Amolops putaoensis Gan, Qin, Lwin, Li, Quan, Liu & Yu, 2020, a newly recorded torrent frog for China

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.


Introduction
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.

Methods
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); snoutlength 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 tympa- num 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.

Molecular analyses
The aligned COI dataset contained a total of 570 nucleotide base pairs (bp), with 214 variable positions and 168 parsi-mony informative sites. The uncorrected pairwise distance (p-distance) between the Gongshan specimens and the holotype of A. putaoensis was only 0.005 (Suppl. material 3). The BI and ML analyses obtained consistent topologies and most nodes were supported well. Both analyses placed the Gongshan specimens and the holotype of A. putaoensis in a single clade, and it was recovered with strong support as sister to the clade containing A. aniqiaoensis, A. kohimaensis, A. adicola, and A. monticola (Fig. 1).

Morphological analyses
The Gongshan specimens closely matched the original description of A. putaoensis by Gan et al. (2020) (Suppl. materials 2, 4), with two notable exceptions. First, the Gongshan specimens have larger body sizes, with male SVL 47.5-51.3 mm, vs. 37.6-40.2 mm in the type series. Second, the Gongshan specimens have a pair of external subgular vocal sacs (Fig. 2G), stated to be internal vocal sacs in the type series by Gan et al. (2020). As this character is not visible in the figures in the original description, we assume that the type series was not calling when collected and this character was therefore overlooked by Gan et al. (2020). The description of A. putaoensis is expanded using the Gongshan specimens, as follows. Expanded diagnosis. A member of the A. monticola group having males (n = 13) with side of head dark with light-colored upper lip stripe extending to axilla; distinct dorsolateral fold; weak to moderately developed supratympanic fold; no outer metatarsal tubercle; circummarginal groove on tip of first finger; SVL 37.6-51.3 mm; head length slightly short or subequal to head width, with HDL : HDW 0.87-1.02; smooth dorsal skin, with dense, tiny dermal granules distributed from posterior margin of nostril to cloaca; tympanum diameter less than half of eye diameter; distinct transverse bands on dorsal surfaces of limbs; venter greyish white, with irregular spots from throat to mid-belly; visible pineal body; vomerine teeth; nuptial pads; external subgular vocal sacs; and maxillary gland.

Amolops putaoensis Gan, Qin, Lwin, Li, Quan, Liu & Yu, 2020
Expanded description. In life, dorsum with smooth skin; dorsum colored in brown with black irregular spots from snout to vent; lateral region lemon yellow to olive green, dorsal and lateral coloration divided by a distinct dorsolat-eral gland extending from posterior margin of eye to near vent; tiny dermal granules extending from posterior margin of nostrils and eyelid to near vent, absent from lateral and ventral regions of body; tarsal and skin folds absent; venter greyish white, with irregular spots from throat to chest. In preservative (75% ethanol), dorsal coloration faded to dark bluish-green and ventral coloration to creamy yellow.
Expanded variation. In life, dorsal coloration varied from yellowish brown to dark brown; lateral coloration varied from lemon yellow to olive green; irregular spots on flank rather distinct or weak; number of transverse bands on forearm ranged from 4-8; and number of transverse bands on tibia ranged from 6-7 (Fig. 3).
Expanded comparisons. Gan et al. (2020) did not compare A. putaoensis with the closely related A. kohimaensis (Fig. 1) and only distinguished A. putaoensis from A. aniqiaoensis on the basis of male body size, a character that does not hold up with the addition of the Gongshan specimens. Its other two relatives, A. monticola and A. adicola (Fig. 1), were poorly known or undescribed, respectively, at the time of the description of A. putaoensis. With the addition of the Gongshan specimens, A. putaoensis can be distinguished from A. kohimaensis by lacking an outer metatarsal tubercle (vs. present); and having distinct dorsolateral folds (vs. absent). Amolops putaoensis can be distinguished from A. aniqiaoensis by having distinct transverse bands on dorsal surfaces of limbs (vs. absent or indistinct patterns); and lacking a "/ \"-shaped mark on chest (vs. present). Amolops putaoensis can be distinguished from A. monticola by lacking an outer metatarsal tubercle (vs. present); and having circummarginal grooves on finger Ⅰ (vs. absent). Amolops putaoensis can be distinguished from A. adicola by having distinct transverse bands on dorsal surfaces of limbs (vs. reticulation); and having ventral spots from throat to mid-belly (vs. absent).
Expanded distribution. Amolops putaoensis is known only from its type locality in Putao County, Kachin State, northern Myanmar, and from our newly recorded locality in Dulongjiang Village, Gongshan County, Yunnan Province, China, approximately 133.7 km northeast of the type locality.

Discussion
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 , and A. monticola and A. adicola from northeastern India (Patel et al. 2021). Unfortunately, both the original description ) 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.