Taxonomic assessment of Zhangixalus puerensis sensu lato identifies two cryptic new species

A taxonomic assessment of the tree frog Zhangixalus puerensis sensu lato found four genetically distinct lineages, two of which were formally described as new species: Zhangixalus liui from Wenshan City in Yunnan, China, and Ha Giang Province, Vietnam, and Zhangixalus orlovi from Yongde County in Lincang City, Yunnan, China. A third lineage from Sa Pa District in Lao Cai Province, Vietnam, remains unnamed because the researchers did not obtain physical or morphological specimens from it.

The study assessed Z. puerensis sensu lato, a name previously applied to frogs recorded across much of Yunnan and in northern Vietnam. Researchers conducted field surveys in Yunnan from 2020 to 2026 and collected specimens at several locations. They combined these specimens and newly generated mitochondrial sequences with sequences obtained from GenBank, including samples from elsewhere in Yunnan and northern Vietnam, to examine the taxonomy and distribution of the broadly recorded species.

For the molecular analysis, the researchers extracted total genomic DNA from liver tissue and amplified and sequenced a fragment of the mitochondrial 16S rRNA gene. They aligned the sequences with MAFFT 7, calculated average uncorrected p-distances in MEGA 12.0.10, and constructed phylogenetic trees through both Bayesian inference and maximum-likelihood analyses. The outgroups were Theloderma albopunctatum, Rhacophorus rhodopus and Leptomantis gauni. Species partitions were also evaluated with the Assemble Species by Automatic Partitioning method, in which lower scores indicate better-supported partitions.

Both phylogenetic analyses divided Z. puerensis sensu lato into four clades, although the relationships among those clades were not resolved because node support was low. The first included samples from Pu’er City, the type locality of Z. puerensis, and from Baoshan City, Dali Prefecture, Yuxi City and Honghe Prefecture, all in Yunnan. The other clades contained samples from Sa Pa District in Lao Cai Province, Wenshan Prefecture in Yunnan together with Ha Giang Province in Vietnam, and Lincang City in Yunnan.

Uncorrected genetic distances among the four clades ranged from 2.2% to 2.6%. These distances were greater than those between several species pairs already recognized in the genus, including Z. dugritei and Z. hui at 0.5%, Z. duboisi and Z. meimontis at 1.6%, and Z. lishuiensis and Z. zhoukaiyae at 1.5%. The ASAP analysis supported the Wenshan, Lincang and Sa Pa samples as three species distinct from Z. puerensis sensu stricto. The authors treated the four lineages as separate species based on the combined molecular results, genetic-distance comparisons and available morphological evidence.

Zhangixalus liui has a male snout-vent length of 42.1–46.4 mm and is distinguished by a head wider than long, heels that do not meet when the legs are positioned at right angles to the body, and a tibiotarsal articulation that reaches the centre or anterior margin of the tympanum when the hind limb is stretched forward. Its fifth toe is longer than its third toe, and a nuptial pad is present on the first finger. The dorsal surface is green with more or fewer dark-edged brown spots, while large black patches occur in the axilla and groin region. The holotype and paratypes were collected in Bozhu Town, Wenshan City, at 2,540 m in April 2025.

The species is also known from Ha Giang Province, Vietnam, based on GenBank sequences that differed by 0.5% from the Wenshan specimens. At its type locality, frogs were found in mud holes or grass beside a small pond on grassland surrounded by montane broadleaved forest. Advertisement calls and foamy egg nests were recorded in April, and the authors presumed a breeding period from April to June. The authors also noted that Z. liui can change its dorsal colour from green with brown spots to brown or brownish-black, with corresponding changes in its ventral spotting.

Zhangixalus orlovi has a smaller male snout-vent length of 34.0–41.3 mm. Its tibiotarsal articulation reaches the posterior margin or centre of the tympanum, its third and fifth toes are approximately equal in length, and nuptial pads occur on the first and second fingers. Its dorsal surface may be green with no spots, a few spots or many dark-edged brown spots, while the posterior thigh is black with many white spots. The holotype and paratypes were collected at Daxueshan Township, Yongde County, at 2,940 m in May 2026. This species is currently known only from that type locality, where advertisement calls and foamy egg nests were observed in April and May.

Z. liui and Z. orlovi can be distinguished from one another by body size, toe proportions, the presence of nuptial pads on the first or first and second fingers, and the size and arrangement of black markings on the axilla, groin and posterior thigh. The study remains limited by its reliance on a single mitochondrial gene fragment. The authors said additional gene fragments, particularly nuclear genes, would be needed for more reliable results. They also cautioned that internal and external vocal sacs can be difficult to distinguish from external appearance alone, while the taxonomic status of populations reported from northern Yunnan remains unresolved.

Subject of Research: Taxonomic and genetic assessment of four lineages within Zhangixalus puerensis sensu lato

Article Title: Taxonomic assessment of Zhangixalus puerensis (He, 1999) sensu lato ( Anura , Rhacophoridae ), with description of two cryptic species

Article References: Liu, S., Bu, C., Deng, T., Bai, J., Luo, B., Hou, M., Li, X., Zhang, J., Li, W., Wang, Z., Feng, Y., Wang, J., & Rao, D. (2026). Taxonomic assessment of Zhangixalus puerensis (He, 1999) sensu lato (Anura, Rhacophoridae), with description of two cryptic species. Biodiversity Data Journal, 14, Article e204046. https://doi.org/10.3897/bdj.14.e204046

Image Credits: AI Generated

DOI: 10.3897/BDJ.14.e204046

Keywords: Zhangixalus puerensis, cryptic species, tree frogs, mitochondrial 16S rRNA, phylogenetics, species delimitation, Yunnan, Vietnam, taxonomy, amphibians, Taxonomic, assessment