Abstract

Karst landscapes across southern China represent global hotspots for vertebrate diversity and endemism, yet amphibian diversity within such habitats remains insufficiently documented. Herein, we describe Boulenophrys calciphila sp. nov., a new species discovered from fragmented karst hills in northern Guangdong Province, China, extending the documented habitat spectrum of the genus in Guangdong to limestone karst terrain. Detailed morphological comparisons coupled with phylogenetic analyses validate its taxonomic distinctness from all known congeners. This new discovery aligns with ongoing records of range-restricted anuran species uncovered from southern Chinese karst formations, reinforcing karst ecosystems as important reservoirs of amphibian diversity. Our results demonstrate that amphibian diversity within poorly-explored karst regions of northern Guangdong is substantially underestimated and call for focused conservation actions to safeguard these microendemic taxa and their unique evolutionary lineages.

Key Words

Boulenophrys calciphila sp. nov., conservation priority, karst habitat, micro-endemism, taxonomy

Introduction

Karst landscapes in southern China are characterised by highly heterogeneous topography, discontinuous surface drainage and complex subterranean hydrological systems (Clements et al. 2006). These features can promote habitat isolation and the formation of narrowly distributed lineages, particularly in amphibians with limited dispersal ability and specific reproductive requirements (Wells 2007; Lin et al. 2022). Recent discoveries of karst-associated amphibians in Guangdong further suggest that these environments may harbour underestimated amphibian diversity (Lin et al. 2022; Song et al. 2025).

The genus Boulenophrys Fei, Ye & Jiang, 2016 represents one of the most diverse and taxonomically complex groups within the subfamily Megophryinae Bonaparte, 1850, currently comprising more than 80 recognised species that are primarily distributed across southern China and northern mainland Southeast Asia (Fei and Ye 2016; Lyu et al. 2023; Frost 2026; Wang et al. 2026). Boulenophrys liboensis (Zhang, Li, Xiao, Li, Pan, Wang, Zhang & Zhou, 2017) is the only known species confirmed to inhabit karst topography (specifically the limestone forests and cave systems of Libo County, Guizhou Province, China), demonstrating that lineages within this genus are capable of successfully colonising highly heterogeneous and calcium-rich substrates distinct from the typical habitats (Zhang et al. 2017). This case suggests the possibility of additional Boulenophrys species occurring in other karst regions. Notably, northern Guangdong Province contains extensive karst formations, covering a considerable portion of the provincial territory. Given that Guangdong Province, China, is one of the recognised centres of Boulenophrys diversity (Wang et al. 2022; Lyu et al. 2023; Wang et al. 2026), yet previous records from this karst region were notably lacking, we conducted herpetological surveys in northern Guangdong and a series of specimens was collected. Based on integrative taxonomic evidence, we herein describe a new species of Boulenophrys, which advances our understanding of adaptive evolution within this genus.

Materials and methods

Morphology

Two adult male specimens (GEP a596 and CIB 122581) and five adult female specimens (GEP a594, 597–599, and SYS a009718) of the genus Boulenophrys were collected from Ruyuan County, northern Guangdong Province, China (Fig. 1). All examined specimens were fixed in 10% buffered formalin and later transferred to 70% ethanol. All studied specimens have been deposited at the Guangdong Polytechnic of Environmental Protection Engineering (GEP) facility in Foshan, Guangdong Province, China; the Museum of Biology, Sun Yat-sen University in Guangzhou, Guangdong Province, China (SYS); and the Herpetological Museum at Chengdu Institute of Biology, Chinese Academy of Sciences (CIB) in Chengdu, Sichuan Province, China.

Figure 1.

Map showing the distribution sites of Boulenophrys calciphila sp. nov.

External measurements were recorded with a digital caliper (Neiko 01407A stainless steel 6-inch digital caliper) to the nearest 0.1 mm. These measurements are as follows: SVL (snout-vent length, from tip of snout to posterior margin of vent); HDL (head length, from tip of snout to the articulation of the jaw); HDW (head width, head width at the commissure of the jaws); SNT (snout length, from 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 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 (forearm or radiulna length, from the flexed elbow to the proximal border of the outer palmar tubercle); FTL (foot length, from distal end of shank to the tip of digit IV); TIB (crus or tibiofibula length, from the outer surface of the flexed knee to the heel). Sex was initially distinguished by the presence of nuptial pads in males and their absence in females and ultimately confirmed through anatomical observation of testes in males and ovaries/eggs in females (Fei et al. 2009). Morphological characters of all recognised species of the genus Boulenophrys used for comparisons were based on information available in the literature (Table 1).

Table 1.

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Literature for morphological characters of 84 recognised species of Boulenophrys.

ID	Boulenophrys spp.	References
1	B. acuta (Wang, Li & Jin, 2014)	Lyu et al. (2023)
2	B. angka (Wu, Suwannapoom, Poyarkov, Pawangkhanant, Xu, Jin, Murphy & Che, 2019)	Wu et al. (2019)
3	B. anlongensis (Li, Lu, Liu & Wang, 2020)	Li et al. (2020); Lyu et al. (2023)
4	B. baishanzuensis (Wu, Li, Liu, Wang & Wu, 2020)	Wu et al. (2020)
5	B. baolongensis (Ye, Fei & Xie, 2007)	Ye et al. (2007); Fei and Ye (2016)
6	B. binchuanensis (Ye & Fei, 1995)	Lyu et al. (2023)
7	B. binlingensis (Jiang, Fei & Ye, 2009)	Fei et al. (2009); Lyu et al. (2023)
8	B. boettgeri (Boulenger, 1899)	Lyu et al. (2023)
9	B. brachykolos (Inger & 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); Lyu et al. (2023)
12	B. congjiangensis (Luo, Wang, Wang, Lu, Wang, Deng & Zhou, 2021)	Lyu et al. (2023)
13	B. changyangensis Li, Shi, Liu, Zhao, Gao & Wang, 2025	Li et al. (2025)
14	B. cheni (Wang & Liu, 2014)	Lyu et al. (2023)
15	B. chishuiensis (Xu, Li, Liu, Wei & Wang, 2020)	Lyu et al. (2023)
16	B. daiyunensis (Lyu, Wang & Wang, 2021)	Lyu et al. (2023)
17	B. dalaolingensis Wang, Chen & Liu, 2025	Wang et al. (2025a)
18	B. daoji (Lyu, Zeng, Wang & Wang, 2021)	Lyu et al. (2023)
19	B. daweimontis (Rao & Yang, 1997)	Rao and Yang (1997)
20	B. daxuemontis Liu, Li, Cheng, Wei, Wang & Cheng, 2025	Liu et al. (2025)
21	B. dongli Wang, Zhan, Xiao, Tan, Chen, Li, Lin, Lyu & Zeng, 2026	Wang et al. (2026)
22	B. dongguanensis (Wang & Wang, 2019)	Lyu et al. (2023)
23	B. dupanglingensis Xiao & Mo, 2025	Xiao et al. (2025)
24	B. elongata Zeng, Wang, Chen, Xiao, Zhan, Li & Lin, 2024	Zeng et al. (2024)
25	B. fengshunensis Wang, Zeng, Lyu & Wang, 2022	Lyu et al. (2023)
26	B. fanjingmontis (Zhang, Liang, Ran & Shen, 2012)	Lyu et al. (2023)
27	B. fansipanensis (Tapley, Cutajar, Mahony, Nguyen, Dau, Luong, Le, Nguyen, Nguyen, Portway, Luong & Rowley, 2018)	Tapley et al. (2018a)
28	B. frigida (Tapley, Cutaja, Nguyen, Portway, Mahony, Nguyen, Harding, Luong & Rowley, 2021)	Tapley et al. (2021)
29	B. gaolanensis Song, Wang, Qi, Wang & Wang, 2024	Song et al. (2024)
30	B. gutu Kuang, Wei & Shi, 2025	Kuang et al. (2025)
31	B. hoanglienensis (Tapley, Cutajar, Mahony, Nguyen, Dau, Luong, Le, Nguyen, Nguyen, Portway, Luong & Rowley, 2018)	Tapley et al. (2018a)
32	B. huangniushiensis Wang, Wu, Song, Huang, Zhuo & Wang, 2025	Wang et al. (2025b)
33	B. hungtai Wang, Zeng, Lyu, Xiao & Wang, 2022	Lyu et al. (2023); Wang et al. (2026)
34	B. hengshanensis Qian, Hu, Mo, Gao, Zhang & Yang, 2023	Qian et al. (2023)
35	B. insularis (Wang, Liu, Lyu, Zeng & Wang, 2017)	Lyu et al. (2023); Wang et al. (2026)
36	B. jiangi (Liu, Li, Wei, Xu, Cheng, Wang & Wu, 2020)	Lyu et al. (2023)
37	B. jingdongensis (Fei & Ye, 1983)	Fei et al. (1983); Lyu et al. (2023)
38	B. jinggangensis (Wang, 2012)	Lyu et al. (2023)
39	B. jiulianensis (Wang, Zeng, Lyu & Wang, 2019)	Lyu et al. (2023)
40	B. kuatunensis (Pope, 1929)	Lyu et al. (2023)
41	B. leishanensis (Li, Xu, Liu, Jiang, Wei & Wang, 2018)	Lyu et al. (2023)
42	B. lushanensis Shen, Zhou & Li, 2025	Shen et al. (2025a)
43	B. lushuiensis (Shi, Li, Zhu, Jiang, Jiang & Wang, 2021)	Lyu et al. (2023)
44	B. lichun Lin, Chen, Li, Peng, Zeng & Wang, 2024	Lin et al. (2024)
45	B. liboensis (Zhang, Li, Xiao, Li, Pan, Wang, Zhang & Zhou, 2017)	Zhang et al. (2017)
46	B. lini (Wang & Yang, 2014)	Lyu et al. (2023)
47	B. lishuiensis (Wang, Liu & Jiang, 2017)	Lyu et al. (2023)
48	B. meihuamontis Luo & Huang, 2025	Luo et al. (2025); Wang et al. (2026)
49	B. minor (Stejneger, 1926)	Lyu et al. (2023)
50	B. mirabilis (Lyu, Wang & Zhao, 2020)	Lyu et al. (2023)
51	B. mufumontana (Wang, Lyu & Wang, 2019)	Lyu et al. (2023)
52	B. nankunensis (Wang, Zeng & Wang, 2019)	Lyu et al. (2023)
53	B. nanlingensis (Lyu, Wang, Liu & Wang, 2019)	Lyu et al. (2023)
54	B. nebulosa Shen & Li, 2025	Shen et al. (2025b)
55	B. obesa (Wang, Li & Zhao, 2014)	Lyu et al. (2023)
56	B. ombrophila (Messenger & Dahn, 2019)	Lyu et al. (2023)
57	B. omeimontis (Liu, 1950)	Lyu et al. (2023)
58	B. palpebralespinosa (Bourret, 1937)	Fei et al. (2009); Lyu et al. (2023)
59	B. pepe (Wang & Zeng, 2024)	Wang et al. (2024)
60	B. puningensis Wang, Zeng, Lyu, Xiao & Wang, 2022	Lyu et al. (2023)
61	B. qianbeinsis (Su, Shi, Wu, Li, Yao, Wang & Li, 2020)	Lyu et al. (2023)
62	B. raoping Wang, Zhan, Xiao, Tan, Chen, Li, Lin, Lyu & Zeng, 2026	Wang et al. (2026)
63	B. rubrimera (Tapley, Cutajar, Mahony, Chung, Dau, Nguyen, Luong & Rowley, 2017)	Tapley et al. (2017, 2018b)
64	B. sangzhiensis (Jiang, Ye & Fei, 2008)	Lyu et al. (2023)
65	B. sanmingensis (Lyu & Wang, 2021)	Lyu et al. (2023)
66	B. shimentaina (Lyu, Liu & Wang, 2020)	Lyu et al. (2023)
67	B. shuichengensis (Tian & Sun, 1995)	Tian and Sun (1995); Tian et al. (2000); Fei and Ye (2016)
68	B. shunhuangensis (Wang, Deng, Liu, Wu & Liu, 2019)	Lyu et al. (2023)
69	B. spinata (Liu & Hu, 1973)	Hu et al. (1973); Lyu et al. (2023)
70	B. tongboensis (Wang & Lyu, 2021)	Lyu et al. (2023)
71	B. tuberogranulata (Shen, Mo & Li, 2010)	Mo et al. (2010); Fei and Ye (2016); Lyu et al. (2023)
72	B. wugongensis (Wang, Lyu & Wang, 2019)	Lyu et al. (2023)
73	B. wuliangshanensis (Ye & Fei, 1995)	Lyu et al. (2023)
74	B. wushanensis (Ye & Fei, 1995)	Ye and Fei (1995); Fei and Ye (2016); Lyu et al. (2023)
75	B. xiangnanensis (Lyu, Zeng & Wang, 2020)	Lyu et al. (2023)
76	B. xianjuensis (Wang, Wu, Peng, Shi, Lu & Wu, 2020)	Lyu et al. (2023)
77	B. xuefengmontis Lyu & Wang, 2023	Lyu et al. (2023)
78	B. yangchunensis Zhao, Lin, Li, Lyu, Zheng, Zeng, Borzée & Wang, 2025	Zhao et al. (2025)
79	B. yangmingensis (Lyu, Zeng & Wang, 2020)	Lyu et al. (2023)
80	B. yaoshanensis Qi, Mo, Lyu, Wang & Wang, 2021	Lyu et al. (2023)
81	B. yezhongensis Liu, Feng, Shen, Li, Cheng, Wei, Wang & Su, 2025	Liu et al. (2025)
82	B. yingdeensis Qi, Lyu, Wang & Wang, 2021	Lyu et al. (2023)
83	B. youran Xiao, Shi & Mo, 2026	Xiao et al. (2026)
84	B. yunkaiensis Qi, Wang, Lyu & Wang, 2021	Lyu et al. (2023)

Phylogeny

Seven muscle tissue samples were derived from the seven adult specimens (GEP a594, GEP a596–599, CIB 122581 and SYS a009718). In total, 96 samples were used for the phylogenetic analyses, encompassing seven newly-sequenced individuals and others downloaded from GenBank (https://www.ncbi.nlm.nih.gov/genbank/). Two samples of the genus Xenophrys Günther, 1864 were used as outgroups (Suppl. material 1). Two partial mitochondrial genes, the 16S ribosomal RNA (16S) and the cytochrome c oxidase 1 (COI), were used for phylogenetic analysis. DNA extraction, PCR amplification and sequencing protocols follow that of Liu et al. (2018). Newly-sequenced data are deposited in GenBase (https://ngdc.cncb.ac.cn/genbase).

We used Clustal X 2.0 (Thompson et al. 1997) to align the sequences, using default parameters. We then used PartitionFinder (Lanfear et al. 2012) to determine the optimal partitioning schemes and we determined that partitioning by gene was optimal for 16S, while partitioning by codon position was optimal for COI, with GTR+I+G identified as the best-fit nucleotide substitution model for all partitions. We constructed the phylogenetic trees using Maximum Likelihood (ML) implemented in RaxmlGUI v.1.3 (Silvestro and Michalak 2012) and Bayesian Inference (BI) using MrBayes v.3.2.4 (Ronquist et al. 2012). For the ML analysis, an optimal tree was obtained and branch supports were evaluated with 1000 rapid bootstrapping replicates. For the BI analysis, two independent runs were conducted for 10,000,000 generations, with sampling every 1000 generations. The first 25% of samples were discarded as burn-in. Convergence was assessed by confirming that the average standard deviation of split frequencies was below 0.01 and that the potential scale reduction factor (PSRF) approached 1.00. Then, the remaining trees were used to create a consensus tree. Nodes having ML bootstrap values (BS) ≥ 70 and BI posterior probabilities (BPP) ≥ 0.90 were considered well supported. Pairwise uncorrected p-distances of both 16S rRNA and COI sequences were separately computed for all samples in MEGA 11, with pairwise deletion applied to gap positions.

Results

Phylogenetic trees reconstructed using both Bayesian Inference (BI) and Maximum Likelihood (ML) methods recovered highly congruent phylogenetic topologies (Fig. 2). The inferred relationships within Boulenophrys species are consistent with previous studies (e.g. Lyu et al. (2023); Lin et al. (2024)). Notably, all seven samples collected from Ruyuan population formed a strongly supported monophyletic lineage (BS = 100, BPP = 1.00). This lineage is clustered with B. yangmingensis, B. shimentaina, B. jiulianensis and B. gutu, although the specific relationships amongst these taxa remained unresolved. Notably, the minimum genetic distance between this lineage and other Boulenophrys species (2.3% of 16S and 7.7% of COI between this lineage and B. changyangensis) exceeds that observed between certain recognised species (Suppl. material 2; pairs exhibiting comparable or lower divergence values than our thresholds are annotated). Furthermore, specimens belonging to this lineage can be diagnosed from all recognised congeners by a suite of stable morphological characters. Given the concordant support from both molecular phylogenetics and morphological comparisons, we conclude that the population from Ruyuan population represents a distinct, previously undescribed species, which we formally describe below.

Figure 2.

Maximum Likelihood tree results from partial DNA sequences of the mitochondrial 16S rRNA and COI gene. Numbers before slashes indicate bootstrap support (BS) and numbers after slashes are Bayesian posterior probabilities (BPP).

Taxonomic account

Boulenophrys calciphila Zeng, Wang, Qi, Song, Lyu & Wang, sp. nov.

https://zoobank.org/D31CB95F-2874-4B7D-ACC2-55A685F88165

Fig. 3, Table 2

Common name.

Limestone Horned Toad (in English) / huī yán jiǎo chán (灰岩角蟾 in Chinese).

Type materials.

Holotype ♂. GEP a596, collected by Zhao-Chi Zeng and Yuan-Hang Li on 16 May 2026 from Hutouyan “虎头岩” (25.037824°N, 113.171132°E, WGS 84; elevation ca. 690 m), Daqiao Town, Ruyuan County, Shaoguan City, Guangdong Province, China.

Paratypes. 1 ♂: CIB 122581 (field number: GEP a595); 4♀: GEP a594, 597–599, same collection data as holotype; 1♀: SYS a009718, collected by Dan-Yang Zhou, Hao-Cong Yang and Han-Ming Song on 15 April 2026 from Wufeng Village “武丰村” (24.902904°N, 113.174518°E, WGS 84; elevation ca. 540 m), Daqiao Town, Ruyuan County, Shaoguan City, Guangdong Province, China.

Etymology.

The specific epithet calciphila combines the Latin calx, genitive calcis (limestone) with the Greek φίλος (phílos) (= loving), in reference to the association of this species with limestone karst habitats.

Diagnosis.

(1) Moderate size (SVL 49.7–52.7 mm in two adult males, 56.3–59.2 mm in five adult females); (2) snout rounded in dorsal view, canthus rostralis well developed, tongue not notched distally; (3) tympanum distinct; (4) vomerine ridges present, vomerine teeth absent; (5) dorsal skin smooth with sparse granules, discontinuous X-shaped ridge on centre of dorsum, discontinuous dorsolateral ridges present, sparse large tubercles on flanks, dorsal limbs with discontinuous transverse ridges and tubercles; (6) outer margin of upper eyelid with a prominent horn-like tubercle, supratympanic fold distinct and narrow, curving posteroventrally to above arm; (7) two metacarpal tubercles distinct, inner one distinctly enlarged, relative finger lengths I < II < IV < III, distinct subarticular tubercle at base of each finger; (8) heels slightly overlapping when hind-limbs folded, tibiotarsal articulation reaching posterior corner of eye; (9) toes with rudimentary webbing and lacking lateral fringes, inner metatarsal tubercle long ovoid, outer one absent, relative toe length I < II < V < III < IV; (10) dorsal surface yellowish-brown to dark brown, with indistinct irregular dark-brown patches and small orange dots, dark-brown V-shaped marking between eyes, dorsal limbs and digits light brown with dark-brown transverse bands; (11) males lacking subgular vocal sacs.

Comparisons.

Since the interspecific phylogenetic affinities of Boulenophrys calciphila sp. nov. with other congeneric taxa are unresolved, we prioritised morphological comparisons between this new taxon and the 21 species recovered within its sister major clade. Morphologically, this new species closely resembles B. mirabilis and can be readily differentiated from the latter by its smaller body size (SVL 49.7–52.7 mm in two adult males, 56.3–59.2 mm in five adult females (vs. 55.8–61.4 mm in two adult males, 68.5–74.8 mm in two adult females); vomerine ridges present (vs. absent); tongue broad and rounded (vs. tongue slender and elongated); tibiotarsal articulation reaching posterior corner of eye when leg stretched forward (vs. reaching centre of eye); toes without lateral fringes (vs. toes with distinct narrow lateral fringes); and dorsolateral ridges present (vs. absent). The new species can be distinguished from the karst dweller B. liboensis by its tongue not notched distally (vs. tongue notched distally), vomerine teeth absent (vs. present) and lateral fringes on toes absent (vs. present). The new species can be easily distinguished from the other 19 congeners by its absent vocal sac in males (vs. present in B. yangmingensis, B. shimentaina, B. jiulianensis, B. gutu, B. congjiangensis, B. leishanensis, B. yaoshanensis, B. dupanglingensis, B. yunkaiensis, B. yangchunensis, B. acuta, B. gaolanensis, B. xuefengmontis, B. shunhuangensis, B. wushanensis, B. tuberogranulata, B. changyangensis, B. baolongensis and B. boettgeri).

Boulenophrys calciphila sp. nov. can be easily distinguished from B. angka, B. anlongensis, B. baishanzuensis, B. binchuanensis, B. binlingensis, B. brachykolos, B. caobangensis, B. cheni, B. chishuiensis, B. daiyunensis, B. dalaolingensis, B. daoji, B. daweimontis, B. daxuemontis, B. dongli, B. dongguanensis, B. elongata, B. fengshunensis, B. fanjingmontis, B. fansipanensis, B. frigida, B. hoanglienensis, B. huangniushiensis, B. hungtai, B. hengshanensis, B. insularis, B. jiangi, B. jingdongensis, B. jinggangensis, B. kuatunensis, B. lushanensis, B. lushuiensis, B. lichun, B. lini, B. lishuiensis, B. meihuamontis, B. minor, B. mufumontana, B. nankunensis, B. nanlingensis, B. nebulosa, B. obesa, B. ombrophila, B. omeimontis, B. palpebralespinosa, B. pepe, B. puningensis, B. qianbeinsis, B. raoping, B. rubrimera, B. sangzhiensis, B. sanmingensis, B. spinata, B. tongboensis, B. wugongensis, B. wuliangshanensis, B. xiangnanensis, B. xianjuensis, B. yezhongensis, B. yingdeensis and B. youran, by its vocal sac in males absent (vs. present). Boulenophrys calciphila sp. nov. can be distinguished from the remaining B. caudoprocta and B. shuichengensis that also lack vocal sac in males, by its vomerine teeth absent (vs. present), lateral fringes on toes absent (vs. wide in B. shuichengensis and narrow on the fourth toe in B. caudoprocta).

Description of holotype.

Adult male. Body size moderate, SVL 49.7 mm. Head length almost equal to head width, HDW/HDL 1.00; snout rounded in dorsal view, projecting, sloping backwards to mouth in profile, protruding well beyond margin of lower jaw; top of head flat; eyes moderate in size, ED/HDL 0.36, pupil vertical, near diamond-shaped; nostril obliquely ovoid; canthus rostralis well developed; loreal region slightly oblique; internasal distance slightly larger than interorbital distance, IND/IOD 1.10; tympanic region oblique, tympanum distinct and visible in dorsal view; tympanum moderate in size, margin clear, upper margin in contact with supratympanic fold, lower margin in contact with upper lip, TD/ED 0.53; large ovoid choanae at base of maxilla; vomerine ridge present, vomerine teeth absent, maxillary teeth present; margin of tongue rounded, not notched distally; subgular vocal sac absent.

Fore-limb moderate in length, RAD/SVL 0.20, HND/SVL 0.24; webbing absent between fingers, lateral fringes absent, relative finger length I < II < IV < III; tips of fingers slightly dilated, round; subarticular tubercles on base of fingers present, distinct; inner metacarpal tubercle observably enlarged, outer one slightly smaller; an underdeveloped nuptial pad present on the dorsal surface of first and second fingers, respectively. Hind-limbs moderate in length, tibiotarsal articulation reaching forwards to posterior corner of eye when hind-limb stretched along body; heels slightly overlapping when flexed hind-limbs held at right angles to body axis; TIB/SVL 0.40, FTL/SVL 0.61; relative toe length I < II < V < III < IV; tips of toes round and slightly dilated; toes with rudimentary webbing, lacking lateral fringes; subarticular tubercles on base of toes present and distinct; inner metatarsal tubercle long ovoid and lacking outer metatarsal tubercle.

Dorsal skin smooth with sparse granules; dense large tubercles on flanks; single horn-like prominent tubercle on edge of upper eyelid; obvious supratympanic fold curving posteroventrally from posterior corner of eye to level above insertion of arm; indistinct discontinuous X-shaped ridge on centre of dorsum, indistinct discontinuous dorsolateral ridges present; dorsal limbs with discontinuous transverse ridges and tubercles; ventral surface smooth; small and distinct pectoral gland closer to axilla; single femoral gland positioned on posterior surface of thigh at mid-point between knee and cloaca.

Colouration of holotype.

In life, dorsal surface of body dark brown with indistinct irregular dark-brown patches and small orange dots, a dark-brown V-shaped marking between eyes. A vertical dark-brown band present below the eye. Tympanum light brown. Dorsal surface of limbs with dark-brown transverse bands. Tubercles on edge of upper eyelids orange. Supratympanic fold greyish-white with orange dots. Iris coppery dark brown. Throat and chest dark brown with numerous greyish-white dots; belly and ventral limbs greyish-brown with greyish-white dots; large dark brown spots on both sides of belly. Pectoral glands and femoral glands greyish-white.

In preservative, dorsal surface of body dark brown, with markings and patches more distinct, orange dots absent. Surface of chest, throat and limbs greyish-brown, greyish-white dots absent; dark brown spots on both sides of belly more distinct. Pectoral glands and femoral glands greyish-white.

Variation.

Morphometric data are summarised in Table 2. The male holotype GEP a596 possesses an underdeveloped nuptial pad on the dorsal surface of first and second fingers, whereas this structure is absent in the male paratype CIB 122581.

Sexual dimorphism.

Males have no subgular vocal sac and exhibit smaller body size compared to females. Mature ova are observed in all female specimens. Male individuals show darker body colouration than females (Fig. 3).

Figure 3.

Life aspect of the male holotype (GEP a596) of Boulenophrys calciphila sp. nov.: lateral view (A1), dorsal view (A2), ventral view (A3), ventral view of hand (A4), ventral view of foot (A5); and the female paratype (GEP a594): lateral view (B1), dorsal view (B2), ventral view (B3), ventral view of hand (B4), ventral view of foot (B5). Photo by Zhao-Chi Zeng.

Table 2.

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Measurements (in mm) of voucher specimens of Boulenophrys calciphila sp. nov., * holotype.

Voucher	CIB 122581	GEP a596 *	GEP a594	GEP a597	GEP a598	GEP a599	SYS a009718	minimum-maximum (mean ± SD)
Sex	♂	♂	♀	♀	♀	♀	♀	Females (n = 5)
SVL	52.7	49.7	56.3	58.5	58.2	58.7	59.2	56.3–59.2 (58.2 ± 1.1)
HDL	16.2	16.9	17.6	18.3	18.5	16.9	18.4	16.9–18.5 (18.0 ± 0.7)
HDW	16.4	17.0	17.7	18.8	18.9	17.6	19.0	17.6–19.0 (18.4 ± 0.7)
SNT	6.4	6.3	6.7	6.2	6.4	6.6	6.8	6.2–6.8 (6.5 ± 0.3)
IND	5.3	5.5	6.0	6.1	6.2	6.4	6.2	6.0–6.4 (6.2 ± 0.2)
IOD	4.9	5.0	5.9	6.0	5.6	5.8	5.6	5.6–6.0 (5.8 ± 0.2)
ED	5.7	6.0	6.7	6.7	7.1	6.9	6.6	6.6–7.1 (6.8 ± 0.2)
TD	2.9	3.2	3.1	3.3	3.1	3.0	2.9	2.9–3.3 (3.1 ± 0.2)
TED	2.4	2.7	2.6	3.0	2.7	2.3	2.5	2.3–3.0 (2.6 ± 0.2)
HND	13.3	12.1	13.1	13.1	14.0	15.0	14.8	13.1–15.0 (14.0 ± 0.9)
RAD	10.6	9.7	11.5	12.5	12.1	13.4	14.3	11.5–14.3 (12.8 ± 1.1)
TIB	22.5	20.4	23.8	24.5	24.5	25.4	25.6	23.8–25.6 (24.8 ± 0.7)
FTL	33.1	30.1	33.6	34.9	36.0	37.5	36.1	33.6–37.5 (35.6 ± 1.4)

Distribution and natural history.

Boulenophrys calciphila sp. nov. is currently known exclusively from two isolated karst mountain areas in Daqiao Town (elevation 540–690 m), northern Guangdong Province, China. All specimens were collected along roadside areas after rainfall (Fig. 4). Males collected in May exhibited absent or underdeveloped nuptial pads. Mature eggs were observed in all female specimens collected in April and May.

Figure 4.

Microhabitat of Boulenophrys calciphila sp. nov. in the karst landscapes of northern Guangdong, China. Photo by Dan-Yang Zhou.

Discussion

Karst landscapes across southern China constitute critical hotspots of amphibian and reptile endemism and diversity, harbouring numerous poorly documented herpetofaunal taxa. Multiple new anuran species have been continuously described from karst habitats within this broad region in recent years (e.g. Mo et al. (2015); Lin et al. (2022); Li et al. (2025); Song et al. (2025)). These cumulative findings demonstrate that structurally complex karst landforms facilitate species diversification and the aggregation of unique amphibian assemblages at regional scales (Wells 2007). The discovery of Boulenophrys calciphila sp. nov. further indicates that karst areas in northern Guangdong remain severely under-sampled, suggesting that amphibian diversity in this zone is considerably underestimated.

Within the genus Boulenophrys, reliably documented karst-obligate species are exceedingly rare (Fei and Ye 2016). Most congeners inhabit montane forest streams with thick topsoil and well-preserved riparian vegetation. By contrast, karst habitats are characterised by discontinuous thin soil, limited permanent surface water and calcareous bedrock, creating harsh abiotic conditions that constrain amphibian persistence. To date, B. liboensis is the sole congener confirmed as an exclusive karst specialist (Zhang et al. 2017), enabling direct ecological comparisons with B. calciphila sp. nov. Notably, the phylogenetic relationships linking our new species to other Boulenophrys taxa remain unresolved in the current phylogenomic dataset. This incomplete phylogenetic framework limits our ability to robustly infer the evolutionary trajectory of karst adaptation within the genus. Even without fully resolved interspecific relationships, however, comprehensive morphological comparisons verify that discrete diagnostic traits clearly separate B. calciphila sp. nov. from all closely-related congeners, including the karst-endemic B. liboensis. Accordingly, while morphological evidence unequivocally supports its status as a distinct new species, targeted future sampling is still necessary to clarify whether karst fragmentation and geographic isolation drive lineage diversification across Boulenophrys. One caveat of the present work is the limited availability of adult male material: merely two adult male vouchers were obtained, in contrast to five adult female specimens. This small male sample hinders thorough quantification of intraspecific morphological variability and sexual dimorphism within B. calciphila sp. nov. We recommend future targeted field sampling across the karst habitats of northern Guangdong Province and adjacent southern Hunan Province to gather more adult male individuals, which will strengthen analyses of morphological variation and reproductive traits in this limestone-adapted species.

Karst ecosystems possess inherently fragile attributes, including shallow soil cover and specialised hydrological systems. Combined with intensive anthropogenic interference, especially limestone quarrying activities, these natural features render amphibian populations residing in karst habitats highly vulnerable to population reduction and local extirpation. As an ecologically vital, yet insufficiently investigated part of terrestrial ecosystems in Guangdong, karst terrain across northern Guangdong calls for precise and targeted conservation strategies. The protection of distinctive karst landforms and associated micro-endemic species is essential to conserve evolutionary resources and sustain the ecological integrity of biodiversity in Guangdong Province.

Acknowledgements

We sincerely thank the editor and reviewers for their valuable comments and constructive suggestions that greatly improved this manuscript. We thank Ai-Lan He (China) for her help in the fieldwork.

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Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Artificial Intelligence (AI) use

The authors accept full responsibility for the content of the manuscript, including the disclosure of any use of AI.

No AI tools were used in the preparation of this manuscript.

Funding

This work was supported by the DFGP Project of Fauna of Guangdong-202115.

Author contributions

Conceptualization: JW. Investigation: YSZ, HCY, YHL, DYZ, SSL, YPY, YYC. Methodology: ZCZ. Supervision: YYW. Validation: HMS, SQ. Writing - original draft: ZCZ, JW. Writing - review and editing: ZTL.

Author ORCIDs

Z.-C. Zeng https://orcid.org/0000-0003-4054-8399

J. Wang https://orcid.org/0000-0003-4249-7767

S. Qi https://orcid.org/0000-0002-2924-6093

H.-M. Song https://orcid.org/0009-0005-8244-1567

S.-S. Lin https://orcid.org/0000-0001-7709-7005

Y.-H. Li https://orcid.org/0009-0009-6543-9245

Z.-T. Lyu https://orcid.org/0000-0002-4891-3859

Data availability

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

Zhao-Chi Zeng and Jian Wang contributed equally to this work.

Supplementary materials

Supplementary material 1

Supplementary Table 1

Zhao-Chi Zeng, Jian Wang, Shuo Qi, Han-Ming Song, Yao-Yu Cai, Yun-Sheng Zhu, Shi-Shi Lin, Yuan-Hang Li, Dan-Yang Zhou, Hao-Cong Yang, Zhi-Tong Lyu, Yi-Ping Yang, Ying-Yong Wang

Data type: xlsx

Explanation note: Collection localities, voucher data and GenBank accession numbers for all samples used in this study.

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 (16.98 kb)

Supplementary material 2

Supplementary Table 2

Zhao-Chi Zeng, Jian Wang, Shuo Qi, Han-Ming Song, Yao-Yu Cai, Yun-Sheng Zhu, Shi-Shi Lin, Yuan-Hang Li, Dan-Yang Zhou, Hao-Cong Yang, Zhi-Tong Lyu, Yi-Ping Yang, Ying-Yong Wang

Data type: xlsx

Explanation note: Genetic distances between members of Boulenophrys (uncorrected p-distance) in 16S rRNA and CO1 sequences, respectively.

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 (83.28 kb)