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Research Article
A new species of Zhangixalus (Anura, Rhacophoridae) from Yunnan, China
expand article infoYuanqiang Pan‡, Mian Hou§, Guohua Yu‡, Shuo Liu|
‡ Guangxi Normal University, Guilin, China
§ Sichuan Normal University, Chengdu, China
| Kunming Natural History Museum of Zoology, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China

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

Academic editor: Umilaela Arifin
© 2024 Yuanqiang Pan, Mian Hou, Guohua Yu, Shuo Liu.
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: Pan Y, Hou M, Yu G, Liu S (2024) A new species of Zhangixalus (Anura, Rhacophoridae) from Yunnan, China. Zoosystematics and Evolution 100(1): 183-197. https://doi.org/10.3897/zse.100.113850
ZooBank: urn:lsid:zoobank.org:pub:FB7E03EC-A2BB-4C9C-9896-20E53A2667DB
Open Access

Abstract

We described herein Zhangixalus yunnanensis sp. nov., a new treefrog species from central and western Yunnan, China, which had previously been confused with Z. nigropunctatus, based on morphological and molecular evidence. Our phylogenetic analyses revealed that the new species is sister to the clade of Z. nigropunctatus and Z. melanoleucus with strong support (100% and 73% for BI and ML, respectively). Our morphological analysis suggested that Z. yunnanensis sp. nov. is distinctly different from all known congeners by the combination of the following morphological characters: black blotches on body flank and hind-limb, medium body size (SVL31.3–36.0 mm in males and 47.6–48.6 mm in females), head wider than long, iris yellowish-brown, dorsum uniformly green, vocal sac external, throat black, webbing greyish and fingers webbed one third and toes webbed half. Additionally, we revealed that the specimens ROM 38011 (Sa Pa, Vietnam) and VNMN 4099 (Son La, Vietnam) are neither Z. dorsoviridis nor Z. nigropunctatus, but probably represent one or two cryptic species of Zhangixalus pending further morphological and molecular data. Including the new species described herein, the genus Zhangixalus currently comprises 42 species, 30 of which are distributed in China with 11 species known from Yunnan. Amongst different zoogeographic regions in Yunnan, south-eastern Yunnan has the highest diversity of Zhangixalus, followed by western Yunnan and southern Yunnan. More studies are required to clarify the species diversity of this genus based on multiple lines of evidence (e.g. morphological and molecular data).

Key Words

cryptic species, treefrog, Zhangixalus, Zhangixalus nigropunctatus

Introduction

The treefrog genus Zhangixalus Li, Jiang, Ren & Jiang, 2019, recently removed from Rhacophorus Kuhl & Van Hasselt, 1822 sensu lato and recognised as a distinct genus, is characterised by medium-to-large body size, absence of dermal folds along limbs, absence of tarsal projections and usual green dorsal colouration (Jiang et al. 2019). The genus is distributed widely in South, East and Southeast Asia and currently contains 41 species (Frost 2023). In China, 29 species of Zhangixalus have been recorded to date, 10 of which are distributed in Yunnan: Z. burmanus (Andersson, 1939), Z. dorsoviridis (Bourret, 1937), Z. duboisi (Ohler, Marquis, Swan & Grosjean, 2000), Z. feae (Boulenger, 1893), Z. franki Ninh, Nguyen, Orlov, Nguyen & Ziegler, 2020, Z. nigropunctatus (Liu, Hu & Yang, 1962), Z. omeimontis (Stejneger, 1924), Z. pachyproctus Yu, Hui, Hou, Wu, Rao & Yang, 2019, Z. puerensis (He, 1999) and Z. smaragdinus (Blyth, 1852) (AmphibiaChina 2023).

Amongst others, the taxonomy of Zhangixalus nigropunctatus is confusing. This species was originally described from western Guizhou, China (Weining and Shuicheng) by Liu et al. (1962) and, afterwards, was recorded from Yunnan (Longling, Longchuan, Yingjiang, Qiaojia, Pingbian, Jinping) (Yang 1991; Zhao and Yang 1997; Fei 1999; Fei et al. 2005, 2009, 2010; Yang and Rao 2008; Yu et al. 2008), Hunan, and Anhui (Fei 1999; Fei et al. 2005, 2009, 2010). Orlov et al. (2001) considered that Z. nigropunctatus is closely allied with Z. dorsoviridis based on the descriptions of the former and, therefore, noted that Z. nigropunctatus was possibly a synonym of Z. dorsoviridis. Yu et al. (2009) revealed that Z. nigropunctatus from Pingbian did not group together with topotypes of Z. nigropunctatus, indicating that misidentification may be involved within Z. nigropunctatus. Li et al. (2012a) revealed that both Z. nigropunctatus and Z. dorsoviridis did not form a monophyletic group. They found that Z. nigropunctatus from the type locality and Longling formed clade II with a sample of Z. dorsoviridis (ROM 38011) from Sa Pa, northern Vietnam and this clade was closely related to Z. chenfui (Liu, 1945). Meanwhile, Z. nigropunctatus from Jinping and Pingbian grouped into clade I with another sample of Z. dorsoviridis from Sa Pa (ROM 38015; see fig. 1 of Li et al. (2012a)). Based on morphological evidence, Li et al. (2012a) referred clade II to true Z. nigropunctatus and referred clade I to Z. dorsoviridis. This finding implies that previous records of Z. nigropunctatus from Jinping and Pingbian belong to Z. dorsoviridis (Zhang et al. 2011) and that Z. nigropunctatus is distributed in northern Vietnam. However, Mo et al. (2016) revealed that the sample ROM 38011 was distantly related to Z. nigropunctatus and it was sister to Z. pinglongensis (Mo, Chen, Liao & Zhou, 2016), indicating that the record of Z. nigropunctatus in northern Vietnam may be misidentified. In addition, Pan et al. (2017) revealed that Z. nigropunctatus recorded from the Dabie Mountains of Anhui Province represents an independent species and named it as Z. zhoukaiyae (Pan, Zhang & Zhang, 2017).

During our recent field surveys in central Yunnan, China, specimens of a treefrog species resembling Zhangixalus nigropunctatus were collected from Xinping County. Amphibian diversity in Yunnan is still poorly understood. In recent years, a number of new amphibian species have been reported from Yunnan, China (e.g. Gan et al. (2020); Du et al. (2022); Wang et al. (2022a); Tang et al. (2023a, 2023b)). Considering the taxonomic history of Z. nigropunctatus and the status of amphibian diversity in Yunnan, we sequenced our newly-collected treefrogs from Yunnan to confirm their identity. Our phylogenetic analyses recovered these specimens as a distinct lineage and are sister to the clade composed of Z. nigropunctatus and Z. melanoleucus Brakels, Nguyen, Pawangkhanant, Idiiatullina, Lorphengsy, Suwannapoom & Poyarkov, 2023. Furthermore, our morphological examination suggested that this lineage differs from Z. nigropunctatus, Z. melanoleucus and other members of Zhangixalus by a series of characters. Herein, we officially describe this lineage as a new species of Zhangixalus.

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 July 2019 and April 2020 at Xinping County, Yunnan, China (Fig. 1) and a total of nine treefrog specimens were collected during the surveys. Specimens were photographed, euthanised, fixed and then stored in 75% ethanol. Liver tissues were preserved in 99% ethanol. Specimens were deposited at Guangxi Normal University (GXNU).

Figure 1. 

Known distribution sites of Zhangixalus yunnanensis sp. nov. in Yunnan, China. The red star represents the type locality of the new species. The map was generated using ArcMap v.10.2 (ESRI Inc.).

Phylogenetic analysis

Total genomic DNA was extracted from liver tissues stored in 99% ethanol. A fragment encoding mitochondrial 12S rRNA, tRNAval and 16S rRNA genes was amplified and sequenced using the primers and protocols of Yu et al. (2019). Seven samples were newly sequenced and all new sequences have been deposited in GenBank under Accession Nos. PP177446 and PP187265‒PP187270 (Table 1). Additionally, 43 homologous sequences of other Zhangixalus species and outgroups were obtained from GenBank (Table 1). Theloderma albopunctatum (Liu & Hu, 1962), Rhacophorus rhodopus Liu & Hu, 1960 and Leptomantis gauni (Inger, 1966) were included in the data as outgroups.

Table 1.
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Species used for phylogenetic analyses in this study (Z. = Zhangixalus).

Species Voucher No. Locality Accession No.
Theloderma albopunctatum ROM 30246 Vietnam AF458148
Rhacophorus rhodopus SCUM 060692L Mengyang, Yunnan, China EU215531
Leptomantis gauni FMNH 273928 Sarawak, Malaysia JX219456
Zhangixalus dulitensis BORNEENSIS09087 Borneo, Malaysia AB847123
Z. pachyproctus KIZ 090148 Puer, Yunnan, China MN613222
Z. smaragdinus KIZ 20160298 Yingjiang, Yunnan, China MN613219
Z. dennysi ROM 30249 Vietnam AF458139
Z. feae SCUM 050642W Hekou, Yunnan, China EU215544
Z. chenfui SCUM 060404L Mt. Omei, Sichuan, China EU215534
Z. jodiae VNMN 07122 Vietnam LC545595
Zhangixalus sp. ROM 38011 Sa Pa, Lao Cai, Vietnam JX219427
VNMN 4099 Son La, Vietnam LC010577
Z. dorsoviridis ROM 38015 Sa Pa, Lao Cai, Vietnam JX219423
Rao060821200 Jinping, Yunnan, China JX219424
YN080446 Pingbian, Yunnan, China JX219425
Rao060821199 Pingbian, Yunnan, China JX219426
KIZ 060821287 Jinping, Yunnan, China EF564563
Z. yaoshanensis NHMG150408 Jinxiu, Guangxi, China MG322122
Z. pinglongensis NHMG201002011 Shiwandashan, Guangxi, China KU170684
Z. nigropunctatus KIZ07061001 Weining, Guizhou, China EU924623
GZ070658 Weining, Guizhou, China JX219430
SCUM 070657L Weining, Guizhou, China EU215533
GXNU YU000361 Weining, Guizhou, China PP187265
GXNU YU000362 Weining, Guizhou, China PP187266
GXNU YU000363 Weining, Guizhou, China PP187267
Z. yunnanensis sp. nov. GXNU YU20160340 Xinping, Yunnan, China PP187268
GXNU YU20160268 Xinping, Yunnan, China PP187269
GXNU YU20160267 Xinping, Yunnan, China PP187270
Rao3494 Longling, Yunnan, China JX219429
Rao3496 Longling, Yunnan, China JX219428
Z. moltrechti SCUM 061106L Lianhuachi, Taiwan, China EU215543
Z. schlegelii - Hiroshima, Japan AB202078
Z. arboreus TTU-R-11748 Japan AF458142
Z. puerensis SCUM 060649L Puer, Yunnan, China EU215542
Z. dugritei SCUM 051001L Baoxing, Sichuan, China EU215541
Z. hui Li01 Zhaojue, Sichuan, China JN688878
Z. hongchibaensis CIB 097687 Wuxi, Chongqing, China JN688883
Z. hungfuensis SCUM 060425L Wenchuan, Sichuan, China EU215538
Z. minimus KIZ 061214YP Mt. Dayao, Guangxi, China EU215539
Z. burmanus SCUM 060614L Mt. Gaoligong, Yunnan, China EU215537
Z. franki VNMN 011687 Ha Giang, Vietnam LC548746
Z. duboisi SCUM 061104L Pingbian, Yunnan, China EU215536
Z. omeimontis SCUM 0606137L Pengxian, Sichuan, China EU215535
Z. zhoukaiyae AHU-RhaDB-120428 Jinzhai, Anhui, China KU601502
HM05293 Anhui, China PP177446
Z. lishuiensis YPX47792 Lishui, Zhejiang, China KY653720
Z. melanoleucus BEI 01010 Phou Samsoum Mt., Xiengkhoang, Laos OQ305233
ZMMU A-7781 Phou Samsoum Mt., Xiengkhoang, Laos OQ305234
BEI 01011 Phou Samsoum Mt., Xiengkhoang, Laos OQ305235
AUP 02507 Phou Samsoum Mt., Xiengkhoang, Laos OQ305236

Sequences were aligned using MUSCLE with default parameters in MEGA v.7.0 (Kumar et al. 2016). Uncorrected pairwise distances (using 16S rRNA sequences) between species were calculated in MEGA v.7.0. The best substitution model was selected using the corrected Akaike Information Criterion (AICc) in jModelTest v.2.1.10 (Darriba et al. 2012). Bayesian Inference was performed in MrBayes v.3.2.6 (Ronquist et al. 2012) under the selected substitution model (GTR + I + G). Two runs were performed simultaneously with four Markov chains starting from a random tree. The chains were run for 3,000,000 generations and sampled every 100 generations. The first 25% of the sampled tree was discarded as burn-in after the standard deviation of split frequencies of the two runs was less than 0.01. The remaining trees were then used to create a consensus tree and to estimate Bayesian posterior probabilities (BPPs). In addition, a Maximum Likelihood (ML) analysis was conducted in raxmlGUI v.2.0 (Edler et al. 2021) with 1000 rapid bootstrap replicates. The node was considered strongly supported with BPP ≥ 0.95 and bootstrap value ≥ 70 (Huelsenbeck and Hillis 1993; Leaché and Reeder 2002).

Morphology

Morphometric data were taken using electronic digital calipers to the nearest 0.1 mm. Morphological terminology followed Yu et al. (2019). 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); upper eyelid width (UEW, maximum width of upper eyelid); eye diameter (ED, diameter of exposed portion of eyeball); distance between nostril and eye (DNE, from nostril to anterior border of eye); tympanum diameter (TD, the greater of tympanum vertical and horizontal diameters); forearm and hand length (FHL, from elbow to tip of third finger); tibia length (TL, distance from knee to heel); foot length (FL, from proximal end of inner metatarsal tubercle to tip of fourth toe); and length of foot and tarsus (TFL, from tibiotarsal joint to tip of fourth toe). Webbing formula followed Myers and Duellman (1982).

Comparative morphological data of other Zhangixalus species were taken from their original descriptions or re-descriptions (Boulenger 1892; Stejneger 1907; Inger 1947; Liu et al. 1962; Lue et al. 1994, 1995; He 1999; Ohler et al. 2000; Harvey et al. 2002; Matsui and Panha 2006; Rao et al. 2006; Bordoloi et al. 2007; Chou et al. 2007; Fei et al. 2009, 2010; Zhang et al. 2011; Li et al. 2012b; Mo et al. 2016; Liu et al. 2017; Pan et al. 2017; Yu et al. 2019; Nguyen et al. 2020; Ninh et al. 2020; Brakels et al. 2023). Multivariate principal component analysis (PCA) was conducted in SPSS v.17.0 (SPSS Inc.), based on the correlation matrix of size-standardised measurements (all measurements divided by SVL) of adult males. Scatter plots of the scores of the first two factors of PCA were used to examine the differentiation between the new species and its closest relatives revealed by phylogenetic analyses (Z. nigropunctatus and Z. melanoleucus). Differences in quantitative characters of adult males between these three species were also evaluated with t-tests in SPSS. In these analyses, Levene’s test was also performed for each character to test for equality of variances. Specimens of Z. nigropunctatus were collected from the type locality and data for Z. melanoleucus were retrieved from Brakels et al. (2023).

Results

Phylogenetic relationship

The obtained sequence alignment was 1971 bp. Phylogenetic analyses revealed that the specimens from Xinping, Yunnan form a distinct clade with two individuals previously identified as Z. nigropunctatus (KIZ Rao3494 and KIZ Rao3496; Li et al. (2012a)) from Longling, Yunnan. This clade was recovered as sister to the clade consisting of Z. nigropunctatus from the type locality and Z. melanoleucus with strong support (Fig. 2). The genetic distances between the novel lineage and other Zhangixalus species ranged from 4.8% to 13.9% in 16S rRNA sequences and the distances between the novel lineage, Z. nigropunctatus and Z. melanoleucus ranged from 5.1%‒5.5% (Suppl. material 1).

Figure 2. 

Bayesian phylogram of Zhangixalus inferred from mitochondrial 12S-tRNA-16S sequences. Numbers above and below branches are Bayesian posterior probabilities and ML bootstrap values (only values above 50% are shown), respectively.

Additionally, KIZ 060821287 and ROM 38011, two samples previously identified as Z. nigropunctatus by Yu et al. (2008) and Li et al. (2012a), respectively, were not grouped together with topotypes of Z. nigropunctatus. KIZ 060821287 was nested within the clade of Z. dorsoviridis from the type locality (Sa Pa, Lao Cai, Vietnam) and Yunnan and this clade was sister to the clade of Z. lishuiensis and Z. zhoukaiyae. ROM 38011 was sister to the lineage of nominal Z. dorsoviridis from Son La, Vietnam (VNMN 4099) and the two together were sister to the clade of Z. yaoshanensis (Liu & Hu, 1962) and Z. pinglongensis with strong support (98% for BI and 83% for ML; Fig. 2).

Morphometric analysis

Morphometric data are summarised in Table 2. Three measurements (FHL, TFL and FL) were not included in PCA analysis because they were not available for Z. melanoleucus. We retained the first two principal components that accounted for 66.99% of the total variance (Table 3). Loadings for PC1, which accounted for 45.21% of the total variance, were most heavily loaded on internarial distance (IND) and Loadings for PC2, which accounted for 21.78%, were most heavily loaded on head length (HL). Differentiation was found along both the PC1 and PC2 axis between the specimens from Xinping and Z. nigropunctatus from the type locality and differentiation was also found along the PC2 axis between the specimens from Xinping and Z. melanoleucus (Fig. 3). The results of PCA analysis revealed distinct morphometric differences in head length and internarial distance between the specimens from Xinping and Z. nigropunctatus from the type locality, as well as in head length between the specimens from Xinping and Z. melanoleucus. Moreover, the t-tests demonstrated that male specimens from Xinping differed significantly (p < 0.05) from male topotypes of Z. nigropunctatus in HL, HW, IND, UEW, TD, DNE and TFL (Table 4) and differed significantly (p < 0.05) from Z. melanoleucus in HL, SL, UEW and ED (Table 5). Additionally, the new lineage is distinguishable from its congeners by body size and the combination of texture and colouration pattern. Therefore, on the basis of the above molecular and morphological evidence, we officially describe them as a new species of the genus Zhangixalus.

Table 2.
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Measurements of Zhangixalus yunnanensis sp. nov. (1‒9), Z. nigropunctatus (10‒17) and Z. melanoleucus (18‒21). Specimens of Z. nigropunctatus were collected from the type locality (Weining, Guizhou) and data of Z. melanoleucus were obtained from Brakels et al. (2023).

ID Voucher no. Sex SVL HL HW SL IND IOD UEW ED TD DNE FHL TL TFL FL
1 GXNU YU20160267 M 31.3 9.7 11.4 4.9 4.0 4.0 2.8 3.5 1.9 2.2 17.3 13.3 21.8 14.9
2 GXNU YU20160268 M 34.0 11.1 12.6 5.3 4.0 4.0 3.2 4.0 2.1 2.2 16.9 13.0 21.6 14.8
3 GXNU YU20160269 M 35.4 11.5 12.5 5.5 4.1 4.1 3.4 4.1 2.1 2.2 18.7 14.5 23.2 15.7
4 GXNU YU20160335 F 48.6 15.1 18.0 7.2 5.6 5.6 4.2 4.9 2.9 3.0 25.5 20.0 32.5 22.5
5 GXNU YU20160336 M 35.3 11.2 13.6 5.4 4.3 4.3 3.5 4.3 2.3 2.2 18.3 14.3 23.5 15.5
6 GXNU YU20160337 M 35.6 11.6 12.9 5.3 4.2 4.2 3.2 4.3 2.2 2.1 18.3 13.9 22.4 15.1
7 GXNU YU20160338 M 34.4 11.3 12.9 5.3 4.3 4.5 3.3 4.1 2.2 2.3 18.1 14.9 22.9 15.6
8 GXNU YU20160340 M 36.0 11.8 13.9 5.5 4.3 4.3 3.6 4.3 2.3 2.2 18.5 14.3 23.5 16.2
9 GXNU YU20160355 F 47.6 14.4 17.2 6.8 5.8 5.7 4.1 5.0 2.9 2.9 25.5 20.5 33.2 23.3
10 KIZ25362 F 39.4 13.2 13.7 5.9 4.2 5.0 3.1 4.1 2.7 2.8 22.1 17.1 27.7 19.0
11 KIZ25367 F 43.7 13.0 13.8 6.1 4.2 4.8 3.0 4.3 2.8 2.8 22.7 17.1 27.5 20.0
12 KIZ25369 M 31.7 11.2 11.4 4.8 3.2 3.7 2.4 3.7 2.2 2.3 16.4 12.8 20.6 14.7
13 KIZ25370 M 33.6 11.5 12.0 5.1 3.4 3.9 2.7 3.9 2.3 2.3 17.4 12.9 21.4 15.8
14 KIZ25372 M 34.8 11.7 12.5 5.1 3.7 4.1 2.6 4.0 2.4 2.4 17.1 13.5 21.6 15.6
15 KIZ25373 M 34.1 11.9 12.1 4.9 3.7 4.0 2.7 3.9 2.5 2.3 18.6 13.4 21.9 15.2
16 KIZ25374 M 34.7 11.7 11.8 5.3 3.3 3.8 2.9 3.6 2.3 2.5 16.8 13.3 21.1 15.4
17 KIZ25375 M 33.2 11.8 11.3 5.1 3.4 4.0 2.7 3.7 2.2 2.2 16.5 11.9 20.4 14.3
18 BEI 01010 M 35.0 13.6 13.3 6.1 4.3 4.7 3.2 4.6 2.3 2.4 - 14.7 - -
19 BEI 01011 M 34.4 12.6 12.2 5.8 4.2 4.1 3.0 4.2 1.9 2.0 - 14.0 - -
20 ZMMU A-7781 M 36.3 13.3 13.4 6.1 4.4 4.8 3.1 4.7 2.2 2.6 - 14.3 - -
21 AUP 02507 M 34.4 12.0 12.0 5.4 4.2 4.2 3.1 4.1 1.9 2.4 - 13.8 - -
Table 3.
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Factor loadings of first two principal components of 10 size-adjusted male morphometric characteristics of Zhangixalus yunnanensis sp. nov., Z. nigropunctatus and Z. melanoleucus.

Character PC1 PC2
Eigenvalue 4.521 2.178
% variation 45.214% 21.778%
HL (head length) 0.124 0.898
HW (head width) 0.708 -0.185
SL (snout length) 0.698 0.527
IND (internarial distance) 0.926 -0.208
IOD (interorbital distance) 0.778 0.402
UEW (width of upper eyelid) 0.713 -0.571
ED (eye diameter) 0.801 0.363
TD (tympanum diameter) -0.577 0.192
DNE -0.323 0.596
TL (tibia length) 0.685 -0.072
Table 4.
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Summary statistics of male specimens (mean ± standard deviation) and results of the t-test between the Z. yunnanensis sp. nov. (n = 7) and Z. nigropunctatus (n = 6) from the type locality. The t-test was performed on the size-adjusted data, except SVL. * = p < 0.05, ** = p < 0.01.

Character Mean ± SD (n = 7) Mean ± SD (n = 6) Levene’s test t-test
Z. yunnanensis sp. nov. Z. nigropunctatus F p-value t p-value
SVL 34.6 ± 1.6 33.7 ± 1.2 0.370 0.555 1.129 0.283
HL 0.323 ± 0.0068 0.346 ± 0.0082 0.793 0.392 -5.420 0.000**
HW 0.371 ± 0.0122 0.352 ± 0.0092 0.388 0.546 3.144 0.009**
SL 0.154 ± 0.0026 0.150 ± 0.0039 2.043 0.181 2.117 0.058
IND 0.121 ± 0.0043 0.102 ± 0.0047 0.016 0.902 7.350 0.000**
IOD 0.122 ± 0.0056 0.116 ± 0.0037 1.862 0.200 1.956 0.076
UEW 0.095 ± 0.0041 0.079 ± 0.0034 0.297 0.597 7.468 0.000**
ED 0.118 ± 0.0034 0.113 ± 0.0049 0.519 0.486 2.266 0.045*
TD 0.062 ± 0.0021 0.069 ± 0.0026 0.010 0.921 -4.966 0.000**
DNE 0.064 ± 0.0038 0.069 ± 0.0025 1.169 0.303 -3.008 0.012*
FHL 0.522 ± 0.0171 0.509 ± 0.0226 0.925 0.357 1.146 0.276
TL 0.406 ± 0.0182 0.385 ± 0.1508 0.556 0.472 2.246 0.046*
FL 0.446 ± 0.0164 0450 ± 0.0143 0.029 0.868 -0.517 0.615
TFL 0.657 ± 0.0223 0.629 ± 0.0167 0.086 0.775 2.576 0.026*
Table 5.
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Summary statistics of male specimens (mean ± standard deviation) and results of the t-test between the Z. yunnanensis sp. nov. (n = 7) and Z. melanoleucus (n = 4) from the type locality. The t-test was performed on the size-adjusted data, except SVL. * = p < 0.05, ** = p < 0.01.

Character Mean ± SD (n = 7) Mean ± SD (n = 4) Levene’s test t-test
Z. yunnanensis sp. nov. Z. melanoleucus F p-value t p-value
SVL 34.6 ± 1.6 35.0 ± 0.9 0.856 0.379 -0.515 0.619
HL 0.323 ± 0.0068 0.368 ± 0.0163 1.420 0.264 -6.502 0.000**
HW 0.371 ± 0.0122 0.363 ± 0.0141 0.239 0.637 0.968 0.358
SL 0.154 ± 0.0026 0.167 ± 0.0072 3.050 0.115 -4.491 0.002**
IND 0.121 ± 0.0043 0.122 ± 0.0007 8.056 0.019 -0.773 0.467
IOD 0.122 ± 0.0056 0.127 ± 0.0074 1.342 0.276 -1.359 0.207
UEW 0.095 ± 0.0041 0.089 ± 0.0027 0.751 0.409 2.753 0.022*
ED 0.118 ± 0.0034 0.126 ± 0.0058 4.303 0.068 -2.737 0.023*
TD 0.062 ± 0.0021 0.059 ± 0.0050 6.278 0.034 1.211 0.300
DNE 0.064 ± 0.0038 0.067 ± 0.0061 0.836 0.384 -1.107 0.297
TL 0.406 ± 0.0182 0.406 ± 0.0110 1.215 0.299 0.057 0.956
Figure 3. 

Scatterplot of principal components 1 and 2 of size-adjusted male morphometric data of Z. yunnanensis sp. nov., Z. nigropunctatus and Z. melanoleucus.

Taxonomic account

Zhangixalus yunnanensis sp. nov.

https://zoobank.org/742755AC-423C-4080-8568-8E7D34700F36
Figs 4, 5, 6 Yunnan tree Frog/ Yún Nán Shù Wā (云南树蛙)

Type material

Holotype : GXNU YU20160340, adult male, collected on 20 April 2020 by Guohua Yu from Mopan Mountain, Xinping County, Yunnan Province, China (23°56'06"N, 102°01'18"E, 2443 m a.s.l.).

Figure 4. 

Views of the holotype (GXNU YU20160340) in life and in preservative. Photos by Guohua Yu.

Paratypes : GXNU YU20160267‒20160269, three adult males, collected on 14 July 2019 from the type locality by Guohua Yu; GXNU YU20160336‒20160338, three adult males, collected on 20 April 2020 by Guohua Yu from the type locality; and two adult females (GXNU YU20160335 and YU20160355), collected on 20 April 2020 by Guohua Yu from the type locality.

Figure 5. 

Views of vomerine teeth, hand and foot of the holotype. Photos by Guohua Yu.

Etymology

The species epithet is named for Yunnan, China, where the species was collected. We suggest the English common name “Yunnan tree frog” and the Chinese common name “Yún Nán Shù Wā (云南树蛙)”.

Figure 6. 

Views of the paratype GXNU YU20160267 in preservative (a, b) and the paratype GXNU YU20160335 in life. Photos by Guohua Yu.

Diagnosis

The new treefrog species is assigned to Zhangixalus by the presence of intercalary cartilage between terminal and penultimate phalanges of digits, Y-shaped distal end of terminal phalanx, tips of digits expanded into large discs bearing circum-marginal grooves, and vomerine teeth present, dermal folds along limbs not significant, tarsal projections absent, green dorsa colouration and medium body size (Jiang et al. 2019). Phylogenetically, the new species is nested within the genus Zhangixalus with strong support (100% for BI and 73% for ML).

Zhangixalus yunnanensis sp. nov. can be distinguished from its congeners by a combination of the following characters: 1) body size medium (SVL 31.3–36.0 mm [34.6 ± 1.6, n = 7] in males and 47.6–48.6 mm [48.1 ± 0.71, n = 2] in females); 2) head wider than long; 3) iris yellowish-brown; 4) tibiotarsal articulation reaching posterior corner of eye; 5) IND/SVL 11.6%‒12. 8% (12.1% ± 0.0043, n =7) in males; 6) HL/SVL 31.0%‒32.8% (32.3% ± 0.0068, n = 7) in males; 7) UEW/SVL 8.9%‒10.00% (9.5% ± 0.0041, n = 7) in males; 8) dorsum uniformly green; 9) black blotches in axilla, groin and posterior part of thigh; 10) vocal sac external, single; 11) throat black; 12) webbing greyish; and 13) fingers webbing formula I2‒2II2‒3III2‒2IV and toes webbing formula I2‒2II1.5‒2.5III2‒3IV3‒1.5V.

Description of holotype

Adult male, body robust, size small (SVL 36.0 mm); HL (11.8 mm) 84.9% of HW (13.9 mm); snout rounded, sloping in profile, protruding beyond margin of lower jaw in ventral view; snout (SL 5.5 mm) longer than eye (ED 4.3 mm); canthus rostralis blunt; lore region oblique, slightly concave; nostril oval, slightly protuberant, located at the middle between snout tip and eye; IND (4.3 mm) equal to IOD and wider than UEW (3.6 mm); pineal spot absent; pupil oval, horizontal; tympanum distinct (TD 2.3 mm), rounded, slightly greater than half of ED, nearly equal to the distance between eye and nostril (DNE 2.2 mm); supratympanic fold distinct, curves from posterior edge of eye to insertion of arm; vomerine teeth in two oblique series touching inner front edge of choanae, separated by space almost equal to length of each series; choanae oval; tongue attached anteriorly and notched posteriorly; single external vocal sac, with a sac slit opening on floor of mouth at each corner.

Relative length of fingers I < II < IV < III; tips of all fingers expanded into discs with circum-marginal grooves; nuptial pad present on first finger; fingers webbed one third, webbing formula I2‒2II2‒3III2‒2IV; lateral fringe on free edge of all fingers; subarticular tubercles prominent and rounded, formula 1, 1, 2, 2; supernumerary tubercles present; inner metacarpal tubercle large, ovoid, outer metacarpal tubercle smaller in size, flattened; white dermal fringe along outer edge of forearm present, not well developed.

Hind limbs relatively short, heels do not meet when legs positioned at right angle to body; tibiotarsal articulation reaching posterior margin of eye; relative length of toes I < II < V < III < IV; tibia (TL 14.3 mm) 39.7% of body size, shorter than foot (FL16.2 mm); tips of toes expanded into discs with circum-marginal grooves, smaller than finger discs; toes webbed, webbing formula I2‒2II1.5‒2.5III2‒3IV3‒1.5V; subarticular tubercles prominent and rounded, formula 1, 1, 2, 3, 2; supernumerary tubercles present; inner metatarsal tubercle oval, prominent; outer metatarsal tubercle absent; white dermal fringe along outer edge of tibia, tarsus and fifth toe.

Dorsal surface of body and head smooth; dorsolateral folds absent; throat smooth; chest, belly and ventral surface of thigh granular; a few white warts around vent.

Colouration in life

Iris yellowish-brown with dark wash; dorsal surface green; side of head and tympanic region green; lower part of flanks cream mottled with greyish-brown; throat black; venter and chest cream-white mottled with yolk yellow; limbs dorsally green and ventrally light yellow; anterior and posterior of thigh light yellow; large black blotches in axilla, groin and posterior part of thigh; white fringes along outer edge from elbow to the fourth finger and from tibiotarsal articulation to the fifth toe; webbing between fingers and toes grey; dorsal surface of discs greyish-brown.

Colouration in preservative

Dorsal surface violet; lower part of flanks and ventral surface of body and limbs white.

Sexual dimorphism

The new species is sexually dimorphic with females being distinctly larger than males (Table 2) and having no vocal sac and nuptial pad. Males have external single subgular vocal sac and light yellow nuptial pad on base of finger I.

Morphological variation

The colour pattern varied amongst individuals. The paratype GXNU YU20160267 has no black blotches on both anterior and posterior parts of the thigh (Fig. 6a, b). The holotype (GXNU YU20160340) and two paratypes (GXNU YU20160267 and GXNU YU20160338) have no black blotches on tibia and tarsal, paratypes GXNU YU20160268 and GXNU YU20160269 have black blotches on tarsal, but have none on tibia and the other four paratypes (GXNU YU20160355, YU20160336, YU20160335 and YU20160337) have black blotches on both tibia and tarsal. The two specimens from Longling have no black blotches on tibia and tarsal. Dorsal surfaces are uniformly green in all types with the exception of GXNU YU20160335, which has small yellow spots scattered on the dorsal surface of the body and limbs (Fig. 6c). In addition, colouration of the new species in life can vary as day period and microhabitat change; dorsum is lighter nocturnally than during the day, with dorsal surfaces appearing light to dark green.

Distribution and ecology

The new species is currently known from the type locality (Mt. Mopan, Xinping, Yunnan) and Longling, Yunnan, China. Breeding was observed from April to July at the type locality. In April, adult males assembled and called on ground beside permanent pools (Fig. 7a) and the eggs were laid in white foam nests deposited in mud near the pool (Fig. 7b); in mid-July, sparse calls of this species were heard. Zhangixalus puerensis and Hyla anectans (Jerdon, 1870) were found at the same site. The Jinping and Pingbian populations previously recorded as Z. nigropunctatus have been re-assigned into Z. dorsoviridis (Li et al. 2012a) and we confirmed that the sample KIZ 060821287, which was collected from Jinping and identified as Z. nigropunctatus (Yu et al., 2008), also belongs to Z. dorsoviridis. Considering that Longchuan and Yingjiang are close to Longling, the populations previously recorded as Z. nigropunctatus from Longchuan and Yingjiang of Yunnan probably belong to the new species pending more data.

Figure 7. 

Habitat of the new species at the type locality (a) and foam nests of the new species (b). Photos by Guohua Yu.

Comparison

Phylogenetically, the new species is closely related to Zhangixalus nigropunctatus and Z. melanoleucus. Zhangixalus yunnanensis sp. nov. can be distinguished from Z. nigropunctatus, with which the new species has previously been confused, by yellowish-brown iris (vs. yellowish-gold; Fig. 8 and Table 6), head obviously wider than long (vs. head width nearly equal to head length), tibiotarsal articulation reaching posterior corner of eye (vs. reaching posterior edge of tympanum), wider internarial space (mean IND/SVL in males 12.1% ± 0.0043 [11.6%‒12.8%, n = 7] vs. 10.2% ± 0.0047 [9.51%‒10.85%, n = 6]), longer hind-limb (mean TFL/SVL in males 65.7% ± 0.0223 [62.92%‒69.65%, n = 7] vs. 62.9% ± 0.0167 [60.8%‒65.0%, n = 6]), shorter head (mean HL/SVL in males 32.3% ± 0.0068 [31.0%‒32.8%, n = 7] vs. 34.6% ± 0.0082 [33.6%‒35.5%, n = 6]), wider upper eyelid (mean UEW/SVL in males 9.5% ± 0.0041 [8.9%‒10.0%, n = 7] vs. 7.9% ± 0.0034 [7.5%‒8.4%, n = 6]), wider head (mean HW/SVL 37.1% ± 0.0122 [35.3%‒38.6%, n = 7] vs. 35.2% ± 0.0092 [34.0%‒36.0%, n = 6]), smaller tympanum (mean TD/SVL 6.2% ± 0.0021 [5.9%‒6.5%, n = 7] vs. 6.9% ± 0.0026 [6.6%‒7.3%, n = 6]) and smaller DNE (mean DNE/SVL 6.4% ± 0.0038 [5.9%‒7.0%, n = 7] vs. 6.9% ± 0.0025 [6.7%‒7.3%, n = 6]) (Tables 1, 4; Fig. 3); and from Z. melanoleucus by yellowish-brown iris (vs. reddish-orange; Fig. 8), shorter head (mean HL/SVL in males 32.3% ± 0.0068 [1.0%‒32.8%, n = 7] vs. 36.8% ± 0.0163 [34.9%‒38.9%, n = 4]), shorter snout (mean SL/SVL in males 15.4% ± 0.0026 [14. 9%‒15.7%, n = 7] vs. 16.7% ± 0.0072 [15.7%‒17.4%, n = 4]), wider upper eyelid (mean UEW/SVL in males 9.5% ± 0.0041 [8.9%‒10.0%, n = 7] vs. 8.9% ± 0.0027 [8.5%‒9.1%, n = 4]) and smaller eye (mean ED/SVL 11.8% ± 0.0034 [11.2%‒12.2%, n = 7] vs. 12.6% ± 0.0058 [11.9%‒13.1%, n = 4]) (Fig. 3; Table 5).

Table 6.
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Morphological comparison between the new species and congeners of Zhangixalus. Characters are: ① dorsum colour: 0 = solid green or green with a few small light spots, 1 = green mottled with brown, 2 = green mottled with black, 3 = brownish; ② black blotches in axilla, groin and thigh: 0 = present, 1 = absent; ③ vocal sac: 0 = external, 1 = internal, 2 = absent; ④ throat: 0 = blackish, 1 = not blackish; ⑤ development of finger web: 0 = webbed one third, 1 = webbed half, 2 = entirely webbed, 3 = rudimentary; ⑥ development of toe web: 0 = webbed half, 1 = entirely webbed, 2 = webbed 2/3, 3 = webbed 1/3. “?” means unknown.

Species Body size ① ② ③ ④ ⑤ ⑥ Webbing color Iris color
Male Female
Z. yunnanensis sp. nov. 31.3–36.0 47.6–48.6 0 0 0 0 0 0 greyish yellowish-brown
Z. achantharrhena 36.5–40.6 47.1 2 1 0 1 1 2 mottled red and green dull red edged with silvery white
Z. chenfui 33–41 46–55 0 1 0 1 0 0 pale brownish-yellow reddish-orange
Z. dorsoviridis 31.3–42.4 37.9–42.8 0 0 1 1 0 0 greyish-brown orange red
Z. dulitensis 38.4–39.7 ? 1 1 ? 1 2 1 webbing between outer two toes red reddish
Z. hungfuensis 30.8–36.8 45.5 0 1 0 1 0 0 greyish greenish
Z. jarujini 33.7–40.0 41.5–46.1 3 1 1 1 0 1 reddish-orange yellow
Z. jodiae 34.8–39.8 ? 0 0 0 1 0 0 cream with black blotches silver
Z. leucofasciatus 35–48.2 ? 0 1 ? 1 2 1 greyish black or greyish-purple yellowish
Z. lishuiensis 34.2–35.8 45.9 0 1 1 1 0 0 golden yellow yellow
Z. melanoleucus 34.4–36.3 53.7 0 0 0 0 0 2 cream with black blotches reddish-orange
Z. minimus 21.3–33.0 31.7–38.3 0 1 0 0 3 3 grey mottled with dark blotches yellowish-gold
Z. moltrechti 36.3–42.4 44.7–54.6 0 0 0 1 1 1 orange red spotted with black red or red brown
Z. nigropunctatus 32.0–37.0 44.0–45.0 0 0 0 0 3 0 greyish yellowish-gold
Z. pinglongensis 32.0–38.5 ? 0 0 0 1 1 1 tangerine silver
Z. puerensis 35.5–41.1 52.0–55.2 1 0 0 0 0 0 grey mottled with dark blotches yellowish-gold
Z. schlegelii 32–43 43–53 0 0 1 1 1 2 purple yellowish-gold
Z. suffry 38.5–52.9 31.5–61.0 0 1 ? 1 2 1 orange red yellowish-gold
Z. taipeianus 30.4–37.9 39.1–43.0 0 1 0 1 3 1 yellow yellowish-gold
Z. wui 35.2–38.2 48.6 1 0 1 1 3 3 greyish-brown yellowish-gold
Z. yaoshanensis 31.6–36.4 49.2–51.1 0 1 0 1 3 1 greyish pale yellowish-gold
Z. zhoukaiyae 27.9–37.1 42.1–44.7 0 0 0 1 1 2 greyish golden-yellow
Z. amamiensis 45–56 65–76 0 0 1 1 0 1 purple greenish
Z. arboreus 42–60 59–82 0 0 1 1 1 1 purple reddish
Z. arvalis 39.0–46.4 59.5–64 0 1 0 1 1 0 pink yellow
Z. aurantiventris 47.8–53.9 ? 0 1 1 1 1 2 orange-red pale yellow
Z. burmanus 54–72 66–82 1 0 1 1 0 2 dull brown greenish
Z. dennysi 68–92 83–109 0 1 1 1 1 1 brown yellowish-gold
Z. duboisi >61.5 ? 1 0 2 1 1 2 black and white dark gold
Z. dugritei 41.5–45.4 57.7–64.3 1 0 0 1 1 2 black and grey yellowish-brown
Z. feae 86–111 68–116 0 1 1 1 2 1 green green-gold
Z. franki 77.9–85.8 ? 1 1 ? 1 2 1 grey bronze
Z. hongchibaensis 46.5–49.7 55.3 1 0 1 1 3 3 grey yellowish-brown
Z. hui 40–45.4 ? 1 0 0 1 3 3 black and grey reddish-brown
Z. omeimontis 52.0–65.5 70.0–79.5 1 0 1 1 1 1 yellowish yellowish-gold
Z. owstoni 42–51 ? 0 0 1 1 1 2 yellow yellowish-gold mottled with silver
Z. pachyproctus 73.4–78.2 102.4 0 1 1 1 2 1 pale blue black bronze
Z. prasinatus 44.8–58.5 63.9–66.9 0 0 0 1 1 1 purple yellowish-gold
Z. prominanus 50.5–51.3 ? 1 1 ? 1 1 1 red reddish
Z. smaragdinus 76.3–79.6 ? 0 1 1 1 2 1 blue black yellowish-gold
Z. viridis 41–54 52–68 0 0 1 1 1 2 red yellow
Z. yinggelingensis 43.0–43.4 ? 0 0 ? 1 0 0 red silver
Figure 8. 

Irises of the new species (a. Photo by Guohua Yu), Z. nigropunctatus (b. Photo by Guohua Yu) and Z. melanoleucus (c. reproduced from Brakels et al. (2023)).

In body size, besides Zhangixalus nigropunctatus and Z. melanoleucus, the new species is relatively similar to Z. achantharrhena (Harvey, Pemberton & Smith, 2002), Z. chenfui, Z. dorsoviridis, Z. dulitensis (Boulenger, 1892), Z. hungfuensis (Liu & Hu, 1961), Z. jarujini (Matsui & Panha, 2006), Z. jodiae (Nguyen, Ninh, Orlov, Nguyen & Ziegler, 2020), Z. leucofasciatus (Liu & Hu, 1962), Z. lishuiensis (Liu, Wang & Jiang, 2017), Z. minimus (Rao, Wilkinsonand & Liu, 2006), Z. moltrechti (Boulenger, 1908), Z. pinglongensis, Z. puerensis, Z. schlegelii (Günther, 1858), Z. suffry (Bordoloi, Bortamuli & Ohler, 2007), Z. taipeianus (Liang & Wang, 1978), Z. wui (Li, Liu, Chen, Wu, Murphy, Zhao, Wang & Zhang, 2012), Z. yaoshanensis and Z. zhoukaiyae (Table 4). The new species can be easily distinguished from Z. achantharrhena, Z. dulitensis, Z. jarujini, Z. puerensis and Z. wui by dorsum uniformly green (vs. green with black and white spots in Z. achantharrhena, yellowish-green with a few purplish dots on head and back and a purplish line round snout in Z. dulitensis, brownish with dark marking in Z. jarujini, green with many reddish-brown blotches edged with dark brown in Z. puerensis and dark yellowish-brown to light green with numerous light-brown spots in Z. wui); from Z. achantharrhena, Z. chenfui, Z. dulitensis, Z. hungfuensis, Z. jarujini, Z. leucofasciatus, Z. lishuiensis, Z. minimus, Z. suffry, Z. taipeianus and Z. yaoshanensis by having black blotches in axilla, groin and posterior part of thigh (vs. absent); from Z. dorsoviridis, Z. jarujini, Z. lishuiensis, Z. schlegelii and Z. wui by vocal sac external (vs. internal); from Z. achantharrhena, Z. chenfui, Z. dorsoviridis, Z. dulitensis, Z. hungfuensis, Z. jarujini, Z. jodiae, Z. leucofasciatus, Z. lishuiensis, Z. moltrechti, Z. pinglongensis, Z. schlegelii, Z. suffry, Z. taipeianus, Z. wui, Z. yaoshanensis and Z. zhoukaiyae by throat black (vs. bright yellow in Z. achantharrhena, purplish flesh in Z. chenfui, yellow in Z. dorsoviridis and Z. taipeianus, cream in Z. hungfuensis and Z. jarujini, greyish in Z. jodiae and Z. yaoshanensis, white in Z. dulitensis, Z. leucofasciatus, Z. lishuiensis, Z. moltrechti and Z. zhoukaiyae, white with slightly grey background in Z. pinglongensis, cream-white in Z. schlegelii and Z. suffry and creamy-white with greyish-brown blotches in Z. wui); from Z. achantharrhena, Z. chenfui, Z. dulitensis, Z. jarujini, Z. jodiae, Z. lishuiensis, Z. minimus, Z. moltrechti, Z. pinglongensis, Z. puerensis, Z. schlegelii, Z. suffry and Z. taipeianus by webbing greyish (vs. having red colour in Z. achantharrhena, Z. dulitensis, Z. jarujini, Z. moltrechti, Z. pinglongensis and Z. suffry, pale brownish-yellow in Z. chenfui, mottled with black blotches in Z. jodiae, Z. minimus and Z. puerensis, purple in Z. schlegelii and yellow in Z. lishuiensis and Z. taipeianus); from Z. achantharrhena, Z. dulitensis, Z. leucofasciatus, Z. moltrechti, Z. suffry, Z. taipeianus and Z. zhoukaiyae by fingers webbed one third and toes webbed half (vs. fingers webbed half in Z. achantharrhena, Z. leucofasciatus and Z. zhoukaiyae, fingers entirely webbed in Z. dulitensis, fingers webbed half and toes webbed entirely in Z. moltrechti, fingers webbed entirely and toes webbed fully in Z. suffry and toes webbed entirely in Z. taipeianus); and from Z. achantharrhena, Z. chenfui, Z. dorsoviridis, Z. dulitensis, Z. jodiae, Z. moltrechti and Z. pinglongensis by iris yellowish-brown (vs. dull red, edged with silvery-white in Z. achantharrhena, orange-red in Z. chenfui and Z. dorsoviridis, reddish in Z. dulitensis, silver in Z. jodiae and Z. pinglongensis and red or reddish-brown in Z. moltrechti). The new species further differs from Z. hungfuensis and Z. wui by nuptial pad present on first finger (vs. present on fingers I and II), from Z. jodiae by lacking orange blotches in the groin, thigh and ventral side of the tibia (vs. present) and from Z. pinglongensis by black blotches on flank and hind-limb being fewer in number and discontinuous (vs. a number of black blotches united to be reticular).

The new species is distinguishable from Zhangixalus amamiensis (Inger, 1947), Z. arboreus (Okada & Kawano, 1924), Z. arvalis (Lue, Lai & Chen, 1995), Z. aurantiventris (Lue, Lai & Chen, 1994), Z. burmanus, Z. dennysi (Blanford, 1881), Z. duboisi, Z. dugritei (David, 1872), Z. feae, Z. franki, Z. hongchibaensis (Li, Liu Chen, Wu, Murphy, Zhao, Wang & Zhang, 2012), Z. hui (Liu, 1945), Z. omeimontis, Z. owstoni (Stejneger, 1907), Z. pachyproctus, Z. prasinatus (Mou, Risch & Lue, 1983), Z. prominanus (Smith, 1924), Z. smaragdinus, Z. viridis (Hallowell, 1861) and Z. yinggelingensis (Chou, Lau & Chan, 2007) by smaller body size (Table 4). Moreover, the new species can be easily distinguished from Z. burmanus, Z. duboisi, Z. dugritei, Z. hongchibaensis, Z. hui, Z. omeimontis and Z. prasinatus by dorsal surface uniformly green and no brown stripe along canthus rostralis and supratympanic fold (vs. dorsal surface green with brown blotches and a brown stripe along canthus rostralis and supratympanic fold or dorsum green with brown stripe along canthus rostralis and supratympanic fold); from Z. amamiensis, Z. arboreus, Z. aurantiventris, Z. burmanus, Z. dennysi, Z. feae, Z. hongchibaensis, Z. omeimontis, Z. owstoni, Z. pachyproctus, Z. smaragdinus, Z. viridis and Z. yinggelingensis by external vocal sac (vs. internal vocal sac); and from Z. arvalis, Z. aurantiventris, Z. dennysi, Z. feae, Z. franki, Z. pachyproctus, Z. prominanus and Z. smaragdinus by having black blotches in axilla, groin and anterior and posterior part of thighs (vs. absent).

Discussion

Zhangixalus nigropunctatus has been recorded widely in central and south-western China (e.g. Fei (1999); Fei et al. (2009, 2010)) and north-western Vietnam (Orlov et al. 2012; Li et al. 2012a). However, previous phylogenetic analyses revealed that records of this species actually involve multiple misidentified populations (Yu et al. 2009; Li et al. 2012a; Mo et al. 2016; Pan et al. 2017). In this study, based on molecular and morphological evidence, we revealed that the Xinping population represents a novel lineage of the genus Zhangixalus and previous records of Z. nigropunctatus from Longling, Yunnan belong to it. This result supports the viewpoint of Dufresnes and Litvinchuk (2022) that some populations assigned to Z. nigropunctatus in Yunnan represent a cryptic species and further improves our understanding of the taxonomy and distribution of Z. nigropunctatus complex.

With the Longling population transferred into Z. yunnanensis sp. nov., there are three records of Z. nigropunctatus left in Yunnan, China according to Yang and Rao (2008) and Fei et al. (2010), including Longchuan, Yingjiang and Qiaojia Counties (Fig. 9). Geographically, Longchuan and Yingjiang are very close to Longling, while Qiaojia is closer to the type locality of Z. nigropunctatus (Weining, Guizhou) than to the known distribution of the new species. Therefore, we presume that the populations of nominal Z. nigropunctatus in Longchuan and Yingjiang likely also belong to the new species and the Qiaojia population probably belongs to true Z. nigropunctatus pending further data.

Figure 9. 

Geographic distribution of Zhangixalus species in Yunnan, China. The map was generated using ArcMap v.10.2 (ESRI Inc.).

In addition to the new species described here, we found that the taxonomy of the samples ROM 38011 and VNMN 4099 needs further investigation. The specimen ROM 38011 was collected from Sa Pa, Vietnam and initially identified as Z. dorsoviridis (Orlov et al. 2001). However, it obviously differs from other individuals of Z. dorsoviridis from Sa Pa (e.g. ROM 38015) by having a darkened vocal sac instead of yellow (Orlov et al. 2001). Li et al (2012a) found that phylogenetically ROM 38011 is closer to Z. nigropunctatus than to other samples of Z. dorsoviridis and transferred it into Z. nigropunctatus. Orlov et al. (2012) also listed Z. nigropunctatus as a member of Vietnamese rhacophorid frogs. However, Mo et al. (2016) recovered the specimen ROM 38011 as sister to Z. pinglongensis. The specimen VNMN 4099 was collected from Son La, Vietnam and was included as single representative of Z. dorsoviridis in Nguyen et al. (2014). In this study, we revealed that these two samples are sister to each other. Moreover, the clade formed by these two samples did not cluster together with the clade containing topotypes of Z. nigropunctatus or the clade containing the topotype of Z. dorsoviridis; instead, it is sister to the clade comprised of Z. pinglongensis and Z. yaoshanensis with strong support. These findings suggest that probably ROM 38011 and VNMN 4099 are neither Z. nigropunctatus nor Z. dorsoviridis, but potentially represent one or two distinct species pending further morphological and molecular data. Consequently, the presumption of Poyarkov et al. (2021) that records of Z. nigropunctatus from Vietnam are a misidentification with Z. dorsoviridis remains debatable. Geographically, the collection site of ROM 38011 (Sa Pa, Vietnam) is adjacent to Yunnan, China. Therefore, it could be expected that this potential cryptic species will also be found in Yunnan.

Including the new species described here, the genus Zhangixalus now contains 42 described species. Amongst these, 30 species are distributed in China and 11 species are known in Yunnan. Yunnan is a mountainous region with an extremely diverse topography and climate, which supports an extremely rich biodiversity and shapes different zoogeographic regions. Generally, six zoogeographic regions were recognised in Yunnan, namely North-western Hengduan Mountains (NHM), Western Hills of Yunnan (WHY), Tropical Hills of Southern Yunnan (THSY), South-eastern Hills of Yunnan (SHY), Northern and Central Yunnan Plateau (NCYP) and North-eastern Hills of Yunnan (NHY) and the three southern zoogeographic regions (WHY, THSY and SHY) located at the northern edge of tropic Asia have the highest overall diversity (Yang and Rao 2008; Wang et al. 2022b). Rhacophorids are conservative in their preferences to ecoregions since they primarily inhabit tropical and subtropical moist broadleaf forests ecoregions (Ellepola and Meegaskumbura 2023). Accordingly, in Yunnan, most members of the genus Zhangixalus are distributed in southern, south-eastern and western Yunnan, with the exceptions of Z. omeimontis and Z. nigropunctatus (Fig. 9). Amongst the three zoogeographic regions, south-eastern Yunnan has the highest diversity of Zhangixalus (six species, namely Z. franki, Z. duboisi, Z. puerensis, Z. dorsoviridis, Z. feae and Z. yunnanensis sp. nov.), followed by western Yunnan (five species including Z. smaragdinus, Z. burmanus, Z. puerensis, Z. yunnanensis sp. nov. and the doubtful records of Z. nigropunctatus in Longchuan and Yingjiang) and southern Yunnan (three species, namely Z. pachyproctus, Z. puerensis and Z. feae) in order (Fig. 9).

Recently, Dufresnes and Litvinchuk (2022) considered Zhangixalus hui as a synonym of Z. dugritei, noted that Z. lishuiensis is likely conspecific with Z. zhoukaiyae and Z. duboisi is likely conspecific with Z. omeimontis and suggested that Z. schlegelii covers additional cryptic species just based on genetic divergence at 16S sequences. In this study, we also revealed low genetic divergence between these sister species at the 16S rRNA gene (1.4% between Z. lishuiensis and Z. zhoukaiyae, 1.4% between Z. duboisi and Z. omeimontis and 0.4% between Z. hui and Z. dugritei; Suppl. material 1). However, we consider that the taxonomic rearrangements of Dufresnes and Litvinchuk (2022) should be treated with caution at the present time because they did not consider morphological data and other genetic factors (e.g. hybridisation and incomplete lineage sorting) that possibly lead to the low divergence between these sister species. For instance, Z. duboisi has no vocal sac according to its original description (Ohler et al. 2000), but Z. omeimontis has an internal single subgular vocal sac (Fei et al. 2010). Therefore, we consider that more studies are required to resolve these taxonomic confusions and clarify the species diversity of the genus Zhangixalus, based on multiple lines of evidence (e.g. morphological and molecular data).

Acknowledgements

Thanks go to Jian Zang for his help with the field surveys. This work was supported by the National Natural Science Foundation of China (32060114), 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) and Guangxi Key Laboratory of Rare and Endangered Animal Ecology, Guangxi Normal University (19-A-01-06).

References

  • AmphibiaChina (2023) The database of Chinese amphibians. Kunming Institute of Zoology (CAS), Kunming, Yunnan, China. https://www.Amphibiachina.org [Accessed 4 October 2023.]
  • Andersson LG (1939) [“1938”] Batrachians from Burma collected by Dr. R. Malaise, and from Bolivia and Ecuador collected by Dr. C. Hammarlund. Arkiv för Zoologi. Stockholm 30(23): 1–24.
  • Blyth E (1852) Report of Curator, Zoological Department. Journal of the Asiatic Society of Bengal 21: 341–358.
  • Bordoloi S, Bortamuli T, Ohler A (2007) Systematics of the genus Rhacophorus (Amphibia, Anura): Identity of red-webbed forms and description of a new species from Assam. Zootaxa 1653(1): 1–20. https://doi.org/10.11646/zootaxa.1653.1.1
  • Boulenger GA (1892) An account of the reptiles and batrachians collected by Mr. C. Hose on Mt. Dulit, Borneo. Proceedings of the Zoological Society of London 1892: 505–508.
  • Boulenger GA (1893) Concluding report on the reptiles and batrachians obtained in Burma by Signor L. Fea dealing with the collection made in Pegu and the Karin Hills in 1887–88. Annali del Museo Civico di Storia Naturale di Genova (Serie 2) 13: 304–347.
  • Bourret R (1937) Notes herpétologiques sur l’Indochine française. XIV. Les batraciens de la collection du Laboratoire des Sciences Naturelles de l’Université. Descriptions de quinze especes ou variétés nouvelles. Annexe au Bulletin Général de l’Instruction Publique. Hanoi 1937: 5–56.
  • Brakels P, Nguyen TV, Pawangkhanant P, Idiiatullina S, Lorphengsy S, Suwannapoom C, Poyarkov NA (2023) Mountain jade: A new high-elevation microendemic species of the genus Zhangixalus (Amphibia: Anura: Rhacophoridae) from Laos. Zoological Research 44: 374–379. https://doi.org/10.24272/j.issn.2095-8137.2022.382
  • Chou WH, Lau MW, Chan BPL (2007) A new treefrog of the genus Rhacophorus (Anura: Rhacophoridae) from Hainan Island, China. The Raffles Bulletin of Zoology 55: 157–165.
  • Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: More models, new heuristics and parallel computing. Nature Methods 9(8): e772. https://doi.org/10.1038/nmeth.2109
  • David A (1872) [“1871”] Rapport adressé a MM. les Professeurs-Administráteurs du Museum d’histoire naturelle. Nouvelles Archives du Muséum d’Histoire Naturelle 7: 75–100.
  • Du LY, Wang J, Liu S, Yu GH (2022) A new cryptic species in the Theloderma rhododiscus complex (Anura, Rhacophoridae) from China-Vietnam border regions. ZooKeys 1099: 123–138. https://doi.org/10.3897/zookeys.1099.80390
  • Dufresnes C, Litvinchuk SN (2022) Diversity, distribution and molecular species delimitation in frogs and toads from the Eastern Palaearctic. Zoological Journal of the Linnean Society 195(3): 695–760. https://doi.org/10.1093/zoolinnean/zlab083
  • Edler D, Klein J, Antonelli A, Silvestro D (2021) RaxmlGUI 2.0: A graphical interface and toolkit for phylogenetic analyses using RAxML. Methods in Ecology and Evolution 12(2): 373–377. https://doi.org/10.1111/2041-210X.13512
  • Ellepola G, Meegaskumbura M (2023) Diversification and biogeography of Rhacophoridae ‒ a model testing approach. Frontiers in Ecology and Evolution 11: e1195689. https://doi.org/10.3389/fevo.2023.1195689
  • Fei L (1999) Atlas of Amphibians of China. Henan Publishing House of Science and Technology, Zhengzhou.
  • Fei L, Ye CY, Jiang JP, Xie F, Huang YZ (2005) An Illustrated Key to Chinese Amphibians. Sichuan Publishing House of Science and Technology, Chengdu.
  • Fei L, Hu SQ, Ye CY, Huang YZ (2009) Fauna Sinica. Amphibia (Vol. 3). Anura Ranidae. Science Press, Beijing.
  • Fei L, Ye CY, Jiang JP (2010) Colored Atlas of Chinese Amphibians. Sichuan Publishing House of Science and Technology, Chengdu.
  • Hallowell E (1861) [“1860”] Report upon the Reptilia of the North Pacific Exploring Expedition, under command of Capt. John Rogers, U.S. N. Proceedings. Academy of Natural Sciences of Philadelphia 12: 480–510.
  • He XR (1999) A new species of the family Rhacophoridae from Yunnan–Polypedates puerensis. Sichuan Journal of Zoology 18: 99–100.
  • Jerdon TC (1870) Notes on Indian herpetology. Proceedings of the Asiatic Society of Bengal 1870: 66–85.
  • Jiang D, Jiang K, Ren J, Wu J, Li J (2019) Resurrection of the genus Leptomantis, with description of a new genus to the family Rhacophoridae (Amphibia: Anura). Asian Herpetological Research 10: 1–12.
  • Kuhl H, Van Hasselt JC (1822) Uittreksels uit breieven van de Heeren Kuhl en van Hasselt, aan de Heeren C. J. Temminck, Th. van Swinderen en W. de Haan. Algemeene Konst-en Letter-Bode 7: 99–104.
  • Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33(7): 1870–1874. https://doi.org/10.1093/molbev/msw054
  • Leaché AD, Reeder TW (2002) Molecular systematics of the eastern fence lizard (Sceloporus undulatus): A comparison of parsimony, likelihood, and Bayesian approaches. Systematic Biology 51(1): 44–68. https://doi.org/10.1080/106351502753475871
  • Li JT, Liu J, Chen YY, Wu JW, Murphy RW, Zhao EM, Wang YZ, Zhang YP (2012b) Molecular phylogeny of treefrogs in the Rhacophorus dugritei species complex (Anura: Rhacophoridae), with descriptions of two new species. Zoological Journal of the Linnean Society 165(1): 143–162. https://doi.org/10.1111/j.1096-3642.2011.00790.x
  • Liang YS, Wang CS (1978) A new tree frog Rhacophorus taipeianus (Anura: Rhacophoridae) from Taiwan (Formosa). Quarterly Journal of the Taiwan Museum 31: 185–202.
  • Liu CC (1945) New frogs from West China. Journal of the West China Border Research Society 15: 28–44. [Series B]
  • Liu CC, Hu SQ (1960) [“1959”] Preliminary report of Amphibia from southern Yunnan. Acta Zoologica Sinica 11: 508–538.
  • Liu CC, Hu SQ (1961) Tailless Amphibians of China. Science Press, Beijing.
  • Liu CC, Hu SQ, Yang FH (1962) Preliminary report of Amphibia from western Kweichow. Acta Zoologica Sinica 14: 381–392.
  • Liu BQ, Wang YF, Jiang K, Chen HM, Zhou JJ, Xu JN, Wu CH (2017) A new treefrog species of the genus Rhacophorus Found in Zhejiang, China (Anura: Rhacophoridae). Chinese Journal of Zoology 52: 361–372.
  • Lue KY, Lai JS, Chen SL (1994) A new species of Rhacophorus (Anura: Rhacophoridae) from Taiwan. Herpetologica 50: 303–308.
  • Lue KY, Lai JS, Chen SL (1995) A new species of Rhacophorus (Anura: Rhacophoridae) from Taiwan. Journal of Herpetology 29(3): 338–345. https://doi.org/10.2307/1564982
  • Matsui M, Panha S (2006) A new species of Rhacophorus from eastern Thailand (Anura: Rhacophoridae). Zoological Science 23(5): 477–481. https://doi.org/10.2108/zsj.23.477
  • Mo YM, Chen WC, Liao X, Zhou SC (2016) A new species of the genus Rhacophorus (Anura: Rhacophoridae) from southern China. Asian Herpetological Research 7: 139–150.
  • Mou YP, Risch JP, Lue KY (1983) Rhacophorus prasinatus, a new tree frog from Taiwan, China (Amphibia, Anura, Rhacophoridae). Alytes 2: 154–162.
  • Myers CW, Duellman WE (1982) A new species of Hyla from Cerro Colorado, and other tree frog records and geographical notes from western Panama. American Museum Novitates 2752: 1–32.
  • Nguyen TT, Matsui M, Eto K, Orlov NL (2014) A preliminary study of phylogenetic relationships and taxonomic problems of Vietnamese Rhacophorus (Anura: Rhacophoridae). Russian Journal of Herpetology 21(4): 274–280.
  • Nguyen TT, Ninh HT, Orlov NL, Nguyen TQ, Ziegler T (2020) A new species of the genus Zhangixalus (Amphibia: Rhacophoridae) from Vietnam. Journal of Natural History 54(1–4): 257–273. https://doi.org/10.1080/00222933.2020.1754484
  • Ninh HT, Nguyen TT, Orlov NL, Nguyen TQ, Ziegler T (2020) A new species of the genus Zhangixalus (Amphibia: Rhacophoridae) from Vietnam. European Journal of Taxonomy 688(688): 1–8. https://doi.org/10.5852/ejt.2020.688
  • Ohler A, Marquis O, Swan SR, Grosjean S (2000) Amphibian biodiversity of Hoang Lien Nature Reserve (Lao Cai Province, northern Vietnam) with description of two new species. Herpetozoa 13: 71–87.
  • Okada Y, Kawano U (1924) On the ecological distribution of two new varieties of Rhacophorus in Japan. Zoological Magazine 36: 104–109. [144–153.]
  • Orlov NL, Lathrop A, Murphy RW, Ho CT (2001) Frogs of the family Rhacophoridae (Anura: Amphibia) in the northern Hoang Lien Mountains (Mount Fan Si Pan, Sa Pa District, Lao Cai Province), Vietnam. Russian Journal of Herpetology 8: 17–44.
  • Orlov NL, Poyarkov NA, Vassilieva AB, Ananjeva NB, Nguyen TT, Sang NN, Geissler P (2012) Taxonomic notes on rhacophorid frogs (Rhacophorinae: Rhacophoridae: Anura) of southern part of Annamite Mountains (Truong Son, Vietnam), with description of three new species. Russian Journal of Herpetology 19(1): 23–64.
  • Pan T, Zhang Y, Wang H, Wu J, Kang X, Qian L, Li K, Zhang Y, Chen J, Rao D, Jiang JP, Zhang B (2017) A New Species of the Genus Rhacophorus (Anura: Rhacophoridae) from Dabie Mountains in East China. Asian Herpetological Research 8: 1–13.
  • Poyarkov NA, Nguyen TV, Popov ES, Geissler P, Pawangkhanant P, Neang T, Suwannapoom C, Orlov NL (2021) Recent progress in taxonomic studies, biogeographic analysis, and revised checklist of amphibians of Indochina. Russian Journal of Herpetology 28(3A): 1–110. https://doi.org/10.30906/1026-2296-2021-2лес8-3A-1-110
  • Rao DQ, Wilkinson JA, Liu HN (2006) A new species of Rhacophorus (Anura: Rhacophoridae) from Guangxi Province, China. Zootaxa 1258: 17–31.
  • Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61(3): 539–542. https://doi.org/10.1093/sysbio/sys029
  • Smith MA (1924) Two lizards and a new tree frog from the Malay Peninsula. Journal of the Federated Malay States Museums 11: 183–186.
  • Stejneger L (1924) Herpetological novelties from China. Occasional Papers of the Boston Society of Natural History 5: 119–121.
  • Tang SJ, Sun T, Liu S, Luo SD, Yu GH, Du LN (2023a) A new species of cascade frog (Anura: Ranidae: Amolops) from central Yunnan, China. Zoological Letters 9(1): 1–15. https://doi.org/10.1186/s40851-023-00214-9
  • Tang SJ, Liu S, Yu GH (2023b) A new species of Nanorana (Anura: Dicroglossidae) from northwestern Yunnan, China, with comments on the taxonomy of Nanorana arunachalensis and Allopaa. Animals 13(21): e3427. https://doi.org/10.3390/ani13213427
  • Wang J, Li J, Du LY, Hou M, Yu GH (2022a) A cryptic species of the Amolops ricketti species group (Anura, Ranidae) from China-Vietnam border regions. ZooKeys 1112: 139–159. https://doi.org/10.3897/zookeys.1112.82551
  • Wang K, Lyu ZT, Wang J, Qi S, Che J (2022b) The updated checklist and zoogeographic division of the reptilian fauna of Yunnan Province, China. Biodiversity Science 30(4): e21326. https://doi.org/10.17520/biods.2021326
  • Yang DT (1991) The Amphiba-Fauna of Yunnan. China Forestry Publishing House, Beijing.
  • Yang DT, Rao DQ (2008) Amphibia and Reptilia of Yunnan. Yunnan Science and Technology Press, Kunming.
  • Yu GH, Rao DQ, Yang JX, Zhang MW (2008) Phylogenetic relationships among Rhacophorinae (Rhacophoridae, Anura, Amphibia), with an emphasis on the Chinese species. Zoological Journal of the Linnean Society 153(4): 733–749. https://doi.org/10.1111/j.1096-3642.2008.00404.x
  • Yu GH, Rao D, Zhang MW, Yang JX (2009) Re-examination of the phylogeny of Rhacophoridae (Anura) based on mitochondrial and nuclear DNA. Molecular Phylogenetics and Evolution 50(3): 571–579. https://doi.org/10.1016/j.ympev.2008.11.023
  • Yu GH, Hui H, Hou M, Wu ZJ, Rao DQ, Yang JX (2019) A new species of Zhangixalus (Anura: Rhacophoridae), previously confused with Zhangixalus smaragdinus (Blyth, 1852). Zootaxa 4711(2): 275–292. https://doi.org/10.11646/zootaxa.4711.2.3
  • Zhang J, Jiang K, Hou M (2011) Rhacophorus dorsoviridis Bourret, a new record of family Rhacophoridae to China. Acta Zootaxonomica Sinica 36(4): 986–989.
  • Zhao EM, Yang DT (1997) Amphibians and Reptiles of the Hengduan Mountains Region. Science Press, Beijing.

Supplementary material

Supplementary material 1 

Genetic distances between Zhangixalus species estimated from 16S sequences

Yuanqiang Pan, Mian Hou, Guohua Yu, Shuo Liu

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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