﻿A new species of Amolops (Amphibia, Anura, Ranidae) from Guizhou Province, China

﻿Abstract The Torrent frogs of the genus Amolops are widely distributed in Nepal and northern India eastwards to southern China and southwards to Malaysia. The genus currently contains 84 species. Previous studies indicated underestimated species diversity in the genus. In the context, a new species occurring from the mountains in the northwestern Guizhou Province, China is found and described based on morphological comparisons and molecular phylogenetic analyses, Amolopsdafangensissp. nov. Phylogenetic analyses based on DNA sequences of the mitochondrial 16S rRNA and COI genes supported the new species as an independent lineage. The uncorrected genetic distances between the 16S rRNA and COI genes in the new species and its closest congener were 0.7% and 2.6%, respectively, which are higher than or at the same level as those among many pairs of congeners. Morphologically, the new species can be distinguished from its congeners by a combination of the following characters: body size moderate (SVL 43.2–46.8 mm in males); head length larger than head width slightly; tympanum distinct, oval; vocal sacs absent; vomerine teeth present; dorsolateral folds weak formed by series of glands; nuptial pads present on the base of finger I; heels overlapping when thighs are positioned at right angles to the body; tibiotarsal articulation reaching the level far beyond the tip of the snout when leg stretched forward.

Guizhou Province is one of the richest areas for amphibians in China and three Amolops species (A. chaochin, A. chunganensis, and A. sinensis) were have been recorded (Amphibia China 2023).During fieldwork in Dafang County, Guizhou Province, some Amolops specimens were collected.By our comparisons, these specimens were different from A. chaochin, A. chunganensis, and A. sinensis by the dorsolateral folds being weak, formed by series of glands, and the presence of a circum-marginal groove on the disc of the first finger.Molecular phylogenetic analyses based on mitochondrial DNA and comprehensive morphological comparisons all indicated that the specimens from Dafang County were an undescribed species, herein described as a new species, Amolops dafangensis sp.nov.

Sampling
Five specimens of Amolops dafangensis sp.nov.including three adult males and two juveniles, were collected from Dafang County, Guizhou Province, China (Fig. 1).All specimens were fixed in 10% buffered formalin for one day, and then transferred to 70% ethanol.Tissue samples were preserved separately in 95% prior to fixation.Specimens collected in this work were all deposited in Maotai Institute (MT), Renhuai City, Guizhou Province, China.

Collection of molecular data
DNA was extracted from tissue using a standard phenol-chloroform extraction protocol (Sambrook et al. 1989).Two mitochondrial genes, partial 16S ribosomal RNA gene (16S) and cytochrome oxidase subunit I (COI), were amplified.
The primers used for 16S were P7 (5'-CGCCTGTTTACCAAAAACAT -3') and P8 (5'-CCGGTCTGAACTCAGATCACGT') following Simon et al. (1994), and that for COI were Chmf4 (5'-TYTCWACWAAYCAYAAAGAYATCGG-3') and Chmr4 (5'-ACYTCRGGRTGRCCRAARAATCA-3') following Che et al. (2012).PCR amplification reactions were performed in a 30 µl reaction with the following cycling conditions: an initial denaturing step at 95 °C for 4 min; 35 cycles of denaturing at 95 °C for 40 s, annealing at 48 °C/46 °C (16S/COI) for 40 s and extending at 72 °C for 70 s, and a final extending step of 72 °C for 10 min.PCR products were purified with spin columns and then were sequenced with both forward and reverse primers same as PCR.Sequencing was conducted using an ABI Prism 3730 automated DNA sequencer in Chengdu TSING KE Biological Technology Co. Ltd. (Chengdu, China).All sequences were deposited in GenBank (for GenBank Accession numbers refer to Table 1).For phylogenetic analyses, we downloaded corresponding sequences for all related species from GenBank according to previous studies (Qian et al. 2023;Tang et al. 2023; for GenBank accession numbers see Table 1).

Phylogenetic analyses and genetic distance
Sequences were assembled and aligned using the Clustalw module in BioEdit 7.0.9.0 (Hall 1999) with default settings.The datasets were checked by eye and revised manually if necessary.Based on the 16S + COI concatenated dataset, phylogenetic analyses were conducted using maximum likelihood (ML) and  Bayesian Inference (BI) methods, implemented in PhyML 3.0 (Guindon et al. 2010) and MrBayes 3.12 (Ronquist and Huelsenbeck 2003), respectively.The best-fit model was obtained by the Bayesian inference criteria (BIC) computed with PartitionFinder 2 (Lanfear et al. 2012).In this analysis, 16S gene and each codon position of COI gene were defined, and Bayesian Inference Criteria was used.As a result, the analysis suggested that the best partition scheme is16S gene/each codon position of COI gene, and selected GTR + G + I model as the best model for each partition.For ML analysis, the bootstrap consensus tree inferred from 1000 replicates was used to estimate nodal supports of inferred relationships on phylogenetic trees.For Bayesian analyses, four Markov chains were run for 50 million generations with sampling every 1000 generations.The first 25% of the trees were discarded, representing the burn-in phase of the analyses, and the remaining trees were used to calculate the Bayesian posterior probabilities.Genetic distance between species of A. mantzorum group were estimated on 16S and COI genes, respectively, based on uncorrected p-distance model using MEGA 6.06 (Tamura et al. 2013).

Morphological comparisons
Morphological measurements were made with dial calipers to nearest 0.1 mm by S-ZL following Fei et al. (2009).In total, twenty morphological characteristics were measured for the adult specimens: ED eye diameter (distance from the anterior corner to the posterior corner of the eye); FL foot length (distance from tarsus to the tip of fourth toe); HDL head length (distance from the tip of the snout to the articulation of jaw); HDW maximum head width (greatest width between the left and right articulations of jaw); HLL hindlimb length (maximum length from the vent to the distal tip of the Toe IV); IND internasal distance (minimum distance between the inner margins of the external nares); IOD interorbital distance (minimum distance between the inner edges of the upper eyelids); LAL length of lower arm and hand (distance from the elbow to the distal end of the Finger IV); ML manus length (distance from tip of third digit to proximal edge of inner palmar tubercle); NED nasal to eye distance (distance between the nasal and the anterior corner of the eye); NSD nasal to snout distance (distance between the nasal the posterior edge of the vent); LW lower arm width (maximum width of the lower arm); SVL snout-vent length (distance from the tip of the snout to the posterior edge of the vent); SL snout length (distance from the tip of the snout to the anterior corner of the eye); TFL length of foot and tarsus (distance from the tibiotarsal articulation to the distal end of the Toe IV); THL thigh length (distance from vent to knee); TL tibia length (distance from knee to tarsus); TW maximal tibia width; TYD maximal tympanum diameter; UEW upper eyelid width (greatest width of the upper eyelid margins measured perpendicular to the anterior-posterior axis).
We also compared the morphological characters of the new taxon with other species of Amolops.Comparative data were obtained from the literature for all species of Amolops (Table 2).

Phylogenetic analyses
The ML and BI phylogenetic trees were constructed based on concatenated DNA sequences of the mitochondrial 16S (425 bp) and COI (606 bp) genes.ML and BI analyses resulted in essentially identical topologies though some basal relationships between clades were not resolved (Fig. 2).The new taxon was indicated as an independent clade.Furthermore, the smallest uncorrected p-distance between Amolops dafangensis sp.nov.and its most closely-related congeners is 0.7% (vs A. sangzhiensis) on 16S gene (Suppl.material 1), and 2.6% (vs A. loloensis) on COI gene (Suppl.material 2), which was higher or at the same level with those among many pairs of congeners, for example, 0.3% between A. sangzhiensis and A. jinjiangensis on the 16S gene, and 3.2% between A. jinjiangensis and A. loloensis on the COI gene.

Morphological comparisons
Morphological measurements are given in Table 3.The new taxon could be identified from its congeners by a series of differences in morphological characters.Diagnosis.Amolops dafangensis sp.nov.resembles members of the A. mantzorum group in the absence of true dorsolateral folds and the presence of a circum-marginal groove on the disc of the first finger.The tarsal fold and tarsal glands are absent, and a nuptial pad is present on the first finger in males (Jiang et al. 2021).
Amolops dafangensis sp.nov.can be distinguished from other congeners by the following characters:  4), body size moderate, SVL 44.7 mm.head length larger than head width slightly (HDL: HDW = 1.02); snout short, rounded in dorsal view, projecting beyond lower jaw; eye large and convex, eye diameter 0.74× of snout length; nostril rounded, between to tip of snout and eyes; internasal distance larger than interorbital distance; tympanum circular, Figure 2. Maximum likelihood (ML) tree of Amolops mantzorum group based on the 16S and CO1 genes.ML bootstrap supports (BS) /Bayesian posterior probability (BPP) were denoted beside each node, and "-" denotes BS < 50% or BPP < 0.60.Samples 1-52 refer to Table 1.distinct, 0.56× of eye diameter; loreal region slightly concave; nares oval; pineal ocellus visible; supratympanic fold extends from back of eye to above shoulder; vomerine teeth present; tongue deeply notched posteriorly; vocal sac absent.
Forelimbs robust (LW/SVL=0.08);lower arm and hand beyond one-second of body length (LAL/SVL=0.51);fingers slender, relative finger lengths I < II < IV < III; finger tips on II-IV dilated to wide cordiform disks with circum-marginal grooves, tip of first finger with small disk but without circum-marginal groove; all fingers without webbing and lateral fringes; subarticular tubercle prominent; supernumerary tubercle indistinct; inner metacarpal tubercle oval, elongate; outer metacarpal tubercles small round; velvety nuptial pad on finger I.
Hindlimbs long, nearly 2× SVL (HLL/SVL = 1.87); tibiotarsal articulation reaching the level far beyond the tip of the snout when leg stretched forward; tibias longer than thigh length, heels overlapped; toes slender, relative lengths I < II < III < V < IV; toes entirely webbed; tips of toes expanded into disc with circum-marginal grooves; outer metatarsal tubercle absent; inner metatarsal tubercle small but well developed.
Skin on dorsum and dorsal surfaces of limbs smooth; dorsolateral folds weak, formed by series of glands been an incomplete line, extending from above shoulder to vent; weak dorsolateral glandular lines; ventral surface of bell and limbs smooth except a few small tubercles on posterior surface of thigh and around vent.Coloration in life.In life, iris pale brown with dark wash; top of head and dorsum golden brown with large rounded black brown and green spots; sides of head with a pale green stripe extending from loreal region to region behind and below eye along upper lip; a black brown band from the tip of the snout through the nostril to an anterior border of the eye, continuing behind the eye to the shoulder; temporal region black brown with green blotches; the flank green with some back brown spots; limbs dorsally golden brown with black brown bands; chest and venter white, throat white with pale brown; ventral surface of anterior forelimbs brown with green spots; finger I and II fresh-colored, finger III and IV brown; ventral surface of hindlimbs fresh-colored (Fig. 3).
Color in preservative.Dorsal surface fade to pale brown with beige brown and black spots on head, flank and on limbs; ventral surface fade to creamy white, marbled with brown on throat and chest (Fig. 4).
Variation.Measurements of all specimens are listed in Table 3.All specimens were very similar in morphology, but in MT DF20230601001 the dorsum was golden brown with few green spots (Fig. 5A); in MT DF20230601003 the dorsum and dorsal surfaces of limbs were green with brown spots (Fig. 5B); in the juvenile specimen MT DF20230601004 the flank was mainly green with black spots and the ventral surface of the throat and chest were white with pale brown spots (Fig. 5C, D).Amolops dafangensis sp.nov. is phylogenetically most closed to A. loloensis and A. sangzhiensis, and the new species could be distinguished from A. loloensis by having a smaller body size in males (adult males SVL 43.2-46.8mm vs 55-62 mm), having distinct tympanum (vs indistinct), tibiotarsal articulation reaching the level far beyond the tip of the snout when leg stretched forward (vs just reaching eye or nostrils), spots on head and dorsum irregular (vs spots on head and dorsum round or oval); differs from A. sangzhiensis by having a larger body size in males (adult males SVL 43.2-46.8mm vs 40.3-40.9mm), having distinct tympanum (vs indistinct),tibiotarsal articulation reaching the level far beyond the tip of the snout when leg stretched forward (vs just reaching nostrils), mouth corner smooth (vs with dense spiny tubercles around the mouth corner).
Etymology.The specific epithet dafangensis refers to the distribution of this species, Dafang County, Guizhou Province, China.We propose the common English name "Dafang cascade frogs" for this species and Chinese name as "Da Fang Tuan Wa (大方湍蛙)".

Discussion
In this study, we describe a new species based on morphological comparisons and molecular phylogenetic analyses; although the genetic distance between the new species and its most closely-related congeners is 0.7% for the 16S gene, the morphological characters differ from those of other species of the genus Amolops.This small genetic difference is likely due to the limited phylogenetic information content in this particular gene fragment (Chan et al. 2022).Speciation usually begins with spatial isolation or adaptation to unique environments without strict isolation (Schilthuizen 2000).Significant spatial isolation and subsequent formation of unique lineages may be due to isolation or longrange dispersal across barriers such as mountains, rivers, or other intervening unsuitable habitats (Mayr 1963;Avise 2000;Rundle and Nosil 2005;Schluter 2009).The geographical distances between Amolops dafangensis sp.nov.and its closely-related congeners A. sangzhiensis and A. loloensis are more than 800 km and 370 km, respectively, and the type locality of the three species are in different mountains: the new species is distributed in Dalou Mountains, A. sangzhiensis in easternmost Wuling Mountains, and A. loloensis in the Daliangshan Mountains, with significantly different biota.Therefore, we speculate that isolation is likely to have promoted speciation between the lineages and led to the evolution of different morphologies between the new species, A. sangzhiensis, and A. loloensis.
In the last five years, 25 new frog species have been described in Guizhou Province, China (Frost 2023).Dafang County is in the northwest of Guizhou Province, China, and there have been few surveys of amphibians in the area over the years.From 2020 to 2023 we conducted five surveys in this region.Only in June 2023 was the new species discovered, and only three adult males and two juveniles were found in a range of ~ 100 meters below the source of the stream.Therefore, we infer that the population of the new species is small.We recommend the new species be assigned as vulnerable (VU) according to the evaluation criteria of the IUCN Red List of threatened Species (IUCN 2012).Future research should focus on determining the distribution and elevational range of the species.

Figure 1 .
Figure 1.Geographical location of the type locality of Amolops dafangensis sp.nov. in Dafang County, Guizhou Province, China.
(1) body size moderate (SVL 43.2 -46.8 mm in males); (2) head length larger than head width slightly; (3) tympanum distinct, oval; (4) vocal sacs absent; (5) vomerine teeth present; (6) dorsolateral folds weak formed by series of glands; (7) nuptial pads present on base of finger I; (8) heels overlapping when thighs are positioned at right angles to the body; tibiotarsal articulation reaching the level far beyond the tip of the snout when leg stretched forward.Description of holotype.Adult male (Figs 3,

Figure 3 .
Figure 3. Photographs of the holotype MT DF20230601002 of Amolops dafangensis sp.nov. in life A dorsal view B ventral view C dorsal view of hand D ventral view of hand E ventral view of foot.

Figure 4 .
Figure 4.The holotype specimen MT DF20230601002 of Amolops dafangensis sp.nov.A dorsal view B ventral view C lateral view D dorsal view of hand E ventral view of hand F ventral view of foot.

Figure 5 .
Figure 5. Color variation in Amolops dafangensis sp.nov.A dorsolateral view of the male specimen MT DF20230601001 B dorsolateral view of the male specimen MT DF20230601003 C dorsolateral view of the juvenile specimen MT DF20230601004 D ventral view of the male specimen juvenile specimen MT DF20230601004

Table 1 .
Information for samples used in molecular phylogenetic analyses in this study.

Table 2 .
References for morphological characters for congeners of the genus Amolops.

Table 3 .
Measurements of the adult specimens of Amolops dafangensis sp.nov.Units are given in mm.See abbreviations for the morphological characters in Materials and methods section.