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Research Article
A new species of the genus Boulenophrys (Anura, Megophryidae) from Guizhou, China
expand article infoJing Liu‡§, Chao-Bo Feng§, Tuo Shen§, Shi-Ze Li§|‡, Yanlin Cheng‡, Gang Wei, Bin Wang|, Haijun Su§
‡ Moutai Institute, Renhuai, China
§ Guizhou University, Guiyang, China
| Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, China
¶ Guiyang College, Guiyang, China

Corresponding author: Bin Wang ( wangbin@cib.ac.cn )

Corresponding author: Haijun Su ( hjsu@gzu.edu.cn )

Academic editor: Günter Gollmann
© 2025 Jing Liu, Chao-Bo Feng, Tuo Shen, Shi-Ze Li, Yanlin Cheng, Gang Wei, Bin Wang, Haijun Su.
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: Liu J, Feng C-B, Shen T, Li S-Z, Cheng Y, Wei G, Wang B, Su H (2025) A new species of the genus Boulenophrys (Anura, Megophryidae) from Guizhou, China. Herpetozoa 38: 117-136. https://doi.org/10.3897/herpetozoa.38.e155430
ZooBank: urn:lsid:zoobank.org:pub:9FDADB27-63C0-41C2-9A42-2E19DDB20F55
Open Access

Abstract

Based on morphological and molecular phylogenetic analyses, a new species of the genus Boulenophrys is described from Yezhong Nature Reserve, Shuicheng County, Guizhou Province, China. Phylogenetic analyses based on the mitochondrial genes 16S rRNA and COI indicate that this new species represents an independent species-level lineage, closely related to B. fanjingmontis, B. qianbeiensis, B. sangzhiensis, and B. spinata. The new species is distinguished from its congeners by a combination of the following characteristics: (1) body size moderate (SVL 41.2–46.2 mm in adult males and 51.8–58.6 mm in adult females); (2) a single small horn-like tubercle at the edge of each upper eyelid; (3) vomerine teeth absent; (4) tongue not notched posteriorly; (5) tympanum distinctly visible, round; (6) toes with rudimentary webbing and narrow lateral fringes; (7) heels overlapping when thighs are positioned at right angles to the body; (8) tibiotarsal articulation reaching the level of the middle of the eye when the leg is stretched forward; (9) a single internal subgular vocal sac in males; (10) in breeding males, the nuptial pads have black nuptial spines on the bases of the first and second fingers.

Key Words

Boulenophrys yezhongensis sp. nov., molecular phylogenetic analysis, morphology, taxonomy

Introduction

The genus Boulenophrys Fei, Ye & Jiang, 2016 comprises 71 recognized species, which are classified within the subfamily Megophryinae (Bonaparte 1850) and are widely distributed in eastern and central China, throughout southeastern Asia, and extending to the islands of the Sunda Shelf and the Philippines, westward to Nepal and northeastern India (Frost 2025). The taxonomic arrangements, especially regarding generic assignments within the group, have been controversial for a long time (e.g., Tian and Hu 1983; Dubois 1987; Rao and Yang 1997; Lathrop 1997; Delorme et al. 2006; Jiang et al. 2008; Fei et al. 2009; Fei and Ye 2016; Chen et al. 2017; Mahony et al. 2017; Qi et al. 2021; Lyu et al. 2023). Recently, Lyu et al. (2023) reviewed the literature comprehensively and conducted morphological and molecular phylogenetic analyses, proposing a revised generic classification for Megophryinae. This classification divides the subfamily into ten genera.

Among these genera, Boulenophrys Fei, Ye and Jiang 2016 is one of the most widely distributed and speciose taxa, and 47 species of this genus have been described in the last decade (Frost 2025). Obviously, the diversity of this genus has been underestimated, and new species continue to be discovered and described (Zeng et al. 2024; Xiao et al. 2025; Frost 2025).

During a recent amphibian survey in the Yezhong Nature Reserve, Guizhou Province, China, eight specimens of Asian horned toads were collected. After molecular phylogenetic analyses and morphological comparisons, the results indicated that the specimens from Yezhong Nature Reserve represent an undescribed taxon within the genus Boulenophrys. Herein, we describe it as a new species.

Materials and methods

Sampling

Five adult males and three adult females of the undescribed species were collected in Yezhong Nature Reserve (26.1638°N, 104.8803°E, 2022 m), Shuicheng County, Guizhou Province, China (Table 1; Fig. 1). In the field, the toads were euthanized by means of isoflurane (Herrel 2001), and the specimens were fixed in 10% buffered formalin and later transferred to 75% ethanol for preservation. The muscle samples used for molecular analysis were preserved in 95% alcohol and stored at −20 °C. The specimens were deposited in the Chengdu Institute of Biology, Chinese Academy of Sciences (CIB, CAS).

Table 1.
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Samples used in molecular phylogenetic analyses in this study.

ID Boulenophrys species Voucher ID Locality 16S rRNA CO1
1 B. yezhongensis sp. nov. CIBSC20240531001 China,Guizhou,Shuicheng PV469438 PV491548
2 B. yezhongensis sp. nov. CIBSC20240531002 China,Guizhou,Shuicheng PV469439 PV491549
3 B. yezhongensis sp. nov. CIBSC20240531004 China,Guizhou,Shuicheng PV469440 PV491550
4 B. yezhongensis sp. nov. CIBSC20240531005 China,Guizhou,Shuicheng PV469441 PV491551
5 B. yezhongensis sp. nov. CIBSC20240531003 China,Guizhou,Shuicheng PV469442 PV491552
6 B. yezhongensis sp. nov. CIBSC20240531008 China,Guizhou,Shuicheng PV469443 PV491553
7 B. yezhongensis sp. nov. CIBSC20240531006 China,Guizhou,Shuicheng PV469444 PV491554
8 B. yezhongensis sp. nov. CIBSC20240531007 China,Guizhou,Shuicheng PV469445 PV491555
9 B. acuta SYS a002266 China, Guangdong, Fengkai KJ579119 MH406122
10 B. angka KIZ 040591 Thailand, Chiang Mai, Doi Inthanon MN508052 /
11 B. anlongensis CIB AL20190531018 China, Guizhou, Anlong MT823184 MT823261
12 B. baishanzuensis CIB QY20200719002 China, Zhejiang, Qingyuan MW001151 MT998292
13 B. baolongensis KIZ 019216 China, Chongqing, Wushan KX811813 KX812093
14 B. binchuanensis KIZ 019441 China, Yunnan, Mt Jizu KX811849 KX812112
15 B. binlingensis KIZ 025807 China, Sichuan, Mt Wawu KX811852 KX812115
16 B. binlingensis SYSa005313 Wawu Shan, Sichuan, China MH406892 MH406354
17 B. binlingensis SYSa005314 Wawu Shan, Sichuan, China MH406893 MH406355
18 B. boettgeri KIZ YPXJK033 China, Fujian, Mt Wuyi KX811814 KX812104
19 B. brachykolos SYS a002258 China, Hong Kong KJ560403 MH406120
20 B. caobangensis IEBR 4385 Vietnam, Cao Bang, Nguyen Binh LC483945 /
21 B. caudoprocta SYS a004293 China, Hunan, Sangzhi MH406796 MH406258
22 B. cheni SYS a004050 China, Jiangxi, Mt Jinggang MF667873 MH406241
23 B. chishuiensis SYS a004953 China, Guizhou, Chishui MH406867 MH406329
24 B. congjiangensis GZNU 20200706003 China, Guizhou, Congjiang MW959773 MW959761
25 B. daiyunensis SYS a007711 China, Fujian, Tong’an MW367054 MW365504
26 B. daoji SYS a006212 China, Zhejiang, Mt Tiantai MW367047 MW365497
27 B. daweimontis KIZ 048997 China, Yunnan, Mt Dawei KX811867 KX812125
28 B. dongguanensis SYS a002007 China, Guangdong, Dongguan MH406654 MH406090
29 B. dupanglingensis HUNU 22SA05 China,Hunan,Dupangling Nature Reserve OP548599 OP550328
30 B. elongata GEPa155 China, Guangdong, Huizhou, Mt Lianhua OR601595 OR597101
31 B. fanjingmontis SYS a004350 China, Guizhou, Mt Fanjing MH406808 MH406270
32 B. fanjingmontis FJS20190728001 China, Guizhou, Mt Fanjing PV469447 PV485343
33 B. fanjingmontis FJS20190728002 China, Guizhou, Mt Fanjing PV469448 PV485344
34 B. fanjingmontis FJS20190728003 China, Guizhou, Mt Fanjing PV469449 PV485345
35 B. fansipanensis AMS R186115 Vietnam, Lao Cai, Sapa MH514887 MW086548
36 B. fengshunensis SYS a004724 China, Guangdong, Fengshun MH406848 MH406310
37 B. frigida AMS R186131 Vietnam, Lao Cai, Bat Xat MT364279 MW086550
38 B. gaolanensis SYS a009225 China: Guangdong: Zhuhai,Gaolan PQ217847 PQ218778
39 B. hengshanensis CSUFT HS210612 China, Hunan, Mt Hengshan ON209291 /
40 B. hoanglienensis VNMN 2018.02 Vietnam, Lao Cai, Sapa MH514889 MW086551
41 B. hungtai SYS a007577 China, Guangdong, Jiexi OL635594 OL634861
42 B. insularis SYS a002171 China, Guangdong, Nan’ao MH406665 MH406105
43 B. jiangi CIB KKS20180722006 China, Guizhou, Kuankuoshui MN107743 MN107748
44 B. jingdongensis SYS a003928 China, Yunnan, Mt Wuliang MH406773 MH406232
45 B. jinggangensis SYS a004028 China, Jiangxi, Mt Jinggang MH406780 MH406239
46 B. jiulianensis SYS a004218 China, Jiangxi, Mt Jiulian MH406790 MH406252
47 B. kuatunensis SYS a003449 China, Jiangxi, Mt Wuyi MF667881 MH406206
48 B. leishanensis KIZ 049172 China, Guizhou, Mt Leigong KX811825 KX812102
49 B. liboensis GNUG 20150813001 China, Guizhou, Libo MF285253 /
50 B. lichun CIB 121428 China, Fujian, Ningde PQ309137 PQ300665
51 B. lini KIZ 07053 China, Jiangxi, Mt Jinggang KX811842 KX812110
52 B. lishuiensis SYS a008445 China, Zhejiang, Liandu OQ180984 OQ180872
53 B. lushuiensis CIB YN201909289 China, Yunnan, Lushui MW001226 MW000913
54 B. minor KIZ YPX37545 China, Sichuan, Dujiangyan KX811895 KX812144
55 B. mirabilis SYS a002289 China, Guangxi, Lingui MH406681 MH406127
56 B. mufumontana SYS a006419 China, Hunan, Mt Mufu MK524107 MK524138
57 B. nankunensis SYS a004503 China, Guangdong, Mt Nankun MH406824 MH406286
58 B. nanlingensis SYS a001962 China, Guangdong, Ruyuan MH406645 MH406081
59 B. obesa SYS a002275 China, Guangdong, Fengkai KJ579123 MH406125
60 B. ombrophila CIB WY18082308 China, Fujian, Mt Wuyi MW001159 MT998300
61 B. omeimontis KIZ 025765 China, Sichuan, Mt Emei KX811884 KX812136
62 B. palpebralespinosa KIZ 011650 Vietnam, Thanh Hoa, Pu Hu KX811889 KX812138
63 B. pepe GEP a207 China, Guangdong, Qingyuan PQ131151 PQ130479
64 B. puningensis SYS a005770 China, Guangdong, Puning OL635585 OL634853
65 B. qianbeiensis CIBTZ20190608015 China,Guizhou,Huanglian Nature Reserve MT651553 MT654520
66 B. qianbeiensis CIBTZ20190608017 China,Guizhou,Huanglian Nature Reserve MT651554 MT654521
67 B. qianbeiensis CIBTZ20160715003 China,Guizhou,Huanglian Nature Reserve MT651555 MT654522
68 B. qianbeiensis CIBKKS20180722002 China,Guizhou,Huanglian Nature Reserve MT651556 MT654523
69 B. qianbeiensis CIBKKS20180722001 China,Guizhou,Huanglian Nature Reserve MT651557 MT654524
70 B. rubrimera AMS R177676 Vietnam, Lao Cai, Sapa MF536419 MW086542
71 B. sangzhiensis SYS a004313 China, Hunan, Sangzhi MH406802 MH406264
72 B. sangzhiensis CIBSZ2012062005 China,Hunan,Badagongshan Nature Reserve MT651558 MT654525
73 B. sangzhiensis CIBSZ2012062008 China,Hunan,Badagongshan Nature Reserve MT651559 MT654526
74 B. sangzhiensis CIBSZ2012062007 China,Hunan,Badagongshan Nature Reserve MH406798 MH406260
75 B. sanmingensis SYS a007057 China, Jiangxi, Mt Magu MW367051 MW365501
76 B. shimentaina SYS a002077 China, Guangdong, Yingde MH406655 MH406092
77 B. shuichengensis CIBDF202100812001 China,Guizhou,Dafang OK017489 OK012066
78 B. shunhuangensis HNNU 16SH04 China, Hunan, Mt Shunhuang MK836027 /
79 B. spinata KIZ 016100 China, Guizhou, Mt Leigong KX811864 KX812119
80 B. spinata CIBLS20190801002 China,Guizhou,Mt Leigong MT651551 MT654518
81 B. spinata CIBLS20190801001 China,Guizhou,Mt Leigong MT651552 MT654519
82 B. spinata ZL-2018 China,Guizhou,Mt Leigong MH406676 MH406116
83 B. tongboensis SYS a003227 China, Jiangxi, Mt Tongbo MH406744 MH406201
84 B. tuberogranulata KIZ YPX10987 China, Hunan, Sangzhi KX811823 KX812095
85 B. wugongensis SYS a004801 China, Jiangxi, Anfu MH406854 MH406316
86 B. wuliangshanensis SYS a003924 China, Yunnan, Mt Wuliang MH406771 MH406230
87 B. wushanensis KIZ YPX47799 China, Chongqing, Wushan KX811835 /
88 B. xiangnanensis SYS a002874 China, Hunan, Shuangpai MH406713 MH406165
89 B. xianjuensis CIB XJ190503 China, Zhejiang, Xianju MN563758 MN563774
90 B. xuefengmontis SYS a007227 China, Hunan, Mt Xuefeng OQ180973 OQ180861
91 B. yangmingensis SYS a002888 China, Hunan, Shuangpai MH406719 MH406171
92 B. yaoshanensis SYS a004878 China, Guangxi, Jinxiu MH406863 MH406325
93 B. yingdeensis SYS a004721 China, Guangdong, Yingde MH406846 MH406308
94 B. yunkaiensis SYS a004694 China, Guangdong, Xinyi MH406845 MH406307
95 Xenophrys mangshanensis SYS a002177 China, Guangdong, Huaiji MH406666 MH406106
96 Xenophrys glandulosa SYS a003757 China, Yunnan, Mt Gaoligong MH406754 MH406213
Figure 1. 

Distribution sites of Boulenophrys yezhongensis sp. nov. and its phylogenetically close species, B. fanjingmontis, B. qianbeiensis, B. sangzhiensis, and B. spinata.

Molecular data and phylogenetic analyses

We obtained sequences from eight specimens of the undescribed species. Three specimens of B. fanjingmontis were sequenced and included in the molecular analyses (Table 1). Total DNA was extracted using a standard phenol-chloroform extraction protocol (Sambrook et al. 1989). Two fragments of the mitochondrial 16S rRNA (16S) and cytochrome oxidase subunit I (COI) genes were amplified. For 16S, the primers P7 (5’-CGCCTGTTTACCAAAAACAT-3’) and P8 (5’-CCGGTCTGAACTCAGATCACGT-3’) were used following Simon et al. (1994), and for COI, Chmf4 (5’-TYTCWACWAAYCAYAAAGAYATCGG-3’) and Chmr4 (5’-ACYTCRGGRTGRCCRAARAATCA-3’) were used following Che et al. (2012). Gene fragments were amplified under the following conditions: an initial denaturation step at 95 °C for 4 min; 36 cycles of denaturation at 95 °C for 30 s, annealing at 52 °C (for 16S) / 47 °C (for COI) for 40 s, and extension at 72 °C for 70 s. Sequencing was conducted using an ABI3730 automated DNA sequencer at Shanghai DNA BioTechnologies Co., Ltd. (Shanghai, China). New sequences were deposited in GenBank (for GenBank accession numbers, see Table 1).

For molecular analyses, a total of 96 available sequence data for congeneric species were downloaded from GenBank (Table 1), primarily from previous studies (Liu et al. 2018; Wang et al. 2024; Song et al. 2024; Xiao et al. 2025). For phylogenetic analyses, sequences from Xenophrys glandulosa (Fei et al. 1990) and X. mangshanensis (Fei et al. 1990) were downloaded (Table 1) and used as outgroups according to Lyu et al. (2023). Sequences were assembled and aligned using the ClustalW module in BioEdit v. 7.0.9.0 (Hall 1999) with default settings. Alignments were checked manually and revised if necessary. Trimming, with gaps partially deleted, was performed using GBLOCKS 0.91b (Castresana 2000). phylogenetic analyses of mitochondrial DNA, the dataset was concatenated with 16S and COI gene sequences. To avoid under- or over-parameterization (Lemmon and Moriarty 2004; McGuire et al. 2007), the best partition scheme and the best evolutionary model for each partition were selected using PARTITIONFINDER v. 1.1.1 (Robert et al. 2012). In this analysis, the 16S gene and each codon position of the COI gene were defined, and the Bayesian Information Criterion was used. The analysis suggested that the best partition scheme is 16S gene/each codon position of the COI gene and selected the GTR + G + I model as the best model for each partition. Phylogenetic analyses were conducted using maximum likelihood (ML) and Bayesian inference (BI) methods, implemented in PhyML v. 3.0 (Guindon et al. 2010) and MrBayes v. 3.12 (Ronquist and Huelsenbeck 2003), respectively. For the ML tree, branch support was estimated from 10,000 nonparametric bootstrap replicates. In BI, two runs, each with four Markov chains, were simultaneously run for 50 million generations with sampling every 1,000 generations. The first 25% of trees were discarded as burn-in, followed by calculations of Bayesian posterior probabilities and a 50% majority-rule consensus of the post-burn-in trees sampled at stationarity. Finally, mean genetic distances between Boulenophrys species based on the uncorrected p-distance model were estimated for the COI and 16S genes using MEGA v. 6.06 (Tamura et al. 2013).

Morphological comparisons

A total of 29 specimens, including five males of the new taxon, five males of B. sangzhiensis, five males of B. fanjingmontis, six males of B. qianbeiensis, and five males of B. spinata, were measured (Table 1 and Suppl. material 1). Additional morphological data were sourced from the literature (Table 2). The terminology and methods followed Fei et al. (2009). Measurements were taken with a dial caliper to the nearest 0.1 mm. Seventeen morphometric characters of adult specimens were measured:

Table 2.
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References for morphological characters for congeners of the genus Boulenophrys.

ID Species References
1 B. acuta Wang, Li & Jin, 2014 Li et al. 2014
2 B. angka Wu, Suwannapoom, Poyarkov, Pawangkhanant, Xu, Jin, Murphy & Che, 2019 Wu et al. 2019
3 B. anlongensisLi, Lu, Liu & Wang, 2020 Li et al. 2020
4 B. baishanzuensis Wu, Li, Liu, Wang & Wu, 2020 Wu et al. 2020
5 B. baolongensis Ye, Fei & Xie, 2007 Ye et al. 2007
6 B. binchuanensis Ye & Fei, 1995 Ye and 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 Boulenger 1899; Lyu et al. 2023
9 B. 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. daiyunensis Lyu, Wang & Wang, 2021 Lyu et al. 2021
16 B. daoji Lyu, Zeng, Wang & Wang, 2021 Lyu et al. 2021
17 B. daweimontis Rao & Yang, 1997 Rao and Yang 1997
18 B. dongguanensis Wang & Wang, 2019 Wang et al. 2019b
19 B. dupanglingensis Xiao & Mo, 2025 Xiao et al. 2025
20 B. elongata Zeng, Wang, Chen, Xiao, Zhan, Li & Lin, 2024 Zeng et al. 2024
21 B. fanjingmontis Zhang, Liang, Ran & Shen, 2012 Zhang et al. 2012
22 B. fansipanensis Tapley, Cutajar, Mahony, Nguyen, Dau, Luong, Le, Nguyen,Nguyen, Portway, Luong & Rowley, 2018 Tapley et al. 2018
23 B. fengshunensis Wang, Zeng, Lyu & Wang, 2022 Wang et al. 2022
24 B. frigida Tapley, Cutaja, Nguyen, Portway, Mahony, Nguyen, Harding, Luong& Rowley, 2021 Tapley et al. 2021
25 B. gaolanensis Song, Wang,Qi, Wang, Wang,2024 Song et al.2024
26 B. hengshanensis Qian, Hu, Mo, Gao, Zhang & Yang, 2023 Qian et al. 2023
27 B. hoanglienensis Tapley, Cutajar, Mahony, Nguyen, Dau, Luong, Le, Nguyen,Nguyen, Portway, Luong & Rowley, 2018 Tapley et al. 2018
28 B. hungtai Wang, Zeng, Lyu, Xiao & Wang, 2022 Wang et al. 2022
29 B. insularis Wang, Liu, Lyu, Zeng & Wang, 2017 Wang et al. 2017a
30 B. jiangi Liu, Li, Wei, Xu, Cheng, Wang & Wu, 2020 Liu et al. 2020
31 B. jingdongensis Fei & Ye, 1983 Fei et al. 1983; Lyu et al. 2023
32 B. jinggangensis Wang, 2012 Wang et al. 2012
33 B. jiulianensis Wang, Zeng, Lyu & Wang, 2019 Wang et al. 2019b
34 B. kuatunensis Pope, 1929 Pope 1929; Lyu et al. 2023
35 B. leishanensis Li, Xu, Liu, Jiang, Wei & Wang, 2018 Li et al. 2018
36 B. liboensis Zhang, Li, Xiao, Li, Pan, Wang, Zhang & Zhou, 2017 Zhang et al. 2017
37 B. lichun Lin, Chen, Li, Peng, Zeng, Wang, 2024 Lin et al.2024
38 B. lini Wang & Yang, 2014 Wang et al. 2014
39 B. lishuiensis Wang, Liu & Jiang, 2017 Wang et al. 2017b
40 B. lushuiensis Shi, Li, Zhu, Jiang, Jiang & Wang, 2021 Shi et al. 2021
41 B. minor Stejneger, 1926 Stejneger 1926; Lyu et al. 2023
42 B. mirabilis Lyu, Wang & Zhao, 2020 Lyu et al. 2020
43 B. mufumontana Wang, Lyu & Wang, 2019 Wang et al. 2019b
44 B. nankunensis Wang, Zeng & Wang, 2019 Wang et al. 2019b
45 B. nanlingensis Lyu, Wang, Liu & Wang, 2019 Lyu et al. 2019
46 B. obesa Wang, Li & Zhao, 2014 Li et al. 2014
47 B. ombrophila Messenger & Dahn, 2019 Messenger et al. 2019
48 B. omeimontis Liu, 1950 Liu 1950; Lyu et al. 2023
49 B. palpebralespinosa Bourret, 1937 Bourret 1937; Lyu et al. 2023
50 B. pepe Wang & Zeng, 2024 Wang et al. 2024
51 B. puningensis Wang, Zeng, Lyu, Xiao & Wang, 2022 Wang et al. 2022
52 B. qianbeiensis Su, Shi, Wu, Li, Yao, Wang & Li, 2020 Su et al. 2020
53 B. rubrimera Tapley, Cutajar, Mahony, Chung, Dau, Nguyen, Luong & Rowley,2017 Tapley et al. 2017
54 B. sangzhiensis Jiang, Ye & Fei, 2008 Jiang et al. 2008
55 B. sanmingensis Lyu & Wang, 2021 Lyu et al. 2021
56 B. shimentaina Lyu, Liu & Wang, 2020 Lyu et al. 2020
57 B. shuichengensis Tian & Sun, 1995 Tian and Sun 1995
58 B. shunhuangensis Wang, Deng, Liu, Wu & Liu, 2019 Wang et al. 2019a
59 B. spinata Liu & Hu, 1973 Hu et al. 1973; Lyu et al. 2023
60 B. tongboensis Wang & Lyu, 2021 Lyu et al. 2021
61 B. tuberogranulatus Shen, Mo & Li, 2010 Mo et al. 2010
62 B. wugongensis Wang, Lyu & Wang, 2019 Lyu et al. 2019b
63 B. wuliangshanensis Ye & Fei, 1995 Ye and Fei 1995
64 B. wushanensis Ye & Fei, 1995 Ye and Fei 1995
65 B. xiangnanensis Lyu, Zeng & Wang, 2020 Lyu et al. 2020
66 B. xianjuensis Wang, Wu, Peng, Shi, Lu & Wu, 2020 Wang et al. 2020
67 B. xuefengmontis Lyu & Wang, 2023 Lyu et al. 2023
68 B. yangmingensis Lyu, Zeng & Wang, 2020 Lyu et al. 2020
69 B. yaoshanensis Qi, Mo, Lyu, Wang & Wang, 2021 Qi et al. 2021
70 B. yingdeensis Qi, Lyu, Wang & Wang, 2021 Qi et al. 2021
71 B. yunkaiensis Qi, Wang, Lyu & Wang, 2021 Qi et al. 2021

ED eye diameter (distance from the anterior corner to the posterior corner of the eye);

FL foot length (distance from tarsus to the tip of the fourth toe);

HDL head length (distance from the tip of the snout to the articulation of the jaw);

HDW maximum head width (greatest width between the left and right articulations of the jaw);

HLL hindlimb length (maximum length from the vent to the distal tip of the Toe IV);

IAE distance between posterior corner of eyes;

IFE distance between anterior corner of eyes;

IND internasal distance (minimum distance between the inner margins of the external nares);

IOD interorbital distance (minimum distance between the inner edges of the upper eyelids);

LAL length of lower arm and hand (distance from the elbow to the distal end of the Finger IV);

LW lower arm width (maximum width of the lower arm);

NED nasal to eye distance (distance between the nasal and the anterior corner of the eye);

NSD nasal to snout distance (distance between the nasal and the posterior edge of the vent);

SVL snout-vent length (distance from the tip of the snout to the posterior edge of the vent);

SL snout length (distance from the tip of the snout to the anterior corner of the eye);

TFL length of foot and tarsus (distance from the tibiotarsal articulation to the distal end of the Toe IV);

THL thigh length (distance from vent to knee);

TL tibia length (distance from knee to tarsus);

TW maximal tibia width;

TYD maximal tympanum diameter;

UEW upper eyelid width (greatest width of the upper eyelid margins measured perpendicular to the anterior-posterior axis).

To reduce the impact of allometry, the ratio of each character to SVL was calculated and log-transformed for subsequent morphometric analyses (Lleonart et al. 2000). One-way analysis of variance (ANOVA) was used to test the significance of differences in morphometric characters between different species. The significance level was set at 0.05. To show the spatial distribution of different species based on morphometric characters, principal component analyses (PCA) were performed. The statistical analyses were conducted using SPSS 21.0 (SPSS Inc., Chicago, IL, USA). The morphology of the new species was also compared with that of all other Boulenophrys species (Suppl. material 1, Table 3). Comparative data were obtained from the literature (Table 2).

Table 3.
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Morphometric comparisons between Boulenophrys yezhongensis sp. nov., B. fanjingmontis, B. qianbeiensis, B. sangzhiensis, and B. spinata. Units given in mm. Abbreviations for the species name: BF, B. fanjingmontis; BQ, B. qianbeiensis; BSZ, B. sangzhiensis; BSP, B. spinata. See abbreviations for morphometric characters in the Materials and Methods section. Significant level at 0.05.

Measurements B. yezhongensis sp. nov B. fanjingmontis B. qianbeiensis B. sangzhiensis B. spinata p-value from ANOVA in male
males (n = 5) females (n = 3) males (n = 5) males (n = 6) males (n = 5) males (n = 5)
Range Mean ± SD Range Mean ± SD Range Mean ± SD Range Mean ± SD Range Mean ± SD Range Mean ± SD sp vs. BSZ sp vs. BQ sp vs. BS sp vs. BF
SVL 41.2–46.2 43.5 ± 1.9 51.8–58.6 54.1 ± 3.9 58.2–67.7 63.2 ± 4.0 49.3–58.2 54.3 ± 3.1 56.1–59.8 58.3 ± 1.6 51.2–56.2 53.9 ± 1.8 0.00 0.00 0.00 0.00
HDL 12.1–13.0 12.6 ± 0.3 14.2–15.2 14.6 ± 0.6 16.1–18.0 17.1 ± 0.8 14.6–17.0 15.6 ± 0.9 16.1–18.0 17.2 ± 1.0 14.3–15.8 14.8 ± 0.6 0.57 0.99 0.10 0.09
HDW 14.3–15.4 14.8 ± 0.4 16.9–17.4 17.2 ± 0.3 20.3–22.9 21.9 ± 1.1 18.3–21.0 19.9 ± 1.1 20.0–21.5 20.8 ± 0.6 18.4–19.1 18.7 ± 0.3 0.03 0.01 0.48 0.38
SL 5.2–5.8 5.5 ± 0.3 6.0–7.1 6.5 ± 0.5 6.7–8.1 7.5 ± 0.5 6.7–7.2 6.9 ± 0.2 6.6–8.2 7.4 ± 0.6 5.7–7.1 6.4 ± 0.6 0.81 0.94 0.20 0.16
TYD 2.8–3.3 3.0 ± 0.2 3.0–3.7 3.4 ± 0.3 3.1–3.6 3.4 ± 0.2 3.2–4.3 3.5 ± 0.4 3.1–3.7 3.4 ± 0.2 2.5–2.9 2.7 ± 0.2 0.01 0.42 0.00 0.00
IFE 6.7–7.5 7.2 ± 0.3 7.5–7.9 7.7 ± 0.2 10.7–11.7 11.0 ± 0.5 7.8–10.9 9.7 ± 1.1 9.4–10.9 10.2 ± 0.5 8.5–9.6 9.1 ± 0.4 0.02 0.10 0.41 0.04
IAE 11.6–12.8 12.2 ± 0.5 14.1–14.6 14.4 ± 0.3 16.8–18.0 17.3 ± 0.5 14.0–16.4 15.5 ± 0.9 16.1–17.3 16.8 ± 0.6 14.0–14.4 14.2 ± 0.2 0.33 0.52 0.04 0.48
NED 2.7–2.9 2.8 ± 0.1 2.8–3.1 3.0 ± 0.1 3.5–4.5 4.1 ± 0.4 2.4–3.4 2.9 ± 0.4 3.0–3.9 3.4 ± 0.4 2.7–3.0 2.8 ± 0.1 0.14 0.02 0.00 0.98
NSD 2.4–2.7 2.5 ± 0.1 2.7–3.0 2.8 ± 0.2 3.5–4.1 3.7 ± 0.2 3.5–4.3 3.8 ± 0.3 3.7–4.6 4.2 ± 0.3 3.4–4.1 3.8 ± 0.3 0.00 0.00 0.01 0.81
IND 5.0–5.6 5.3 ± 0.2 5.5–6.0 5.8 ± 0.3 6.5–7.5 7.2 ± 0.4 5.7–7.5 6.7 ± 0.6 6.8–7.7 7.2 ± 0.3 5.8–6.3 6.0 ± 0.2 0.00 0.00 0.02 0.01
IOD 3.9–4.9 4.4 ± 0.4 5.2–5.6 5.3 ± 0.2 5.1–6.1 5.7 ± 0.4 3.7–5.3 4.5 ± 0.6 4.7–5.8 5.1 ± 0.5 4.2–5.3 4.9 ± 0.4 0.00 0.00 0.00 0.00
ED 4.3–5.2 4.9 ± 0.3 5.4–6.2 5.7 ± 0.4 6.3–7.1 6.7 ± 0.3 5.4–6.9 6.3 ± 0.6 6.1–7.4 6.7 ± 0.5 5.1–6.0 5.6 ± 0.3 0.31 0.15 0.18 0.15
UEW 3.8–4.3 4.0 ± 0.2 4.6–5.3 5.0 ± 0.3 5.1–5.9 5.4 ± 0.3 5.1–6.2 5.6 ± 0.4 5.1–6.1 5.7 ± 0.4 4.5–6.1 5.0 ± 0.7 0.10 0.02 0.72 0.10
LAL 18.8–21.3 19.7 ± 1.0 23.3–25.0 23.9 ± 0.9 27.1–29.4 28.7± 1.0 20.4–25.4 24.0 ± 1.8 26.2–28.2 26.9 ± 0.8 24.0–25.1 24.3 ± 0.4 0.39 0.38 0.94 0.69
LW 2.5–3.1 2.9 ± 0.2 2.9–3.3 3.1 ± 0.2 5.5–6.3 5.9 ± 0.4 5.7–7.4 6.6 ± 0.6 6.1–6.7 6.4 ± 0.2 5.5–7.4 6.3 ± 0.7 0.00 0.00 0.00 0.00
HLL 56.5–70.6 63.5 ± 5.2 76.3–84.3 80.1 ± 4.0 100.8–109.3 105.7 ± 3.2 76.9–93.7 87.8 ± 6.1 97.3–105.0 100.3 ± 2.8 85.7–99.0 91.8 ± 5.3 0.00 0.01 0.00 0.00
THL 17.4–20.5 18.6 ± 1.2 21.7–24.4 23.1 ± 1.4 29.7–34.6 32.3 ± 1.8 24.1–28.4 26.6 ± 1.8 27.9–31.6 29.6 ± 1.6 26.1–29.8 27.6 ± 1.4 0.00 0.00 0.00 0.00
TL 19.3–22.4 20.6 ± 1.2 24.6–26.0 25.1 ± 0.8 32.8–35.2 34.1 ± 1.0 25.0–32.4 29.2 ± 2.6 31.6–32.6 32.2 ± 0.4 28.9–30.4 29.6 ± 0.6 0.00 0.00 0.00 0.00
TW 4.2–4.7 4.4 ± 0.2 5.1–5.2 5.2 ± 0.0 6.9–7.8 7.4 ± 0.3 7.0–8.7 8.0 ± 0.6 7.3–8.1 7.7 ± 0.3 6.0–8.0 7.0 ± 0.9 0.00 0.00 0.01 0.00
TFL 26.6–32.4 29.1 ± 2.4 35.1–38.0 36.5 ± 1.5 43.9–47.9 45.7 ± 1.6 34.5–44.0 40.4 ± 3.6 42.9–46.1 44.2 ± 1.3 39.2–43.1 40.9 ± 1.6 0.00 0.02 0.00 0.05
FL 15.7–19.7 17.6 ± 1.5 22.1–23.2 22.6 ± 0.6 29.4–32.5 31.4 ± 1.2 24.5–29.4 27.5 ± 2.9 26.7–29.9 28.9 ± 1.3 26.4–29.6 28.1 ± 1.2 0.00 0.00 0.00 0.00

Results

Phylogenetic analyses

A total of 537 base pairs (bp) of the 16S gene and 558 bp of the COI gene were concatenated into a single 1,095 bp sequence.

ML and BI trees of the mitochondrial DNA dataset presented nearly identical topologies. Although the relationships of many lineages were unresolved (Fig. 2), major nodes in both tree types showed strong support, defined here as Bayesian posterior probabilities (BPP) ≥ 0.95 and maximum likelihood (ML) ultrafast bootstrap values ≥ 95% (Wheeler 2016). In the mitochondrial DNA trees, all samples of the genus Boulenophrys in this study clustered within one group, forming a single lineage with high nodal support (BPP = 1.00 and BS = 100%). There was almost no divergence between sequences within this lineage, and the lineage was sister to a clade composed of B. fanjingmontis, B. qianbeiensis, B. sangzhiensis, and B. spinata, with high support.

Figure 2. 

Maximum likelihood (ML) tree of the genus Boulenophrys reconstructed based on the 16S rRNA and COI gene sequences. Bayesian posterior probability/ML bootstrap supports were denoted beside each node. Samples 1–96 refer to Table 1.

Genetic distances based on the 16S and COI genes, calculated using the uncorrected p-distance model, showed that divergence within the undescribed species was below 0.1%. The COI gene distance between the undescribed species and its closest related species, B. qianbeiensis, was 2.5%; 5.3% with B. sangzhiensis; 5.8% with B. fanjingmontis; and 5.9% with B. spinata. For the 16S gene, the closest genetic distance was 0.6% with B. binlingensis, 0.9% with B. qianbeiensis, 0.9% with B. fanjingmontis, 1.1% with B. sangzhiensis, and 1.4% with B. spinata (see Suppl. materials 2, 3).

Morphological analyses

The results of the ANOVA tests based on several morphometric characteristics revealed significant differences (p < 0.05; Table 3) between male individuals of the new species and males of B. qianbeiensis, B. fanjingmontis, B. sangzhiensis, and B. spinata. In the PCA for males, the first two principal components accounted for 61.9% of the total variance (Table 4). Loadings for PC1, which explained 40.5% of the total variance, were most heavily weighted on IND, HLL, TL, TW, TFL, and FL, while loadings for PC2, which explained 21.4%, were most heavily weighted on TYD and IAE. Differentiation was evident along the PC1 axis between the newly collected specimens and B. qianbeiensis, B. sangzhiensis, B. spinata, and B. fanjingmontis (Fig. 3). Detailed morphological comparisons revealed discrete diagnostic characters distinguishing the undescribed taxon from its congeners. Based on the molecular phylogenetic analyses and morphological comparisons (see Suppl. material 4), the specimens from Yezhong Nature Reserve represent a new species, which is described as follows.

Table 4.
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Factor loadings of the first four principal components for 21 size-adjusted male morphometric characteristics of Boulenophrys yezhongensis sp. nov., B. sangzhiensis, B. spinata, B. qianbeiensis and B. fanjingmontis.

Character PC1 PC2 PC3 PC4
Eigenvalue 8.497 4.495 2.117 1.274
% variation 40.462 21.406 10.083 6.066
SVL 0.510 -0.606 -0.013 0.417
HDL 0.108 0.751 0.103 -0.155
HDW 0.681 0.613 0.036 0.005
SL 0.103 0.693 -0.005 0.180
NED -0.403 0.154 0.715 0.127
NSD 0.740 0.274 -0.238 -0.336
IND 0.820 0.057 -0.222 0.073
IOD -0.730 0.350 0.354 -0.301
ED 0.300 0.686 -0.110 0.411
UEW 0.351 0.515 -0.323 -0.205
LAL 0.338 0.194 0.794 -0.063
LW 0.792 -0.099 -0.376 -0.255
HLL 0.843 -0.232 0.391 -0.030
THL 0.756 -0.417 0.176 0.062
TL 0.907 -0.158 0.260 -0.054
TW 0.882 0.211 -0.183 -0.049
TFL1 0.868 -0.022 0.318 -0.189
FL 0.889 -0.210 0.182 -0.210
TYD -0.307 0.806 -0.104 -0.056
IFE 0.532 0.247 -0.024 0.660
IAE 0.276 0.804 0.210 0.100
Figure 3. 

Plots of the first principal component (PC1) versus the second (PC2) for males of Boulenophrys yezhongensis sp. nov. and B. fanjingmontis, B. qianbeiensis, B. sangzhiensis, and B. spinata.

Taxonomic account

Boulenophrys yezhongensis sp. nov.

https://zoobank.org/34D3EB46-4E41-4EAA-8DC4-066BC038437E

Type material.

Holotype • CIB SC20240531005 (Figs 4, 5), adult male, from Yezhong Nature Reserve, Shuicheng County, Guizhou Province, China (26.163426°N, 104.880083°E, ca. 2022 m a. s. l.), collected by Jing Liu on 31 May 2024.

Figure 4. 

Male holotype CIB SC20240531005 of Boulenophrys yezhongensis sp. nov. in life. A. Dorsal view; B. Ventral view; C. Dorsal view of hand; D. Ventral view of hand; E. Ventral view of foot.

Figure 5. 

The holotype specimen CIB SC20240531005 of Boulenophrys yezhongensis sp. nov. in preservative. A. Dorsal view; B. Ventral view; C. Dorsal view of hand; D. Ventral view of hand; E. Ventral view of foot.

Paratypes • Seven adult specimens (4 males and 3 females) from the same place as holotype. Four males (CIB SC20240531001– 4) collected on 31 May 2024 by Shize Li and Chaobo Feng. • Three females (CIB SC20240531006–8) collected on 31 May 2024 by Jing Liu and Shize Li.

Diagnosis.

Boulenophrys yezhongensis sp. nov. is assigned to the genus Boulenophrys based on molecular phylogenetic analyses and the following generic diagnostic characters: snout shield-like; projecting beyond the lower jaw; canthus rostralis distinct; chest glands small and round, closer to the axilla than to midventral line; femoral glands on rear part of thigh; vertical pupils; and the presence of subarticular tubercles at the base of each finger (Fei et al. 2016; Lyu et al. 2023).

Boulenophrys yezhongensis sp. nov. can be distinguished from its congeners by a combination of the following morphological characters: body size moderate (SVL 41.2–46.2 mm in adult males and 51.8–58.6 mm in adult females); a small horn-like tubercle at edge of each upper eyelid; vomerine teeth absent; tongue not notched posteriorly; tympanum distinctly visible, round; two metacarpal tubercles on hand; toes with rudimentary webbing and narrow lateral fringes; heels overlapping when thighs are positioned at right angles to the body; tibiotarsal articulation reaching the level to the middle of eye when leg stretched forward; a single internal subgular vocal sac in adult males; in breeding males, nuptial pads with black nuptial spines on the dorsal bases of the first and second fingers.

Description of holotype.

CIB SC20240531005 (Figs 4, 5). SVL 44.4 mm; head width greater than head length (HDW/HDL ratio about 1.2); snout obtusely pointed, protruding well beyond the margin of the lower jaw in ventral view; loreal region vertical and concave; canthus rostralis well-developed; top of head flat in dorsal view; eye large, eye diameter 40.0% of head length; pupils vertical; nostril orientated laterally, closer to snout than eye; tympanum distinct, TYP/EYE ratio 0.6; vomerine ridges present and vomerine teeth absent; margin of tongue smooth, not notched behind.

Forelimbs slender and comparatively short relative to those of congeneric species; length of lower arm and hand 44.4% of SVL; fingers robust; relative finger lengths: II < IV< I < III; digit tips globular, without lateral fringes; distinct subarticular tubercles at finger bases; two prominent metacarpal tubercles, oval-shaped, the inner larger than the outer.

Hindlimbs slender (HLL/SVL 1.47); heels overlapping when thighs at right angles to body, tibiotarsal articulation reaching mid-eye when leg stretched forward; tibia longer than thigh; relative toe lengths: I < II < V < III < IV; toes round, slightly dilated; subarticular tubercles at toe base; toes with rudimentary webbing, narrow and barely visible lateral fringe; inner metatarsal tubercle oval-shaped; outer metatarsal tubercle absent.

Dorsal skin rough, with numerous brownish-yellow granules; several large warts scattered on flanks; tubercles on the dorsum forming a weak V-shaped ridge; two discontinuous dorsolateral parallel ridges on either side of the V-shaped ridges; tubercles on flanks, dorsal thighs, and tibias; supratympanic fold distinct; white granules with black hard spines on temporal fold region and adjacent areas.

Ventral surface smooth with numerous white granules; pectoral glands indistinct; femoral glands on rear of thighs; inner thighs and shanks with white granules, each tipped with single black hard spine.

Coloration of holotype in life.

(Fig. 4). Inverted triangular brown speckle between eyes; dorsal V-shaped ridges dotted with brown speckles; dorsal surfaces of the thigh and shank characterized by four prominent dark brown transverse bands; upper and lower lips with alternating dark brown and white vertical bars; belly light orange-red; large white blotch and small grey blotch at center, small white blotches and large black patches on sides (from discontinuous line); posterior limb venters orange, thickly covered with numerous granules; palms, soles, and digit tips purple-grey; femoral glands pure white; iris copper-brown; throat and anterior chest light purple-brown.

Coloration of holotype in preservation.

(Fig. 5). Dorsal surface faded to brown; inverted triangular brown speckle between eyes and V-shaped ridges on dorsum indistinct; throat and anterior chest light brownish-tan, abdomen pale orange-red with central white spot, lateral small white speckles interspersed with large black patches; ventral hindlimbs with white tubercles, males with black hardened spines.

Variation.

In CIB SC20240531002 (Fig. 6A): Overall color relatively dark; indistinct transverse stripes between thighs and shanks; black spines on white granules sparse, color not prominent. In CIB SC20240531004 (Fig. 6B): Dorsal parallel ridges in reticular pattern. In CIB SC20240531006 (Fig. 6C), tympanic membrane small, color indistinct; dorsal parallel ridges obscure, with only one ridge discernible. In CIB SC20240531008 (Fig. 6D): dorsum dark brownish; markings abundant and broad; wide transverse stripes on thighs and shanks; dorsal wart granules small, several large ones on flanks.

Figure 6. 

Color variation in Boulenophrys yezhongensis sp. nov. in life. A. Dorsolateral view of adult male CIB SC20240531003; B. Dorsolateral view of adult male CIB SC20240531002; C1. Dorsolateral view of adult female CIB SC20240531006; C2. Ventral view of adult female CIB SC20240531006; D1. Dorsolateral view of adult female CIB SC20240531007; D2. Ventral view of adult female CIB SC20240531007.

Secondary sexual characters.

Adult males (SVL 41.2–46.2 mm) are smaller than adult females (SVL 51.8–58.6 mm). Adult males have a single subgular vocal sac. In breeding males, there are dark black nuptial pads at the bases of the first and second fingers, and these pads are covered with fine, dense black nuptial spines. Moreover, white granules are present on the inner thighs, around the anus, the temporal fold region, and these granules are adorned with black hard spines. In contrast, females possess only white granules.

Comparisons.

(Suppl. material 4) Boulenophrys yezhongensis sp. nov. is phylogenetically closest to B. fanjingmontis, B. qianbeiensis, B. sangzhiensis, and B. spinata (Fig. 7). The new species is easily distinguished from B. fanjingmontis by its slightly smaller body size: in adult males, the SVL is 41.2–46.2 mm (vs. 58.2– 63.6 mm in the latter), and in adult females, the SVL is 51.8–58.6 mm (vs. 62.8–72.2 mm in the latter); tongue not notched posterior (vs. tongue feebly notched behind in the latter); having narrow lateral fringes on toes (vs. wide lateral fringes in the latter); breeding males have dark black nuptial pads at the bases of the first and second fingers, and these pads are covered with fine and dense black nuptial spines (vs. obvious black nuptial spines, spines on nuptial pads are larger and sparser in the latter); the values of morphological characteristics such as SVL, IND, IOD, LW, HLL, THL, TL, TW, TFL, FL, TYD, and IFE are significantly smaller in the new species than those in B. fanjingmontis (Table 3).

Figure 7. 

Comparison of the morphological characteristics of adult male Boulenophrys yezhongensis sp. nov. and its closely related species. A1–A5. Boulenophrys yezhongensis sp. nov.; B1–B5. B. sangzhiensis; C1–C5. B. qianbeiensis; D1–D5. B. spinata; E1–E5. B. fanjingmontis.

The new species differs from B. qianbeiensis by the following characters: having a slightly smaller body size, in adult males SVL 41.2–46.2 mm (vs. 49.3–58.2 mm in B. qianbeiensis), in adult females SVL 51.8–58.6 mm (vs. 65.2 mm in B. qianbeiensis); Breeding males have dark black nuptial pads at the bases of the first and second fingers, and these pads are covered with fine and dense black nuptial spines (vs. obvious black nuptial spines, spines on nuptial pads are larger and sparser in the latter); vomerine ridges separated and weak, vomerine teeth absent (vs. vomerine ridges and vomerine teeth present and distinct in the latter); tongue not notched posterior (vs. tongue feebly notched behind in the latter); toes with rudimentary webbing (vs. toes one quarter webbed in the latter); having narrow lateral fringes on toes(vs. wide lateral fringes in the latter); tibiotarsal articulation reaching the middle of eye when leg stretched forward (vs. reaching the level between tympanum and eye in the latter); the values of morphological characteristics such as SVL, HDW, NED, NSD, IND, IOD, UEW, LW, HLL, THL, TL, TW, TFL and FL are significant smaller in the new species than those in B. qianbeiensis (Table 3).

The new species differs from B. sangzhiensis by the following characters: having a slightly smaller body size, in adult males SVL 41.2–46.2 mm (vs. 55.7–62.2 mm in the latter), in adult females SVL 51.8–58.6 mm (vs. 62.2–76.3 mm in the latter); breeding males have dark black nuptial pads at the bases of the first and second fingers, and these pads are covered with fine and dense black nuptial spines (vs. obvious black nuptial spines, spines on nuptial pads are larger and sparser in the latter); tongue not notched posterior (vs. tongue feebly notched behind in the latter);the values of morphological characteristics such as SVL, HDW, NSD, IND, IOD, LW, HLL, THL, TL, TW, TFL, FL, TYD and IFE are significant smaller in the new species than those in B. sangzhiensis (Table 3).

The new species differs from B. spinata by the following characters: having a slightly smaller body size, in adult males SVL 41.2–46.2 mm (vs. 47.2–54.4 mm in the latter), in adult females the SVL is not significant different; tongue not notched posterior (vs. tongue feebly notched behind in the latter); toes with rudimentary webbing (vs. toes one quarter webbed in the latter); having narrow lateral fringes on toes(vs. wide lateral fringes in the latter); breeding males have dark black nuptial pads at the base of the first and second fingers, and these pads are covered with fine and dense black nuptial spines (vs. B. spinata has obvious black nuptial spines, and the spines on its nuptial pads are larger and sparser); the values of morphological characteristics such as SVL, TYD, IAE, NED, NSD, IND, IOD, LW, HLL, THL, TL, TW, TFL and FL are significant smaller in the new species than those in B. spinata (Table 3).

By having medium body size, Boulenophrys yezhongensis sp. nov. differs from B. acuta, B. angka, B. baishanzuensis, B. binchuanensis, B. boettgeri, B. cheni, B. congjiangensis, B. daiyunensis, B. daoji, B. daweimontis, B. elongata, B. frigida, B. gaolanensis, B. hungtai, B. jinggangensis, B. jiulianensis, B. kuatunensis, B. lichun, B. lishuiensis, B. lushuiensis, B. mufumontana, B. nankunensis, B. obesa, B. ombrophila, B. pepe, B. puningensis, B. rubrimera, B. sanmingensis, B. shimentaina, B. shunhuangensis, B. tongboensis, B. wugongensis, B. wuliangshanensis, B. wushanensis, B. xianjuensis, B. yangmingensis and B. yingdeensis (minimum SVL > 41.0 mm in the new species vs. maximum SVL < 37.0 mm in the latter), and differs from B. caudoprocta, B. fanjingmontis, B. jingdongensis, B. liboensis, B. mirabilis, B. omeimontis, B. qianbeiensis, B. sangzhiensis and B. shuichengensis (maximum SVL < 46.0 mm in the new species vs. minimum SVL > 50.0 mm in the latter).

By vomerine teeth being absent, Boulenophrys yezhongensis sp. nov. differs from B. brachykolos, B. caudoprocta, B. daiyunensis, B. daweimontis, B. dongguanensis, B. elongata, B. fansipanensis, B. fengshunensis, B. frigida, B. hoanglienensis, B. insularis, B. jingdongensis, B. jinggangensis, B. jiulianensis, B. liboensis, B. nankunensis, B. nanlingensis, B. omeimontis, B. palpebralespinosa, B. puningensis, B. qianbeiensis, B. rubrimera, B. shimentaina, B. tongboensis, and B. yingdeensis (vs. present in the latter).

By the vomerine ridges present, Boulenophrys yezhongensis sp. nov. differs from B. baolongensis, B. binchuanensis, B. boettgeri, B. chishuiensis, B. congjiangensis, B. daoji, B. dupanglingensis, B. gaolanensis, B. hengshanensis, B. hungtai, B. leishanensis, B. lichun, B. lishuiensis, B. minor, B. mirabilis, B. ombrophila, B. sanmingensis, B. shunhuangensis, B. tuberogranulatus, B. wuliangshanensis, B. wushanensis, and B. xuefengmontis (vs. absent in the latter).

By a small horn-like tubercle present at the edge of each upper eyelid, Boulenophrys yezhongensis sp. nov. differs from B. acuta, B. caudoprocta, B. jinggangensis, B. liboensis, B. mirabilis, and B. palpebralespinosa (vs. having a prominent and elongated tubercle in the latter).

By tongue not notched posteriorly, Boulenophrys yezhongensis sp. nov. differs from B. baolongensis, B. binlingensis, B. boettgeri, B. cheni, B. fanjingmontis, B. insularis, B. jingdongensis, B. jiulianensis, B. kuatunensis, B. liboensis, B. lushuiensis, B. minor, B. nanlingensis, B. omeimontis, B. pepe, B. qianbeiensis, B. sangzhiensis, B. sanmingensis, B. shuichengensis, B. spinata, and B. tongboensis (vs. tongue feebly notched behind in the latter).

By toes with narrow lateral fringes, Boulenophrys yezhongensis sp. nov. differs from B. angka, B. baolongensis, B. brachykolos, B. caobangensis, B. chishuiensis, B. daweimontis, B. dongguanensis, B. dupanglingensis, B. fansipanensis, B. fengshunensis, B. frigida, B. gaolanensis, B. hengshanensis, B. hoanglienensis, B. hungtai, B. insularis, B. jiangi, B. jiulianensis, B. leishanensis, B. lichun, B. lishuiensis, B. major, B. nankunensis, B. obesa, B. ombrophila, B. pepe, B. puningensis, B. shunhuangensis, B. tongboensis, B. tuberogranulata, B. wugongensis, B. wuliangshanensis, B. xuefengmontis, B. yaoshanensis, B. yingdeensis, and B. yunkaiensis (vs. lacking lateral fringes on toes). differs from B. binchuanensis, B. boettgeri, B. cheni, B. fanjingmontis, B. jingdongensis, B. liboensis, B. lini, B. palpebralespinosa, B. qianbeiensis, B. rubrimera, B. sanmingensis, B. shuichengensis, B. spinata, and B. xiangnanensis (vs. having lateral wide fringes on toes in the latter).

By toes with rudimentary webbing, Boulenophrys yezhongensis sp. nov. differs from B. baishanzuensis, B. baolongensis, B. daweimontis, B. elongata, B. fansipanensis, B. frigida, B. gaolanensis, B. hengshanensis, B. hungtai, B. kuatunensis, B. lichun, B. lishuiensis, B. ombrophila, B. pepe, B. rubrimera, B. shunhuangensis, B. tongboensis, B. wuliangshanensis, and B. xuefengmontis (vs. toes lacking webbing in the latter) and differs from B. jingdongensis, B. palpebralespinosa, B. qianbeiensis, B. shuichengensis, B. spinata, and B. wushanensis (vs. toes with one-fourth in the latter).

By heels overlapping when thighs are positioned at right angles to the body, Boulenophrys yezhongensis sp. nov. differs from B. acuta, B. brachykolos, B. daoji, B. dongguanensis, B. fengshunensis, B. gaolanensis, B. hengshanensis, B. hungtai, B. insularis, B. kuatunensis, B. lichun, B. nankunensis, B. obesa, B. ombrophila, B. pepe, B. puningensis, B. shuichengensis, and B. wugongensis (vs. not meeting in the latter). differs from B. binchuanensis, B. elongata, B. minor, B. xiangnanensis, and B. xuefengmontis (vs. just meeting in the latter).

By tibiotarsal articulation reaching to the level to the middle of eye when leg stretched forward, Boulenophrys yezhongensis sp. nov. differs from B. cheni, B. congjiangensis, B. daweimontis, B. lini, B. obesa, B. shunhuangensis and B. yangmingensis (vs. reaching the anterior corner of the eye or beyond eye or nostril and tip of snout in the latter); and differs from B. brachykolos, B. chishuiensis, B. daoji, B. dongguanensis, B. fengshunensis, B. hungtai, B. insularis, B. jiangi, B. kuatunensis, B. lishuiensis, B. mufumontana, B. nankunensis, B. puningensis, B. qianbeiensis, B. shimentaina, B. xiangnanensis, B. xuefengmontis, B. yingdeensis and B. yunkaiensis (vs. reaching the level between tympanum and eye or reach the tympanum or be even closer in the latter).

By having an internal single subgular vocal sac in males, Boulenophrys yezhongensis sp. nov. differs from B. caudoprocta and B. shuichengensis (vs. vocal sac absent in the latter).

Distribution and habitats.

Boulenophrys yezhongensis sp. nov. is known from the type locality, Yezhong Nature Reserve, Shuicheng County, Guizhou Province, China, at elevations of approximately 2022 m. The individuals of the new species were frequently found on stones in the streams or gorges surrounded by evergreen broadleaved forest (Fig. 8), and two sympatric amphibian species, i.e., Rana chaochiaoensis and Quasipaa boulengeri, were found.

Figure 8. 

Habitats of Boulenophrys yezhongensis sp. nov. in the type locality, Yezhong Nature Reserve, Shuicheng County, Guizhou Province, China. A. Landscape of montane forests in the type locality; B. A mountain stream where Boulenophrys yezhongensis sp. nov. were encountered.

Etymology.

The specific epithet yezhongensis refers to the type locality of the species. We propose the common English name “Yezhong Horned Toad” and the Chinese name “Yě Zhōng Jiǎo Chán (野钟角蟾)” for this species.

Discussion

Speciation in amphibians often occurs through a combination of spatial isolation and ecological specialization, particularly in montane regions where topographic complexity creates micro-refugia (Katharina et al. 2019; Schilthuizen 2000). Our integrative study demonstrates that Boulenophrys yezhongensis sp. nov. meets the criteria for species delimitation, despite its low 16S genetic divergence (0.6%) from B. binlingensis. While such minimal mitochondrial divergence might raise questions about species status, recent studies have shown that comparable or even lower interspecific genetic divergences have been recorded among anuran species with significant morphological and ecological differences in the genus Zhangixalus (e.g., the 16S genetic divergence between Z. hui and Z. dugritei is 0.58%; Pan et al. 2025). The congruence between discrete morphological characters (e.g., body size, webbing reduction, nuptial spine distribution) and bioclimatic niche partitioning strongly supports the recognition of this lineage as a distinct evolutionary unit under the framework of integrative taxonomy (Cheng et al. 2025).

The spatial isolation driving this speciation event is mediated by multiple geographic barriers. The new species is restricted to a single peak (2,022 m) within the Wumeng Mountain Range, separated from the nearest congeneric populations (B. qianbeiensis and B. fanjingmontis, at 1,400–1,800 m) by 12 km of intervening low-elevation valleys (<1,000 m) crossed by the deeply incised Beipan River (Fig. 1). This phylogeographic pattern mirrors speciation mechanisms observed in other East Asian montane frogs, where altitudinal stratification and riverine barriers synergistically prevent gene flow (Li et al. 2015). The sharp elevational transition (>800 m vertical difference) between populations creates a thermal discontinuity equivalent to a 4–5 °C mean annual temperature differential – a critical threshold for amphibian physiological isolation (Wang et al. 2021; Jin et al. 2022). Furthermore, the karstic topography of the Yezhong region features extensive exposed limestone formations between populations, creating an ecological filter that could limit dispersal for these stream-dependent amphibians, as in other karst-endemic species (Wen et al. 2022).

This This microendemic species’ confinement to a <0.5 km² stream system highlights the vulnerability of montane amphibians to environmental change. Its reproductive strategy – breeding in perennial cascades with stable low temperatures (14–16 °C) – suggests specialized adaptation to cold stenothermy, a trait increasingly recognized as both a speciation driver and extinction risk factor in tropical montane ecosystems (Kissel et al. 2018). Based on current surveys, B. yezhongensis sp. nov. is restricted to a limited area within the Yezhong Nature Reserve, with no populations detected in adjacent low-elevation regions. Following the IUCN Red List criteria (IUCN 2012) and considering its extremely small extent of occurrence (<20 km²) and single known location, we recommend classifying this species as Vulnerable (VU).

Institutional review board statement

All animal protocols in this study were approved by the Management Office of Yezhong Nature Reserve, Guizhou Province (project number: P52230020243100013). The Animal Care and Use Committee of Guizhou University provided full approval for the research protocol (approval number: EAE-GZU-20244-T1228).

Acknowledgments

This work was supported by the West Light Foundation of the Chinese Academy of Sciences (Grant No. 2021XBZG_XBQNXZ_A_006), the National Natural Science Foundation of China (Nos. 32270498, 31960099, 32260136, and 32070426), Guizhou Provincial Science and Technology Projects (Nos. ZK[2022]540 and [2023]099), and high-level personnel research start-up funding projects of the Moutai Institute (Nos. mygccrc[2022]055, mygccrc[2022]067, and mygccrc[2022]083).

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1Jing Liu and Chao-Bo Feng have contributed equally to this work.

Supplementary materials

Supplementary material 1 

Morphometric data used for morphological characteristic analysis

Jing Liu, Chao-Bo Feng,Tuo Shen, Shi-Ze Li, Yanlin Cheng, Gang Wei, Bin Wang, Haijun Su

Data type: xlsx

Explanation note: Measurements of the adult specimens of Boulenophrys yezhongensis sp. nov. Units are in mm. See abbreviations for the morphological 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 (14.69 kb)
Supplementary material 2 

Genetic distance of 16S sequences

Jing Liu, Chao-Bo Feng,Tuo Shen, Shi-Ze Li, Yanlin Cheng, Gang Wei, Bin Wang, Haijun Su

Data type: xls

Explanation note: Uncorrected p-distances between the Boulenophrys species based on the 16S gene sequences.

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 (70.50 kb)
Supplementary material 3 

Genetic distance of COI sequences

Jing Liu, Chao-Bo Feng,Tuo Shen, Shi-Ze Li, Yanlin Cheng, Gang Wei, Bin Wang, Haijun Su

Data type: xls

Explanation note: Uncorrected p-distances between the Boulenophrys species based on the COⅠ gene sequences.

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 (66.00 kb)
Supplementary material 4 

Diagnostic characters separating Boulenophrys yezhongensis sp. nov. from other species of Boulenophrys

Jing Liu, Chao-Bo Feng,Tuo Shen, Shi-Ze Li, Yanlin Cheng, Gang Wei, Bin Wang, Haijun Su

Data type: xlsx

Explanation note: Morphological characteristic differences in Boulenophrys are used for identification.

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 (25.31 kb)
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