90urn:lsid:arphahub.com:pub:4EBD0208-8328-5D69-8C44-EC50939C0967HerpetozoaHerpetozoa1013-44252682-955XPensoft Publishers10.3897/herpetozoa.33.e6021460214Research ArticleAmphibiaAnuraRhacophoridaeBiodiversity & ConservationAsiaChinaMyanmarFirst record of Rhacophorusverrucopus Huang, 1983 from MyanmarLiuShuohttps://orcid.org/0000-0001-7825-30061LwinYe Htethttps://orcid.org/0000-0002-7812-13772QuanRuichang23LiSonglis@mail.kiz.ac.cn1Kunming Natural History Museum of Zoology, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, ChinaKunming Institute of Zoology, Chinese Academy of SciencesKunmingChinaSoutheast Asia Biodiversity Research Institute, Chinese Academy of Sciences, Yezin, Nay Pyi Taw 05282, MyanmarSoutheast Asia Biodiversity Research Institute, Chinese Academy of SciencesNay Pyi TawMyanmarCenter for Integrative Conservation, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Mengla, Yunnan 666303, ChinaXishuangbanna Tropical Botanical Garden, Chinese Academy of SciencesMenglaChina
Corresponding author: Song Li (lis@mail.kiz.ac.cn)
Academic editor: G.Gollmann
202001122020332072115CB1CF7B-EA3D-51A2-8F02-D624C2C361406A7AD27F-91CA-4E9F-AA9C-0B5AB864021B43123853110202019112020Shuo Liu, Ye Htet Lwin, Ruichang Quan, Song LiThis 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.http://zoobank.org/6A7AD27F-91CA-4E9F-AA9C-0B5AB864021B
We report the first country record of Rhacophorusverrucopus Huang, 1983 from Myanmar, based on one specimen collected from Htamanthi Wildlife Sanctuary, Sagaing Division. Morphologically, the specimen shows good agreement with the original description of R.verrucopus and phylogenetically, it is clustered with the specimen of R.verrucopus from Medog, Tibet, China with strong support. This is also the first record of R.verrucopus from outside of China.
Rhacophorus Kuhl & van Hasselt, 1822 occurs in the tropical and temperate zones of East, South and Southeast Asia and, recently, was partitioned into three genera including Rhacophorus, Leptomantis Peters, 1867 and Zhangixalus Li, Jiang, Ren & Jiang, 2019 (in Jiang et al. 2019). Now the genus Rhacophorus includes 44 recognised species with a distribution range from India, Bangladesh, Bhutan, Myanmar, Thailand, Laos, Cambodia, Vietnam, Malaysia, Indonesia and Philippines, as well as extreme southern and south-western China (mainly in Hainan, Guangxi, Yunnan and Tibetan) (Jiang et al. 2019; Frost 2020). Rhacophorusverrucopus, a species which was described and named from Medog, Tibet, China, was previously known only from the type locality (Huang 1983; AmphibiaChina 2020; Frost 2020).
Myanmar is an important component of the Indo-Burma biodiversity hot-spot and its northern region lies at a biogeographic crossroads where the faunas of China, Indochina, India and Himalaya converge (Wogan et al. 2008). In recent years, the researchers of Southeast Asia Biodiversity Research Institute, Chinese Academy of Science have found many new species and new records of animals and plants in northern Myanmar (Li and Quan 2017). During our field survey in northern Myanmar in 2019, a specimen of Rhacophorus with a small and elongated body and distinct tarsal projections was collected. Molecular comparison indicated this individual to be R.verrucopus. Herein, we describe this new record for Myanmar in detail.
Materials and methods
Field surveys were conducted in Htamanthi Wildlife Sanctuary, Sagaing Division, Myanmar. The specimen was collected and euthanised with ethyl acetate and then fixed in 75% ethanol for storage after taking photographs. Liver tissue sample was preserved in 99% ethanol for molecular analysis. The specimen was deposited in Southeast Asia Biodiversity Research Institute, Chinese Academy of Sciences (abbreviation: SEABRI; address: Yezin, Nay Pyi Taw, Myanmar).
Total genomic DNA was extracted from liver tissue. Tissue sample was digested using proteinase K and subsequently purified, following standard phenol/chloroform isolation and ethanol precipitation. A fragment of encoding partial 16S rRNA gene was amplified using primer pairs 16Sar/16Sbr (Palumbi et al. 1991). Polymerase chain reaction (PCR) amplifications were performed in 50 μl reactions using the following cycling conditions: initial denaturing step at 95 °C for 4 min; 35 cycles of denaturing at 94 °C for 60 s, annealing at 51 °C for 60 s and extending at 72 °C for 60 s; and a final extension step of 72 °C for 10 min. Sequencing was conducted directly using the corresponding PCR primers. The new sequence was deposited in GenBank under accession number MW275978. Outgroups were selected according to Jiang et al. (2019) and Nguyen et al. (2020). Homologous and outgroup sequences were obtained from GenBank (Table 1).
Species used for molecular phylogenetic analysis.
Species
Voucher
Locality
Accession No.
Ingroup
Rhacophorusannamensis
VNMN 4092
Gia Lai, Kon Ka Kinh, Vietnam
LC010568
VNMN 4090
Dak Nong, Nam Nung, Vietnam
LC010566
Rhacophorusbipunctatus
CAS235303
Bee Hoe, Chin, Myanmar
JX219444
CAS229913
Putao, Kachin, Myanmar
JX219445
Rhacophoruscalcaneus
VNMN 4093
Dak Lac, Chu Yang Sin, Vietnam
LC010573
KIZ 528
Bi Doup, Lam Dong, Vietnam
JX219450
Rhacophorusexechopygus
VNMN 4107
Gia Lai, Kon Ka Kinh, Vietnam
LC010585
VNMN 4108
Gia Lai, Kon Ka Kinh, Vietnam
LC010586
Rhacophorushelenae
AMS R 173230
Binh Thuan, Vietnam
JQ288087
UNS 00450
Dong Nai, Vietnam
JQ288088
Rhacophorushoabinhensis
IEBR A.2016.18
Hoa Binh, Vietnam
LC331096
VNMN A.2016.16
Hoa Binh, Vietnam
LC331097
Rhacophoruskio
VNMN 4110
Gia Lai, Kon Ka Kinh, Vietnam
LC010589
VNMN 4111
Ha Giang, Bac Quang, Vietnam
LC010590
Rhacophorusnigropalmatus
Rao081203
Malaysia
JX219438
Rao081204
Malaysia
JX219437
Rhacophorusorlovi
VNMN 3067
Ha Tinh, Huong Son, Vietnam
LC010598
VNMN 4115
Nghe An, Pu Huong, Vietnam
LC010600
Rhacophorusrhodopus
SCUM 060692L
Mengyang, Yunnan, China
EU215531
X219440
Lvchun, Yunnan, China
JX219440
Rhacophorusrobertingeri
VNMN 4123
Gia Lai, Kon Ka Kinh, Vietnam
LC010613
VNMN 3446
Kon Tum, Kon Plong, Vietnam
LC010615
Rhacophorusspelaeus
IEBR A.2011.1
Khammouan, Lao
LC331095
Rhacophorustranslineatus
Rao6237
Medog, Tibet, China
JX219449
Rhacophorusverrucopus
6254 Rao
Medog, Tibet, China
JX219436
SEABRI2019120056
Htamanthi, Sagaing, Myanmar
MW275978
Outgroup
Zhangixalusduboisi
VNMN 4102
Sa Pa, Lao Cai, Vietnam
LC010580
Zhangixalusdugritei
SCUM 051001L
Baoxing, Sichuan, China
EU215541
Leptomantisgauni
FMNH273928
Sarawak, Bintulu, Malaysia
JX219456
Leptomantispenanorum
ZRC 1.12116
Sarawak, Bintulu, Malaysia
JN377350
Buergeriabuergeri
IABHU 41011
Hiroshima, Japan
AB127977
Sequences were aligned using ClustalW with default parameters in MEGA 7 (Kumar et al. 2016). Uncorrected pairwise distances between species were calculated in MEGA 7 with the parameters Transitions + Transversions, Uniform rates and Pairwise deletion. The best substitution model GTR+G was the Bayesian Information Criterion (BIC) in jModelTest 2.1.7 (Darriba et al. 2012). Bayesian Inferences were performed in MRBAYES v3.2.6 (Ronquist et al. 2012). Two runs were performed simultaneously with four Markov chains starting from the random tree. The chains were run for 1 000 000 generations and sampled every 100 generations. The first 25% of the sampled trees were discarded as burn-in after the standard deviation of split frequencies of the two runs was less than 0.01. The remaining trees were then used to create a consensus tree and to estimate Bayesian posterior probabilities (BPPs). Maximum Likelihood analysis was performed in RaxmlGUI 1.5 (Silvestro and Michalak 2012) and nodal support values were estimated by 1,000 rapid bootstrap replicates.
Measurements were taken with a digital caliper to the nearest 0.1 mm. Morphological terminology followed Fei et al. (2009). Measurements included: snout-vent length (SVL, from tip of snout to vent); head length (HL, from tip of snout to rear of jaw); head width (HW, width of head at widest point); snout length (SL, from tip of snout to anterior border of eye); internarial distance (IND, distance between nares); interorbital distance (IOD, minimum distance between upper eyelids); eye diameter (ED, diameter of exposed portion of eyeball); tympanum diameter (TD, greater of tympanum vertical and horizontal diameters); distance from nostril to eye (DNE, from nostril to anterior border of eye); forearm and hand length (FHL, from elbow to tip of third finger); tibia length (TL, distance from knee to heel); and foot length (FL, from proximal end of inner metatarsal tubercle to tip of fourth toe).
Results
The obtained sequence alignment for the 16S gene was 510 bp long. SEABRI2019120056 clustered with R.verrucopus from Medog, Tibet, China with strong support (Fig. 1). The genetic distance between SEABRI2019120056 and R.verrucopus from Medog, Tibet, China was 1.2% (Table 2). Therefore, we considered SEABRI2019120056 belongs to R.verrucopus.
Maximum Likelihood tree of partial Rhacophorus species inferred from 16S rRNA gene sequences (Numbers before slashes indicate Bayesian posterior probabilities and numbers after slashes indicate bootstrap support for Maximum Likelihood analyses. The symbol “–” represents values below 60). The specimen collected from Myanmar is indicated by red.
https://binary.pensoft.net/fig/481768
Divergence (P-distance; %) between and within homologous species and Rhacophorusverrucopus estimated from 16S gene sequences.
Adult female (SEABRI2019120056) collected on 21 December 2019 by the local guides from Htamanthi Wildlife Sanctuary, Sagaing Division, Myanmar (25°21'59"N, 95°22'59"E, 90 m elevation).
The specimen (SEABRI2019120056) collected from Myanmar in life. A. Dorsal view; B. ventral view; C. lose-up view of anterior part of thigh; D. close-up view of posterior part of thigh; E. close-up view of hand; F close-up view of foot.
Morphological characters of the specimen from Myanmar agreed well with the original description of Huang (1983) and subsequent descriptions of Fei et al. (2009, 2012). Body small and elongate, SVL 52.0 mm; head length (HL 17.6 mm) larger than width (HW 15.7 mm); snout slightly sharp, canthus rostralis distinct, loreal region slightly oblique; nostril at tip of snout, internarial distance (IND 4.3 mm) narrower than interorbital distance (IOD 9.3 mm); tympanum (TD 3.1 mm) rounded, slightly larger than half eye diameter (ED 5.4 mm), separated from eye by 1.6 mm; pupil transverse, eye diameter larger than half snout length (SL 7.7 mm). Vomerine teeth present; tongue attached anteriorly, deeply notched posteriorly.
Forearm and hand length (FHL 17.8 mm) no more than half SVL; relative length of fingers I < II < IV < III; tips of all fingers expanded into discs with transverse grooves, disc of first finger small, disc of third finger largest, its width nearly equal to tympanum; webbing formula I 1-1 II 0-1 III 1-1/2 IV; subarticular tubercles distinct, formula 1, 1, 2, 2; rows of tubercles present on palms; inner metacarpal tubercle large and flat, outer metacarpal tubercle absent.
Hind limbs long, tibia (TL 24.2 mm) about half SVL and longer than thigh (22.9 mm) and foot (FL 20.4 mm); relative length of toes I < II < III < V < IV; tips of toes expanded into discs, smaller than those of fingers; webbing formula I 0-0- II 0-1+ III 0-1 IV 1-0 V, subarticular tubercles distinct, formula 1, 1, 2, 3, 2; rows of tubercles present on pes; inner metatarsal tubercle flat, outer metatarsal tubercles absent.
Skin of dorsum smooth; supratympanic fold thin and distinct; outer edge of forearm with light coloured granules arranged in serrated shape; dermal calcars present on heels forming tarsal projections; granules above vent forming transverse skin fold; ventral skin covered with small flat granules.
Colour in life.
Colour pattern similar to the holotype of R.verrucopus. Dorsal surface greyish-yellow with scattered small brownish-black spots; limbs with very indistinct transverse stripes; each side of thigh and inner sides of shank, tarsus and foot orangish-red; webbing between fingers orangish-yellow, webbing between toes orangish-red; ventral surface greyish-white, belly sides light yellow; pupil black, iris greyish-yellow.
Ecological notes.
The specimen was found at night on a bush approximately 0.8 m above the ground near a large river. Several eggs were visible through the skin of the belly. The eggs were yellow and large. The breeding period of this species remains unknown.
Discussion
Morphologically, the specimen of R.verrucopus from Myanmar shows good agreement with the original description, except for minor differences in colouration. In the original description, the colour of the upper eyelids was greyish-brown and there were greyish-brown transverse bands present on limbs. However, the upper eyelids of the specimen from Myanmar were the same colour as the dorsal surface and the transverse bands on the limbs were nearly invisible.
Rhacophorusverrucopus was known previously only from Tibet, China. This is the first record of R.verrucopus from Myanmar and from outside of China (Fig. 3). According to the original description, this species inhabited the area between 850 m and 1500 m elevation in the type locality (Huang 1983). The new location in Myanmar is approximately 440 km away from the type locality in China and the altitude (90 m elevation) of the new location is much lower than the type locality.
The field survey in Myanmar was undertaken at the invitation of the Republic of the Union of Myanmar, Ministry of Natural Resources and Environmental Conservation, Forest Department, Forest Research Institute. We thank them for the invitation. Thanks also to the staff and the local guides of Myanmar for their help in the field.
ReferencesAmphibiaChina (2020) The database of Chinese amphibians. Electronic Database. http://www.amphibiachina.org [Accessed on 20 Oct 2020]DarribaDTaboadaGLDoalloRPosadaD (2012) jModelTest 2: more models, new heuristics and parallel computing.9: 772–772. https://doi.org/10.1038/nmeth.2109FeiLHuSQYeCYHuangYZ (2009) Science Press, Beijing, 957 pp.FeiLYeCYJiangJP (2012) Sichuan Publishing House of Science and Technology, Chengdu, 620 pp.FrostDR (2020) Amphibian Species of the World: an Online Reference. Version 6.1. Electronic Database. https://amphibiansoftheworld.amnh.org/index.php [Accessed on 20 October 2020]HuangYZ (1983) A new species of flying frog from Xizang – Rhacophorusverrucopus.2(4): 63–65.JiangDCJiangKRenJLWuJLiJT (2019) Resurrection of the genus Leptomantis, with description of a new genus to the family Rhacophoridae (Amphibia: Anura).10: 1–12. https://doi.org/10.16373/j.cnki.ahr.180058KuhlHHasseltJC van (1822) Uittreksels uit breieven van de Heeren Kuhl en van Hasselt, aan de Heeren C. J. Temminck, Th. van Swinderen en W. de Haan.7: 99–104.KumarSStecherGTamuraK (2016) MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets.33(7): 1870–1874. https://doi.org/10.1093/molbev/msw054LiSQQuanRC (2017) Taxonomy is the cornerstone of biodiversity conservation – SEABRI reports on biological surveys in Southeast Asia.38(5): 213–214. https://doi.org/10.24272/j.issn.2095-8137.2017.061NguyenTTNinhHTOrlovNNguyenTQZieglerT (2020) A new species of the genus Zhangixalus (Amphibia: Rhacophoridae) from Vietnam.54: 257–273. https://doi.org/10.1080/00222933.2020.1754484PalumbiSRMartinARomanoSMcMillanWSticeLGrabowskiG (1991) University of Hawaii Press, Honolulu, 94 pp.PetersWCH (1867) Herpetologische Notizen.1867: 13–37.RonquistFTeslenkoMvan der MarkPAyresDLDarlingAHöhnaSLargetBLiuLSuchardMAHuelsenbeckJP (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space.61: 539–542. https://doi.org/10.1093/sysbio/sys029SilvestroDMichalakI (2012) raxmlGUI: a graphical front-end for RAxML.12(4): 335–337. https://doi.org/10.1007/s13127-011-0056-0WoganGOUVindumJVWilkinsonJAKooMSSlowinskiJBWinHThinTKyiSOoSLwinKSheinA (2008) New country records and range extensions for Myanmar Amphibians and Reptiles.33(1): 83–96.