The specimens were collected by hand at night during a herpetological expedition, with the permission of the reserve’s management and protection bureau. All four individuals, catalogued as KIZ2023080 through KIZ2023083, were adult males gathered on 22 July 2023 at an elevation of 1,430 metres above sea level. Liver tissues were stored in analytical pure ethanol and the whole specimens were preserved in 75 percent ethanol, before being deposited at the Kunming Natural History Museum of Zoology at the Kunming Institute of Zoology, Chinese Academy of Sciences. Morphological measurements were taken with a digital caliper to the nearest 0.1 millimetre, following the methodology used in the species’ original description.

The identification rested on two independent lines of evidence: morphology and genetics. In terms of appearance, the Chinese specimens match the original description of A. truongi closely, with only a few minor variations. The males are relatively small, with snout-vent lengths of 39.3 to 39.9 millimetres. Their heads are moderately long and longer than wide, the snout is relatively long and projects beyond the lower jaw, and the tympanum, the external eardrum, is distinct. The dorsolateral fold is clearly visible, the supratympanic fold is indistinct, and the skin of the head and body is smooth with only a few very small tubercles on the temporal sides of the head, above the tibiae and near the vent. The fingers bear expanded discs with circum-marginal grooves, webbing between the fingers is absent, and adult males carry a glandular nuptial pad on the first finger together with an external vocal sac.

Colouration in life also fits the species. The dorsal sides of the head and body are green or olive brown with some black dots, the lateral side of the head and the tympanum are dark brown or black, and a white stripe runs from the tip of the snout to the shoulder on each side. The iris is pale gold, the throat, chest and belly are cream with some brown dots, and the vocal sac is orange or light yellow. The ventral surfaces of the fore-limbs are light red or flesh-coloured, and the toe webbing is dark brown. These details, recorded from the living animals, helped confirm the assignment alongside the genetic data.

The molecular analysis targeted a fragment of the mitochondrial NADH dehydrogenase subunit 2 gene, ND2, which was amplified by polymerase chain reaction and sequenced following the protocols of the original species description. The new sequences were deposited in GenBank, and additional ND2 sequences for comparison were obtained from the same database, covering a broad sample of Amolops species from China, Vietnam, Laos, Thailand, India and Myanmar. The sequences were aligned with ClustalW in MEGA X, and uncorrected pairwise genetic distances were calculated in the same software. The best-fit substitution models were selected with the Akaike Information Criterion in ModelFinder, and phylogenies were estimated both by Maximum Likelihood in IQ-TREE, with nodal support from 1,000 ultrafast bootstrap replicates, and by Bayesian Inference in MrBayes, with Markov chains run for 1,000,000 generations.

Both phylogenetic methods produced the same topology, with slightly different support values at some nodes. Crucially, the sequences from the Chinese specimens clustered together with the type specimens of A. truongi from Vietnam with strong support. The uncorrected p-distance between the Chinese and Vietnamese specimens was only 0.4 percent, a small genetic divergence consistent with the two sets of samples belonging to the same species. By comparison, the same matrix places A. truongi at distances of several percent to more than 17 percent from other Amolops species included in the analysis, underscoring how distinct the species is from its relatives and how close the new Chinese material is to the Vietnamese type series.

There were, however, a few morphological differences worth noting, and these prompted the authors to revise the species’ diagnosis. In the Chinese specimens, the distance from the nostril to the snout tip is equal to or slightly greater than the distance from the eye to the nostril, whereas the original description recorded the nostril-to-snout distance as smaller than the eye-to-nostril distance. The upper eyelid width is narrower than the interorbital distance in the Chinese specimens, while the original description described the upper eyelid as wider than the interorbital distance. In addition, the original description gave the dorsum colour as light grey, but some of the Chinese specimens have a green dorsum. On the basis of these variants, the authors provided an updated diagnosis of the species, combining the original description with the new data. The revised diagnosis gives snout-vent lengths of 37.5 to 41.3 millimetres in adult males and 61.5 to 62.5 millimetres in adult females, and confirms characters such as the presence of vomerine teeth, a distinct round tympanum, smooth skin, an indistinct supratympanic fold, a distinct dorsolateral fold, the toe webbing formula I0–1/2II0–1III0–1IV1–0V, and the presence of an external vocal sac and a nuptial pad on the first finger in adult males.

The new collection site lies in Yunnan Huanglianshan National Nature Reserve, in the south of Yunnan Province, an area the authors note borders Vietnam. The reserve contains a variety of terrain and vegetation types and has a long-established reputation for rich species diversity; in recent years it has yielded several newly described species, including the frogs Leptobrachella aspera and Raorchestes huanglianshan and the plants Agapetes heana and Primula zhengyii. Given that the reserve sits directly on the Vietnamese border, the authors write that it is not surprising to find species previously considered to be distributed only in Vietnam. The discovery also updates the national tally: previously 52 species of Amolops had been recorded in China, 20 of them in Yunnan, and the addition of A. truongi raises those figures to 53 species in China and 21 in Yunnan. Amolops is the most speciose genus within the family Ranidae, with 85 species currently recognised, distributed from the southern and eastern Himalayas to Peninsular Malaysia.

The new locality also adds ecological detail. According to the original description, the type specimens in Vietnam were found on trees or limestone cliffs near a stream, at an altitude of 1,360 metres above sea level. The newly collected Chinese specimens, by contrast, were all found on herbaceous plants near a river, at 1,430 metres, a difference in altitude the authors describe as not significant. The authors also attempted to gather information on the species’ breeding biology, but with limited success: during the surveys they collected only males, did not find females or tadpoles, and did not hear the courtship calls of males. Because the original description did not mention courtship calls or the breeding season either, the breeding habits of the species remain undetermined, and the authors note that more field observations are needed to understand its habits and ecology.

As a small point of nomenclature, the authors suggest the Chinese common name shan luo tuan wa, written in Chinese characters as 山罗湍蛙, deriving from the type locality of Son La Province in Vietnam. The study’s broader significance lies in how quickly a species described from a single locality can turn out to cross international borders once nearby, poorly surveyed mountain regions are sampled. With the genetic and morphological evidence now in hand, A. truongi is formally recognised as part of China’s amphibian fauna, and the borderlands of southern Yunnan continue to demonstrate their value as a reservoir of frog diversity that is still being documented.

Subject of Research: First record of the frog species Amolops truongi in China, based on morphological and mitochondrial DNA evidence from Yunnan Province.

Article Title: First record of Amolops truongi Pham, Pham, Ngo, Sung, Ziegler & Le, 2023 ( Anura , Ranidae ) from China

Article References: Liu, S., Mo, M., Guo, J., Lu, Y., Wang, W., Zhang, W., Rao, D., & Li, S. (2024). First record of Amolops truongi Pham, Pham, Ngo, Sung, Ziegler & Le, 2023 (Anura, Ranidae) from China. Herpetozoa, 37, 149-156. https://doi.org/10.3897/herpetozoa.37.e121610

Image Credits: AI Generated

DOI: 10.3897/herpetozoa.37.e121610

Keywords: Amolops truongi, cascade frogs, Ranidae, Yunnan, Vietnam, new species record, mitochondrial ND2, phylogenetics, herpetology, biodiversity, Huanglianshan National Nature Reserve, taxonomy