We report the first country record of Rhacophorus verrucopus 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.

16S rRNA, Htamanthi Wildlife Sanctuary, new record, tree frog

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). Rhacophorus verrucopus, 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.

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).

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).

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.

Figure 1. 

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.

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.

Rhacophorus verrucopus 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.

Figure 3. 

Collection site of Rhacophorus verrucopus (black dot) in Myanmar and the type locality (black star) in Tibet, China.

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.