A new species of cascade frog (Anura: Ranidae: Amolops) from central Yunnan, China
Zoological Letters volume 9, Article number: 15 (2023)
A new species of the genus Amolops, Amolops ailao sp. nov., is described from central Yunnan, China. The new species belongs to the A. mantzorum species group. Phylogenetic analyses based on the combination of mitochondrial 16S rRNA, COI, and cytb genes revealed that the new species is the sister taxon to Amolops ottorum with strong support. Genetically, the new species differs from A. ottorum by 5.0% in cytb sequences. Morphologically, the new species can be distinguished from known congeners by the combination of the following characters: true dorsolateral folds absent, but dorsolateral folds formed by series of glands present; circummarginal groove on tip of first finger absent; body size small (males SVL 33.0–35.1 mm and female SVL 41.3 mm); HW/SVL 0.32‒0.35; UEW/SVL 0.08‒0.10; THL/SVL 0.52‒0.56; vomerine teeth absent; interorbital distance narrower than internarial distance; tympanum distinct, less than half eye diameter; supratympanic fold present, indistinct; a pair of large tubercles on sides of cloaca; tibiotarsal articulation reaching beyond anterior corner of eye; and vocal sac absent. The cladogenesis events within the A. mantzorum group rapidly occurred from Pliocene 4.23 Mya to Pleistocene 1.2 Mya, coinciding with the recent intensive uplift of the Qinghai-Tibetan Plateau since the Pliocene. Combining findings in this study with the most recent taxonomic progress, we consider that there are 20 known Amolops species in Yunnan, China, accounting for the highest proportion of amphibian diversity of Yunnan, and five of them belong to the A. mantzorum group. Among different subfauna and water systems in Yunnan, the species diversity of Amolops in northwestern Yunnan and Nu River Basin is highest.
The cascade frogs of genus Amolops Cope, 1865  inhabit rocky streams or waterfalls, enabled by abdominal suckers in larvae and enlarged digital discs in adults , and are widely distributed from Nepal and northern India eastwards to China and southwards to Malaysia . The species diversity in Amolops has been poorly understood owing to morphological conservation [4, 5], and efforts relying on molecular data during the last decade have greatly improved our understanding of the taxonomy and species diversity of this genus, with a high number of new species having been discovered (e.g., [3, 4, 6,7,8,9,10,11,12,13]). So far, as the most speciose genus within the family Ranidae, the genus Amolops contains 79 species , which can be allocated 10 species groups . In China, a total of 50 Amolops species have been recorded  and most of them have been assigned to eight species groups, namely Amolops chayuensis group, Amolops daiyunensis group, Amolops hainanensis group, Amolops mantzorum group, Amolops monticola group, Amolops marmoratus group, Amolops viridimaculatus group, and Amolops ricketti group, based on morphological and molecular evidence [4, 7, 9, 11, 16,17,18,19,20,21,22,23,24].
The A. mantzorum species group was defined based on the absence of true dorsolateral folds (not formed by incomplete series of glands), circummarginal groove on the tip of first finger, tarsal fold and tarsal glands absent, and nuptial pad present on first finger in males [11, 16, 17, 25]. It was comprised of 11 species [11, 24], namely Amolops mantzorum (David, 1872) , Amolops granulosus (Liu and Hu, 1961) , Amolops loloensis (Liu, 1950) , Amolops lifanensis (Liu, 1945) , Amolops xinduqiao Fei, Ye, Wang, and Jiang, 2017 , Amolops jinjiangensis Su, Yang, and Li, 1986 , Amolops tuberodepressus Liu and Yang, 2000 , Amolops sangzhiensis Qian, Xiang, Jiang, Yang, and Gui, 2023 , Amolops shuichengicus Lyu and Wang, 2019 , Amolops ottorum Pham, Sung, Pham, Le, Zieger, and Nguyen, 2019 , and Amolops minutus Orlov and Ho, 2007 . Recently, A. xinduqiao was placed into synonymy of A. mantzorum as a subspecies . Thus, currently the A. mantzorum species group contains 10 species, of which two (A. ottorum and A. minutus) are only known from northwestern Vietnam and seven are known from southwestern China .
Yunnan is located in southwestern China and harbors a rich amphibian fauna in terms of species count and endemism. It has been known that there are four members of the A. mantzorum group in Yunnan, i.e., A. jinjiangensis, A. loloensis, A. mantzorum, and A. tuberodepressus . In recent years, a series of new species or new records of Amolops have been discovered intensively from southwestern China [2, 5, 7, 11, 18, 20, 22,23,24,25, 34,35,36], suggesting that species diversity of Amolops in the region still remains underestimated and probably more species would be found. During recent field surveys in central Yunnan, China, we collected seven specimens of an Amolops species that morphologically resemble some members of the A. mantzorum group in that they lack a circummarginal groove on tip of the first finger and have folds formed by incomplete series of glands along the dorsolateral junction of the body (hereafter dorsolateral glandular folds). Molecular and morphological comparison supported that these specimens differ from other members of the genus Amolops. Thus, we considered them to represent a new Amolops species.
Materials and methods
Specimens were collected at Mt. Ailao, Xinping County, Yunnan Province, China (Fig. 1) by Guohua Yu in May 2019 and July 2019, and by Shuo Liu in June 2022. Specimens were photographed, euthanized, fixed, and then stored in 75% ethanol. Liver tissues were preserved in 99% ethanol. Specimens were deposited at Guangxi Normal University (GXNU) and Kunming Institute of Zoology, Chinese Academy of Sciences (KIZ).
Morphometric data were taken using digital calipers to the nearest 0.1 mm. Morphological terminologies follow Fei et al. . Measurements included: snout-vent length (SVL, from tip of snout to vent); head length (HL, from tip of snout to rear of jaws); head width (HW, width of head at its 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); upper eyelid width (UEW, maximum width of upper eyelid); eye diameter (ED, diameter of exposed portion of eyeball); tympanum diameter (TD, the greater of tympanum vertical and horizontal diameters); forearm and hand length (FHL, from elbow to tip of third finger); thigh length (THL, from vent to knee); tibia length (TL, from knee to heel); foot length (FL, from proximal end of inner metatarsal tubercle to tip of fourth toe); length of foot and tarsus (TFL, from tibiotarsal joint to tip of fourth toe); and horizontal diameter of digital disc of third finger (F3DSC). Comparative morphological data of congeners were taken from their original descriptions or re-descriptions [2, 3, 6,7,8,9,10,11,12,13, 17,18,19,20, 22,23,24,25, 27,28,29,30,31,32, 34, 35, 37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59].
A multivariate principal component analysis (PCA) was conducted using SPSS 17.0 (SPSS Inc., USA) based on a correlation matrix of size-standardized measurements (all measurements divided by SVL). Scatter plots of the first two PCA factors were used to examine the differentiation between the new species and A. ottorum, which was recovered as the sister to the new species by phylogenetic analyses (see below). The measurements of A. ottorum were obtained from its original description .
Total genomic DNA was extracted from liver tissues. Tissue samples were digested using proteinase K, and subsequently purified following a standard phenol/chloroform isolation and ethanol precipitation. Fragments encoding partial 16S rRNA (16S), partial cytochrome oxidase subunit I (COI), and partial cytochrome b (cytb) genes were amplified using primer pairs L2188  /16H1 , Chmf4/Chmr4 , and F1/R3 , respectively. PCR amplifications were performed in 50 µl reactions using the following cycling conditions: an initial denaturing step at 95 °C for 4 min; 35 cycles of denaturing at 94 °C for 60s, annealing at 46, 49, or 51 °C for 60s (46 °C for COI, 49 °C for cytb, and 51 °C for 16S), and extending at 72 °C for 60s; and a final extending step of 72 °C for 10 min. Sequencing was conducted directly using the corresponding PCR primers. All new sequences have been deposited in GenBank under Accession Nos. MN650737–MN650749, MN650751‒MN650757, OP879227, OP880242, and OP887035 (Table 1). Homologous sequences of 25 congeners were obtained from GenBank. Two Odorrana species were included as outgroups according to Ngo et al.  and their sequences were also downloaded from GenBank.
Sequences were aligned using MUSCLE with the default parameters in MEGA 7 . Uncorrected pairwise distances between species were calculated in MEGA 7. Because sequences of the three genes are not all available for each species (Table 1), we prepared a combined dataset of the three genes for phylogenetic analyses. The best substitution model was selected using the Corrected Akaike Information Criterion (AICc) in jMODELTEST v. 2.1.6  via Cipres Science Gateway . Bayesian inference was performed in MRBAYES 3.2.6  based on the selected substitution model (GTR + I + G). Two runs were performed simultaneously with four Markov chains starting from random tree. The chains were run for 3,000,000 generations and sampled every 100 generations. Burn-in was checked using the program Tracer v.1.6 . The first 25% of the sampled trees were discarded as burn-in and then the remaining trees were used to create a consensus tree and to estimate Bayesian posterior probabilities (BPPs). In addition, a maximum likelihood (ML) analysis was conducted in RAXML-HPC v.8.2.12  with 1000 rapid bootstrap replicates.
We estimated the lineage divergence times using an uncorrelated lognormal relaxed molecular clock model in BEAST v. 1.8.0 . The birth-death process was chosen as the tree prior because of the mixed inter- and intraspecies sampling in the dataset sets . Recently, the crown age for Amolops was inferred to be 25.01 Ma (95% HPD: 21.31‒28.58) by Wu et al. . Therefore, the time of most recent common ancestor (TMRCA) of the genus Amolops (25.01 ± 2.2 Ma) was used as a secondary calibration point based on Wu et al. . A run of 20 million generations was conducted by sampling every 1000 generations. The effective sample sizes of values of parameters were examined using Tracer v.1.6 . The maximum clade credibility tree was constructed in TreeAnnotator v.1.8.0  and was visualized in FigTree v.1.4.0 (from http://tree.bio.ed.ac.uk/software/figtree).
Phylogeny and divergence time estimation
Alignments of 16S, COI, and cytb genes were 884 bp, 676 bp, and 998 bp in length, respectively. The newly collected individuals from Mt. Ailao, Yunnan, China represented a distinct lineage nested in the clade of members of the A. mantzorum group, and it was recovered as the sister taxon to A. ottorum with strong supports (Fig. 2). Genetic distances (p-distance) between this lineage and other species in the A. mantzorum group ranged from 1.9% (vs. A. sangzhiensis) to 8.3% (vs. A. lifanensis) in 16S (Table 2), from 4.0% (vs. A. mantzorum mantzorum) to 14.2% (vs. A. lifanensis) in COI (Table 2), and from 5.0% (vs. A. ottorum) to 14.4% (vs. A. lifanensis) in cytb (Table 3). The specimen IOZ4373, which was identified as A. jinjiangensis in Lu et al. , was not clustered together with topotypes of A. jinjiangensis (Fig. 2).
The dating analysis revealed that A. lifanensis split from all other members of the A. mantzorum species group ca. 7.59 Mya (95% HPD: 4.6‒10.67 Mya) and then the lineage divergence within the A. mantzorum species group mainly occurred from 4.23 Mya (95% HPD: 2.56‒6.1 Mya) to 1.2 Mya (95% HPD: 0.53‒1.98 Mya) (Fig. 3). The divergence between the novel lineage from Mt. Ailao and A. ottorum occurred 2.2 Mya (95% HPD: 1.1‒3.37 Mya).
Morphological measurements are given in Table 4. We retained the first two principal components, which had eigenvalues above 1.0 and accounted for 64.035% of total variance (Table 5). Loadings for PC1, which accounted for 34.26% of the total variance, were most heavily loaded on HW, UEW, ED, and THL (load factor > 0.7), and differentiation was found along the PC1 axis between the new species and A. ottorum (Fig. 4), indicating that the new species differs from A. ottorum by wider head, wider upper eyelid, larger eye, and longer femur. The second principal component (PC2) accounted for 29.78% of the total variance, but no clear separation was observed along this axis between the new species and A. ottorum. In addition, the new lineage can be distinguishable from its congeners by body size and the combination of texture and coloration pattern.
GXNU YU000004, an adult male, collected on 12 May 2019 by Guohua Yu from Mt. Ailao (23°56′58.20″N, 101°29′51.66″E, 2043 m above sea level; Fig. 1), Xinping County, Yunnan Province, China.
GXNU YU000001–YU000003, three adult males, collected at same time as the holotype from the type locality by Guohua Yu; GXNU YU20160273 and GXNU YU20160274, two adult males, collected from the type locality by Guohua Yu on 17 July 2017; and KIZ 2022041, an adult female, collected from the type locality by Shuo Liu on 22 June 2022.
The specific epithet is named for the type locality, Ailao Mt., Xinping County, Yunnan Province, China. We suggest the English common name “Ailao cascade frog” and the Chinese common name “Āi Láo Tūan Wā (哀牢湍蛙)”.
Morphologically, Amolops ailao sp. nov. resembles members of the A. mantzorum group in the absence of true dorsolateral folds and circummarginal groove on the disc of the first finger, and further resembles A. jinjiangensis and A. shuichengicus in the presence of folds formed by incomplete series of glands along the dorsolateral junction of the body (dorsolateral glandular folds). Phylogenetically, a clade consisting of the new species, A. mantzorum, A. sangzhiensis, A. jinjiangensis, A. granulosus, A. loloensis, A. tuberodepressus, A. shuichengicus, and A. ottorum was strongly supported (Clade I; Fig. 2). Amolops ailao sp. nov. can be distinguished from its congeners by the combination of the following characters: (1) body size small (SVL 33.0–35.1 mm in males and 41.3 mm in female); (2) HW/SVL 0.32‒0.35; UEW/SVL 0.08‒0.10; THL/SVL 0.52‒0.56; (3) vomerine teeth absent; (4) tympanum distinct, less than half eye diameter; (5) supratympanic fold present, indistinct; (6) true dorsolateral folds absent, but dorsolateral glandular folds distinct; (7) absence of circummarginal groove on the disc of the first finger; (8) tibiotarsal articulation reaching beyond anterior corner of eye; (9) dorsal surface smooth with no white spines; (10) a pair of large tubercles on sides of cloaca; 11) vocal sac absent; 12) toes fully webbed except the fourth; 13) interorbital space narrower than internarial space.
Description of holotype
Adult male (SVL 33.6 mm); head slightly longer (HL 11.0 mm) than wide (HW 10.8 mm); snout obtusely pointed, projecting beyond margin of lower jaw in ventral view, rounded in profile; canthus rostralis distinct, curved; loreal region sloping, concave; nostril oval, lateral, slightly protuberant; internarial distance (IND 3.7 mm) greater than interorbital distance (IOD 3.3 mm); upper eyelid width (UEW 3.0 mm) slightly narrower than interorbital distance; pineal spot present; pupil oval, horizontal; tympanum distinct, rounded, less than half eye diameter; supratympanic fold indistinct; vomerine teeth absent; choanae oval; tongue attached anteriorly, cordiform, notched posteriorly; vocal sac opening absent.
Forelimbs robust, relative length of fingers I < II < IV < III; tips of outer three fingers expanded into discs with circummarginal grooves, relative size of discs: 1 < 2 < 3 = 4; nuptial pads present on finger I; webbing between fingers absent; subarticular tubercles prominent and rounded, formula 1, 1, 2, 2; supernumerary tubercles present; thenar (inner metacarpal) tubercle oval; outer metacarpal tubercle single, rounded.
Hindlimbs long, heels overlapping when legs at right angle to body, tibiotarsal articulation reaching beyond anterior corner of eye; tibia length (TL 20.1 mm) longer than forearm and hand length (FHL17.6 mm), thigh length (THL 18.4 mm), and foot length (FL 19.8 mm); relative length of toes I < II < III < V < IV; all toe tips expanded into discs with circummarginal grooves; webbing between toes well developed, two third web, webbing formula I1–1.5II1–1.5III1–2IV2–1V; subarticular tubercles distinct, formula 1, 1, 2, 3, 2; inner metatarsal tubercle prominent, oval; outer metatarsal tubercle absent; supernumerary tubercles absent.
True dorsolateral folds absent, but folds formed by incomplete series of glands along dorsolateral junction of body (dorsolateral glandular folds) present, extending from rear of eye to groin; skin smooth, with a few flattened tubercles on flanks and dorsal surface of limbs; a few small tubercles on posterior surface of thigh and around vent; a pair of relatively large tubercles at the side of the anus; ventral surface smooth; a rictal gland.
Color of holotype
In life, iris light 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 short brown stripe below the green stripe on the loreal region; 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 a green blotch; flanks green with few back brown and light yellow spots, a golden brown patch below dorsolateral glands; rictal gland pale green; limbs dorsally golden brown with black brown bands; anterior and posterior of forelimb and thigh black brown, mottled with green and light green blotches; throat, chest, and venter creamy white, mottled with light green and marbled with gray on throat and chest; venter of limbs flesh-colored, scattered with light green spots; web orange yellow.
In preservative, color faded. Dorsal surface light brown with beige brown and gray blue spots on head and body and beige brown bands on limbs; ventral surface white, marbled with brown on throat and chest.
Body size of males smaller than that of female; nuptial pads present on the base of finger I in males.
Color of dorsal surfaces varied among specimens. Ground color of dorsal surfaces of the holotype and three paratypes (GXNU YU000002, GXNU YU000003, and KIZ2022041) is brown, and ground color of dorsal surfaces of remaining paratypes (GXNU YU20160273, GXNU YU20160274, and GXNU YU000001) is green. No large black brown spot on dorsum and flanks of GXNU YU20160273.
Distribution and ecology
The new species is only known from the type locality. It was found on leaves or small branches less than 2 m above the ground along a mountain stream at night (Fig. 9) from May to July. All male types have nuptial pads on first finger and the female paratype (KIZ 2022041) is pregnant with eggs, suggesting that the breeding season may be from May to July. No tadpoles were collected for the new species. Amolops tuberodepressus was also encountered during surveys at the type locality.
Within the A. mantzorum group, the new species can be distinguished from its sister taxon, A. ottorum, by smaller body size (female SVL 41.3 mm vs. 47.5‒48.2 mm in females), wider head (HW/SVL 0.32‒0.35 in the new species vs. HW/SVL 0.31 in A. ottorum), wider upper eyelid (UEW/SVL 0.08‒0.10 in the new species vs. 0.07 in A. ottorum), larger eye (ED/SVL 0.12‒0.14 vs. 0.12), and longer femur (THL/SVL 0.52‒0.56 vs. 0.49), the presence of dorsolateral glandular folds (versus absent), the presence of tubercles on flanks and limbs (versus absent), and the presence of a pair of large tubercles on sides of cloaca (versus absent). Amolops tuberodepressus also occurred at the type locality of the new species. Amolops ailao sp. nov. can be easily distinguished from A. tuberodepressus by smaller body size (males SVL 33.0–35.1 and female SVL 41.3 mm vs. males SVL 44.3–56.7 and females SVL 60.8–71.1 mm), vomerine teeth absent (vs. present), dorsolateral glandular folds present (versus absent), a pair of large tubercles on sides of cloaca (vs. absent), and tibiotarsal articulation reaching beyond anterior corner of eye (vs. tibiotarsal articulation reaching beyond tip of snout).
The new species is distinguishable from A. minutus by vomerine teeth absent (vs. strongly developed), vocal sac absent (vs. paired well-developed vocal sacs), and tympanum less than half eye diameter (vs. TD/ED mean 0.52 in males and mean 0.58 in females). The new species differs from the other seven members of the A. mantzorum species group (A. sangzhiensis, A. shuichengicus, A. mantzorum, A. granulosus, A. jinjiangensis, A. lifanensis, and A. loloensis) by vomerine teeth absent (vs. present) and smaller body size, males SVL 33.0–35.1 mm and female SVL 41.3 mm (vs. males SVL 40.3–40.9 mm and females SVL 52.6–57.7 mm in A. sangzhiensis, males SVL 34.6–39.6 mm and females SVL 48.5–55.5 mm in A. shuichengicus, males SVL 41.2–57.5 mm and females SVL 48.5–72.0 mm in A. mantzorum, males SVL 36.3–41.8 mm and female SVL 51.9 mm in A. granulosus, males SVL 43.0–52.0 mm and females SVL 54–66.4 mm in A. jinjiangensis, males SVL 52–56 mm and females SVL 61.0–79.0 in A. lifanensis, and males SVL 54.5–62.0 mm and females SVL 69.5–77.5 mm in A. loloensis).
Amolops ailao sp. nov. further differs from A. mantzorum, A. lifanensis, and A. loloensis by the presence of distinct dorsolateral glandular folds (vs. absent); from A. mantzorum, A. jinjiangensis, A. lifanensis, and A. loloensis by tympanum distinct (vs. obscure or invisible); and from A. granulosus by dorsal surface smooth with no white spines (vs. dorsal surface rough with spines) and vocal sac absent (vs. a pair of internal subgular vocal sacs).
Amolops ailao sp. nov. further differs from A. jinjiangensis by dorsal surfaces smooth (vs. skin coarse, with many rounded tubercles on head side, body side, and posterior part of dorsum); from A. lifanensis by toes fully webbed except the fourth (vs. webs fully developed to the bases of all toe disks); and from A. loloensis by interorbital space narrower than internarial space (vs. interorbital space about equal to the internarial space).
The A. monticola group contains 23 members, namely A. adicola Patel, Garg, Das, Stuart, and Biju, 2021 , A. akhaorum Stuart, Bain, Phimmachak, and Spence, 2010 , A. aniqiaoensis Dong, Rao, and Lü, 2005 , A. archotaphus (Inger and Chanard, 1997) , A. bellulus Liu, Yang, Ferraris, and Matsui, 2000 , A. chakrataensis Ray, 1992 , A. chaochin Jiang, Ren, Lyu, and Li, 2021 , A. chunganensis (Pope, 1929) , A. compotrix (Bain, Stuart, and Orlov, 2006) , A. cucae (Bain, Stuart, and Orlov, 2006) , A. daorum (Bain, Lathrop, Murphy, Orlov, and Ho, 2003) , A. deng Jiang, Wang, and Che, 2020 , A. iriodes (Bain and Nguyen, 2004) , A. kohimaensis Biju, Mahony, and Kamei, 2010 , A. mengdingensis Yu, Wu, and Yang, 2019 , A. mengyangensis Wu and Tian, 1995 , A. monticola (Anderson, 1871) , A. nyingchiensis Jiang, Wang, Xie, Jiang, and Che, 2016 , A. putaoensis Gan, Qin, Lwin, Li, Quan, Liu, and Yu, 2020 , A. truongi Pham, Pham, Ngo, Sung, Ziegler, and Le, 2023 , A. tuanjieensis Gan, Yu, and Wu, 2020 , A. vitreus (Bain, Stuart, and Orlov, 2006) , and A. wenshanensis Yuan, Jin, Li, Stuart, and Wu, 2018 . Amolops ailao sp. nov. can be distinguished from these species by absence of true dorsolateral folds (vs. present). The new species can be further distinguished from A. adicola, A. akhaorum, A. aniqiaoensis, A. archotaphus, A. chaochin, A. chunganensis, A. compotrix, A. cucae, A. daorum, A. iriodes, A. kohimaensis, A. mengdingensis, A. mengyangensis, A. monticola, A. putaoensis, A. truongi, A. tuanjieensis, A. vitreus, and A. wenshanensis by vocal sac absent (vs. present); and from A. adicola, A. akhaorum, A. aniqiaoensis, A. archotaphus, A. bellulus, A. chakrataensis, A. chaochin, A. chunganensis, A. compotrix, A. cucae, A. deng, A. iriodes, A. kohimaensis, A. mengdingensis, A. mengyangensis, A. nyingchiensis, A. putaoensis, A. truongi, A. tuanjieensis, A. vitreus, and A. wenshanensis by vomerine teeth absent (vs. present).
Amolops ailao sp. nov. is distinguishable from A. chayuensis Sun, Luo, Sun, and Zhang, 2013 , the sole member of the A. chayuensis group, by true dorsolateral folds absent (vs. present), vocal sacs absent (vs. present), and vomerine teeth absent (vs. present).
The A. viridimaculatus group contains 14 species based on recent taxonomic studies [11, 13, 57], namely A. beibengensis Jiang, Li, Zou, Yan, and Che, 2020 , A. chanakya Saikia, Laskar, Dinesh, Shabnam, and Sinha, 2022 , A. formosus (Günther, 1876) , A. himalayanus (Boulenger, 1888) , A. kaulbacki (Smith, 1940) , A. longimanus (Andersson, 1939) , A. medogensis Li and Rao, 2005 , A. nidorbellus Biju, Mahony, and Kamei, 2010 , A. pallasitatus Qi, Zhou, Lyu, Lu, and Li, 2019 , A. senchalensis Chanda, 1987 “1986” , A. tawang Saikia, Laskar, Dinesh, Shabnam, and Sinha, 2022 , A. wangyali Mahony, Nidup, Streicher, Teeling, and Kamei, 2022 , A. wangyufani Jiang, 2020 , and A. viridimaculatus (Jiang, 1983) . The new species can be distinguished from these species by vomerine teeth absent (vs. present), glands in compete series along dorsolateral junction present (vs. absent), and smaller body size (vs. male SVL 75.8 mm and females SVL 90.2–93.2 mm in A. beibengensis, male SVL 76.4 mm in A. chanakya, males SVL 61.3–63.1 mm and females SVL 79.4–83.7 mm in A. formosus, male SVL 80 mm in A. himalayanus, males SVL 70–72 mm in A. kaulbacki, male SVL ca. 95 mm and females SVL72.4–96.9 mm in A. medogensis, males SVL 76.4–82.3 mm and females SVL 85.4–98 mm in A. nidorbellus, female SVL 70.6–72.3 mm in A. pallasitatus, male SVL 46.2 mm in A. senchalensis, male SVL 82.5 mm in A. tawang, males SVL 71.4–76.7 mm and females SVL 80.5–89.6 mm in A. wangyali, males SVL 68.3–69.0 mm and female SVL 83.4 mm in A. wangyufani, males SVL 72.7–82.3 mm and female SVL 83.0–94.3 mm in A. viridimaculatus).
The Amolops marmoratus group contains 13 species, namely A. afghanus (Günther, 1858) , A. assamensis Sengupta, Hussain, Choudhury, Gogoi, Ahmed, and Choudhury, 2008 , A. gerbillus (Annandale, 1912) , A. indoburmanensis Dever, Fuiten, Konu, and Wilkinson, 2012 , A. jaunsari Ray, 1992 , A. latopalmatus (Boulenger, 1882) , A. mahabharatensis Khatiwada, Shu, Wang, Zhao, Xie, and Jiang, 2020 , A. marmoratus (Blyth, 1855) , A. nepalicus Yang, 1991 , A. panhai Matsui and Nabhitabhata, 2006 , A. siju Saikia, Sinha, Shabnam, and Dinesh, 2023 , A. terraorchis Saikia, Sinha, Laskar, Shabnam, and Dinesh, 2022 , and A. yarlungzangbo Jiang, Wang, Li, Qi, Li, and Che, 2020 . The new species differs from these species by circummarginal groove on disc of finger I absent (vs. present), vomerine teeth absent (vs. present), and vocal sac absent (vs. present with the exception of A. siju).
The new species differs from A. spinapectoralis Inger, Orlov, and Darevsky, 1999 , the sole member of the A. spinapectoralis group, by vomerine teeth absent (vs. present), circummarginal groove on disc of finger I absent (vs. present), and vocal sac absent (vs. present).
The A. larutensis group contains four species, namely A. australis Chan, Abraham, Grismer, and Grismer, 2018 , A. cremnobatus Inger and Kottelat, 1998 , A. gerutu Chan, Abraham, Grismer, and Grismer, 2018 , and A. larutensis (Boulenger, 1899) . The new species can be distinguished from these species by vomerine teeth absent (vs. present), circummarginal groove on disc of finger I absent (vs. present), and vocal sac absent (vs. present).
The A. ricketti group contains eight species, namely A. shihaitaoi Wang, Li, Du, Hou, and Yu, 2022 , A. sinensis Lyu, Wang, and Wang, 2019 , A. ricketti (Boulenger, 1899) , A. wuyiensis (Liu and Hu, 1975) , A. yunkaiensis Lyu, Wang, Liu, Zeng, and Wang, 2018 , A. albispinus Sung, Wang, and Wang, 2016 , A. yatseni Lyu, Wang, and Wang, 2019 , and A. tonkinensis (Ahl, 1927 “1926’’) . The new species differs from these species by circummarginal groove on disc of finger I absent (vs. present), dorsolateral glandular folds present (vs. absent), and nuptial pad without conical or papillate nuptial spines (vs. present).
The A. daiyunensis group contains three species, namely A. daiyunensis (Liu and Hu, 1975) , A. hongkongensis (Pope and Romer, 1951) , and A. teochew Zeng, Wang, Lyu, and Wang, 2021 . The new species differs from them by circummarginal groove on disc of finger I absent (vs. present), dorsolateral glandular folds present (vs. absent), and vocal sac absent (vs. present).
The A. hainanensis group contains two members, namely A. hainanensis (Boulenger, 1900)  and A. torrentis (Smith, 1923) . The new species can be distinguished from them by dorsolateral glandular folds absent (vs. absent), circummarginal groove on disc of finger absent (vs. present), and nuptial pad present in males (vs. absent).
The new species is distinguishable from A. binchachaensis Rao, Hui, Ma, and Zhu, 2022 “2020” , which has not been assigned to any species group, by true dorsolateral folds absent (vs. present) and circummarginal groove on disc of finger I absent (vs. present).
Based on molecular and morphological evidence, we find a novel lineage belonging to the A. mantzorum species group from central Yunnan, China and describe it as a new species. This finding brings the number of species of the A. mantzorum group to 11. The phylogenetic relationships within the clade consisting of all members of the A. mantzorum group with the exception of A. lifanensis (labelled as clade I) were not well resolved and most basal branches in this clade are short (Fig. 2), suggesting that this group might have undergone a rapid speciation process. This inference was supported by the analysis of divergence dating, which revealed that the cladogenesis events within the A. mantzorum group mainly occurred from Pliocene 4.23 Mya (95% HPD: 2.56‒6.1 Mya) to Pleistocene 1.2 Mya (95% HPD: 0.53‒1.98 Mya) (Fig. 3). It is generally believed that the southeastern margin of the Qinghai-Tibet Plateau has experienced rapid and recent uplift since the Pliocene [93,94,95] and there were two phases of recent intense uplift occurring between 0.6 and 3.4 Mya , which caused dramatic habitat and climatic changes (e.g., reorganization of drainage  and formation of fluvial system ) and created environmental conditions (new habitats, dispersal barriers, etc.) that increase the rate at which species divide and evolve to form new ones . Additionally, so far A. minutus has never been included in phylogenetic analysis. Thus, more studies are necessary to unveil the phylogenetic relationships within the A. mantzorum species group.
The phylogenetic position of A. jinjiangensis in previous studies is controversial. Lu et al.  found that A. jinjiangensis is closely related to A. mantzorum, but recently Lyu et al.  and Wang et al.  revealed that A. jinjiangensis is the sister taxon to A. loloensis. The samples of A. jinjiangensis in Lu et al.  came from Zhongdian in Yunnan and Yajiang in Sichuan, while samples of A. jinjiangensis in Lyu et al.  and Wang et al.  contained topotypes from Deqin, Yunnan. In this study, with inclusion of samples from both Zhongdian and the type locality (Deqin, Yunnan), we found that A. jinjiangensis contains two clades. The clade containing topotypes was recovered as the sister to A. loloensis, whereas the clade containing the sample from Zhongdian was nested in the clade of A. mantzorum (Fig. 2), indicating that the samples from Zhongdian in Lu et al.  actually do not belong to A. jinjiangensis but belong to A. mantzorum. Amolops mantzorum is comprised of four sub-lineages and the central lineage refers to A. mantzorum mantzorum according to Frost . Thus, additional studies are needed to name the northern lineage and the lineage containing samples from Zhongdian in Yunnan.
Thanks to the extremely complicated topography and climatic condition in Yunnan, which promoted rapid divergence and speciation in small and isolated populations , Yunnan is the region with highest amphibian species diversity in China . Of the 649 amphibian species known from China, about one-third (220 species belonging to 51 genus) are distributed in Yunnan (Fig. 10). Combining findings in this study with most recent taxonomic progress [5, 13, 35, 36], we consider that so far there are 20 Amolops species known from Yunnan, accounting for the highest proportion of amphibian diversity of Yunnan (ca. 9.1%; Fig. 11) and including five members of the A. mantzorum group (namely, A. ailao sp. nov., A. jinjiangensis, A. loloensis, A. tuberodepressus, and A. mantzorum), one member of the A. marmoratus group (namely A. afghanus), eight members of the A. monticola group (namely A. bellulus, A. daorum, A. deng, A. iriodes, A. mengdingensis, A. mengyangensis, A. putaoensis, A. tuanjieensis, and A. wenshanensis), two members of the A. viridimaculatus group (namely A. viridimaculatus, A. kaulbacki), one member of the A. ricketti group (namely A. shihaitaoi), one member of the A. chayuensis group (A. chayuensis), and A. binchachaensis, which has not yet been assigned to any species group but likely belongs to the A. monticola group owing to the fact that it has true dorsolateral folds and head side dark with white upper lip stripe. Among different parts of Yunnan, the species diversity of Amolops in northwestern Yunnan was the highest (eight species, namely A. bellulus, A. binchachaensis, A. chayuensis, A. deng, A. jinjiangensis, A. kaubacki, A. mantzorum, A. putaoensis), followed by western Yunnan (six species, namely A. afghanus, A. bellus, A. mengdingensis, A. tuanjieensis, A. tuberodepressus, and A. viridimaculatus), central Yunnan (four species, namely A. daorum, A. tuberdepressus, A. viridimaculatus, and A. ailao sp. nov.), and southeastern Yunnan (three species, namely A. iriodes, A. shihaitaoi, and A. wenshanensis) in order, while southern and northeastern Yunnan have only one species each (Fig. 12). Accordingly, species diversity of Amolops in the Nu River Basin is highest (seven species, namely A. bellulus, A. binchachaensis, A. chayuensis, A. deng, A. kaulbacki, A. tuberodepressus, and A. viridimaculatus), followed by the Lancang River Basin (six species, namely A. daorum, A. mengdingensis, A. mengyangensis, A. tuanjieensis, A. tuberodepressus, and A. viridimaculatus), the Red River Basin (five species, namely A. iriodes, A. shihaitaoi, A. tuberodepressus, A. wenshanensis, and A. ailao sp. nov.), the Dulong River Basin (three species, namely A. chayuensis, A. kaulbacki, and A. putaoensis) and the Jinsha River Basin (three species, namely A. jinjiangensis, A. loloensis, and A. mantzorum), while there are two species (A. afghanus and A. viridimaculatus) in the Basins of the Ying River and the Ruili River (Fig. 12), both of which flow to the Irrawaddy River. Amolops ailao sp. nov. is sympatric with A. tuberodepressus at the type locality, but it is easy to distinguish them because the new species has smaller body size and dorsolateral glandular folds and lacks vomerine teeth. Amolops mantzorum was widely recorded from central, southwestern, and northwestern Yunnan [15, 17, 101]. Certainly some of these records actually apply to A. tuberodepressus or A. jinjiangensis because they were once placed into the synonymy of A. mantzorum by Fei et al. [17, 101]. Studies based on additional sampling will be necessary to clarify the species boundary within the A. mantzorum species group in Yunnan, China.
In summary, based on morphological and molecular evidence, we revealed a new cascade frog species belonging to the A. mantzorum species group. The new species Amolops ailao sp. nov. is only found in Mt. Ailao, central Yunnan, China and has considerable variation of color pattern. Including A. ailao sp. nov., now there are 20 Amolops species known from Yunnan, China and five of them belong to the A. mantzorum species group. We also revealed that the samples of A. jinjiangensis in Lu et al.  are misidentification of A. mantzorum. The A. mantzorum species group has undergone a rapid speciation process since the Pliocene, coinciding with the recent rapid uplift of the Qinghai-Tibetan Plateau since the Pliocene. Among different subfauna and water systems in Yunnan, northwestern Yunnan and Nu River Basin harbor the highest species diversity of Amolops. The findings in this study improve our understanding of the species diversity of the genus Amolops. More studies are necessary to unveil the phylogenetic relationships and species boundaries within the A. mantzorum species group.
Availability of data and materials
All data generated or analyzed during this study are included in this published article. Sequences are deposited in GenBank, NCBI.
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We are grateful to Jian Zang and Chunsheng Du for assistance during the surveys.
This work was supported by the National Natural Science Foundation of China (32060114, 31872212), Guangxi Natural Science Foundation Project (2022GXNSFAA035526), and Key Laboratory of Ecology of Rare and Endangered Species and Environmental Protection (Guangxi Normal University), Ministry of Education (ERESEP2022Z04), and Guangxi Key Laboratory of Rare and Endangered Animal Ecology, Guangxi Normal University (19-A-01-06).
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Tang, S., Sun, T., Liu, S. et al. A new species of cascade frog (Anura: Ranidae: Amolops) from central Yunnan, China. Zoological Lett 9, 15 (2023). https://doi.org/10.1186/s40851-023-00214-9
- Amolops mantzourm
- Species group
- The Qinghai-Tibetan Plateau
- 16S rRNA