Zoosyst. Evol. 100 (1) 2024, 183-197 | DOI 10.3897/zse.100.113850 ee BERLIN A new species of Zhangixalus (Anura, Rhacophoridae) from Yunnan, China Yuangiang Pan’*, Mian Hou’, Guohua Yu!*, Shuo Liu* 1 Key Laboratory of Ecology of Rare and Endangered Species and Environmental Protection, Guangxi Normal University, Ministry of Education, Guilin 541004, China 2 Guangxi Key Laboratory of Rare and Endangered Animal Ecology, College of Life Science, Guangxi Normal University, Guilin 541004, China 3 College of Continuing (Online) Education, Sichuan Normal University, Chengdu 610068, Sichuan, China 4 Kunming Natural History Museum of Zoology, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China https://zoobank. org/FB7E03EC-A2BB-4C9C-98 96-20E53A2667DB Corresponding authors: Guohua Yu (yugh2018@126.com); Shuo Liu (liushuo@mail.kiz.ac.cn) Academic editor: Umilaela Arifin # Received 8 October 2023 Accepted 30 January 2024 Published 20 Februarry 2024 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. 1s distinctly different from all known congeners by the combination of the following morphological characters: black blotches on body flank and hind-limb, me- dium 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 recorded to date, 10 of which are distributed in Yunnan: Z. burmanus (Andersson, 1939), Z. dorsoviridis (Bour- The treefrog genus Zhangixalus Li, Jiang, Ren & Jiang, 2019, recently removed from Rhacophorus Kuhl & Van Hasselt, 1822 sensu lato and recognised as a distinct ge- nus, is characterised by medium-to-large body size, ab- sence of dermal folds along limbs, absence of tarsal pro- jections 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 ret, 1937), Z. duboisi (Ohler, Marquis, Swan & Gros- jean, 2000), Z. feae (Boulenger, 1893), Z. franki Ninh, Nguyen, Orlov, Nguyen & Ziegler, 2020, Z. nigropunc- tatus (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_ni- gropunctatus is confusing. This species was originally Copyright Pan, Y. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distri- bution, and reproduction in any medium, provided the original author and source are credited. 184 described from western Guizhou, China (Weining and Shuicheng) by Liu et al. (1962) and, afterwards, was recorded from Yunnan (Longling, Longchuan, Yingji- ang, 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 to- gether with topotypes of Z. nigropunctatus, indicating that misidentification may be involved within Z. nigro- punctatus. Li et al. (2012a) revealed that both Z. nigro- punctatus and Z. dorsoviridis did not form a monophylet- ic 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 Viet- nam 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 Viet- nam. 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. nigropuncta- tus 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, Chi- na, specimens of a treefrog species resembling Zhangix- alus 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. nigro- punctatus and the status of amphibian diversity in Yun- nan, we sequenced our newly-collected treefrogs from Yunnan to confirm their identity. Our phylogenetic anal- yses recovered these specimens as a distinct lineage and are sister to the clade composed of Z. nigropunctatus and Z. melanoleucus Brakels, Nguyen, Pawangkhanant, Idi- iatullina, Lorphengsy, Suwannapoom & Poyarkov, 2023. Furthermore, our morphological examination suggested that this lineage differs from Z. nigropunctatus, Z. melan- oleucus and other members of Zhangixalus by a series of characters. Herein, we officially describe this lineage as a new species of Zhangixalus. zse.pensoft.net Pan, Y. et al.: A new species of Zhangixalus from China 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 col- lected 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). Phylogenetic analysis Total genomic DNA was extracted from liver tissues stored in 99% ethanol. A fragment encoding mitochon- drial 12S rRNA, tRNA“ and 16S rRNA genes was ampli- fied 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 un- der Accession Nos. PP177446 and PP187265—PP 187270 (Table 1). Additionally, 43 homologous sequences of oth- er 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. Sequences were aligned using MUSCLE with default parameters in MEGA v.7.0 (Kumar et al. 2016). Uncor- rected pairwise distances (using 16S rRNA sequences) between species were calculated in MEGA v.7.0. The best substitution model was selected using the correct- ed 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) un- der 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 gener- ations. 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 strong- ly 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 cal- ipers to the nearest 0.1 mm. Morphological terminology Zoosyst. Evol. 100 (1) 2024, 183-197 98°0'0"E 100°0'0"E 102°0'0"E 29°0'0"N 28°0'0"N 27°0'0"N 26°0'0"N 25°0'0"N 24°0'0"N 23°0'0"N4 Legend Elevation Value High : 6456 22°0'0"N 1 = Low : 76 185 104°0'0"E 106°0'0"E 108°0'0"E 110°0'0"E 60 Kilometers 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.). 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 dis- tance (IND, distance between nares); interorbital distance (OD, minimum distance between upper eyelids); upper eyelid width (UEW, maximum width of upper eyelid); eye diameter (ED, diameter of exposed portion of eye- ball); distance between nostril and eye (DNE, from nos- tril to anterior border of eye); tympanum diameter (TD, the greater of tympanum vertical and horizontal diame- ters); 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 meta- tarsal 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 measure- ments (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 phylogenet- ic analyses (Z. nigropunctatus and Z. melanoleucus). Differences in quantitative characters of adult males be- tween these three species were also evaluated with t-tests in SPSS. In these analyses, Levene’s test was also per- formed 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. Phyloge- netic analyses revealed that the specimens from Xinping, Yunnan form a distinct clade with two individuals previous- ly 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 zse.pensoft.net 186 Pan, Y. et al.: A new species of Zhangixalus from China Table 1. Species used for phylogenetic analyses in this study (Z. = Zhangixalus). Species Voucher No. Theloderma albopunctatum ROM 30246 Rhacophorus rhodopus SCUM 060692L Leptomantis gauni FMNH 273928 Zhangixalus dulitensis BORNEENSISO9087 Z. pachyproctus KIZ 090148 Z. smaragdinus KIZ 20160298 Z. dennysi ROM 30249 Z. feae SCUM 050642W Z. chentui SCUM 060404L Z. jodiae VNMN 07122 Zhangixalus sp. ROM 38011 VNMN 4099 Z. dorsoviridis ROM 38015 Rao060821200 YNO80446 Ra0060821199 KIZ 060821287 Z. yaoshanensis NHMG150408 Z. pinglongensis NHMG201002011 Z. nigropunctatus KIZ07061001 GZ070658 SCUM 070657L GXNU YU000361 GXNU YU000362 GXNU YU000363 Z. yunnanensis sp. nov. GXNU YU20160340 GXNU YU20160268 GXNU YU20160267 Rao3494 Ra03496 Z. moltrechti SCUM 061106L Z. schlegelii - Z. arboreus TTU-R-11748 Z. puerensis SCUM 060649L Z. dugritei SCUM 051001L Z. hui LiO1 Z. hongchibaensis CIB 097687 Z. hungfuensis SCUM 060425L Z. minimus KIZ 061214YP Z. burmanus SCUM 060614L Z. franki VNMN 011687 Z. duboisi SCUM 061104L Z. omeimontis SCUM 0606137L Z. zhoukaiyae AHU-RhaDB-1 20428 HM05293 Z. lishuiensis YPX47792 Z. melanoleucus BEI 01010 ZMMU A-7781 BEI 01011 AUP 02507 between the novel lineage, Z. nigropunctatus and Z. melan- oleucus ranged from 5.1%-—5.5% (Suppl. material 1). 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. KI1Z, 060821287 was nested within the clade of Z. dorsoviridis from the type locality (Sa Pa, Lao Cai, Vietnam) and Yun- nan 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 zse.pensoft.net Locality Accession No. Vietnam AF458148 Mengyang, Yunnan, China EU215531 Sarawak, Malaysia JX219456 Borneo, Malaysia AB847123 Puer, Yunnan, China MN613222 Yingjiang, Yunnan, China MN613219 Vietnam AF458139 Hekou, Yunnan, China EU215544 Mt. Omei, Sichuan, China EU215534 Vietnam LC545595 Sa Pa, Lao Cai, Vietnam JX219427 Son La, Vietnam LCO10577 Sa Pa, Lao Cai, Vietnam JX219423 Jinping, Yunnan, China JX219424 Pingbian, Yunnan, China JX219425 Pingbian, Yunnan, China JX219426 Jinping, Yunnan, China EF564563 Jinxiu, Guangxi, China MG322122 Shiwandashan, Guangxi, China KU1 70684 Weining, Guizhou, China EU924623 Weining, Guizhou, China JX219430 Weining, Guizhou, China EU215533 Weining, Guizhou, China PP187265 Weining, Guizhou, China PP187266 Weining, Guizhou, China PP187267 Xinping, Yunnan, China PP187268 Xinping, Yunnan, China PP187269 Xinping, Yunnan, China PP187270 Longling, Yunnan, China JX219429 Longling, Yunnan, China JX219428 Lianhuachi, Taiwan, China EU215543 Hiroshima, Japan AB202078 Japan AF458142 Puer, Yunnan, China EU215542 Baoxing, Sichuan, China EU215541 Zhaojue, Sichuan, China JN688878 Wuxi, Chongqing, China JN688883 Wenchuan, Sichuan, China EU215538 Mt. Dayao, Guangxi, China EU215539 Mt. Gaoligong, Yunnan, China EU215537 Ha Giang, Vietnam LC548746 Pingbian, Yunnan, China EU215536 Pengxian, Sichuan, China EU215535 Jinzhai, Anhui, China KU601502 Anhui, China PP177446 Lishui, Zhejiang, China KY653720 Phou Samsoum Mt., Xiengkhoang, Laos 0Q305233 Phou Samsoum Mt., Xiengkhoang, Laos 0Q305234 Phou Samsoum Mt., Xiengkhoang, Laos 0Q305235 Phou Samsoum Mt., Xiengkhoang, Laos 0Q305236 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 mea- surements (FHL, TFL and FL) were not included in PCA analysis because they were not available for Z. melano- leucus. 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 Zoosyst. Evol. 100 (1) 2024, 183-197 187 Table 2. Measurements of Zhangixalus yunnanensis sp. nov. (1-9), Z. nigropunctatus (10-17) and Z. melanoleucus (18-21). Spec- imens 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 10D UEW ED TD DNE FHL TL TFL FL 1 GXNUYU20160267 M 31.3 9.7 11.4 4.9 4.0 4.0 2.8 3:5 1.9 Zid 17.3 133 218 149 2 GXNUYU20160268 M 84.00 TIL | AAG or 4.0 4.0 op 4.0 eal. 2.2 16.9 130 216 148 3 GXNU YU20160269 M 35.4 11.5 | be2e5) 5S 4.1 4.1 3.4 4.1 Ark 22 18.7 14.5 23:2 15.7 4 GXNUYU20160335 =F 48.6 15.1 18.0 EZ 5.6 5:6 4.2 4.9 2.9 3.0 2510. 120.0" © BP 225 5 GXNU YU20160336 M 35:3. 12 ‘13:6 5.4 4.3 4.3 35 4.3 2.3 22 13s Tas. °23.5— 15:5 6 GXNU YU20160337 M goo. Fle eo a3 4.2 4.2 os2 4.3 2.2 al 18.3 139 224 15.1 7 GXNU YU20160338 M 344 11.3 12.9 5.3 4.3 4.5 a3 4.1 ore. 2.3 18.1 149 229 15.6 8 GXNU YU20160340 M ao SelB~ 3.9 325 4.3 4.3 3.6 4.3 Le 2i2 18.5 ‘143. 23:5 16.2 9 GXNUYU20160355 =F 47.6 144 17.2 6.8 5.8 Bf 4.1 5.0 2.9 2.9 25,5 20:5. 83:2) ~ 233 10 KIZ25362 F 39.4 1327 S27. 5.9 4.2 5.0 SPal 4.1 27 2.8 22.1 17.1 Le ie 19.0 11 KIZ25367 F ASF LS 38 6.1 4.2 4.8 3.0 4.3 2.8 2.8 LAF el Fil AUS | gp COO I? KIZ25369 M Sif 11h2 6114 4.8 3.2 SF: 2.4 D7 2 2.3 16.4 128 206 147 13 KIZ25370 M 33%. 11.5 °12:0 5.1 3.4 3.9 fod 3.9 Ao 2.3 17.4 129 214 158 14 KIZ25372 M 34.8 11.7 15 5.1 pee 4.1 2.6 4.0 2.4 2.4 17.1 ge p38) 216 15.6 15 KIZ25373 M 34.1 11.9 12.1 4.9 37 4.0 2.7 3.9 25 2.3 18.6 13.4 21.9 15.7 16 KIZ25374 M 34.7 11.7 11.8 5.3 Baa 3.8 29 3.6 233 2.5 16.8 1333 2 el 15.4 17 KIZ25375 M 33.2 11.8 113 oul 3.4 4.0 2:1 a7 2:2 22. 16.5 11.9 20.4 14.3 18 BEI 01010 M S5O.- 0 Ube = TeS 6.1 4.3 4.7 a2 4.6 2.3 2.4 - 14.7 19 BEI 01011 M 34.4 126 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 Las 13.4 6.1 44 4.8 Bal 4.7 was 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 Theloderma albopunctatum Rhacophorus rhodopus 100 Leptomantis gauni 100 Z. dulitensis ze Z. feae 100 Z. pachyproctus 100 99 Z. smaragdinus Zz. dennysi Zhangixalus Z. moltrechti 100 100 Zz. schlegelii 100 99 Z. arboreus Z. puerensis 100] go Z dugritei 100) OOL 7 ju 2 100} 0 Z. hongchibaensis 100 Z. hungfuensis 190 sis Z. minimus ool 2: Sranki hog Z. duboisi 100| 22 — 2. omeimontis 100 98 Z. burmanus O4 1 Z. lishuiensis 100| ; AHU-RhaDB- 120428 Fchiiiab 1o0_|1007L GXNU HM0S293 Bieta ad ROM 38015 (Sa Pa, Vietnam) oo Ly KIZ 060821287 (Jinping, Yunnan) —_ 100}| Rao060821200 (Jinping, Yunnan) Z. dorsoviridis | Rao060821199 (Pingbian, Yunnan) YN080446 (Pingbian, Yunnan) Z. chenfui Z. jodiae 100__| 9g Z. nigropunctatus ROM 38011 (Sa Pa, Vietnam) 100_| |g] LZ, dorsoviridis VNMN 4099 (Son La, Vietnam) mi 83 iho Z. yaoshanensis 31 Z. pinglongensis = KIZ Rao3494 (Longling, Yunnan)” 63 109 | - KIZ Rao3496 (Longling, Yunnan) 100 |) GXNU YU20160268 (Xinping, Fy oes iad Pace aaee } GXNU YU20160340 (Xinping, Yu wi 4 tango hod GXNU YU20160267 (Xinping, Yu 3 oo [AUP 02507 100 | eC Z. melanoleucus a BEI 01011 KIZ 07061001 (Weining, Guizhou) 100_|/ SCUM 070657L (Weining, Guizhou) | GZ070658 (Weining, Guizhou) Z ni nih GXNU YU000362 (Weining, Guizhou) “ “8’°P#7@1as aide GXNU YU000363 (Weining, Guizhou) 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. zse.pensoft.net 188 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. ni- gropunctatus 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 differ- ences 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 spec- imens 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). Ad- ditionally, the new lineage is distinguishable from its con- geners by body size and the combination of texture and colouration pattern. Therefore, on the basis of the above molecular and morphological evidence, we officially de- scribe them as a new species of the genus Zhangixalus. Table 3. 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 PCl PC2 Eigenvalue 4.521 2ATS % 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 lOD (interorbital distance) 0.778 0.402 UEW (width of upper eyelid) O713 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 Pan, Y. et al.: A new species of Zhangixalus from China V Z. melanoleucus @ Z. yunnanensis sp. nov. A Z. nigropunctatus PC1 (45.214%) -1.00000 0.00000 1.00000 2.00000 PC2 (21.778%) 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-4080-8568-8E7D34700F 36 Figs 4-6 Yunnan tree Frog/ Yun Nan Shu Wa (zs Fa Py HEE) Type material. Holotype: GXNU YU20160340, adult male, collected on 20 April 2020 by Guohua Yu from Mo- pan Mountain, Xinping County, Yunnan Province, China (23°56'06"N, 102°01'18"E, 2443 m a.s.1.). Paratypes: GXNU YU20160267—20160269, three adult males, collected on 14 July 2019 from the type lo- cality by Guohua Yu; GXNU YU20160336—20160338, Table 4. 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 ¢-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) Z. yunnanensis sp. nov. SVL 34.6 + 1.6 Sor/et 1,2 Z. nigropunctatus HL 0.323 + 0.0068 0.346 + 0.0082 HW 0.371 + 0.0122 0.352 + 0.0092 SL 0.154 + 0.0026 0.150 + 0.0039 IND 0.121 + 0.0043 0.102 + 0.0047 lOD 0.122 + 0.0056 0.116 + 0.0037 UEW 0.095 + 0.0041 0.079 + 0.0034 ED 0.118 + 0.0034 0.113 + 0.0049 TD 0.062 + 0.0021 0.069 + 0.0026 DNE 0.064 + 0.0038 0.069 + 0.0025 FHL 0.522 + 0.0171 0.509 + 0.0226 TL 0.406 + 0.0182 0.385 + 0.1508 FL 0.446 + 0.0164 0450 + 0.0143 TEL 0.657 + 0.0223 0.629 + 0.0167 zse.pensoft.net Levene’s test t-test F p-value t p-value 0.370 0.555 1.129 0.283 0.793 0.392 -5.420 0.000** 0.388 0.546 3.144 0.009** 2.043 0.181 2.117 0.058 0.016 0.902 7.350 0.000** 1.862 0.200 1.956 0.076 0.297 0.597 7.468 0.000** 0.519 0.486 2.266 0.045* 0.010 0.921 -4.966 0.000** 1.169 0.303 -3.008 0.012* 0.925 O:35/ 1.146 0.276 0.556 0.472 2.246 0.046* 0.029 0.868 0.517 0.615 0.086 0.775 2.5/6 0.026* Zoosyst. Evol. 100 (1) 2024, 183-197 189 Table 5. Summary statistics of male specimens (mean + standard deviation) and results of the ¢-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 lOD 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 2:193 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 ieee: 0.299 0.057 0.956 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. 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 Chi- nese common name “Yun Nan Shu Wa (zs BA Py REY”. 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 dig- its expanded into large discs bearing circum-marginal grooves, and vomerine teeth present, dermal folds along limbs not significant, tarsal projections absent, green dor- sa colouration and medium body size (Jiang et al. 2019). Phylogenetically, the new species is nested within the ge- nus 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 pos- terior 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 [2—2112—31112—21V and toes webbing formula 12-2111 .5—2.5 1112-31 V3-1.5V. 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, horizon- tal; tympanum distinct (TD 2.3 mm), rounded, slightly greater than half of ED, nearly equal to the distance be- tween 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 ante- riorly and notched posteriorly; single external vocal sac, with a sac slit opening on floor of mouth at each corner. Relative length of fingers I < I Ne eran. ey Se es Poe? <= ea ge Sis videos ~ ; me Figure 7. Habitat of the new species at the type locality (a) and foam nests of the new species (b). Photos by Guohua Yu. tions 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. nigropunc- tatus (Yu et al., 2008), also belongs to Z. dorsoviridis. Considering that Longchuan and Yingjiang are close to Longling, the populations previously recorded as Z. ni- gropunctatus from Longchuan and Yingjiang of Yunnan probably belong to the new species pending more data. 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 yel- lowish-brown iris (vs. yellowish-gold; Fig. 8 and Ta- ble 6), head obviously wider than long (vs. head width nearly equal to head length), tibiotarsal articulation reaching posterior corner of eye (vs. reaching posteri- or 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 Met > ok. > ae oa zse.pensoft.net 192 Table 6. 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 = rudi- mentary; (6) development of toe web: 0 = webbed half, 1 = entirely webbed, 2 = webbed 2/3, 3 = webbed 1/3. “?” means unknown. O®@O®™O® © Species Z. yunnanensis sp. nov. Z. achantharrhena Z. chentui Z. dorsoviridis Z. dulitensis Z. hungfuensis Z. jaryjini Z. jodiae Z. . lishuiensis . melanoleucus 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)). NNNNNNNNNNNNNNNNNNNNNNNNN'N leucofasciatus minimus . moltrechti . nigropunctatus . pinglongensis puerensis . schlegelii suffry . taipeianus wul yaoshanensis . zhoukalyae amamiensis arboreus arvalis . aurantiventris burmanus dennysi duboisi dugritei feae franki . hongchibaensis hui . omeimontis Z. ts Z. re Lf. ve Z. owstoni pachyproctus prasinatus prominanus smaragdinus viridis yinggelingensis zse.pensoft.net Body size Male Female 31.3-36.0 47.6-48.6 36.5-40.6 47.1 33-41 46-55 31.3-42.4 37.9-42.8 38.4-39.7 ? 30.8-36.8 45.5 33.7-40.0 41.5-46.1 34.8-39.8 ? 35-48.2 7 34.2-35.8 45.9 34.4-36.3 Doar 21.3-33.0 31.7-38.3 36.3-42.4 44,7-54.6 32.0-37.0 44.0-45.0 32.0-38.5 ? 35.5-41.1 52.0-55.2 32-43 43-53 38.5-52.9 31.5-61.0 30.4-37.9 39.1-43.0 35.2-38.2 48.6 31.6-36.4 49.2-51.1 27.9-37.1 42.1-44.7 45-56 65-76 42-60 59-82 39.0-46.4 59.5-64 47.8-53.9 e 54-72 66-82 68-92 83-109 >61.5 ? 41.5-45.4 57.7-64.3 86-111 68-116 77.9-85.8 £ 46.5-49.7 5673 40-45.4 ? 52.0-65.5 70.0-79.5 42-51 ? 73.4-78.2 102.4 44.8-58.5 63.9-66.9 50.5-51.3 ? 76.3-79.6 ? 41-54 52-68 43.0-43.4 ? oOoOrrcdrdancooocorrdv9”cororerouoqoclodrodcdrr coo ov OF OFFOrRFF OFF OO Re i ke i i i i i i i i i i i i i ep ep ph pt OH OF OO PR BP HP HHP HP HP HH CS OrFRNFRrRNRFRFRWWNHENMNFRFRROF FF OrFRWwWWNHFrFOODOFWFWOODONDAUDOAOUDON ODO F CO ONMFrrFrRrRNRrRWWRERNRNNFT,NNOODOFRYNFWRrRRYNM ODF VDeFwWwWN DEF ODOFWOrF OO NM CO Pan, Y. et al.: A new species of Zhangixalus from China Webbing color greyish mottled red and green pale brownish-yellow greyish-brown webbing between outer two toes red greyish reddish-orange cream with black blotches greyish black or greyish-purple golden yellow cream with black blotches grey mottled with dark blotches orange red spotted with black greyish tangerine grey mottled with dark blotches purple orange red yellow greyish-brown greyish greyish purple purple pink orange-red dull brown brown black and white black and grey green grey grey black and grey yellowish yellow pale blue black purple red blue black red red lris color yellowish-brown dull red edged with silvery white reddish-orange orange red reddish greenish yellow silver yellowish yellow reddish-orange yellowish-gold red or red brown yellowish-gold silver yellowish-gold yellowish-gold yellowish-gold yellowish-gold yellowish-gold pale yellowish-gold golden-yellow greenish reddish yellow pale yellow greenish yellowish-gold dark gold yellowish-brown green-gold bronze yellowish-brown reddish-brown yellowish-gold yellowish-gold mottled with silver bronze yellowish-gold reddish yellowish-gold yellow silver Zoosyst. Evol. 100 (1) 2024, 183-197 [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 tym- panum (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). In body size, besides Zhangixalus nigropunctatus and Z. melanoleucus, the new species is relatively sim- ilar to Z. achantharrhena (Harvey, Pemberton & Smith, 2002), Z. chenfui, Z. dorsoviridis, Z. dulitensis (Bouleng- er, 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. mini- mus (Rao, Wilkinsonand & Liu, 2006), Z. moltrechti (Bou- lenger, 1908), Z. pinglongensis, Z. puerensis, Z. schlegelii (Gunther, 1858), Z. suffry (Bordoloi, Bortamuli & Ohler, 2007), Z. taipeianus (Liang & Wang, 1978), Z. wui (L1, Liu, Chen, Wu, Murphy, Zhao, Wang & Zhang, 2012), Z. yaoshanensis and Z. zhoukaiyae (Table 4). The new Species can be easily distinguished from Z. achanthar- rhena, 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 yel- lowish-brown to light green with numerous light-brown spots in Z. wui); from Z. achantharrhena, Z. chenfui, Z. dulitensis, Z. hungfuensis, Z. jarujini, Z. leucofascia- tus, 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. dorsovir- idis, 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. zhoukaiy- ae by throat black (vs. bright yellow in Z. achantharrhe- na, purplish flesh in Z. chenfui, yellow in Z. dorsoviridis tos and Z. taipeianus, cream in Z. hungfuensis and Z. jaru- Jini, greyish in Z. jodiae and Z. yaoshanensis, white in Z. dulitensis, Z. leucofasciatus, Z. lishuiensis, Z. mol- trechti and Z. zhoukaiyae, white with slightly grey back- ground 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. chen- fui, Z. dulitensis, Z. jarujini, Z. jodiae, Z. lishuiensis, Z. minimus, Z. moltrechti, Z. pinglongensis, Z. pueren- sis, 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. sufjry, pale brownish-yellow in Z. chenfui, mottled with black blotches in Z. jodiae, Z. minimus and Z. puer- ensis, 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. achantharrhe- na, Z. leucofasciatus and Z. zhoukaiyae, fingers entire- ly webbed in Z. dulitensis, fingers webbed half and toes webbed entirely in Z. moltrechti, fingers webbed entire- ly 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. mol- trechti 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 I), 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 Zhangix- alus 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 (Da- vid, 1872), Z. feae, Z. franki, Z. hongchibaensis (L1, 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). More- over, 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 sur- face 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. aurantiven- zse.pensoft.net 194 tris, Z. burmanus, Z. dennysi, Z. feae, Z. hongchibaensis, Z. omeimontis, Z. owstoni, Z. pachyproctus, Z. smarag- dinus, Z. viridis and Z. yinggelingensis by external vocal sac (vs. internal vocal sac); and from Z. arvalis, Z. au- rantiventris, Z. dennysi, Z. feae, Z. franki, Z. pachyproc- tus, 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 in- volve 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 nov- el 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 Lit- vinchuk (2022) that some populations assigned to Z. ni- gropunctatus 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. yun- nanensis sp. nov., there are three records of Z. nigro- punctatus left in Yunnan, China according to Yang and Rao (2008) and Fei et al. (2010), including Longchuan, 98°0'0"E 100°0'0"E 102°0'0"E 29°0'0"N 28°0'0"N 27°0'0"N 26°0'0"N 25°0'0"N 24°0'0"N 23°0'0"N7 Legend Elevation Value 22°0'0"N High : 6456 ~ Low : 76 Pan, Y. et al.: A new species of Zhangixalus from China Yingjiang and Qiaojia Counties (Fig. 9). Geographical- ly, Longchuan and Yingjiang are very close to Longling, while Qiaojia is closer to the type locality of Z. nigro- punctatus (Weining, Guizhou) than to the known distri- bution 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. nigro- punctatus pending further data. 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 in1- tially 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 hav- ing 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. nigropuncta- tus. 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 col- lected 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 104°0'0"E 106°0'0"E 108°0'0"E 110°0'0"E @ Z. yunnanensis sp. nov. @ Z. puerensis © Z. burmanus © Z dorsoviridis @ Z. duboisi @ Z feae @ Z franki @ Z. pachyproctus © Z. smaragdinus © Z nigropunctatus @ Z omeimontis 60 Kilometers Figure 9. Geographic distribution of Zhangixalus species in Yunnan, China. The map was generated using ArcMap v.10.2 (ESRI Inc.). zse.pensoft.net Zoosyst. Evol. 100 (1) 2024, 183-197 with strong support. These findings suggest that probably ROM 38011 and VNMN 4099 are neither Z. nigropunc- tatus nor Z. dorsoviridis, but potentially represent one or two distinct species pending further morphological and molecular data. Consequently, the presumption of Poyar- kov et al. (2021) that records of Z. nigropunctatus from Vietnam are a misidentification with Z. dorsoviridis re- mains debatable. Geographically, the collection site of ROM 38011 (Sa Pa, Vietnam) is adjacent to Yunnan, China. Therefore, 1t could be expected that this potential cryptic species will also be found in Yunnan. Including the new species described here, the ge- nus Zhangixalus now contains 42 described species. Amongst these, 30 species are distributed in China and 11 species are known in Yunnan. Yunnan is a mountain- ous region with an extremely diverse topography and climate, which supports an extremely rich biodiversi- ty and shapes different zoogeographic regions. Gen- erally, 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 ecore- gions (Ellepola and Meegaskumbura 2023). According- ly, 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. ni- gropunctatus (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. yunnanen- sis 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. ni- gropunctatus in Longchuan and Yingjiang) and southern Yunnan (three species, namely Z. pachyproctus, Z. puer- ensis 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 spe- cies 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 consid- er that the taxonomic rearrangements of Dufresnes and Litvinchuk (2022) should be treated with caution at the present time because they did not consider morphologi- cal data and other genetic factors (e.g. hybridisation and 195 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 de- scription (Ohler et al. 2000), but Z. omeimontis has an internal single subgular vocal sac (Fei et al. 2010). 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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 | Genetic distances between Zhangixalus species estimated from 16S sequences Authors: Yuanqiang Pan, Mian Hou, Guohua Yu, Shuo Liu Data type: xls Copyright notice: 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 us- ers 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. Link: https://do1.org/10.3897/zse.100.113850.suppl1 zse.pensoft.net