Limestone frog hidden across China–Vietnam border turns up in Yunnan karst forests

Deep in the limestone forests of south-eastern Yunnan Province, close to the border with Vietnam, three unassuming female frogs collected at night near a cave entrance have quietly rewritten the map of a species that, until very recently, was known from a single mountaintop reserve in northern Vietnam. The frogs, examined by a team of Chinese herpetologists and now reported in the Biodiversity Data Journal, belong to Odorrana nagao, a moss-green cascade frog described only in 2026 from the karst forests of Tuyen Quang Province. Their identification, confirmed by mitochondrial DNA sequencing and detailed morphological comparison, extends the species’ known range roughly 75 kilometres northwards across an international border and adds a new member to China’s already remarkable amphibian fauna.

The discovery matters far beyond a simple country tick. Odorrana nagao belongs to the O. lipuensis species group, a phylogenetically distinct lineage of frogs whose members are intimately tied to limestone landscapes — karst streams, cave mouths and subterranean aquatic habitats. All six species currently recognised in the group have been described since 2015, a rate of discovery that suggests scientists are only now beginning to catalogue the diversity sheltered within the fragmented, rugged and often dangerously inaccessible karst blocks of southern China and northern Vietnam. With the new record, the number of Odorrana species known from China rises from 47 to 48, and Yunnan — the province with the highest documented amphibian richness in the country — now harbours 17 species of the genus.

The genus Odorrana, the Asian cascade frogs, sprawls across an enormous range: from north-eastern India and southern mainland China, through mainland Southeast Asia, to the Malay Peninsula and the Greater Sunda Islands, and eastwards to Hainan, Taiwan and the Ryukyu Archipelago. Most species live along forested streams, but several have evolved a striking affinity for limestone. That affinity is no accident of geography. Karst systems are environmentally heterogeneous and fragmented, scattered across the landscape like islands, which restricts dispersal and promotes local differentiation of populations. Add the sheer difficulty of surveying many limestone areas, and it becomes clear why the amphibian diversity of these terrains remains incompletely documented.

The trail to the new record began in May 2026, during fieldwork in Laoshan Provincial Nature Reserve in Malipo County, Yunnan, at an elevation of 1,030 metres above sea level. Three adult female Odorrana, referable to the O. lipuensis species group on external appearance, were collected after complete darkness on limestone rocks near the entrance of a cave — a microhabitat closely matching that reported at the species’ type locality in Vietnam. The specimens were euthanised with tricaine methanesulphonate, fixed in buffered formalin and preserved in 75% ethanol in the herpetological collection of Sun Yat-sen University in Guangzhou, with thigh-muscle tissue retained in 95% ethanol for molecular work.

In the laboratory, the team amplified partial fragments of two mitochondrial markers — the 16S ribosomal RNA gene and the cytochrome c oxidase subunit I gene, COI — following established primers and protocols. The final concatenated alignment spanned 1,630 base pairs, comprising 1,036 base pairs of 16S and 594 of COI, and included 23 ingroup samples of Odorrana plus two samples of the related genus Nidirana as outgroups. Phylogenetic relationships were inferred under both Maximum Likelihood and Bayesian Inference, with substitution models selected separately for 16S and the three COI codon positions using the Bayesian information criterion. Nodal support was assessed with 1,000 SH-like approximate likelihood-ratio test replicates and 1,000 ultrafast bootstrap replicates, alongside Bayesian posterior probabilities from two independent MrBayes runs of 100 million generations each.

The results were unambiguous. The six sampled species of the O. lipuensis species group formed a well-supported lineage distinct from other Odorrana, with combined support values of 1.00 for Bayesian posterior probability, 99.4 for SH-aLRT and 100 for ultrafast bootstrap. Within that lineage, the three Yunnan specimens clustered with two specimens from the type series of O. nagao from Tuyen Quang Province in a strongly supported clade (1.00/98.3/99). Genetic distances sealed the case: the Kimura two-parameter distance between the Chinese frogs and the Vietnamese type series was just 0.38% for 16S and 1.73–1.90% for COI, whereas the distance to O. feii, the genetically closest sampled congener, was 2.11% for 16S and 6.25% for COI. The three Chinese specimens showed almost no divergence among themselves — 0 to 0.19% for 16S, with identical COI sequences.

Morphology agreed with the molecules. The three females are of moderate size, with snout–vent lengths of 56.6, 57.0 and 59.5 millimetres. Their heads are longer than wide, their tympana distinct, round and roughly two-thirds of the eye diameter, and their snouts obtusely rounded in dorsal view, projecting beyond the lower jaw. Their fingers bear the group’s signature features — the discs of Fingers III and IV prominently enlarged and transversely oval, with the disc of Finger IV the widest, and the tip of Finger I lacking a lateroventral groove. Their toe webbing follows the diagnostic formula I0–1II0–1III0–1⅓IV1⅓–¼V, and the tibio-tarsal articulation reaches the tip of the snout when the hind-limb is adpressed along the body. In life, the dorsum is moss-green to olive-green with irregular dark-brown blotches, the flanks brownish-green with darker mottling, the upper lip mottled brown and cream, the iris golden brown and the ventral surfaces creamy-white. No diagnostically relevant differences separate them from the type series.

The Chinese specimens do, however, push the species’ known physical limits. They are larger than the females of the Vietnamese type series, which measured 47.1–54.7 millimetres in snout–vent length, extending the known female range of the species to 47.1–59.5 millimetres. The record also raises the documented upper elevational limit by 257 metres, from 773 metres at the type locality to 1,030 metres in Yunnan. Mature ovarian oocytes were present in all three females collected in May, hinting at reproductive activity during this period, though the species’ breeding season, reproductive microhabitat and larval ecology remain unknown. Minor variation was noted in the ratio of internarial to interorbital distance, which is slightly greater than one in the Chinese specimens but was reported as 0.94 in the male holotype — a difference the authors attribute to intraspecific variation.

Biogeographically, the record is entirely plausible. Malipo County and the type locality in Cham Chu Nature Reserve lie within the same transboundary limestone belt, unseparated by any obvious major ecological or geographical barrier. The country record is new, but the presence of the species in southern China is not unexpected and probably reflects incomplete survey coverage in the border region rather than a genuine distributional gap. Extensive limestone formations and potentially suitable karst forests occur between and around the two known localities, and additional populations may well occur in Tuyen Quang and Cao Bang Provinces of northern Vietnam, in Wenshan Prefecture of south-eastern Yunnan, and in other karst landscapes along the international border. The finding adds to a growing body of evidence that the herpetofaunas on either side of the China–Vietnam border are closely connected, with recent studies repeatedly revealing comparable transboundary distributions and previously unrecognised diversity.

Conservation questions remain wide open. Odorrana nagao is currently known from only two confirmed localities, both within protected areas — an encouraging circumstance, but one that does not by itself demonstrate stable populations or the absence of threats. Nothing is yet known about population size, trends, reproductive success or connectivity between the two populations, and the species’ extent of occurrence and area of occupancy cannot be estimated reliably. Potential threats to limestone ecosystems, including quarrying, forest degradation, tourism development and hydrological alteration, have not been evaluated across its possible range. The team provisionally recommends assessing the species as Data Deficient under the IUCN Red List Categories and Criteria, and calls for targeted surveys across the limestone landscapes between and surrounding the two confirmed localities. Notably, while the present manuscript was under peer review, a second team independently published the first record of O. nagao from China, based on a single female collected in Tianbao Town, Malipo County, in 2021 — a study the authors acknowledge as preceding their account, with the two works complementing one another and jointly strengthening the evidence for the species’ occurrence in south-eastern Yunnan. Coordinated cross-border surveys integrating voucher specimens, morphology, molecular data and acoustic observations, the authors argue, will be essential for refining the distribution of O. nagao — and may yet reveal overlooked populations or undescribed diversity within this enigmatic limestone lineage.

Subject of Research: Taxonomy and Biodiversity

Article Title: Range extension of Odorrana nagao ( Anura , Ranidae ) to south-western China, with comments on the limestone-associated Odorrana lipuensis species group

Article References: Song, H.-M., Zhou, D.-Y., Qi, S., Lu, M.-X., Poyarkov, N., Nguyen, T., & Wang, Y.-Y. (2026). Range extension of Odorrana nagao (Anura, Ranidae) to south-western China, with comments on the limestone-associated Odorrana lipuensis species group. Biodiversity Data Journal, 14, Article e213369. https://doi.org/10.3897/bdj.14.e213369

Image Credits: AI Generated

DOI: 10.3897/BDJ.14.e213369

Keywords: Range, extension, Odorrana, nagao, Anura, Ranidae, south-western, China, comments, limestone-associated, lipuensis, species