoligong Mountain Range. This result confirms the underestimated amphibian diversity of the Gaoligong Mountain Range, especially in Tongbiguan Provincial Nature Reserve, where multiple sympatric congeneric species occur, including four species of Leptobrachella, five species and a putative new species of Xenophrys, and three species of Polypedates. These findings also highlight the need for future research to investigate the mechanisms of sympatric and syntopic coexistence.

Key Words

Asian Leaf Litter Toad, frog, Leptobrachella albomarginata sp. nov., sympatric distribution, Tongbiguan Provincial Nature Reserve

Introduction

The Asian leaf litter toad genus Leptobrachella Smith, 1925 (Megophryidae; Leptobrachiinae) occurs widely in the mountain forests of southern China, mainland Indochina, peninsular Malaysia, and the island of Borneo (Frost 2024). The genus is characterized by having a relatively small body size, vomerine teeth absent, the presence of an inner metacarpal tubercle, rounded fingertips, and macro-glands on the supra-axillary and femoral glands (Dubois 1980; Ohler et al. 2011; Rowley et al. 2013). Given their small body size, conserved morphology and high degree of sympatry, the species diversity of Leptobrachella is likely underestimated (e.g. Chen et al. 2018, 2020; Wu et al. 2022). More than half of the recognized species were described in the past decade by utilizing morphological, molecular, and bioacoustic data (e.g. Lin et al. 2022; Luo et al. 2022; Liu et al. 2023; Luong et al. 2023; Matsui et al. 2023; Shi et al. 2023; Chen et al. 2024; Frost 2024; Li et al. 2024). Currently, the genus contains 107 recognized species, of which 43 have been recorded from China, and 23 were described in the past five years, mostly from southwestern China (AmphibiaChina 2024; Frost 2024).

Gaoligong Mountain Range is a sub-range located in the western part of China’s Yunnan Province adjoining northern Myanmar, spanning approximately 600 km. It is drained by the Salween River on the east and the Irrawaddy River on the west, and characterized by its rugged terrain of deep valleys and high mountains, with elevations ranging from 210 m to 5128 m a.s.l. The unique geographic location and extreme topographic relief, together with its wide altitudinal and latitudinal range, have produced extreme differences in climate. The diverse climate and topography of the mountain has resulted in extremely high levels of biodiversity, forming a crossroad of three global biodiversity hotspots (i.e., Indo-Burma, Himalaya, and mountains of Southwest China) (Myers et al. 2000; Mittermeier et al. 2011). The range is one of the biologically richest places on Earth (Mittermeier et al. 2005; Chan et al. 2019). Several cryptic and novel herpetological species have been discovered and described in recent years (e.g. Lee et al. 2024; Yu et al. 2024; Wu et al. 2023, 2024a,b), indicating that amphibian diversity in the region may still be largely underestimated.

In recent years, we carried out a series of biodiversity surveys in Yingjiang County, from 2023 to 2024 and collected three specimens of Leptobrachella, which differed from known species in the genus by both morphological and molecular characteristics. Herein, we describe them as a new species.

Materials and methods

Sampling

During herpetological surveys at Tongbiguan Town, Yingjiang County, Yunnan, China conducted between 2023–2024 (Fig. 1), two adult specimens (one male and one female) of Leptobrachella were collected and photographed. In addition, one larvae was collected. After taking photographs, the adult frogs were euthanized using benzocaine. Liver tissues were taken from the specimens and preserved in 95% ethanol at -80 °C for DNA extraction. The specimens were then fixed in 10% formalin and after 24 hours stored in 75% ethanol. The procedures for tissue sampling and specimen fixation follow the protocols detailed in Chen et al. (2021). The larvae was directly stored as a whole in 75% alcohol for molecular experiments. All the newly collected specimens were deposited in the herpetological collection of the Museum of the Kunming Institute of Zoology (KIZ), Chinese Academy of Sciences (CAS). Research protocols were approved by the Ethics Committee of the Kunming Institute of Zoology, Chinese Academy of Science (IACUC no.: IACUC-OE-2021-07-001).

Figure 1.

Collection localities (yellow stars) of Leptobrachella albomarginata sp. nov. specimens from Tongbiguan Provincial Nature Reserve, Yunnan, China used in this study.

Morphological characters

Morphological characters were obtained for both adult specimens. All measurements were recorded with digital calipers to the nearest 0.1 mm. Morphological terminology and methods adhered to the guidelines of Fei et al. (2009). Measurements included the following: snout–vent length (SVL): measured from tip of snout to vent; head length (HDL): measured from tip of snout to rear of jaw; maximum head width (HDW): measured width of head at its widest point; snout length (SNT): measured from tip of snout to anterior corner of ocular aperture; eye diameter (ED): diameter of exposed portion of eyeball; width of upper eyelid (UEW): (maximum width of upper eyelid); tympanum diameter (TD): measured as maximal diameter of tympanum; distance from nostril to eye (DNE): distance from the front of the eye to the center of the nostril; distance from the center of the nostril to the tip of the snout (SN); internarial distance (IND): distance between nares; distance from anterior edge of tympanum to posterior corner of eye (TEY); interorbital distance (IOD): measured at narrowest point between eyes on top of the head; forearm length (FAL): measured from the elbow to the wrist; diameter of lower arm (LAD); forearm and hand length (FHL): distance from elbow to the tip of the third finger; hand length (HL): distance from the posterior end of the inner metacarpal tubercle to tip of third finger; hindlimb length (HLL); foot length (FL): distance from the proximal end of inner metatarsal tubercle to the tip of fourth toe; thigh length (THL): from the cloaca to the knee; tarsus length (TAL): measured as the distance from knee to heel; first to fourth finger length (FLI-IV): from the tip of the finger to its base where it joins the adjacent finger; IMTL (inner metatarsal tubercle length); PEC (maximum diameter of pectoral gland); FEM (maximum diameter of femoral gland); and SUP (maximum diameter of supra-axillary gland). Adult males and females were distinguished by the presence of vocal sacs (males), or eggs or enlarged oviducts (females).

DNA extraction, PCR and sequencing

Genomic DNA was extracted from the collected tissue samples using standard phenol-chloroform protocols (Sambrook et al. 1989). We amplified and sequenced a partial fragment of the mitochondrial ribosomal RNA gene (16S rRNA) using the primers 16SAR (Forward) and 16SBR (Reverse) (Kocher et al. 1989). The PCR amplification process was carried out in a 20-μl reaction volume. Polymerase Chain Reaction (PCR) condition followed was initial denaturation at 95 °C for 4 minutes, followed by 35 cycles of denaturation at 94 °C for 45 seconds, annealing at 55 °C for 45 seconds and extension at 72 °C for 1 minute, and a final extending step of 72 °C for 10 minutes. Sequencing was conducted using an ABI 3730xl DNA automated sequencer (Applied Biosystems, UK). All sequences were assembled from forward and reverse reads and edited manually using AutoSeqMan (Sun 2018). New sequences were deposited in the GenBank (Suppl. material 1).

To construct a phylogeny for Leptobrachella, trees were inferred using Maximum Likelihood (ML) and Bayesian Inference (BI) based on 16S rRNA. Since the new species belong to Clade A of Chen et al. (2018), we included all species from Clade A along with all species described after the publication of Chen et al. (2018). The homologous sequences of related species in the genus Leptobrachella, and those of the outgroups Leptobrachium huashen and Megophrys montana, were downloaded from GenBank (Suppl. material 1). Sequences were aligned using the default parameters implemented in MEGA v6.0.6 (Tamura et al. 2013). Alignments were visually checked by eye for accuracy and trimmed to minimize missing characters in MEGA v.6.0.6 (Tamura et al. 2013). Uncorrected pairwise distances (p) of 16S rRNA were calculated using MEGA v6.0.6 with complete deletion of missing data and gaps (Tamura et al. 2013). For BI analyses, JMODELTEST v2.1.7 (Darriba et al. 2012) was used to select an appropriate nucleotide substitution model. Based on the Bayesian Information Criterion (BIC; Posada 2008), the GTR+I+G model was chosen as the optimal nucleotide substitution model. BI analysis was implemented by the CIPRES web server (Miller et al. 2010), with Markov Chain Monte Carlo (MCMC) for 10,000,000 generations. Trees were sampled every 1000 generations and the initial 25% of trees were discarded as burn-in. Convergence was assessed by the average standard deviation of split frequencies (less than 0.01) and the ESS values (greater than or equal to 200) in Tracer v.1.5 (Rambaut and Drummond 2009). ML analysis was performed using RAxML-HPC BlackBox v.8.2.10 with the GTRGAMMA model of molecular evolution, implemented by the CIPRES web server (Miller et al. 2010).

Results

The aligned sequence matrix of the 16S rRNA gene contained 91 individuals with 516 base pairs (bps) (including outgroups). Amongst the 516 sites, 253 were established as conserved sites and 257 were considered variable sites, of which 211 were found to be potentially parsimony-informative sites (including outgroups).

ML and BI analyses yielded near-identical topologies, with relatively high nodal support values for most terminal nodes. The monophyly of Leptobrachella was strongly supported (BPP = 1, BS = 97), forming three major clades (clades A–C, Fig. 2). Our new samples from Tongbiguan Provincial Nature Reserve nested in Clade A, which strongly clustered into a lineage (BI = 1, ML = 100; Fig. 2) and clustered with L. tamdil, L. khasiorum, and L. yingjiangensis with good support (BI = 1, ML = 78; Fig. 2).

Figure 2.

Phylogram of Leptobrachella derived from analyses of 16S rRNA mitochondrial gene. Nodal support values with Bayesian posterior probability (BPP) > 0.95 / ML inferences (ML-BS) > 70 are shown near the respective nodes. A “-” denotes a Bayesian posterior probability < 0.95 and bootstrap support < 70. Nodes without any numbers or symbols represent low support values (BS < 75% and BPP < 0.95). New samples for the present study are indicated in red bold font.

The new population showed obvious genetic divergence from its congeners. The minimum uncorrected genetic distance was 9.1% between our species and L. tamdil, L. khasiorum, and L. graminicola (Suppl. material 2). These levels of pairwise divergence of the 16S rRNA gene far exceeded the threshold of 3.0% previously proposed by Vences et al. (2005) as a reliable indicator for identifying candidate new species in frogs.

Moreover, morphologically these specimens were distinguished from all other species of Leptobrachella by a series of taxonomically important diagnostic characters. Thus, we describe these specimens as a new species of Leptobrachella.

Taxonomic account

Leptobrachella albomarginata Wu, Yu, Kilunda, Murphy & Che, sp. nov.

https://zoobank.org/630BA976-31C1-4D96-AD45-4F6DAF801B07

Figs 3, 4, 5, Table 1

Type material.

Holotype : • KIZ 050905, an adult female from Tongbiguan Provincial Nature Reserve, Yingjiang County, Yunnan, China (24.56355°N, 97.58719°E, 1,634 m a.s.l.), collected on 20 April 2023 by Zhong-Bin Yu, Dong An, Yu-Xuan Wu, and Xian-Kun Huang.

Paratype : • KIZ 056551, an adult male from Tongbiguan Provincial Nature Reserve, Yingjiang County, Yunnan, China (24.63669°N, 97.59515°E, 1,318 m a.s.l.), collected on 30 April 2024 by Zhong-Bin Yu, Peng Yang, Dong An, and Xian-Rong Wu.

Etymology.

The name refers to reverse-triangle markings and ˄-shaped marking with a white lining on dorsum of the new species: the specific epithet “albus” is a Latin adjective which means “white”, and “marginis” is Latin adjective for “border, lining”. We propose the English common name “White-lined Leaf Litter Toad” and the Chinese common name “Bái Yuán Zhǎng Tū Chán (白缘掌突蟾)”.

Diagnosis.

Leptobrachella albomarginata sp. nov. can be distinguished from its congeners by the following combination of morphological characters: (1) body size small (SVL 26.5 mm in one adult male, 32.5 mm in one female); (2) tibiotarsal articulation reaches the middle eye; (3) tongue with a shallow notch at the posterior tip; (4) heels meeting; (5) toes with rudimentary webbing and narrow lateral fringes; (6) relative finger lengths: I ≈ II < IV < III; (7) reverse-triangle markings and a ˄-shaped marking with a white lining in dorsal view; (8) black and bluish-white marbling all over ventral surfaces of throat, chest and belly; (9) flanks with distinct irregular black spots; (10) iris bicolored, upper 1/3 of the iris being coppery, lower 2/3 silvery gray.

Description of the holotype

(measurements in Table 1). KIZ 050905, adult female, body size small (SVL 32.5 mm), head longer (HDL 12.7 mm) than wide (HDW 11.8 mm); head triangular in dorsal view; snout short (SNT/HDL 37.0%), snout bluntly rounded in profile and obtusely pointed in dorsal view, projecting slightly beyond margin of the lower jaw; oval-shaped nostril dorsolaterally positioned, situated slightly below canthus, closer to tip of snout (SN 1.8 mm) than to anterior margin of eye (DNE 2.8 mm); loreal region oblique and slightly concave; canthus rostralis distinct; eyes large (ED/HDL 18.9%), eye diameter smaller than snout length (ED/SNT 51.1%); pupil vertical; eye diameter (ED 2.4 mm) less than snout length (SNT 4.7 mm); tympanum distinct, round; tympanic rim distinctly elevated relative to skin of temporal region; upper margin of tympanum in contact with supratympanic ridge; interorbital space (IOD 2.8 mm) flat, less than internarial distance (IND 3.5 mm) and width of upper eyelid (UEW 3.6 mm); vomerine teeth absent; tongue with shallow notch at posterior tip; supratympanic ridge distinct, extending from posterior corner of eye to supra-axillary gland (Fig. 3).

Figure 3.

Holotype of Leptobrachella albomarginata sp. nov. (KIZ 050905) in life. A. Lateral view; B. Lateral view of head; C. Dorsal view; D. Ventral view. Photos by Zhong-Bin Yu.

Table 1.

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XLSX

Measurements (in mm) of type series of Leptobrachella. Bold font and an asterisk (*) indicate the holotype.

	KIZ 050905*	KIZ 056551
Sex	♀	♂
SVL	32.5	26.5
HDL	12.7	10.1
HDW	11.8	9.1
SNT	4.7	4.1
SNT/HDL	37.0%	40.6%
ED	4.2	3.6
IOD	2.8	3.2
UEW	3.6	2.6
IND	3.5	3.0
DNE	2.8	2.2
SN	1.8	1.5
ED	2.4	1.6
ED/HDL	18.9%	15.8%
ED/SNT	51.1%	39.0%
TEY	1.3	1.1
FHL	16.5	13.5
HL	7.9	7.1
LAD	2.1	1.7
FAL	6.5	6.8
HLL	47.3	40.2
THL	12.7	11.8
TAL	14.6	12.1
TAL/SVL	44.9%	45.7%
FL	13.1	10.8
IMTL	0.9	1.2
PEC	0.8	0.9
FEM	0.8	1.0
SUP	0.6	1.3

Forelimbs thin, slender; forearm shorter than hand, not enlarged (FAL 6.5 mm, HL 7.9 mm); fingertips round, slightly swollen, almost equal to phalange width; relative finger lengths: I ≈ II < IV < III; subarticular tubercles absent on fingers; supernumerary tubercles absent; finger webbing absent; lateral fringes absent; a large, round inner metacarpal tubercle, distinctly separated from small, laterally compressed outer metacarpal tubercle (Figs 3, 4).

Figure 4.

Holotype of Leptobrachella albomarginata sp. nov. (KIZ 050905) in preservative. A. Dorsal view; B. Ventral view; C. Ventral view of hand; D. Ventral view of toe. Photos by Zhong-Bin Yu.

Hindlimbs long, tibia slightly shorter than half of the snout-vent length (TAL/SVL ratio 44.9%); tibiotarsal articulation reaches the middle eye when hindlimb is stretched along the side of the body; heels meeting when hind limbs are flexed and held perpendicular to body; tips of toes rounded, slightly swollen; relative length of toes: IV > III > V > II > I; subarticular tubercles indistinct under the base of II and III toe; narrow lateral fringes present on all toes; rudimentary webbing between toes; inner metatarsal tubercle distinct and prolonged (IMTL 0.9 mm, 2.8% SVL), outer metatarsal tubercle absent (Figs 3, 4).

Skin on dorsum shagreened, lacking enlarged tubercles or warts; the back and surfaces of limbs scattered with fine tubercles and short longitudinal folds; upper arms and upper eyelid covered by small tubercles; tiny reddish warts on flanks; ventral skin smooth; pectoral gland laterally compressed, indistinct, not easy to find, about 0.8 mm in diameter; femoral glands small, oval, about 0.8 mm in diameter, located on posteroventral surfaces of thighs, closer to knee than to vent; supra-axillary gland raised, about 0.6 mm in diameter; ventrolateral glands present, dorsolaterally compressed forming an incomplete line (Fig. 3).

Color of the holotype in life. Dorsum brown backgrounding, with small, distinct darker brown markings and spots; large reverse-triangle dark brown markings with white lining between anterior corner of eyes, connected to the ˄-shaped marking with white lining between axillae; a dark ˄-shaped stripe with white lining on rear part of dorsal surface; upper lip with dark brown vertical bars; supratympanic ridge reddish and large black marking under supratympanic ridge from posterior corner of eye to supra-axillary glands; most of tympanum black; transverse dark brown bars on dorsal surface of fingers and toes, lower arms, tarsus, thighs and tibia; supra-axillary gland coppery orange; three distinct dark blotches and several small black spots on flanks from groin to axilla; ventral surface of throat, chest, and belly creamy white; black and bluish-white marbling all over ventral surfaces of throat, chest and belly; ventral surfaces of limbs black, covered with bluish-white marbling; ventrolateral glands, pectoral glands and femoral glands white; iris bicolored, upper 1/3 of the iris being coppery, lower 2/3 silvery gray, with black reticulations throughout (Fig. 3).

Color of holotype in preservative. After one year of storage in 75% alcohol, dorsum of the body and limbs fade to dark brown; transverse bars on limbs still distinct; three distinct dark blotches and several small black spots on flanks still clear; dark-brown, inverse reverse-triangle marking, connected to the ˄-shaped marking and a dark ˄-shaped stripe with a white lining on the rear part of the dorsal surface distinctly visible; elbow to upper arm distinctly creamy white in color on the dorsum; ventral surfaces of throat, chest and belly dull white with well-discernable marbling; inner metatarsal tubercle, inner metatarsal tubercle, supra-axillary, femoral and pectoral glands fading to gray (Fig. 4).

Description of the paratype

(measurements in Table 1). KIZ 056551, adult male, body size small (SVL 26.5 mm), head longer (HDL 10.1 mm) than wide (HDW 9.1 mm); head triangular in dorsal view; snout short (SNT/HDL 40.6%), snout bluntly rounded in profile and obtusely pointed in dorsal view, projecting slightly beyond margin of the lower jaw; oval-shaped nostril dorsolaterally positioned, situated slightly below canthus, closer to tip of snout (SN 1.5 mm) than to anterior margin of eye (DNE 2.2 mm); loreal region oblique and slightly concave; canthus rostralis indistinct; eyes large (ED/HDL 15.8%), eye diameter slightly smaller than snout length (ED/SNT 39.0%); pupil vertical; eye diameter (ED 1.6 mm) less than snout length (SNT 4.1 mm); tympanum distinct, round; tympanic rim distinctly elevated relative to skin of temporal region; upper margin of tympanum in contact with supratympanic ridge; interorbital space (IOD 3.2 mm) flat, slightly larger than internarial distance (IND 3.0 mm) and width of upper eyelid (UEW 2.6 mm); vomerine teeth absent; tongue with shallow notch at posterior tip; supratympanic ridge distinct, extending from posterior corner of eye to supra-axillary gland; male with internal subgular vocal sacs, vocal sac openings slit-like, small, paired, located posteriolaterally on mouth floor (Fig. 5).

Figure 5.

Paratype of Leptobrachella albomarginata sp. nov. (KIZ 056551). A. Laterodorsal view in life; B. Dorsal view in preservative; C. Ventral view in preservative; D. Ventral view of head in preservative; E. Ventral view of hand in preservative; F. Ventral view of toe in preservative. Photos by Zhong-Bin Yu.

Forelimbs thin, slender; forearm shorter than hand, not enlarged (FAL 6.8 mm, HL 7.1 mm); fingertips round, slightly swollen, almost equal to phalange width; nuptial pad absent; relative finger lengths: I ≈ II < IV < III; subarticular tubercles absent on fingers; supernumerary tubercles absent; finger webbing absent; lateral fringes absent; a large, round inner metacarpal tubercle, distinctly separated from small, laterally compressed outer metacarpal tubercle (Fig. 5).

Hindlimbs long, tibia slightly shorter than half of the snout-vent length (TAL/SVL ratio 45.7%); tibiotarsal articulation reaches the middle eye when hindlimb is stretched along the side of the body; heels meeting when hind limbs are flexed and held perpendicular to body; tips of toes rounded, slightly swollen; relative length of toes: IV > III > V > II > I; subarticular tubercles absent; narrow lateral fringes present on all toes; rudimentary webbing between toes; inner metatarsal tubercle distinct and prolonged (IMTL 1.2 mm, 4.5% SVL), outer metatarsal tubercle absent (Fig. 5).

Skin on dorsum shagreened; the back and surfaces of limbs scattered with fine tubercles and short longitudinal folds; upper arms and upper eyelid covered by small tubercles; tiny reddish warts on flanks; ventral skin smooth; pectoral gland laterally compressed, indistinct, not easy to find, about 0.9 mm in diameter; femoral glands small, oval, about 1.0 mm in diameter, located on posteroventral surfaces of thighs, closer to knee than to vent; supra-axillary gland raised, about 1.3 mm in diameter; ventrolateral glands present, dorsolaterally compressed forming an incomplete line (Fig. 5).

Color of the paratype in life. Dorsum brown backgrounding; large reverse-triangle dark brown markings with a white lining between anterior corner of eyes, connected to the ˄-shaped marking with a white lining between axillae; a dark 人-shaped stripe with a white lining on the rear part of dorsal surface; upper lip with dark brown vertical bars; supratympanic ridge reddish and large black marking under supratympanic ridge from posterior corner of eye to supra-axillary glands; most of tympanum black; transverse dark brown bars on dorsal surface of fingers and toes, lower arms, tarsus, thighs and tibia; supra-axillary gland coppery orange; one distinct dark blotch and several small black spots on flanks from groin to axilla (Fig. 5A).

Color of paratype in preservative. After six months of storage in 75% alcohol, dorsum of the body and limbs fade to dark brown; transverse bars on limbs still distinct; one distinct dark blotch and several small black spots on flanks still clear; dark-brown, inverse reverse-triangle marking, connected to the ˄-shaped marking and a dark 人-shaped stripe with white lining on the rear part of dorsal surface distinctly visible; elbow to upper arm distinctly creamy white on the dorsum; ventral surfaces of throat, chest and belly dull white with well-discernable marbling; inner metatarsal tubercle, supra-axillary, femoral and pectoral glands fading to gray (Fig. 5B–F).

Morphological variation.

The paratype matches the overall characters of the holotype (see Table 1). The female specimen is relatively larger than the male (SVL 32.5 mm in a single adult female and 26.5 mm in a single adult male). Male with internal subgular vocal sacs, vocal sac openings slit-like, small, paired, located posteriolaterally on mouth floor. The size of the dark blotches on flanks is variable: KIZ 050905 has three distinct dark blotches and several small black spots on flanks; KIZ 056551 has a relatively small blotch and several small black spots on flanks.

Description of the larvae.

KIZ 051631, an early metamorph at stages 44–46 was collected on 28 April 2023 (Gosner 1960). A young froglet with a relatively elongate body, bluntly rounded snout, and slender limbs with thin, delicate digits. After one year of storage in 75% alcohol, the ground dorsal coloration of the body and limbs now dark brown; dorsal parts of limbs with transverse dark brown bars; ventral surfaces of throat, chest and limbs creamy white (Suppl. material 3).

Distribution and ecology.

Leptobrachella albomarginata sp. nov. is only known from Tongbiguan Provincial Nature Reserve, Tongbiguan Town, Yingjiang County, Yunnan, China. The new species inhabits montane streams surrounded by shrubland at elevations of approximately 1300–1600 m (Fig. 6). The breeding season of this species is likely in April as the female collected during this month was gravid. This species occurs sympatrically, if not syntopically with congeners L. purpurus, L. yingjiangensis, and L. ventripunctata. In addition, other frog species also found at the site include Nanorana aenea, Limnonectes longchuanensis, and Jingophrys feii.

Figure 6.

Habitat at the type locality of Leptobrachella albomarginata sp. nov. at Tongbiguan Provincial Nature Reserve, Yingjiang County, Yunnan, China. (Photo by Zhong-Bin Yu).

Comparisons.

Phylogenetically, the matrilineal genealogy assigns Leptobrachella albomarginata sp. nov. to clade A. Thus, we compared Leptobrachella albomarginata sp. nov. to all other recognized species of clade A (Fei et al. 1990; Lathrop et al. 1998; Ohler et al. 2000, 2011; Das et al. 2010; Sengupta et al. 2010; Rowley et al. 2010, 2012, 2015, 2017; Jiang et al. 2013; Sung et al. 2014; Yang et al. 2016, 2018; Yuan et al. 2017; Hou et al. 2018; Wang et al. 2018, 2019, 2020, 2022; Chung et al. 2019; Chen et al. 2019, 2020, 2021a,b,c, 2023, 2024; Luo et al. 2020; Lyu et al. 2020; Qian et al. 2020; Li et al. 2020, 2024; Cheng et al. 2021; Nguyen et al. 2021; Wu et al. 2021; Shi et al. 2021, 2023; Lin et al. 2022; Luo et al. 2022; Luong et al. 2023; Liu et al. 2023; Matsui et al. 2023; Suppl. material 2).

Leptobrachella albomarginata sp. nov. differs from L. bijie by relative finger lengths: I ≈ II < IV < III (vs. relative finger lengths: I = II = IV < III), reverse-triangle markings and ˄-shaped marking with white lining (vs. lack of white lining); from L. jinyunensis by iris bicolored, upper 1/3 of the iris being coppery, lower 2/3 silvery gray (vs. iris gold above, gradually silver bellow), reverse-triangle markings and ˄-shaped marking with white lining (vs. lack of white lining); from L. chishuiensis by relative finger lengths: I ≈ II < IV < III (vs. relative finger lengths: II < IV < I < III), tibiotarsal articulation reaches the middle eye (vs. reaches the tympanum), reverse-triangle markings and ˄-shaped marking with white lining (vs. lack of white lining); from L. jinshaensis by reverse-triangle markings and ˄-shaped marking with white lining (vs. lack of white lining), black and bluish-white marbling all over ventral surfaces of throat, chest and belly (vs. absent); from L. suiyangensis by reverse-triangle markings and ˄-shaped marking with white lining (vs. lack of white lining), black and bluish-white marbling all over ventral surfaces of throat, chest and belly (vs. ventral part with distinct or indistinct light brown speckling); from L. purpuraventra by ventral surface of throat, chest, and belly creamy white (vs. ventral surface gray purple), relative finger lengths: I ≈ II < IV < III (vs. relative finger lengths: I = II = IV < III), reverse-triangle markings and ˄-shaped marking with white lining (vs. lack of white lining); from L. bourreti by a smaller body size, SVL 32.5 mm in one female (vs. 42.0–45.0 mm in adult females), nostrils closer to tip of snout than to anterior margin of eye (vs. nostrils closer to eye than to tip of snout); from L. dong by reverse-triangle markings and ˄-shaped marking with white lining (vs. lack of white lining), eye diameter smaller than snout length (vs. eye diameter longer than snout length); from L. dushanensis by tibiotarsal articulation reaches the middle eye (vs. reaches the interior corner of the eye), reverse-triangle markings and ˄-shaped marking with white lining (vs. lack of white lining); from L. graminicola by tibiotarsal articulation reaches the middle eye (vs. reaches the anterior edge of eye), narrow lateral fringes present on all toes (vs. wide lateral fringes), reverse-triangle markings and ˄-shaped marking with white lining (vs. lack of white lining); from L. wulingensis by iris bicolored, upper 1/3 of the iris being coppery, lower 2/3 silvery gray (vs. iris bicolored with bright orange or golden upper half, fades to silver in lower half), reverse-triangle markings and ˄-shaped marking with white lining (vs. lack of white lining); from L. dorsospina by a larger body size, SVL 32.5 mm in one female (vs. 25.0–26.4 mm in three adult females), relative finger lengths: I ≈ II < IV < III (vs. relative finger lengths: I < IV < II < III), reverse-triangle markings and ˄-shaped marking with white lining (vs. lack of white lining); from L. niveimontis by a larger body size, SVL 32.5 mm in one female (vs. 28.5–28.7 mm in three adult females), reverse-triangle markings and ˄-shaped marking with white lining (vs. lack of white lining), tibiotarsal articulation reaches the middle eye (vs. reaches beyond eye); from L. yeae by reverse-triangle markings and ˄-shaped marking with white lining (vs. lack of white lining), heels just meeting (vs. heels partially overlapped); from L. yunyangensis by relative finger lengths: I ≈ II < IV < III (vs. relative finger lengths: I < II = IV < III), heels just meeting (vs. heels overlapped), tibiotarsal articulation reaches the middle eye (vs. reaches beyond the anterior corner of the eye); from L. alpina by tibiotarsal articulation reaches the middle eye (vs. reaches anterior corner of the eye), reverse-triangle markings and ˄-shaped marking with white lining (vs. lack of white lining), ventrolateral glands forming a discontinuous line (vs. continuous); from L. purpurus by iris bicolored, upper 1/3 of the iris being coppery, lower 2/3 silvery gray (vs. upper half orange yellow, lower half sliver white), relative finger lengths: I ≈ II < IV < III (vs. relative finger lengths: I = II = IV < III), reverse-triangle markings and ˄-shaped marking with white lining (vs. lack of white lining), narrow lateral fringes present on all toes (vs. wide lateral fringes); from L. eos by a smaller body size, SVL 32.5 mm in one female (vs. 40.7 mm in one adult female), nostril closer to tip of snout than to anterior margin of eye (vs. nostril closer to eye than to tip of snout), narrow lateral fringes present on all toes (vs. wide lateral fringes); from L. oshanensis by toes with rudimentary webbing and narrow lateral fringes (vs. absent), relative finger lengths: I ≈ II < IV < III (vs. relative finger lengths: I < II = IV < III); from L. korifi by tibiotarsal articulation reaches the middle eye (vs. reaches beyond the anterior corner of the eye), heels just meeting (vs. heels overlapped), subarticular tubercles indistinct under the base of II and III toe (vs. subarticular tubercles at base of each toe); from L. sinorensis by heels just meeting (vs. heels overlapped), reverse-triangle markings and ˄-shaped marking with white lining (vs. lack of white lining), tibiotarsal articulation reaches the middle eye (vs. reaches beyond the anterior corner of the eye); from L. murphyi by iris bicolored, upper 1/3 of the iris being coppery, lower 2/3 silvery gray (vs. upper half orange, lower half sliver white), tibiotarsal articulation reaches the middle eye (vs. reaches beyond eye), black and bluish-white marbling all over ventral surfaces of throat, chest and belly (vs. ventral surface of belly creamy white with small spots on the margin); from L. tengchongensis by iris bicolored, upper 1/3 of the iris being coppery, lower 2/3 silvery gray (vs. iris not bicolored, uniformly dark brown and scattered with minute, coppery reticulations throughout), a larger body size, SVL 32.5 mm in one female (vs. 28.8–28.9 mm in two adult females), reverse-triangle markings and ˄-shaped marking with white lining (vs. lack of white lining); from L. tamdil by relative finger lengths: I ≈ II < IV < III (vs. relative finger lengths: IV < I < II < III), head longer than wide (vs. head wider than long), iris bicolored, upper 1/3 of the iris being coppery, lower 2/3 silvery gray (vs. top third of iris bright orange, rest of iris grayish-cream), narrow lateral fringes present on all toes (vs. wide lateral fringes); from L. khasiorum by head longer than wide (vs. head wider than long), relative finger lengths: I ≈ II < IV < III (vs. relative finger lengths: IV < I < II < III), heels just meeting (vs. heels widely separated), iris bicolored, upper 1/3 of the iris being coppery, lower 2/3 silvery gray (vs. top third of iris bright orange, rest of iris yellowish-cream), narrow lateral fringes present on all toes (vs. wide lateral fringes); from L. yingjiangensis by dermal fringes in fingers absent (vs. narrow to moderate dermal fringes present on 2^nd to 4^th fingers), narrow lateral fringes present on all toes (vs. wide lateral fringes), iris bicolored, upper 1/3 of the iris being coppery, lower 2/3 silvery gray (vs. iris bicolored, upper half orange yellow, lower half sliver white), tibiotarsal articulation reaches the middle eye (vs. reaches anterior corner of the eye); from L. namdongensis by relative finger lengths: I ≈ II < IV < III (vs. relative finger lengths: I < II = IV < III), reverse-triangle markings and ˄-shaped marking with white lining (vs. lack of white lining); from L. petrops by toes with rudimentary webbing (vs. toes lacking webbing), tibiotarsal articulation reaches the middle eye (vs. reaches anterior edge of eye), reverse-triangle markings and ˄-shaped marking with white lining (vs. lack of white lining), iris bicolored, upper 1/3 of the iris being coppery, lower 2/3 silvery gray (vs. iris gold in lower half and copper in upper half); from L. puhoatensis by tibiotarsal articulation reaches the middle eye (vs. reaches anterior edge of eye), black and bluish-white marbling all over ventral surfaces of throat, chest and belly (vs. ventral surfaces deep reddish brown, faint white speckling on chest and belly); from L. liui by black and bluish-white marbling all over ventral surfaces of throat, chest and belly (vs. absent), reverse-triangle markings and ˄-shaped marking with white lining (vs. lack of white lining), narrow lateral fringes present on all toes (vs. wide lateral fringes); from L. mangshanensis by iris bicolored, upper 1/3 of the iris being coppery, lower 2/3 silvery gray (vs. iris bicolored, bright orange upper, grayish cream below), tibiotarsal articulation reaches the middle eye (vs. reaches anterior margin of snout); from L. verrucosa by heels just meeting (vs. heels not meeting), tibiotarsal articulation reaches the middle eye (vs. reaches anterior corner of eye); from L. yunkaiensis by black and bluish-white marbling all over ventral surfaces of throat, chest and belly (vs. surface of throat creamy white and scattered with small whitish dots, belly pinkish and scattered with small brown speckling), reverse-triangle markings and ˄-shaped marking with white lining (vs. lack of white lining), narrow lateral fringes present on all toes (vs. wide lateral fringes); from L. shimentaina by black and bluish-white marbling all over ventral surfaces of throat, chest and belly (vs. ventral surface grayish pink, with distinct hazy brown speckling on chest and ventrolateral flanks), reverse-triangle markings and ˄-shaped marking with white lining (vs. lack of white lining); from L. maoershanensis by tibiotarsal articulation reaches the middle eye (vs. reaches snout), relative finger lengths: I ≈ II < IV < III (vs. relative finger lengths: II < I < IV < III), reverse-triangle markings and ˄-shaped marking with white lining (vs. lack of white lining); from L. bashaensis by tibiotarsal articulation reaches the middle eye (vs. reaches snout), reverse-triangle markings and ˄-shaped marking with white lining (vs. lack of white lining); from L. laui by relative finger lengths: I ≈ II < IV < III (vs. relative finger lengths: I < IV < II < III), tibiotarsal articulation reaches the middle eye (vs. reaches anterior margin of eye), narrow lateral fringes present on all toes (vs. wide lateral fringes), iris bicolored, upper 1/3 of the iris being coppery, lower 2/3 silvery gray (vs. iris uniformly coppery orange), black and bluish-white marbling all over ventral surfaces of throat, chest and belly (vs. ventral surface of chest and belly opaque cream white with little brown dusting along the margins of ventrolateral glands); from L. phiaoacensis by black and bluish-white marbling all over ventral surfaces of throat, chest and belly (vs. throat, chest and belly pinkish white with dark brown specking), reverse-triangle markings and ˄-shaped marking with white lining (vs. lack of white lining); from L. flaviglandulosa by tibiotarsal articulation reaches the middle eye (vs. reaches beyond eye), reverse-triangle markings and ˄-shaped marking with white lining (vs. lack of white lining), iris bicolored, upper 1/3 of the iris being coppery, lower 2/3 silvery gray (vs. iris distinctly bicolored, typically golden-orange in upper half, fading to whitish gray in lower half); from L. aerea by narrow lateral fringes present on all toes (vs. very weak lateral fringes), tibiotarsal articulation reaches the middle eye (vs. reaches to tip of snout), iris bicolored, upper 1/3 of the iris being coppery, lower 2/3 silvery gray (vs. iris bronze), reverse-triangle markings and ˄-shaped marking with white lining (vs. lack of white lining); from L. pelodytoides by narrow lateral fringes present on all toes (vs. very weak lateral fringes), rudimentary webbing between toes (vs. 1/3 toe webbing); from L. minima by narrow lateral fringes present on all toes (vs. absent), iris bicolored, upper 1/3 of the iris being coppery, lower 2/3 silvery gray (vs. iris dark golden in upper part, gray in lower part); from L. feii by tibiotarsal articulation reaches the middle eye (vs. reaches beyond eye), black and bluish-white marbling all over ventral surfaces of throat, chest and belly (vs. throat, chest and belly pinkish white with dark brown specking on belly periphery), reverse-triangle markings and ˄-shaped marking with white lining (vs. lack of white lining); from L. aspera by narrow lateral fringes present on all toes (vs. very weak lateral fringes), reverse-triangle markings and ˄-shaped marking with white lining (vs. lack of white lining); from L. ventripunctata by reverse-triangle markings and ˄-shaped marking with white lining (vs. lack of white lining), ventrolateral glands forming a discontinuous line (vs. continuous); from L. guinanensis by a smaller body size, SVL 32.5 mm in one female (vs. 38.7–41.8 mm in adult females), rudimentary webbing between toes (vs. 1/3 toe webbing), heels just meeting (vs. heels not meeting), black and bluish-white marbling all over ventral surfaces of throat, chest and belly (vs. ventral surface creamy white without dark brown spots); from L. shiwandashanensis by heels just meeting (vs. heels not meeting), tibiotarsal articulation reaches the middle eye in female (vs. reaches the shoulder in females), toes with narrow lateral fringes and rudimentary webbing (vs. without webbing and lateral fringes on toes); from L. wuhuangmontis by tongue with shallow notch at posterior tip (vs. tongue deeply notched behind), black and bluish-white marbling all over ventral surfaces of throat, chest and belly (vs. ventral surface grayish-white mixed with tiny white and black dots); from L. wumingensis by toes with narrow lateral fringes and rudimentary webbing (vs. absence of toe webbing and lateral fringes), tongue with shallow notch at posterior tip (vs. tongue with a deep notch at posterior tip), black and bluish-white marbling all over ventral surfaces of throat, chest and belly (vs. belly with tiny creamy white spots, throat creamy white with tiny light brown spots); from L. phiadenensis by head longer than wide (vs. head wider than long), black and bluish-white marbling all over ventral surfaces of throat, chest and belly (vs. throat, chest and belly white with dark specking on outer margins); from L. shangsiensis by narrow lateral fringes present on all toes (vs. very weak lateral fringes), head longer than wide (vs. head wider than long); from L. nyx by a smaller body size, SVL 32.5 mm in one female (vs. 37.0–41.0 mm in adult females), narrow lateral fringes present on all toes (vs. absent), flanks with distinct irregular black spots (vs. poorly distinct spots on flanks); from L. damingshanensis by narrow lateral fringes present on all toes (vs. very weak lateral fringes), relative finger lengths: I ≈ II < IV < III (vs. relative finger lengths: I < II < IV < III), black and bluish-white marbling all over ventral surfaces of throat, chest and belly (vs. absent); from L. nahangensis by a smaller body size, SVL 26.5 mm in one adult male (vs. 40.8 mm in one adult male), narrow lateral fringes present on all toes (vs. absent), tongue with shallow notch at posterior tip (vs. tongue deeply notched), iris bicolored, upper 1/3 of the iris being coppery, lower 2/3 silvery gray (vs. iris gold uniformly distributed with minute black, reticulations); from L. pluvialis by tibiotarsal articulation reaches the middle eye (vs. reaches nostril), relative finger lengths: I ≈ II < IV < III (vs. relative finger lengths: I < II = IV < III), narrow lateral fringes present on all toes (vs. absent); from L. zhangyapingi by a smaller body size, SVL 26.5 mm in one male (vs. 45.8–52.5 mm in seven adult males), heels just meeting (vs. heels widely separated), flanks with distinct irregular black spots (vs. absent), narrow lateral fringes present on all toes (vs. wide lateral fringes); from L. sungi by a smaller body size, SVL 26.5 mm in one male (vs. 48.3–52.7 mm in adult males), three distinct dark blotches and several small black spots on flanks (vs. absent or small); from L. firthi by three distinct dark blotches and several small black spots on flanks (vs. absent), narrow lateral fringes present on all toes in female (vs. absent in females); and from L. isos by three distinct dark blotches and several small black spots on flanks (vs. absent), tibiotarsal articulation reaches the middle eye (vs. reaches to nostril), narrow lateral fringes present on all toes in male (vs. wide in males).

Discussion

The amphibian diversity of Tongbiguan Provincial Nature Reserve is underestimated. In recent years, a series of new record genera, species, and new species records of amphibians have been reported from there, such as the genus Nasutixalus (Yang and Chan 2018), L. yingjiangensis (Yang et al. 2018), Xenophrys dehongensis (Lyu et al. 2023), Xenophrys yingjiangensis (Wu et al. 2024a), and Polypedates teraiensis (Yu et al. 2024). In addition, some studies have shown that cryptic species exist in this region (e.g. Chen et al. 2017, 2018; Wu et al. 2024a). These instances highlight that the true amphibian diversity of the reserve is substantially underestimated. Our new finding of L. albomarginata sp. nov. further confirms this view. Thus, intensifying field surveys with increased research efforts in this region will likely result in further discoveries of yet unknown lineages and new amphibian species. Our study reveals that species diversity within Leptobrachella remains largely underexplored. The discovery of L. albomarginata sp. nov. results in 108 species of Leptobrachella, 44 of which occur in China, and now seven on the Gaoligong Mountain Range.

The occurrence of co-distributed species may also be one of the reasons for the underestimation of species diversity in this region. Currently, multiple co-distributed species occur in the reserve. During our field work, four adult species of Leptobrachella (L. purpurus, L. yingjiangensis, L. ventripunctata, and L. albomarginata sp. nov.) were found syntopically at the same site and same time. A similar sympatric distribution pattern has also been reported, including four species and a putative new species of Xenophrys (X. dehongensis, X. glandulosa, X. periosa, X. yingjiangensis, and X. sp.; Wu et al. 2024a) and three species of Polypedates (P. braueri, P. impresus, and P. teraiensis; Yu et al. 2024). Future explorations and investigations need to treat species identifications with caution. Furthermore, studies should integrate life history data (e.g., advertisement call and breeding season) with genomic data to explore the drivers and mechanisms of sympatric coexistence and understand how these sympatric species interact and adapt to their respective niches.

Acknowledgments

This work was supported by the National Key R & D Program of China (2022YFC2602500), Science and Technology Basic Resources Investigation Program of China (Grant No. 2021FY100200); National Natural Science Foundation of China (NSFC 32100371); Yunnan Applied Basic Research Projects (No. 202301AT070312, 202301AT070431); Yunnan Revitalization Talent Support Program Yunling Scholar Project, China’s Biodiversity Observation Network (Sino-BON), and the Animal Branch of the Germplasm Bank of Wild Species, CAS (Large Research Infrastructure Funding). We thank Dong An, Peng Yang, Xian-Kun Huang, and Xian-Rong Wu for their help in the field. We thank the Tongbiguan Provincial Nature Reserve for their support in undertaking field surveys and specimen collections.

References

AmphibiaChina (2024) The Database of Chinese Amphibians. Kunming Institute of Zoology (CAS), Kunming, Yunnan, China. Electronic Database. http://www.amphibiachina.org [accessed 31 December 2024]

Chan BPL, Bi Z, Duan SZ (2019) Introduction to a four-year biodiversity survey of Tengchong Section of Gaoligongshan National Nature Reserve, in the footsteps of pioneering naturalists in western Yunnan, China. Journal of Threatened Taxa 11(11): 14391−14401. https://doi.org/10.11609/jott.4438.11.11.14391-14401

Chen JM, Zhou WW, Poyarkov Jr NA, Stuart BL, Brown RM, Lathrop A, Wang YY, Yuan ZY, Jiang K, Hou M, Chen HM, Suwannapoom C, Nguyen SN, Duong TV, Papenfuss TJ, Murphy RW, Zhang YP, Che J (2017) A novel multilocus phylogenetic estimation reveals unrecognized diversity in Asian horned toads, genus Megophrys sensu lato (Anura: Megophryidae). Molecular Phylogenetics and Evolution 106: 28–43. https://doi.org/10.1016/j.ympev.2016.09.004

Chen JM, Poyarkov NA, Suwannapoom C, Lathrop A, Wu YH, Zhou WW, Yuan ZY, Jin JQ, Chen HM, Liu HQ, Nguyen TQ, Nguyen SN, Duong TV, Eto K, Nishikawa K, Matsui M, Orlov NL, Stuart BL, Brown RM, Rowley JJ, Murphy RW, Wang YY, Che J (2018) Large scale phylogenetic analyses provide insights into unrecognized diversity and historical biogeography of Asian leaf-litter frogs, genus Leptolalax (Anura: Megophryidae). Molecular Phylogenetics and Evolution 124: 162–171. https://doi.org/10.1016/j.ympev.2018.02.020

Chen WC, Liao X, Zhou SC, Mo YM (2019) A new species of Leptobrachella (Anura: Megophryidae) from southern Guangxi, China. Zootaxa 4563: 67–82. https://doi.org/10.11646/zootaxa.4563.1.3

Chen JM, Xu K, Poyarkov NA, Wang K, Yuan ZY, Hou M, Suwannapoom C, Wang J, Che J (2020) How little is known about “the little brown frogs”: description of three new species of the genus Leptobrachella (Anura: Megophryidae) from Yunnan Province, China. Zoological Research 41(3): 292–313. https://doi.org/10.24272/j.issn.2095-8137.2020.036

Chen JM, Suwannapoom C, Wu YH, Poyarkov NA, Xu K, Pawangkhanant P, Che J (2021a) Integrative taxonomy reveals a new species of Leptobrachella (Anura: Megophryidae) from the mountains of northern Thailand. Zootaxa 5052(2): 41–64. https://doi.org/10.11646/zootaxa.5052.2.2

Chen WC, Peng W, Pan W, Liao N, Liu Y, Huang Y (2021b) A new species of Leptobrachella Smith 1925 (Anura: Megophryidae) from southern Guangxi, China. Zootaxa 5020: 581–596. https://doi.org/10.11646/zootaxa.5020.3.8

Chen WC, Yu GD, Cheng ZY, Meng T, Wei H, Zhou GY, Lu YW (2021c) A new species of Leptobrachella (Anura: Megophryidae) from central Guangxi, China. Zoological Research 42: 783–788. https://doi.org/10.24272/j.issn.2095-8137.2021.179

Chen WC, Peng WX, Li P, Yu GD (2023) A new species of the genus Leptobrachella Smith 1925 (Amphibia, Anura, Megophryidae) from Guangxi, China. ZooKeys 1178: 1–16. https://doi.org/10.3897/zookeys.1178.106038

Chen WC, Li P, Peng WX, Liu YJ, Huang Y (2024) The fourth species of Leptobrachella (Anura, Megophryidae) found at Shiwandashan National Nature Reserve, Guangxi, China. ZooKeys 1192: 257–279. https://doi.org/10.3897/zookeys.1192.98352

Cheng YL, Shi SC, Li J, Liu J, Li SZ, Wang B (2021) A new species of the Asian leaf litter toad genus Leptobrachella Smith, 1925 (Anura, Megophryidae) from northwest Guizhou Province, China. ZooKeys 1021: 81–107. https://doi.org/10.3897/zookeys.1021.60729

Chung HV, Tao NT, Vinh LQ, Truong NQ, Jiang J (2019) A new species of Leptobrachella Smith 1925 (Anura: Megophryidae) from Thanh Hoa Province, Vietnam. Raffles Bulletin of Zoology 67: 536–556. https://doi.org/10.26107/RBZ-2019-0042

Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: More models, new heuristics and parallel computing. Nature Methods 9(8): 772–772. https://doi.org/10.1038/nmeth.2109

Das I, Tron RKL, Rangad D, Hooroo RN (2010) A new species of Leptolalax (Anura: Megophryidae) from the sacred groves of Mawphlang, Meghalaya, north-eastern India. Zootaxa 2339(1): 44–56. https://doi.org/10.11646/zootaxa.2339.1.2

Dubois A (1980) Notes sur la systématique et la répartition des amphibiens anoures de Chine et des régions avoisinantes. IV. Classification générique et subgénérique des Pelobatidae Megophryinae. Bulletin Mensuel de la Société Linnéenne de Lyon 49: 469–482. https://doi.org/10.3406/linly.1980.10444

Fei L, Hu SQ, Ye CY, Huang YZ (2009) Fauna Sinica. Amphibia Anura. Beijing: Science Press, 957 pp. [in Chinese]

Frost DR (2024) Amphibian Species of the World: An Online Reference. Version 6.1. https://amphibiansoftheworld.amnh.org/ [accessed 31 December 2024]

Gosner KL (1960) A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica 16(3): 183–190.

Hou Y, Zhang M, Hu F, Li S, Shi S, Chen J, Mo X, Wang B (2018) A new species of the genus Leptolalax (Anura, Megophryidae) from Hunan, China. Zootaxa 4444(3): 247–266. https://doi.org/10.11646/zootaxa.4444.3.2

Jiang K, Yan F, Suwannapoom C, Chomdej S, Che J (2013) A new species of the genus Leptolalax (Anura: Megophryidae) from northern Thailand. Asian Herpetological Research 4(2): 100–108. https://doi.org/10.3724/SP.J.1245.2013.00100

Kocher TD, Thomas WK, Meyer A, Edwards SV, Pääbo S, Villablanca FX, Wilson AC (1989) Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proceedings of the National Academy of Sciences of the USA 86(16): 6196–6200. https://doi.org/10.1073/pnas.86.16.6196

Lathrop A, Murphy RW, Orlov NL, Ho CT (1998) Two new species of Leptolalax (Anura: Megophryidae) from northern Vietnam. Amphibia-Reptilia 19: 253–267. https://doi.org/10.1163/156853898X00160

Lee PS, Liu B, Ouyang M, Ai RD, Liu XL, He YH, Huang PQ, Li YC, Naveen RS, Yuan ZY, Chen JM (2024) Hidden in the bamboo: A new parachuting frog (Rhacophoridae, Rhacophorus) from the borderlands of western China, with comments on the taxonomy of R. rhodopus. Zoosystematics and Evolution 100(3): 851–862. https://doi.org/10.3897/zse.100.120224

Li SZ, Liu J, Wei G, Wang B (2020) A new species of the Asian leaf litter toad genus Leptobrachella (Amphibia, Anura, Megophryidae) from southwest China. ZooKeys 943: 91–118. https://doi.org/10.3897/zookeys.943.51572

Li S, Li W, Cheng Y, Liu J, Wei G, Wang B (2024) Description of a new Asian Leaf Litter Toad of the genus Leptobrachella Smith, 1925 (Anura, Megophryidae) from southern Guizhou Province, China. Biodiversity Data Journal 12: e113427. https://doi.org/10.3897/BDJ.12.e113427

Lin SS, Li YH, Lu YH, Su HL, Wu SB, Zhang QQ, Mo MJ, Xiao XJ, Pan Z, Pan H, Zeng ZC, Wang J (2022) A new species of the genus Leptobrachella (Anura, Megophryidae) from northwestern Guangdong Province, China. Herpetozoa 35: 165–178. https://doi.org/10.3897/herpetozoa.35.e89981

Liu J, Shi S, Li S, Zhang M, Xiang S, Gang W, Wang B (2023) A new Asian leaf litter toad of the genus Leptobrachella (Amphibia, Anura, Megophryidae) from central south China. ZooKeys 1149: 103–134. https://doi.org/10.3897/zookeys.1149.85895

Luo T, Xiao N, Gao K, Zhou J (2020) A new species of Leptobrachella (Anura, Megophryidae) from Guizhou Province, China. ZooKeys 923: 115–140. https://doi.org/10.3897/zookeys.923.47172

Luo T, Wang W, Peng D, Lei B, Deng H, Ji S, Huang H, Zhou J (2022) A new species of the Asian Leaf Litter Toad genus Leptobrachella (Amphibia, Anura, Megophryidae) from Chongqing City, Southwest China. Asian Herpetological Research 13(2): 75–95. https://doi.org/10.16373/j.cnki.ahr.210052

Luong AM, Van Hoang C, Ziegler T, Nguyen TQ (2023) Two new species of Leptobrachella Smith 1925 (Amphibia: Megophryidae) from Cao Bang Province, Vietnam. Zootaxa 5369(3): 301–335. https://doi.org/10.11646/zootaxa.5369.3.1

Lyu JC, Dai LL, Wei PF, He YH, Yuan ZY, Shi WL, Zhou SL, Ran SY, Kuang ZF, Guo X, Wei G, Yuan G (2020) A new species of the genus Leptobrachella Smith, 1925 (Anura, Megophryidae) from Guizhou, China. 1008: 139–157. https://doi.org/10.3897/zookeys.1008.56412

Lyu ZT, Qi S, Wang J, Zhang SY, Zhao J, Zeng ZC, Wan H, Yang J, Mo Y, Wang Y (2023) Generic classification of Asian Horned Toads (Anura: Megophryidae: Megophryinae) and monograph of Chinese species. Zoological Research 44: 380–450. https://doi.org/10.24272/j.issn.2095-8137.2022.372

Miller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES Science Gateway for inference of large phylogenetic trees. Gateway Computing Environments Workshop (GCE), 2010: 1–8. https://doi.org/10.1109/GCE.2010.5676129

Matsui M, Panha S, Eto K (2023) Two New Species of Leptobrachella from Northern Thailand (Amphibia, Anura, Megophryidae). Current Herpetology 42(1): 83–97. https://doi.org/10.5358/hsj.42.83

Mittermeier RA, Gil PR, Hoffmann M, Pilgrim J, Brooks T, Mittermeier CG, Lamoreux J, da Fonseca GAB (2005) Hotspots Revisited: Earth’s Biologically Richest and Most Endangered Terrestrial Ecoregions. Mexico City: Conservation International, 392 pp.

Mittermeier RA, Turner WR, Larsen FW, Brooks TM, Gascon C (2011) Global biodiversity conservation: the critical role of hotspots. In: Zachos FE, Habel JC. Biodiversity Hotspots: Distribution and Protection of Conservation Priority Areas. Berlin: Springer, 3–22. https://doi.org/10.1007/978-3-642-20992-5_1

Myers N, Mittermeier RA, Mittermeier CG, Da Fonseca GA, Kent J (2000) Biodiversity hotspots for conservation priorities. Nature 403(6772): 853−858. https://doi.org/10.1038/35002501

Nguyen LT, Tapley B, Nguyen CT, Van Luong H, Rowley JJ (2021) A new species of Leptobrachella (Anura, Megophryidae) from Mount Pu Ta Leng, northwest Vietnam. Zootaxa 5016(3): 301–332. https://doi.org/10.11646/zootaxa.5016.3.1

Ohler A, Marquis O, Swan SR, Grosjean S (2000) Amphibian biodiversity of Hoang Lien Nature Reserve (Lao Cai Province, northern Vietnam) with description of two new species. Herpetozoa 13: 71–87.

Ohler A, Wollenberg KC, Grosjean S, Hendrix R, Vences M, Ziegler T, Dubois A (2011) Sorting out Lalos: description of new species and additional taxonomic data on megophryid frogs from northern Indochina (genus Leptobrachella, Megophryidae, Anura). Zootaxa 3147: 1–83. https://doi.org/10.11646/zootaxa.3147.1.1.

Posada D (2008) jModelTest: Phylogenetic model averaging. Molecular Biology and Evolution 25(7): 1253–1256. https://doi.org/10.1093/molbev/msn083

Qian TY, Xia X, Cao Y, Xiao NW, Yang DD (2020) A new species of Leptobrachella (Anura: Megophryidae) Smith, 1925 from Wuling Mountains in Hunan Province, China. Zootaxa 4816(4): 491–526. https://doi.org/10.11646/zootaxa.4816.4.4

Rambaut A, Drummond AJ (2009) Tracer. Version 1.5. http://tree.bio.ed.ac.uk/software/tracer/ [accessed 25 January 2021]

Rowley JJ, Dau VQ, Cao TT (2017) A new species of Leptolalax (Anura: Megophryidae) from Vietnam. Zootaxa 4273(1): 61–79. https://doi.org/10.11646/zootaxa.4273.1.5

Rowley JJL, Stuart BL, Richards SJ, Phimmachak S, Sivongxay N (2010) A new species of Leptolalax (Anura: Megophryidae) from Laos. Zootaxa 2681: 35–46. https://doi.org/10.11646/zootaxa.2681.1.3

Rowley JJL, Hoang HD, Dau VQ, Le DTT, Cao TT (2012) A new species of Leptolalax (Anura: Megophryidae) from central Vietnam. Zootaxa 3321: 56–68. https://doi.org/10.11646/zootaxa.3321.1.4

Rowley JJL, Dau QV, Nguyen TT (2013) A new species of Leptobrachella (Anura: Megophryidae) from the highest mountain in Indochina. Zootaxa 3737(4): 415–428. https://doi.org/10.11646/zootaxa.3737.4.5

Rowley JJL, Stuart BL, Neang T, Hoang HD, Dau VQ, Nguyen TT, Emmett DA (2015) A new species of Leptolalax (Anura: Megophryidae) from Vietnam and Cambodia. Zootaxa 4039: 401–417. https://doi.org/10.11646/zootaxa.4039.3.1

Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual. 2^nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Habor, NY.

Sengupta S, Sailo S, Lalremsanga HT, Das A, Das I (2010) A new species of Leptolalax (Anura: Megophryidae) from Mizoram, north-eastern India. Zootaxa 2406(1): 56–68. https://doi.org/10.11646/zootaxa.2406.1.3

Shi S, Hou Y, Song Z, Jiang J, Wang B (2021) A new leaf litter toad of Leptobrachella Smith, 1925 (Anura, Megophryidae) from Sichuan Province, China with supplementary description of L. oshanensis. Asian Herpetological Research 12(2): 143–166. https://doi.org/10.16373/j.cnki.ahr.200118

Shi S, Shen T, Wang X, Jiang J, Wang B (2023) Multiple data sources reveal a new Asian leaf litter toad of Leptobrachella Smith, 1925 (Anura, Megophryidae) from southwestern China. Asian Herpetological Research 14(1): 65–94. https://doi.org/10.16373/j.cnki.ahr.220065

Sun YB (2018) FasParser2: a graphical platform for batch manipulation of tremendous amount of sequence data. Bioinformatics 34(14): 2493–2495. https://doi.org/10.1093/bioinformatics/bty126

Sung YH, Yang J, Wang Y (2014) A new species of Leptolalax (Anura: Megophryidae) from southern China. Asian Herpetological Research 5(2): 80–90. https://doi.org/10.3724/SP.J.1245.2014.00080

Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution 30(12): 2725–2729. https://doi.org/10.1093/molbev/mst197

Vences M, Thomas M, Van der Meijden A, Chiari Y, Vieites DR (2005) Comparative performance of the 16S rRNA gene in DNA barcoding of amphibians. Frontiers in Zoology 2(1): 1–12. https://doi.org/10.1186/1742-9994-2-5

Wang J, Yang J, Li Y, Lyu Z, Zeng Z, Liu Z, Ye Y, Wang Y (2018) Morphology and molecular genetics reveal two new Leptobrachella species in southern China (Anura, Megophryidae). ZooKeys (776): 105–137. https://doi.org/10.3897/zookeys.776.22925

Wang J, Li YL, Li Y, Chen HH, Zeng YJ, Shen JM, Wang YY (2019) Morphology, molecular genetics, and acoustics reveal two new species of the genus Leptobrachella from northwestern Guizhou Province, China (Anura, Megophryidae). ZooKeys 848: 119–154. https://doi.org/10.3897/zookeys.848.29181

Wang J, Lyu ZT, Qi S, Zeng ZC, Zhang WX, Lu LS, Wang YY (2020) Two new Leptobrachella species (Anura, Megophryidae) from the Yunnan-Guizhou Plateau, southwestern China. ZooKeys 995: 97–125. https://doi.org/10.3897/zookeys.995.55939

Wang J, Qi S, Dai KY, Lyu ZT, Zeng ZC, Chen HH, Li YQ, Zhao YY, Wang YZ, Wang YY (2022) A new Leptobrachella species (Anura, Megophryidae) from South China, with comments on the taxonomic status of L. chishuiensis and L. purpurus. Zoosystematics and Evolution 98(1): 165–180. https://doi.org/10.3897/zse.98.73162

Wu YH, Pawangkhanant P, Chen JM, Gao W, Suwannapoom C, Che J (2021) Confirmation of Leptobrachella ventripunctata (Fei, Ye & Li, 1990), based on molecular and morphological evidence in Thailand. Biodiversity Data Journal 9: e74097. https://doi.org/10.3897/BDJ.9.e74097

Wu YH, Chen JM, Pawangkhanant P, Yothawut C, Karuno AP, Suwannapoom C, Che J (2022) Distribution extension of Leptobrachella eos (Ohler, Wollenberg, Grosjean, Hendrix, Vences, Ziegler & Dubois, 2011): first record from Thailand. Herpetozoa 35: 25–32. https://doi.org/10.3897/herpetozoa.35.e78627

Wu YH, Hou SB, Yuan ZY, Jiang K, Huang RY, Wang K, Liu Q, Yu ZB, Zhao HP, Zhang BL, Chen JM, Wang LJ, Stuart BL, Chambers EA, Wang YF, Gao W, Zou DH, Yan F, Zhao GG, Fu ZX, Wang SN, Jiang M, Zhang L, Ren JL, Wu YY, Zhang LY, Yang DC, Jin JQ, Yin TT, Li JT, Zhao WG, Murphy RW, Huang S, Guo P, Zhang YP, Che J (2023) DNA barcoding of Chinese snakes reveals hidden diversity and conservation needs. Molecular Ecology Resources 23(5): 1124–1141. https://doi.org/10.1111/1755-0998.13784

Wu YH, Yu ZB, Chen JM, Kilunda FK, Zhang DC, Zuo CS, Zuo AR, Duan ZP, Che J (2024a) A field survey on the genus Xenophrys (Amphibia, Megophryidae) confirms underestimated diversity in the Gaoligong Mountains, with the description of a new species. Zoosystematics and Evolution 100(3): 1041–1052. https://doi.org/10.3897/zse.100.127635

Wu YH, Yu ZB, Lu CQ, Zhang YP, Dong WJ, Liu XL., Kilunda FK, Xiong Y, Jiang YF, Ouyang H, Fu ZX, He YB, Yuan ZY, Che J (2024b) A new species of the genus Amolops (Amphibia: Ranidae) and the first national record of Amolops vitreus from China. Vertebrate Zoology 74: 343–357. https://doi.org/10.3897/vz.74.e108013

Yang J, Chan BPL (2018) A new phytotelm-breeding treefrog of the genus Nasutixalus (Rhacophoridae) from western Yunnan of China. Zootaxa 4388: 191–206. https://doi.org/10.11646/zootaxa.4388.2.3

Yang JH, Wang YY, Chen GL, Rao DQ (2016) A new species of the genus Leptolalax (Anura: Megophryidae) from Mt. Gaoligongshan of western Yunnan Province, China. Zootaxa 4088(3): 379–394. https://doi.org/10.11646/zootaxa.4088.3.4

Yang J, Zeng ZC, Wang Y (2018) Description of two new sympatric species of the genus Leptolalax (Anura: Megophryidae) from western Yunnan of China. PeerJ 6(e4586): 1–32. https://doi.org/10.7717/peerj.4586

Yu ZB, Kilunda FK, Wang K, Cao YY, Wu CL, Duan ZP, Zuo CS, Zhang DC, Wu YH, Che J (2024) The first discovery of Polypedates teraiensis (Dubois, 1987) (Rhacophoridae, Anura) in China. Biodiversity Data Journal 12: e127029. https://doi.org/10.3897/BDJ.12.e127029

Yuan Z, Sun R, Chen J, Rowley JJL, Wu Z, Hou S, Wang S, Che J (2017) A new species of the genus Leptolalax (Anura: Megophryidae) from Guangxi, China. Zootaxa 4300(4): 551–570. https://doi.org/10.11646/zootaxa.4300.4.5

Yun-He Wu and Zhong-Bin Yu contributed equally to this work.

Supplementary materials

Supplementary material 1

Localities and voucher data for all specimens used in this study

Yun-He Wu, Zhong-Bin Yu, Shen-Pin Yang, Zheng-Pan Duan, An-Ru Zuo, Ding-Can Zhang, Robert W. Murphy, Jing Che

Data type: xlsx

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Download file (59.43 kb)

Supplementary material 2

Average uncorrected p-distances (percentage) among Leptobrachella species

Yun-He Wu, Zhong-Bin Yu, Shen-Pin Yang, Zheng-Pan Duan, An-Ru Zuo, Ding-Can Zhang, Robert W. Murphy, Jing Che

Data type: xlsx

Explanation note: Average uncorrected p-distances (percentage) among Leptobrachella species calculated from 16S rRNA gene sequences (below the diagonal) and standard error estimates (above the diagonal). The ingroup mean uncorrected p-distances are shown on the diagonal.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Download file (32.44 kb)

Supplementary material 3

Larvae of Leptobrachella albomarginata sp. nov. (KIZ 051631) in preservative

Yun-He Wu, Zhong-Bin Yu, Shen-Pin Yang, Zheng-Pan Duan, An-Ru Zuo, Ding-Can Zhang, Robert W. Murphy, Jing Che

Data type: jpg

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Download file (764.28 kb)

﻿Abstract

Key Words

﻿Introduction

﻿Materials and methods

﻿Sampling

﻿Morphological characters

﻿DNA extraction, PCR and sequencing

﻿Results

﻿Taxonomic account

﻿ Leptobrachella albomarginata Wu, Yu, Kilunda, Murphy & Che, sp. nov.

Type material.

Etymology.

Diagnosis.

Description of the holotype

Description of the paratype

Morphological variation.

Description of the larvae.

Distribution and ecology.

Comparisons.

﻿Discussion

﻿Acknowledgments

﻿References

Supplementary materials

Abstract

Introduction

Materials and methods

Sampling

Morphological characters

DNA extraction, PCR and sequencing

Results

Taxonomic account

Leptobrachella albomarginata Wu, Yu, Kilunda, Murphy & Che, sp. nov.

Discussion

Acknowledgments

References