Currently, the Treefrog of the genus Rhacophorus Kuhl & Van Hasselt, 1822 contains 44 nominal species recognized, distributed widely across South and Southeast Asia, including India, Bangladesh, Myanmar, Indochina, Malaysia, Indonesia, and Brunei, as well as extreme Southern and southwestern China (Jiang et al. 2019; Poyarkov et al. 2021; Frost 2024). Even so, the species diversity in the genus was indicated to be much underestimated and many cryptic species have not been described until now and this underscores the need for additional studies (Kropachev et al. 2022; Li et al. 2022). Presently, 16 nominal Rhacophorus species are recorded from Vietnam, including: R. annamensis Smith; R. calcaneus Smith; R. exechopygus Inger, Orlov & Darevsky; R. helenae Rowley, Tran, Hoang & Le; R. hoabinhensis Nguyen, Pham, Nguyen, Ninh & Ziegler; R. hoanglienensis Orlov, Lathrop, Murphy & Ho; R. kio Ohler & Delorme; R. larissae Ostroshabov, Orlov & Nguyen; R. marmoridorsum Orlov; R. orlovi Ziegler & Köhler; R. rhodopus Liu & Hu; R. robertingeri Orlov, Poyarkov, Vassilieva, Ananjeva, Nguyen, Nguyen & Geissler; R. trangdinhensis Kropachev, Evsyunin, Orlov & Nguyen; R. vanbanicus Kropachev, Orlov, Ninh & Nguyen; R. vampyrus Rowley, Le, Tran, Stuart & Hoang; and R. viridimaculatus Ostroshabov, Orlov & Nguyen (Poyarkov et al. 2021; Kropachev et al. 2022; Frost 2024).

Among the Treefrogs occurring in the Indochinese region of South-East Asia, the Rhacophorus bipunctatus complex is characterized by red web on feet, green or reddish-brown dorsal colour, presence of black spots at axillary region and skin folds above the anus, currenly including three species: R. bipunctatus Ahl, R. rhodopus, and R. napoensis Li, Liu, Yu & Sun (Bordoloi et al. 2007; Li et al. 2022). In the past few decades the phylogenetic relationship between R. bipunctatus and R. rhodopus has attracted considerable controversy (eg. Yu et al. 2008a; Li et al. 2012; Nguyen et al. 2014; Chan et al. 2018; Chen et al. 2022). The distribution of R. bipunctatus has now been restricted to Northeastern India, Bangladesh, Bhutan, and Northern and Western Myanmar, whereas R. rhodopus is known from Southern China, Central and Southern Myanmar, Thailand, Laos, Northern southwards Langbian Plateau Vietnam, Cambodia, and Peninsular Malaysia (Chan et al. 2018; Nguyen et al. 2020a; Poyarkov et al. 2021; this study).

The Napo Treefrog, Rhacophorus napoensis was recently described based on five males originating from Napo County, Baise City, Guangxi Autonomous Region, China (type locality) belonging to the R. bipunctatus complex (Li et al. 2022). To date, this species was known only from its type locality, which is approximately four kilometers northeast (straight line) of the Chinese/Vietnamese border. The species is characterized by: comparatively medium body size (SVL 38.6–43.6 mm in males); head width greater than head length, snout pointed, loreal region oblique, tympanum distinct, maxillary teeth distinct, tongue cordiform, external single subgular vocal sac, tibiotarsal articulation reaches the snout, tibia length is greater than foot length and slightly greater than half of snout-vent length, and single outer metatarsal tubercle is flat (Li et al. 2022).

Futhermore, the Red-webbed Treefrog Rhacophorus rhodopus was reported to be widely distributed in the Northern southwards Langbian Plateau of Vietnam, including the following provinces: Ha Giang (Bac Me District [hereafter – Dist.]), Bac Giang (Tay Yen Tu Nature Reserve [hereafter – NR]), Ha Noi (Soc Son Dist.), Dien Bien [Muong Nhe NR], Lai Chau (Sin Ho Dist.), Lao Cai (Van Ban NR), Son La (Coipa NR, Sop Cop NR, Xuan Nha NR), Phu Tho (Xuan Son National Park [hereafter – NP]) Thanh Hoa (Xuan Lien NR), Nghe An (Pu Hoat NR, Pu Huong NR, Pu Mat NP), Ha Tinh (Vu Quang NP), Quang Binh (Phong Nha-Ke Bang NP), Quang Tri (Bac Huong Hoa NR), Thua Thien-Hue (Sao La NR), Quang Nam (Tay Giang Dist.), Kon Tum (Kon Plong Dist.), Gia Lai (Kon Ka Kinh NP, Kon Chu Rang NR), Lam Dong (Biduop-Nui Ba NP, Cat Tien NP, Di Linh Dist.), Binh Thuan (Nui Ong NR) (Nguyen et al. 2009; Hecht et al. 2013; Nguyen et al. 2014; Rowley et al. 2014; Nguyen et al. 2015b; Pham et al. 2016; Pham et al. 2017a,b; Luong et al. 2021; our data). However, the records of the Rhacophorus rhodopus in Vietnam are mainly based on morphological data, with very few molecular phylogeny investigations being examined. According to Nguyen et al. (2014) the Vietnamese populations from Bac Giang, Thanh Hoa, and Nghe An provinces are not conspecific with true Rhacophorus rhodopus from Yunnan Province, China (the province of the type locality of the species).

To address this, we examined specimens Rhacophorus spp. resembling R. rhodopus from Nghe An Province deposited in the Zoological Collections of Duy Tan University (DTU, Vietnam), Hong Duc University (HDU, Vietnam), and Zoological Museum of Lomonosov Moscow State University (ZMMU, Russia). Our results confirm that the specimens previously identified as R. rhodopus from Bac Giang and Nghe An provinces based both on morphological and molecular data, as well as the specimens from Thanh Hoa and Ha Tinh pronvices based on molecular data only, should be re-identified as Rhacophorus napoensis. We herein formally confirm the occurrence of R. napoensis in Vietnam, update the distribution of this species, provide additional data on its natural history and revise its diagnostic characters.

The ML and BI analyses recovered trees with similar topologies (Fig. 2). With respect to the position of R. napoensis, our phylogenetic results largely conform to those of Nguyen et al. (2014), Chan et al. (2018), Chen et al. (2022), Li et al. (2022). The sequences related to the R. bipunctatus complex formed six highly divergent lineages as follows: the lineage from Southern Yunnan, China represents the true R. rhodopus; the lineage from Northern Myanmar and Eastern India represents the true R. bipunctatus; the lineage from Guangxi, China and Northeastern Vietnam represents R. napoensis; and the other three lineages from Western Yunnan, China; Xizang, China; and Hainan, China as well as Gia Lai, Vietnam, respectively, represent three unnamed species (Fig. 2).

The sequences from Bac Giang, Thanh Hoa, Nghe An, and Ha Tinh provinces, Vietnam and the sequences of the type series of R. napoensis formed a highly supported clade, and there are two sub-clades within this clade. One sub-clade contains the sequences of the type series of R. napoensis and the sequences from Bac Giang Province; the other sub-clade contains the sequences from Thanh Hoa, Nghe An, and Ha Tinh provinces. The uncorrected pairwise distance between these two sub-clades was 1.8% (Table 3).

Furthermore, we also examined the morphology of Rhacophorus spp. specimens from Tay Yen Tu NR in Bac Giang Province as well as Pu Hoat NR Pu Huong NR, and Pu Mat NP in Nghe An Province and found morphological similarities with R. napoensis. These results support our hypothesis that previous records of R. rhodopus in Northern and North-Central Vietnam (Bac Giang, Thanh Hoa, Nghe An, and Ha Tinh provinces), should be referred to as R. napoensis. Therefore, we extend the distribution Rhacophorus napoensis to Vietnam and provide an expanded diagnosis and description.

Rhacophorus napoensis was known previously only from Guangxi Autonomous Region, China. Based on our data and the available literature, this study provides the first records, of R. napoensis outside of China, from Vietnam (Bac Giang, Thanh Hoa, Nghe An, and Ha Tinh provinces). These new findings highlight that our knowledge of the herpetofauna of Vietnam, particularly of the border region between China and Vietnam (see Poyarkov et al. 2021, 2023; Nguyen et al. 2024), is still incomplete and that additional field research is required.

Presently, the range of Rhacophorus bipunctatus s. str. should be restricted to Northeastern India, Bangladesh, Bhutan, and Northern and Western Myanmar, while the range of R. rhodopus s. str. should be restricted to Southern China, Central and Southern Myanmar, Thailand, Northern and Northwestern Laos, Northwestern Vietnam, Cambodia, and Western (Peninsular) Malaysia (Bordoloi et al. 2007; Chan et al. 2018; Chen et al. 2022; this study). Consequently, herein we speculate that the records of these two species from the southeastern part of Yunnan Province and areas except Napo County in Guangxi Autonomous Region of China as reported by Yu et al. (2008a), Fei et al. (2009, 2012), AmphibiaChina (2024); Northern and North-Central Vietnam, including Ha Giang, Ha Noi, Lai Chau, Lao Cai, Son La, Phu Tho, Quang Binh, and Quang Tri provinces, which as reported by Nguyen et al. (2008), Nguyen et al. (2009), Hecht et al. (2013), Nguyen et al. (2014), Pham et al. (2016), Pham et al. (2017a,b), Tran & Le (2019, 2021), and Luong et al. (2021), and from central Laos in Nakai-Nam Theun NP, Khammuoan Province as reported based on specimen NCSM 85400 (http://portal.vertnet.org/o/ncsm/ncsm-herp?id=2d83b252-923a-4689-866b-2253f1821e00); in Viengthong, Bolikhamxay Province as reported based on specimen NCSM 80909 (http://portal.vertnet.org/o/ncsm/ncsm-herp?id=22b8590b-786d-4bab-8cbc-a2761263dc4e); and Phou Samsoum Mt. in Xiangkhoang Province as reported by Nguyen et al. (2020a) should be assigned to R. napoensis. Re-examinations of specimens of R. bipunctatus and/or R. rhodopus from Southeastern region of Yunnan Province and areas except Napo County of Guangxi in China, Northern and North-Central Vietnam as well as from Central Laos are required to verify these distributions.

On other hand, although reported to be widely distributed in Vietnam,the actual distribution of R. rhodopus in Vietnam is still quite vague. According to literature, we only confirm the record Rhacophorus rhodopus in Northwest Vietnam based on morphological evidence by Nguyen et al. (2015b). Nguyen et al. (2015b) reported on the R. rhodopus from Muong Nhe NR, Dien Bien Province with SVL 33.3–36.4 mm (n=5) in males, 45.7–49.0 mm (n=3) in females, TbL/SVL 0.52–0.53 in males, 0.51–0.53 in females. These ratios are consistent with the identification characteristics of the species R. rhodopus (see Suppl. material 1: table S2) and this location Muong Nhe NR is very close to the type locality of R. rhodopus (123 km from northwest of Mengyang Town, Yunnan Province, China). Therefore, we recommend that the population at Muong Nhe NR, Dien Bien Province, Vietnam is still assigned to the species R. rhodopus, although future molecular studies are required to verify this.

Futhermore, the Rhacophorus namdaphaensis Sarkar & Sanyal, 1985 (type locality: Miao, Tirap District, Arunachal Pradesh Sate, India) was subsequently considered as a junior synonym of R. rhodopus according to Bordoloi et al. (2007). However, the individuals were found in Arunachal Pradesh State, in northeastern India, far from the type locality of R. rhodopus in southwestern China, and according to our observations, the morphology of the individuals of this species from Arunachal Pradesh seems to be more closely related to R. bipunctatus than to R. rhodopus (see Suppl. material 1: fig. S1D) with the presence of yellow pigmentation at the upper and lower portions of the iris, which are considered diagnostic characteristics of R. bipunctatus (see Suppl. material 1: figs. S1, S2). In addition, the area of distribution of R. namdaphaensis is within the range of R. bipunctatus (Arunachal Pradesh Sate, India). Therefore, additional sampling of R. namdaphaensis at the type locality, with subsequent molecular and morphological analyses should be conducted to elucidate its taxonomic status. Futhermore, our molecular results confirm that previous findings of populations of R. cf. rhodopus from the Kon Tum-Gia Lai Plateu, Vietnam together with those from Hainan, China, as well as R. cf. rhodopus from Xizang, China, likely represent cryptic species in this complex, as revealed by other recent studies (Nguyen et al. 2014; Chan et al. 2018; Chen et al. 2022; Li et al. 2022). Therefore, future molecular and morphological studies and phylogenetic studies are required to clarify the taxonomy of the R. bipunctatus and R. rhodopus species complexes.

According to the original description of Rhacophorus napoensis by Li et al. (2022), this species is characterized by head width greater than head length (HW>HL), tibia length is slightly greater than a half of snout-vent length (TbL/SVL 0.50–0.51 [avg. 0.50, n=5] in males), and by the absence of nuptial pad in males. However, the specimens from Vietnam examined in this study have head width almost equal to head length (HW≥HL), tibia length less than a half of snout-vent length (TbL/SVL 0.45–0.49 [avg. 0.47, n=9] in males, 0.43–0.46 [avg. 0.44, n=2] in females); nuptial pad is present on finger I in males. Our results suggest that these characters appear to be quite variable, so they cannot be used as diagnostic characters of R. napoensis.

Rhacophorus napoensis is to date reliably known from seven localities including two national parks, four nature reserves in Northern and North-Central Vietnam, as well as one narrow region in Southern Guangxi Autonomous Region, China. It is an arboreal species recorded from closed-canopy evergreen forests and forest edges in lowland and montane regions of rainforest. The main threats to this species in Vietnam are habitat loss and degradation, and harvesting for food (TV Nguyen pers. obs.). Though the actual extent of distribution, population trends, reproductive behaviour and ecology of this species remain poorly known, given the available information, we suggest Rhacophorus napoensis be considered of Least Concern (LC) following IUCN’s Red List categories (IUCN 2019).

The new record of Rhacophorus napoensis in this study raises the known number of the genus Rhacophorus species in Vietnam to 17 with nine species being endemic in this country namely: R. helenae, R. hoabinhensis, R. hoanglienensis. R. larissae, R. marmoridorsum, R. trangdinhensis, R. vanbanicus, R. vampyrus, and R. viridimaculatus (Poyarkov et al. 2021; Frost et al. 2024; this study). Despite recent progress, the taxonomy and distribution of the species of Rhacophorus species in Vietnam still remain unclear, especially of the species belonging to R. hoanglienensis-orlovi species complex (including: R. hoanglienensis. R. larissae, R. marmoridorsum, R. trangdinhensis, R. vanbanicus, and R. viridimaculatus); many of these species lack molecular data and their taxonomic status remains questionable. Further field surveys and taxonomic efforts both in Vietnam and in the adjacent parts of Southeast Asia and China will likely reveal additional lineages within the widely-distributed and insufficiently sampled species of the genus Rhacophorus.