Zoosyst. Evol. 100 (2) 2024, 625-643 | DOI 10.3897/zse.100.118879 eee BERLIN Underestimated species diversity within the Rhacophorus rhodopus and Rhacophorus bipunctatus complexes (Anura, Rhacophoridae), with a description of a new species from Hainan, China Shangjing Tang'*, Fanrong Xiao*, Shuo Liu*, Lijun Wang?, Guohua Yu! 2, Lina Du? 1 Key Laboratory of Ecology of Rare and Endangered Species and Environmental Protection (Guangxi Normal University), Ministry of Education, Guilin 541004, China 2 Guangxi Key Laboratory of Rare and Endangered Animal Ecology, College of Life Science, Guangxi Normal University, Guilin 541004, China 3 Ministry of Education Key Laboratory for Ecology of Tropical Islands & Key Laboratory of Tropical Animal and Plant Ecology of Hainan Province, College of Life Sciences, Hainan Normal University, Haikou 571158, China 4 Kunming Natural History Museum of Zoology, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China https://zoobank. org/675C D047-159E-4363-A0A6-ECD9549A 98 9B Corresponding authors: Guohua Yu (yugh2018@126.com); Lina Du (Dulina@mailbox.gxnu.edu.cn) Academic editor: Umilaela Arifin # Received 16 January 2024 # Accepted 6 April 2024 Published 21 May 2024 Abstract Taxonomy and species boundaries within the Rhacophorus rhodopus and Rhacophorus bipunctatus complexes are very confusing. In this study, we attempt to delimit the species boundaries and test the currently accepted taxonomic assignments within these two complexes based on newly collected samples and previously published data across their distributions. Phylogenetic analyses revealed that the R. rhodopus and R. bipunctatus complexes consisted of six distinct clades (labeled A-F) that diverged from each other by genetic distances (p-distance) ranging from 5.3% to 9.2% in 16S rRNA sequences, and accordingly analyses of species delimitation placed them into six species, of which three correspond to known species (R. rhodopus, R. bipunctatus, and R. na- poensis) and three represent different cryptic species. Rhacophorus rhodopus (Clade C) is distributed in southern Yunnan, China, northern Laos, and northern and central Thailand; R. bipunctatus (Clade F) is distributed in northeastern India and western and northern Myanmar; and R. napoensis (Clade B) is distributed in Guangxi, China and northern Vietnam. Based on both molecular and morphological evidence, we described the clade consisting of samples from Hainan, China and central Vietnam (Clade A) as a new species, Rhacophorus giongica sp. nov. There are two cryptic species requiring additional morphological studies: one only contains samples from Motuo, Xizang, China (Clade E), and the other is distributed in western Yunnan, China, central Myanmar, central Thailand, and Malaysia (Clade D). Additionally, our results supported the idea that some old GenBank sequences of R. reinwardtii need to be updated with the correct species name. Key Words Cryptic species, Hainan, Rhacophorus giongica sp. nov., Species complex, Species delimitation Introduction Rhacophorus Kuhl & Van Hassalt, 1822, a genus of the family Rhacophoridae that originated ca. 19.3—33.0 mil- lion years ago (O’Connell et al. 2018; Chen et al. 2020; Ellepola and Meegaskumbura 2023), is widely distribut- ed in India, Bhutan, China (Xizang, Yunnan, Guanegx1, Hainan), Myanmar, Thailand, Laos, Cambodia, Vietnam, Indonesia (Sumatra, Sulawesi), Philippines, and Kali- mantan (Frost 2023). It is characterized by medium or large body size, intercalary cartilage between the end of the finger and penultimate phalanges of digits, Y-shaped distal end of terminal phalanx, tip of digits expanded into rounded disks with circum-marginal grooves, web Copyright Tang, S. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 626 Tang, S. et al.: Species diversity within R. rhodopus and R. bipunctatus complexes between fingers, horizontal pupil, skin not co-ossified to skull, absence of upper eyelid projections and presence of tarsal projections in most species, extensive dermal folding usually on forearm and tarsus, anal folds, and brown or green dorsal color (Li et al. 2012; Pan et al. 2017; Jiang et al. 2019), and currently it contains 43 spe- cies excluding Rhacophorus verrucopus Huang, 1983, which was considered a synonym of Rhacophorus tuber- culatus Anderson, 1871 by Che et al. (2020). In China, there are eight Rhacophorus species, namely Rhacoph- orus bipunctatus Ahl, 1927; Rhacophorus kio Ohler & Delorme, 2006; Rhacophorus laoshan Mo, Jiang, Xie & Ohler, 2008; Rhacophorus napoensis Li, Liu, Yu & Sun, 2022; Rhacophorus orlovi Ziegler & Kohler, 2001; Rhacophorus rhodopus Liu & Hu, 1960; Rhacophorus translineatus Wu, 1977; and Rhacophorus tuberculatus (Anderson, 1871) (AmphibiaChina 2023). Rhacophorus rhodopus is mainly distributed in north- eastern India to Myanmar (Kachin State, Shan State), Thailand, Laos, Vietnam (Lao Cai, Ha Tinh, Bac Giang, Quang Binh, Lai Chau, Quang Tri, Thua Thien Hue, Kon Tum, Gia Lai, Lam Dong, and Dong Nai), south- ern China (southeast Tibet, southern Yunnan, northeast- ern Guangxi, Hainan), and Peninsular Malaysia (Frost 2023). This species was originally described by Liu and Hu (1960) based on specimens from Mengyang, Yun- nan, China. It is characterized by reddish-brown dorsal color, pointed snout, smooth head skin, black spots at axillary region, and bright scarlet webs, resembling R. bipunctatus, a species originally discovered from north- eastern India (type locality: Khasi Hills) and later widely recorded from Bangladesh (e.g. Reza and Mukul 2009; Hakim et al. 2020), Cambodia (e.g. Ohler et al. 2002; Stuart and Emmett 2006; Neang and Holden 2008), Thailand (Taylor 1962; Chan-ard 2003; Chan-ard et al. 2011), Malaysia (Leong and Lim 2003; Grismer et al. 2006), Vietnam (Nguyen et al. 2005; Bain et al. 2007), Laos (Stuart 2005), Myanmar (Zug and Mulcahy 2020; Zug 2022), and China (Fei 1999; Fei et al. 2004, 2009, 2010; Che et al. 2020). The disputes over the taxonomy of R. rhodopus and R. bipunctatus have been going on for many years. In- ger et al. (1999) compared R. bipunctatus from northern and central Vietnam with R. rhodopus and concluded that the two species are synonymous. However, Bor- doloi et al. (2007) considered that this conclusion is not reliable owing to the fact that Inger et al. (1999) did not compare the specimens from Vietnam with topotypes of R. bipunctatus, and suggested that records of R. bi- punctatus from Thailand and Vietnam actually refer to R. rhodopus. Nguyen et al. (2008) also suggested that all re- cords of R. bipunctatus in Vietnam should be classified as R. rhodopus. Fei et al. (2009) considered that R. rhodopus from the type locality obviously differs from R. bipunc- tatus from northern India in body size and color pattern, so they suggested maintaining the validity of R. rhodopus and considered that R. bipunctatus from Vietnam is more similar to R. rhodopus from China. zse.pensoft.net Analysis of molecular data can more accurately test the taxonomic hypothesis based on morphology (Jablonski and Finarelli 2009). Previous molecular phylogenetic analyses have demonstrated that the tax- onomy of R. rhodopus and R. bipunctatus complex- es (Chan et al. 2018) is complicated. Yu et al. (2007, 2008) found that R. rhodopus is not monophyletic, and R. rhodopus from Hainan, China is closer to R. bipunc- tatus collected from Vietnam. Li et al. (2012) found that samples of R. rhodopus from Hainan, China and Vietnam form a clade that did not cluster together with the clade containing R. rhodopus from the type local- ity, indicating that R. rhodopus from Hainan, China and Vietnam likely represents a cryptic species. Nguy- en et al. (2014) also revealed that R. rhodopus is not monophyletic since R. rhodopus from Vietnam did not cluster together with the clade of samples from Yunnan and Malaysia. Dang et al. (2015) revealed two distinct lineages within R. rhodopus from Yunnan and consid- ered that one of them could be a cryptic species. More- over, Chan et al. (2018) suggested tentatively moving Malaysian R. bipunctatus to R. rhodopus and revealed that R. rhodopus from Vietnam is composed of two dis- tinct clades, one only containing samples from Vietnam and one containing samples from Vietnam and Hainan, China. Over all, these previous molecular phylogenetic analyses revealed that R. bipunctatus and R. rhodopus represent two complexes (Chan et al. 2018), and at least two cryptic species may exist in the R. rhodopus com- plex: one occurs in Hainan, China and Vietnam, and one is only known from Vietnam. Recently, Li et al. (2022) described a new species resembling R. rhodopus from Guangxi, China (R. napoensis). This finding raises an- other question. That is, whether R. napoensis is conspe- cific with one of the two potential cryptic species within the R. rhodopus complex mentioned above. Addition- ally, Li et al. (2012) and Che et al. (2020) found that R. bipunctatus, which is likely restricted to northeastern India, Myanmar, and Xizang, China (Fei et al. 2009; Chan et al. 2018; Poyarkov et al. 2021), is not mono- phyletic since samples from Motuo, Xizang, China formed a clade whereas samples from northern Myan- mar formed another clade. Because no samples from India were included in these two studies, it 1s necessary to investigate which of these two clades represents the true R. bipunctatus by employing R. bipunctatus sam- ples from northeastern India. In this study, we attempt to delimit the species bound- ary and test the currently accepted taxonomic assignments within R. rhodopus and R. bipunctatus complexes based on newly collected and previously sequenced samples across their distributions. Our results revealed that there are probably three cryptic species, and one of them was described as a new species herein based on morphologi- cal and molecular evidence. The samples from northern Vietnam belong to R. napoensis, and populations from central Vietnam are conspecific with the new species de- scribed here. Zoosyst. Evol. 100 (2) 2024, 625-643 Materials and methods Sampling This study was carried out in accordance with the ethical guidelines issued by the Ethics Committee of Guangxi Normal University (permit number: GXNU- 202308-010). A total of 58 individuals of R. rhodo- pus and R. bipunctatus complexes collected from 33 sites across China, Vietnam, Laos, Thailand, Malay- sia, Myanmar, and India (Fig. 1) were included in this study. Of the 58 samples, 18 were collected and se- quenced by this study, and the homologous sequences of the other 40 individuals were downloaded from Gen- Bank (Table 1). All newly collected specimens in this 92° O-0CE 96°0'0"E 30°0'0"N 26°0'0"N 22°0'0"N 18°0'0"N 14°0'0"N 10°0'0"N 6°0'0"N 100°0'0"E 627 study were deposited at Guangxi Normal University (GXNU). Rhacophorus norhayatiae Chan & Grismer, 2010, Rhacophorus reinwardtii (Schlegel, 1840), Rha- cophorus borneensis Matsui, Shimada & Sudin, 2013, Rhacophorus helenae Rowley, Tran, Hoang & Le, 2012, R. kio, Rhacophorus lateralis Boulenger, 1883, and Rhacophorus nigropalmatus Boulenger, 1895 were included in this study, and Leptomantis gauni (Inger, 1966), Zhangixalus smaragdinus (Blyth, 1852), Buer- geria buergeri (Temminck & Schlegel, 1838), Kurix- alus idiootocus (Kuramoto & Wang, 1987), Chiro- mantis rufescens (Gunther, 1869), Nyctixalus pictus (Peters, 1871), and Theloderma albopunctatum (Liu & Hu, 1962) were selected as the outgroup according to Yu et al. (2019) and Li et al. (2022). 104°0'0"E 108°0'0"E 112°0'0"E @ Clade A (RX. giongica sp. nov.) ® Clade B (2. napoensis) @ Clade C (R. rhodopus) ® Clade D @ Clade E -*. | @ Clade F (R. bipunctatus) Figure 1. Map showing the collection sites of samples of the R. rhodopus and R. bipunctatus complexes used in this study. Sites are labeled as in Table 1, and the star represents the type locality of R. giongica sp. nov. in Hainan, China. zse.pensoft.net 628 Tang, S. et al.: Species diversity within R. rhodopus and R. bipunctatus complexes Table 1. Species used in phylogenetic analyses of this study. Species Voucher Locality (ID) Accession No. Buergeria buergeri TTU-R-11759 Japan AF458122 Nyctixalus pictus FMNH 231094 Lahad Datu, Sabah, Malaysia AF458135 Theloderma albopunctatum ROM 30246 Vietnam AF458148 Chiromantis rufescens CAS 207601 Bioko Norte Province, Equatorial Guinea AF4581 26 Kurixalus idiootocus CAS 211366 Taipei, Taiwan, China AF458129 Zhangixalus smaragdinus HM05292 Xima, Yingjiang, Yunnan, China MN613221 Leptomantis gauni FMNH 273928 Bintulu, Sarawak, Malaysia JX219456 Rhacophorus borneensis BORN 22411 Sabah, Maliau Basin, Malaysia AB781694 R. helenae UNS 00451 Binh Thuan, Vietnam JQ288090 R. kio KUHE 55165 Xuan Lien, Than Hoa, Vietnam AB781695 R. lateralis - Mudigere, India AB530548 R. nigropalmatus Ra0081204 Malaysia JX219437 R. norhayatiae NNRn Johor, Endau Rompin, Malaysia AB728191 R. reinwardtii NMBE 1056517 Batang Ai NP, Sarawak, Malaysia JN377366 R. reinwardtii Rao081205 Malaysia JX219443 R. reinwardtii ENS 16447 (UTA) Sumatra, Bandung, Indonesia KY886335 R. reinwardtii ENS 16179 (UTA) Java, Patuha, Indonesia: KY886328 R. giongica sp. nov. GXNU HN110501 Diaoluo Mountain, Hainan, China (1) OP740711 R. giongica sp. nov. GXNU HN110502 Diaoluo Mountain, Hainan, China (1) OP740712 R. giongica sp. nov. GXNU HN110503 Diaoluo Mountain, Hainan, China (1) OP740713 R. giongica sp. nov. GXNU YU000691 Yinggeling, Hainan, China (2) PP115440 R. giongica sp. nov. GXNU YU000693 Yinggeling, Hainan, China (2) PP115441 R. giongica sp. nov. GXNU YU000696 Yinggeling, Hainan, China (2) PP115442 R. giongica sp. nov. GXNU YU000697 Yinggeling, Hainan, China (2) PP115443 R. giongica sp. nov. GXNU YU000698 Yinggeling, Hainan, China (2) PP115444 R. giongica sp. nov. VNMN:4117 K’ Bang, Gia Lai, Vietnam (3) LC010604 R. giongica sp. nov. FMNH253114 Ankhe Dist, Gia Lai, Vietnam (4) GQ204716 R. napoensis GXNU YU000171 Napo, Guangxi, China (5) ON217796 R. napoensis GXNU YU000173 Napo, Guangxi, China (5) ON217798 R. rhodopus VNMN:4118 Yen Tu, Bac Giang, Vietnam (6) LCO10605 R. bipunctatus AMNH-A 161418 Huon Son Reserve, Ha Tinh, Vietnam (7) AY843750 R. rhodopus VNMN:4120 Pu Huong, Nghe An, Vietnam (8) LC010609 R. rhodopus VNMN:4121 Thanh Hoa, Vietnam (9) LC010608 R. rhodopus clone 5 Mengyang, Yunnan, China (10) EF646366 R. rhodopus SCUM 060692L Mengyang, Yunnan, China (10) EU215531 R. rhodopus GXNU HPO18 Jiangcheng, Yunnan, China (11) OP740717 R. rhodopus KIZ060821 229 Lvchun, Yunnan, China (12) EF564574 R. rhodopus clone 3 Lvchun, Yunnan, China (12) EF646364 R. rhodopus 2004.0409 Long Nai Khao, Phongsali, Laos (13) KR828049 R. rhodopus 2006.2519 Ban Vang Thong, Louangphrabang, Laos (14) KR828069 R. rhodopus K3353 Ban Keng Koung, Louangphrabang, Laos (14) KR828071 R. rhodopus K3046 Doi Chiang Dao, Chiang Mai, Thailand (15) KR828066 R. rhodopus K3085_1 Mae Lao-Mae Sae Wildlife Sanctuary, Chiang Mai, Thailand (16) KR828067 R. rhodopus 0909Y3 Phu Hin Rong Kla NP, Phitsanulok, Thailand (1 7) KR828052 R. rhodopus 0906Y5 Phu Hin Rong Kla NP, Phitsanulok, Thailand (1 7) KR828078 R. rhodopus O0954Y Thung Salaeng Luang NP, Phetchabun, Thailand (18) KR828061 R. rhodopus O955Y Thung Salaeng Luang NP, Phetchabun, Thailand (18) KR828062 R. rhodopus 1000Y Khao Ang Rui Ni wildlife sanctuary, Chachoengsao, Thailand (19) KR828065 R. rhodopus clone 4 Jingdong, Yunnan, China (20) EF646365 R. rhodopus KIZ060821248 Jingdong, Yunnan, China (20) EF564575 R. rhodopus KIZ060821175 Yongde, Yunnan, China (21) EF564573 R. rhodopus clone 2 Yongde, Yunnan, China (21) EF646363 R. rhodopus K|Z587 Longling, Yunnan, China (22) EF564577 R. rhodopus KIZ589 Longling, Yunnan, China (22) EF564578 R. rhodopus GXNU 039927 Longchuan, Yunnan, China (23) OP740718 R. rhodopus GXNU 039928 Longchuan, Yunnan, China (23) OP740719 R. rhodopus GXNU YU20160263 Mengding, Yunnan, China (24) PP106375 R. rhodopus GXNU YU20160264 Mengding, Yunnan, China (24) PP106376 R. rhodopus GXNU YU000492 Menglian, Yunnan, China (25) OP740720 R. rhodopus GXNU YU000493 Menglian, Yunnan, China (25) OP740721 zse.pensoft.net Zoosyst. Evol. 100 (2) 2024, 625-643 629 Species Voucher Locality (ID) Accession No. R. rhodopus GXNU YU000485 Xiding, Menghai, Yunnan, China (26) OP740714 R. rhodopus GXNU YU000486 Xiding, Menghai, Yunnan, China (26) OP740715 R. rhodopus GXNU YU000487 Xiding, Menghai, Yunnan, China (26) OP740716 R. rhodopus USNM:Herp:587063 Kandawgyi National Gardens, Mandalay, Myanmar (27) MG935991 R. rhodopus 0937Y1 Kui Buri NP, Prachuap Khiri Khan, Thailand (28) KR828056 R. rhodopus 0937Y4 Kui Buri NP, Prachuap Khiri Khan, Thailand (28) KR828058 R. bipunctatus KUHE:53375 Genting, Pahang, Malaysia (29) LC010569 R. bipunctatus KIZ016380 Motuo, Xizang, China (30) MW111517 R. bipunctatus YPX40427 Motuo, Xizang, China (30) MW111518 R. rhodopus L06245 Motuo, Xizang, China (30) JX219441 R. rhodopus L062456 Motuo, Xizang, China (30) JX219442 R. bipunctatus CAS229913 Nagmung Township, Putao District, Kachin State, Myanmar (31) JX219445 R. bipunctatus CAS235303 Mindat Township, Mindat District, Chin State, Myanmar (32) JX219444 R. bipunctatus PUCZM/IX/SL360 Mizoram, Inida (33) MHO087073 R. bipunctatus PUCZM/IX/SL612 Mizoram, India (33) MHO087076 Molecular analyses and species delimitation The total genomic DNA of the specimens was extracted from liver tissue preserved in 99% ethanol. Tissue sam- ples were digested with proteinase K and purified using standard phenol/chloroform separation and ethanol pre- cipitation. A fragment encoding partial 12S rRNA, com- plete tRNA”, and partial 16S rRNA (16S) was amplified using the protocol of Yu et al. (2019) and the primer pair L1091 (Kocher et al. 1989)/16H1 (Hedges 1994). Se- quencing was conducted using the corresponding PCR primers and the internal primer Rh-int (Yu et al. 2019). All new sequences have been deposited in GenBank un- der accession Nos. OP740711—OP740721, PP106375— PP106376, and PP115440—PP115442 (Table 1). Sequences were aligned in MEGA v. 7 (Kumar et al. 2016) using the MUSCLE option with the default param- eters. The uncorrected pairwise distances (p-distances) between species were calculated in MEGA v. 7. The best substitution model (GTR + I + G) was selected in JMOD- ELTEST v. 2.1.10 (Darriba et al. 2012) based on the cor- rected Akaike Information Criterion (AICc). Bayesian phy- logenetic inference and maximum likelihood analysis were performed based on the best model. Bayesian phylogenetic inference was performed using MrBayes v. 3.2.6 (Ronquist et al. 2012). Two runs were performed simultaneously with four Markov chains starting from a random tree. The chain was run for 3,000,000 generations and sampled every 100 generations. When the average standard deviation of the split frequency was less than 0.01, the first 25% of the sampled trees were discarded as burn-in, and the remain- ing trees were used to create a consensus tree and estimate the Bayesian posterior probabilities (BPPs). The maximum likelihood analysis was conducted using raxmlGUI 2.0 (Edler et al. 2020) with 1000 bootstrap replicates. We used Assemble Species by Automatic Partitioning (ASAP; Puillandre et al. 2021) and multirate PTP (mPTP; Kapli et al. 2017) to delineate the species boundary within the R. rhodopus and R. bipunctatus complexes based on 16S rRNA sequences. For the ASAP method, the substi- tution model of p-distances was used to compute the dis- tances under the default values for other parameters. We selected the partition with the lowest ASAP-score as the best partition, according to Puillandre et al. (2021). The mPTP analysis was conducted in mPTP v. 0.2.5 using a maximum likelihood tree generated from 16S sequences by raxmlGUI 2.0. For this analysis, 10 different runs were performed with the following settings: mcmce run of 50 million generations, samples every 1000 generations, and the first 10 million generations were discarded as burn-in. Morphology As the molecular phylogenetic and species delimitation analyses revealed that species diversity in the R. rhodo- pus and R. bipunctatus complexes was underestimated and Hainan populations represent one of the three putative Species (see below), we further conducted morphological analyses to confirm its taxonomic status and to officially de- scribe it. The other two putative species were not included in morphological analyses owing to the fact that not enough morphometric data on them is available for the time being. Morphometric data were taken using electronic digital calipers to the nearest 0.1 mm. The terminology followed Fei (1999). Measurements included the following: snout- vent length (SVL); head length (HL); head width (HW); snout length (SL); internarial distance (IND); interorbit- al distance (IOD); upper eyelid width (UEW); diameter of eye (ED); diameter of tympanum (TD); distance from nostril to eye (DNE); length of forearm and hand (FHL); tibia length (TL); length of tarsus and foot (TFL); and foot length (FL). Comparative morphological data of con- generic species were obtained from published literature (Liu and Hu 1960; Ohler and Delorme 2006; Bordoloi et al. 2007; Fei et al. 2009, 2012; Chan and Grismer 2010; Rowley et al. 2012; Matsui et al. 2013; Li et al. 2022). Measurements were corrected for size (measurements divided by SVL). We used the f-test in SPSS v. 17.0 (SPSS Inc., Chicago, IL, USA) to evaluate the differences in quantitative characters of adult males between Hain- an populations and its two relatives (R. rhodopus and R. napoensis) because the Hainan populations were once placed in R. rhodopus and both the clade containing Hain- zse.pensoft.net 630 Tang, S. et al.: Species diversity within R. rhodopus and R. bipunctatus complexes an populations and the clade of R. napoensis occur in Viet- Results nam (see below). Principal component analyses (PCA) were conducted based on a correlation matrix of size-cor- rected measurements of males using SPSS v. 17.0. Scatter plots of the first two PCA factors were used to examine the morphological differentiation between specimens from Hainan, R. rhodopus, and R. napoensis. Females were not included as a separate analysis for both the test and PCA analysis owing to the small sample size (n = 2; one female from Hainan and one female of R. rhodopus). Phylogeny and species delimitation The BI and ML analyses yielded similar topologies, and both analyses revealed that there are six distinct clades in the R. rhodopus and R. bipunctatus complex- es (Clades A-F; Fig. 2): Clade A contains samples from Hainan, China (sites 1 and 2) and Gia Lai, Vietnam (sites 3 and 4); Clade B contains types of R. napoensis Buergeria buergeri 100 Nyctixalus pictus 100 Theloderma albopunctatum Chiromantis rufescens 64 Kurixalus idiootocus 59 96 Leptomantis gauni 75 Zhangixalus smaragdinus 100 R. lateralis 94 R. nigropalmatus 100 R. helenae 100 j 100 100 R. kio d 1 CAS229913 (Kachin, Myanmar; 31) Too Igor ©CAS235303 (Chin, Myanmar; 32) Clade F 68 100) PUCZM/TX/SL360 (Mizoram, India; 33) | R. bipunctatus 50 PUCZM/LX/SL612 (Mizoram, India; 33) ogy AMNH-A 161418 (Ha Tinh, Vietnam; 7) Rar VNMN:4120 (Nghe An, Vietnam; 8) Clade B VNMN:4121 (Thanh Hoa, Vietnam; 9) , Loop WNMN:4118 (Bac Giang, Vietnam; 6) R. napoensis Tooy GXNU YU000171 (Guangxi, China; 5) 100 81 GXNU YU000173 (Guangxi, China; 5) FMNH253114 (Gia Lai, Vietnam; 4) VNMN:4117 (Gia Lai, Vietnam; 3) 100 |; GXNU YU000691 (Hainan, China; 1) 100 | ff GXNU HN110501 (Hainan, China; 1) GXNU HN110503 (Hainan, China; 1) | Clade A GXNU YU000696 (Hainan, China; 2) rete GRNU HIN 10502: (Hainan, China ty [ones eee ney. GXNU YU000693 (Hainan, China; 2) 100 GXNU YU000697 (Hainan, China; 2 91 GXNU YU000698 (ainan: China; 53 100 R. borneensis BORN 22411 bel R. reinwardtii NMBE 1056517 100 + R. norhayatiae 91] 100! R. reinwardtii Rao081205 61 100 - R. reinwardtii ENS 16447 TTA} Sumatra 99 L R. reinwardtii ENS 16179 (UTA) Java 109 | -06245 (Xizang, China; 30) 99 100 | 1062456 (Xizang, China; 30) Clade E 69 YPX40427 (Xizang, China; 30) R. ‘bi tatus’ KIZ016380 (Xizang, China; 30) iia ate 1000Y (Chachoengsao, Thailand; 19) 95| - 0954Y (Phetchabun, Thailand; 18) 99 81| + O955Y (Phetchabun, Thailand; 18) 86 V§ _- 0906Y5 (Phitsanulok, Thailand; 17) 0909Y3 (Phitsanulok, Thailand; 17) 69 K3085-1 (Chiang Mai, Thailand: 16) | Clade C 100 || SCUM 060692L (Yunnan, China; 10) R. rhodopus 100, HPO18 (Yunnan, China; 11) or vi clone 3 (Yunnan, China; 12) 00 clone 5 (Yunnan, China; 10) KIZ060821229 (Yunnan, China; 12) K3353 (Louangphrabang, Laos; 14) 2004.0409 (Phongsali, Laos; 13) K3046 (Chiang Mai, Thailand; 15) 2006.2519 (Louangphrabang, Laos; 14) r- KUHE 53375 (Pahang, Malaysia; 29) 100 | KIZ 039928 (Yunnan, China; 23) 9 0937Y4 (Kui Buri NP, Thailand; 28) 0937Y1 (Kui Buri NP, Thailand; 28) USNM:Herp:587063 (Mandalay; 27) YU000493 (Yunnan, China; 25) YU000485 (Yunnan, China; 26) YU000492 (Yunnan, China; 25) YU000486 (Yunnan, China; 26) YU000487 (Yunnan, China; 26) Clade D clone 2? (Yunnan, China; 21) clone 4 (Yunnan, China; 20) K1IZ060821175 (Yunnan, China; 21) KIZ587 (Yunnan, China; 22) KIZ589 (Yunnan, China; 22) KIZ060821248 (Yunnan, China; 20) KIZ 039927 (Yunnan, China; 23) YU20160263 (Yunnan, China; 24) ee YU20160264 (Yunnan, China; 24) Figure 2. Bayesian phylogenetic tree of R. rhodopus and R. bipunctatus complexes and related species inferred from 12S rRNA, tRNA“, and 16S rRNA genes. The numbers above and below the branches are Bayesian posterior probabilities (BPP) and maximum likelihood (ML) bootstrap values, respectively (only values greater than 50% are shown). R. ‘rhodopus’ zse.pensoft.net Zoosyst. Evol. 100 (2) 2024, 625-643 (site 5) and samples from northern Vietnam (Bac Gi- ang, Ha Tinh, Nghe An, and Thanh Hoa; sites 6-9); Clade C contains R. rhodopus from the type locality (Mengyang, Yunnan, China; site 10) and samples from southern Yunnan (Jiangcheng and Lvchun; sites 11 and 12), northern Laos (Phongsali and Louangphra- bang; sites 13 and 14), and northern and southeastern Thailand (Chiang Mai, Phitsanulok, Phetchabun, and Chachoengsao; sites 15—19); Clade D contains samples from western and southwestern Yunnan, China (Jing- dong, Yongde, Longling, Longchuan, Mengding, Men- glian, and Xiding; sites 20-26), Myanmar (Mandalay; site 27), Thailand (Prachuap Khiri Khan; site 28), and Malaysia (Pahang; site 29); Clade E is consisted of X1- zang population (site 30) that was previously identified as R. rhodopus or R. bipunctatus. Clade F is consist- ed of R. bipunctatus from northern (Kachin State; site 31) and western (Chin State; site 32) Myanmar and northeastern India (Mizoram; site 33). All phylogenet- ic analyses supported that clades C, D, and E form a monophyly, and clade C is sister to clade D. Clade F was recovered as sister to the clade of R. helenae and R. kio with moderate support, and clade A was recov- ered as sister to clade B with weak support. The sequences of specimens under the name R. rein- wardtii in GenBank did not form monophyly. The two specimens that came from Malaysia (NMBE 1056517 and Rao081205) clustered together with R. borneensis and R. norhayatiae, respectively, and the two speci- mens that came from Indonesia (Java and Sumatra) formed a clade. Genetically, the pairwise distances between the six clades in R. rhodopus and R. bipunctatus complexes ranged from 5.3% to 9.2%, which is greater than the distance between R. kio and R. helenae (4.5%), the dis- tance between R. helenae and R. borneensis (4.9%), and the distances between R. norhayatiae, R. borneensis, and R. reinwardtii (4.0%—4.8%; Table 2). The best partition (score = 2.50) obtained by the ASAP species delimitation analysis grouped all samples of R. rhodopus and R. bipunctatus complexes used in this study into six species completely corresponding to 631 the six distinct clades (A—F) mentioned above (Fig. 3a), with a distance threshold of about 4% (Fig. 3b). The clade consisting of R. borneensis and the specimen un- der the name R. reinwardtii (NMBE 105617), the clade containing R. norhayatiae and the specimen under the name R. reinwardtii (Rao081205), and the clade com- prising of R. reinwardtii from Indonesia were identified as three different species (Fig. 3a). These operational taxonomic units were also supported by the mPTP anal- ysis (Fig. 4). Morphological study Morphological data are summarized in Table 3. The t-tests revealed that the male specimens from Hainan differ significantly (p < 0.05) from male topotypes of R. rhodopus in tympanum diameter (TD), upper eye- lid width (UEW), and distance between nostril and eye (DNE; Table 4), and differ from R. napoensis in body size (SVL), head length (AL), internarial distance (IND), and distance between nostril and eye (Table 5). PCA analysis on Hainan populations and R. rhodopus revealed that the first two principal components accounted for 66.13% of the total variance (Table 6), and loadings for PC2 were heavily loaded on TD and UEW, which separated Hainan samples from R. rhodopus along the PC2 axis (Fig. 5a). PCA analysis on Hainan populations and R. napoensis showed that loadings for PC2 were heavily loaded on HL, which can effectively separate Hainan populations from R. napoensis along the PC2 axis (Fig. 5b). Addi- tionally, morphological comparison indicated that spec- imens from Hainan can be distinguished from known members of R. rhodopus and R. bipunctatus complexes and other related species of Rhacophorus by a series of characters (see below). Based on the above molecular and morphological evidence, we considered that misidentifications were in- volved in the R. rhodopus and R. bipunctatus complex- es, and herein, the clade consisting of specimens from Hainan and central Vietnam (Clade A) is described as a new species. Table 2. Mean uncorrected pairwise distances (%) between clades of Rhacophorus rhodopus and R. bipunctatus complexes and related species based on 16S rRNA sequences. ID Species 1 2 3 1 Clade A (R. giongica sp. nov.) 2 Clade B (R. napoensis) 6.0 3 Clade C (R. rhodopus) 6.7 7.6 4 Clade D (R. ‘rhodopus’) 8.0 73 5:3 5 Clade E (R. ‘bipunctatus’) 7.4 6.0 7.9 6 Clade F (R. bipunctatus) 9.2 7.0 8:7 f- R. helenae 7.9 LAL 9.6 8 R. kio 8.2 FE 9.9 9 R. norhayatiae 7.4 7.0 7.5 10 R. borneensis 6.1 5.8 8.0 11 R. reinwardtii 5.7 53 7.1 4 5 6 7 8 g 10 6.7 8.3 ee Tee) 6.8 7.4 8.4 8.2 8.7 4.5 6.5 1h; 25 vias} 8.3 6.2 6.2 8.6 49 6.8 4.8 29) 3.2 6.8 6.3 LD 4.0 45 zse.pensoft.net 632 Tang, S. et al.: Species diversity within R. rhodopus and R. bipunctatus complexes Table 3. Measurements (mm) of Rhacophorus qiongica sp. nov., R. rhodopus, and R. napoensis. Species Voucher SEX SVL HL HW SL _ IND IOD UEW ED TD ODNE FHL TL TFL FL Rhacophorus giongica sp. nov. GXNUYUOO0O690 M 35.5 11.4 13.1 54 3.7 46 3.7 47 23 £428 (17.3 181 25.3 16.5 Rhacophorus giongica sp. nov. GXNU YU000691 M 37.8 12.0 13.2 55 38 42 36 47 2.3 26 182 186 25.6 16.7 Rhacophorus giongica sp. nov. GXNU YU000693 M 37.8 12.3 134 56 39 44 39 47 22 28 185 21.0 27.5 17.6 Rhacophorus giongica sp. nov. GXNU YU000696 M_ 36.1 11.1 12.7 54 39 41 37 48 20 28 417.8 18.7 25.8 16.6 Rhacophorus giongica sp. nov. GXNUYU000697 M 35.1 11.1 12.7 52 38 40 38 47 #19 2.7 17.2 17.2 24.3 15.6 Rhacophorus giongica sp. nov. GXNUYUO00698 F 49.3 146 168 74 48 55 42 54 26 3.7 23.6 249 34.3 23.1 Rhacophorus giongica sp. nov. GXNUHN110501 M 38.2 11.5 136 54 36 44 35 48 2.3 2.7 #%17.3 17.9 24.5 16.2 Rhacophorus giongica sp. nov. GXNUHN110502 M 38.1 11.0 136 52 3.7 42 3.5 47 22 28 181 19.2 25.5 168 Rhacophorus giongica sp. nov. GXNUHN110503 M 37.8 11.7 13.7 51 36 43 3.8 #47 21 £27 180 184 25.1 16.8 Rhacophorus rhodopus GXNU YU090185 M 33.1 10.7 115 50 35 38 26 40 21 £2.11 #164 165 224 148 Rhacophorus rhodopus GXNU YU0S0I86. M 33:6. 11.2 124: 4.8> 3.8. 41) 28 42 2:3 -2:3 17.0 17 232 155 Rhacophorus rhodopus GXNUYU090187 M 334 10:6 12:07 46 3.6 40 30 -43 20 2:1 164 165 22.8 148 Rhacophorus rhodopus GXNU YU090188 M 38.7 11.8 13.8 55 3.9. 43 32 49 25 25 180 183 254 168 Rhacophorus rhodopus GXNU YU090189 F 50.2 149 176 75 54 59 41 #56 3.3 3.3 256 244 354 23.6 Rhacophorus rhodopus GXNU YU090190 M 37.4 11.7 128 52 40 44 26 40 24 2.5 182 18.5 26.5 17.2 Rhacophorus rhodopus GXNU YU090194 M 35.5 108 125 49 38 41 #28 40 25 24 17.0 164 23.4 15.6 Rhacophorus napoensis GXNU YU000169 M 39.9 12.8 14.1 57 43 44 40 49 21 #28 19.9 19.2 284 18.7 Rhacophorus napoensis GXNU YU000170 M 44.2 15.3 16.2 69 47 47 45 #53 25 3.0 20.1 20.8 29.2 19.2 Rhacophorus napoensis GXNU YUOO0O171 M 41.2 145 153 64 46 46 43 52 25 28 20.7 19.9 28.7 19.0 Rhacophorus napoensis GXNU YUO00172 M 39.7 13.1 142 59 43 #43 41 52 23 28 18.9 19.1 26.7 17.4 Rhacophorus napoensis GXNU YU000173 M 41.4 13.9 15.2 63 45 45 40 49 23 2.9 206 20.5 283 18.7 Nb. of subsets [21] [20] [15] [13] Asap score [4.5] [3.5] [5.0] [5.0] a Rank [3] VNMN 4117 [J GXNU YU000691 GXNU HN110501 GXNU HN110503 GXNU YU000693 [2] {3] [4] AMNH-A 161418 VNMN 4120 VNMN 4121 GXNU YU000171 GXNU YU000173 VNMN 4118 YPX40427 KIZ 016380 L062456 L06245 BORN 22411 Clade E_ Clade A (2. giongica sp. nov.) Clade B (R. napoensis) R. borneensis + R. reinwardtii (NMBE 1056517) NMBE 1056517 ! NNRn _R norhayatiae + R. reinwardtii (Rao081205 Rao081205 ENS 16447 (UTA) R. reinwardtii ENS 16179 (UTA) UNS 00451 R. helenae R. kio KUHE 55165 KIZ 060821175 GXNU YU20160264 GXNU YU20160263 USNM Herp 587063 KUHE 53375 0937Y4 0937Y1 GXNU 039928 clone 5 SCUM 060692L GXNU HP018 clone 3 K3353 K3046 KIZ 060821229 CAS 235303 CAS 229913 Dist, Dt Clade D Clade C (R. rhodopus) Clade F (2. bipunctatus) Figure 3. ASAP species delimitation based on 16S rRNA sequences used in this study. The best partition with the lowest score is highlighted with a red frame. zse.pensoft.net Zoosyst. Evol. 100 (2) 2024, 625-643 633 Table 4. Results of the t-test between male specimens of Rhacophorus qiongica sp. nov. and R. rhodopus based on the size-adjusted data except SVL. Character Mean = SD (n = 8) Mean = SD (n = 6) Levene’s test t-test R. qiongica sp. nov. R. rhodopus (clade C) F p-value t p-value SVL hl teil 35:3'£'2.3 4.016 0.068 1.825 0.093 HL 0.311 + 0.012 0.316 + 0.011 0.022 0.883 -0.816 0.430 HW 0.358 + 0.006 0.354 + 0.009 0.871 0.369 0.774 0.454 SL 0.144 + 0.006 0.142 + 0.005 1.036 0.329 0.872 0.400 IND 0.101 + 0.005 0.107 + 0.004 2.199 0.164 -2.094 0.058 lOD 0.115 + 0.006 0.117 + 0.004 0.143 0.712 -0.470 0.647 UEW 0.100 + 0.006 0.080 + 0.007 0.019 0.891 5.581 0.000* ED 0.128 + 0.005 0.120 + 0.009 3.071 0.093 Zalay 0.055 TD 0.058 + 0.004 0.065 + 0.004 0.021 0.887 -3.488 0.004* DNE 0.074 + 0.004 0.066 + 0.002 Leal? 0.293 4.948 0.000* FHL 0.481 + 0.013 0.487 + 0.014 0.034 0.857 -0.892 0.390 TL 0.503 + 0.026 0.488 + 0.018 0.392 0.543 1.181 0.261 TFL 0.687 + 0.030 0.679 + 0.020 1.908 0.192 0.601 0.559 FL 0.448 + 0.014 0.447 + 0.011 0.539 0.477 0.106 0.917 UNS 00451 R. helenae KUHE 55165 R. kio FMNH 253114 One VNMN 4117 Clade A (R. giongica sp. nov.) GXNU HN110501 GXNU HN110503 GXNU YU000691 GXNU YU000696 GXNU HN110502 GXNU YU000693 GXNU YU000697 GXNU YU000698 1.00 GXNU YU000173 Clade B (R. napoensis) CaUale. AMNH-A 161418 VNMN 4121 VNMN 4120 1.00 KIZ016380 Clade E 1.062456 0.99 L06245 YPX40427 ENS 16447 (UTA) R. reinwardtii 0.62 ENS 16179 (UTA) R. reinwardtii 0.71 NNRn R. norhayatiae : Rao081205 R. reinwardtii 0.98 NMBE 1056517 R. reinwardtii BORN 22411 R. borneensis 1000Y K3085-1 SCUM 060692L GXNU HP018 KIZ060821229 0.98 Clade C (R. rhodopus) Gane 2006.2519 K3046 2004.0409 1.00 K3353 clone 5 0906Y5 0909Y3 0.69 0954Y O0955Y KUHE 53375 GXNU 039927 KIZ589 KIZ060821248 KIZ587 GXNU YU20160264 GXNU YU160263 Clade D KIZ060821175 clone 2 clone 4 GXNU YU000492 GXNU YU000485 GXNU YU000493 GXNU YU000487 GXNU YU000486 USNM Herp 587063 0937Y4 0937Y 1 GXNU 039928 Clade F (R. bipunctatus) CAS229913 CAS235303 PUCZM/TX/SL360 PUCZM/IX/SL612 Figure 4. mPTP species delimitation based on ML tree generated from 16S rRNA sequences. The support values above the branches indicate the fraction of sampled delimitations in which a node was part of the speciation process. zse.pensoft.net 634 2.000004 @ R. giongica sp. nov. 4 R. rhodopus 1.00000 5 ral / / \ \ \ \ 0.000004 | a sa \ \a \ PCI (41.014%) -1.000005 - -2.00000 4 1.00000 -2.00000 - 1.00000 0.00000 PC2 (25.116%) 2.00000 Tang, S. et al.: Species diversity within R. rhodopus and R. bipunctatus complexes 2.000004 © R. giongica sp. nov. . F b « R. napoensis fo 1.00000 es / e \ iH \ J e \ / \ je \ > / \eut \ xy | | \ So | a) | A i 1 g.00000 4 } | a a | | | Ty \ f —* | | | _ \ i \ | = = \e | -1.000005 =. \ | \ / | a, T T 0.00000 1.00000 PC2 (24.674%) -2.00000 4 T T -2.00000 -1.00000 2.00000 Figure 5. The scatter plot of the principal component analysis based on size-adjusted morphological data from males of the new species plus R. rhodopus (a) and data from males of the new species plus R. napoensis (b). Table 5. Results of the t-test between male specimens of Rhacophorus qgiongica sp. nov. and R. napoensis based on the size-adjusted data except SVL. Character Mean + SD (n = 8) Mean + SD (n = 5) R. qiongica sp. nov. R. napoensis (clade B) SVL 37H Zales 41.3+1.8 HL 0.311 + 0.012 0.337 + 0.012 HW 0.358 + 0.006 0.363 + 0.007 SL 0.144 + 0.006 0.151 + 0.005 IND 0.101 + 0.005 0.109 + 0.002 lOD 0.115 + 0.006 0.109 + 0.002 UEW 0.100 + 0.006 0.101 + 0.003 ED 0.128 + 0.005 0.124 + 0.005 TD 0.058 + 0.004 0.057 + 0.003 DNE 0.074 + 0.004 0.069 + 0.001 FHL 0.481 + 0.013 0.486 + 0.020 TL 0.503 + 0.026 0.482 + 0.009 TFL 0.687 + 0.030 0.685 + 0.020 FL 0.448 + 0.014 0.451 + 0.015 Table 6. Factor loadings of first two principal components of 13 size-adjusted morphometric characteristics of male specimens of Rhacophorus qiongica sp. nov., R. rhodopus, and R. napoensis. Character R. qiongica sp. nov. R. qiongica sp. nov. and and R. rhodopus R. napoensis PCl PC2 PCl PC2 Eigenvalue 5.332 3.265 4.845 3.208 % variation 41.014% 25.116% 37.268% 24.674% HL 0.724 -0.399 0.425 -0.852 HW 0.354 0.351 0.270 -0.506 SL 0.682 0.413 0.590 -0.597 IND 0.604 -0.577 0.729 -0.573 lOD 0.603 -0.269 0.437 0.615 UEW 0.459 0.853 0.785 -0.185 ED 0.419 OAS 0.458 0.460 TD -0.111 -0.790 0.063 0.186 DNE 0.507 0.675 0.500 0.738 FHL 0.791 -0.409 0.775 -0.029 TL 0.794 0.112 0.544 0.529 TFL 0.891 -0.079 0.907 0.217 FL 0.885 -0.246 0.849 0.108 zse.pensoft.net Levene’s test t-test F p-value t p-value 0.053 0.823 -5.006 0.000* 0.019 0.892 -3.778 0.003* 0.364 0.559 -1.420 0.183 0.365 0.558 -1.897 0.084 7.240 0.021 -3.387 0.008* 1.146 0.307 2.267 0.045* 4.071 0.069 -0.536 0.603 0.012 0.916 1.480 0.167 0.282 0.606 0.892 0.391 4.361 0.061 2.756 0.019* 2.125 0.173 -0.572 0.579 2.803 0.122 LF05 0.116 1.570 0.236 0.156 0.879 0.003 0.957 -0.317 0.757 Taxonomy Rhacophorus qiongica sp. nov. https://zoobank.org/182E48F4-9743-4B7F-A 825-FA63499F 1 5F2 Figs 6-9 Rhacophorus rhodopus — Fei 1999; Fei et al. 2004, 2009, 2010, 2012; Shi 2011; Nguyen et al. 2014. Rhacophorus bipunctatus — Orlov et al. 2002. Type material. Holotype. GXNU YU000691, adult male, collected on 14 July 2023 by Lingyun Du from Diaoluo Mt., Lingshui, Hainan, China (18°43'28"N, 109°52'12"E, ca914ma.s.1.). Paratypes. GXNU YU000690, an adult male, collect- ed at the same time as the holotype from the type local- ity by Lingyun Du and Jiaqi Luo; GXNU HN110501— HN110503, three adult males, collected on 20 July 2021 by Fanrong Xiao from the type locality; and three adult males (GXNU YU000693, GXNU YU000696, GXNU Zoosyst. Evol. 100 (2) 2024, 625-643 YU000697) and an adult female (GXNU YU000698) collected on 11 July 2023 by Qiumei Mo and Chunyi Pang from Yinggeling, Hainan, China (19°2'24"N, 109°34'12"E, ca 670 m a.s.l.). Etymology. The specific name giongica is derived from Qiong (#8), referring to Hainan, China, and mean- ing good and beautiful in Chinese. The specific name means that this species is very beautiful, and in China, it is distributed in Hainan. We suggest the English com- mon name “Hainan flying frog” and the Chinese common name “Ey pt HE (Qiong Shu Wa)”. Diagnosis. The new species is assigned to Rhacoph- orus by the presence of intercalary cartilage between terminal and penultimate phalanges of digits, terminal phalanges of fingers and toes Y-shaped, the tip of the digits expanded into disks with circummarginal grooves, fingers webbed, tarsal projections present, dermal folds along the forearm and tarsus present, and a horizontal pupil (Jiang et al. 2019). Rhacophorus qiongica sp. nov. differs from its congeners by a combination of the follow- ing characters: 1) medium body size (adult males SVL 35.1-38.2 mm); 2) dorsal surface red brown; 3) entire web between fingers and toes; 4) webbing between toes purely scarlet; 5) small black blotches on flank; 6) bands on limbs distinct; 7) throat smooth; 8) absence of dermal appendage on snout tip; 9) absence of small black spots on head sides; 10) palm smooth without small tubercles; and 11) tibiotarsal articulation reaching beyond eye. Description of holotype. Adult male, body size medi- um (SVL 37.8 mm); head width (HW 13.2 mm) longer than head length (HL 12.0 mm); snout blunt pointed, sloping in profile, protruding beyond the margin of lower jaw in ven- tral view; snout length (SL 5.5 mm) longer than diameter of eye (ED 4.4 mm); canthus rostralis distinct, curved; lo- real region oblique, concave; nostril oval, lateral, slightly protuberant, slightly closer to tip of snout than to eye; in- ternarial space (IND 3.8 mm slightly smaller than interor- bital distance (IOD 4.2 mm), nearly equal to the width of the upper eyelid (UEW 3.6 mm); pupil horizontal; pineal ocellus absent; tympanum distinct (TD 2.3 mm), round- ed, about half eye diameter (ED 4.4 mm); supratympanic fold narrow, flat; tongue cordiform, attached anteriorly, notably notched posteriorly; choanae oval; vomerine teeth 635 present in two series, touching the inner front edges of the choanae; an internal single subgular vocal sac; a vocal sac opening on the floor of the mouth at each corner. Forelimbs thin, length of forearm and hand (FHL 18.2 mm) is about half snout-vent length; relative length of fingers I < II < IV < II; tips of all fingers expand into discs with circummarginal and transverse ventral grooves, disc of finger I smaller than discs of other fin- gers; entire web between fingers, webbing formula: 12—2111—1.5011—1IV; subarticular tubercles rounded and prominent, formula 1, 1, 2, 2, proximal one smaller than distal one on the third and fourth fingers; supernumerary tubercles below the base of finger absent; metacarpal tu- bercle single, inner, oval and prominent (Fig. 7). Hindlimbs slender and long, heels overlapping when legs at right angle to body, tibiotarsal articulation reach- ing beyond eye; tibia length (TL 18.6 mm) nearly equal to length of forearm and hand (FHL 18.2 mm), longer than foot length (FL 16.7 mm), and shorter than length of tar- sus and foot (TFL 25.6 mm); relative length of toes I < II