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A study using preserved specimens, observations of living frogs, and mitochondrial DNA has confirmed that the Napo treefrog, Rhacophorus napoensis, lives in Vietnam for the first time. The confirmed records come from four Vietnamese provinces: Bac Giang, Thanh Hoa, Nghe An, and Ha Tinh. Researchers carefully examined 11 Vietnamese specimens, including nine adult males and two adult females. They measured body parts and studied colors and physical features. They also examined mitochondrial 16S rRNA DNA sequences, which helped them place the frogs within the treefrog family tree.

The findings also show that several frogs previously reported in Vietnam as Rhacophorus rhodopus are more accurately identified as R. napoensis. Scientists first officially described R. napoensis in 2022, based on five males collected in Napo County, Baise City, in China's Guangxi Autonomous Region. The original collection site is only about four kilometres from the Vietnamese border in a straight line. However, before this study, the species had only been recorded in Guangxi.

The new Vietnamese records connect the species' known distribution across the international border and greatly extend its known range from the small area previously known in southern China. The southernmost confirmed Vietnamese location is in the Huong Son area of Vu Quang National Park, about 520 kilometres in a straight line from the original collection site. The species may live across a larger area because suitable forest exists between and beyond the places where researchers found it. However, the study confirms specific locations rather than establishing the frog's full range.

The researchers combined studies of preserved frogs with genetic evidence because most earlier Vietnamese records of the red-webbed treefrog were based mainly on physical appearance, and very few had been studied genetically. They examined 39 specimens identified as R. bipunctatus, R. napoensis, or R. rhodopus. These specimens came from northern and central Vietnam, northern and central Myanmar, and southern China. The researchers compared their measurements with information published in earlier taxonomic studies.

The researchers used digital calipers to measure body parts and limbs to the nearest 0.01 millimetre. They then rounded the measurements to 0.1 millimetre. They determined the frogs' sex by examining their reproductive organs after dissection. For the DNA analysis, they lined up mitochondrial 16S rRNA sequences using MAFFT, calculated uncorrected genetic distances using MEGA 11, and built evolutionary trees using two methods: maximum likelihood and Bayesian analysis. They used Zhangixalus dennysi and Z. dugritei as outgroups to help place the frogs in the evolutionary tree. They also produced a new DNA sequence from a specimen collected in Tay Yen Tu Nature Reserve.

The maximum-likelihood and Bayesian analyses produced similar evolutionary trees. Both placed the Vietnamese frog populations together with the original group of R. napoensis specimens from Guangxi. The larger group containing R. napoensis had strong statistical support and split into two smaller groups. One contained DNA sequences from the original specimens and frogs from Bac Giang. The other contained frogs from Thanh Hoa, Nghe An, and Ha Tinh.

The genetic difference between these two smaller groups was 1.8%, based on uncorrected pairwise distance. More broadly, the DNA sequences assigned to the Rhacophorus bipunctatus group formed six strongly different genetic lineages. These were 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 results therefore revealed several genetically distinct groups among frogs that had often been given species names based on their outward appearance.

The Vietnamese frogs also closely matched the updated description of R. napoensis. Adult males measured 38.6–43.6 millimetres from the tip of the snout to the rear end of the body, while the two females measured 54.9 and 55.8 millimetres. Their identifying features included a pointed snout with a clear bulge at its tip, a round eardrum, a heart-shaped tongue with a notch at the back, one external vocal sac under the throat, fully webbed fingers and toes, and a small, flat bump on the inner side of the foot near the ankle.

When the hind leg was stretched forward along the body, the ankle joint reached the snout. Living frogs had reddish-brown or grayish-brown backs, yellowish-white undersides, two or three black spots near the armpits, and reddish-orange webbing between their fingers and toes without black spots. Their heads were almost as wide as they were long. The ratio of head width to head length ranged from 1.00 to 1.05 in males and from 1.04 to 1.05 in females.

The study revises three features described in the original species description because the larger sample from Vietnam showed that these features can vary. In Vietnamese males, head width was about equal to head length or only slightly greater, rather than always being greater. In most Vietnamese males, the tibia, or lower leg bone, was less than half the length of the body from snout to rear end. The tibia-to-body ratios ranged from 0.45 to 0.49, with an average of 0.47. The earlier description had reported values ranging from 0.50 to 0.51.

Adult males also had a nuptial pad on the first finger, even though the original species description said this feature was absent. The authors conclude that head width, the ratio of tibia length to body length, and the presence of a nuptial pad vary too much to reliably identify R. napoensis. Instead, the updated description uses a wider combination of features, including body proportions, head shape, eardrum, tongue, vocal sac, webbing, colors, and the position of the foot bump.

Field observations show that this treefrog lives in trees and shrubs in forest areas between 181 and 1,360 metres above sea level. In Tay Yen Tu Nature Reserve, researchers found frogs on trees and bushes about 0.5–1.5 metres above the ground, beside pooled water in secondary forest. In Pu Hoat Nature Reserve, they found frogs in a tree about 0.5–2.0 metres above the ground, more than ten metres from the nearest stream and beside a puddle in secondary forest.

At Pu Mat National Park, frogs called from leaves and branches 1–2.5 metres above the ground near still water between 19:00 and 21:00. A call recorded in August at Pu Hoat contained two types of calls, each with one or two notes. The first type had 3–29 pulses, while the second had 4–26 pulses. The strongest sound frequencies ranged from 1.9 to 2.3 kilohertz.

During the breeding season from May to August at Pu Mat National Park, researchers observed the frogs breeding in rain-filled pools and still water within streams. The frogs made foam nests on tree branches hanging over shallow, flowing water. Other treefrogs from the Rhacophoridae family lived in the same areas, including Rhacophorus kio and R. orlovi, as well as species from the genera Theloderma, Zhangixalus, Kurixalus, Polypedates, Gracixalus, Raorchestes, and Rohanixalus.

The species was found in secondary forest and evergreen forest, including both lowland and mountain habitats. However, observations cover only certain places and seasons. Researchers still know little about the full range of habitats it uses, its population size and changes over time, its breeding behaviour, and its general ecology. The authors identify habitat loss, forest damage, and collection for food as the main possible threats to the species in Vietnam.

The findings change what scientists know about the ranges of species in this difficult-to-classify group. The authors limit the confirmed range of R. bipunctatus to northeastern India, Bangladesh, Bhutan, and northern and western Myanmar. They limit 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 also suggest, with some uncertainty, that many records previously identified as these two species in other parts of northern and north-central Vietnam, central Laos, southeastern Yunnan, and other areas of Guangxi may actually be R. napoensis.

This suggestion remains a hypothesis that needs to be tested by examining specimens again, using both DNA and physical features. In contrast, measurements of frogs from Muong Nhe Nature Reserve in Dien Bien, Vietnam, match R. rhodopus. The researchers therefore provisionally keep these frogs under that name until future genetic studies can confirm their identity. With the Vietnamese records accepted, the number of recognized Rhacophorus species in Vietnam rises from 16 to 17, with nine species found only in that country.

The authors suggest that R. napoensis may qualify for a preliminary Least Concern assessment because it is now known from seven locations across northern and north-central Vietnam and southern Guangxi. However, its actual range, population trends, and breeding ecology remain insufficiently understood. The study also leaves several questions unanswered, including the status of R. namdaphaensis, the identities of three unnamed genetic lineages in the R. bipunctatus–R. rhodopus group, and the evolutionary relationships among several Vietnamese members of the R. hoanglienensis–orlovi group. For treefrog research more broadly, the main result is the confirmation that a recently described species also occurs outside its original range in China. The findings also show that more field surveys and taxonomic research are needed to determine where different Rhacophorus species live and how to identify them across Vietnam and nearby parts of Southeast Asia.

Subject of Research: A study of physical features and mitochondrial DNA that re-examines Rhacophorus napoensis and confirms 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, body measurements, taxonomic reassessment, species distribution, forest amphibians, redescription, significant range extension