A new species of Boulenophrys (Anura, Megophryidae) from northern Jiangxi, China

A new species of Boulenophrys (Anura, Megophryidae) from northern Jiangxi, China Deming Shen1, Haiying Zhou1, Chaoyang Wang1, Jun Xu2, Shi Xu2, Yifeng Zhong1, Zhenyu Wang1, Hao Cai1, Yahui Wu1, Meicai Wei1, Yankuo Li1 1 College of Life Sciences, Jiangxi Normal University, Nanchang 330022, China 2 Administration of Lushan National Nature Reserve, Jiujiang 332900, China https://zoobank.org/22135BBD-1048-4C9C-817F-2A2A2A96CC85 Corresponding authors: Meicai Wei ([email protected]); Yankuo Li ([email protected]) Academic editor: Umilaela Arifin ♦ Received 25 July 2025 ♦ Accepted 5 November 2025 ♦ Published 18 November 2025 Abstract A new species of the genus Boulenophrys (Anura, Megophryidae), Boulenophrys nebulosa sp. nov., is described from Mt. Lushan, Jiangxi Province, China. Phylogenetic analyses based on mitochondrial COI and 16S rRNA genes show that the new species forms a sister lineage to B. mufumontana. Morphologically, B. nebulosa sp. nov. differs from all known congeners by a unique combination of characters and from its closest relative, B. mufumontana, by the following combination of characters: (1) relative finger lengths I < II < IV < III; (2) indistinct subarticular tubercle at the base of each finger; (3) larger male body size (SVL 31.8–32.3 mm); (4) heels just meeting when hindlimbs are folded; (5) tibiotarsal articulation reaching the center of the eye when the leg is stretched forward; and (6) vomerine teeth present. This study expands the known distribution of Boulenophrys in Jiangxi and provides baseline data for biodiversity conservation efforts in Mt. Lushan. Key Words Asian horned toads, biodiversity, Boulenophrys nebulosa sp. nov., taxonomy Introduction The genus Boulenophrys Fei, Ye & Jiang, 2016 (Megophryinae) currently comprises 78 recognized species (Table 1). Its members are distributed across the Himalayas, northeastern India, southern China, and Southeast Asia (Lyu et al. 2023; AmphibiaWeb 2025; Frost 2025). Despite its broad range, recent integrative studies have revealed extensive cryptic diversity within Boulenophrys, much of which is not discernible by morphology alone (Liu et al. 2018). These findings have spurred taxonomic revisions within the subfamily Megophryinae, with Lyu et al. (2023) proposing a ten-genus system that affirms Boulenophrys as the most species-rich lineage. In Jiangxi, known Boulenophrys species are primarily described from the Luoxiao Mountains along the Hunan border and the Wuyi Mountains along the Fujian border. However, the northern part of Jiangxi, including the topographically isolated granitic massif of Mt. Lushan in the Yangtze River Basin, has received limited herpetological attention. This suggests that the species diversity of Boulenophrys in this region may still be underestimated. Historically, two Boulenophrys populations have been recorded in Mt. Lushan: B. lushanensis Shen, Zhou & Li (Shen et al. 2025), and another first reported by Yang et al. (2007), initially identified as Megophrys minor brachykolos (Inger & Romer, 1961) (currently B. brachykolos). However, once thought to be widely distributed in southern China and northern Vietnam (Fei et al. 2009), B. brachykolos has been restricted to Hong Kong and Shenzhen based on voucher specimens and molecular data (Lyu et al. 2023). For instance, the reZoosyst. Evol. 101 (4) 2025, 2251–2266|DOI 10.3897/zse.101.166556 Copyright Shen, D. et al. 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. zse.pensoft.net Shen, D. et al.: A new species of Boulenophrys (Anura, Megophryidae) from northern Jiangxi, China 2252 Table 1. References for morphological characters of 78 recognized species of Boulenophrys. ID Boulenophrys Species References 1B. acuta Wang, Li & Jin, 2014 Li et al. 2014 2B. angka Wu, Suwannapoom, Poyarkov, Pawangkhanant, Xu, Jin, Murphy & Che, 2019 Wu et al. 2019 3B. anlongensis Li, Lu, Liu & Wang, 2020 Li et al. 2020 4B. baishanzuensis Wu, Li, Liu, Wang & Wu, 2020 Wu et al. 2020 5B. baolongensis Ye, Fei & Xie, 2007 Ye et al. 2007 6B. binchuanensis Ye & Fei, 1995 Ye and Fei 1995; Lyu et al. 2023 7B. binlingensis Jiang, Fei & Ye, 2009 Fei et al. 2009; Lyu et al. 2023 8B. boettgeri Boulenger, 1899 Boulenger 1899; Lyu et al. 2023 9B. brachykolos Inger & Romer, 1961 Inger and Romer 1961; Lyu et al. 2023 10 B. caobangensis Nguyen, Pham, Nguyen, Luong & Ziegler, 2020 Nguyen et al. 2020 11 B. caudoprocta Shen, 1994 Shen. 1994 12 B. cheni Wang & Liu, 2014 Wang et al. 2014 13 B. chishuiensis Xu, Li, Liu, Wei & Wang, 2020 Xu et al. 2020 14 B. congjiangensis Luo, Wang, Wang, Lu, Wang, Deng & Zhou, 2021 Luo et al. 2021 15 B. changyangensis Li, Shi, Liu, Zhao, Gao, & Wang, 2025 Li et al. 2025 16 B. daiyunensis Lyu, Wang & Wang, 2021 Lyu et al. 2021 17 B. dalaolingensis Wang, Chen & Liu, 2025 Wang et al. 2025a 18 B. daoji Lyu, Zeng, Wang & Wang, 2021 Lyu et al. 2021 19 B. daweimontis Rao & Yang, 1997 Rao and Yang 1997 20 B. daxuemontis Liu, Li, Cheng, Wei, Wang & Cheng, 2025 Liu et al. 2025b 21 B. dongguanensis Wang & Wang, 2019 Wang et al. 2019a 22 B. dupanglingensis Xiao & Mo, 2025 Xiao et al. 2025 23 B. elongata Zeng, Wang, Chen, Xiao, Zhan, Li & Lin, 2024 Zeng et al. 2024 24 B. fanjingmontis Zhang, Liang, Ran & Shen, 2012 Zhang et al. 2012 25 B. fansipanensis Tapley, Cutajar, Mahony, Nguyen, Dau, Luong, Le, Nguyen,Nguyen, Portway, Luong & Rowley, 2018 Tapley et al. 2018 26 B. fengshunensis Wang, Zeng, Lyu & Wang, 2022 Wang et al. 2022 27 B. frigida Tapley, Cutaja, Nguyen, Portway, Mahony, Nguyen, Harding, Luong& Rowley, 2021 Tapley et al. 2021 28 B. gaolanensis Song, Wang,Qi, Wang, Wang,2024 Song et al.2024 29 B. gutu Kang, Wei & Shi, 2025" Kang et al. 2025 30 B. hengshanensis Qian, Hu, Mo, Gao, Zhang & Yang, 2023 Qian et al. 2023 31 B. hoanglienensis Tapley, Cutajar, Mahony, Nguyen, Dau, Luong, Le, Nguyen,Nguyen, Portway, Luong & Rowley, 2018 Tapley et al. 2018 32 B. huangniushiensis Wang, Wu, Song, Huang, Zhuo, & Wang, 2025 Wang et al. 2025b 33 B. hungtai Wang, Zeng, Lyu, Xiao & Wang, 2022 Wang et al. 2022 34 B. insularis Wang, Liu, Lyu, Zeng & Wang, 2017 Wang et al. 2017a 35 B. jiangi Liu, Li, Wei, Xu, Cheng, Wang & Wu, 2020 Liu et al. 2020 36 B. jingdongensis Fei & Ye, 1983 Fei et al. 1983; Lyu et al. 2023 37 B. jinggangensis Wang, 2012 Wang et al. 2012 38 B. jiulianensis Wang, Zeng, Lyu & Wang, 2019 Wang et al. 2019a 39 B. kuatunensis Pope, 1929 Pope 1929; Lyu et al. 2023 40 B. leishanensis Li, Xu, Liu, Jiang, Wei & Wang, 2018 Li et al. 2018 41 B. liboensis Zhang, Li, Xiao, Li, Pan, Wang, Zhang & Zhou, 2017 Zhang et al. 2017 42 B. lichun Lin, Chen, Li, Peng, Zeng, Wang, 2024 Lin et al.2024 43 B. lini Wang & Yang, 2014 Wang et al. 2014 44 B. lushanensis Shen, Zhou & Li, 2025 Shen et al. 2025 45 B. lishuiensis Wang, Liu & Jiang, 2017 Wang et al. 2017b 46 B. lushuiensis Shi, Li, Zhu, Jiang, Jiang & Wang, 2021 Shi et al. 2021 47 B. minor Stejneger, 1926 Stejneger 1926; Lyu et al. 2023 48 B. mirabilis Lyu, Wang & Zhao, 2020 Lyu et al. 2020 49 B. mufumontana Wang, Lyu & Wang, 2019 Wang et al. 2019a 50 B. nankunensis Wang, Zeng & Wang, 2019 Wang et al. 2019a 51 B. nanlingensis Lyu, Wang, Liu & Wang, 2019 Wang et al. 2019a 52 B. obesa Wang, Li & Zhao, 2014 Li et al. 2014 53 B. ombrophila Messenger & Dahn, 2019 Messenger et al. 2019 54 B. omeimontis Liu, 1950 Liu 1950; Lyu et al. 2023 55 B. palpebralespinosa Bourret, 1937 Bourret 1937; Lyu et al. 2023 56 B. pepe Wang & Zeng, 2024 Wang et al. 2024 57 B. puningensis Wang, Zeng, Lyu, Xiao & Wang, 2022 Wang et al. 2022 58 B. qianbeiensis Su, Shi, Wu, Li, Yao, Wang & Li, 2020 Su et al. 2020 59 B. rubrimera Tapley, Cutajar, Mahony, Chung, Dau, Nguyen, Luong & Rowley,2017 Tapley et al. 2017 60 B. sangzhiensis Jiang, Ye & Fei, 2008 Jiang et al. 2008 61 B. sanmingensis Lyu & Wang, 2021 Lyu et al. 2021 62 B. shimentaina Lyu, Liu & Wang, 2020 Lyu et al. 2020 63 B. shuichengensis Tian & Sun, 1995 Tian and Sun 1995 64 B. shunhuangensis Wang, Deng, Liu, Wu & Liu, 2019 Wang et al. 2019b 65 B. spinata Liu & Hu, 1973 Hu et al. 1973; Lyu et al. 2023 66 B. tongboensis Wang & Lyu, 2021 Lyu et al. 2021 Zoosyst. Evol. 101 (4) 2025, 2251–2266 zse.pensoft.net 2253 cord from Mt. Dayaoshan, Guangxi, was revised and described as the new species B. yaoshanensis Qi, Mo, Lyu, Wang & Wang, 2021, and the record from Mt. Hengshan, Hunan, was revised as the new species B. hengshanensis Qian, Hu, Mo, Gao, Zhang & Yang, 2023. Consequently, all previous records of B. brachykolos from other regions are now considered questionable and need to be carefully re-examined. Notably, the population of B. brachykolos from Mt. Lushan has not yet been evaluated using genetic or detailed morphological analyses, leaving its taxonomic identity unresolved. Herpetological surveys on Mt. Lushan since 2021 have led to the collection of several horned toad specimens. Morphological examinations revealed that these individuals could not be classified as any known species of the genus. While they share a superficial resemblance to B. mufumontana, they exhibit distinct differences in a combination of morphological characters, most notably in the relative length of their fingers. To further elucidate their taxonomic status, we sequenced 16S and COI mitochondrial gene fragments and performed phylogenetic analyses. Integrative evidence from phylogeny and morphology supports the recognition of the Mt. Lushan population as a distinct evolutionary lineage. Based on this evidence, we formally describe it as a new species herein. Materials and methods Sampling Fieldwork was conducted at Mt. Lushan, China. Seven adult specimens and one tadpole specimen were collected in April, June, and November 2021. After capture, the specimens were euthanized with tricaine methanesulfonate (MS-222). Liver or thigh muscle tissues were taken for molecular experiments, preserved in 95% ethanol, and stored at –70 °C. Whole specimens were fixed in 75% ethanol. All specimens and tissue samples were deposited in the Animal Museum of the Life Sciences College, Jiangxi Normal University (JXNU), Nanchang, Jiangxi Province, China. All animal protocols were reviewed and approved by the Animal Ethical and Welfare Committee of Jiangxi Normal University. Morphological analyses Morphological terminology and character definitions followed Lyu et al. (2023) and subsequent taxonomic revisions of Boulenophrys. External measurements were taken using a digital caliper (Neiko 01407A, stainless steel, 6-inch) to the nearest 0.1 mm. The following measurements were recorded: snout–vent length (SVL), head length (HDL), head width (HDW), snout length (SNT), internasal distance (IND), interorbital distance (IOD), eye diameter (ED), tympanum diameter (TD), tympanum–eye distance (TED), hand length (HND), forearm length (RAD), tibial length (TIB), and foot length (FTL). Sex was determined by the presence of secondary sexual characteristics, including internal vocal sac openings or nuptial pads in males, and by direct observation of courtship calling when possible (Fei et al. 2009). The presence or absence of nuptial pads was verified under a dissecting microscope. Comparative morphological data for congeners were compiled from original species descriptions and taxonomic revisions (Table 1), ensuring consistency in character definitions and measurement standards. For the single tadpole of the undescribed species, the following morphometric characters were measured: maximum body height (BH), maximum body width (BW), interocular distance (IOS), mouth width (MW), snout length (SL), snout-to-spiraculum distance (SS), snout–vent length (SVL), tail height (TAH), tail length (TAL), maximum width of the tail base (TBW), and total length (TOL). The morphometric measurements were statistically analyzed using Python 3.11 with the pandas, numpy, scikit-learn, and matplotlib packages. For analyses, all measurements were ln-transformed to normalize and reduce variance, and then scaled to remove allometric effects of body size using the following equation: Xa = Xln − β × (SVLln − SVLm), where Xa = adjusted value, Xln = ln-transformed measurement, β = unstandardized regression coefficient for each species, SVLln = ln-transformed SVL, and SVLm = overall average SVLln of all samples. The size-adjusted measurements were then standardized using z-score normalization to ensure comparability across variables. Principal component analysis (PCA) was performed to examine morphological variation among species using the PCA function from scikit-learn. Since sexual dimorphism is ID Boulenophrys Species References 67 B. tuberogranulatus Shen, Mo & Li, 2010 Mo et al. 2010 68 B. wugongensis Wang, Lyu & Wang, 2019 Wang et al. 2019a 69 B. wuliangshanensis Ye & Fei, 1995 Ye and Fei 1995 70 B. wushanensis Ye & Fei, 1995 Ye and Fei 1995 71 B. xiangnanensis Lyu, Zeng & Wang, 2020 Lyu et al. 2020 72 B. xianjuensis Wang, Wu, Peng, Shi, Lu & Wu, 2020 Wang et al. 2020 73 B. xuefengmontis Lyu & Wang, 2023 Lyu et al. 2023 74 B. yangmingensis Lyu, Zeng & Wang, 2020 Lyu et al. 2020 75 B. yaoshanensis Qi, Mo, Lyu, Wang & Wang, 2021 Qi et al. 2021 76 B. yezhongensis Liu, Feng, Shen, Li, Cheng, Wei, Wang & Su, 2025 Liu et al. 2025a 77 B. yingdeensis Qi, Lyu, Wang & Wang, 2021 Qi et al. 2021 78 B. yunkaiensis Qi, Wang, Lyu & Wang, 2021 Qi et al. 2021 zse.pensoft.net Shen, D. et al.: A new species of Boulenophrys (Anura, Megophryidae) from northern Jiangxi, China 2254 common in frogs, we compiled a dataset using male specimens from the type series of B. mufumontana (Wang et al. 2019a) and the undescribed species for PCA. DNA sequencing and molecular analyses Genomic DNA was extracted from tissue samples using the Universal Genomic DNA Kit (CWBIO, Beijing, China) following the manufacturer’s protocol for animal tissue. Two mitochondrial gene fragments, the partial 16S ribosomal RNA coding sequence (16S rRNA) and the partial cytochrome c oxidase subunit I gene (COI), were amplified in 50 µL reactions. Primer pairs and PCR protocols followed Liu et al. (2018). For the phylogenetic analyses, sequences of 78 Boulenophrys species were obtained from GenBank, except for B. changyangensis, B. dalaolingensis, and B. gutu due to unavailable data. Two outgroup species, Xenophrys glandulosa and X. mangshanensis, were also included (Suppl. material 1: table S1). All sequences were aligned using the MUSCLE algorithm with default parameters. Phylogenetic analyses were conducted based on a concatenated mitochondrial dataset comprising COI and 16S rRNA genes (total length: 1,236 bp). Both Maximum likelihood (ML) and Bayesian inference (BI) methods were employed to construct phylogenetic trees. The ML analysis was performed in IQ-TREE v3.0.1, with the best-fit substitution model (TVM+F+R5) selected using ModelFinder. Branch support was assessed using 1,000 ultrafast bootstrap replicates. The BI analysis was conducted in MrBayes v3.2 (Ronquist et al. 2012), using gene partitions defined by PartitionFinder 2 (Lanfear et al. 2017) (16S: 1–554; COI: 555–1236). Markov Chain Monte Carlo (MCMC) sampling was run for 2,000,000 generations, with trees sampled every 1,000 generations and the first 25% discarded as burn-in. Convergence was considered achieved when the average standard deviation of split frequencies dropped below 0.01. Acoustic analyses Advertisement calls of the undescribed species were recorded from the holotype (JXNU 21042833) on 15 June 2021 at a distance of 0.4 m on Mt. Lushan, Jiujiang, Jiangxi Province, China (ambient temperature: 22.3 °C, relative humidity: 85%). Digital recordings were made using a TASCAM DR05X recorder (96 kHz sampling rate, 24-bit depth) with a built-in microphone. The acoustic analysis implemented a Python 3.9 pipeline using librosa for audio processing, noisereduce for spectral denoising, and scipy.signal.find_peaks for pulse detection. Core procedures included amplitude-threshold segmentation at a 0.013 ratio to isolate calls, spectral peak identification for pulse quantification, and dominant frequency extraction via a 512-point Hann-windowed FFT. All parameters were measured following Tapley et al. (2021). Results Phylogenetic trees reconstructed using BI and ML methods showed highly consistent topologies. The ML tree is shown in Fig. 1, with node support considered strong when bootstrap values (BS) ≥ 70 or Bayesian posterior probabilities (BPP) ≥ 0.90. Samples of the undescribed species formed a strongly supported monophyletic clade in both analyses (BS = 100; BPP = 1.0). This clade was consistently recovered as sister to B. mufumontana (BS = 97; BPP = 1.0). Genetic distances based on the COI and 16S genes, calculated using the uncorrected p-distance model, showed that divergence within the undescribed species was below 0.6% for COI and below 0.4% for 16S. The COI gene distance between the undescribed species and its closest related species, B. mufumontana, was 8.2%; 10.8% with B. caudoprocta; 11.4% with B. boettgeri; and 12.1% with B. liboensis. For the 16S gene, the closest genetic distance was 1.5% with B. boettgeri, 1.7% with B. mufumontana, 2.7% with B. caudoprocta, and 3.2% with B. liboensis (Suppl. material 1: tables S2, S3). Morphologically, in the PCA analysis, the extracted components PC1 and PC2 explained 62.30% and 25.92% of the variance, respectively, accounting for 88.23% cumulatively. As shown in the scatter plot of PC1 and PC2 (Fig. 2), the undescribed species and B. mufumontana are separated without overlapping. Moreover, the undescribed species can be distinguished from all recognized congeners by a unique combination of morphological characters (see Taxonomic account below). Supported by both phylogenetic and morphological evidence, we formally describe it below as a new species. Taxonomic account Boulenophrys nebulosa Shen & Li, sp. nov. https://zoobank.org/6BC99CDF-720B-41EA-AE1E-C0216AD3EF6B Fig. 3, Table 2 Type materials. Holotype. • JXNU 21042833 (Fig. 3A–F), adult male, collected by Deming Shen, Haiying Zhou, Yifeng Zhong, Chaoyang Wang, Hao Cai, Jun Xu and Shi Xu on 28 April 2021 from Mt. Lushan (29°32.56'N, 115°57.58'E; ca. 970 m a.s.l.), Lushan City, Jiujiang, Jiangxi, China. Paratypes. (n = 6) • JXNU 21042832 (Fig. 4A–C), JXNU 21042834, adult males, and JXNU 21042810, adult female, collected by Deming Shen, Haiying Zhou, Yifeng Zhong, Chaoyang Wang, Hao Cai, Jun Xu and Shi Xu on 28 April 2021 from Mt. Lushan (29°31.87'N, 115°55.87'E; ca. 660 m a.s.l.); • JXNU 21061501(Fig. 4D–F), JXNU 21061502, JXNU 21061504, adult females, collected by same collectors on 15 June 2021 from the type locality (29°32.56'N, 115°57.58'E; ca. 970 m a.s.l.). Other material examined. • One tadpole (JXNU 21110702; Fig. 5) collected by Haiying Zhou on 2 November 2021. Etymology. The specific epithet nebulosa (feminine of Latin nebulosus) refers to the frequently mist-shrouded Zoosyst. Evol. 101 (4) 2025, 2251–2266 zse.pensoft.net 2255 montane forests above 600 m on Mt. Lushan, Jiangxi Province, China. This name not only reflects the species’ habitat preference but also alludes to the region’s famous “Cloud & Fog Tea” (云雾茶, yún wù chá) speciality. The English vernacular name “Yunwu Horned Toad” is here proposed, with the corresponding Chinese name “云雾角蟾” (Yúnwù Jiǎochán). Diagnosis. This species can be distinguished from its congeners by the combination of following morphological characteristics: (1) adult males, SVL 31.8–32.3 mm (n = 3); mufumontana sp. nov.nebulosa glandulosa mangshanensis acuta gaolanensis lushuiensis fansipanensis chishuiensis minor daxuemontis jiangi caudoprocta jinggangensis lushanensis leishanensis palpebralespinosa frigida hoanglienensis JXNU 21042810 boettgeri liboensis tongboensis xuefengmontis tuberogranulata congjiangensis wuliangshanensis JXNU 21061504 SYS a006390 SYS a006392 SYS a006419 SYS a006391 lishuiensis brachykolos elongata mirabilis shunhuangensis baolongensis wushanensis rubrimera JXNU 21061501 daoji xianjuensis lichun yaoshanensis hengshanensis JXNU 21110702 wugongensis dupanglingensis yunkaiensis jiulianensis caobangensis jingdongensis JXNU 21042832 dongguanensis nankunensis baishanzuensis sanmingensis fengshunensis shimentaina yangmingensis angka anlongensis JXNU 21042833 hungtai insularis cheni daiyunensis lini nanlingensis yingdeensis binchuanensis daweimontis omeimontis JXNU 21061502 JXNU 21042834 ombrophila kuatunensis puningensis huangniushiensis xiangnanensis binlingensis obesa pepe qianbeiensis yezhongensis spinata sangzhiensis fanjingmontis 100/1 93/1 94/- -/- 90/- 78/- 100/1 -/- 100/1 99/1 95/- 100/1 94/- 83/1 99/- 97/1 80/- 100/1 85/- -/- -/- 94/0.98 91/- 100/1 100/1 93/- 94/1 99/1 99/1 95/0.93 -/- -/- 96/- -/- -/- 99/- 89/- -/- -/- 98/1 -/- 72/- 97/1 74/- -/- 78/- -/- -/- 100/1 98/- -/- -/- 100/1 99/1 -/- 86/- 100/1 99/- -/- -/- 100/1 85/- 70/- 99/- 100/1 90/0.97 -/- 100/1 94/- 100/- 100/1 100/1 100/1 99/- 100/1 100/0.98 Xenophrys Boulenophrys 0.02 Tree scale Figure 1. The maximum likelihood topology inferred from partial DNA sequences of the mitochondrial 16S rRNA and COI genes. Bootstrap support values and Bayesian posterior probabilities are shown at the tree nodes. A dash denotes bootstrap support below 70 or Bayesian posterior probability below 0.9. zse.pensoft.net Shen, D. et al.: A new species of Boulenophrys (Anura, Megophryidae) from northern Jiangxi, China 2256 Figure 2. Principal component analysis based on the morphometric measurements of Boulenophrys nebulosa sp. nov. and B. mufumontana. A. Scatter plot of PC1 and PC2; B. Loadings plot of PC1 and PC2. Figure 3. Holotype of Boulenophrys nebulosa sp. nov. (JXNU 21042833) in life: A. Lateral view; B. Dorsal view; C. Ventral view; D. Volar and dorsal views of left hand; E. Plantar view of left foot; F. Edge of tongue. Photos by Deming Shen. Table 2. Measurements (mm) of adult specimens in the type series of Boulenophrys nebulosa sp. nov. JXNU 21042832 JXNU 21042833 JXNU 21042834 JXNU 21042810 JXNU 21061504 JXNU 21061502 JXNU 21061501 Sex Male Male Male Female Female Female Female SVL 31.8 32.2 32.3 37.1 36.5 35 37.3 HDL 10.3 10.6 10.4 12.1 11.5 11.6 11.7 HDW 9.9 9.6 10.3 11.1 11.5 11 11.6 SNT 3.1 3.8 3.4 4.1 4.2 3.9 3.9 IND 3.2 3.3 3.3 3.5 3.6 3.2 3.6 IOD 3.2 2.8 2.8 3 3.6 3.2 3.7 ED 3.8 3.7 4.1 4.1 4.3 4 4.4 TD 1.8 1.5 1.5 2.2 2.1 1.7 1.8 TED 1.1 1.2 1.1 1.4 1 1.4 1.9 HND 6.7 7 7 7.9 7.5 7.5 7.4 RAD 7.3 7.7 7.7 7.9 8.1 8.5 8.6 TIB 14.3 14.5 14.6 16 15.2 16.3 16.7 FTL 13.7 12.7 14.4 14.3 14.4 14.8 14.6 Zoosyst. Evol. 101 (4) 2025, 2251–2266 zse.pensoft.net 2257 adult females, SVL 35.0–37.3 mm (n = 4); (2) canthus rostralis well developed, tongue not notched posteriorly; (3) tympanum distinct; (4) vomerine ridges weak; vomerine teeth present; (5) outer margin of the upper eyelid bears a small, prominent horn-like tubercle; supratympanic fold distinct, narrow, curving posteroventrally to above the arm; (6) relative finger lengths I < II < IV < III; indistinct subarticular tubercle on base of each finger; Figure 4. Paratypes of Boulenophrys nebulosa sp. nov. in life. Male paratype (JXNU 21042832): A. Lateral view; B. Dorsal view; C. Ventral view. Female paratype (JXNU 21061501): D. Lateral view; E. Dorsal view; F. Ventral view. Photos by Deming Shen. zse.pensoft.net Shen, D. et al.: A new species of Boulenophrys (Anura, Megophryidae) from northern Jiangxi, China 2258 (7) heels meeting when hindlimbs folded; tibiotarsal articulation reaching center of eye when leg stretched forward; (8) toes with narrow fringes and only rudimentary webbing; (9) rough dorsal skin, a V-shaped, X-shaped or Y-shaped ridge is present on central back of trunk, dorsal surface bears short, irregular and weak ridges, with scattered tubercles and granules, dorsolateral ridges absent; several tubercles on flanks and dorsal hindlimbs; ventral skin smooth with granules; (10) dorsal surface yellowish brown; hollow brown triangular marking between eyes; brown V-shaped, X-shaped, or Y-shaped marking on central back of trunk; dorsal limbs and digits with dark brown transverse bands; and (11) dense nuptial spines on dorsal bases of fingers I and II in breeding adult males, subgular vocal sac present in males. Description of holotype. JXNU 21042833, adult male, body size small, SVL 32.2 mm; head width slightly smaller than head length, HDW/HDL 0.91; snout rounded in dorsal view, projecting, sloping backward to mouth in profile, protruding well beyond margin of lower jaw; top of head flat; eyes large, ED 0.35 of HDL, pupil vertical, ovoid in natural state; nostril obliquely ovoid; canthus rostralis well developed; loreal region slightly oblique; internasal distance larger than interorbital distance; tympanic region oblique, tympanum moderate in size, margin clear, upper margin in contact with supratympanic fold, TD/ED 0.41; large ovoid choanae at base of maxilla; vomerine ridge weak, vomerine teeth present, maxillary teeth present; margin of tongue rounded, not notched distally; presence of subgular vocal sac. Figure 5. Tadpole specimen of Boulenophrys nebulosa sp. nov. (JXNU 21110702) in preservative: A. Lateral view; B. Ventral view; C. Dorsal view. Photos by Deming Shen. Zoosyst. Evol. 101 (4) 2025, 2251–2266 zse.pensoft.net 2259 Forearm length 0.24 of SVL, hand 0.22 of SVL; webbing absent between fingers, lateral fringes absent, relative finger length I < II < IV < III; tips of fingers slightly dilated, round; indistinct subarticular tubercles present; inner metacarpal tubercle observably enlarged, outer one indistinct; single nuptial pad bearing nuptial spines present on dorsal surface of first and second fingers, respectively. Hindlimbs short, heels meeting when flexed hindlimbs are held at right angles to the body axis; tibiotarsal articulation reaching the center of the eye when the hindlimb is stretched forward along the body; tibiofibular length 0.45 of SVL, foot length 0.39 of SVL; relative toe length I < II < V < III < IV; tips of toes rounded and slightly dilated; toes with narrow lateral fringes and rudimentary webbing; subarticular tubercles indistinct at the base of each toe; inner metatarsal tubercle long and ovoid; outer metatarsal tubercle absent. Dorsal skin smooth with granules; a discontinuous Y-shaped ridge present on the central dorsum of trunk, discontinuous longitudinal ridges present laterally; several rounded tubercles present on the flanks and dorsal surfaces of the hindlimbs; a prominent, small horn-like tubercle located on the upper eyelid; supratympanic fold distinct, extending from the posterior corner of the eye to above the insertion of the forelimb; tympanum distinct; ventral skin smooth to weakly granular, with spinose tubercles surrounding the cloacal region; pectoral gland small and distinct, located closer to the axilla; a single large femoral gland present on the posterior surface of the thigh, situated approximately midway between the knee and the cloaca. Coloration of holotype. In life, dorsum yellowish brown, with a hollow brown triangular interorbital marking between eyes and a distinct Y-shaped brown marking at the center of the dorsum, surrounded by indistinct brown netlike pattern; dorsal surfaces of limbs yellowish brown, each bearing several brown transverse bars; axillary region and adjacent lateral flank light yellowish brown; tubercles on the upper eyelids orange; a vertical brown stripe present below the eye, extending from the lower margin of the eye to the upper lip; ventral surface of throat and chest greyish brown, bearing irregular dark brown and white blotches; ventral surfaces of limbs, manus and pes purple, with white blotches; abdominal surface greyish white, scattered with creamy white and orange spots; inner and outer metatarsal tubercles and inner metacarpal tubercle pale purple; pectoral white, digits and femoral glands greyish white; iris light yellowish brown. In preservative, the dorsum fades from yellowish brown to greyish, while brown markings, including a Y-shaped interscapular marking, a triangular interorbital marking and transverse bands on the dorsal surfaces of the forelimbs and hindlimbs, darken to greyish brown and become more conspicuous. The ventral surfaces fade to greyish white, making the original white spots and blotches more prominent. Variation. Morphometric data are given in Table 2. All paratypes conform to the holotype in key diagnostic characters but differ in dorsal patterning, ranging from specimens with an indistinct brown netlike pattern as in the holotype to those bearing only an X-shaped interscapular marking and a triangular interorbital marking (e.g., JXNU 21042832). Female paratypes lack nuptial pads and keratinized spines, which are present in males. Tadpole description. The tadpole was confirmed as Boulenophrys nebulosa sp. nov. by molecular phylogenetic analyses. The following tadpole description is based on the specimen JXNU 21110702 at Gosner Stage 29 (Fig. 5). Body depressed and longitudinally oval; TOL 28.0 mm; body width longer than body height, BW/BH 1.3; eyes dorsolateral, pupils round; nostril near eyes; spiracle on the left side of the body and distinct; tail approximately 2.3 times as long as snout-vent length; tail height greater than body height, TAH/BH 1.2; dorsal fin arising just behind tail base, highest near mid-length, tapering gradually to bluntly pointed tip; oral disk terminal; lips expanded, directed upward, forming umbelliform structure; lateral corners of oral disc fold medially, turning upward when not fully extended. In preservative, the background color of the head and trunk fades from brown to dark brown, interspersed with dark-brown chromatophores, and becomes progressively paler toward the tail tip. Areas lacking white pigmentation contain fewer melanocytes. The belly bears dense white spots, especially concentrated on the chest, while the lateral surfaces of the tail are covered with yellowish-brown spots. BH 2.7; BW 3.6; IOD 2.5; MW 1.6; SL 1.8; SS 3.2; SVL 8.5; TAH 3.3; TAL 19.5; TBW 1.4; TOL 28.0. Advertisement call. Call descriptions are based on the vocalizations of the male holotype JXNU 21042833. Spectrograms and waveforms are shown in Fig. 6. A total of 13 call groups were analyzed. Each call had an average duration of 120.3 ± 7.4 ms (110.3–135.6 ms) and an average inter-call interval of 487.6 ± 48.8 ms (415.9– 573.4 ms). Calls were repeated at a mean rate of 2.05 calls/s (constant repetition rate). Each call consisted of 31.7 ± 2.1 pulses (28–35 pulses). The dominant frequency averaged 3.70 ± 0.08 kHz (3.59–3.84 kHz). Comparisons. Morphological comparisons between the proposed new species and other recognized species of the genus Boulenophrys are provided (Table 3). Boulenophrys nebulosa sp. nov. is phylogenetically most closely related to B. mufumontana, forming a well-supported sister clade. However, the new species can be readily distinguished from B. mufumontana by the following combination of characters: 1) relative finger lengths I < II < IV < III (vs. II = IV < I < III); 2) indistinct subarticular tubercle on base of each finger (vs. distinct subarticular tubercle); 3) larger male body size, SVL 31.8–32.3 mm (vs. 30.1–30.8 mm); 4) heels just meeting when hindlimbs folded (vs. heels overlapping); 5) tibiotarsal articulation reaching the center of eye when leg stretched forward (vs. reaching region between posterior margin of tympanum and center of eye); 6) vomerine teeth present (vs. vomerine teeth absent). zse.pensoft.net Shen, D. et al.: A new species of Boulenophrys (Anura, Megophryidae) from northern Jiangxi, China 2266 Supplementary material 1 Additional information Authors: Deming Shen, Haiying Zhou, Chaoyang Wang, Jun Xu, Shi Xu, Yifeng Zhong, Zhenyu Wang, Hao Cai, Yahui Wu, Meicai Wei, Yankuo Li Data type: xlsx Explanation note: table S1. Sample list of genus Boulenophrys and outgroups for phylogenetic analysis in this study. table S2. Uncorrected p-distance (%) among Boulenophrys species from the COI. table S3. Uncorrected p-distance (%) among Boulenophrys species from the 16S. Copyright notice: 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. Link: https://doi.org/10.3897/zse.101.166556.suppl1