Zoosyst. Evol. 100 (3) 2024, 851-862 | DOI 10.3897/zse.100.120224 25D oe > PENSUFT. pdt Hidden in the bamboo: A new parachuting frog (Rhacophoridae, Rhacophorus) from the borderlands of western China, with comments on the taxonomy of R. rhodopus Ping-Shin Lee**, Ben Liu!*, Meng Ouyang?, Ren-Da Ai*, Xiao-Long Liu’, Yan-Hong He, Ping-Qian Huang", Ying-Chun Li*, R. S. Naveen®®, Zhi- Yong Yuan°, Jin-Min Chen! 1 The Anhui Provincial Key Laboratory of Biodiversity Conservation and Ecological Security in the Yangtze River Basin, College of Life Sciences, Anhui Normal University, Wuhu 241000, Anhui, China Key Laboratory for Conserving Wildlife with Small Populations in Yunnan, Southwest Forestry University, Kunming 650224, Yunnan, China Key Laboratory of Freshwater Fish Reproduction and Development Ministry of Education, College of Life Science, Southwest University, Chongqing, 400715, China Gaoligong Mountain Forest Ecosystem Observation and Research Station of Yunnan Province, Yunnan, China 5 Sdlim Ali Centre for Ornithology and Natural History, Anaikatty, Coimbatore, Tamil Nadu, India 6 EDGE of Existence Programme, Conservation and Policy, Zoological Society of London, London, NW1 4RY, UK https://zoobank. org/2245A 35B-E010-455D-82AC-DE9B327F 4440 Corresponding authors: Jin-Min Chen (chenjinminkiz@126.com); Zhi- Yong Yuan (yuanzhiyongkiz@126.com) Academic editor: Umilaela Arifin # Received 4 February 2024 # Accepted 3 June 2024 Published 21 June 2024 Abstract The Gaoligong Mountains are characterized by large variations in elevation and topography, which support high levels of biodiversi- ty and endemism that remain largely understudied. Herein, based on the integration of morphological comparisons and phylogenetic reconstruction, we describe a new species of Rhacophorus from the northern Gaoligong Mountains, Yunnan Province, China. The new species, Rhacophorus dulongensis sp. nov., is morphologically distinguishable from its congeners based on the differences in body size, head length, tibia length, snout and tongue shape, toe webbing formula and coloration, ventral skin texture and coloration, dorsal pattern and coloration, body macroglands, iris coloration, and pattern of markings on flanks. Phylogenetically, it differs from its congeners by uncorrected p-distances of >4.8% for the 16S rRNA gene fragment. Rhacophorus dulongensis sp. nov. is likely to be found in Myanmar, considering its type locality lies close to the China-Myanmar border. The phylogenetic analysis revealed that the “widespread” species, R. rhodopus, 1s a species complex and a composite of five distinct lineages. The results revealed that R. napoensis is also found in Vietnam, making it a new country record for Vietnam. Interestingly, R. dulongensis sp. nov. likely breeds in bamboo, a hidden behavioral characteristic that makes them easy to overlook. Given the ongoing habitat loss and degrada- tion in the region, further biological exploration is urgently needed in the Gaoligong Mountains as a biodiversity reservotr. Key Words Biodiversity hotspot, frog, Gaoligong Mountains, new record species, new species, systematics * These authors contributed equally to this work. Copyright Lee, P.-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. 852 Lee, P.-S. et al.: A new frog of Rhacophorus and the comments on R. rhodopus taxonomy Introduction The Gaoligong Mountains, situated longitudinally along the border of China and Myanmar, are at the conver- gence of three key biodiversity hotspots: Indo-Burma, the Himalaya, and the Mountains of Southwest China (Myers et al. 2000). These mountains extend north- south in the western part of Yunnan Province, China, and are drained by the Irrawaddy River on the west and the Salween River on the east (Chaplin 2005). Due to its large variation in elevation, multiple latitudinal belts, and complex topography, which provided a wide range of neighboring climatic and structural niches, this re- gion represents a classic example of a montane hotspot for biodiversity and endemicity (Ricketts et al. 2005). Recently, the rate of new species discovery from the Gaoligong Mountains has been high, including new primates (e.g., Geissmann et al. 2011; Fan et al. 2017; Hu et al. 2022; Li et al. 2024). Despite this, amphibi- ans and reptiles in the northern Gaoligong Mountains have remained poorly studied because of difficulties in accessing the region previously (Liu et al. 2021; Wu et al. 2021), suggesting that our knowledge of the region’s herpetofaunal diversity is incomplete. Currently, the frog genus Rhacophorus Kuhl and Van Hasselt, 1822, contains 43 species, distributed wide- ly across Asia, including India, Myanmar, Bangladesh, Laos, Thailand, Vietnam, Cambodia, Malaysia, Brunet, and Indonesia, as well as southern China (Jiang et al. 2019; Frost 2024). At present, the following eight spe- cies of Rhacophorus have been recorded from China: R. bipunctatus Ahl, 1927; R. kio Ohler & Delorme, 2006; R. laoshan Mo, Jiang, Xie, & Ohler, 2008; R. napoensis Li, Liu, Yu, & Sun, 2022; R. orlovi Ziegler & Kohler, 2001; R. rhodopus Liu & Hu, 1960; R. translineatus Wu, 1977; and R. tuberculatus (Anderson, 1871) (Che et al. 2020; Naveen et al. 2023; AmphibiaChina 2024). Recent studies suggest that the overall species richness of Rha- cophorus is underestimated (Kropachev et al. 2022; Li et al. 2022). The reevaluations of the “widespread” species and the survey of unexplored areas are likely to reveal overlooked diversity. During the recent herpetological surveys in Dulong- jiang Village, northern Gaoligong Mountains, Yunnan Province, China, we collected unidentified specimens of a Rhacophorus population, which differed from other congeneric members by both morphological and molec- ular characteristics. As a result, we herein describe it as a new species. Materials and methods Sampling During a field survey at Dulongjiang Village, Gongshan County, Nujiang Prefecture, Yunnan Province, China, zse.pensoft.net in February 2022 (Fig. 1), two adult specimens (one male and one female) of Rhacophorus were collected and photographed. Sex was determined by the pres- ence of internal vocal sac openings and the presence of eggs in the abdomen, as observed via external in- spection. The specimens were then euthanized, fixed in 10% formalin, and subsequently stored in 75% ethanol for 24 hours. Liver tissues were taken and preserved in 95% alcohol. The procedures for DNA tissue sampling and specimen fixation follow the protocols detailed in Chen et al. (2021). Voucher specimens were deposited at Anhui Normal University (ANU), China. The proto- cols (No. SYDW-20130814-71) of the Animal Care and Ethics Committee were followed for the proper treat- ment of animals. DNA extraction, PCR, and sequencing Total genomic DNA was extracted from liver tissue stored in 95% ethanol using the standard phenol-chloroform ex- traction protocol (Sambrook et al. 1989). The partial se- quences of the mitochondrial 16S rRNA gene from the new samples were amplified and sequenced using the primers in Yu et al. (2019). PCR amplification was per- formed in a 25 ul reaction volume with the following cycling conditions: initial denaturation step at 95 °C for 5 min, 35 cycles of denaturation at 95 °C for 1 min, an- nealing at 55°C for 1 min, extension at 72 °C for 1 min, and final extension at 72 °C for 10 min. PCR products were purified using the Gel Extraction Mini Kit (Watson BioTechnologies, Shanghai, China) and then sequenced in both directions using the BigDye Terminator Cycle Sequencing Kit on an ABI PRISM 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Newly gen- erated sequences were deposited in GenBank (Table 1). Phylogenetic analyses Newly obtained nucleotide sequences were first assem- bled and edited using DNASTAR LASERGENE 7.1. To obtain the phylogenetic relationships among Rhacopho- rus, homologous sequences of all Rhacophorus species available in the NCBI GenBank were downloaded (Ta- ble 1). Our final dataset included 37 described species of Rhacophorus. In cases of geographically widespread spe- cies, multiple samples from different localities were in- cluded. Zhangixalus burmanus and Z. wui were chosen as outgroups based on previous phylogenetic studies (Jiang et al. 2019). New sequences incorporated with the data retrieved from GenBank were aligned using MUSCLE 3.8 (Edgar 2004) and then inspected by eye for accuracy and trimmed to minimize missing characters in MEGA11 (Tamura et al. 2021). The aligned length of the complete 16S rRNA data matrix was 969 bps. The phylogenetic reconstruction was performed us- ing Bayesian (BI) analyses and maximum likelihood Zoosyst. Evol. 100 (3) 2024, 851-862 96°0'0"E 98°0'0"E 100°0'0"E 30°0'0"N 28°0'0"N 24°0'0"N 22°0'0"N 7 ie +f 'é ss Ae : ‘ 20°0'0"N - a cm. 96°0'0"E 98°0'0"E 100°0'0"E 853 102°0'0°E 104°0'0"E 106°0'0"E 30°0'0"N _— cee | 28°0'0"N 4 sa 26°0'0"N -£24°0'0"N 22°0'0"N 4 20°0'0"N E 102°0'0"E 106°0'0" Figure 1. Map showing the collection site and type locality of Rhacophorus dulongensis sp. nov. (A; red circle) and R. rhodopus (B; green square). (ML) methods for the 16S rRNA gene. The best-fit model of evolution for 16S rRNA was determined us- ing the Bayesian Information Criterion (BIC) by j;Mod- elTest 2.1.7 (Darriba et al. 2012). The BI analyses were conducted in MrBayes 3.2 (Ronquist and Huelsenbeck 2003) with 30 million generations and sampled every 1000 generations. Convergence was assessed in Tracer 1.5 (Rambaut and Drummond 2009) based on having an average standard deviation of split frequencies less than 0.01 and ESS values greater than 200. We excluded the first 25% of trees before the log-likelihood scores stabi- lized as burn-in. The ML analyses were performed using RAXxML-HPC BlackBox 8.2.10 (Stamatakis 2014) on the CIPRES Science Gateway (Miller et al. 2010). The analyses used the proportion of invariable sites estimat- ed from the data and 1000 bootstrap pseudoreplicates under the GTR+gamma model. Nodes in the trees were considered well-supported when ML bootstrap support (BS) was => 75% and Bayesian posterior probabili- ties (PP) were > 0.95. Uncorrected pairwise distances (p-distances) between species were calculated in MEGA 11 (Tamura et al. 2021). Morphology and morphometrics Measurements were recorded to the nearest 0.1 mm with digital calipers by Renda Ai following Fei et al. (2009) and Li et al. (2022). The measurements taken were as follows: SVL (snout-vent length, the length from the tip of snout to vent); HL (head length, the length from the tip of snout to the posterior edge of the mandibular joint); HW (head width, the maximum distance between two sides of the head); SL (snout length, the length from the tip of snout to anterior border of eye); INS (interna- sal space, the distance between the inner edges of the left and right nostrils); IOS (interorbital space, the nar- rowest distance between the medial edges of the left and right upper eyelids); UEW (width of upper eyelid, the maximum width of upper eyelid); ED (diameter of eye, the diameter of the eye parallel to the body axis); TD (diameter of tympanum, the maximum diameter of tym- panum); DNE (distance from nostril to eye, the length from the anterior border of the eye to the inner edge of the ipsilateral nostril); SN (distance from snout to nostril, the length from the tip of snout to the inner edge of the zse.pensoft.net 854 Lee, P.-S. eta |.: A new frog of Rhacophorus and the comments on R. rhodopus taxonomy Table 1. Information for samples used in the molecular phylogenetic analyses in this study. New sequences generated for the present study are PP574166—PP574167. Species Voucher Locality GenBank No. Ingroup 1 R. dulongjiang sp. nov. ANU010645 Dulongjiang Village, Yunnan, China PP574166 2 R. dulongjiang sp. nov. ANU010646 Dulongjiang Village, Yunnan, China PP574167 3 R. annamensis KIZ1196 Binh Thuan, Vietnam JX219446 4 R. baluensis FM235958 Tambunan District, Sabah, Malaysia KC961089 5 R. barisani UTAA-61235 Gunung Dempo, Provinsi Sumatera Selatan, Indonesia KC701714 6 R. bengkuluensis UTAA-62770 Kabupaten Lampung Barat, Lampung, Sumatra KM212948 7 R. bipunctatus CAS229913 Au Yin Gacamp, Kachin State, Myanmar JX219445 8 R. bipunctatus PUCZM/IX/SL612 Mizoram, India MHO087076 9 R. borneensis BORN:22410 Maliau Basin, Sabah, Malaysia AB781693 10 ~~. calcadensis SDB.2011.291 Kadalar, Idukki, Kerala KC571276 11 PR. calcaneus KIZ528 Bi Doup National Park, Lam Dong, Vietnam JX219450 12. R.catamitus ENS7657 Sumatra, Indonesia JF748387 13. =~. edentulus MZB:Amp:30875 Gunung Katopasa, Sulawesi, Indonesia MH751448 14 R. exechopygus VNMN:4107 Gia Lai, Vietnam LC010585 15 R. gauni FMNH273928 Bintulu Division, Sarawak, Malaysia JX219456 16 =~. georgii MZB:Amp:23395 Suaka Marga Satwa Nantu, Sulawesi, Indonesia MH751453 17. ~~. helenae NAPO3164 Ba Ria-Vung Tau, Vietnam KX139175 18 R. hoabinhensis IEBRA.2016.18 Hoa Binh, Vietnam LC331096 19 __‘R. indonesiensis MZB:Amp:23619 Birun Village, Jambi Province, Indonesia AB983367 20 ~=R.kio KUHE:55167 Than Hoa, Vietnam AB781697 21 RF. laoshan 1705014 Guangxi, China MW149528 22 PR. lateralis RBRLO50709-35,36,37 Mudigere, India AB530548 23 =F. malabaricus Release Madikeri, India AB530549 24 =F. margaritifer ENS16162 Tilu, Indonesia KX398889 25 FR. modestus ENS16853 Samosir, Sumatra, Indonesia KX398904 26 ~=R.monticola RMB1236 Mt. Lompo Batang, Sulawesi , Indonesia AY326060 27 FR. napoensis GXNUYU000171 Napo, Guangxi, China ON217796 28 FR. nigropalmatus Rao081204 Malaysia JX219437 29 ~~ FR. norhayatii KUHEUNL Endau Rompin, Johor, Malaysia AB728191 30_—s*#R... orlovi LJTR44 Maguan, Yunan, China KC465840 31 sR. pardalis FMNH273245 Bintulu Division, Sarawak, Malaysia JX219453 32 RP. poecilonotus ENS16480 Sibayak, Sumatra, Indonesia KX398920 33. RR. pseudomalabaricus SDB.2011.1010 Kadalar, Kerala, India KC593855 34 sR. reinwardtii ENS16179 Patuha, Java, Indonesia KY886328 35 R. rhodopus (Lineage 1 KIZYPX20553 Mengyang, Yunnan, China MW133350 36 —_—-R. rhodopus (Lineage 2 Loc08007018 Longchuan, Yunnan, China JX219439 37 R. rhodopus (Lineage 2 K|IZ587 Longling, Yunnan, China EF564577 38 R. rhodopus (Lineage 3 L062456 Médog, Tibet, China JX219442 39 __R. rhodopus (Lineage 4 SN030035 Hainan, China EU215529 40 R. rhodopus (Lineage 4 VNMN:4117 Gia Lai, Vietnam LC010604 41 R. rhodopus (Lineage 5 VNMN:4118 Bac Giang, Vietnam LC010605 42 R. robertingeri VNMN:4123 Gig Lai, Vietnam LC010613 43 R. rufipes FMNH272858 Bintulu Division, Sarawak, Malaysia JX219455 44 R. spelaeus IEBRA.2011.1 Khammouan, Laos Pes3T095 45 _ R. translineatus KIZ06648 Médog, Tibet, China MW111521 46 iR.. tuberculatus KIZ014154 Médog, Tibet, China MW111522 Outgroup 47 Zhangixalus burmanus SCUM060614L Mt. Gaoligong, Yunnan, China EU215537 48 Zhangixalus wui CIB097690 Lichuan, Hubei, China JN688880 ipsilateral nostril); LAHL (length of lower arm and hand, the length from elbow joint to the tip of the third finger); HAL (hand length, from proximal end of outer palmar tubercle to tip of the third finger); TYE (distance from anterior margin of tympanum to posterior corner of eye); HLL (hindlimb length, measured as length of straight- ened hindlimb from groin to tip of fourth toe); THL zse.pensoft.net (thigh length, the length from vent to knee); TIL (tibia length, the length from knee to ankle); TFL (length of the foot and tarsus, the length from the tibial tarsal joint to the tip of the fourth toe); FL (foot length, the length from the proximal end of the medial metatarsal process to the tip of the fourth toe). The description of webbing formula followed Guayasamin et al. (2006). Zoosyst. Evol. 100 (3) 2024, 851-862 Results Morphology The two collected specimens were assigned to the genus Rhacophorus based on the following morphological char- acters: intercalary cartilage between terminal and penul- timate phalanges of digits present, Y-shaped distal end of terminal phalanx, finger tips expanding into large disks bearing circum-marginal grooves, webbing exists be- tween all fingers, skin not co-ossified with the skull, pu- pil horizontal, extensive dermal folds exist on the limbs, and dorsal color predominantly brown or green (Li et al. 2012; Jiang et al. 2019; AmphibiaChina 2024). The samples of Rhacophorus collected in Dulongjiang Village, Gongshan County, Nujiang Prefecture, Yunnan Province were reliably differentiated from all known con- geners based on the body size, head length, tibia length, snout and tongue shape, toe webbing formula and color- ation, ventral skin texture and coloration, dorsal pattern and coloration, body macroglands, iris coloration, and pattern of markings on flanks, which supported the rec- ognition of the new species. Phylogenetics and genetic divergence The BI and ML analyses resulted in essentially identical topologies (Fig. 2). Although the basal relationships within Rhacophorus were not well resolved, our analyses revealed two major clades, denoted A and B, within Rhacophorus. Clade A contained the majority of species of Rhacopho- rus and was widely distributed across Southeast Asia and southwestern China. Our new samples from Dulongjiang Village belong to Clade A, which strongly clustered into a lineage (BI = 1.00, ML = 100; Fig. 2) and clustered with R. tuberculatus, R. orlovi, and R. spelaeus from the eastern Himalayas, southwestern China, and northern Indochina with strong support (BI = 1.00, ML = 100; Fig. 2). 855 The putative new species from Dulongjiang Village showed obvious genetic divergence from other conge- ners. When compared with closely related recognized congeners, the minimum uncorrected genetic distance was 4.8% between the clade from Dulongjiang Village and R. tuberculatus (Table 2). These levels of pairwise divergence of the 16S rRNA gene obviously exceeded the accepted threshold of species-level genetic divergence in anurans (3.0%; Vences et al. 2005) and are greater than some known interspecific distances for Rhacophorus (Ta- ble 2). In addition, the analysis showed that R. rhodopus is not monophyletic and is a composite of five phyloge- netically distinct lineages (Fig. 2). The sequence diver- gence in the 16S rRNA gene among the five lineages of R. rhodopus is 4.7-9.1% (Table 2). The samples of R. rhodopus from Bac Giang, Vietnam (Lineage 5; GenBank accession numbers: LC010605; Fig. 2) strongly clustered with R. napoensis with only 0.2% sequence divergence. Taken together, our results indicated that the observed molecular divergence of the newly discovered population of Rhacophorus from Dulongjiang Village is concordant with stable differences in diagnostic morphological char- acter, which distinguish it from all known congeners (see Comparisons). Thus, upon combining morphological and molecular lines of evidence, we herein describe this dis- tinct lineage of Rhacophorus as a new species. Taxonomic account Rhacophorus dulongensis Chen, Lee & Yuan, sp. nov. https://zoobank.org/976AF923-DDF9-4E9A-BF72-0B2141DF493C Figs 3, 4 Type materials. Holotype. ANUO10645, adult male, collected from Dulongjiang Village, Gongshan Coun- ty, Nujiang Prefecture, Yunnan Province, China (27.7838°N, 98.3248°E, 1620 m as.l.; Fig. 1) on 15 February 2022, by Renda Ai. Paratypes. ANU010646, Table 2. Sequence divergences based on uncorrected p-distances (%) of 16S rRNA between the new species and its close congeners of Rhacophorus used in this study. Species al 2 5 4 5 1 R. dulongensis sp. nov. 2 R. rhodopus (Lineage 1) 15.0 3 R. rhodopus (Lineage 2) 11.6 4.8 4 R. rhodopus (Lineage 3) 13.6 8.2 6.0 5 R. rhodopus (Lineage 4) 12.4 74 9.1 68 6 R.rhodopus (Lineage 5) 10.5 8.1 7.7 4.7 6.5 7 R. napoensis age "772 6G" Lie OS 8 R. tuberculatus AS) 1338) 10:7 21270-4140 9 R. orlovi 16 Jaa eDLS s13.3°) 1335 10 R. spelaeus 8.6) W283" 139-6! PE33 11 R. bipunctatus 14.1 10.2 66 85 84 12 R. poecilonotus LOs2* 6829 7.6. eee) Or 13 R. barisani 95 84 7.5 67 9.4 14 R. bengkuluensis L238 O2-.bOs EG le 15 R. margaritifer 113 “OF "Gil ~ 8:37 2 10.7 6 iL 8 9 10 11 12 #13 14 «15 0.2 9:3. 12:0 Ih apes BT bao eae | TG 16 °S:8- 347 58 77 130 14.0 11.3 O07 ie "Fol. AOR PIO:o.. 3:0 OAS Sree roy Sop LOe = Geo 2a VO 39.3 12 LO AO 29:8" Bbrst eat 10:7 9.3 11.0 11.2 109 10.1 64 66 43 zse.pensoft.net 856 Lee, P.-S. et al.: A new frog of Rhacophorus and the comments on R. rhodopus taxonomy % JX219445 MH087076 * KX139175 R. helenae AB781697 R. kio * LC010605 J R. rhodopus (Lineage 5; Bac Giang, Vietnam) ON217796 fl R. napoensis «, EU215529 LC010604 KY886328 R. reinwardtii JX219442 R. rhodopus (Lineage 3; Motuo, Xizang, China) AB781693 R. borneensis = AB728191 R. norhayatii «| EF564577 bd JX219439 MW133350 R. rhodopus (Lineage 1; Type locality) R. bipunctatus | R. rhodopus (Lineage 4; Hainan, China; Gia Lai, Vietnam) R. rhodopus (Lineage 2; Western Yunnan, China) - KC701714 R. barisani * KX398920 R. poecilonotus KX398904 R. modestus JX219437 R. nigropalmatus ‘* KX398889 R. margaritifer KM212948 R. bengkuluensis JF748387 R. catamitus AB983367 R. indonesiensis aj ae eae R. dulongensis sp. nov. A * Yuan21333 * * MW111522 R. tuberculatus * KC465840 R. orlovi LC331095 R. spelaeus LC010613 R. robertingeri LC331096 R. hoabinhensis MW149528 R. laoshan * JX219446 R. annamensis LC010585 R. exechopygus Rhacophorus KC961089 R. baluensis JX219450 R. calcaneus * MW111521 R. translineatus * KC593855 R. pseudomalabaricus AB530549 R. malabaricus JX219453 R. pardalis AB530548 R. lateralis * MH751453 R. georgii * MH751448 R. edentulus B AY326060 R. monticola Figure 2. Phylogram of Rhacophorus inferred from a 16S rRNA mtDNA gene fragment. JX219455 R. rufipes KC571276 R. calcadensis JX219456 R. gauni 0.05 603 99 denotes high support by bootstrap support values (BS > 75%) and Bayesian posterior probabilities (BPP > 0.95): nodes without any numbers or symbols represent low support values (BS < 75% and BPP < 0.95). Outgroup taxa are not shown. The inserted photo (acknowledgment to Renda Ai) shows Rhacophorus dulongensis sp. nov. Scale bars represent the number of substitutions per site. The new samples for the present study are indicated by the red font. R. rhodopus is framed by the purple shade. adult female, collected at the same time as the holotype from the type locality by Renda A1. Etymology. The specific epithet “dulongensis” is giv- en as a noun in apposition and refers to the name of the Dulongjiang Village, where the new species occurs. We suggest the English common name “Dulongjiang tree frog” and the Chinese common name “7727 RE” (du long jiang shu wa). Diagnosis. Rhacophorus dulongensis sp. nov. can be distinguished from its congeners by the following com- bination of morphological characters: (1) body size small (SVL 31.7 mm in male; 35.3 mm in female); (2) head length longer than head width; (3) tibia length shorter than half of snout-vent length; (4) third finger disk small- er in diameter than tympanum; (5) snout pointed without a distinct bulge; (6) the tibiotarsal articulation reaches the eye when hindlimb is stretched along the side of the body; (7) dorsal surface of body uniformly green, and dorsal surface of limbs brown with irregular green patch- es; (8) belly mostly yellowish, rough, and granular (9) large black warts present on ventral surface of thigh near vent; (10) webs between toes red and webbing formula on toes: 11°-1?9111--2'71M1*-2'91V2'9-1!V; (11) black spots at axillary region absent; (12) vomerine teeth weakly developed: (13) iris darkgoldenrod; (14) maxillary teeth distinct; (15) tongue notably notched posteriorly. zse.pensoft.net Description of holotype. Adult male, body size small (SVL 31.7 mm); head length (HL 9.8 mm) longer than head width (HW 8.7 mm); snout pointed, protruding from the margin of the lower jaw, longer (SL 4.6 mm) than diameter of eye (ED 3.5 mm); canthus rostralis dis- tinct; loreal region oblique; nostril small, closer to tip of snout than to eye; interorbital space (IOS 2.8 mm) longer than internasal space (INS 2.5 mm) and width of upper eyelid (UEW 2.1 mm); pupil horizontal and iris dark- goldenrod; pineal ocellus absent; tympanum rounded and distinct, diameter of tympanum (TD 1.7 mm) shorter than half of eye diameter (ED 3.5 mm), internasal space (INS 2.5 mm) and interorbital space (IOS 2.8 mm); supratym- panic fold distinct; maxillary teeth distinct; vomerine teeth weak; internal single subgular vocal sac; vocal sac Openings small, slit-like; tongue heart-shaped, attached anteriorly, with distinct notch at posterior end; choanae oval (Table 3). Forelimbs thin, slender and not very long; length of lower arm and hand (LAHL 13.4 mm) shorter than half snout-vent length (SVL 31.7 mm); relative length of fin- gers: III>IV>II>I; tips of fingers expanded into discs; finger webbing formula: I2*-2'°1I1*-2771112'?-1?°TV; sub- articular tubercles distinct, blunt and round; third finger disk shorter than diameter of tympanum (TD 1.7 mm); supernumerary tubercles below the base of finger present; Zoosyst. Evol. 100 (3) 2024, 851-862 857 10 mm a. Figure 3. Holotype of Rhacophorus dulongensis sp. nov. (ANU010645) in life. A. Dorsal view; B. Ventral view; C. Dorso-lateral view; D. Ventral surface of the thigh and the cloacal region; E. Plantar view of the left foot; F. Thenar view of the right hand. The blue arrow in D points to large black warts. Photos by Renda Ai. Table 3. Measurements of the type series of Rhacophorus dulongensis sp. nov. and R. turpes (all measurements are in mm). N/A indicates that data are lacking for that morphological index. Abbreviations are defined in the text. Species R. dulongensis sp. nov. R. turpes Holotype Paratype Syntype Paratype Voucher Nos. ANU010645 ANU010646 BMNH1940.6.1.30 BMNH1974.832 Sex Male Female Female Female SVL 31.7 lop! 37.4 35.8 HL 9.8 10.5 12.2 10.8 HW O./ 8.8 oy. 9.8 SL 4.6 4.3 4.9 5.4 INS 2.5 2.3 2.8 rl lOS 2.8 3H 4.3 4.1 UEW 2:1 23 2.4 2.0 ED oo 3.4 3.5 3.0 TD LZ ee] 2.4 2.8 SN 12 Tsp. N/A N/A LAHL 13.4 13.8 N/A N/A HAL 7.9 8.5 o.2 8.6 RYE 0.6 0.8 1.1 1.2 HLL 49.1 48.9 N/A N/A THL 157 14 14.9 Le: TIL 14.6 15.1 16.9 18.3 TFL 19.3 19.8 N/A N/A FL 13.1 12.6 10.8 10.4 DNE Zieh Bae ad 33 nuptial pads absent; inner metacarpal tubercle distinct, 31.7 mm) and thigh length (THL 15.2 mm); tibiotarsal large and oval (Fig. 3; Table 3). articulation reaches the eye when hindlimb is stretched Hindlimbs slender, relatively long, tibia length (TIL along the side of the body; heels overlapping when 14.6 mm) shorter than half of snout-vent length (SVL held at right angles to the body; relative length of toes: zse.pensoft.net 858 Lee, P.-S. et al.: A new frog of Rhacophorus and the comments on R. rhodopus taxonomy Figure 4. Type series of Rhacophorus dulongensis sp. nov. in preservation. A. ANU010645; B. ANU010645; AJ. Dorsal view; A2. Ventral view; B1. Dorsal view; B2. Ventral view. The blue arrows in A2 and B2 point to large black warts. Photos by Renda A1. IV>V>IH>UFI; tips of toes expanded into discs; subar- ticular tubercles on all toes round, distinct, and protuber- ant; entire web between toes; toes webbing formula: I1°- 177771 --2!7NT1*-2'71V2"3-1'7V; inner metatarsal tubercle small; outer metatarsal tubercle absent (Fig. 3; Table 3). The dorsal surface of body smooth and uniformly green; dorsal surface of limbs brown with irregular green patches; the skin of throat, chest, and ventral surface of tibia, foot, and tarsus smooth; black dusting present on the margin of the throat; the belly mostly yellowish and rough; large black warts present on ventral surface of thigh near vent; dermal fringe along the outer sides of limbs indistinct; webs between toes red; black spots at axillary region absent. Color of holotype in preservative. Dorsal color dark- ened; ventral surface faded to creamy white; brown dust- ing present on ventral surfaces of throat. The patterns of dark spots and markings all over the body are the same as in life. Large black warts on the ventral surface of the thigh near the vent are more distinct (Figs 3, 4). Male secondary sexual characteristics. Nuptial pad and lineae masculinae were not observed. Morphological variation. The paratype matches the overall characters of the holotype (Table 3; Fig. 4). Female larger than male (SVL 35.3 mm in one female zse.pensoft.net and 31.7 mm in one male). The male has small slit-like vocal sac openings. Dusting pattern on the belly, chest, throat, and ventral surfaces of limbs varies individually. Female has more distinct brown dusting on the venter than male (Figs 3, 4). Distribution and ecology. Rhacophorus dulongensis Sp. nov. 1s presently known only from its type locality, near Dulongjiang Village, Gongshan County, Nujiang Prefecture, Yunnan Province, China. Both individuals of the new species were found inside bamboo internodes along rocky streams in well-preserved montane evergreen broadleaf forest (Fig. 5). Vocal recordings and tadpoles of this new species were not collected. Comparisons. Rhacophorus dulongensis sp. nov. is distinguishable from all other species of Rhacophorus by a combination of features of body size, head length, tib- ia length, snout and tongue shape, toe webbing formula and coloration, ventral skin texture and coloration, dorsal pattern and coloration, body macroglands, iris coloration, and pattern of markings on flanks (Tao et al. 2014; Fei et al. 2009; Li-et al. 2012;Che-et al. 2020; Li-et al. 2022; Naveen et al. 2023). In particular, R. dulongensis sp. nov. can be easily distinguished from its four morphologically and phylo- genetically close congeners (R. tuberculatus, R. orlovi, Zoosyst. Evol. 100 (3) 2024, 851-862 859 eae Paty 4 \ | Figure 5. Habitat of Rhacophorus dulongensis sp. nov. at the type locality in Dulongjiang Village, Yunnan Province, China. B, C. show the native bamboo species, Cephalostachyum virulentum, where the new species inhabits. Photos by Ying-Chun L1. R. spelaeus, and R. turpes; Figs 2, 6; Table 3). Specifical- ly, it differs from R. tuberculatus by having green dorsum coloration in life (vs. uniform brown dorsum coloration), different webbing formula on toes (11°-177II1--2'7III1*- 2 22 Pa vs eso Tee a2 V2) as well as the absence of a prominent calcar at tibio-tarsal articulation (vs. presence) and the absence of dark stripes on the hindlimb (vs. presence). R. dulongensis sp. nov. differs from R. orlovi by hav- ing smaller head (HL/SVL = 0.30-0.31 vs. HL/SVL = 0.38-0.41), different webbing formula on toes (I1°-1?°1I1- 2! TTT 1-2'91V2'9-1'2V vs. 11 °-2°T1°-2° HT 1°-2°TV2°- 1°V), green dorsal coloration in life (vs. reddish brown), hindlimb without transverse stripes (vs. limbs with trans- verse stripes), as well as the presence of large black warts on the ventral surface of the thigh (vs. absence) and the absence of spotting on flanks (vs. presence). R. dulongensis sp. nov. differs from R. spelaeus by having smaller body size in males (SVL 31.7 vs. 38.9- 43.1 mm), different webbing formula on toes (I1°-1?°II1- -2!? TTT 1*-2!71V2'9-1'!8V vs. 10-1110-1/2111-0V1/2-1/2V), green dorsal coloration in life (vs. grey-brown coloration in life), ventral surface of belly yellowish (vs. light gray), as well as the presence of vomerine teeth (vs. absence). Specifically, R. dulongensis sp. nov. differs from R. turpes by having hindlimbs without dark stripes (vs. hindlimbs with dark stripes), by different webbing for- mulawonatoes *CilPale Mye-2 7120 IV OP Nias. 1119-1 !771'7-1?971T1°- 1!7TV2°-1°V), =vomerine — teeth weak and small (vs. vomerine teeth distinct and large), dermal projection on the heel poorly developed (vs. well-developed), having a relatively longer foot length (FL/SVL ratio 0.36—0.41 in R. dulongensis sp. nov. Vs. 0.29 in R. turpes), numerous and large black warts on ventral surface of thigh (vs. few and small in R. turpes), as well as the absence of small black spots in male (vs. presence in R. turpes) (Fig. 6). In addition, R. dulongensis sp. nov. further differs from R. rhodopus, the notoriously “widespread” species of Rhacophorus in China and Indochina, by head length longer than head width (vs. head length almost equal to head width), third finger disk shorter than diameter of tympanum (vs. third finger disk longer than diameter of tympanum), tibia length shorter than half of snout-vent length (vs. tibia length about half of snout-vent length), as Well as the absence of the black spots at axillary region (vs. presence), the absence of dermal calcars on vent (vs. present), and the absence of transverse stripes on hind- limb (vs. presence). Among the species that are geographically close to R. dulongensis sp. nov., it distinctly differs from R. bipunc- tatus by having distinct tympanum (vs. indistinct), head length longer than head width (vs. head length almost equal to head width), as well as the absence of dermal calcars on heels (vs. presence), and the absence of black spots at axillary region (vs. presence). R. dulongensis sp. nov. differs from R. translineatus by having smaller body size (SVL 31.7 mm in male, 35.3 mm in female vs. 49.4— 54.1 mm in males, 61.5—65.2 mm in females), different webbing formula on toes (I1°-1731I1--2!71I1*-2'71V2'2- 1'8V vs. IO-OII0-OII0-0-IVO-OV), as well as the absence of transverse dark brown lines on the back (vs. presence), and the absence of an appendage on the tip of snout (vs. presence). R. dulongensis sp. nov. differs from R. sub- ansiriensis by having smaller body size in male (SVL 31.7 mm vs. 37.0—39.0 mm), head length longer than head width (vs. head length shorter than head width), as well as the absence of dark cross bands on limbs (vs. presence), and the absence of spots on flanks (vs. presence). For the remaining congeners, R. dulongensis sp. nov. differs from R. kio by having smaller body size (SVL 31.7 mm in male, 35.3 mm in female vs. 58.0—-79.1 mm in male, 82.6—88.9 mm in female), different webbing on toes (entirely webbed vs. fully webbed), red web (vs. web zse.pensoft.net 860 Lee, P.-S. et al.: A new frog of Rhacophorus and the comments on R. rhodopus taxonomy Figure 6. Type series of Rhacophorus turpes. A. The illustration of R. turpes in the original literature (Smith 1940); B, C. The syn- type of R. turpes (BMNH1940.6.1.30) in the dorsal and ventral views, respectively (Photos by R. S. Naveen). with black spot and orange yellow distal zone), as well as the absence of pointed dermal flap on heels (vs. pres- ence), and the absence of black marking on flanks (vs. presence). R. dulongensis sp. nov. differs from R. laoshan by having fifth finger longer than third finger (vs. equal), different webbing formula on toes (I1°-177II1--2'7III1*- 2'31V2'3-1'9V vs. T1-2!111-2!711-2'1V2-1V), limbs without broad transverse stripes (vs. limbs with broad transverse stripes), and uniform green dorsum coloration in life (vs. chocolate brown dorsum coloration with wide dark cross-shaped mark). R. dulongensis sp. nov. differs from R. napoensis by having smaller body size in male (SVL 31.7 mm vs. 38.6-43.6 mm), head width shorter than head length (vs. head width longer than head length), the tibiotarsal articulation reaches the eye (vs. the tibio- tarsal articulation reaches the snout), as well as the ab- sence of a distinct bulge on the tip of snout (vs. presence), the absence of black spots at axillary region (vs. pres- ence), and the absence of horizontal banding on dorsum and dorsal surface of limbs (vs. presence). Lastly, R. dulongensis sp. nov. can be distinguished by its smaller body size in male (SVL 31.7 mm vs. 53.16 mm in R. barisani, 40.5-46.7 mm in R. bengkuluensis; 50.9 mm in R. borneensis; 60.0 mm in R. helenae; 49.9 mm in R. larissae; 64.7 mm in R. norhayatiae; 66.8 mm in R. pseudomalabaricus, over 58.0 mm in R. reinward- tit; 49.5—68.2 mm in R. malabaricus; 46.3 mm in R. par- dalis, 45.9-46.4 mm in R. exechopygus), green dorsum coloration in life (vs. brown in R. barisani, R. margari- tifer and R. vanbanicus; brown with darker cross bands in R. bengkuluensis and R. catamitus,; yellowish grey with brown blotches in R. hoabinhensis, reddish brown with irregular dark brown blotches in R. indonesiensis; zse.pensoft.net X-shaped blotch on the anterior part of the back in R. monticola, cream-colored in R. marmoridorsum, dark grey or brownish-grey in R. calcadensis; yellowish grey in R. hoabinhensis), head longer than head width (vs. head wider than long in R. calcaneus, R. baluensis, R. trangdinhensis and R. viridimaculatus), third finger disk shorter than diameter of tympanum (vs. third finger disk longer than diameter of tympanum in R. calcane- us, R. annamensis, R. hoanglienensis, R. exechopygus, R. robertingeri and R. baluensis);, red webs between toes (vs. black webs in R. borneensis, proximally black and distally greenish webs in R. helenae; yellow webs in R. edentulus), tibiotarsal articulation reaches the eye (vs. tibiotarsal articulation reaches the tip of the snout in R. nigropalmatus, tibiotarsal articulation reaches beyond the tip of snout in R. georgii); absence of narrow dark cross- streaks on the limbs (vs. presence in R. bifasciatus), ab- sence of a white streak on each side of body (vs. presence in R. lateralis), absence of a large triangular calcar heel (vs. presence in R. robertinger’). Discussion Our study further reveals the underestimation of species diversity and the taxonomic dispute in Rhacophorus. The discovery of R. dulongensis sp. nov. brings the total number of Rhacophorus species to 44 and the number of Rhacophorus known from China to 9. Besides, this study uncovers that the notoriously “widespread” spe- cies, R. rhodopus, is not monophyletic and is a com- posite of five phylogenetically distinct lineages (Fig. 2), as also revealed previously (e.g., Li et al. 2012; Li Zoosyst. Evol. 100 (3) 2024, 851-862 et al. 2022). The divergence among the five lineages of R. rhodopus exceeds the threshold of species-level ge- netic divergence in anurans (3.0%; Vences et al. 2005) and is greater than some known interspecific distances for Rhacophorus (Table 2). Specifically, in addition to R. rhodopus from the type locality (Lineage 1), lineages 2—4 are likely to represent three new species (Fig. 2). Lineage 5 of R. rhodopus from northern Vietnam (Tao et al. 2014) is conspecific with R. napoensis with only 0.2% sequence divergence (Fig. 2; Table 2). Rhacopho- rus napoensis is currently only known from Baise City, Guangxi Province, China (Li et al. 2022; Frost 2024). Thus, our results revealed that R. napoensis is also found in Vietnam, making it a new country record for Vietnam, and we would formally correct the identity of LCO10605 from Vietnam as R. napoensis. Detailed field investiga- tions and more integrative studies, 1.e., combining mor- phological, acoustic, and molecular data, are warranted to resolve these taxonomic disparities. In addition, interestingly, both individuals of R. dulon- gensis sp. nov. were found inside the hollow internodes of the native bamboo species, Cephalostachyum virulentum. This behavior is a non-random event because its sister species, R. tuberculatus, has been reported to use bam- boo internodes as breeding sites (Che et al. 2020). More- over, this behavior has been reported in other genera of Rhacophoridae, such as Raorchestes. They usually enter the bamboo internodes through narrow openings and de- posit eggs inside the bamboo. This elusive habit makes them easy to overlook (Seshadri and Bickford 2018; Che et al. 2020). However, the overharvesting of bamboo 1s threatening the survival of these bamboo nesting frogs (Seshadri et al. 2015). How this novel reproductive mode evolved is still unclear and needs further study. Lastly, our study further highlights the lack of knowl- edge about the cryptic diversity in the Gaoligong Moun- tains. There was no previous record of the genus Rha- cophorus from Dulongjiang Village (Yuan et al. 2022; AmphibiaChina 2024), where the new species was dis- covered in this study. Our finding and other recent dis- coveries (e.g., Yang et al. 2016; Chen et al. 2020; Liu et al. 2021; Wu et al. 2021) revealed that the increased ex- peditions to remote, previously unexplored areas largely contribute to the high discovery rate of new species in the Gaoligong Mountains. Besides, considering that the continuous forest habitats stretch across the internation- al borders in this region, we expect that the new species found in this study may also occur in the adjacent regions of Myanmar. Further studies are needed to investigate the true extent of the occurrence of R. dulongensis sp. nov. Therefore, we propose that R. dulongensis sp. nov. be considered data deficient (DD) following the IUCN’s Red List categories. With the increasing anthropogenic pres- sures and changing climatic conditions in the Gaoligong Mountains (Yang et al. 2019; Chen et al. 2020), focal bio- diversity surveys and transboundary collaboration along the Chinese-Myanmar border are needed to detect more overlooked biodiversity and reinforce its value. 861 Acknowledgements This work was supported by the National Natural Science Foundation of China (NSFC 31900323), and Anhui Pro- vincial Key Laboratory of the Conservation and Exploita- tion of Biological Resources (692001) to J.M.C.; NSFC 32001222 to PS.L.; the Fundamental Research Funds for the Central Universities (SWU-KR22014, SWU 5330500880), NSFC32170478, NSFC32370478; and the “Youth Top Talent Program of Chongqing” (CQYC 20220510893) to Z.Y.Y. References AmphibiaChina (2024) The database of Chinese amphibians. Kunming Institute of Zoology (CAS), Kunming, Yunnan, China. http://www. amphibiachina.org/ [Accessed on: 2024-1-31] Chaplin G (2005) Physical geography of the Gaoligong Shan Area of southwest China in relation to biodiversity. Proceedings of the Cali- fornia Academy of Sciences 56: 527—556. Che J, Jiang K, Yan F, Zhang Y (2020) Amphibians and Reptiles in Ti- bet-Diversity and Evolution [In Chinese with English abstracts and species descriptions]. Chinese Academy of Sciences, Science Press, Beijing, China, 385-393 pp. Chen JM, Xu K, Poyarkov NK, Wang K, Yuan ZY, Hou M, Suwannap- oom C, Wang J, Che J (2020) How little is known about “the little brown frogs”: Description of three new species of the genus Lep- tobrachella (Anura, Megophryidae) from Yunnan Province, China. Zoological Research 41: 292. https://doi.org/10.24272/j.issn.2095- 8137.2020.036 Chen JM, Jin JQ, Wu YH, Hou SB, Liu S, Che J (2021) Protocols for DNA Tissue Sampling and Specimen Fixation of Amphibians. Bio- 101: e1010656. https://doi.org/10.21769/BioProtoc.1010656 [In Chinese] Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: More models, new heuristics and parallel computing. Nature Methods 9(8): 772-772. https://doi.org/10.1038/nmeth.2109 Edgar RC (2004) MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research 32(5): 1792— 1797. https://doi.org/10.1093/nar/gkh340 Fan PF, He K, Chen X, Ortiz A, Zhang B, Zhao C, Li YQ, Zhang HB, Kimock C, Wang WZ, Groves C, Turvey ST, Roos C, Helgen KM, Jiang XL (2017) Description of a new species of Hoolock gibbon (Primates, hylobatidae) based on integrative taxonomy. American Journal of Primatology 79(5): e22631. https://doi.org/10.1002/ ajp.22631 Fei L, Hu SQ, Ye CY, Huang YZ (2009) Fauna Sinica. Amphibia Vol. 2 Anura. Science Press, Beijing. [In Chinese] Frost DR (2024) Amphibian Species of the World: an Online Reference. Version 6.0. Electronic Database. American Museum of Natural History, New York. http://research.amnh.org/herpetology/amphibia/ index.html [Accessed on: 2024-1-31] Geissmann T, Lwin N, Aung SS, Aung TN, Aung ZM, Hla TH, Grindley M, Momberg F (2011) A new species of snub-nosed monkey, genus Rhinopithecus Milne-Edwards, 1872 (Primates, Colobinae), from northern Kachin state, northeastern Myanmar. American Journal of Primatology 73(1): 96-107. https://do1.org/10.1002/ajp.20894 zse.pensoft.net 862 Lee, P.-S. et al Guayasamin JM, Bustamante MR, Almeida-Reinoso D, Funk WC (2006) Glass frogs (Centrolenidae) of Yanayacu Biological Sta- tion, Ecuador, with the description of a new species and com- ments on centrolenid systematics. Zoological Journal of the Linnean Society 147(4): 489-513. https://doi.org/10.1111/.1096- 3642 .2006.00223.x Hu T, Peng S, Zhou XX, Zheng YM, Cong YY, Hu GW (2022) Two new species of /mpatiens (Balsaminaceae) from Gaoligong Moun- tains, Yunnan, China. Phytotaxa 566(3): 268-278. https://doi. org/10.11646/phytotaxa.566.3.2 Jiang DC, Jiang K, Ren JL, Wu J, Li JT (2019) Resurrection of the genus Leptomantis, with description of a new genus to the family Rhacophoridae (Amphibia, Anura). Asian Herpetological Research 10: 1-16. https://do1.org/10.16373/).cnki.ahr. 180058 Kropachev II, Evsyunin AA, Orlov NL, Nguyen TT (2022) A New Spe- cies of Rhacophorus Genus (Anura, Rhacophoridae, Rhacophori- nae) from Lang Son Province, Northern Vietnam. Russian Journal of Herpetology 29(1): 35-46. https://doi.org/10.30906/1026-2296- 2022-29- 1-35-46 Li JT, Li Y, Murphy RW, Rao DQ, Zhang YP (2012) Phylogenetic reso- lution and systematics of the Asian tree frogs, Rhacophorus (Rhaco- phoridae, Amphibia). Zoologica Scripta 41(6): 557-570. https://doi. org/10.1111/j.1463-6409.2012.00557.x Li J, Liu S, Yu G, Sun T (2022) A new species of Rhacophorus (Anu- ra, Rhacophoridae) from Guangxi, China. ZooKeys 1117: 123-138. https://doi.org/10.3897/zookeys.1117.85787 Li Q, Li XY, Hu WQ, Song WY, He SW, Wang HJ, Hu ZC, Li MC, Onditi KO, Chen ZZ, Pu CZ, Xiong Y, Rao CH, Zhang FY, Zuo CS, Jiang XL (2024) Mammals of Gaoligong Mountain in China: Diver- sity, distribution, and conservation. Zoological Research. Diversity and Conservation 1(1): 3-19. Liu XL, He YH, Wang YF, Beukema W, Hou S, Li YC, Che J, Yuan ZY (2021) A new frog species of the genus Odorrana (Anura, Ra- nidae) from Yunnan, China. Zootaxa 4908(2): 263-275. https://do1. org/10.11646/zootaxa.4908.2.7 Miller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES Sci- ence Gateway for inference of large phylogenetic trees. 2010 gate- way computing environments workshop (GCE). Ieee, New Orleans, 1-8. https://dot.org/10.1109/GCE.2010.5676129 Myers N, Mittermeier RA, Mittermeier CG, Da Fonseca GA, Kent J (2000) Biodiversity hotspots for conservation priorities. Nature 403(6772): 853-858. https://doi.org/10.1038/35002501 Naveen RS, Liu S, Chandramouli SR, Babu S, Karunakaran PV, Ku- mara HN (2023) Redescription of Rhacophorus tuberculatus (An- derson, 1871) and the validity of Rhacophorus verrucopus Huang, 1983. Herpetozoa (Wien) 36: 325-333. https://doi.org/10.3897/her- petozoa.36.e113656 Rambaut A, Drummond AJ (2009) Tracer v1.5. http://beast.bio.ed.ac. uk/Tracer Ricketts TH, Dinerstein E, Boucher T, Wikramanayake E (2005) Pin- pointing and preventing imminent extinctions. Proceedings of the National Academy of Sciences of the United States of America 102(51): 18497-18501. https://doi.org/10.1073/pnas.0509060102 zse.pensoft.net . Anew frog of Rhacophorus and the comments on R. rhodopus taxonomy Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics (Oxford, England) 19(12): 1572-1574. https://do1.org/10.1093/bioinformatics/btg 1 80 Sambrook J, Fritsch E, Maniatis T (1989) Molecular cloning: a labora- tory manual (2™ edition). Cold spring harbor laboratory press. Seshadri KS, Bickford DP (2018) Faithful fathers and crooked cannibals: The adaptive significance of parental care in the bush frog Raorches- tes chalazodes, Western Ghats, India. Behavioral Ecology and So- ciobiology 72(1): 1-14. https://doi.org/10.1007/s00265-017-2420-3 Seshadri KS, Gururaja KV, Bickford DP (2015) Breeding in bamboo: A novel anuran reproductive strategy discovered in rhacophorid frogs of the Western Ghats, India. Biological Journal of the Lin- nean Society, Linnean Society of London 114(1): 1-11. https://doi. org/10.1111/bij.12388 Smith MA (1940) The amphibians and reptiles obtained by Mr. Ron- ald Kaulback in Upper Burma. Records of the Indian Museum 42: 465-486. https://doi.org/10.26515/rzsi/v42/13/1940/16243 1 Stamatakis A (2014) RAxML version 8: A tool for phylogenetic analy- sis and post-analysis of large phylogenies. Bioinformatics (Oxford, England) 30(9): 1312-1313. https://doi.org/10.1093/bioinformatics/ btu033 Tamura K, Stecher G, Kumar S (2021) MEGA11: Molecular Evolution- ary Genetics Analysis Version 11. Molecular Biology and Evolution 38(7): 3022-3027. https://doi.org/10.1093/molbev/msab120 Tao NT, Matsui M, Eto K, Orlov NL (2014) A preliminary study of phylogenetic relationships and taxonomic problems of Vietnamese Rhacophorus (Anura, Rhacophoridae). Russian Journal of Herpetol- ogy 21: 274-280. Vences M, Thomas M, Van der Meijden A, Chiari Y, Viettes DR (2005) Comparative performance of the 16S rRNA gene in DNA barcod- ing of amphibians. Frontiers in Zoology 2(1): 1-12. https://doi. org/10.1186/1742-9994-2-5 Wu YH, Liu XL, Gao W, Wang YF, Li YC, Zhou EE, Yuan ZY, Che J (2021) Description of a new species of Bush frog (Anura, Rhaco- phoridae, Raorchestes) from northwestern Yunnan, China. Zootaxa 4941(2): 239-258. https://doi.org/10.11646/zootaxa.4941.2.5 Yang J, Wang Y, Chen G, Rao D (2016) A new species of the genus Lep- tolalax (Anura, Megophryidae) from Mt. Gaoligongshan of western Yunnan Province, China. Zootaxa 4088(3): 379-394. https://doi. org/10.11646/zootaxa.4088.3.4 Yang Y, Ren G, Li W, Huang Z, Lin AK, Garber P, Ma C, Yi S, Momberg F, Gao Y, Wang X, Li G, Behie A, Xiao W (2019) Identi- fying transboundary conservation priorities in a biodiversity hotspot of China and Myanmar: Implications for data poor mountainous regions. Global Ecology and Conservation 20: e00732. https://doi. org/10.1016/j.gecco.2019.e00732 Yu GH, Hui H, Hou M, Wu ZJ, Rao DQ, Yang JX (2019) A new spe- cies of Zhangixalus (Anura, Rhacophoridae), previously confused with Zhangixalus smaragdinus (Blyth, 1852). Zootaxa 4711(2): 275-292. https://doi.org/10.11646/zootaxa.4711.2.3 Yuan ZY, Chen JM, Wu YH, Li XQ, Che J (2022) Revision of the list of amphibian species in Yunnan Province. Shengwu Duoyangxing 30(4): 21470. https://do1.org/10.17520/biods.2021470