2 urn:lsid:arphahub.com:pub:45048D35-BB1D-5CE8-9668-537E44BD4C7E urn:lsid:zoobank.org:pub:91BD42D4-90F1-4B45-9350-EEF175B1727A ZooKeys ZK 1313-2989 1313-2970 Pensoft Publishers 10.3897/zookeys.1197.104851 104851 Research Article Amphibia Animalia Anura Chordata Rhacophoridae Vertebrata Taxonomy Asia Vietnam Zhangixalus thaoae sp. nov., a new green treefrog species from Vietnam (Anura, Rhacophoridae) Nguyen Tao Thien nguyenthientao@gmail.com https://orcid.org/0000-0002-5640-4536 1 Writing - review and editing Nguyen Huy Hoang https://orcid.org/0000-0002-6284-5813 1 Ninh Hoa Thi https://orcid.org/0009-0002-8133-432X 1 Le Linh Tu Hoang https://orcid.org/0009-0003-0887-2786 1 Bui Hai Tuan https://orcid.org/0000-0003-4065-7229 1 Orlov Nikolai https://orcid.org/0000-0003-4401-348X 2 Hoang Chung Van https://orcid.org/0000-0002-0709-974X 3 Ziegler Thomas https://orcid.org/0000-0002-4797-609X 4 5 Institute of Genome Research, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Hanoi, Vietnam Vietnam Academy of Science and Technology Hanoi Vietnam Department of Herpetology, Zoological Institute, Russian Academy of Sciences, 199034, St. Petersburg, Russia Zoological Institute, Russian Academy of Sciences St. Petersburg Russia Forest Resources and Environment Centre, 300 Ngoc Hoi Road, Thanh Tri, Hanoi, Vietnam Forest Resources and Environment Centre Hanoi Vietnam AG Zoologischer Garten Köln, Riehler Strasse 173, D-50735 Cologne, Germany AG Zoologischer Garten Köln Cologne Germany Institute of Zoology, University of Cologne, Zülpicher Straße 47b, D-50674 Cologne, Germany University of Cologne Cologne Germany

Corresponding author: Tao Thien Nguyen (nguyenthientao@gmail.com)

Academic editor: Angelica Crottini

 2024 08 04 2024 1197 93 113 CC6A2014-441D-5D7D-91C8-0C02022A4B87 E33270AB-994B-4438-974C-1BE11A4FF3A4 11 04 2023 10 03 2024 Tao Thien Nguyen, Huy Hoang Nguyen, Hoa Thi Ninh, Linh Tu Hoang Le, Hai Tuan Bui, Nikolai Orlov, Chung Van Hoang, Thomas Ziegler 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. http://zoobank.org/E33270AB-994B-4438-974C-1BE11A4FF3A4

We describe a new treefrog species from Lao Cai Province, northwestern Vietnam. The new species is assigned to the genus Zhangixalus based on a combination of the following morphological characters: (1) dorsum green, smooth; body size medium (SVL 30.1–32.2 in males); (2) fingers webbed; tips of digits expanded into large disks, bearing circum-marginal grooves; (3) absence of dermal folds along limbs; (4) absence of supracloacal fold and tarsal projection. The new species can be distinguished from its congeners by: (1) dorsal surface of the head and body green without spots; (2) axilla and groin cream with a black blotch; (3) ventral cream without spot; (4) chin creamy with grey marbling; anterior part of the thigh and ventral surface of tibia orange without spots; posterior parts of thigh orange with a large black blotch; (5) ventral side of webbing orange with some grey pattern (6) iris red-bronze, pupils black; (7) finger webbing formula I1¼-1¼II1-2III1-1IV, toe webbing formula I½-½II0-1½III¼-1¾IV1¾-½V. Phylogenetically, the new species is nested in the same subclade as Z. jodiae, Z. pinglongensis, and Z. yaoshanensis, with genetic distances ranging from 3.23% to 4.68%. The new species can be found in evergreen montane tropical forests at an elevation of about 1,883 m a.s.l. This new discovery brings the number of known genus Zhangixalus species to 42 and the number of species reported from Vietnam to 10.

 16S rRNA gene Lao Cai Province molecular phylogeny morphology new species Citation

Nguyen TT, Nguyen HH, Ninh HT, Le LTH, Bui HT, Orlov N, Hoang CV, Ziegler T (2024) Zhangixalus thaoae sp. nov., a new green treefrog species from Vietnam (Anura, Rhacophoridae). ZooKeys 1197: 93–113. https://doi.org/10.3897/zookeys.1197.104851

 Introduction

The genus Zhangixalus Li, Jiang, Ren & Jiang, 2019 currently contains 42 species, with a wide distribution in northeastern India, Nepal, Bhutan, southern China, Myanmar, northern Thailand, Laos, northern Vietnam, Taiwan, and Japan, and south to Indonesia, Brunei, and Malaysia (Frost 2024). Brakels et al. (2023) revealed two major clades in the genus Zhangixalus in their phylogenetic analysis of the group; these include a continental Southeast Asian and East Asian species group and a Sundaland group. Brakels et al. (2023) also included six species in the Z. chenfui group, distributed in China, Vietnam, and Laos, including Zhangixalus chenfui (Liu, 1945), Z. jodiae (Nguyen, Ninh, Orlov, Nguyen & Ziegler, 2020), Z. melanoleucus Brakels, Nguyen, Pawangkhanant, Idiiatullina, Lorphengsy, Suwannapoom & Poyarkov, 2023, Z. nigropunctatus (Liu, Hu & Yang, 1962), Z. pinglongensis (Mo, Chen, Liao & Zhou, 2016), and Z. yaoshanensis (Liu & Hu, 1962).

Among the nine Zhangixalus species reported from Vietnam, Z. dennysi (Blanford, 1881) has been reported from southeastern China to northeastern Vietnam, Z. dorsoviridis (Bourret, 1937) from northwestern Vietnam and southern China; Z. duboisi (Ohler, Marquis, Swan & Grosjean, 2000) from the Hoang Lien range in Vietnam and China; Z. feae (Boulenger, 1893) from Myanmar to the Tay Nguyen Plateau, Vietnam; Z. franki Ninh, Nguyen, Orlov, Nguyen & Ziegler, 2020 exclusively from Ha Giang Province, Vietnam; Z. hungfuensis (Liu & Hu, 1961) from Lao Cai Province, Vietnam, and Sichuan and Guangxi provinces, China; Z. jodiae Nguyen, Ninh, Orlov, Nguyen & Ziegler, 2020 from Ha Giang Province, Vietnam; Z. pachyproctus Yu, Hui, Hou, Wu, Rao & Yang, 2019 from northern Vietnam, Yunnan Province, China, and Prachuap Khiri Khan, Thailand; and Z. puerensis (He, 1999) from Lao Cai and Ha Giang provinces, Vietnam, and Yunnan, China (Frost 2024).

During our 2019 fieldwork in Y Ty Commune, Bat Xat District, Lao Cai Province, Vietnam, we collected a series of tree frogs that morphologically resembled Z. yaoshanensis, a species known from Guangxi Province, China (Liu and Hu 1962). However, the newly discovered population from Vietnam differs from Z. yaoshanensis by the following distinct morphological characteristics: size medium (SVL 30.1–32.2 mm in males); dorsum green without spots, venter cream without spots; flank, axilla, and posterior thigh cream with large black blotches. Furthermore, our phylogenetic analysis shows that the Lao Cai Province population is nested in the same subclade with Z. jodiae, Z. pinglongensis, and Z. yaoshanensis in the Z. chenfui group.

The pairwise distance from the newly collected species to the congeners of Zhangixalus species ranges from 3.23% (compared to Z. pinglongensis) to 10.83% (compared to Z. smaragdinus). Meanwhile, the genetic distance among species of Zhangixalus ranges from 0.49% (Z. dugritei and Z. hui) to 11.89% (Z. pachyproctus and Z. yaoshanensis). This demonstrates that the treefrog from Y Ty Commune is a distinct taxon, with a genetic distance of at least 3.23% from other Zhangixalus species.

Based on the morphological characters and molecular information, we describe here the unnamed Zhangixalus species from Lao Cai Province, Vietnam, as a new species.

 Materials and methods Repositories, Institutional acronyms, or Institutional abbreviations

IEBR Institute of Ecology and Biological Resources

ROM Royal Ontario Museum

 Sampling

The field survey was conducted from 25 April to 1 May 2019 by C.V. Hoang and A.M. Luong in Y Ty Commune, Bat Xat District, Lao Cai Province, northwestern Vietnam. Geographic coordinates and elevations were obtained using a Garmin GPSMAP 76CSX (using the WGS84 datum). After the frogs were photographed alive, three specimens of the new species (IEBR A 5136, IEBR A 5137 and IEBR A 5138) (Table 1) were anaesthetized and euthanized in a closed vessel with a piece of cotton wool containing ethyl acetate (Simmons 2002), fixed in 80% ethanol for 5 h, and then transferred to 70% ethanol for permanent storage. Liver-tissue samples were preserved separately in 96% ethanol before fixation and subsequently deposited in the collection of IEBR.

Samples of Zhangixalus and other species were used for DNA analysis in this study.

No. Scientific name Voucher Locality GenBank no. Source
1. Rhacophorus kio VN.2018.84 Kon Tum, Vietnam This study
2. R. kio VN.2018.83 Kon Tum, Vietnam This study
3. Zhangixalus chenfui RaoZT0806013 Zhaotong, Yunnan, China JX219431 Li et al. 2012b
4. Z. chenfui Li05 Mt. Omei, Sichuan, China JX219432 Li et al. 2012b
5. Z. dennysi ML.2019.1 Vinh Phuc, Vietnam This study
6. Z. dennysi ML.2019.2 Vinh Phuc, Vietnam This study
7. Z. dorsoviridis YN080446 Jinping, Yunnan JX219425 Li et al. 2012b
8. Z. dorsoviridis Yt.2018.16 Lao Cai, Vietnam This study
9. Z. dorsoviridis YT 2018 6 Lao Cai, Vietnam This study
10. Z. duboisi VNMN7079 Ha Giang, Vietnam This study
11. Z. duboisi VNMN010243 Lai Chau, Vietnam This study
12. Z. dugritei LJT 051002 Sichuan, China JN688872 Li et al. 2012a
13. Z. dugritei LJT 051008 Sichuan, China JN688873 Li et al. 2012a
14. Z. feae HB.2014.28 Hoa Binh, Viet Nam This study
15. Z. feae VNMN05859 Lai Chau Viet Nam This study
16. Z. franki VNMN 011686 Ha Giang, Vietnam LC548745 Ninh et al. 2020
17. Z. franki VNMN 011687 Ha Giang, Vietnam LC548746 Ninh et al. 2020
18. L Z. hongchibaensis CIB 097696 Chongqing, China JN688882 Li et al. 2012a
19. Z. hongchibaensis CIB 097687 Chongqing, China JN688883 Li et al. 2012a
20. Z. hui SCUM 0504111 L Sichuan, China JN688877 Li et al. 2012a
21. Z. hui SCUMLi 01 Sichuan, China JN688878 Li et al. 2012a
22. Z. hungfuensis SCUM 060425L Sichuan, China EU215538 Li et al. 2008
23. Z. hungfuensis SCUM 060424 L Sichuan, China JN688879 Li et al. 2012a
24. Z. jodiae VNMN 07121 Ha Giang, Vietnam LC545594 Nguyen et al. 2020
25. Z. jodiae VNMN 07122 Ha Giang, Vietnam LC545595 Nguyen et al. 2020
26. Z. lishuiensis YPX47794 Lishui, Zhejiang, China KY653719 Liu et al. 2017
27. Z. lishuiensis YPX47792 Lishui, Zhejiang, China KY653720 Liu et al. 2017
28. Z. melanoleucus BEI 01010 Phou Samsoum Mt., Xiengkhoang, Laos OQ305233 Brakels et al. 2023
29. Z. melanoleucus BEI 01011 Phou Samsoum Mt., Xiengkhoang, Laos OQ305235 Brakels et al. 2023
30. Z. melanoleucus ZMMU A7781 Phou Samsoum Mt., Xiengkhoang, Laos OQ305234 Brakels et al. 2023
31. Z. nigropunctatus GZ070658 Weining, Guizhou, China JX219430 Li et al. 2012b
32. Z. nigropunctatus Li06 Weining, Guizhou, China JX219433 Li et al. 2012b
33. Z. omeimontis Li02 Sichuan, China JX219420 Li et al. 2012b
34. Z. omeimontis RaoZT0806010 Sichuan, China JX219419 Li et al. 2012b
35. Z. pachyproctus TQ.2018.72 Tuyen Quang, Viet Nam This study
36. Z. pachyproctus VNMN:1299 Nghe An, Vietnam LC545592 This study
37. Z. pinglongensis NHMG201002011 Guangxi, China KU170684 Mo et al. 2016
38. Z. pinglongensis NHMG201002003 Guangxi, China KU170683 Mo et al. 2016
39. Z. puerensis VNMN 010284 Lai Chau, Viet Nam This study
40. Z. puerensis SCUM 060648 L Yunnan, ChinaYunnan, China JN688884 Li et al. 2012a
41. Z. schlegelii KUHE 44531 Okayama, Japan LC369670 Matsui et al. 2018
42. Z. schlegelii Genbank Hiroshima, Japan NC007178 Sano et al. 2005
43. Z. smaragdinus RAO6241 Tibet, China JX219411 Li et al. 2012b
44. Z. smaragdinus CAS 224708 Nagmung, Putao District, Kachin, Myanmar MN613214 Yu et al. 2019
45. Z. wui CIB 097685 Hubei, China JN688881 Li et al. 2012a
46. Z. wui CIB 097690 Hubei, China JN688880 Li et al. 2012a
47. Z. yaoshanensis NHMG150408 Guangxi, China MG322122 Chen et al. 2018
48. Z. yaoshanensis NHMG150404 Guangxi, China MG322121 Chen et al. 2018
49. Z. yinggelingensis HN2018002 Hainan, China MW192130 Genbank
50. Z. yunnanensis Rao3494 Longling, Yunnan, China JX219429 Li et al. 2012b
51. Z. yunnanensis Rao3496 Longling, Yunnan, China JX219428 Li et al. 2012b
52. Z. zhoukaiyae AHURhaDb-150420-03 Anhui, China KU601499 Pan et al. 2017
53. Z. zhoukaiyae AHURhaDb-150420-01 Anhui, China KU601500 Pan et al. 2017
54. Zhangixalus thaoae sp. nov. ROM38011 Lao Cai, Vietnam JX219427 Li et al. 2012b
55. Zhangixalus thaoae sp. nov. IEBR A 5136 Lao Cai, Vietnam LC762092 This study
56. Zhangixalus thaoae sp. nov. IEBR A 5137 Lao Cai, Vietnam LC762093 This study
57. Zhangixalus thaoae sp. nov. IEBR A 5138 Lao Cai, Vietnam LC762094 This study
Molecular data and phylogenetic analyses

We used the protocols of Kuraishi et al. (2013), modified by Nguyen et al. (2015a), for DNA extraction, amplification, and sequencing. Fragments of the 16S rRNA mitochondrial DNA gene were amplified using the same primers used by Kuraishi et al. (2013).

For the phylogenetic analyses, 55 sequences of 26 Zhangixalus species were combined with two sequences of Rhacophorus kio as outgroups (Table 1).

Chromas Pro software (Technelysium Pty Ltd, Tewantin, Australia) was used to edit the sequences, which were aligned using MAFFT v. 7 (Katoh and Standley 2013) with default settings. We then checked the initial alignments by eye and adjusted them slightly. Phylogenetic trees were constructed using IQ-TREE v. 1.6.12 (Nguyen et al. 2015b) while maximum-likelihood bootstrap support (MLBS) was evaluated by ultrafast bootstrap approximation with 1000 replicates (Hoang et al. 2018) (ML). Prior to Bayesian analyses, we chose the optimum substitution models for entire sequences by using ModelFinder implemented in IQ-TREE based on the Bayesian information criterion (BIC) (Kalyaanamoorthy et al. 2017). According to ModelFinder, the best-fit model for ML analysis was TIM2+F+I+G4. Because the TIM2 model and F parameter are not implemented in MrBayes, we selected the next best-fit model for our Bayesian-inference (BI) analysis, which was the general time reversible model (GTR; Tavaré 1986) with a proportion of invariable sites and a gamma shape parameter (Alpha 0.215). The BI phylogenetic construction was done in MrBayes v. 3.2.7a (Ronquist et al. 2012) with two independent runs of four Markov Chains for 10,000,000 generations. A tree was sampled every 100 generation, and a consensus topology was calculated for 75,001 trees after discarding the first 25,000 trees using the relative burn-in option (25% of trees discarded). We checked parameter estimates and convergence using TRACER v. 1.5 (Rambaut and Drummond 2009). We regarded tree nodes in the ML tree with bootstrap values of 95% or greater as sufficiently resolved (Hoang et al. 2018), and nodes with a BPP of 95% or greater as significant in the BI analysis (Leaché and Reeder 2002).

 Morphological characters

A total of 34 measurements were taken with digital calipers to the nearest 0.1 mm (Nguyen et al. 2016). Abbreviations are as follows: SVL: snout–vent length, HW: head width (across angle of jaws), HL: head length (from back of mandible to tip of snout), MND: distance from jaw angle to nostril, MFED: distance from back of mandible to front of the eye, MBED: distance from back of mandible to back of the eye, SNL: snout length (from anterior corner of eye to tip of snout), ED: eye diameter, UEW: maximum width of upper eyelid, IND: internarial distance, IOD: interorbital distance (minimal distance between orbits), AED: distance between anterior corners of eyes, PED: distance between posterior corners of eyes, NS: distance from nostril to tip of snout, EN: distance from anterior corner of eye to nostril, TYD: maximal tympanum diameter, TED: distance from anterior margin of tympanum to posterior corner of eye, Ua: upper arm length (from axilla to elbow), Fa: lower arm and hand length (from elbow to tip of third finger), NPL: nuptial pad length, F1–4: length of fingers I–IV (from basis of finger to tip of finger), FeL: thigh length from vent to knee, TbL: tibia length from knee to tarsus, TbW: maximal tibia width, FL: foot length from tibiotarsal joint to tip of fourth toe, T1–5: length of toes I–V, IML: inner metatarsal tubercle length. Terminology for describing the webbing formula followed Glaw and Vences (2007). Sex was determined by the presence of nuptial pads and gonadal inspection.

 Results Phylogenetic analyses

The aligned 16S sequences yielded a total of 1,033 characters. Of 1033 nucleotide sites, 309 were variable and 295 were parsimony informative within the analysed Zhangixalus species. Nucleotide frequencies were A = 37.7%, T = 24.4%, C = 20.7%, and G = 17.2% (data for ingroup only). Our phylogenetic analyses employing ML and BI methods yielded identical topologies, and only the BI tree is presented in Fig. 1.

10.3897/zookeys.1197.104851.figure1 EF6B69F3-73F8-5C8B-881E-4DAB172578C6

BI tree from a 1033 bp sequence of mitochondrial 16S rRNA gene of Zhangixalus and outgroup species. Numbers above and below branches are Bayesian posterior probabilities (BPP) and ML bootstrap. For GenBank accession numbers, refer to Table 1.

https://binary.pensoft.net/fig/1021506

Phylogenetically, the undescribed species of Zhangixalus from Y Ty Commune, Bat Xat District, Lao Cai Province, Vietnam was clustered with seven species in the Z. chenfui group with a well-supported node (both 100% in the ML and BI analyses). Furthermore, the undescribed Zhangixalus species was found to be most closely related to a clade consisting of Z. pinglongensis and Z. yaoshanensis, with significantly high support value in the BI analysis (99%) and a high support value from ML analysis (94%). The genetic distance among the examined sequences ranges from 3.23% (between Zhangixalus sp. from Lao Cai Province and Z. pinglongensis) to 8.10% (between Z. chenfui and Z. yaoshanensis) (Table 2).

Uncorrected pairwise distances (p-distance) among Zhangixalus species analysed.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
1. Zhangixalus thaoae sp. nov. 0.00–0.19
2. Z. chenfui 7.42 0.00
3. Z. dennysi 10.72 11.12–11.32 0.19
4. Z. dorsoviridis 8.59–8.69 9.29–9.38 9.76–40.15 0.39–0.78
5. Z. duboisi 7.62–7.91 8.69–8.89 9.07–9.27 4.88–5.27 0.29
6. Z. dugritei 8.02–8.99 8.22–8.32 9.86–10.16 6.05–6.54 4.40–4.59 0.11
7. Z. feae 8.81–8.99 9.00–9.09 8.30–8.50 9.00–9.38 8.31–8.40 8.02–8.21 0.00
8. Z. franki 7.81–7.97 8.50–8.70 9.07–9.51 5.18–569 2.73–2.90 4.88–5.18 8.21–8.70 0.00
9. Z. hongchibaensis 8.71–8.90 9.21–9.30 10.95–11.24 6.55–7.04 5.09–5.38 3.62–3.81 8.72–8.90 5.57–5.91 0.10
10. Z. hui 8.21–8.31 8.22 9.86–10.06 6.35–6.54 4.50–4.59 0.49–0.59 8.12–8.21 4.79–4.87 3.91–4.01 0.00
11. Z. hungfuensis 8.62–8.72 9.21 10.08–10.27 5.97–6.16 5.09–5.19 3.71–3.81 8.81–8.90 5.19–5.50 3.52–3.62 3.62 0.00
12. Z. jodiae 3.90 7.32 10.32–10.52 8.98–9.27 7.41–7.61 7.81–7.91 8.31–8.40 7.71–7.76 8.90–8.99 8.01 8.90 0.00
13. Z. lishuiensis 9.09–9.19 8.61 9.47 4.00–4.20 4.50–4.59 5.87–5.96 7.93–8.02 4.69–4.97 6.56–6.65 5.87 5.48 9.08 0.00
14. Z. melanoleucus 6.43 7.22 10.81–11.00 8.29–8.68 8.20–8.39 7.71–7.81 8.80–8.89 7.61–7.86 8.70–8.80 7.91 8.71 7.21 8.89 0.00
15. Z. nigropunctatus 5.56–5.76 6.93 10.33–10.53 9.08–9.28 8.11–8.30 8.11–8.20 8.41–8.50 7.71–7.96 9.29–9.38 8.30 9.00 5.65 8.90 5.46 0.00
16. Z. omeimontis 7.91–8.11 8.79–8.89 9.17–9.56 4.98–5.18 1.27–1.37 4.50–4.59 8.21–8.30 3.03–3.11 5.38–5.58 4.40 4.99–5.09 7.32 4.40–4.59 8.19–8.59 8.30 0.20
17. Z. pachyproctus 10.54–10.83 10.94–11.13 10.04 9.76–10.34 9.46–9.66 9.96–10.25 7.62–7.82 9.76–10.13 10.36–10.65 9.86–10.06 10.18 10.82–11.01 9.28–9.47 10.92–11.11 10.44–10.63 9.37–9.56 0.19
18. Z. pinglongensis 3.23–3.33 7.24 10.36 8.80–8.90 7.72–7.82 8.02–8.12 8.62–8.71 7.83–7.88 8.81–8.91 8.22 8.42 4.01 8.61 6.74 5.68 7.82–7.92 10.65–10.85 0.00
19. Z. puerensis 7.92–8.71 8.22–8.62 9.29–9.77 6.35–6.74 4.89–5.28 3.42–3.61 7.73–8.12 5.08–5.59 3.82–4.79 3.32–3.42 3.62–3.82 7.81–8.21 5.68–6.16 8.21–8.30 8.69–8.90 4.79–4.89 9.58–9.86 7.93–8.13 2.83
20. Z. schlegelii 8.50–9.20 9.00–10.11 10.25–10.65 7.32–7.43 5.87–6.16 6.05–6.65 7.92–8.32 5.96–6.53 6.16–6.56 5.57–6.06 6.07–6.17 8.69–9.38 6.84–7.14 8.20–8.40 8.89–9.38 5.87–6.26 9.28–9.58 9.00–9.40 5.86–6.36 1.17
21. Z. smaragdinus 10.73–10.83 10.94 10.33 10.93–11.32 10.54 10.74–10.84 8.79–8.80 10.63–11.07 11.44–11.53 10.84 11.06 10.43 10.35 10.82 10.54 10.44 8.38 10.75 10.36–10.45 9.96–10.17 0.00
22. Z. wui 8.61–8.81 9.01–9.11 9.87–10.17 6.65–6.94 4.99–5.09 3.52–3.71 8.91–9.10 5.18–5.49 3.91–4.10 3.23–3.32 2.84–2.94 8.90–8.99 6.56–6.65 8.41–8.50 8.99–9.09 4.99–5.09 10.39–10.65 8.91–9.01 4.11–4.30 6.26–6.56 11.53–11.63 0.10
23. Z. yaoshanensis 3.80–4.68 7.62–8.10 10.53–11.10 9.07–9.85 7.90–8.68 8.50–9.28 9.29–9.77 7.90–8.79 9.68–10.36 8.69–9.38 9.30–9.88 4.68–5.36 9.18–9.67 6.82–7.79 5.95–6.53 8.29–8.88 11.21–11.89 3.62–4.30 8.69–9.58 9.18–9.97 10.82–11.40 9.38–10.07 0.88
24. Z. yinggelingensis 8.81–8.91 8.42 10.76–10.86 7.34–7.44 6.17 5.97–6.07 8.54–8.63 6.37–6.54 7.90–8.68 6.07 6.67 9.20 7.05 8.71 8.52 6.07–6.17 10.37–10.57 8.43 6.08–6.17 7.84–8.83 11.06 7.06 8.81–9.10
25. Z. yunnanensis 6.54 6.93 10.23–10.43 8.40–8.50 8.49–8.68 7.82–7.92 8.20–8.21 8.01–8.39 8.90–9.00 7.82 8.71 6.43 8.50 4.97 5.46 8.20–8.29 10.73–10.93 6.74 8.31–8.41 8.31–8.61 9.85 8.71–8.81 6.83–7.41 8.52
26. Z. zhoukaiyae 8.61–8.81 8.52–8.62 9.68–9.78 3.81–4.01 4.40–4.60 5.68–5.87 8.04–8.23 4.79–5.19 6.56–6.76 5.58–5.68 5.39–5.49 8.41–8.50 1.56–1.66 8.60–8.70 8.61–8.71 4.21–4.50 8.90–9.19 8.42–8.52 5.68–6.07 6.65–6.86 9.68–9.78 6.56–6.66 8.90–9.48 6.56–6.66 8.32–8.41 0.10
Taxonomic account Animalia Anura Rhacophoridae 69706455-064C-5527-8D62-BEC5AD7D25A2 Zhangixalus thaoae https://zoobank.org/DB9E5E8E-3E66-4AB1-B837-8FB8D485E982 sp. nov. Fig. 2 Material examined.

Holotype: Vietnam • ♂; Y Ty Commune, Bat Xat District, Lao Cai Province, Northwestern Vietnam; 22°37'17.6"N, 103°37'23.5"E; 1883 m a.s.l.; 01 May 2019; C. V. Hoang and A. M. Luong leg.; IEBR A 5136; GenBank: LC762092.1. Paratypes: Vietnam • 2 ♂; same locality as for holotype; same geo-coordinates; same altitude; same collection date; same collectors; IEBR A 5137, 5138; GenBank: LC762093.1, LC762094.1.

 Diagnosis.

The new species is placed in the genus Zhangixalus based on some morphological characters: dorsum green and smooth; body size medium (SVL 30.1–32.2 in males); fingers webbed; tips of digits expanded into large disks, bearing circum-marginal grooves; absence of dermal folds along limbs; absence of supracloacal fold and tarsal projection (Fig. 2).

10.3897/zookeys.1197.104851.figure2 F25BB097-83D8-527D-9E34-FFFCFAD65D1A

Adult male holotype (IEBR A 5136) of Zhangixalus thaoae sp. nov., in life, from Y Ty Commune, Bat Xat District, Lao Cai Province, northwestern Vietnam.

https://binary.pensoft.net/fig/1021507

The new species is distinguished from its congeners by a combination of the following characteristics: 1) dorsal surface of head and body green without spots; 2) axilla and groin cream with a black blotch; 3) ventrum cream-colored without spots; 4) chin cream, with grey marbling; anterior part of thigh and ventral surface of tibia orange, without blotch; posterior parts of thigh orange with a large, black blotch; 5) ventral side of webbing orange, with some grey; 6) iris red-bronze, pupils black. 7) finger webbing formula I1¼-1¼II1-2III1-1IV, and toe webbing formula I½-½II0-1½III¼ -1¾IV1¾-½V.

 Description of the holotype

(male). Size medium (SVL 32.2 mm), body robust; head slightly compressed, head length nearly equal to the width (HW 12.1 mm, HL 11.7 mm), convex above; snout round, slightly protruding beyond lower jaw in lateral view, and longer than the horizontal diameter of eye (SNL 5.4 mm, ED 4.2 mm); canthus rostralis round, loreal region oblique, concave; interorbital distance wider than internarial distance and upper eyelid (IOD 4.3 mm, IND 3.1 mm, UEW 3.0 mm); distance between anterior corners of eyes about 69.30% of the distance between posterior corners of eyes; nostrils round, without lateral flap of skin, closer to tip of snout than to eye; pupil oval, horizontal; tympanum distinct, round, about half the size of eye diameter, and twice greater than distance between tympanum and eye (TYD 2.1, TYE 1.0); pineal ocellus and spinules on upper eyelid absent; vomerine teeth well developed, in two oblique ridges; choanae round; tongue deeply notched posteriorly; supratympanic fold weakly.

Forelimbs robust, upper arm short, nearly one-half of hand length (Ua 6.2 mm, Fa 14.6 mm), dermal fringe along the outer edge of forearm absent; relative finger lengths I<II<V<III; tips of fingers with enlarged discs with distinct circum-marginal grooves; disc of finger III approximately 1.5 times of the width of finger III (fd3/fw3 1.5), greater than tympanum diameter (fd3/TYD 1.27); webbing formula I1¼-1¼II1-2III1-1IV, subarticular tubercles distinct, blunt, round, formula 1, 1, 2, 2; nuptial pads prominent, oval, smooth.

Hindlimbs long and thin, heels overlapping when held at right angles to the body; tibia length about four times greater than tibia width (TbL 13.9 mm, TbW 3.1 mm), longer than thigh length (FeL 13.3 mm), shorter than foot length (FL 19.0 mm); relative toe lengths I<II<III<V<IV; tips of toes with enlarged discs with distinct circum-marginal grooves, discs slightly smaller than those of fingers; webbing formula I½-½II0-1½III¼-1¾IV1¾-½V; subarticular tubercles distinct, blunt, round, formula 1, 1, 2, 3, 2; inner metatarsal tubercle small (IML 1.6 mm); dermal ridge along the outer edge of tibia and tarsus absent; dermal projection at tibiotarsal articulation absent.

Skin texture: dorsal surface of head and body smooth; supratympanic fold weakly developed, throat and chest smooth, belly rough; ventral surface of fore- and hindlimbs smooth.

Coloration in life: iris red-bronze, pupil black; dorsal surface of head and body green without spots; dorsal surface of fore and hind limbs green, upper side of fingers II and II and toes I, II, and III yellow, all tip of fingers and toes yellow; axilla cream and groin cream with a black blotch; anterior part of thigh and ventral surface of tibia orange without spots; posterior parts of thigh orange with a large black blotch; lower jaw cream, with grey marbling, and throat region white; ventral side of webbing orange with some gray pattern, nuptial pads grey.

Coloration in preservative: As in life, but with green dorsal surface fading to dark blue; and ventral side of body, limbs, and upper side of fingers I and II, upper side of toes I, II, and III fading to light yellow.

 Variation.

Ground color of dorsum light green; ventral surface cream, without spots. The size of blotches in the axilla, groin, and posterior thigh region of the paratype is smaller than in the holotype (Fig. 3). For measurements of the type series, see Table 3.

Measurements (in mm) of Zhangixalus thaoae sp. nov.

Number IEBR A 5136 IEBR A 5137 IEBR A 5138
Sex Male (holotype) Male Male
SVL 32.2 32.0 30.1
HW 12.1 11.7 12.8
HL 11.7 11.3 13.1
MND 9.5 9.8 10.8
MFED 7.5 7.2 10.6
MBED 3.8 4.5 4.1
SNL 5.4 5.1 5.4
ED 4.2 3.4 3.9
UEW 3.0 3.0 3.5
IND 3.1 3.9 4.0
IOD 4.3 4.6 4.7
AED 7.0 7.1 7.5
PED 10.1 10.4 11.5
NS 3.1 2.8 3.2
EN 2.9 2.6 3.2
TYD 2.1 2.0 2.5
TED 1.0 1.2 1.4
Ua 6.2 6.1 7.0
Fa 14.6 16.1 16.6
F1 3.0 4.0 3.1
F2 4.2 5.2 5.4
F3 6.1 6.4 7.2
F4 4.8 5.7 5.3
FeL 13.3 13.2 14.0
TbL 13.9 13.9 14.0
TbW 3.4 3.1 3.3
FL 19.0 20.3 21.8
T1 3.6 5.0 4.5
T2 6.2 7.4 6.8
T3 9.2 10.3 10.7
T4 11.8 13.5 13.6
T5 10.0 10.9 11.3
IML 1.6 1.8 1.7
HW/HL 1.03 1.04 0.97
SNL/HW 0.44 0.43 0.43
NS/EN 1.08 1.07 0.99
ED/SNL 0.78 0.66 0.72
TYE/TYD 0.46 0.62 0.57
TYD/ED 0.50 0.59 0.64
HAL/FLL 2.36 2.64 2.37
TBL/TL 1.05 1.05 1.01
10.3897/zookeys.1197.104851.figure3 8366FB2E-7F2C-520E-B3DB-CBEDA847E01B

The variation of morphological characteristic of Zhangixalus thaoae sp. nov. IEBR A 5136 is the holotype, and the two remaining samples are paratypes.

https://binary.pensoft.net/fig/1021508 Etymology.

The species is named after the first author’s wife, Nguyen Thi Thanh Thao, as a token of gratitude for her understanding and strong support of his research activity. We recommend Thao’s Tree Frog as the English common name and Ếch cây thảo as the Vietnamese common name.

 Male secondary sexual characters.

Male specimens with nuptial pad present on base of the finger I and external single subgular vocal sac.

 Natural history notes.

Specimens were collected between 19:00 and 24:00h on a branch about 1 m above the ground. The ground consisted of mountain soil and puddles, and there was a small stream about 2 m away (Fig. 4A). The habitat was an undisturbed evergreen forest on a granite mountain (Fig. 4B). Other tree frogs that were found at the site were Polypedates sp., of the P. leucomystax species complex; Zhangixalus duboisi (Ohler, Marquis, Swan & Grosjean, 2000); Hyla annectans (Jerdon, 1870); and Gracixalus gracilipes (Bourret, 1937). Females, larval stages, and eggs of the new species are unknown.

10.3897/zookeys.1197.104851.figure4 B0C116E8-28E3-54E4-AB96-69FD272665D9

Habitat of the new species in Y Ty Commune, Bat Xat District, Lao Cai Province A collection site with a small puddle B the stream near the biotope of the holotype.

https://binary.pensoft.net/fig/1021509 Distribution.

Zhangixalus thaoae sp. nov. is currently known only from the type locality (Fig. 5). The species was recorded at an elevation of approximately 1,880 m a.s.l.

10.3897/zookeys.1197.104851.figure5 B9060A90-241B-58D0-87B8-FDCA275BE418

The distribution of species of the Zhangixalus chenfui group.

https://binary.pensoft.net/fig/1021510 Conservation status.

The new species is expected to be found in the evergreen forest of Guangxi Province, southern China, because the terrain there consists mostly of granite mountains, but in Yunnan Province, China, which contains mostly limestone terrain, the species is not expected. However, the geographic distribution of the species needs to be confirmed by further studies. Because there is a lack of information on the species’ abundance and distribution, we suggest that it be considered as Data Deficient following IUCN Red List categories (IUCN 2023).

 Comparisons.

We compare Zhangixalus thaoae sp. nov. with other species of Zhangixalus occurring in Vietnam and elsewhere.

The new species mostly resembles Z. yaoshanensis by the combination of the following characteristics: head as long as wide in Zhangixalus thaoae sp. nov. (HL/HW 0.96–1.02), posterior parts of thigh orange with a large black blotch, ventral surface of tibia orange, iris red-bronze with black pupil in Zhangixalus thaoae sp. nov. vs head wider than long (HL/HW = 0.83); posterior thigh surface and ventral surface of tibia red-orange without spot, iris pale yellowish gold with a network of fine dark gold reticulations in Z. yaoshanensis (Chen et al. 2018).

Zhangixalus thaoae sp. nov. can be distinguished from other species in the genus Zhangixalus by its smaller size (SVL 30.1–32.2 mm) vs SVL >50 mm in the following species: 47.0–70.5 (Wilkinson and Rao 2004; Ohler 2009; Jiang et al. 2016) in Z. burmanus; 68–92 mm (Fei et al. 2010) in Z. dennysi; 53.1–67.2 mm (Ohler et al. 2000; Orlov et al. 2001; Ziegler et al. 2014) in Z. duboisi; 68–116 mm (Fei et al. 2010) in Z. feae (Boulenger, 1893); 77.9–85.8 mm (Ninh et al. 2020) in Z. franki; 52–66 mm (Fei et al. 2010), 52–65 mm (Liu 1950) in Z. omeimontis; 74.2 mm (Luu and Calame 2014), 73.4–78.2 mm in Z. pachyproctus (Yu et al. 2019); and 76.3–79.6 mm in Z. smaragdinus (Yu et al. 2019).

Zhangixalus thaoae sp. nov. can be distinguished from other Zhangixalus species of a similar size in having a different coloration pattern: dorsum green without blotches or spots in the new species vs light or dark green with many white or brown spots or blotches in various sizes in Z. dugritei (David 1872; Li et al. 2012a), Z. hongchibaensis (Li et al. 2012a), Z. hui (Liu 1945), and Z. wui (Li et al. 2012a).

Zhangixalus arboreus has a green dorsum with numerous dark spots (Okada and Kawano 1924), which is absent in Zhangixalus thaoae sp. nov.

Zhangixalus achantharrhena has a green dorsum, without spots, and a cream venter, without the brown flecks of the new species (Harvey et al. 2002).

Zhangixalus arvalis has a white line along flanks, which is absent in Z. thaoae sp. nov. (Lue et al. 1995).

Zhangixalus chenfui has a brown ventrum, with a dark-brown pattern, not cream-colored, without a dark-brown pattern, as in Zhangixalus thaoae sp. nov. (Liu 1945).

Zhangixalus dorsoviridis has cream flanks with many black blotches and the anterior thigh is orange with some irregularly sized black circles (Bourret 1937) (Figs 6C, D, 7C, D). In Z. thaoae sp. nov., the flanks are cream with one single black spot and the posterior thigh is orange with a single large black blotch (Figs 6A, B, 7A, B).

10.3897/zookeys.1197.104851.figure6 3738C89E-35B2-59B9-8E47-526B168F5B67

Dorsal and ventral views of three Zhangixalus species in life (except for F, which was immediately photographed after the specimen was anesthetized) A, BZhangixalus thaoae sp. nov. IEBR.A 5136 C, DZ. dorsoviridis VNMN 06156 E, FZ. jodiae VNMN 07122.

https://binary.pensoft.net/fig/1021511

Zhangixalus dulitensis is pea-green dorsally with some white dots, the head and back have purplish dots, there is purplish line from eye to eye around the snout and passing through the nostrils, and there is reddish-brown patch on each eyelid (Boulenger 1892; Haas et al. 2012). Zhangixalus thaoae sp. nov. does not have this color pattern.

Zhangixalus jarujini has a reddish-brown dorsum with irregular dark-brown markings, while Z. thaoae sp. nov. has a green dorsum without any markings.

Zhangixalus jodiae has black and orange blotches interposed on anterior, posterior part of thighs and ventral surface of tibia (Figs 6E, F, 7E, F); Z. thaoae sp. nov. does not have this color pattern, but rather a large, black blotch on an orange background (Figs 6A, B, 7A, B).

10.3897/zookeys.1197.104851.figure7 985F2641-0527-5530-8205-ABC99F1B3015

Dorsal and ventral views of three Zhangixalus species in preservative A, BZhangixalus thaoae sp. nov IEBR.A 5136 C, DZ. dorsoviridis VNMN 06156 E, FZ. jodiae VNMN 07122.

https://binary.pensoft.net/fig/1021512

Zhangixalus leucofasciatus exhibits a cream axilla without dark spots, in contrast to a black blotch on the cream axilla of Z. thaoae sp. nov.; additionally, Z. leucofasciatus possesses a white stripe along the upper lip, body and limbs, a feature that is absent in Z. thaoae sp. nov. (Liu and Hu 1962; Fei et al. 2010).

Zhangixalus pinglongensis has flanks, anterior and posterior surfaces of the thigh covered with black blotches and white spots (Mo et al. 2016). In contrast, Z. thaoae sp. nov. lacks similar black blotches with white spots on its flanks, anterior and posterior surfaces of the thighs.

Zhangixalus minimus is characterized by a dark-brown mottling pattern on its hands and feet, which is absent in Z. thaoae sp. nov. (Rao et al. 2006).

Zhangixalus melanoleucus is whitish cream with an irregular black pattern on the ventral surface of its thighs, shanks, dorsal surfaces of the feet and fingers I, II, and III (Brakels et al. 2023). Meanwhile, in Z. thaoae sp. nov., the ventral surface of the thighs, shanks, dorsal surfaces of the feet and fingers I, II, and III are orange, without any irregular black pattern.

Zhangixalus moltrechti has a red-orange anterior and posterior thigh with multiple dark spots, while the thigh in Z. thaoae sp. nov. has an orange anterior with no spots and an orange posterior with a single large blotch (Boulenger 1908; Fei et al. 2010). Furthermore, the webbings of the foot in Z. moltrechti are red-orange, with dark spots, while those in Z. thaoae sp. nov. are yellow with no spots.

Zhangixalus nigropunctatus has yellow flanks and posterior thigh with some black blotches, in contrast to Z. thaoae sp. nov., in which the flank is cream-colored and the posterior thigh is orange, both featuring a single black spot (Liu et al. 1962; Fei et al. 2010). In Z. nigropunctatus, there is also a white stripe along the flanks and limbs, separating the dorsal and ventral sides, which is a feature that is absent in Z. thaoae sp. nov.

Zhangixalus schlegelii has flanks and groin without spots, while in Z. thaoae sp. nov. these each have a large black blotch (Günther 1858). A white stripe along the flanks and limbs, separating the dorsal and ventral sides, is present in Z. schlegelii but absent in Z. thaoae sp. nov. Furthermore, there is a prominent supratympanic fold in Z. schlegelii, which is only weakly visible in Z. thaoae sp. nov.

Zhangixalus yinggelingensis has green dorsal head, body and limbs, adorned with a small number of very fine white spots (Chou et al. 2007), which are absent in Z. thaoae sp. nov. In addition, the supratympanic fold in Z. yinggelingensis is prominent, whereas it is weakly visible in Z. thaoae sp. nov.

Zhangixalus yunnanensis has greyish webbings and yellowish-brown iris, which are respectively orange and red-bronze in Z. thaoae sp. nov. (Pan et al. 2024). Furthermore, Z. yunnanensis exhibits a somewhat broader head (IND/IOD 0.96-1), which is proportionally smaller relative to the body (HL/SVL 0.31-0.33) compared to the head of Z. thaoae sp. nov. (IND/IOD 0.72-0.85, HL/SVL 0.37-0.43).

Zhangixalus zhoukaiyae has yellowish posterior thigh with irregular greyish blotching, whereas the posterior thigh of Z. thaoae sp. nov. is orange without spots. In Z. zhoukaiyae, the pupil is dark charcoal-grey and the iris is golden-yellow, while in Z. thaoae sp. nov. the pupil is black, and the iris is red-bronze (Pan et al. 2017).

A more detailed comparison of morphological differences between Z. thaoae and other members in the Z. chenfui group can be found in Table 4.

Comparison of morphology character of species within Z. chenfui group.

Species SVL Dorsum color Vomerine teeth Ventral color Flank coloration Snout shape The color pattern of the thigh Fingers web formula Toes web formula Source
Zhangixalus thaoae sp nov. 30-32.2 (M) Smooth, and green without spots Present Lower jaw cream with grey and throat region white belly cream without spots Flank cream with a black blotch Rounded The anterior part of the thigh is orange without spots; the posterior part of the thigh is orange with a large black blotch I1¼-1¼II1-2III1-1IV I½-½II0-1½III¼ -1¾IV1¾-½V This study
Z. chenfui 33-41 (M) 48-55 (F) Skin with granules above Present Ventral brown with dark-brown pattern Flanks orange with blotches, have a black strip along flank isolated upper and lower part of the body Rounded Anterior of thigh orange with blotches – – Liu et al. 1945; Fei et al. 2009
Z. jodiae 36.1–39.8 (M) Dorsal surface of the head and body green without spots Present Lower jaw region greyish, chest and belly cream Flank cream, axilla, and groin with large black blotches Rounded The dorsal surface is green without spots, the front-rear parts of the thigh, and the ventral surface of the tibia black with orange blotches I1-1II1-1III2-1IV I1-1II½-1III0-1½IV1-½V Nguyen et al. 2021
Z. melanoleucus 34.4-36.3 (M) 53.7 (F) Dorsum smooth and uniform green with several dark and light-green spots Present Throat grey with dark grey margins, chest and belly immaculate white Flank white, covered by an irregular black pattern, groin cream Rounded The anterior and posterior surfaces of the thighs are white cream covered with an irregular black pattern, ventral surfaces of the thighs cream I2½-3II2-3 III2¼- 2IV I2-2½II1-2III1-2 IV2-1V Brakels et al. 2023
Z. nigropunctatus 32.0–37.0 (M) 44-45(F) Dorsum smooth and green _ Lower jaw grey, chest and belly white Flanks orange with blotches has a white strip along isolated dorsal and ventral side Rounded Dorsal surface green, anterior and posterior surfaces of thighs orange and has some black blotches on posterior of thigh – – Fei et al. 2009
Z. pinglongensis 32.0–38.5 (M) Dorsum smooth and green Present Lower jaw grey, chest and belly cream Flank covered with black blotches with white spots or white spots with a faint orange tint Rounded The anterior and posterior surfaces of the thigh covered with black blotches with white spots or white spots with a faint orange tint I 1-–1-II 1+–1+III2-–2-IV I1+–1-II 2+–2-III 2+–3-IV 3-–2+V Mo et al. 2016
Z. yaoshanensis 31.6–36.4 (M) 51.1 (F) The dorsal surface is smooth and green, with or without faint spots, Present Throat grey in males, ventral green without spots The posterior surface of the flanks is orange-red without spots Point Dorsal surface green with or without faint green spots, anterior and posterior surface of thighs orange-red without spots I1-–1-II1–1- III1+–2-IV I 1+–1-II1+–1+III 2+–2 IV2+–2+V Chen et al. 2018
Z. yunnanensis 31.3-36.0 (M) 47.6-48.6 (F) Smooth and green Present Throat black Cream mottled with greyish brown Rounded Black blotches in axilla, groin, and posterior part of thigh I1–1II1–2III1–1IV I1–1II0.5–2.5III1–2IV2–0.5IV Pan et al. 2024
Discussion

Zhangixalus nigropunctatus was first recorded in Weining District, Guizhou Province, China by Liu et al. (1962) in 1962. Based Orlov et al. (2001) study of a series of specimens collected from Fan Si Pan Mountain, in the northern part of the Hoang Lien Mountains, Sa Pa district, Lao Cai Province, they suggested that Z. nigropunctatus might be a junior synonym of Z. dorsoviridis, a species originally described by Bourret (1937) from Sa Pa District, Lao Cai Province, Vietnam. Li et al. (2012b) investigated the phylogenetic relationships of the genus Rhacophorus sensu lato and showed that a specimen identified as Z. nigropunctatus in the collection of Orlov et al. (2001; ROM 38011) was nested in the same subclade as Z. nigropunctatus from China (Weining, Guizhou) (Pan et al. 2024). Although the genetic distance between them was greater than 5% and there was no specimen description, Orlov et al. (2001) concluded that both Z. dorsoviridis and Z. nigropunctatus occur in Sa Pa, Lao Cai Province (see also Pan et al. 2017).

The phylogenetic results of our study reveal that the specimen JX219427 from (ROM 38011; Orlov et al. 2001), which was originally classified as belonging in the Z. nigropunctatus clade of Li et al. (2012b), is in fact Z. thaoae with a genetic distance of only 0.19 compared with the three other newly found specimens. Furthermore, Z. thaoae, Z. dorsoviridis, and Z. nigropunctatus show high genetic distance of at least 5.5%. This large genetic distance makes it unlikely that by using genetic data, the taxonomic assignment of a specimen into the three aforementioned species can be confused. Although molecular data indicates that ROM 38011 belongs to Z. thaoae, detailed morphological data to support such assignment is not available; neither Orlov et al. (2001) nor Li et al. (2012b) provided a detailed description of that specimen, and Orlov et al. (2001) did not provide a figure of ROM 38011. However, Orlov et al. (2001), noted that ROM 38011 had an “obviously darkened” median vocal sac which differed from the other specimens they examined.

Overall, there is evidence that the specimen ROM 38011 had been misidentified as either Z. dorsoviridis or Z. nigropunctatus and that Z. thaoae is a valid new taxon. Based on our reclassification of ROM 38011 as Z. thaoae and lack of other known records, we herein formally exclude Z. nigropunctatus from the amphibian fauna of Vietnam. Zhangixalus nigropunctatus is only known from Yunnan Province (Pingbian), Anhui Province (Yuei), Guizhou Province, (Weining and Leishan), and Hunan Province (Sangzhi and Chengbu), China (Li et al. 2012b; Pan et al. 2017, 2024).

In addition to the new species described in this paper, Nguyen et al. (2020) previously described Z. jodiae and assigned earlier records of Z. dorsoviridis from Ha Giang Province to Z. jodiae. Thus, two species belonging to the Z. chenfui group of green treefrogs, Z. jodiae and Zhangixalus thaoae, are currently known from Vietnam.

 Acknowledgements

We are grateful to the directorates of the Forest Protection Department of Lao Cai Province for their support of our fieldwork and for issuing relevant permits. We thank MA. Luong (IEBR) for supporting in the field and TN. Nguyen for supporting in laboratory. This research was partially funded by the State Themes of ZISP 122031100282-2 and grant no.075-15-2021-1069 by the Ministry of Science and Higher Education of the Russian Federation to Nikolai Orlov.

 Additional information Conflict of interest

The authors have declared that no competing interests exist.

 Ethical statement

No ethical statement was reported.

 Funding

This research is funded by the Vietnam Academy of Science and Technology (VAST) under grant number NCXS.01.03/23–25.

 Author contributions

Writing – original draft: TTN. Writing – reviewing and editing: TTN, NO, TZ. Funding acquisition & Supervision: HHN, TTN. Investigation – sample collection: CVH. Formal analysis – phylogenetic & morphological analysis: HTN, LTHL, HTB.

 Author ORCIDs

Tao Thien Nguyen https://orcid.org/0000-0002-5640-4536

Huy Hoang Nguyen https://orcid.org/0000-0002-6284-5813

Hoa Thi Ninh https://orcid.org/0009-0002-8133-432X

Linh Tu Hoang Le https://orcid.org/0009-0003-0887-2786

Hai Tuan Bui https://orcid.org/0000-0003-4065-7229

Nikolai Orlov https://orcid.org/0000-0003-4401-348X

Chung Van Hoang https://orcid.org/0000-0002-0709-974X

Thomas Ziegler https://orcid.org/0000-0002-4797-609X

 Data availability

All of the data that support the findings of this study are available in the main text.

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