A New Species of the Genus Boulenophrys (Anura, Megophryidae) from Southern Hunan Province, Central China

1. Introduction

The subfamily Megophryinae is endemic to Asia, with a distribution spanning the Himalayas, northeastern India, southern China, and extending southwards to the islands of Southeast Asia [1,2,3]. The classification of Megophryinae has been a subject of debate for decades. Molecular phylogenetic studies consistently indicate that it is a monophyletic group [4,5,6]. However, recent years have been numerous alternations in the generic relationships and divisions within this subfamily [4,5,6,7,8,9,10]. Lyu et al. proposed a new classification system, identifying ten genera: Atympanophrys Tian and Hu, 1983, Brachytarsophrys Tian and Hu, 1983, Grillitschia Dubois, Ohler, and Pyron, 2021, Sarawakiphrys Lyu and Wang, 2023, Jingophrys Lyu and Wang, 2023, Xenophrys Günther, 1864, Megophrys Kuhl and Van Hasselt, 1822, Pelobatrachus Beddard, 1907, Ophryophryne Boulenger, 1903, Boulenophrys Fei, Ye, and Jiang, 2016[7,10,11,12,13,14,15,16]. Among these, Boulenophrys is the most diverse, comprising 69 recognized species to date (Table 1).

Dupangling National Nature Reserve is located in the southwest of Yongzhou City, Hunan Province, within the Nanling Mountains. During herpetological surveys conducted in 2022, we discovered an undescribed species in Daoxian County and Jiangyong County. Based on the diagnostic characters established by Lyu et al.[10], these specimens belong to the genus Boulenophrys, however, they could not be assigned to any recognized species due to their unique combination of morphological characteristics. Subsequent molecular phylogenetic analysis confirmed that these specimens represent a distinct evolutionary lineage. Accordingly, we describe them here as a new species within the genus Boulenophrys.

2. Materials and Methods

2.1. Sampling

Field surveys were conducted in Dupangling National Nature Reserve, Hunan Province, China (Figure 1), in April 2022. A total of ten specimens were newly collected. All specimens were initially fixed in 10% formalin and then transferred to 75% ethanol for storage. Before being fixed in formalin, liver tissues were sampled from four specimens and preserved in 95% ethanol for DNA extraction. Nine specimens were deposited in the Animal Museum of Life Sciences College of Hunan Normal University (HUNU), and one museum specimen was preserved in 75% ethanol and deposited at Chengdu Institute of Biology (CIB), Chinese Academy of Sciences, Chengdu City, Sichuan Province, China.

2.2. Molecular and Phylogenetic Analyses

Genomic DNA was extracted from 95% ethanol-preserved liver tissues using a TSINGKE DNA extraction kit. The mitochondrial genes, specifically the partial 16S ribosomal RNA gene (16S rRNA) and partial cytochrome c oxidase 1 gene (COI) were sequenced for three samples (HUNU 22SA05, HUNU 22SA07, HUNU 22SA08 and CIB2016050802). Polymerase Chain Reaction (PCR) amplifications were conducted following Liu et al. [5]. All PCR products were sequenced on an ABI 3730 XL Genetic Analyzer at the Sangon Biotech Co., Ltd (Shanghai), and the resulting sequences were deposited in GenBank.

For phylogenetic analyses, sequences were obtained from GenBank, including 68 known Boulenophrys species (except B.shuichengensis for which no sequence information is availablle) and two outgroup species (Xenophrys glandulosa and X. mangshanensis) (Table S1). All sequences were aligned using the Clustal W algorithm with default parameters [54] and trimmed with the partial gaps deletion in MEGA 11 [55]. In highly variable regions, all gaps were removed. The best-fit evolutionary model (GTR+G+I) was selected using PartitionFinder v. 2.1.1 under the AIC criterion [56]. The sequenced data were analyzed using Bayesian inference (BI) with MrBayes 3.2 [57], and phylogenetic trees were generated using maximum likelihood (ML) methods implemented in PhyML v. 3.0[58]. The BI analysis was conducted with 10,000,000 generations, sampling every 1000 generations, with the first 25% of samples discarded as burn-in, resulting in a potential scale reduction factor (PSRF) of < 0.005. In the ML analysis, a bootstrap consensus tree inferred from 1000 replicates was generated.

2.3. Morphological Analysis

The definition of morphological characters follows to Lyu et al. [10]. The following measurements were taken with digital calipers to the nearest 0.1 mm : SVL, snout-vent length (from the tip of snout to the posterior edge of the vent); HDL, head length (from the tip of snout to the articulation of the jaw); HDW, head width (head width at the commissure of the jaws); SNT, snout length (from the tip of snout to the anterior corner of the eye); IND, internasal distance (distance between nares); IOD, interorbital distance (minimum distance between upper eyelids); ED, eye diameter (from the anterior corner of the eye to posterior corner of the eye); TD, tympanum diameter (horizontal diameter of tympanum); TED, tympanum-eye distance (from the anterior edge of tympanum to posterior corner of the eye); HND, hand length (from the proximal border of the outer palmar tubercle to the tip of digit III); RAD, radio-ulna length (from the flexed elbow to the proximal border of the outer palmar tubercle); FTL, foot length (from the distal end of the shank to the tip of digit IV); TIB, tibial length (from the outer surface of the flexed knee to the heel). The webbing was expressed using the webbed formula[59], where Roman numerals refer to the fingers and toes. Sex was determined by direct observation of calls or the presence of internal vocal sac openings in males. Presence or absence of nuptial pads was examined under a dissecting microscope. Diagnostic characters distinguishing all 69 species of Boulenophrys are shown in Table 2.

3. Results

3.1. Molecular Phylogenetic Analyses

A total of 540 base pair (bp) of 16S and 636 bp of COI, were concatenated seriatim into a single1196 bp single sequence. Phylogenetic analyses using ML and BI methods produced nearly identical results, with most major nodes well supported on both the ML and Bayesian trees. The Bayesian tree is presented in (Figure 2), with ML bootstrap values (BS) ≥ 70 and BI posterior probabilities (BPP) ≥ 0.90 considered indicative of strong support for major nodes. In different analyses, the newly collected samples were clustered into a monophyletic lineage with robust nodal support (BS 100, BPP 1.00) and exhibited low intraspecific genetic divergence (mean p-distances 4.2 % in COI). Additionally, these specimens can be distinguished from all congeners by a unique combination of morphological characters (see Taxonomic account below).

3.2. Morphological Comparisons

Comparative data of Boulenophrys dupanglingensis sp. nov. with other recognized species of the genus Boulenophrys are listed in Table 2.

Boulenophrys dupanglingensissp. nov. is phylogenetically closed to B. yunkaiensis. It differs from B. yunkaiensis by (1) relatively shorter shanks, TIB/SVL 0.37–0.39 (0.38 ± 0.01, n=7) in adult males (vs. TIB/SVL 0.40–0.48, 0.44 ± 0.04, n=3), 0.32–0.36 in adult female(0.34± 0.02, n=3) (vs. TIB/SVL 0.42–0.46 in adult, 0.44 ±0.02, n=3 for B. yunkaiensis); (2) distinct supernumerary tubercles below the base of I and II toes (vs. indistinct under all toes in B. yunkaiensis); (3) rough dorsum skin with several dense small granules on the flanks (vs. sparse larger granules on the flanks) (Figure 3). These characteristics make Boulenophrys dupanglingensis sp. nov. easily distinguished from B. yunkaiensis.

With relatively longer shanks, and heels overlapping when the flexed hindlimbs are held at right angles to the body axis, Boulenophrys dupanglingensis sp. nov. is easily distinguished from the following 16 congeners, B. acuta, B. brachykolos, B. daoji, B. dongguanensis, B. fengshunensis, B. hungtai, B. hengshanesis, B. insularis, B. kuatunensis, B.lichun, B. nankunensis, B. obesa, B. ombrophila, B. puningensis and B. wugongensis (vs. all of which have relatively shorter shanks with the heels not meeting), B. xuefengmontis (vs. just meeting).

Having an unnotched tongue, Boulenophrys dupanglingensis sp. nov. can be distinguished from the following 9 congeners, B. baolongensis, B. binlingensis, B. boettgeri, B. cheni, B. lushuiensis, B. minor, B.mufumontana, B.qianbeiensis and B. sanmingensis (vs. tongue notched posteriorly in these species).

Having a subarticular tubercle present at the base of each finger, differs from the following 7 congeners, B. angka, B. chishuiensis, B. leishanensis, B. lishuiensis, B. tuberogranulatu, B. wuliangshanensis and B. yaoshanensis (vs. just the first and second finger present or not a finger in sight).

Lacking vomerine teeth, vomerine ridge present, Boulenophrys dupanglingensis sp. nov. differs from the following 15 congeners, B. daiyunensis, B. daweimontis, B.elongata, B. fansipanensis, B. frigida, B. hoanglienensis, B. jinggangensis, B. jiulianensis, B. nanlingensis, B. palpebralespinosa, B.pepe, B. rubrimera, B. shimentaina, B. tongboensis, and B. yingdeensis (vs. presence of vomerine teeth in these species).

Lacking lateral fringes on toes, Boulenophrys dupanglingensis sp. nov. differs from the following 9 congeners, B. anlongensis, B. baishanzuensis, B. binchuanensis, B. congjiangensis, B. lini, B. xiangnanensis, B. xianjuensis, and B. yangmingensis (vs. presence of lateral fringes on toes in these species); and from B. wushanensis (vs. presence of wide lateral fringes on toes in males while lacking in females).

In compared to other congeners, Boulenophrys dupanglingensis sp. nov. has a smaller body size with SVL 37.6–40.2 mm (38.9 ±1.3, n=7) in adult males and SVL 41.8–45.9mm (43.7 ±2.1, n=3) in adult females. It is significantly different from the 9 congeners whose SVL ≥ 50 mm in adult males or females, including B. caudoprocta (81.3 mm in males), B.fangjingmontis (58.6–63.8 in males), B. jingdongensis (53.0–56.5 mm in males), B. liboensis (60.5–67.7 mm in males), B. mirabilis (55.8–61.4 mm in males), B. omeimontis (56.0–59.5 mm in males), B. sangzhiensis (53.0–60.8 mm in male), B. shuichengensis(102.0–118.3 mm in males), and B. spinata (47.2–54.4 mm in males).

For the remaining congeners, Boulenophrys dupanglingensis sp. nov. can be further distinguished by having two subarticular tubercle at the base of each finger (vs. three metacarpal tubercles in B.jiangi), and tibio-tarsal articulation reaching forward between anterior margin of tympanum and posterior corner of eye (vs. tibio-tarsal articulation reaching to tip of snout in B. shunhuangensis, or the middle of eye in B. caobangensis)

3.3. Taxonomic Account

Based on the results of molecular phylogenetic and morphological comparisons, the specimens distinct from all other congeners of the Boulenophrys. The description of this new species is provided below.

Boulenophrys dupanglingensis Xi & Mo, sp. nov.

Dupangling Horned Toad (in English)/ (都庞岭角蟾 in Chinese)

Figures 4, Figures 5

Holotype. Adult male, HUNU 22SA01 (Figure 4), collected by Xiaoyang Mo on 20 April 2022, from Dajiangyuan Village (111.36477082º E, 25.45793412º N; 380 m a.s.l.), Dupangling National Nature Reserve, Daoxian County, Yongzhou City, Hunan Province, China.

Paratypes: nine adults: six adult males (HUNU 22SA02–06, CIB2016050802) from the same locality as the holotype on 21 April 2022 and 23 June 2016; one adult female (HUNU 22SA07), which was collected from Daguyuan Village, Jiangyong County (111.337095º E, 25.435091º N; 442 m a.s.l.) on 23 April 2022 (Figure 5); two adult females (HUNU 22SA08–09), which were collected from Daguyuan Village, Jiangyong County (112.245310º E, 25.328928º N; 451 m a.s.l.) on 24 April 2022. The Paratypes collected by Xiaoyang Mo, Bin Wang, Jiayan Xi, Shengchao Shi, Qi Li, Hui Li, Leqiang Zhu, Ayinuer Maimaiti, Yalan Xu, and Fan Zhou.

Etymology: The specific epithet dupanglingensis refers to the type locality of the new species, Dupangling National Nature Reserve, Southern Hunan Province, China.

Diagnosis. (1) small body size, SVL 37.6–40.2 mm (38.9 ± 1.3, n = 7) in adult males and SVL 41.8–45.9 mm (43.6 ± 2.1, n = 3) in adult females; (2) tympanum boundary clear, TD/ED 0.48–0.57 in males, 0.47–0.57 in females; (3) presence of small horn-like tubercle at the edge of upper eyelid; (4) vomerine ridge present, vomerine teeth absent; (5) margin of tongue rounded, not notched posteriorly; (6) dorsal skin rough, a discontinuous “V”-shaped ridge with two discontinuous dorsolateral ridges on two side on the back, dense tubercles on skin of ventral surface of dorsal shank and thigh, spiny tubercles surrounding the cloaca; (7) hindlimbs slender, heels overlapping when the flexed hindlimbs are held at right angles to the body axis, tibio-tarsal articulation reaching forward between anterior margin of tympanum and posterior corner of eye when leg stretched forward; (8) relative finger length IV < II < I < III, a subarticular tubercle present at the base of each finger; (9)supernumerary tubercles below the base of I and II toes distinct; (10) toes without lateral fringes and with only rudimentary webbing, webbing formula: I1 – 1^-II1 – 2^-III2 – 3IV3^- – 2V.

Coloration of holotype in life. Forearms and hindlimbs are marked with dark brown transverse bands; supratympanic ridges are light ivory with dark spot in the posterior edge; a dark vertical band extend below the eye, from the inferior margin of the eye to the upper lip; numerous brown patches scattered across the lateroventral surface of flanks; groin red-orange; ventral surface of throat and chest are light salmon in color with dark brown patches and dark orange blotches; a distinct longitudinal stripe on throat; ventral surface of body light salmon in color with brown patches and white spots; ventral surface of limbs light salmon in color with dark brown spots and blotches; ventral surfaces of hands and feet are brown, with tips of digits pale brown; metacarpal tubercle and metatarsal tubercles are reddish; Pectoral glands and femoral glands are white; Iris yellowish brown.

Coloration of holotype in preservative. Dark brown faded to greyish brown dorsally. Triangular marking is present between the eyes, accompanied by a “V” shaped marking on the mid-dorsum, transverse bands on dorsal sides of the forearms and hind limbs become indistinct. The ventral surface faded to greyish white, with all previously distinct bands and spots becoming less pronounced or indistinguishable.

Variation and sexual dimorphism. Measurement data for the type series are listed in Table 3. Morphological diagnostic characters observed in all paratypes were consistent with those of the holotype. However, there were variations in coloration and stripe patterns between individuals (Figure 4). For example, adult male paratype (HUNU 22SA03) with a smaller body size (SVL 37.8 mm); has light brown back and a black-brown belly; ventral surfaces of limbs primarily black-brown; have a prominent black spot at the right upper eyelids; In contrast, adult female paratype (HUNU 22SA09) with a larger body size (SVL 41.8mm); had a yellowish brown dorsal coloration; chest and anterior abdomen exhibited dark orange blotches on a light brown background; ventral surfaces of limbs were predominantly orange red; fewer markedly enlarged tubercles were present on the flanks.

Sexual Size Dimorphism: Females (SVL 43.6 ± 2.1 mm, n = 3) are distinctly larger than males (SVL 38.8 ± 0.9 mm, n = 6). Adult males possess a single internal subgular vocal sac. Breeding males develop prominent black nuptial spines on dorsal surfaces at the bases of first and second fingers.

Reproductive Characteristics: The eggs are creamy white, with an approximate diameter of 2.6 mm. The observed clutch size consisted of approximately 258 eggs. (Female, HUNU 22SA08, Figure 6B)

Distribution and habitats. Currently, Boulenophrys dupanglingensis sp. nov. is known only from Dupangling National Nature Reserve, southern Hunan, China. Individuals were found in evergreen secondary forest, near mountain streams, and within leaf litter on the forest floor at elevations from 380–450 m (Figure 7). Male calls were recorded during fieldwork conducted from April to June, suggesting that their breeding season corresponds to this period. The collected female specimens contained mature, creamy white oocytes. No tadpoles were observed during the fieldwork.

4. Discussion

In this study, we described a new species of the genus Boulenophrys from Hunan Province, which brings the total number of recognized Boulenophrys species in Hunan Province to 10, including B. caudoprocta, B. dupanglingensis sp. nov., B.hengshanensi, B. mufumontana, B.Sangzhiensis, B. shunhuangensis, B. tuberogranulatus, B. xiangnanensis, B. xuefengmontis, B. yangmingensis [10,33,60].

The Nanling Mountain range spans the provinces of Guangxi, Hunan, Guangdong and Jiangxi, forming a natural barrier between the Yangtze River and Pearl River basins. This region exhibits high species diversity, strong forest dependence, and overlapping distributions of the Boulenophry genus [36]. These patterns highlight the need for further research into speciation processes, niche differentiation, and coexistence mechanisms is warranted [23]. Moreover, Climatic fluctuations, habitat diversity, and the dynamics of montane forests likely drive diversification within Boulenophrys, influencing the evolution of complex phenotypes.

5. Conclusions

Combining the molecular phylogenetic and morphological analysis results, a new Boulenophry species, B. dupanglingensis sp. nov. is described from Yongzhou City, Hunan Province, China. This research brings the total number of recognized Boulenophrys species in Hunan Province to 10. This discovery highlights the biodiversity within the Nanling Mountain range and further emphasizes the importance of the region as a biodiversity hotspot.