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Research Article
Integrative taxonomic evidence for a new species of genus Gracixalus (Anura, Rhacophoridae) from Mao'er Mountain, Guangxi, China
expand article infoXiangjian Wu‡, Yuanqiang Pan‡, Ju Chen‡, Jianping Ye§, Guohua Yu‡, Tongxiang Zou‡
‡ Guangxi Normal University, Guilin, China
§ Maoershan National Natural Reserve, Guilin, China

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

Corresponding author: Tongxiang Zou ( zoutongx@126.com )

Academic editor: Umilaela Arifin
© 2025 Xiangjian Wu, Yuanqiang Pan, Ju Chen, Jianping Ye, Guohua Yu, Tongxiang Zou.
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, Chen J, Ye J, Yu G, Zou T (2025) Integrative taxonomic evidence for a new species of genus Gracixalus (Anura, Rhacophoridae) from Mao'er Mountain, Guangxi, China. Zoosystematics and Evolution 101(4): 2039-2053. https://doi.org/10.3897/zse.101.161448
ZooBank: urn:lsid:zoobank.org:pub:3A74C63D-B826-436A-B1C1-17803DC8FC65
Open Access

Abstract

It has been shown that the taxonomy of Gracixalus jinxiuensis species complex is very confusing. In this study, we tested for the taxonomy of Gracixalus species from Mao’er Mountain, Guangxi, China, which was once recorded as G. jinxiuensis, based on molecular, morphological, and bioacoustics evidence. Phylogenetic analyses based on mitochondrial 16S rRNA sequences indicated that the samples from Mao’er Mountain form a distinct lineage closely related to G. jinxiuensis, G. huaping, and G. weii, and they were assigned into an independent operational taxonomic unit by analysis of species delimitation. Morphologically, the lineage from Mao’er Mountain can be distinguished from known congeners by a combination of the following characters: SVL 27.5–33.2 mm in males and 38.1‒39.7 mm in females; dorsal surface beige to brown, rough with a few tubercles on dorsum; internal vocal sac; linea masculina absent; nuptial pads present on fingers I and II; tibiotarsal articulation reaching center of eye; heels overlapping when legs at right angle to body; and white tubercles on temporal region, edge of upper and lower jaw, flank, and dorsal surface of limbs. In addition, bioacoustics analyses showed that the advertisement calls of the population from Mao’er Mountain also differ from advertisement calls of other Gracixalus species. Therefore, we officially described the lineage from Mao’er Mountain as a new species and named it as Gracixalus liusanjieae sp. nov. herein. Including the new species described in this study, now the genus Gracixalus contains 23 species, 12 of which are distributed in China with six known in Guangxi.

Key Words

Bioacoustics analysis, Gracixalus jinxiuensis, species complex, 16S rRNA, species delimitation

Introduction

The genus Gracixalus Delorme, Dubois, Grosjean & Ohler, 2005 has a wide distribution in the mountain forests of Thailand, Myanmar, Laos, Vietnam, India, and southern and southwestern China (Frost 2024). This genus is characterized by having small to medium body size, intercalary cartilage between terminal and penultimate phalanges of digits present, tips of digits enlarged to discs bearing circum-marginal grooves, vomerine teeth absent, inner (first and second) and outer (third and fourth) fingers not opposable, and an inversed Y-shaped dark brown marking on the dorsum (Fei 1999; Yu et al. 2019; Tran et al. 2023). Currently, the genus Gracixalus contains 22 species and more than half of them were described from southern China and Vietnam in the last decade based on morphological and molecular data (e.g. Liu et al. 2025; Luo et al. 2025), implying that the species diversity of the genus Gracixalus likely remains underestimated owing to conserved morphology. As of now, 11 species of the genus Gracixalus have been recorded from China and five are distributed in Guangxi, namely G. gracilipes (Bourret, 1937), G. jinxiuensis (Hu, 1978), G. nonggangensis Mo, Zhang, Luo, Zhou & Chen, 2013, G. tianlinensis Chen, Bei, Liao, Zhou & Mo, 2018, and G. huaping Luo, Zhang, Pan & Yu, 2025.

Mao’er Mountain, a part of Yuechengling Mountain range of Nanling Mountain system, is located in northeastern Guangxi and is famous for the headstream of Lijiang River and the top peak of South China. Its elevations range from 280 m to 2142 m a.s.l and it is characterized by rugged terrain with deep valleys and high mountains. The extreme topographic relief, together with its wide altitudinal range, have produced extreme differences in climate. The diverse climate and topography of the mountain have resulted in extremely high levels of biodiversity. Currently, according to Jiang et al. (2006), Mo et al. (2014) and AmphibiaChina (2024), more than 40 amphibian species including G. jinxiuensis are known from Mao’er Mountain.

Gracixalus jinxiuensis was originally described from Dayao Mountain, Jinxiu, Guangxi, China (Hu et al. 1978) and was once recorded widely from China (Guangxi, Hunan, Yunnan), Vietnam, and Laos. However, it has been revealed by studies in recent years that misidentifications were involved in this species and some records of this species actually belong to other species (e.g. Yu et al. 2019; Luo et al. 2025), indicating that the taxonomic status of other records of G. jinxiuensis also needs further investigation.

During recent fieldwork in Maoershan National Natural Reserve, Guilin, Guangxi, we collected six specimens of a small-sized tree frog resembling G. jinxiuensis. We conducted phylogenetic analyses, morphological comparison, and bioacoustics analyses to ascertain their identity. Phylogenetic analyses recovered these specimens as a distinct lineage of genus Gracixalus, and morphological examination and bioacoustics analyses demonstrated that this lineage is distinguished from known members of the genus Gracixalus by a series of characters. Herein, we officially describe the lineage consisted of samples from Mao’er Mountain as a new species of Gracixalus.

Materials and methods

Sampling

This study was carried out in accordance with the ethical guidelines issued by the Ethics Committee of Guangxi Normal University. Field surveys were conducted in April 2024, August 2024, and April 2025 under the permissions of Maoershan National Natural Reserve, and a total of six specimens of the genus Gracixalus were collected from Mao’er Mountain during the surveys (Fig. 1). Specimens were photographed, euthanized, fixed, and then stored in 75% ethanol. Liver tissues were preserved in 99% ethanol. Specimens were deposited at Guangxi Normal University (GXNU).

Figure 1. 

The distribution of Gracixalus species in Guangxi, China. The red circle represents the type locality of the new species (Mao’er Mountain). The map was produced using ArcMap v.10.8.1.

Phylogenetic analyses and species delineation

Genomic DNA was extracted from the collected liver tissue samples using standard phenol-chloroform protocols. We amplified and sequenced a partial fragment of mitochondrial 16S ribosomal RNA gene (16S rRNA) using the primer pair L2188 (Matsui et al. 2006)/16H1 (Hedges 1994) and the protocol of Yu et al. (2010). All new sequences have been deposited in GenBank under accession Nos. PV746479‒PV746484 (Table 1). Homologous sequences of known species of Gracixalus were retrieved from GenBank. We selected Rhacophorus borneensis Matsui, Shimada & Sudin,2013 and Kurixalus idiootocus (Kuramoto & Wang, 1987) as outgroup according to Luo et al. (2025) and sequences of them were also downloaded from GenBank.

Table 1.
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Species used for phylogenetic analyses in this study.

Species Locality Voucher no. GenBank no. References
Rhacophorus borneensis Sabah, Malaysia BORN 22410 AB781693 Matsui et al. (2013)
Kurixalus idiootocus Taiwan, China KUHE 12979 AB933306 Nguyen et al. (2014)
Gracixalus yunnanensis Bada, Menghai, Yunnan, China KIZ 20160216 MK234877 Yu et al. (2019)
Gracixalus yunnanensis Xuelin, Lancang, Yunnan, China KIZ 20160222 MK234878 Yu et al. (2019)
Gracixalus yunnanensis Fudong, Lancang, Yunnan, China KIZ 20160226 MK234880 Yu et al. (2019)
Gracixalus yunnanensis Fazhanhe, Lancang, Yunnan, China KIZ 20160228 MK234881 Yu et al. (2019)
Gracixalus sapaensis Lao Cai, Vietnam CIB XM-439 GQ285670 Li et al. (2009)
Gracixalus sapaensis Lao Cai, Vietnam KUHE 46401 LC011938 Matsui et al. (2015)
Gracixalus sapaensis Lai Chau, Vietnam IEBR 2351 EU871425 Nguyen et al. (2008)
Gracixalus sapaensis Lao Cai, Vietnam MNHN 1999.5961 AY880503 Matsui et al. (2017)
Gracixalus quangi Nghe An, Vietnam AMS R173417 JN862539 Rowley et al. (2011)
Gracixalus supercornutus Kon Tum, Vietnam AMS R173887 JN862545 Rowley et al. (2011)
Gracixalus lumarius Kon Tum, Vietnam AMS R 176202 KF918412 Rowley et al. (2014)
Gracixalus jinggangensis Mt. Jinggang, Jiangxi, China SYS a003186 KY624587 Zeng et al. (2017)
Gracixalus seesom Kanchanaburi, Thailand KUHE 35084 LC011932 Matsui et al. (2015)
Gracixalus quyeti Cha Noi, Vietnam VNUH 160706 EU871428 Nguyen et al. (2008)
Gracixalus sp. Wenshan, Yunnan, China 03320Rao GQ285669 Li et al. (2009)
Gracixalus gracilipes Lao Cai, Vietnam AMS R 177672 KT374014 Rowley et al. (2015)
Gracixalus gracilipes Pingbian, Yunnan, China 060821196Rao GQ285668 Li et al. (2009)
Gracixalus ziegleri Yen Bai, Vietnam MCC.2018.35 LC642813 Le et al. (2021)
Gracixalus ziegleri Yen Bai, Vietnam MCC.2018.15 LC642812 Le et al. (2021)
Gracixalus trieng Kon Tum Province, Vietnam AMS R176206 MT328246 Rowley et al. (2020)
Gracixalus trieng Kon Tum Province, Vietnam UNS 00342/AMS R176205 MT328245 Rowley et al. (2020)
Gracixalus tianlinensis Guangxi, China NHMG 1705016 MH117961 Chen et al. (2018)
Gracixalus tianlinensis Guangxi, China NHMG 1705015 MH117960 Chen et al. (2018)
Gracixalus guangdongensis Hunan, China CIB HN201108200 LC011936 Matsui et al. (2015)
Gracixalus guangdongensis Guangdong, China SYS a004902 MG520193 Wang et al. (2018)
Gracixalus guangdongensis Guangdong, China SYS a005750 MG520197 Wang et al. (2018)
Gracixalus medogensis Medog, Xizang, China KIZ010956 MW023606 Xu et al. (2020)
Gracixalus huaping Huaping, Guangxi, China GXNU YU000480 PP083976 Luo et al. (2025)
Gracixalus huaping Huaping, Guangxi, China GXNU YU000511 PP083977 Luo et al. (2025)
Gracixalus huaping Huaping, Guangxi, China GXNU YU000512 PP083978 Luo et al. (2025)
Gracixalus huaping Huaping, Guangxi, China GXNU YU000513 PP083979 Luo et al. (2025)
Gracixalus huaping Huaping, Guangxi, China GXNU YU000514 PP083980 Luo et al. (2025)
Gracixalus jinxiuensis Jinxiu, Guangxi, China KIZ 060821013 EF564524 Yu et al. (2008)
Gracixalus jinxiuensis Jinxiu, Guangxi, China KIZ 061210YP EU215525 Li et al. (2008)
Gracixalus jinxiuensis Jinxiu, Guangxi, China SYS a002183 KY624585 Zeng et al. (2017)
Gracixalus jinxiuensis Jinxiu, Guangxi, China SYS a002182 KY624584 Zeng et al. (2017)
Gracixalus truongi Tuan Giao District, Dien Bien Province, Vietnam IEBR A.5006 OP750514 Tran et al. (2023)
Gracixalus truongi Tuan Giao District, Dien Bien Province, Vietnam IEBR A.5005 OP750513 Tran et al. (2023)
Gracixalus nonggangensis Guangxi, China NHMG111024 JX841317 Mo et al. (2013)
Gracixalus nonggangensis Guangxi, China NHMG20091009 JX841319 Mo et al. (2013)
Gracixalus nonggangensis Guangxi, China NHMG200910010 JX841320 Mo et al. (2013)
Gracixalus ananjevae Nghe An, Vietnam VNMN 03012 JN862546 Rowley et al. (2011)
Gracixalus patkaiensis Northeast India WII-ADA1352 OQ940026 Boruah et al. (2023)
Gracixalus patkaiensis Northeast India WII-ADA1353 OQ940027 Boruah et al. (2023)
Gracixalus weii Leigongshan, Guizhou, China MT LS20240517001 PP852208 Liu et al. (2025)
Gracixalus weii Leigongshan, Guizhou, China MT LS20240517002 PP852209 Liu et al. (2025)
Gracixalus weii Leigongshan, Guizhou, China MT LS20240518009 PP852212 Liu et al. (2025)
Gracixalus weii Leigongshan, Guizhou, China MT LS20240518010 PP852213 Liu et al. (2025)
Gracixalus liusanjieae sp. nov. Mao’er Mountain, Guangxi, China GXNU YU000795 PV746479 This study
Gracixalus liusanjieae sp. nov. Mao’er Mountain, Guangxi, China GXNU YU000796 PV746480 This study
Gracixalus liusanjieae sp. nov. Mao’er Mountain, Guangxi, China GXNU YU000962 PV746481 This study
Gracixalus liusanjieae sp. nov. Mao’er Mountain, Guangxi, China GXNU YU000978 PV746482 This study
Gracixalus liusanjieae sp. nov. Mao’er Mountain, Guangxi, China GXNU YU000979 PV746483 This study
Gracixalus liusanjieae sp. nov. Mao’er Mountain, Guangxi, China GXNU YU000980 PV746484 This study

Sequences were aligned in MEGA v.11 (Tamura et al. 2021) using MUSCLE with the default parameters. Alignments were visually checked by eye for accuracy and trimmed to minimize missing characters. Uncorrected pairwise distances were calculated in MEGA v. 11. The best substitution model (GTR + I + G) was selected using the corrected Akaike Information Criterion (AICc) in jMODELTEST v. 2.1.10 (Darriba et al. 2012), and then we used maximum likelihood and Bayesian inference to construct the phylogeny of the genus Gracixalus. The maximum likelihood analysis was performed in RaxmlGUI v. 2.0 (Edler et al. 2021) with 1000 rapid bootstrap replicates. Bayesian inference was conducted in MrBayes v. 3.2.6 (Ronquist et al. 2012) based on the selected substitution model. Two runs were performed simultaneously with four Markov chains starting from a random tree. The chains were run for 3000,000 generations and sampled every 100 generations. Convergence and burn-in were checked using the program Tracer v. 1.6. (Rambaut et al. 2014). The first 25% of the sampled trees were discarded as burn-in, and the remaining trees were used to create a consensus tree and to estimate Bayesian posterior probabilities (BPPs).

We conducted analysis of species delimitation using the method of Assemble Species by Automatic Partitioning (ASAP; Puillandre et al. 2020) and the model of simple distance (p-distance). The partitioning with the lowest ASAP score was chosen as the best according to Puillandre et al. (2020).

Morphological analysis

Morphometric data were taken using electronic digital calipers to the nearest 0.1 mm. The morphological terminologies followed Yu et al. (2019). A total of 13 characters were measured, including snout-vent length (SVL), head length (HL), head width (HW), snout length (SL), internarial distance (IND), interorbital distance (IOD), eye diameter (ED), upper eyelid width (UEW), tympanum diameter (TD), forearm and hand length (FHL), tibia length (TL), length of tarsus and foot (TFL), and foot length (FL).The webbing formula followed Myers and Duellman (1982).

We used the t-test in SPSS v. 17.0 to evaluate the differences in quantitative characters of adult males between the new species and its closest relatives revealed by phylogenetic analyses. Principal component analyses (PCA) were conducted based on a correlation matrix of size-corrected measurements of males in SPSS. Scatterplots of the first two PCA factors were used to present the morphological differentiation.

Bioacoustics analysis

Advertisement calls were recorded using SONY PCM-A10 digital sound recorder in the field. Calls of an individual of the new species were recorded on 26 April 2025 at ambient temperature of 15 °C, calls from an individual of G. jinxiuensis were recorded in Jinxiu, Guangxi, China on 10 June 2024 at ambient temperature of 22 °C, and calls of an individual of G. huaping were recorded in Huaping, Guangxi, China on 4 May 2022 at ambient temperature of 20 °C. The sound files were sampled at 44.1 kHz with 16 bits in depth. Number of notes, call duration, interval of calls, frequency range, and dominant frequency were measured using Raven Pro 1.6 (www.birds.cornell.edu/raven). The bioacoustics data of G. guangdongensis Wang, Zeng, Liu & Wang, 2018, G. jinggangensis Zeng, Zhao, Chen, Chen, Zhang & Wang, 2017, G. nonggangensis, and G. tianlinensis were obtained from Wang et al. (2018), Zeng et al. (2017), and Chen et al. (2018), respectively, for comparison.

Results

Phylogenetic relationship and species delineation

After cutting off both ragged sides, length of the obtained alignment of 16S rRNA sequence is 547 bp. Both maximum likelihood analysis and Bayesian inference recovered the samples of Gracixalus from Mao’er Mountain, Guangxi, China as a distinct lineage. This lineage is closely related to G. weii Liu, Peng, Wang, Feng, Shen, Li, Chen, Su & Tang, 2025 and the clade of G. jinxiuensis and G. huaping with strong support, although the relationships between them were not resolved well (Fig. 2). The genetic distances (uncorrected p-distance) between this novel linage and its congeners ranged from 1.8% (vs. G. weii) to 13.5% (vs. G. lumarius Rowley, Le, Dau, Hoang & Cao, 2014) (Suppl. material 1).

Figure 2. 

Bayesian phylogram of Gracixalus inferred from 547 bp of 16S rRNA gene. Numbers above and below branches are Bayesian posterior probabilities and ML bootstrap values (only values above 50% are shown), respectively.

The best partition (score = 4.0) obtained by the ASAP analysis placed the samples of Gracixalus included in this study into 23 operational taxonomic units (OUT), one of which corresponds to the lineage consisted of samples from Mao’er Mountain and other 22 OUT correspond to 21 known species and an unnamed lineage from Yunnan (Fig. 3).

Figure 3. 

ASAP species delimitation in genus Gracixalus based on 16S sequences. The best partition with the lowest score is highlighted with a red frame.

Morphology

Morphometric data are summarized in Table 2. The t-tests revealed that the male specimens from Mao’er Mountain differ significantly (p < 0.05) from males of G. huaping in snout length (SL), internarial distance (IND), and tibia length (TL; Table 3); from males of G. jinxiuensis in forearm and hand length (FHL) and foot length (FL; Table 4); and from males of G. weii in head length (HL), snout length (SL), internarial distance (IND), upper eyelid width (UEW), eye diameter (ED), tympanum diameter (TD), tibia length (TL), length of tarsus and foot (TFL), and foot length (FL; Table 5).

Table 2.
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Measurements (mm) of Gracixalus liusanjieae sp. nov., G. jinxiuensis, G. huaping, and G. weii. The data of G. weii was obtained from Liu et al. (2025).

Character Gracixalus liusanjieae sp. nov. G. jinxiuensis G. huaping G. weii
Male (n = 4) Female (n = 2) Male (n = 4) Male (n = 3) Female (n = 4) Male (n = 5) Female (n = 2)
SVL 27.5–33.2 (30.8 ± 2.4) 38.1–39.7 27.4–30.8 (29.6 ± 1.5) 26.6–28.8 (28.0 ± 1.2) 29.8–32.8 (31.5 ± 1.3) 30.1–34.0 (31.9 ± 1.4) 35.2–36.0
HL 9.8–11.4 (10.5 ± 0.7) 13.1–13.6 9.9–11.2 (10.4 ± 0.6 9.3–9.9 (9.7 ± 0.3) 10.5–11.7 (11.0 ± 0.5) 9.5–10.8 (10.3 ± 0.5) 11.3–11.8
HW 10.2–12.1 (11.5 ± 0.9) 13.6–14.0 10.7–11.5 (11.0 ± 0.4) 10.3–10.9 (10.7 ± 0.3) 11.0–12.4 (11.7 ± 0.6) 11.6–12.8 (12.1 ± 0.4) 12.4–13.1
SL 3.6–4.2 (4.1 ± 0.3) 5.1–5.4 4.0–5.0 (4.6 ± 0.5) 3.0–3.2 (3.1 ± 0.1) 3.5–3.8 (3.7 ± 0.1) 4.7–5.1 (4.9 ± 0.2) 5.2–5.4
IND 3.1–3.6 (3.3 ± 0.2) 3.6–3.9 2.6–3.5 (3.1 ± 0.4) 3.0–3.4 (3.2 ± 0.2) 3.2–3.6 (3.4 ± 0.2) 3.8–4.5 (4.1 ± 0.3) 4.0–4.8
IOD 3.2–3.7 (3.5 ± 0.2) 4.4–4.5 3.5–4.3 (3.7 ± 0.4) 3.2–3.4 (3.3 ± 0.1) 3.4–3.8 (3.6 ± 0.2) 3.4–4.2 (3.7 ± 0.4) 3.7–4.4
UEW 2.3–3.3 (2.9 ± 0.4) 3.3–3.4 2.6–2.9 (2.7 ± 0.1) 2.5–2.7 (2.6 ± 0.1) 2.6–3.2 (2.9 ± 0.3) 2.3–2.9 (2.5 ± 0.2) 2.7–3.0
ED 3.8–4.0 (3.9 ± 0.1) 4.2–4.3 3.7–4.1 (3.9 ± 0.2) 3.2–3.3 (3.2 ± 0.1) 3.3–3.6 (3.5 ± 0.1) 3.3–3.7 (3.6 ± 0.2) 4.4–4.9
TD 1.5–1.7 (1.6 ± 0.1) 2.0–2.2 1.5–2.0 (1.7 ± 0.2) 1.5–1.6 (1.5 ± 0.1) 1.7–1.9 (1.8 ± 0.1) 2.2–2.6 (2.4 ± 0.2) 2.7–3.4
FHL 13.9–17.4 (15.7 ± 1.4) 18.4–19.6 13.4–14.7 (14.2 ± 0.6) 13.6–14.7 (14.2 ± 0.6) 15.7–16.4 (16.0 ± 0.3) 15.9–17.6 (16.7 ± 0.7) 18.0–18.2
TL 13.3–15.8 (14.7 ± 1.1) 18.2–18.5 13.8–14.7 (14.1 ± 0.4) 13.2–14.7 (14.0 ± 0.8) 14.9–15.7 (15.3 ± 0.4) 14.9–17.2 (15.8 ± 0.9) 17.3–17.4
TFL 18.2–22.8 (21.0 ± 2.0) 25.4–26.6 18.2–20.5 (19.2 ± 1.0) 18.6–20.4 (19.5 ± 0.9) 21.8–22.4 (22.0 ± 0.3) 21.9–23.7 (22.6 ± 1.2) 24.5–24.6
FL 12.1–15.5 (14.0 ± 1.4) 17.3–17.4 11.5–13.4 (12.4 ± 0.8) 11.8–13.0 (12.4 ± 0.6) 14.0–14.5 (14.2 ± 0.2) 12.1–14.8 (13.6 ± 1.0) 14.7–15.2
Table 3.
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Results of t-test between males of G. liusanjieae sp. nov. and G. huaping based on the size-corrected data except SVL (*p < 0.05).

Character Mean ± SD (n = 4) Mean ± SD (n = 3) Levene’s test t-test
G. liusanjieae sp. nov. G. huaping F p-value t p-value
SVL 30.8 ± 2.4 28.0 ± 1.2 0.699 0.441 1.841 0.125
HL 0.342 ± 0.011 0.345 ± 0.005 1.485 0.277 -0.501 0.638
HW 0.372 ± 0.010 0.381 ± 0.006 0.681 0.447 -1.448 0.207
SL 0.131 ± 0.004 0.112 ± 0.001 3.489 0.121 8.754 0.000*
IND 0.107 ± 0.004 0.117 ± 0.001 4.290 0.093 -3.570 0.016*
IOD 0.112 ± 0.011 0.118 ± 0.002 2.483 0.176 -0.872 0.423
UEW 0.094 ± 0.008 0.092 ± 0.004 1.415 0.288 0.398 0.707
ED 0.127 ± 0.010 0.116 ± 0.004 0.667 0.451 1.872 0.120
TD 0.052 ± 0.003 0.055 ± 0.002 0.541 0.495 -1.390 0.223
FHL 0.509 ± 0.014 0.507 ± 0.006 1.749 0.243 0.208 0.844
TL 0.476 ± 0.007 0.501 ± 0.008 0.128 0.735 -4.214 0.008*
TFL 0.681 ± 0.126 0.695 ± 0.015 0.150 0.715 -1.397 0.221
FL 0.452 ± 0.011 0.442 ± 0.009 0.201 0.673 1.142 0.305
Table 4.
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Results of t-test between males of G. liusanjieae sp. nov. and G. jinxiuensis based on the size-corrected data except SVL (*p < 0.05).

Character Mean ± SD (n = 4) Mean ± SD (n = 4) Levene’s test t-test
G. liusanjieae sp. nov. G. jinxiuensis F p-value t p-value
SVL 30.8 ± 2.4 29.6 ± 1.5 0.445 0.529 0.846 0.430
HL 0.342 ± 0.011 0.351 ± 0.013 1.112 0.332 -1.092 0.317
HW 0.372 ± 0.010 0.371 ± 0.015 0.763 0.416 0.077 0.941
SL 0.131 ± 0.004 0.154 ± 0.021 21.325 0.004 -2.096 0.122
IND 0.107 ± 0.004 0.103 ± 0.008 0.661 0.447 0.994 0.359
IOD 0.112 ± 0.011 0.126 ± 0.013 0.767 0.415 -1.603 0.160
UEW 0.094 ± 0.008 0.092 ± 0.003 3.742 0.101 0.408 0.697
ED 0.127 ± 0.010 0.133 ± 0.008 0.100 0.763 -0.904 0.401
TD 0.052 ± 0.003 0.058 ± 0.006 0.803 0.405 -1.804 0.121
FHL 0.509 ± 0.014 0.478 ± 0.011 0.098 0.765 3.582 0.012*
TL 0.476 ± 0.007 0.477 ± 0.020 1.763 0.232 -0.029 0.978
TFL 0.681 ± 0.126 0.648 ± 0.024 1.114 0.332 2.365 0.056
FL 0.452 ± 0.011 0.418 ± 0.014 0.096 0.767 3.877 0.008*
Table 5.
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Results of t-test between males of G. liusanjieae sp. nov. and G. weii based on the size-corrected data except SVL (*p < 0.05).

Character Mean ± SD (n = 4) Mean ± SD (n = 5) Levene’s test t-test
G. liusanjieae sp. nov. G. weii F p-value t p-value
SVL 30.8 ± 2.4 31.9 ± 1.4 0.871 0.382 0.867 0.415
HL 0.342 ± 0.011 0.324 ± 0.009 0.001 0.982 -2.678 0.032*
HW 0.372 ± 0.010 0.380 ± 0.013 0.139 0.720 1.052 0.328
SL 0.131 ± 0.004 0.153 ± 0.003 0.014 0.909 9.223 0.000*
IND 0.107 ± 0.004 0.128 ± 0.005 0.194 0.673 6.404 0.000*
IOD 0.112 ± 0.011 0.117 ± 0.009 0.053 0.824 0.771 0.466
UEW 0.094 ± 0.008 0.079 ± 0.008 0.015 0.906 -2.758 0.028*
ED 0.127 ± 0.010 0.112 ± 0.006 0.150 0.710 -2.863 0.024*
TD 0.052 ± 0.003 0.074 ± 0.004 0.072 0.796 10.020 0.000*
FHL 0.509 ± 0.014 0.522 ± 0.018 0.773 0.409 1.182 0.276
TL 0.476 ± 0.007 0.495 ± 0.011 0.417 0.539 2.841 0.025*
TFL 0.681 ± 0.126 0.709 ± 0.015 1.638 0.242 2.957 0.021*
FL 0.452 ± 0.011 0.426 ± 0.018 1.716 0.232 -2.487 0.042*

PCA analysis illustrated that the first two principal components accounted for 62.30% of the total variance (Table 6). Loadings for PC1 accounted for 40.65% of the total variance and were most heavily loaded on IND and TFL, which can separate the Mao’er Mountain population from G. jinxiuensis, G. huaping, and G. weii (Fig. 4). Loadings for PC2 accounted for 21.65% and were heavily loaded on SL and FL, which showed that generally the Mao’er Mountain population can be separated from G. weii and G. jinxiuensis by having shorter snout and longer foot (Table 6, Fig. 4).

Figure 4. 

Scatterplot of principal components 1 and 2 of size-adjusted morphometric data of Gracixalus liusanjieae sp. nov., G. huaping, G. jinxiuensis, and G. weii.

Table 6.
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Factor loadings of first two principal components of 12 size-adjusted morphometric characteristics of Gracixalus liusanjieae sp. nov., G. jinxiuensis, G. huaping, and G. weii.

Character PC1 PC2
Variance 4.878 2.597
% of var. 40.653 21.645
Cumulative % of var. 40.653 62.298
HL –0.728 –0.125
HW 0.453 –0.011
SL 0.089 0.839
IND 0.908 0.023
IOD –0.208 0.664
UEW –0.645 –0.437
ED –0.734 0.298
TD 0.768 0.515
FHL 0.769 –0.192
TL 0.632 –0.088
TFL 0.856 –0.376
FL –0.027 –0.840

Additionally, in terms of advertisement calls, the Mao’er Mountain population can be distinguished from other species by number of notes, call duration, or absence of harmonics (see below). Therefore, based on the integrative evidence of morphological, molecular, and bioacoustics analyses, we consider that the samples of Gracixalus from Mao’er Mountain, Guangxi, China represent a new species and describe it herein.

Taxonomic account

Gracixalus liusanjieae sp. nov.

https://zoobank.org/6F021E10-7516-4719-A7AF-3A67D15F2254
Figs 5, 6, 7, 8

Type material.

Holotype. • GXNU YU000978, an adult male collected on 4 April 2025 by Guohua Yu, Yuanqiang Pan, and Xiangjian Wu from Maoershan National Natural Reserve, Guilin, Guangxi, China (25.911°N, 109.466°E, 1573 m elevation).

Paratypes. • Two adult males (GXNU YU000795, GXNU YU000796) collected on 26 April 2024 by Guohua Yu, Yuanqiang Pan, and Xiangjian Wu, • an adult male (GXNU YU000962) collected on 13 August 2024 by Tongxiang Zou, and • two adult females (GXNU YU000979 and GXNU YU000980) collected on 26 April 2025 by Guohua Yu, Yuanqiang Pan, and Xiangjian Wu. All paratypes were collected from the type locality.

Etymology.

The specific epithet is named for Sanjie Liu, referring to a famous woman in Chinese ancient legend who came from Guangxi and who was known for her exceptional singing talent. The specific name means that the new species is distributed in Guangxi and its advertisement calls are melodic. We suggested “Maoershan small tree frog” for the common English name and “刘三姐纤树蛙 (Liú Sān Jiě Xiān Shù Wā)” for the common Chinese name.

Diagnosis.

The new species is assigned to genus Gracixalus based upon the following set of morphological characters: tips of digits enlarged to discs bearing circum-marginal grooves, vomerine teeth absent, inner (first and second) and outer (third and fourth) fingers not opposable, and an inversed Y-shaped dark brown marking on the dorsum (Fei 1999; Yu et al. 2019; Tran et al. 2023).

The new species can be distinguished from its congeners by a combination of the following morphological characters: (1) SVL 27.5–33.2 mm in males and 38.1‒39.7 mm in females; (2) dorsal surface beige to brown; (3) dorsal surface rough with a few flatten tubercles on dorsum; (4) internal vocal sac in males, vocal sac opening slitted; (5) throat relatively smooth with barely visible tubercles; (6) finger webbing rudimentary; (7) linea masculina absent; (8) snout rounded; (9) tibiotarsal articulation reaching center of eye; (10) tibiotarsal projection absent; (11) nuptial pads present on fingers I and II; (12) heels slightly overlapping when legs at right angle to body; (13) ventral surface translucent, creamy white with dark blotches; (14) belly granular.

Description of holotype.

Small size (SVL 33.2 mm); head wider (HW 12.1 mm) than long (HL 11.4 mm); snout rounded, slightly projecting beyond margin of lower jaw in ventral view, rounded in profile; canthus rostralis rounded; nostril oval, protuberant, closer to tip of snout than eye; loreal region oblique, slightly concave; interorbital distance (IOD 3.7 mm) nearly equal to internarial distance (IND 3.6 mm) and upper eyelid width (UEW 3.3 mm); eye large, horizontal diameter (ED 3.8 mm) slightly shorter than snout length (SL 4.2 mm); pupil oval, horizontal; tympanum distinct (TD 1.6 mm), slightly smaller than half of ED; supratympanic fold distinct, extending from posterior corner of eye to above insertion of arm; vomerine teeth absent; tongue notched posteriorly; single internal vocal sac, a pair of vocal sac slits on floor of mouth at both corners.

Forelimbs relatively strong; forearm and hand length (FHL 17.4 mm) longer than 50% of SVL; relative finger lengths I < II < IV < III; finger webbing rudimentary; tips of all fingers expanded into discs with circum-marginal grooves; subarticular tubercles prominent and rounded, formula 1, 1, 2, 2, the proximal one smaller than the distal one on fingers III and IV; two metacarpal tubercles, the outer divided into two; nuptial pads white, present on the base of the first and the second fingers.

Heels meeting when legs at right angle to body; the relative lengths of the toes I < II < III < V < IV; tibiotarsal articulation reaching the middle of the eye when hindlimb adpressed to body; tip of toes expanded into discs with circum-marginal grooves; toe discs slightly smaller than finger discs; half web between toes, webbing formula I2–2⅓II1½–3III1½–2⅔IV2⅓–1⅔V; subarticular tubercles prominent, round, formula 1, 1, 2, 3, 2; a few supernumerary tubercles present; inner metatarsal tubercle oval, outer metatarsal tubercle absent.

Dorsal surface rough, sparsely scattered with a few small flatten tubercles on the upper eyelids, head, dorsum, and limbs; white tubercles scattered on temporal region, edge of upper and lower jaw, and dorsal surface of limbs; flank rough, scattered with white tubercles; throat relatively smooth with barely visible tubercles; chest and belly granular, and ventral surface of thighs rough scattered with white tubercles.

Coloration of holotype. In life, dorsal surface beige; a dark brown inverted Y-shaped marking on dorsal surface covering interorbital region, bifurcating into two branches on shoulder and extending posteriorly; dorsal surface of limbs beige with dark brown stripes; anterior and posterior parts of thigh light yellow; ventral surface of thigh light yellow mottled with light purple; discs of fingers and toes light yellow; flanks beige; throat and chest creamy white with dark speckles; iris bronze (Fig. 5).

Figure 5. 

Views of the holotype of Gracixalus liusanjieae sp. nov. (voucher no. GXNU YU000978) in life. Nuptial pads were highlighted with arrows (Photo by Guohua Yu).

In preservative, color faded but pattern same as in life; dorsal surface gray-brown with a dark brown inverted Y-shaped marking; dark brown stripes on limbs; ventral surface dirty white with dark speckles (Fig. 6).

Figure 6. 

Dorsal and ventral views of the holotype of Gracixalus liusanjieae sp. nov. (voucher no. GXNU YU000978) in preservative (Photo by Xiangjian Wu).

Sexual dimorphism. Females are larger than males in body size. Males have white nuptial pads on the base of fingers I & II (Figs 5, 7C) and internal single subgular vocal sac.

Figure 7. 

Paratypes GXNU YU000979 (A), GXNU YU000980 (B), and GXNU YU000795 (C) in life (A, B. Photo by Yuanqiang Pan; C. Photo by Guohua Yu).

Morphological variation. The dorsal surface of paratypes GXNU YU000795 and GXNU YU000979 is dark brown, whereas the dorsal surface of other types is beige or brown (Figs 7, 8). Paratypes GXNU YU000962 and GXNU YU000980 have less dark blotches on ventral surface (Fig. 8). In addition, the inverted Y-shaped marking on the dorsum in paratype GXNU YU000980 is discontinuous (Fig. 8).

Figure 8. 

Dorsal and ventral surfaces of paratypes GXNU YU000796 (A), GXNU YU000795 (B), GXNU YU000962 (C), GXNU YU000980 (D), and GXNU YU000979 (E) in preservative (Photo by Xiangjian Wu).

Advertisement call. The advertisement calls of the new species consist of two notes (Fig. 9A). The call duration of the new species is 0.66‒0.80 s (0.74 ± 0.04; Table 7) and the peak frequency is 2.58 kHz. The frequency range is 2.2‒2.9 kHz and the interval between calls ranges from 5.6 s to 18.2 s (8.9 ± 2.9).

Figure 9. 

Waveforms and spectrograms of the advertisement calls of Gracixalus liusanjieae sp. nov. (A), G. jinxiuensis (B), and G. huaping (C).

Table 7.
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The measurements of the advertisement calls of G. liusanjieae sp. nov., G. jinxiuensis, and G. huaping.

Species Number of calls Number of notes Call duration (s) Call interval (s) Peak frequency (kHz) Frequency range (kHz)
G. liusanjieae sp. nov. 20 2 0.66–0.80 (0.74 ± 0.04) 5.6–18.2 (8.9 ± 2.9) 2.58 2.25–2.95
G. jinxiuensis 32 2 0.46–0.61 (0.51 ± 0.03) 0.99–14.43 (3.78 ± 2.66) 2.50 2.08–2.95
G. huaping 3 1 0.61–0.69 (0.64 ± 0.04) 7.18–10.03 2.89 2.10–3.37

Distribution and ecology. Currently, the new species is known only from the type locality. The new species inhabits bamboo forest (Fig. 10) and enters breeding season in early April. Eggs with gel nests were found on the broken branches placed on the bottles that were used as artificial breeding trap for surveillance of tree frogs (Fig. 10), and calls were heard during the surveys. Zhangixalus minimus (Rao, Wilkinson & Liu, 2006) was also found at the type locality.

Figure 10. 

Habitat of Gracixalus liusanjieae sp. nov. at the type locality (A) and eggs of the new species laid on broken branches (C) that was placed on an artificial breeding trap (B) (A, B. Photo by Guohua Yu; C. Photo by Yuanqiang Pan).

Measurements of holotype

(in mm). SVL 33.2, HL 11.4, HW 12.1, SL 4.2, IND 3.6, IND 3.7, UEW 3.3, ED 3.8, TD 1.6, FHL 17.4, TL 15.8, TFL 22.8, FL 15.5.

Comparisons.

Phylogenetically, the new species is closer to G. jinxiuensis, G. huaping, and G. weii than to other species. Gracixalus liusanjieae sp. nov. is distinguishable from G. jinxiuensis, with which the new species has previously been confused, by nuptial pads distinct, white, present on the base of fingers I and II (vs. nuptial pads not distinct, light yellow, present on the base of finger I), longer forearm and hand (FHL/SVL 0.509 ± 0.0136 vs. 0.4779 ± 0.0107), longer foot (FL/SVL0.452 ± 0.0113 vs. 0.417 ± 0.0135), and longer duration of advertisement calls (0.74 ± 0.04 vs. 0.51 ± 0.03; Table 7); from G. weii by nuptial pads present on fingers I and II (vs. on finger I), larger body size in females (SVL 38.1‒39.7 mm vs. 35.2‒36.0 mm), longer head (HL/SVL 0.342 ± 0.011 vs. 0.324 ± 0.009), shorter snout (SL/SVL 0.131 ± 0.0037 vs. 0.153 ± 0.0034), narrower internarial distance (IND/SVL 0.107 ± 0.004 vs. 0.128 ± 0.005), shorter tibia (TL/SVL 0.476 ± 0.007 vs. 0.4945 ± 0.011), shorter tarsus and foot (TFL/SVL 0.681 ± 0.123 vs. 0.709 ± 0.015), and longer foot (FL/SVL 0.452 ± 0.011 vs. 0.426 ± 0.018); and from G. huaping by bigger body size (SVL 27.5–33.2 mm in males and 38.1‒39.7 mm in females vs. SVL 26.6‒28.8 mm in males and 29.8‒32.8 mm in females), linea masculina absent (vs. present), vocal sac opening slitted (vs. oval and rim of the opening dark brown), nuptial pads present on fingers I and II (vs. on finger I), and advertisement calls composed of two notes (vs. one note; Fig. 9).

The new species can be distinguished from G. ananjevae (Matsui & Orlov, 2004) by nuptial pads present on fingers I and II (vs. on finger I), finger webbing rudimentary (vs. finger webbing formula I2‒2II2‒basalIII3‒2.75IV) and the presence of white granules around anus and on limbs (vs. absent); from G. carinensis (Boulenger, 1893) by smaller body size (SVL 27.5–31.5 mm in males and 38.1‒39.7 mm in females vs. 30.2–38.1 mm in males and 40.3‒41.8 mm in females), less developed toe webbing (Fig. 11), and tubercles on dorsum present (vs. absent); from G. gracilipes by bigger body size (SVL 27.5–31.5 mm in males and 38.1‒39.7 mm in females vs. 20–24 mm in males and ca. 30 mm in females), snout rounded (vs. triangularly pointed), and dorsal surface beige to brown (vs. greenish); from G. guangdongensis by nuptial pads present on fingers I and II (vs. on finger I), bigger body size in females (38.1‒39.7 mm vs. 34.9‒35.4 mm), and longer duration of advertisement calls (0.74 ± 0.04 vs. 0.56 ± 0.07); from G. jinggangensis by heels overlapping (vs. just meeting) and calls composed of two notes with no harmonics (vs. three or four notes with multiple harmonics); from G. lumarius by smaller body size (SVL 27.5–31.5 mm in males vs. 38.9–41.6 mm in males), dorsal surface rough sparsely scattered with flatten tubercles (vs. dorsal surface of head and back covered in dense and distinctive white conical tubercles), nuptial pads present on fingers I and II (vs. on finger I), and supratympanic fold distinct (vs. indistinct); and from G. medogensis (Ye & Hu, 1984) by dark brown mark beginning between eyes bifurcated into two wide branches after the back of shoulders (vs. bifurcated into two relatively narrow branches before the back of shoulders), dorsal color beige to brown (vs. grass green), finger webbing rudimentary (vs. absent), linea masculine absent (vs. present), and nuptial pads present on fingers I and II (vs. on finger I).

Figure 11. 

Ventral views of foot of Gracixalus liusanjieae sp. nov. (A. GXNU YU000796, photo by Guohua Yu) and G. carinensis (B. Reproduced from Matsui et al. 2017).

Gracixalus liusanjieae sp. nov. differs from G. nonggangensis by dorsal surface beige to brown with dark-brown mark (vs. yellowish-olive with dark-green mark), nuptial pads on fingers I and II (vs. absent), iris bronze (vs. olive), and advertisement calls composed of two notes (vs. 40‒72 notes); from G. patkaiensis Boruah, Deepak, Patel, Jithin, Yomcha & Das, 2023 by bigger body size (SVL 27.5–31.5 mm in males vs. 23.6‒26.5 mm in males), dorsal surface beige to brown (vs. green with brown spots), vocal sac internal (vs. external), and nuptial pads on fingers I and II (vs. on finger I); from G. quangi Rowley, Dau, Nguyen, Cao & Nguyen, 2011 by larger body size (SVL 27.5–31.5 mm in males vs. 21.4‒24.5 mm in males), dorsal surface beige to brown (vs. olive-green), vocal sac internal (vs. external), snout rounded (vs. triangularly pointed), nuptial pads present on fingers I and II (vs. on finger I), and tibiotarsal projection absent (vs. present); and from G. quyeti (Nguyen, Hendrix, Böhme, Vu & Ziegler, 2008) by dorsal surface beige to brown (vs. brownish to moss-green), head wider than long (vs. head longer than wide), tibiotarsal articulation reaching center of eye (vs. reaching to snout), supratympanic fold distinct (vs. indistinct), and forelimb, dorsal parts of thighs, tibia, and foot brown with dark brown bands (vs. moss-green with dark brown bands).

The new species differs from G. sapaensis Matsui, Ohler, Eto & Nguyen, 2017 by nuptial pads present on fingers I and II (vs. on finger I) and ventral surface of throat and chest creamy white (vs. light yellow); from G. seesom Matsui, Khonsue, Panha & Eto, 2015 by bigger body size (SVL 27.5–31.5 mm in males vs. 21.6–23.0 mm in males), snout rounded (vs. triangularly pointed), dorsal surface rough scattered with tubercles (vs. smooth), dorsal surface beige to brown with dark brown inverted Y-shaped marking (vs. tan), and nuptial pads present on fingers I and II (vs. absent); from G. supercornutus (Orlov, Ho & Nguyen, 2004) by bigger body size (SVL 27.5–31.5 mm in males vs. 22.0–24.1 mm), dorsal surface beige to brown (vs. dorsum green with brown spots), snout rounded (vs. pointed), white patch on temporal region absent (vs. present), dorsal surface nearly smooth (vs. considerable bigger horn-like projections in supraorbital area, around cloaca, and on dorsum, forelimbs and hindlimbs), and tibiotarsal projection absent (vs. present); and from G. tianlinensis by vocal sac internal (vs. external), ventral of thigh rough with tubercles (vs. ventral surface of limbs smooth), finger webbing rudimentary (vs. absent), and calls lacking harmonic (vs. present).

Gracixalus liusanjieae sp. nov. differs from G. trieng Rowley, Le, Hoang, Cao & Dau, 2020 by smaller body size (SVL in males 27.5–31.5 mm vs. 37.2–41.4 mm), presence of white tubercles on temporal region, edge of upper and lower jaw, flank, and dorsal surface of limbs (vs. absent), and throat and chest white with dark specks (vs. yellow or yellowish brown with pinkish mottling); from G. truongi Tran, Pham, Le, Nguyen, Ziegler & Pham, 2023 by smaller body size (SVL 27.5–31.5 mm in males vs. 32.2–33.1 mm in males), dorsal surface beige to brown (vs. moss green with grey), and nuptial pads present on fingers I and II (vs. on finger I); from G. yunnanensis Yu, Li, Wang, Rao, Wu & Yang, 2019 by vocal sac internal (vs. external), linea masculine absent (vs. present), and nuptial pads present on fingers I and II (vs. on finger I); and from G. ziegleri Le, Do, Tran, Nguyen, Orlov, Ninh & Nguyen, 2021 by nuptial pads present on fingers I and II (vs. on finger I), skin of throat relatively smooth (vs. granular), and tibiotarsal articulation reaching center of eye (vs. reaching tip of snout).

Discussion

The species diversity in the genus Gracixalus erected by Delorme et al. (2005) was underestimated. In recent years, a series of new species of this genus has been reported from China and Indochina, such as G. guangdongensis (Wang et al. 2018), G. tianlinensis (Chen et al. 2018), G. yunnanensis (Yu et al. 2019), G. ziegleri (Le et al. 2021), G. patkaiensis (Boruah et al. 2023), G. trieng (Rowley et al. 2020), G. weii (Liu et al. 2025), and G. huaping (Luo et al. 2025). Some of these recently described species (e.g. G. yunnanensis, G. guangdongensis, and G. huaping) have been confused with G. jinxiuensis, a species was once recorded widely in China, Vietnam, and Laos (Orlov et al. 2004; Nguyen et al. 2008; Shen 2014; Matsui et al. 2015, 2017; Rowley et al. 2011; Tran et al. 2023), implying that the species diversity of G. jinxiuensis species complex is underestimated and more studies are needed to clarify the taxonomic status of other records of G. jinxiuensis (Luo et al. 2025). Our finding of the new species G. liusanjieae sp. nov. from Mao’er Mountain, Guangxi further confirms this viewpoint and indicates that more efforts on the taxonomy of G. jinxiuensis species complex will likely result in further discoveries of yet unknown lineages.

Including the new species G. liusanjieae sp. nov., the genus Gracixalus now contains 23 species, 12 of which are distributed in China, and six in Guangxi (Fig. 1). 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). In recent years, a series of new species and new records of amphibian and reptile were discovered in Guangxi, especially in its northeastern part that geographically belongs to Nanling Mountains area (e.g., Lyu et al. 2019; Chen et al. 2022; Pan et al. 2025). The Nanling Mountains are the largest mountain range as well as an important natural geographical boundary between southern and central subtropical China (Zhou et al. 2018). Serving as refugium, evolutionary museum, and geographic barrier for various floral and faunal taxa (López-Pujol et al. 2011; Gong et al. 2016; Tian et al. 2018; Hu et al. 2021; Mi et al. 2021; Wang et al. 2023) and recognized as a biodiversity hotspot, the region of Nanling Mountains has exceptional species richness and is characterized by exceptionally high levels of species endemism for animals (Wang et al. 2024; Xu et al. 2024). In the case of amphibian biodiversity in China, the hotspot of Nanling Mountains is second only to the hotspot of Southwest Yunnan Mountains (Xu et al. 2024). The Nanling Mountains are composed of a series of discontinuous mountains separated by basins, terraces, plains, and hills with low altitude (Miao et al. 2020), which might have promoted the divergence and speciation of species with low dispersal ability that adapted to relatively high altitude such as Gracixalus species in this region (G. huaping, G. jinxiuensis, and G. liusanjieae sp. nov.).

Acknowledgements

We are deeply indebted to Zhilin Chen for his assistance with sample collection and thank the staff from Maoershan National Nature Reserve for their help during the field surveys. This work was supported by the National Natural Science Foundation of China (32460128, 32060114) and Guangxi Natural Science Foundation Project (2022GXNSFAA035526).

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Xiangjian Wu and Yuanqiang Pan contributed equally to this work.

Supplementary material

Supplementary material 1 

Genetic distance (uncorrected p-distance) between Gracixalus species estimated from 16S sequences

Xiangjian Wu, Yuanqiang Pan, Ju Chen, Jianping Ye, Guohua Yu, Tongxiang Zou

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.
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