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Research Article
Integrative taxonomic evidence for a new species of the short-legged toads Brachytarsophrys (Anura, Megophryidae) from Guangxi, China
expand article infoXiangjian Wu‡, Yuanqiang Pan‡, Wei Xiao‡, Ju Chen‡, Guohua Yu‡, Xinkui Wei‡
‡ Guangxi Normal University, Guilin, China

Corresponding author: Guohua Yu ( yugh2018@126.com )

Corresponding author: Xinkui Wei ( wxk1282025@163.com )

Academic editor: Umilaela Arifin
© 2025 Xiangjian Wu, Yuanqiang Pan, Wei Xiao, Ju Chen, Guohua Yu, Xinkui Wei.
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: Wu X, Pan Y, Xiao W, Chen J, Yu G, Wei X (2025) Integrative taxonomic evidence for a new species of the short-legged toads Brachytarsophrys (Anura, Megophryidae) from Guangxi, China. Zoosystematics and Evolution 101(4): 1369-1382. https://doi.org/10.3897/zse.101.157834
ZooBank: urn:lsid:zoobank.org:pub:68884383-5514-40E5-BBCC-A6DF9BE93BDE
Open Access

Abstract

Currently, the genus Brachytarsophrys contains nine species. In this study, we describe a new species of Brachytarsophrys, named Brachytarsophrys guilinensis sp. nov., from northeastern Guangxi, China, based on morphological, molecular, and acoustic evidence. This new species can be distinguished from other known congeners by the combination of the following morphological characters: (1) smaller body size (SVL 70.0‒81.9 mm in eight adult males); (2) head wider than long, with head width nearly 1.5 times the length and about half of the SVL; (3) tongue broad and round, feebly notched; (4) male with nuptial pad on the dorsal surface of the first and second fingers; (5) hind limbs relatively short and robust, heels not meeting when legs are positioned at a right angle to the body; (6) tibiotarsal articulation reaching the angle of the mouth when hind limbs are extended forward alongside the body; (7) absence of outer metatarsal tubercle, with the inner metatarsal tubercle oval and approximately equal to the first toe; (8) fingers without webbing; toes with incomplete webbing, webbing formula: I1 - 2II1 - 2III1½ - 3-IV3- - 1½V; (9) lateral fringes on toes wide; (10) several warts on the outer side of the upper eyelid, with one being relatively larger. Phylogenetic analyses based on mitochondrial 16S rRNA, COI, and Cytb genes revealed that this new species is more closely related to B. popei and B. orientalis than to other known species, with strong Bayesian posterior probability and moderate bootstrap support. More studies are necessary to clarify the taxonomy and species diversity of the genus Brachytarsophrys in Guangxi, China.

Key Words

Acoustic, phylogenetic analysis, species diversity, taxonomy

Introduction

The genus Brachytarsophrys Tian & Hu, 1983, commonly known as short-legged toads, is characterized by large body size; thickset and stout habitus; head enormous and extremely depressed, with head width approximately twice the skull length; presence of a transverse groove defining the head behind; tympanum hidden; maxillary teeth present; pupil vertical; upper eyelid bearing several conical tubercles, one elongated, forming a conical or flattened horn; hindlimbs short and strongly thickset, heels not meeting and separated by a greater distance; toes with webbing and fringes (Li et al. 2020). The genus is widely distributed in southern China, Myanmar, Vietnam, Laos, and northern Thailand, and currently comprises nine species (Frost 2025). In China, seven species have been recorded: B. chuannanensis Fei, Ye & Huang, 2001; B. feae (Boulenger, 1887); B. orientalis Li, Lyu, Wang & Wang, 2020; B. platyparietus Rao & Yang, 1997; B. popei Zhao, Yang, Chen, Chen & Wang, 2014; B. qiannanensis Li, Liu, Yang, Wei & Su, 2022; and B. wenshanensis He, Huang, Li, Chen & Yuan, 2024 (AmphibiaChina 2025). Of these seven species, only B. platyparietus is recognized in Guangxi (Li et al. 2020; AmphibiaChina 2025).

During field surveys conducted in September 2024 in Guangxi, China, we collected eight specimens of Brachytarsophrys. Both morphological and phylogenetic analyses confirmed that these specimens represent an undescribed species of Brachytarsophrys, suggesting that the species diversity within the genus Brachytarsophrys has been underestimated.

Materials and methods

Sampling

This study was carried out in accordance with the ethical guidelines issued by the Ethics Committee of Guangxi Normal University. A total of eight specimens of the genus Brachytarsophrys were collected in September 2024 from Wantian Township, Lingui District, Guilin City, Guangxi Zhuang Autonomous Region, China (Fig. 1). All specimens were photographed, euthanized, fixed, and stored in 75% ethanol. Liver tissues were preserved in 99% ethanol. All specimens were deposited at Guangxi Normal University (GXNU).

Figure 1. 

Map showing the distribution of the Brachytarsophrys species in Guangxi. The red star indicates the type locality of Brachytarsophrys guilinensis sp. nov. The “?” in the legend indicates that the species identity is yet to be confirmed. 1: Longlin; 2: Jingxi; 3: Shangsi; 4: Wuming; 5: Yulin; 6: Jinxiu; 7: Hezhou; 8: Huanjiang; 9: Luocheng; 10: Rongshui; 11: Longsheng; 12: Ziyuan; 13: Xing’an (Fei et al. 2009, 2012; Mo et al. 2014). The map was produced using ArcMap v.10.8.1.

Morphological analysis

Morphometric data were taken using electronic digital calipers to the nearest 0.1 mm. Morphological terminology followed Fei (1999). Measurements included: snout–vent length (SVL, from tip of snout to vent); head length (HL, from tip of snout to rear of jaws); head width (HW, width of head at its widest point); snout length (SL, from tip of snout to anterior corner of eye); internarial distance (IND, distance between nares); interorbital distance (IOD, minimum distance between upper eyelids); eye diameter (ED, diameter of exposed portion of eyeball); hand length (HAL, from outer palm to the end of third finger); forearm length (FOL, from proximal end of outer palm protrusion to elbow joint); tibia length (TL, distance from knee to heel); and foot length (FL, from proximal end of inner metatarsal tubercle to tip of fourth toe). The webbing formula followed Myers and Duellman (1982).

Comparative morphological data of known Brachytarsophrys species were taken from their original and subsequent descriptions (Boulenger 1887, 1889; Smith 1921; Taylor 1962; Rao and Yang 1997; Fei and Ye 2001; Fei et al. 2009; Zhao et al. 2014; Li et al. 2020, 2022; He et al. 2024).

Multivariate principal component analysis (PCA) was conducted in SPSS v.25.0 (SPSS Inc.) based on the correlation matrix of size-standardized measurements (all measurements divided by SVL) of adult males. Scatter plots of the scores of the first two principal components were used to examine differentiation between the new species and its closest relatives as revealed by phylogenetic analyses (Brachytarsophrys popei only; data for B. orientalis were not available). Differences in quantitative characters of adult males between these two species were also evaluated with t-tests in SPSS. For each character, Levene’s test was performed to assess equality of variances.

Acoustic analysis

Acoustic data were recorded in the field using a Sony PCM-A10 recorder. Ambient temperature and humidity were measured using a digital hygrothermograph (DELIXI). Recordings were resampled at 44.1 kHz and 16-bit resolution and analyzed using Adobe Audition (Adobe Inc.). The software Praat v.6.2.14 (Boersma 2001) was used to obtain waveforms and sonograms. Terminology and description of advertisement call analyses followed Köhler et al. (2017).

Phylogenetic analysis

Total genomic DNA was extracted from liver tissue preserved in 99% ethanol. Three mitochondrial genes – 16S ribosomal RNA (16S rRNA), cytochrome oxidase subunit I (COI), and cytochrome b (Cytb) – were amplified and sequenced. For 16S rRNA, primers L2188 and 16H1 were used following Matsui and Panha (2006) and Hedges (1994), respectively; for COI, primers Chmf4 and Chmr4 were used following Che et al. (2012); and for Cytb, primers PFGlu14140L and PFThr15310 were used following Zhang et al. (2013). Gene fragments were amplified in a 25 μL volume with the following PCR conditions: initial denaturation at 95 °C for 4 min; 35 cycles of denaturation at 95 °C for 30 s, annealing at 49 °C–54 °C (54 °C for 16S rRNA, 49 °C for COI, and 50 °C for Cytb) for 40 s, and extension at 72 °C for 70 s. PCR products were sequenced using an ABI PRISM 3730 automatic sequencer. New sequences have been deposited in GenBank (Table 1). A total of 105 homologous sequences from all known Brachytarsophrys species, as well as an outgroup, were downloaded from GenBank (Table 1). Atympanophrys shapingensis (Liu, 1950) was included as the outgroup.

Table 1.
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Species used for phylogenetic analyses in this study (B. = Brachytarsophrys).

Species Locality Voucher No. 16S COI Cytb References
B. guilinensis sp. nov. Guilin, Guangxi, China GXNU YU000903 PV819239 PV817819 PV820718 This study
B. guilinensis sp. nov. Guilin, Guangxi, China GXNU YU000904 PV819240 PV817820 PV820719 This study
B. guilinensis sp. nov. Guilin, Guangxi, China GXNU YU000905 PV819241 PV817821 PV820720 This study
B. guilinensis sp. nov. Guilin, Guangxi, China GXNU YU000906 PV819242 PV817822 PV820721 This study
B. guilinensis sp. nov. Guilin, Guangxi, China GXNU YU000907 PV819243 PV817823 PV820722 This study
B. guilinensis sp. nov. Guilin, Guangxi, China GXNU YU000908 PV819244 PV817824 PV820723 This study
B. guilinensis sp. nov. Guilin, Guangxi, China GXNU YU000909 PV819245 PV817825 PV820724 This study
B. guilinensis sp. nov. Guilin, Guangxi, China GXNU YU000910 PV819246 PV817826 PV820725 This study
B. orientalis Longnan County, Jiangxi, China SYS a004225 OQ180989 MT162625 MT162650 Li et al. (2020); Lyu et al. (2023)
B. orientalis Longnan County, Jiangxi, China SYS a004226 – MT162626 MT162651 Li et al. (2020)
B. orientalis Longnan County, Jiangxi, China SYS a004227 – MT162627 MT162652 Li et al. (2020)
B. orientalis Longnan County, Jiangxi, China SYS a004228 – MT162628 MT162653 Li et al. (2020)
B. orientalis Longnan County, Jiangxi, China SYS a004486 – MT162629 MT162654 Li et al. (2020)
B. orientalis Longnan County, Jiangxi, China SYS a005451 – MT162632 MT162655 Li et al. (2020)
B. orientalis Shanghang County, Fujian, China SYS a003249 – MT162623 MT162648 Li et al. (2020)
B. orientalis Nanjing County, Fujian, China SYS a003340 – MT162624 MT162649 Li et al. (2020)
B. carinense Doi Chiang Dao, Chiang Mai, Thailand K3001 KR827713 KR087626 – Grosjean et al. (2015)
B. carinense Omkoi, Chiang Mai, Thailand KIZ 024170 – MT162640 MT162663 Li et al. (2020)
B. carinense Mae Surin NP., Mae Hong Son, Thailand KIZ 024429 – MT162641 MT162664 Li et al. (2020)
B. carinense Thong Pha Phum, Kanchanaburi, Thailand KIZ 024640 – MT162642 MT162665 Li et al. (2020)
B. chuannanensis Hejiang County, Sichuan, China SYS a004926 MH406901 MH406364 MH407194 Liu et al. (2018)
B. chuannanensis Hejiang County, Sichuan, China SYS a004927 MH406902 MH406365 MH407195 Liu et al. (2018)
B. feae Jingdong County, Yunnan, China SYS a003912 MH406899 MH406362 MH407192 Liu et al. (2018)
B. feae Jingdong County, Yunnan, China SYS a003913 MH406900 MH406363 MH407193 Liu et al. (2018)
B. feae Huangcaoling, Yunnan, China KIZ 046706 KX811810 KX812056 – Chen et al. (2017)
B. intermedia Krong Pa, Gia Lai, Vietnam ROM 23794 – MT162643 MT162666 Li et al. (2020)
B. platyparietus Dayao county, Yunnan, China SYS a005919 OQ180990 MT162633 MT162656 Li et al. (2020); Lyu et al. (2023)
B. platyparietus Longlin County, Guangxi, China SYS a002236 – MT162622 MT162647 Li et al. (2020)
B. platyparietus Tongren City, Guizhou, China YPX43968 – MT162644 MT162667 Li et al. (2020)
B. platyparietus Xinping County, Yunnan, China SYS a007774 – MT162634 MT162657 Li et al. (2020)
B. platyparietus Xinping County, Yunnan, China SYS a007775 – MT162635 MT162658 Li et al. (2020)
B. platyparietus Xinping County, Yunnan, China SYS a007776 – MT162636 MT162659 Li et al. (2020)
B. platyparietus Xinping County, Yunnan, China SYS a007777 – MT162637 MT162660 Li et al. (2020)
B. platyparietus Shiping County, Yunnan, China SYS a007790 – MT162638 MT162661 Li et al. (2020)
B. platyparietus Yanbian County, Sichuan, China SYS a007853 – MT162639 MT162662 Li et al. (2020)
B. popei Yanling County, Hunan, China SYS a001864 KM504256 MH406361 MH407191 Zhao et al. (2014); Liu et al. (2018)
B. popei Yanling County, Hunan, China SYS a001865 KM504257 MT162620 MT162645 Zhao et al. (2014); Li et al. (2020)
B. popei Yanling County, Hunan, China SYS a001866 KM504258 MT162621 MT162646 Zhao et al. (2014); Li et al. (2020)
B. popei Mt.Jinggang, Jiangxi, China SYS a001876 KM504253 – – Zhao et al. (2014)
B. popei Mt.Jinggang, Jiangxi, China SYS a001877 KM504254 – – Zhao et al. (2014)
B. popei Mt.Jinggang, Jiangxi, China SYS a001878 KM504255 – – Zhao et al. (2014)
B. popei Mt.Jinggang, Jiangxi, China SYS a004209 MK524124 MK524155 – Wang et al. (2019)
B. popei Mt.Jinggang, Jiangxi, China SYS a001485-1 KM504252 – – Zhao et al. (2014)
B. popei Tiantaishan, Mangshan, Hunan, China CSUFT T10115 ON209276 – – –
B. popei Tiantaishan, Mangshan, Hunan, China CSUFT T10117 ON209284 – – –
B. popei Nanling Nature Reserve, Guangdong, China SYS a000589 KM504251 – – Zhao et al. (2014)
B. popei Nanling Nature Reserve, Guangdong, China YTP34994 PV819247 PV819270 PV820726 This study
B. popei Nanling Nature Reserve, Guangdong, China YTP34995 PV819248 PV819271 PV820727 This study
B. popei Nanling Nature Reserve, Guangdong, China YTP34996 PV819249 PV819272 PV820728 This study
B. qiannanensis Libo County, Guizhou, China CIB LB20210806053 OK104099 OK104052 OK127913 Li et al. (2022)
B. qiannanensis Libo County, Guizhou, China CIB LB20210806054 OK104100 OK104053 OK127914 Li et al. (2022)
B. qiannanensis Libo County, Guizhou, China CIB LB20210806055 OK104101 OK104054 OK127915 Li et al. (2022)
B. qiannanensis LiboCounty, Guizhou, China CIB LB20210806056 OK104102 OK104055 OK127916 Li et al. (2022)
B. wenshanensis Xichou, Yunnan, China SWU 0004072 PP153337 PP155195 PP156671 He et al. (2024)
B. wenshanensis Xichou, Yunnan, China SWU 0002976 PP153338 PP155198 PP156670 He et al. (2024)
B. wenshanensis Xichou, Yunnan, China Yuan 25290 PP153339 PP155197 PP156669 He et al. (2024)
B. wenshanensis Xichou, Yunnan, China Yuan 25291 PP153336 PP155196 PP156668 He et al. (2024)
Atympanophrys shapingensis Hongya County, Sichuan, China SYS a005310 MH406890 MH406352 MH407182 Liu et al. (2018)

–: Not available.

Sequences were aligned using MUSCLE in MEGA v.7.0 (Kumar et al. 2016) with default parameters. Uncorrected pairwise distances between Brachytarsophrys species were calculated for each locus in MEGA v.7.0. The best nucleotide substitution model for the combined dataset was selected using the Akaike Information Criterion (AIC) in PartitionFinder v.2.1.1 (Robert et al. 2017), with GTR+G+I identified as the best-fit model for all three genes. Bayesian inference was performed using the combined data in MrBayes v.3.2.6 (Ronquist et al. 2012) under the selected model. Two runs were performed, each with four Markov chains starting from a random tree. Chains were run for 5,000,000 generations and sampled every 1,000 generations. The first 25% of sampled trees were discarded as burn-in after the standard deviation of split frequencies between runs dropped below 0.01. The remaining trees were used to generate a consensus tree and to estimate Bayesian posterior probabilities (BPPs). Maximum likelihood phylogenetic analysis was conducted in IQ-TREE v.2.2.0 (Nguyen et al. 2015), with nodal support estimated using 5,000 ultrafast bootstrap replicates. Species delimitation analysis was performed using the p-distance model based on COI sequences on the ASAP webserver (https://bioinfo.mnhn.fr/abi/public/asap/) (Puillandre et al. 2021).

Results

Morphological study

Morphological data were summarized in Table 2. Two measurements (UEW and FHL) were not included in PCA because they were not obtainable for Brachytarsophrys popei. The t-tests revealed that male specimens from Guilin significantly (p < 0.05) differed from male specimens of B. popei from Jiangxi, Guangdong, and Yanling, Hunan, in HL, HW, SL, IOD, ED, FOL, and FL (Table 3). We retained the first two principal components that accounted for 71.794% of the total variance (Table 4). Loadings for PC1, which account for 51.580% of the total variance, were most heavily loaded on HL, HW, SL, ED, FOL, and FL. Loadings for PC2, which account for 20.214% of the total variance, were most heavily loaded on IND and TL. Differentiation was found along the PC1 axis between the specimens from Guilin and B. popei (Fig. 2). This result indicates that specimens from Guilin differ from B. popei in several characteristics, such as shorter and narrower head, smaller eye diameter, shorter snout length, forearm length, and foot length. Along the PC2 axis, no differentiation was observed between the specimens from Guilin and B. popei.

Table 2.
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Measurements (mm) of Brachytarsophrys guilinensis sp. nov. (1‒8) and B. popei (9‒21). Data of B. popei were obtained from Zhao et al. (2014).

ID Voucher No. Sex SVL HL HW SL IND IOD ED HAL FOL TL FL
1 GXNU YU000903 M 77.3 25.3 36.9 8.7 8.4 11.1 8.2 19.7 15.4 32.0 34.1
2 GXNU YU000904 M 74.6 22.6 33.3 8.4 7.4 11.0 7.1 18.7 14.3 28.0 30.4
3 GXNU YU000905 M 76.2 26.2 39.0 8.6 8.2 10.9 8.3 19.9 15.7 31.9 32.7
4 GXNU YU000906 M 76.4 25.7 37.9 8.9 7.7 11.7 8.4 19.4 15.6 31.7 32.4
5 GXNU YU000907 M 73.7 22.7 33.8 8.4 7.3 10.7 7.1 19.1 14.2 28.5 30.5
6 GXNU YU000908 M 71.0 21.6 33.5 8.4 7.4 10.4 6.6 19.1 13.9 28.7 30.6
7 GXNU YU000909 M 70.0 21.5 31.8 8.1 7.3 10.3 6.6 18.9 13.7 27.9 30.6
8 GXNU YU000910 M 81.9 27.8 40.4 9.8 8.3 11.4 8.6 21.4 16.5 32.4 35.4
9 SYS a001874 M 79.8 33.2 40.1 10.3 7.4 12.0 11.1 20.8 17.5 32.2 47.4
10 SYS a001876 M 75.1 32.3 38.6 9.4 7.8 11.2 9.6 19.3 17.4 32.2 46.3
11 SYS a001877 M 83.5 33.4 39.9 10.0 8.4 14.0 10.6 20.6 17.0 32.4 48.4
12 SYS a001878 M 75.8 32.9 39.5 9.9 7.9 11.6 9.2 20.6 15.6 30.4 45.3
13 SYS a001864 M 78.4 32.7 40.8 10.2 8.0 11.9 9.9 21.6 17.1 32.2 49.5
14 SYS a001865 M 78.4 31.0 37.2 9.7 7.9 11.2 8.9 19.4 17.0 30.4 45.0
15 SYS a001866 M 79.5 34.2 41.6 9.6 8.7 13.4 10.0 20.5 17.5 33.5 49.7
16 SYS a001867 M 72.9 30.8 36.3 8.9 7.5 12.2 7.8 19.1 16.1 28.9 43.9
17 SYS a000583 M 70.7 29.8 36.9 8.5 7.0 11.1 7.9 18.6 15.7 28.0 42.0
18 SYS a000584 M 72.6 30.6 38.7 9.4 7.7 12.3 8.0 18.8 15.9 29.4 45.8
19 SYS a000585 M 79.3 33.2 40.0 10.2 7.7 11.8 9.7 20.2 17.9 30.1 46.6
20 SYS a000588 M 76.5 32.3 40.0 9.8 7.6 11.7 8.4 19.3 18.0 30.8 46.2
21 SYS a000589 M 76.8 32.5 38.4 9.2 7.4 11.1 9.3 18.8 16.8 29.4 43.8
Table 3.
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Summary statistics of male specimens (mean ± standard deviation) and results of the t-test between Brachytarsophrys guilinensis sp. nov. (n = 8) and B. popei (n = 13). The t-test was performed on the size-adjusted data, except for SVL. * = p < 0.05.

Character Mean ± SD (n = 8) Mean ± SD (n = 13) Levene’s test t-test
B. guilinensis sp. nov. B. popei F p-value t p-value
SVL 75.1 ± 3.7 76.9 ± 3.5 0.010 0.920 -1.069 0.299
HL 0.321 ± 0.0175 0.419 ± 0.0110 7.039 0.016 -14.276 0.000*
HW 0.476 ± 0.0232 0.509 ± 0.0180 0.720 0.407 -3.614 0.002*
SL 0.115 ± 0.0025 0.125 ± 0.0043 7.102 0.015 -6.658 0.000*
IND 0.103 ± 0.0038 0.101 ± 0.0044 0.023 0.881 1.112 0.280
IOD 0.146 ± 0.0041 0.156 ± 0.0095 7.807 0.012 -3.362 0.004*
ED 0.101 ± 0.0072 0.120 ± 0.0093 0.253 0.621 -4.994 0.000*
HAL 0.260 ± 0.0067 0.258 ± 0.0091 0.561 0.463 0.547 0.591
FOL 0.198 ± 0.0054 0.220 ± 0.0086 0.244 0.627 -6.359 0.000*
TL 0.401 ± 0.0150 0.400 ± 0.0144 0.067 0.798 0.099 0.922
FL 0.427 ± 0.0112 0.601 ± 0.0205 2.986 0.100 -21.874 0.000*
Table 4.
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Factor loadings of first two principal components of 10 size-adjusted male morphometric characteristics of Brachytarsophrys guilinensis sp. nov. and B. popei. Absolute values of loading greater than 0.75 in boldface. Abbreviations are defined in Materials and methods.

Character PC1 PC2
Eigenvalue 5.158 2.021
% variation 51.580% 20.214%
HL 0.973 -0.114
HW 0.853 0.310
SL 0.853 -0.119
IND -0.108 0.875
IOD 0.554 0.192
ED 0.811 -0.146
HAL 0.022 0.619
FOL 0.886 -0.117
TL 0.275 0.819
FL 0.959 -0.084
Figure 2. 

Scatterplot of principal components 1 and 2 of size-adjusted male morphometric data of Brachytarsophrys guilinensis sp. nov. and B. popei.

Phylogenetic relationship

The obtained sequence alignments for 16S rRNA, COI, and Cytb genes resulted in a combined dataset of 2181 bp in length. Phylogenetic analyses revealed that the specimens from Guilin, Guangxi, form a distinct clade with strong support (100% and 100% for BI and ML, respectively), and this clade was closer to Brachytarsophrys popei and B. orientalis than to other known species, with strong Bayesian posterior probability and moderate bootstrap support (100% and 88% for BI and ML, respectively) (Fig. 3). Genetic distances (uncorrected p-distances) between this novel lineage and other Brachytarsophrys species ranged from 2.1% to 8.9% in 16S rRNA sequences (Suppl. material 1), from 3.5% to 15.8% in COI sequences (Suppl. material 2), and from 3.7% to 15.5% in Cytb sequences (Suppl. material 3). The results of species delimitation obtained by the ASAP analysis indicated that the specimens from Guilin represent an operational taxonomic unit within the genus Brachytarsophrys (Fig. 4). In addition, it was revealed that B. popei is composed of two distinct lineages, one containing samples from the type locality (Yanling, eastern Hunan) and Jinggang Mountain, western Jiangxi, and one containing samples from Mangshan (southern Hunan) and adjacent Nanling Nature Reserve, Guangdong (Fig. 3). Furthermore, the analysis of ASAP species delimitation assigned these two lineages to two operational taxonomic units (Fig. 4).

Figure 3. 

Bayesian phylogram of Brachytarsophrys inferred from 16S rRNA, COI, and Cytb genes. Numbers above and below branches are Bayesian posterior probabilities and ML bootstrap values (only values above 50% are shown), respectively.

Figure 4. 

Species delimitation results obtained by ASAP based on COI sequences. Rank 1 represents the best partition, identifying 11 species within the Brachytarsophrys genus.

Based on the combination of morphological and phylogenetic evidence mentioned above, we consider that the individuals from Wantian Township, Lingui District, Guilin City, Guangxi Zhuang Autonomous Region, China, represent a new species of the genus Brachytarsophrys, which we describe herein.

Taxonomic account

Brachytarsophrys guilinensis sp. nov.

https://zoobank.org/8AE13F98-2D19-4B78-A9F8-A4BB1DE906E5
Figs 5, 6, 7, 8, 11

Type material.

Holotype • GXNU YU000904, an adult male (Fig. 5), collected on 6 September 2024 by Guohua Yu, Yuanqiang Pan, and Wei Xiao from Wantian Township, Lingui District, Guilin City, Guangxi Zhuang Autonomous Region, China (25°31'53.54"N, 110°2'43.12"E, 289 m a.s.l; Fig. 1).

Figure 5. 

Dorsolateral view of the holotype of Brachytarsophrys guilinensis sp. nov. (GXNU YU000904) in life (A) and dorsal (B) and ventral (C) views of the holotype of the species in preservative.

Paratypes • GXNU YU000903 and YU000905‒YU000910, seven adult males, collected on 6 September 2024 by Guohua Yu, Yuanqiang Pan, and Wei Xiao from the type locality.

Etymology.

The species name guilinensis is derived from the type locality of this species, Guilin City, Guangxi Zhuang Autonomous Region, China. We suggest the English common name “Guilin Short-legged Toad” and the Chinese common name “Guì Lín Duǎn Tuǐ Chán (桂林短腿蟾)”.

Diagnosis.

Brachytarsophrys guilinensis sp. nov. could be distinguished from its congeners by the combination of the following morphological characters: (1) smaller body size (SVL 70.0‒81.9 mm in eight adult males); (2) head wider than long, head width nearly 1.5 times the length, and about half of the SVL; (3) tongue broad and round, feebly notched; (4) male with nuptial pad present on the dorsal surface of the first and second fingers; (5) hind limbs relatively short and robust, heels do not meet when legs positioned at right angle to body; (6) tibiotarsal articulation reaches the angle of mouth when the hind limbs are extended forward alongside the body; (7) absence of outer metatarsal tubercle, inner metatarsal tubercle oval and approximately equal to first toe; (8) fingers without webbing, toes have webbing but incomplete, webbing formula: I1 - 2II1 - 2III1½ - 3-IV3- - 1½V; (9) lateral fringes on toes wide; (10) several warts on the outer side of the upper eyelid, with one being relatively larger.

Description of holotype.

Adult male; body robust, SVL 74.6 mm; head enormous, extremely wide and flat, about 1.5 times as broad as long, and nearly one-half as broad as the SVL; snout short, rounded in dorsal view, slightly protruding beyond margin of lower jaw in ventral view, sloping in profile; canthus rostralis distinct; loreal region sloping, slightly concave; nostrils oval, much closer to eye than to snout tip; interorbital distance (11.0 mm) significantly wider than the width of upper eyelid, and wider than internasal distance (7.4 mm); occipital region slightly uplifted, forming two oval bulges and a longitudinal concave groove along the middle line across occiput; a distinct transverse groove behind the head; pupil oval, vertical; tympanum hidden; choanae large, circular, partly concealed by the maxillary shelves; vomerine teeth present in two series, touching inner front edges of choanae, separated by distance larger than length of each series; tongue broad and round, feebly notched posteriorly; an internal single subgular vocal sac with a vocal sac opening at each corner of mouth.

Forelimbs short and robust; relative length of fingers I = II < IV < III; tips of fingers round, feebly dilated; no lateral fringes and webbing between fingers; nuptial pad on dorsal surface of the first and second fingers; inner metacarpal tubercle large, oval; outer metacarpal tubercle inconspicuous (Fig. 6A).

Figure 6. 

Ventral view of hand (A) and foot (B) and lateral view (C) of the holotype of Brachytarsophrys guilinensis sp. nov. (GXNU YU000904) in preservative.

Hindlimbs relatively short and robust; heels do not meet when legs positioned at right angle to body; tibiotarsal articulation reaching angle of mouth when hind limbs are extended forward alongside the body; tips of toes round, moderately dilated; relative length of toes I < II < V < III < IV; toes have webbing but incomplete, webbing formula: I1 - 2II1 - 2III1½ - 3-IV3- - 1½V; lateral fringes on toes wide; subarticular tubercle absent; inner metatarsal tubercle distinct, oval, approximately equal to first toe; outer metatarsal tubercle absent (Fig. 6B).

Dorsal and lateral skin rough, covered with some slightly larger black glandular warts and small granules (Fig. 6C); several warts on the outer side of the upper eyelid, with one being relatively larger; ventral skin smooth, with some white granules scattered on abdomen and ventral sides of the limbs; pectoral glands distinct, light yellow, with one on each side near axilla; femoral glands smaller than pectoral glands, white, with one on each side of rear of thigh.

Coloration of holotype in life.

Iris red; dorsal surface yellow-brown, scattered with yellow patches (Fig. 5A); black stripe between eyes, patch to back of stripe black; tympanic region brown; dorsal glandular warts black; limbs dorsal sides with multiple black stripes; ventral surface dark brown, scattered with yellow patches and white granules; ventral sides of limbs with white granules; pectoral gland light yellow; femoral gland white.

Coloration of holotype in preservative.

Dorsal skin dark grey, scattered with grey-white patches; interocular stripe black, patch to back of stripe black; limbs dorsal stripes black (Fig. 5B); ventral patches grey-white; abdomen and limbs ventral granules white; pectoral gland white (Fig. 5C); femoral gland white.

Sexual dimorphism.

Adult males have a nuptial pad on the dorsal surface of the first and second fingers. Additionally, males have an internal single subgular vocal sac with a vocal sac opening at each corner of the mouth.

Variation.

Coloration of three paratypes (GXNU YU000906, GXNU YU000908, and GXNU YU000909) is similar to holotype, paratype GXNU YU000907 has a lighter coloration, three paratypes (GXNU YU000903, GXNU YU000905, and GXNU YU000910) have a brown coloration (Fig. 7). Most paratypes share same characteristic as the holotype, having a broad black stripe between the eyes, whereas GXNU YU000903 and GXNU YU000910 lack this stripe (Fig. 7). The femoral glands are distinct in the holotype and most paratypes, but they are indistinct in GXNU YU000906 (Fig. 8).

Figure 7. 

Variation in dorsal skin coloration of Brachytarsophrys guilinensis sp. nov. in preservative.

Figure 8. 

Difference in the femoral gland between holotype (GXNU YU000904, A) and paratype (GXNU YU000906, B). The femoral gland is indicated by the red circle.

Advertisement call.

The call description is based on recording of an individual calling in a stream, with the ambient air temperature at 20 °C. Each call consisted of 9 to 18 (mean 13.5 ± 6.4; n = 2) notes, with a dominant frequency of 1507‒1550 Hz (mean 1528.5 ± 30.4; n = 2). Call duration was 3856.5‒7895.7 ms (mean 5876.1 ± 2856.2; n = 2). Call interval was 72.7 s. Each note had a duration of 164.9–224.9 ms (mean 199.7 ± 17.2; n = 27), and the intervals between notes were 183.3–439.9 ms (mean 254.4 ± 59.6; n = 25). Distinct amplitude variations within each note showed an overall trend of increasing and then decreasing in intensity (Fig. 9).

Figure 9. 

Visualization of advertisement calls of Brachytarsophrys guilinensis sp. nov. A. Waveform showing one note; B. Sonogram showing one note; C. Waveform showing 9 notes of one call; D. Sonogram showing 9 notes of one call (A and B are the same note, and C and D are the same call).

Distribution and ecology.

The new species is currently only known from Wantian Township, Lingui District, Guilin City, Guangxi Zhuang Autonomous Region, China. The species inhabits a stream within a subtropical evergreen broad-leaved forest. The water in the stream is shallow (Fig. 10). During early September, male individuals were discovered on or beneath rocks (Fig. 11), and advertisement calls were heard, indicating that the new species is in breeding season.

Figure 10. 

Microhabitat of Brachytarsophrys guilinensis sp. nov. at the type locality.

Figure 11. 

Brachytarsophrys guilinensis sp. nov. beneath rocks (Photo by Guohua Yu).

Comparisons.

The new species can be distinguished from Brachytarsophrys popei by the shallow notching at the posterior end of the tongue (vs. deep notching), distinct canthus rostralis (vs. indistinct canthus rostralis), iris red (vs. iris brownish), head width about 1.5 times head length (vs. head width about 1.2 times head length), first finger equal in length to second (vs. first finger longer than second), inner metatarsal tubercle approximately equal to first toe (vs. inner metatarsal tubercle longer than first toe), and webbing formula I1 - 2II1 - 2III1½ - 3-IV3- - 1½V (vs. I1½ - 2II1½ - 3III2½ - 3⅔IV3⅔ - 2V) (Zhao et al. 2014; Li et al. 2020). Furthermore, the PCA analysis suggested that the new species also differs from B. popei by a series of morphometric characters associated with head and limbs, such as shorter and narrower head, smaller eye diameter, shorter snout length, forearm length, and foot length.

The new species can be distinguished from Brachytarsophrys orientalis by distinct canthus rostralis (vs. indistinct canthus rostralis), iris red (vs. iris brownish), head width about 1.5 times head length (vs. head width about 1.2 times head length), first finger equal in length to second (vs. first finger longer than second), and webbing formula I1 - 2II1 - 2III1½ - 3-IV3- - 1½V (vs. I1½ - 2II1½ - 3III2½ - 4IV4 - 2V) (Li et al. 2020).

Brachytarsophrys guilinensis sp. nov. can be distinguished from other known congeners by the following characteristics: smaller body size, SVL of adult males 70‒80 mm (vs. SVL of adult males 80‒90 mm in B. wenshanensis and SVL of adult males > 90 mm in B. carinense, B. intermedia, B. chuannanensis, B. feae, and B. platyparietus) (Boulenger 1889; Smith 1921; Taylor 1962; Rao and Yang 1997; Fei and Ye 2001; Fei et al. 2009; Li et al. 2020, 2022; He et al. 2024); head width about 1.5 times head length (vs. head width about 1.7 times head length in B. qiannanensis and head width about 1.2 times head length in B. platyparietus and B. wenshanensis) (Rao and Yang 1997; Fei et al. 2009; Li et al. 2020, 2022; He et al. 2024); male with nuptial pad on dorsal surface of the first and second fingers (vs. male with nuptial pad on dorsal surface of the first finger in B. qiannanensis, and male lacking nuptial pad in B. wenshanensis) (Li et al. 2022; He et al. 2024); tibiotarsal articulation reaching angle of mouth when hind limbs are extended forward alongside the body (vs. only reaching behind angle of the mouth in B. feae) (Boulenger 1887; Fei et al. 2009; Li et al. 2020); inner metatarsal tubercle approximately equal to first toe (vs. inner metatarsal tubercle longer than first toe in B. feae) (Boulenger 1887; Fei et al. 2009; Li et al. 2020); lateral fringes on toes wide (vs. lateral fringes on toes narrow in B. wenshanensis) (He et al. 2024). Moreover, the new species can be distinguished from B. chuannanensis, B. feae, B. platyparietus, B. qiannanensis, and B. wenshanensis by webbing formula I1 - 2II1 - 2III1½ - 3-IV3- - 1½V (vs. I1½ - 2++II2 - 3++III3 - 4IV4++ - 2½V in B. chuannanensis; I2 - 2++II2- - 3III2⅔ - 4IV4 - 2⅔V in B. feae; I1½ - 2+II1½ - 3III2⅓ - 3⅔IV3⅔ - 2-V in B. platyparietus; I1 - 2II2- - 3-III2½ - 4-IV4 - 2-V in B. qiannanensis; and I2- - 2++II2- - 3++III2½ - 4IV4+ - 2⅔V in B. wenshanensis) (Fei et al. 2009; Li et al. 2020, 2022; He et al. 2024).

Finally, Brachytarsophrys guilinensis sp. nov. differs from B. qiannanensis in dominant frequency, call duration, and call interval (Table 5).

Table 5.
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Acoustic comparisons between Brachytarsophrys guilinensis sp. nov. and B. qiannanensis.

Dominant frequency (Hz) Call durations (ms) Call intervals (s) Temperature (°C) References
B. guilinensis sp. nov. 1507–1550 3856.5–7895.7 72.7 20 This study
B. qiannanensis 1640–2330 7690–11330 10.98–15.67 18 Li et al. 2022

Discussion

Guangxi is a part of the Indo-Burma Biodiversity Hotspot and is second only to Yunnan in amphibian species diversity in China (Tang et al. 2023). Including the new species described from northeastern Guangxi in this study, there are two Brachytarsophrys species recognized from Guangxi, China; the other one is B. platyparietus, which was confirmed from Mt. Jinzhong, Longlin County, in northwestern Guangxi (Li et al. 2020; AmphibiaChina 2025).

Rao and Yang (1997) considered that short-legged toads from Longsheng and Jinxiu counties belong to Brachytarsophrys platyparietus, which was once treated as a synonym of B. carinensis and was recorded from Huanjiang, Luocheng, Hezhou, Rongshui, Wuming, Longsheng, Jinxiu, Yulin, Xing’an, and Ziyuan counties in Guangxi (as B. carinensis; Fei et al. 2009; Mo et al. 2014). However, the taxonomy of Brachytarsophrys from northeastern Guangxi has never been confirmed by molecular study. In this study, we revealed that Brachytarsophrys from Wantian Township, Guilin, northeastern Guangxi, is neither B. carinensis nor B. platyparietus but represents a new species. Therefore, probably other records of Brachytarsophrys from adjacent localities in northeastern Guangxi (Xing’an, Longsheng, and Ziyuan counties; Fei et al. 2009) may also apply to B. guilinensis sp. nov., pending additional data, and the records of B. carinensis or B. platyparietus from Jinxiu in central Guangxi and Yulin in southeastern Guangxi (Rao and Yang 1997; Fei et al. 2009) also need to be clarified. In addition, Fei et al. (2009, 2012) and Mo et al. (2014) recorded B. feae from Jingxi County in western Guangxi and Shangsi County in southern Guangxi. However, in China this species is currently recognized only from Yunnan (Li et al. 2020; AmphibiaChina 2025), so the taxonomy of records of B. feae from Jingxi and Shangsi also remains unclear. Overall, more studies are necessary to investigate the species diversity of the genus Brachytarsophrys in Guangxi, China.

It is interesting that Brachytarsophrys popei is composed of two distinct lineages, and they were placed into two taxonomic units by the analysis of species delimitation (Fig. 4). Considering that the phylogenetic relationship between these two lineages and B. orientalis was not well resolved (Fig. 3), and no distinct morphometric difference was found between these two lineages (Guangdong population vs. Yanling and Jiangxi populations; Fig. 2) in this study, future studies should incorporate more molecular and morphological data to clarify the phylogeny and taxonomy of them.

Including the new species Brachytarsophrys guilinensis sp. nov., the genus Brachytarsophrys now comprises ten recognized species. Previous studies have supported that this genus can be divided into two major lineages (e.g., Li et al. 2022; Lyu et al. 2023; He et al. 2024): one is distributed in the southern part of the Indochinese Peninsula and contains B. carinense and B. intermedia (B. carinense group; Lyu et al. 2023), and one contains the other species and is distributed in southern China (B. orientalis group; Lyu et al. 2023). This study confirms the existence of two major groups within this genus and indicates that the new species belongs to the B. orientalis group.

Acknowledgments

We are deeply indebted to Prof. Yingyong Wang for his kindly sharing of the morphological data of Brachytarsophrys popei and to Prof. Zhiyong Yuan for generously providing tissue samples of B. popei. Thanks also go to Xinmin Zhang for his assistance in sample collection. This work was supported by the National Natural Science Foundation of China (32460128), the Guangxi Natural Science Foundation Project (2022GXNSFAA035526), and the Key Laboratory of Ecology of Rare and Endangered Species and Environmental Protection (Guangxi Normal University), Ministry of Education (ERESEP2022Z04).

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Supplementary materials

Supplementary material 1 

Genetic distances between members of Brachytarsophrys (uncorrected p-distance) in 16S rRNA sequences

Xiangjian Wu, Yuanqiang Pan, Wei Xiao, Ju Chen, Guohua Yu, Xinkui Wei

Data type: xls

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 (29.00 kb)
Supplementary material 2 

Genetic distances between members of Brachytarsophrys (uncorrected p-distance) in COI sequences

Xiangjian Wu, Yuanqiang Pan, Wei Xiao, Ju Chen, Guohua Yu, Xinkui Wei

Data type: xls

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 (29.00 kb)
Supplementary material 3 

Genetic distances between members of Brachytarsophrys (uncorrected p-distance) in Cytb sequences

Xiangjian Wu, Yuanqiang Pan, Wei Xiao, Ju Chen, Guohua Yu, Xinkui Wei

Data type: xls

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 (29.00 kb)
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