Amolops putaoensis is a recently described torrent frog species from A. monticola group that is known only from its type locality, northern Myanmar. We compared morphology and mitochondrial DNA sequence data from ten recently collected adult male specimens from the upper Dulong River System in Gongshan County, Yunnan Province, China, to the original description of A. putaoensis. Both datasets strongly supported referring the Chinese specimens to A. putaoensis, extending the known range of this species by approximately 133.7 km distance into China. Molecular phylogenetic analyses recovered A. putaoensis to be closely related to A. aniqiaoensis, A. kohimaensis, A. monticola, and A. adicola. We use the newly collected Chinese specimens to expand the morphological description of the species.

Dulong River System, herpetofauna, national new record, Ranidae

The torrent or cascade frogs of the genus Amolops Cope, 1865 inhabit swift, rocky streams in mountainous regions of Asia, across Indo-Burma, including northeastern India, Southeast Asia, and southern continental China. Species groups in this genus were revised to ten (A. chayuensis group, A. daiyunensis group, A. hainanensis group, A. larutensis group, A. mantzorum group, A. marmoratus group, A. monticola group, A. ricketti group, A. spinapectoralis group, A. viridimaculatus group) by Jiang et al. (2021), among these, the A. monticola group currently contains 22 species within a monophyly. As a newly discovered monticola complex species, A. putaoensis Gan, Qin, Lwin, Li, Quan, Liu & Yu (2020) has been indicated to occur only around its type locality in northern Myanmar.

Our fieldwork in early July 2019 in the upper Dulong River system in Gongshan County, Yunnan Province, China (27.669949°N, 98.268219°E, 1281 m elev.), near to its border with Tibet Province, revealed ten adult males of Amolops near a shallow sandy-bottom stream with rocky banks (Fig. 1). All specimens were humanely handled and euthanized, then preserved in 75% ethanol, with liver tissues transferred into 95% ethanol and stored at -20℃ for molecular analysis. Voucher specimens were deposited in the Kunming Institute of Zoology (KIZ), Yunnan, China. In this study, taxonomy of these specimens was investigated based on morphological comparison and molecular phylogenetic analyses. Both morphological and molecular datasets corroborate referring these specimens to the recently described A. putaoensis that was known only from northern Myanmar, extending the known range of A. putaoensis by a distance of approximately 133.7 km, and representing a new addition to the known anuran fauna of China.

Figure 1. 

Known localities and molecular phylogenetic relationships of Amolops putaoensis. A. The type locality (yellow star) at Putao County, Kachin State, Myanmar and the new locality (red star) at Dulongjiang Village, Gongshan County, Yunnan Province, China. Map generated by ArcGIS Pro; B. Phylogenetic tree based on Bayesian inference of a fragment of the mitochondrial COI gene. Branches support values of posterior probability (PP) and bootstrap supports (BS) were performed, where the strong supports found in represented Circles represent very strong nodal support of posterior probabilities (PP) of 1.0 and bootstrap support (BS) from a separate maximum likelihood analysis of 100, and squares represent strong nodal support of PP ≤ 0.95 and BS ≤ 70, with lower support values not shown.

Morphological terminology follows Fei et al. (2009). All 10 adult male specimens were measured (Suppl. material 1) using slide calipers to the nearest 0.1 mm: snout-vent length (SVL); head length from the tip of snout to rear of the jaws (HDL); maximum head width at commissure corner of jaws (HDW); internarial distance (IND); snout-length from the tip of snout to the anterior corner of the eye (SNT); horizontal diameter of exposed portion of the eyeball (EYE); interorbital distance at the narrowest point (IOD); horizontal diameter of tympanum (TMP); tympanum-eye distance from the anterior edge of tympanum to posterior corner of the eye (TEY); femur length from the vent to outer edge of knee (FEM); hand length from the base of palm to the tip of finger Ⅲ (HND); maximum width of finger Ⅲ digital disc (F3DSC); foot length (FTL); manus length from the tip of third digit to proximal edge of inner palmar tubercle (ML); pes length from the tip of toe Ⅳ to proximal edge of inner metatarsal tubercle (PL); maximum width of upper eyelid (UEW); forearm and hand length (FAHL); maximum width of forearm (FAW); length of tarsus and foot (TFL); width of finger Ⅱ digital disc (F2D); and width of toe Ⅳ digital disc (T4D).

Genomic DNA was extracted from liver tissues of two individuals (KIZ 035035, KIZ 035037) using the KaTaRa DNA purification kit. A fragment of the mitochondrial cytochrome C oxidase subunit 1 gene (COI, Che et al. 2012) was amplified by the polymerase chain reaction (PCR). Amplified PCR products were sequenced using an ABI 3730 automated sequencer. Newly-generated sequences were edited to consistent length using MEGA X (Kumar et al. 2018) and deposited in GenBank under accession numbers OP831318 and OP831319 (vouchered specimens see Suppl. material 2).

Homologous sequences of 21 species in the A. monticola group were obtained from GenBank, including the holotype of A. putaoensis. Homologous sequences of A. chayuensis (A. chayuensis group), A. xinduqiao and A. lifanensis (both in the A. mantzorum group) were also downloaded from GenBank for use as outgroups (Suppl. material 2). The newly-generated and downloaded sequences were aligned by MEGA X. Mean pairwise uncorrected genetic distances (p-distances) between the Gongshan specimens and other closely-related Amolops species were calculated by MEGA X. Phylogenetic trees were reconstructed using Bayesian inference (BI) by MrBayes 3.2.7a. and the Maximum Likelihood (ML) criterion performed by RAxML NG v0.9.0. (Kozlov et al. 2019). The GTR+I+G model was selected as the best substitution model for each codon position by JModelTest 2 (Guindon and Gascuel 2003; Darriba et al. 2012). The BI analyses used Metropolis Coupled Markov Chain Monte Carlo (MCMC) with three heated chains and one cold chain for 5,000,000 generations and sampled every 1,000 generations, with every 1,500 generations with the first 25% of samples discarded as burn-in. The potential scale reduction factor (PSRF > 1) and the average standard deviation of split frequencies (ASDSF < 0.01) were used to evaluate topological and branch-length convergences, respectively. Confidence in tree nodes was assessed by posterior probabilities (PP) (Huelsenbeck and Ronquist 2001). ML analyses were performed with nodal support based on 1,500 bootstrap (BS) replicates. Relationships with PP ≥ 0.95 and BS ≥ 70 were considered to be strongly supported.

Our morphological and mitochondrial DNA data strongly support referring the newly collected specimens at Gongshan County, Yunnan Province, China as the new national record of Amolops putaoensis. Our study extends the geographic range of the species northeast into China and demonstrates that A. putaoensis is closely related to A. kohimaensis from northeastern India (Biju et al. 2010), A. aniqiaoensis from Medog, Tibet, China (Che et al. 2020), and A. monticola and A. adicola from northeastern India (Patel et al. 2021). Unfortunately, both the original description (Gan et al. 2020) and our study did not find females or larvae of A. putaoensis, or document advertisement calls of males. Hence, additional research into the natural history of A. putaoensis is warranted.

We thank Ge Gao, Jun Sun, and Long-Jie Guo from Gaoligongshan National Nature Reserve for helped our fieldworks. This work was supported by grants from Fundamental Research Funds for the Central Universities (SWU 5330500880), the Second Tibetan Plateau Scientific Expedition and Research Program (2019QZKK0501), the Ministry of Science and Technology of China (2021FY100203), Young Talent Project of China Association for Science and Technology (2019–2021QNRC001), Yunnan Fundamental Research Project (202001AW070016, 202005AC160046), the Digitalization, Development and Application of Biotic Resource (202002AA100007), and the Animal Branch of the Germplasm Bank of Wild Species, Chinese Academy of Sciences (the Large Research Infrastructure Funding).