Napo treefrog confirmed in Vietnam, extending its documented range

A taxonomic study combining preserved specimens, live-animal observations and mitochondrial DNA has confirmed the Napo treefrog, Rhacophorus napoensis, in Vietnam for the first time. The verified records come from four Vietnamese provinces: Bac Giang, Thanh Hoa, Nghe An and Ha Tinh. Eleven Vietnamese specimens, including nine adult males and two adult females, were examined through detailed morphometrics, coloration and anatomical characters, while mitochondrial 16S rRNA sequences placed them within the broader treefrog phylogeny.

The findings also show that several frogs previously reported in Vietnam as Rhacophorus rhodopus are better identified as R. napoensis. R. napoensis was formally described in 2022 from five males collected in Napo County, Baise City, Guangxi Autonomous Region, China. Its type locality lies only about four kilometres from the Vietnamese border in a straight line, but until this study the species had been recorded nowhere beyond Guangxi.

The Vietnamese evidence now bridges the international boundary and adds a substantial longitudinal extension to a distribution previously represented by one narrow area in southern China. The southernmost confirmed Vietnamese site is in the Huong Son area of Vu Quang National Park, about 520 airline kilometres from the type locality. The true biological range may be larger because suitable forest lies between and beyond the sampled sites, although the study documents the confirmed occurrences rather than establishing a complete range.

The analytical approach combined preserved specimens with genetic evidence because many Vietnamese records of the red-webbed treefrog had rested primarily on morphology, with very few molecular investigations available. The researchers examined 39 specimens assigned to R. bipunctatus, R. napoensis or R. rhodopus from northern and central Vietnam, northern and central Myanmar, and southern China. They compared their measurements with data in the taxonomic literature.

Body and limb measurements were taken with digital calipers to the nearest 0.01 millimetre and then rounded to 0.1 millimetre, while sex was determined by gonadal inspection after dissection. For the molecular analysis, the investigators aligned mitochondrial 16S rRNA sequences in MAFFT, calculated uncorrected genetic distances in MEGA 11 and reconstructed trees through both maximum-likelihood and Bayesian methods. Zhangixalus dennysi and Z. dugritei served as outgroups, and a new sequence was generated from a specimen collected in Tay Yen Tu Nature Reserve.

Maximum-likelihood and Bayesian analyses produced similar trees and placed the sampled Vietnamese populations with the Guangxi type series of R. napoensis. The encompassing R. napoensis clade was highly supported and contained two sub-clades. One contained the type-series sequences and the Bac Giang sequences; the other contained specimens from Thanh Hoa, Nghe An and Ha Tinh.

The uncorrected pairwise distance between these two sub-clades was 1.8%. More broadly, the sequences assigned to the Rhacophorus bipunctatus complex formed six highly divergent lineages: true R. bipunctatus from northern Myanmar and eastern India; true R. rhodopus from southern Yunnan; R. napoensis from Guangxi and northeastern Vietnam; and three unnamed lineages from western Yunnan, Xizang, and Hainan together with Gia Lai. The analysis therefore identified several genetically distinct lineages within a complex in which external appearance had often been used to assign names.

The Vietnamese specimens also closely matched the revised morphology of R. napoensis. Adult males measured 38.6–43.6 millimetres in snout–vent length, while the two females measured 54.9 and 55.8 millimetres. Diagnostic features included a pointed snout with a distinct terminal bulge, a rounded tympanum, a cordiform tongue notched at the rear, a single external subgular vocal sac, fully webbed fingers and toes, and a small, flat inner metatarsal tubercle.

The tibiotarsal joint reached the snout when the hindlimb was adpressed against the body. Living animals had a reddish- or gray-brown dorsum, yellowish-white underside, two or three black axillary blotches, and reddish-orange finger and toe webbing without black spotting. Their head was nearly equal in width and length, with the head-width-to-head-length ratio ranging from 1.00 to 1.05 in males and from 1.04 to 1.05 in females.

The study revises three traits proposed in the original description because they varied in the larger Vietnamese sample. Head width in the Vietnamese males was approximately equal to, or only slightly greater than, head length, rather than consistently greater. Tibia length was less than half of snout–vent length in most Vietnamese males, with ratios of 0.45–0.49 and an average of 0.47, rather than the previously reported range of 0.50–0.51.

Adult males also possessed a nuptial pad on the first finger, contrary to its absence in the original diagnosis. The authors conclude that head width, tibia-to-body ratio and nuptial-pad presence are too variable to serve as diagnostic characters of R. napoensis. The revised account instead emphasizes a broader set of features, including body proportions, head shape, tympanum, tongue, vocal sac, webbing, coloration and the position of the metatarsal tubercle.

Field observations reveal an arboreal frog associated with forest habitats at elevations from 181 to 1,360 metres above sea level. In Tay Yen Tu Nature Reserve, individuals were found on trees and bushes about 0.5–1.5 metres above the ground beside ponded water in secondary forest. Frogs in Pu Hoat Nature Reserve were found in a tree about 0.5–2.0 metres above ground, more than ten metres from the nearest stream and beside a puddle in secondary forest.

At Pu Mat National Park, animals called from leaves and branches 1–2.5 metres above ground near standing water between 19:00 and 21:00. A call recorded in August at Pu Hoat comprised two call types, each containing one or two notes. The first type had 3–29 pulses, the second had 4–26 pulses, and the dominant frequency was 1.9–2.3 kilohertz.

During the May-to-August breeding season in Pu Mat National Park, the frogs were observed breeding in rain pools and standing water within streams. Foam nests were created on tree branches overhanging shallow, moving water. Several other rhacophorid treefrogs occurred alongside R. napoensis, including Rhacophorus kio and R. orlovi, together with species of Theloderma, Zhangixalus, Kurixalus, Polypedates, Gracixalus, Raorchestes and Rohanixalus.

The species was associated with secondary and evergreen forest, including lowland and montane settings. These observations remain limited to particular sites and seasons, so the breadth of its habitat use, population size, trends, reproductive behaviour and general ecology are still poorly known. The authors identify habitat loss, forest degradation and harvesting for food as principal potential threats in Vietnam.

The findings reshape known ranges within a taxonomically difficult group. The authors restrict the confirmed range of R. bipunctatus to northeastern India, Bangladesh, Bhutan, and northern and western Myanmar, and the confirmed range of R. rhodopus to southern China, central and southern Myanmar, Thailand, northern and northwestern Laos, northwestern Vietnam, Cambodia, and western Peninsular Malaysia. They tentatively suggest that many records previously assigned to those species elsewhere in northern and north-central Vietnam, central Laos, southeastern Yunnan and other parts of Guangxi may instead belong to R. napoensis.

That proposal remains a hypothesis requiring re-examination of specimens, including molecular and morphological analyses. By contrast, the population at Muong Nhe Nature Reserve in Dien Bien, Vietnam, has measurements consistent with R. rhodopus and is provisionally retained under that name pending future genetic study. With the Vietnamese records accepted, Vietnam’s recognized Rhacophorus species total rises from 16 to 17, nine of them endemic to the country.

The authors suggest a preliminary Least Concern assessment for R. napoensis because the frog is now known from seven localities across northern and north-central Vietnam and southern Guangxi, although its actual range, population trends and reproductive ecology remain insufficiently characterized. The study also leaves several questions open, including the status of R. namdaphaensis, the identity of three unnamed lineages in the R. bipunctatus–R. rhodopus complex, and the relationships among several Vietnamese members of the R. hoanglienensis–orlovi group. For the broader treefrog fauna, the central result is the confirmation of a newly described species outside its original Chinese range and evidence that additional surveys and taxonomic work are needed to resolve the distribution and identities of Rhacophorus species across Vietnam and neighboring parts of Southeast Asia.

Subject of Research: Morphological and mitochondrial reassessment of Rhacophorus napoensis confirming its first records in Vietnam

Article Title: Redescription and a significant range extension of Rhacophorus napoensis Li, Liu, Yu & Sun, 2022 ( Amphibia , Anura , Rhacophorinae ) with the first record from Vietnam

Article References: Nguyen, T. V., Liu, S., Wilkinson, J. A., Tran, T. G., Tran, P. N., Trofimets, A. V., Dau, V. Q., & Poyarkov, N. A. (2024). Redescription and a significant range extension of Rhacophorus napoensis Li, Liu, Yu & Sun, 2022 (Amphibia, Anura, Rhacophorinae) with the first record from Vietnam. Herpetozoa, 37, 123-136. https://doi.org/10.3897/herpetozoa.37.e122317

Image Credits: AI Generated

DOI: 10.3897/herpetozoa.37.e122317

Keywords: Rhacophorus napoensis, Napo treefrog, Vietnam, Guangxi, treefrogs, mitochondrial 16S, morphometrics, taxonomic reassessment, species distribution, forest amphibians, Redescription, significant