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Research Article
A new toad of Oreolalax Myers & Leviton, 1962 (Anura, Megophryidae) from Sichuan Province, southwest China
expand article infoYin Meng Hou‡§|, Pu Yang Zheng§#, Hao Qi Yu§#, Bin Wang§, Xiao Hong Chen‡|, Feng Xie§
‡ Henan Normal University, Xinxiang, China
§ Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, China
| The Observation and Research Field Station of Taihang Mountain Forest Ecosystems of Henan Province, Xinxiang, China
¶ University of Chinese Academy of Sciences, Beijing, China
# Sichuan University, Chengdu, China

Corresponding author: Feng Xie ( xiefeng@cib.ac.cn )

Academic editor: Luis Ceríaco
© 2024 Yin Meng Hou, Pu Yang Zheng, Hao Qi Yu, Bin Wang, Xiao Hong Chen, Feng Xie.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Hou YM, Zheng PY, Yu HQ, Wang B, Chen XH, Xie F (2024) A new toad of Oreolalax Myers & Leviton, 1962 (Anura, Megophryidae) from Sichuan Province, southwest China. ZooKeys 1212: 109-127. https://doi.org/10.3897/zookeys.1212.122222
ZooBank: urn:lsid:zoobank.org:pub:9996A35A-9485-4906-AD8B-52BEABE11B16
Open Access

Abstract

A new species of the genus Oreolalax Myers & Leviton, 1962 is described from Sichuan Province, southwest China. Molecular phylogenetic analyses based on mitochondrial gene sequences clustered the new species as an independent clade nested with O. rugosus, O. liangbeiensis, and O. major. The new species could be distinguished from its congeners by a combination of the following characters: body size moderate (39.8–52.8 mm in male); head broad; tympanum absent; interorbital region with dark triangular pattern; 1/3 toes webbed, with broad lateral fringes, belly smooth, brown yellow or medium yellow scattered variable brown spots; skin on dorsum relatively rough with fine tiny and large warts granules; middle pectoral glands are evident in males; flanks with dark-brown warts granules; upper surface of limbs with dark bars; and iris orange above and creamy-white below. The new species inhabits subtropical alpine scrub and swamp.

Key words

16S, COI, Hengduan Mountains, new species, Oreolalax species, taxonomy

Introduction

Oreolalax Myers & Leviton (1962) belongs to the family Megophryidae Bonaparte (1850) (Amphibian, Anura), Leptobrachiinae Dubois (1980). In 1962, Myers and Leviton established the genus using Oreolalax pingii as the type species, including four species, Oreolalax pingii Liu, 1943, Oreolalax popei Liu, 1947, Oreolalax rugosus Liu, 1943 and Oreolalax schmidti Liu, 1947. With the deepening of morphological, ecological and molecular studies, the taxonomic status of the genus has been gradually established (Tian and Jiang 1986; Fei et al. 1989, 1990; Dubois and Ohler 1998; Fu et al. 2007; Pyron and Wiens 2011).

Based on morphological differences, molecular divergence, and phylogenetic placement, Oreolalax sterlingae Nguyen, Phung, Le, Ziegler & Böhme, 2013 from northeast Vietnam and Oreolalax longmenmontis Hou, Shi, Hu, Deng, Jiang, Xie & Wang, 2020 from eastern Hengduan Mountains have been described, showing controversial internal cladistic relationships within the genus (Nguyen et al. 2013; Hou et al. 2020). As of now, 19 species have been recorded in this genus, distributed in southwest China and the northernmost part of Vietnam (Frost 2024), inhabiting mountain streams at 700–3300 m a.s.l. (Wei et al. 2009; Fei et al. 2012).

Hengduan Mountains, located in the southeastern part of Qinghai-Tibet Plateau, have a complex terrain and significant vertical climate changes. The mountain ecosystem in this region boasts a diverse range of habitats, capturing global attention for its biodiversity, and its speciation and protection have been of concern. During the field surveys in 2023 in Yanyuan City, Sichuan Province (Prov.), southeastern Hengduan Mountains, China, we collected eight adult and seven tadpole Oreolalax specimens. Our detailed morphological comparisons and molecular phylogenetic analyses indicated that these specimens should represent an undescribed species. Herein we describe it as a new species.

Materials and methods

Sample

Through a field survey in June 2023, a total of 15 samples of the undescribed species including eight adult males and seven tadpoles were collected nocturnally from Shuhe town, Yanyuan County, Sichuan Province, China (Suppl. material 1; Fig. 1). Taxonomic assignments of tadpoles were confirmed by molecular results. After taking photographs, the toads and tadpoles were euthanized using isoflurane, and the specimens were fixed and preserved in 75% ethanol. Tissue samples were taken and preserved separately in 95% ethanol prior to fixation. Specimens were deposited in Chengdu Institute of Biology, Chinese Academy of Sciences (CIB, CAS).

Figure 1. 

Location of the type locality of Oreolalax yanyuanensis sp. nov., Shuhe town, Sichuan Province, China.

Molecular phylogenetic analyses

Genomic DNA from each specimen collected in this work was extracted using a TIANamp Genomic DNA Kit by TIANGEN (BEIJING) BIOTECH, China. Fragments of the mitochondrial genes 16S rRNA and cytochrome c oxidase I (COI) genes were amplified. Primer sequences were retrieved from the literature for 16S (Simon et al. 1994) and COI (Che et al. 2012). PCR amplifications for the 16S/COI gene were performed in a 25 mL volume reaction with the following conditions: an initial denaturing step at 95 °C for 4 min; 36 cycles of denaturing at 95 °C for 40 s, annealing at 55 °C/50 °C for 40 s and extending at 72 °C for 70 s, and a final extending step of 72 °C for 10 min. Sequencing was conducted using an ABI3730 automated DNA sequencer at Sangon Biotechnologies Co., Ltd. (Shanghai, China).

For phylogenetic comparisons, Scutiger boulengeri Bedriaga, 1898 was selected as an outgroup. Sequences of 16 Oreolalax species and outgroups were downloaded from GenBank, and seven of Oreolalax species (O. rugosus, O. pingii, Oreolalax major Liu & Hu, 1960, O. schmidti, Oreolalax liangbeiensis Liu, Hu & Fei, 1979, Oreolalax granulosus Fei, Ye & Chen, 1990, and O. popei) were uploaded to GenBank in this study (Accession numbers: PP272909–PP272951, Table 1).

Table 1.
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Information for samples used in molecular phylogenetic analyses in this study. “–” represents a lack of data.

Species Voucher Locality GenBank accession number
16S CO1
Oreolalax yanyuanensis sp. nov. CIBSH20230603kd01 Yanyuan, Liangshan, Sichuan, China PP272915 PP272937
O. yanyuanensis CIBSH20230603kd02 Yanyuan, Liangshan, Sichuan, China PP272916 PP272938
O. yanyuanensis CIBSH20230603kd03 Yanyuan, Liangshan, Sichuan, China PP272917 PP272939
O. yanyuanensis CIBSH20230603kd04 Yanyuan, Liangshan, Sichuan, China PP272918 PP272940
O. yanyuanensis CIBSH20230603kd05 Yanyuan, Liangshan, Sichuan, China PP272919 PP272941
O. yanyuanensis CIBSH20230603kd06 Yanyuan, Liangshan, Sichuan, China PP272920 PP272942
O. yanyuanensis CIBSH20230603kd07 Yanyuan, Liangshan, Sichuan, China PP272921 PP272943
O. yanyuanensis CIBSH20230603016 Yanyuan, Liangshan, Sichuan, China PP272922 PP272944
O. yanyuanensis CIBSH20230603017 Yanyuan, Liangshan, Sichuan, China PP272923 PP272945
O. yanyuanensis CIBSH20230603018 Yanyuan, Liangshan, Sichuan, China PP272924 PP272946
O. yanyuanensis CIBSH20230603019 Yanyuan, Liangshan, Sichuan, China PP272925 PP272947
O. yanyuanensis CIBSH20230603020 Yanyuan, Liangshan, Sichuan, China PP272926 PP272948
O. yanyuanensis CIBSH20230603021 Yanyuan, Liangshan, Sichuan, China PP272927 PP272949
O. yanyuanensis CIBSH20230603023 Yanyuan, Liangshan, Sichuan, China PP272928 PP272950
O. yanyuanensis CIBSH20230603024 Yanyuan, Liangshan, Sichuan, China PP272929 PP272951
O. multipunctatus CIB2013wb091 Emei, Sichuan, China NC_037382 NC_037382
O. xiangchengensis CIB20130642 Xiangcheng, Sichuan, China MH727696 MH727696
O. lichuanensis – Hubei, China KU096847 KU096847
O. rhodostigmatus – Suiyang, Guizhou, China MF770485 MF770485
O. jingdongensis – Xujiaba, Jingdong, Yunnan, China MF953479 MF953479
O. omeimontis CIBEMS18061205 Emei, Sichuan, China MN688660 OP247647
O. nanjiangensis CIBSCNJNJ2006004 Shibatan, Nanjiang, Sichuan, China MN688658 –
O. sterlingae IEBR A.2012.1 Sa Pa, Lao Cai, Vietnam KC569981 –
O. longmenmontis CIB20180526001 Pengzhou, Sichuan, China MN688670 OP247644
O. rugosus CIBSCJFGYC201301 Zhaojue, Liangshan, Sichuan, China PP272909 PP272930
O. pingii CIBSC20130521004 Zhaojue, Liangshan, Sichuan, China PP272910 PP272931
O. major CIBEM1824 Emei, Sichuan, China MN688655 PP272932
O. schmidti CIBEM1820 Emei, Sichuan, China PP272911 PP272933
O. liangbeiensis WG20180538 Puxiong, Yuexi, Sichuan, China PP272912 PP272934
O. granulosus CIBYN20130305023 Ailoushan, Jingdong, Yunnan, China PP272913 PP272935
O. popei CIB2020061508 Baoxing, Ya’an, Sichuan, China PP272914 PP272936
Scutiger boulengeri GGS-PBX2-16 Kangding, Sichuan, China OK584750 OK544538

Sequences were assembled and aligned using Mega v. 7.0 (Kumar et al. 2016) A total of 1084 bp (16S+COI gene) sequences were obtained from the Oreolalax species. The maximum likelihood method (ML) and Bayesian inference (BI) method were used to construct gene trees to analyze the intraspecific phylogenetic relationship of species using PhyloSuite v. 1.2.2 (Zhang et al. 2020). The best substitution models were inferred by the corrected Akaike information criterion (AICc) using the ModelFinder module in PhyloSuite software (BI: (GTR+I+G model for all partition); IQ: (GTR+F+R2: 16S, TPM2u+F+I+G4: COI)); in the ML analyses, we performed 1000 ultrafast bootstrap replicates based on the IQ-TREE. In the BI analyses, two runs each with four Markov chains were run for 20 million iterations with sampling every 1000 generations. The first 25% of generations were removed at the “burn-in” stage followed by calculation of Bayesian posterior probabilities and the 50% majority-rule consensus of the post burn-in trees sampled at stationarity. Trees were visualized with the FigTree v. 1.4.2 program (Rambaut 2016). Finally, genetic distance was calculated with the pairwise uncorrected p-distance model between Oreolalax species on the COI gene using MEGA v. 7 (Kumar et al. 2016).

Morphological analyses

Measurements were made with a digital caliper to the nearest 0.1 mm. The terminology and methods followed Fei et al. (2009) and Watters et al. (2016). Twenty-three morphometric characters were measured for adults: SVL (snout–vent length), direct line distance from tip of snout to posterior margin of vent; HW (head width), at the widest point of the jaws angle; IOD (interorbital distance), the shortest distance between the anterior corners of the orbits; HL (head length), from the posterior of the jaws to the tip of the snout; ED (eye diameter), horizontally from the anterior to posterior corner of the eye; SL (snout length), distance from the tip of the snout to the anterior corner of the eye; UEW (upper eyelid width), greatest width of the upper eyelid margins, measured perpendicular to the anterior-posterior axis; IND (internarial distance), shortest distance between the inner margins of the nostrils; EN (eye–nostril distance), from anterior corner of the eye to the posterior margin of the nostril; NS (snout–nostril length), distance from the center of the external nares to the tip of the snout; LAHL (length of lower arm and hand), the length from the elbow to the end of the third finger; FAW (forearm width), greatest width of the forearm; THL (thigh length), distance from the vent to the knee; TL (tibia length), distance from the outer surface of the flexed knee to the heel/tibiotarsal inflection; TW (tibia width), maximum width of tibia along its length; FL (foot length), from the base of the inner metatarsal tubercle to the tip of Toe IV; LFT (length of foot and tarsus), the length from the tibial appendicular joint to the end of the fourth toe; Toe4L (toe IV length), from the metatarsal tubercle to the tip of Toe IV; Fin1L (finger I length), from the proximal edge of the palmar tubercle to the tip of the Finger I; Fin3L (finger III length), from the proximal edge of the palmar tubercle to the tip of the Finger III; IMT (inner metatarsal tubercle length), the greatest length of the inner metatarsal tubercle; IPTL (inner palmar tubercle length), maximum length of the inner palmar tubercle; OPTL (outer palmar tubercle length), maximum length of the outer palmar tubercle, measured parallel along forearm axis.

Thirteen morphometric characters were measured for tadpoles: BH (maximum body height); BW (maximum body width); SVL (snout–vent length); MW (mouth width), distance between two corners of mouth; SL (snout length), distance from the tip of the snout to the anterior corner of the eye; SS (snout to spiraculum), distance from spiraculum to the tip of the snout; ED (maximum eye diameter); IND (internasal distance),minimum distance between nostrils; IOD (interocular distance), minimum distance between eyes; TAH (tail height), maximum height between upper and lower edges of tail; TAL (tail length), distance from base of vent to the tip of tail; TBW (maximum width of tail base); TOL (total length), distance from the tip of the snout to the tip of tail.

Results

Molecular phylogenetic analyses

Phylogenetic results based on 16S and COI genes showed that the topological structures obtained by BI and ML analyses resulted in essentially identical topologies (Fig. 2). All samples of the undescribed species occupied an independent monophyly and were closely related to O. rugosus, O. major, and O. liangbeiensis. Genetic distances on the COI gene between all samples of the undescribed species were 0.0%–0.3%. The undescribed species is closest to O. rugosus on genetic distance (4.5%), being higher than, or at the same level, as many pairs of substantial species, such as O. liangbeiensis vs O. major (3.8%), O. liangbeiensis vs O. rugosus (3.3%) (Suppl. material 2).

Figure 2. 

Bayesian inference (BI) tree of Oreolalax species based on the mitochondrial 16S and COI gene sequences. ML bootstrap support/Bayesian posterior probability is denoted beside node. The “*” represents posterior probability ≤0.5/ bootstrap value ≤50.

Morphological analyses

We also compared morphological characters of the undescribed species with other Oreolalax species. Comparative morphological data were obtained from the literature for Oreolalax chuanbeiensis Tian, 1983 (Tian 1983), O. granulosus (Fei et al. 1990), Oreolalax jingdongensis Ma, Yang & Li, 1983 (Yang et al. 1983), O. liangbeiensis (Liu et al. 1979), Oreolalax lichuanensis Liu, Hu & Fei, 1979 (Liu et al. 1979), O. major (Liu and Hu 1960), Oreolalax multipunctatus Wu, Zhao, Inger & Shaffer, 1993 (Wu et al. 1993), Oreolalax nanjiangensis Fei & Ye, 1999 (Fei et al. 1999), Oreolalax omeimontis Liu & Hu, 1960 (Liu and Hu 1960), O. pingii (Liu 1943), O. popei (Liu 1947), Oreolalax puxiongensis Liu, Hu & Fei, 1979 (Liu et al. 1979), Oreolalax rhodostigmatus Liu, Hu & Fei, 1979 (Liu et al. 1979), O. rugosus (Liu 1943), O. schmidti (Liu 1947), O. sterlingae (Nguyen et al. 2013), Oreolalax weigoldi Vogt, 1924 (Vogt 1924), and Oreolalax xiangchengensis Fei & Huang, 1983 (Fei and Huang 1983), and O. longmenmontis (Hou et al. 2020). Specimens were examined for comparison: 16 Oreolalax specimens (holotype, paratype, and topotype) from Chengdu Institute of Biology, Chinese Academy of Sciences (CIB, CAS) (Suppl. material 3).

Oreolalax yanyuanensis sp. nov.

https://zoobank.org/D770076A-DB34-4045-AEF9-FFFBC2FBA5D8
Figs 3, 4, 5, 6

Type material

Holotype. • CIBSH20230603020 (Fig. 3), adult male, collected by F. Xie from Shuhe town, Yanyuan county, Sichuan Province (27.473443°N, 101.789624°E, 3108 m a.s.l.) China. Paratypes. • Seven adult males collected from a small stream of Shuhe town (27.475205°N, 101.789108°E, 3127 m a.s.l.); on June 3, 2023 (CIBSH20230603016–19, CIBSH20230603021, CIBSH20230603023–24) by P.Y. Zheng, H.Q. Yu and F. Xie.

Figure 3. 

Photos of the holotype CIBSH20230603020 of Oreolalax yanyuanensis sp. nov. A dorsal view B ventral view; the white arrow indicates the position of the pectoral gland C lateral view D ventral view of hand E ventral view of foot.

Other specimens

• Seven tadpoles (CIBSH20230603kd01–07) collected from a small stream of Shuhe town (27.466241°N, 101.786933°E, 3032 m a.s.l.); on June 3, 2023; by P.Y. Zheng, H.Q. Yu and F. Xie.

Diagnosis

The new species is assigned to genus Oreolalax based on following characters: maxillary teeth prominent; back rough, scattered with large warts, covered with oval black spots; pupil vertical; tongue moderately broad, notched behind; femoral glands prominent; pectoral and axillary gland present in males.

The new species differs from its congeners by a combination of the following characters: body size moderate 39.8–52.8 mm in male; head broad; tympanum hidden; distinct black spots present on flanks; toes 1/3 webbed, with broad lateral fringes; dorsal body deep brown or yellowish-brown; belly smooth, middle yellow, scattered fine mottling, abdominal margin more spotted; skin on dorsum rough with dense and varied size granules, warts are covered with dark spots or no spots; iris light orange or light yellow above, creamy silver white bellow; and middle pectoral glands are evident in males.

Holotype description

(Fig. 3). Body size moderate (SVL 47.4 mm). Head width greater than length (HW 17.6 mm, HL 16.2 mm); maxillary teeth developed, without vomerine teeth or acoustic sac; snout bluntly rounded in dorsal view, slightly projecting over lower jaw, longer than eye diameter (SL 6.3 mm, ED 5.9 mm); canthus rostralis indistinct, interorbital distance (IOD 5.0 mm) wider than internarial distance (IND 3.5 mm), distinctly larger than upper eyelid (UEW 3.0 mm); nostrils oval, closer to tip of snout than eyes (EN 3.1 mm, NS 3.0 mm); no tympanic membrane; supratympanic fold broad; tongue moderately broad, notched behind; pupil vertical.

Fingers moderate, relative finger lengths: I < II < IV < III; finger tips slightly dilated; subarticular tubercles absent; inner palmar tubercle large, nearly rounded, outer palmer tubercle small, oval, completely separated.

Hindlimbs relatively long, length 177% of body length; shank length subequal to thigh length, slightly shorter than foot length (THL 23.4 mm, TL 23.8 mm, FL 24.4 mm); heels partially overlap when thighs are positioned at right angles to the body and tibia-tarsal articulation reaches the middle eye when leg stretched; toes 1/3 webbed with distinct fringes; inner metatarsal tubercle long oval, small.

In life, dorsal body and head rough, back with moderate sparse granules, relatively small warts on head; with dark-brown triangular between eyes; dorsal arms and hindlimbs with small granules and bumps; distinct warts cover the fold and posterior of snout. Ventral skin smooth; pectoral glands flat; pectoral glands evident, chest spines and finger spines not visible; femoral glands slightly swollen, distinct on posterior thigh. Small verrucous granules around the anus.

Large brown markings on dorsum, dark brown triangular pattern between eyes; large markings dorsum brown; ventral skin medium yellow, with scattered little dark speckling; supratympanic fold dark brown; lateral head and flanks brown with dark patches; throat mixed pink and orange yellow, margin with small beige warts; chest pink and the pectoral glands medium yellow; forelimbs covered with black irregular spots; dorsal limbs yellowish-brown, the spots and stripes of dorsal upper arms and tibiotarsal articulation black; ventral arms, thigh, tibia medium yellow with flesh marking; upper iris light orange yellow, lower iris creamy white, both parts embedded black mesh lines.

In preservative (75% ethanol), dorsal body and head dark grey; irregular spots in forelimbs, black longitudinal stripes on hindlimbs; ventral surface beige, throat and arms beige white; with grayish-brown speckling; mandibular margin warts white; pectoral glands and ventral of the hindlimb beige yellow, scattered black spots; hand and feet dark grey, finger tips and palms grayish-white, inner metatarsal tubercle grey; lateral grey on snout and undereye, patches black; skins beneath supratympanic fold dark grey, flanks grey, covered with creamy white warts, black spots around the edges of warts. Perianal warts and femoral gland creamy-white (Fig. 4).

Figure 4. 

The holotype CIBSH20230603020 of Oreolalax yanyuanensis sp. nov. in preservative A dorsal view B ventral view C lateral view D dorsal view of the left hand E ventral view of the left foot.

Tadpoles

Measurements see Suppl. material 4. Description based on preserved tadpole CIBSH20230603kd01 at Gosner stage 37 (TOL 62.3 mm, BL 20.6 mm) (Fig. 5). The mouth is located below the rostral end; labial tooth row formula I:(5+5) /(5+5):I; the upper lip papillae is large, the central missing length is close to 3 papillae position, the lower lip papillae is small and pointed; and there are small teeth on the auxiliary processes of the oral corner; jaw sheaths strong, serrated, the lip teeth are daggerlike; body elliptical in dorsal view, body width is 113% of height; snout rounded, eye positioned dorsolateral; SL 29% of BL; eyes relatively small, ED 8.3% of BL; nostrils near oval; tail long and muscled, TAL 210% of BL; TAH 113% of BH; TBW 44% of BW; SS 53% of BL. Body dark brown in the back and lateral view, creamy yellow in the ventral; tail brown; the single opening of the spiracle lateral, without a free distal tube; tail end blunt; faint brown cloud spots faintly visible on upper caudal fin, caudal fin light and broad.

Figure 5. 

Photos for the tadpole CIBSH20230603kd01 in preservative of Oreolalax yanyuanensis sp. nov. A dorsal view B ventral view C lateral view D ventral view of head.

Variation

Measurements variation of specimens see Suppl. material 1 (unit in mm). Half of the individuals had faint markings on the abdomen (CIBSH20230603016, CIBSH20230603018, CIBSH20230603021, CIBSH20230603023), and half had more markings on the abdomen (CIBSH20230603017, CIBSH20230603019, CIBSH20230603020, CIBSH20230603022). Color of each specimen was brownish-yellow (CIBSH20230603018, CIBSH20230603023), medium brown (CIBSH20230603016, CIBSH20230603019, CIBSH20230603021, CIBSH20230603022), or dark brown (CIBSH20230603017, CIBSH20230603020). Dorsal markings are obvious in most individuals (CIBSH20230603016–22), except one (CIBSH20230603023). Lateral markings are obvious in most individuals (CIBSH20230603016–21, CIBSH20230603023), except one (CIBSH20230603022). Color of warts covering the back was yellowish-brown (CIBSH20230603022), light brown (CIBSH20230603023), brown (CIBSH20230603016, CIBSH20230603020), dark brown (CIBSH20230603018, CIBSH20230603019), black (CIBSH20230603017, CIBSH20230603021). Wart size was relatively small in some individuals (CIBSH20230603017, CIBSH20230603020, CIBSH20230603021, CIBSH20230603023) and relatively large in others (CIBSH20230603016, CIBSH20230603018, CIBSH20230603022, CIBSH20230603023). Half of the specimens had dark temporal folds (CIBSH20230603017, CIBSH20230603018, CIBSH20230603020, CIBSH20230603021), in others the folds were generally lighter (CIBSH20230603016, CIBSH20230603019, CIBSH20230603022, CIBSH20230603023).

Measurements variation of tadpoles see Suppl. material 4. The dorsal color of CIBSH20230606kd01, CIBSH20230606kd03–04, CIBSH20230606kd05–06 is nearly black, while numbers CIBSH20230606kd02 and CIBSH20230606kd07 are dark brown. Light brown cloud spots on the upper caudal fin, ranged from faintly visible (CIBSH20230606kd01, CIBSH20230606kd03) to obvious (CIBSH20230606kd02, CIBSH20230606kd04–07).

Comparisons

In Oreolalax, 19 species occur in southwest China and northern Vietnam. Oreolalax yanyuanensis sp. nov. could be easily distinguished from them by several characters (Suppl. material 5). By having moderate body size (39.8–52.8 mm) in males, the new species differs from O. major (vs. 59.2–68.7 mm), O. popei (vs. 60.0–69.0 mm), O. rhodostigmatus (vs. 57.5–73.5 mm), O. sterlingae (vs. 36.8 mm), and O. weigoldi (vs. 58.2 mm).

By having head width > head length, the new species differs from O. chuanbeiensis, O. nanjiangensis (vs. head width ≈ head length), O. weigoldi (vs. head width = head length), O. multipunctatus, O. popei, O. rhodostigmatus, and O. schmidti (vs. head width < head length).

By having no tympanum, the new species differs from O. liangbeiensis, O. major, O. longmenmontis, O. sterlingae, O. chuanbeiensis, O. multipunctatus, O. nanjiangensis, O. pingii, O. popei, O. puxiongensis, O. schmidti, O. weigoldi (vs. hidden), O. lichuanensis, O. omeimontis (vs. concealed or slightly visible), and O. rhodostigmatus (vs. rather visible).

By having 1/3 toe webbing, the new species differs from O. puxiongensis, O. schmidti (vs. no webbing), O. longmenmontis, O. sterlingae, O. lichuanensis, O. multipunctatus, O. nanjiangensis, O. omeimontis, O. pingii, O. popei, O. rhodostigmatus (vs. rudimentary), O. xiangchengensis, and O. weigoldi (vs. well webbed).

By having triangular pattern between eyes, the new species differs from O. rugosus, O. liangbeiensis, O. major, O. xiangchengensis, O. sterlingae, O. chuanbeiensis, O. granulosus, O. lichuanensis, O. nanjiangensis, O. pingii, O. popei, O. rhodostigmatus, and O. weigoldi (vs. no triangular pattern).

By having middle spiny patches on the chest, the new species differs from O. liangbeiensis, O. major, O. xiangchengensis, O. chuanbeiensis, O. granulosus, O. weigoldi, O. omeimontis (vs. large patches), O. pingii, O. rhodostigmatus, O. jingdongensis, O. lichuanensis (vs. relatively large patches), O. longmenmontis, O. sterlingae, O. multipunctatus, O. nanjiangensis, and O. popei (vs. small patches).

By having dark bars on the limbs, the new species can differ from O. rugosus (vs. no or irregular), O. xiangchengensis, O. pingii, and O. puxiongensis (vs. no).

By having brown yellow or medium yellow scattered variable brown spots on the belly, the new species can differ from O. rugosus (creamy yellow or yellow, no spots), O. liangbeiensis (creamy white without any spots), O. xiangchengensis (light brown, no spots), O. sterlingae (cream with dark marbling), O. granulosus (yellow-white or with fine light gray veins), O. lichuanensis (purplish with dark brown flecks), O. multipunctatus (grey brown, with few or without spots), O. nanjiangensis (without dark spots), O. pingii (gray-white, no spots), O. popei (brown-red, fully covered with small gray-brown spots), O. puxiongensis (grayish-yellow, no spots), O. rhodostigmatus (grayish-brown, no spots), O. schmidti (entirely purple-yellow, no spots), O. weigoldi (light brown with dark cloudy spots on ventrolateral), and O. longmenmontis (flesh red and greyish-white with some black speckles).

Oreolalax yanyuanensis is genetically closest to O. rugosus, O. liangbeiensis and O. major. In addition to the morphological differences (Fig. 6; Suppl. material 5), the new species distinctly differs from these three in measurement proportions. The new species can differ from O. rugosus by having larger LFT, TL, and smaller IN, IOD, UEW, TW. The new species distinctly differs from O. liangbeiensis by having larger HL, HW, ED, LAHL, TL, LFT and smaller SL, IOD. The new species distinctly differs from O. major by having larger HW, ED, TL, LFT and smaller SL, IN, IOD (Table 2).

Table 2.
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Morphometric comparisons between Oreolalax yanyuanensis sp. nov. and its relatives. Shaded values represent a ratio of body measurements to SVL that differs between these three species and the new species.

Measurements/SVL (%) Oreolalax yanyuanensis sp. nov. O. rugosus O. liangbeiensis O. major
8♂♂ 10♂♂ 20♂♂ 6♂♂
HL 35.9 (34.2–37.4) 35.4 33.3 35.4
HW 37.6 (36.6–39.5) 37.5 35.2 35.8
SL 14.3 (13.2–15.3) 14.6 15.5 15.5
IND 9.0 (7.4–10.3) 10.4 9.8 10.4
IOD 10.1 (9.2–10.6) 11.5 10.9 10.8
ED 11.9 (11.5–12.5) 12.2 10.1 10.5
UEW 8.4 (6.3–9.4) 9.9 9.2 9.0
FAW 10.3 (8.9–12.4) 11.5 10.9 10.8
LAHL 54.6 (51.2–58.0) 51.6 51.0 55.0
TL 51.3 (49.6–53.2) 47.8 45.0 48.0
TW 11.3 (9.2–13.6) 13.7 12.3 11.1
LFT 77.5 (74.3–80.2) 67.5 69.0 73.8
FL 50.8 (47.1–54.6) 48.1 48.7 50.9
Figure 6. 

Specimen photos of Oreolalax yanyuanensis sp. nov. and its relative species A, B dorsal and ventral view of the holotype CIB25333 of O. rugosus C, D dorsal and ventral view of the topotype CIB24678 of O. liangbeiensis E, F dorsal and ventral view of the topotype CIB24695 of O. major G, H dorsal and ventral view of the topotype CIBSH20230603020 of Oreolalax yanyuanensis sp. nov. Scale bar: equal to 10 mm.

In elevational distribution, the new species (occurring between 3000–3200 m) can be distinguished from some Oreolalax species occurring below 3000 m (a.s.l.) as follows: O. major (vs. 1600–2000 m), O. longmenmontis (vs. 1300–1450 m), O. chuanbeiensis (vs. 2000–2200 m), O. granulosus (vs. 2300–2450 m), O. jingdongensis (vs. 2300–2450 m), O. lichuanensis (vs. 1790–1840 m), O. multipunctatus (vs. 1800–1920 m), O. nanjiangensis (vs. 1600–1856 m), O. omeimontis (vs. 1050–1800 m), O. popei (vs. 1000–2000 m), O. puxiongensis (vs. 2600–2900 m), O. rhodostigmatus (vs. 700–1790 m), O. schmidti (vs. 1700–2400 m), and O. sterlingae (vs. 2900 m).

Etymology

The specific epithet “yanyuan” refers to the type locality of the species, Yanyuan County, Sichuan Province. We suggested the common name as “Yanyuan toothed toad”, and the Chinese name as “Yan Yuan Chi Chan (盐源齿蟾)”.

Distribution and ecology

Oreolalax yanyuanensis sp. nov. is currently only known from the type locality, Shuhe town, Yanyuan county, Sichuan Prov., China at elevations of 3000–3200 m. The new species inhabits subtropical alpine scrub and swamp, and was found in small montane streams (Fig. 7). The breeding season is currently uncertain; it is speculated that it breeds in April or May based on the tadpole development stage. Four sympatric amphibian species (Bombina maxima Boulenger, 1905 (Boulenger 1905), Rana chaochiaoensis Liu, 1946 (Liu 1946), Panophrys binchuanensis Ye & Fei, 1995 (Ye and Fei 1995) and Nanorana sichuanensis Dubois, 1987 (Dubois 1987 “1986”)) were found in the same habitat.

Figure 7. 

Habitats of Oreolalax yanyuanensis sp. nov. in southeastern Hengduan Mountains region, Sichuan Province, China A, B, C adults’ habitats D tadpoles’ habitat.

Discussion

Hengduan Mountains is part of the hotspot defined as “Mountains of Southwest China”, and its biodiversity conservation has attracted much attention (CEPF 2024). At present, most Oreolalax species are distributed in the eastern Hengduan Mountains and the surrounding mountains of the Sichuan Basin at an altitude of 700–3550 m. More than half of Oreolalax species is listed as “threatened” by IUCN due to habitat loss, habitat degradation, logging, tourism development, and invasive species (Fei and Ye 2016; Jiang et al. 2016; IUCN 2024). Twelve species of Oreolalax are listed as vulnerable, near threatened, endangered, or critically endangered (IUCN 2024). Furthermore, some species have only been recorded at their type locality, and their habitat is not covered by any biodiversity conservation network, even with the high extinction risk (e.g., O. puxiongensis). Also, O. longmenmontis, which has not yet been evaluated by IUCN, faces low population and habitat loss (Hou et al. 2020). Assessments of habitat status, breeding activity, population size and dynamics are needed for these groups, especially for the newly discovered (e.g., O. yanyuanensis sp. nov.) and data deficient species (e.g., O. weigoldi).

The auditory system is critical for animals’ survival and reproduction. Studies have shown that the thin air and low air density at high altitudes lead to slow sound speed, and animals singing in anoxic environment at high altitudes will consume a lot of energy, posing a threat to survival (Liao and Liu 2008; Wen 2014). Some amphibians have reduced investment in acoustic communication, resulting in structural degradation of acoustic communication organs which may be adapted to the high-altitude environment (Lehr and Trueb 2007; Wen 2014). In Oreolalax, the tympanic membranes are hidden or absent in most groups except O. rhodostigmatus. Some high-altitude groups (>3000 m), such as O. rugosus and O. xiangchengensis, showed the absence of columella, while the low-altitude groups showed a developed columella (Wei et al. 2009). Oreolalax yanyuanensis is distributed at high altitudes (3000–3200 m), but it is not clear whether the ear structure is degraded. Due to the limited number of specimens, we will use Micro-CT scans to explore the middle ear structure in the future.

In June 2023, the individuals of O. yanyuanensis found in the wild apparently lacked finger spines and chest spines, and no female individuals have been collected, so it is possible that its breeding season had ended. Based on its morphological characteristics, number and size of tadpoles, it is speculated that O. yanyuanensis may reproduce in April or May. Further studies are needed to investigate its reproductive behavior and population dynamics.

Conclusions

Based on morphological and molecular evidence, we revealed a new toad belonging to the Oreolalax species group—O. yanyuanensis sp. nov. The new species is so far only known from Shuhe town, Yanyuan County, south Sichuan Prov., China. The findings in this study improve our understanding of species diversity in the genus Oreolalax. More studies are necessary to uncover the population size, reproductive ecology, and habitat status to better protect the new Oreolalax species.

Acknowledgements

We thank for Zhonghao Luo for taking photos; Yanjun Zhu and Junjie Hu for their help on picture processing and Yuanfei Wang for his help in specimen preservation.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This work was supported by the National Natural Science Foundation of China (No. 32170428), China Biodiversity Observation Networks (Sino BON– Amphibian and Reptile).

Author contributions

YMH designed the study, wrote the original draft, performed analyses and data visualization and curation. PYZ and HQY collected samples and performed data curation. BW performed data curation and participated in methodological guidance. XHC conceptualized, reviewed and edited the manuscript. FX collected samples, acquired habitat information, performed supervision, writing-review and editing, and acquired funding. All authors read and approved the final manuscript.

Author ORCIDs

Yin Meng Hou https://orcid.org/0000-0002-4216-9678

Bin Wang https://orcid.org/0000-0001-6036-5579

Feng Xie https://orcid.org/0000-0001-9532-9586

Data availability

All of the data that support the findings of this study are available in the main text or Supplementary Information.

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Supplementary materials

Supplementary material 1 

Measurements of Oreolalax yanyuanensis sp. nov.

Yin Meng Hou, Pu Yang Zheng, Hao Qi Yu, Bin Wang, Xiao Hong Chen, Feng Xie

Data type: xlsx

Explanation note: Units in mm. See abbreviations for characters in the Materials and methods section.

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 (12.02 kb)
Supplementary material 2 

Uncorrected p-distance between Oreolalax species on the COI gene

Yin Meng Hou, Pu Yang Zheng, Hao Qi Yu, Bin Wang, Xiao Hong Chen, Feng Xie

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 (13.65 kb)
Supplementary material 3 

Examined specimens in this study

Yin Meng Hou, Pu Yang Zheng, Hao Qi Yu, Bin Wang, Xiao Hong Chen, Feng Xie

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 (11.76 kb)
Supplementary material 4 

Measurements of tadoples of Oreolalax yanyuanensis sp. nov.

Yin Meng Hou, Pu Yang Zheng, Hao Qi Yu, Bin Wang, Xiao Hong Chen, Feng Xie

Data type: xlsx

Explanation note: Units in mm. See abbreviations for morphometric characters in Materials and methods section.

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 (13.47 kb)
Supplementary material 5 

Morphological differences between males of Oreolalax yanyuanensis sp. nov. and males of its congeners

Yin Meng Hou, Pu Yang Zheng, Hao Qi Yu, Bin Wang, Xiao Hong Chen, Feng Xie

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 (12.35 kb)
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