Description of two cryptic species of the Amolopsricketti group (Anura, Ranidae) from southeastern China

Abstract Two cryptic species, which were previously reported as Amolopsricketti, are revealed on the basis of significant morphological and genetic divergences. Amolopssinensissp. n. from central Guangdong, northeastern Guangxi and southwestern Hunan can be distinguished by the longitudinal glandular folds on the skin of the shoulders and other character combinations. Amolopsyatsenisp. n. from the coastal hills of west Guangdong can be distinguished by the dense tiny round translucent, or white, spines on the dorsal skin of the body, dorsal and dorsolateral skin of the limbs, and other character combinations. The phylogenetic interrelationships of the A.ricketti group have been inferred as (A.wuyiensis + A.ricketti) + (A.yunkaiensis + (A.albispinus + (A.sinensissp. n. + A.yatsenisp. n.))). This work indicates that the current records of A.ricketti might be a species complex composed of multiple species, and further work is needed to figure out this puzzle.


Introduction
The torrent frog genus Amolops Cope, 1865, which is comprised of 55 species, is widespread from the southern and eastern Himalayas, eastward to southeastern mainland China, and southward to Malay Peninsula (Frost 2018). Of these, 28 species from China were assigned into six species groups (Fei et al. 2009(Fei et al. , 2017Sun et al. 2013;Lu et al. 2014;Jiang et al. 2016;Sung et al. 2016;Yuan et al. 2018;Lyu et al. 2018). Among them, the A. ricketti group is a monophyletic species group containing four recognized species from southeastern China (Fei et al. 2012;Sung et al. 2016;Lyu et al. 2018): A. yunkaiensis Lyu, Wang, Liu, Zeng & Wang, 2018 from southwestern Guangdong, A. albispinus Sung, Wang & Wang, 2016 from Shenzhen City, Guangdong, A. wuyiensis (Liu & Hu, 1975) distributed in Fujian, Zhejiang, Anhui and Jiangxi in eastern China, and A. ricketti (Boulenger, 1899) reported to be widely distributed in Sichuan, Chongqing, Yunnan, Guizhou, Hubei, Hunan, Jiangxi, Fujian, Guangdong and Guangxi in southern China and even to northern and central Indochina. It is worth noting that the species A. albispinus and A. yunkaiensis were recognized as other known species for a long time and recognition as new species represented the beginning of uncovering the cryptic diversity within the A. ricketti group (Sung et al. 2016;Lyu et al. 2018).
During our herpetological surveys in Guangdong, Guangxi and Hunan provinces in southeastern China, we have collected a series of Amolops specimens which were recorded as A. ricketti (Fei et al. 2009(Fei et al. , 2012Li et al. 2011). However, morphological examinations indicated that these specimens belong to two different undescribed species that can be markedly and reliably distinguished from all congeners, especially from specimens of A. ricketti collected from the type locality, Mt. Wuyi, Fujian. Molecular analyses further well supported the morphological result, showing that these specimens formed two lineages within the A. ricketti group; in this study we describe them as two new species of genus Amolops.

Sampling
A total of 28 muscle samples of the two new species were collected for molecular analyses, encompassing six from Zhongshan City, Guangdong, two from Shangchuan Island, Guangdong, two from Mt. Gudou, Guangdong, three from Ehuangzhang Nature Reserve, Guangdong, two from Yunkaishan Nature Reserve, Guangdong, seven from Shimentai Nature Reserve, Guangdong, two from Mt. Nankun, Guangdong, two from Mt. Dupangling, Guangxi, one from Mt. Yangming, Hunan, and one from Mt. Hengshan, Hunan. In addition, 36 samples from nine known species of the genus Amolops, namely A. albispinus, A. ricketti, A. wuyiensis, A. yunkaiensis, A. daiyunensis (Liu & Hu, 1975), A. hongkongensis (Pope & Romer, 1951), A. hainanensis (Boulenger, 1900), A. torrentis (Smith, 1923) and A. chunganensis (Pope, 1929), were also collected and incorporated into our dataset. All muscle samples were attained from euthanasia specimens and then preserved in 95% ethanol and stored at -40 °C. Detail information for these materials is shown in Table 1 and Figure 1.

Phylogenetic analyses
DNA sequences were aligned by the Clustal W algorithm with default parameters (Thompson et al. 1997) and trimmed with the gaps partially deleted in MEGA 6 (Tamura et al. 2013), while within highly variable regions, all gaps were removed. Two gene segments, 637 base pairs (bp) of CO1 and 1032 bp of 16S, were concatenated seriatim into a 1669-bp sequence, and further divided into two partitions based upon each gene. Two partitions were tested respectively in jmodeltest v2.1.2 (Darriba et al. 2012) with Akaike and Bayesian information criteria, all resulting in the best-fitting nucleotide substitution models of GTR + I + G. Sequenced data were analyzed using Bayesian inference (BI) in MrBayes 3.2.4 (Ronquist et al. 2012) and maximum likelihood (ML) in RaxmlGUI 1.3 (Silvestro and Michalak 2012). Two independent runs were conducted in a BI analysis, each of which was performed for 2,000,000 generations and sampled every 1000 generations with the first 25% samples discarded as burn-in, resulting a potential scale reduction factor (PSRF) of < 0.01. In ML analysis, the bootstrap consensus tree inferred from 1000 replicates was used to represent the evolutionary history of the taxa analyzed. Pairwise distances (p-distance) were calculated in MEGA 6 using the uncorrected p-distance model.

Morphology
We obtained diagnostic characters of known species of the genus Amolops from the literature for comparison. In addition, a total of 67 museum specimens of A. ricketti group were examined for comparison, which are listed in Appendix 1. Measurements follow Fei et al. (2009) and Lyu et al. (2018), and were taken with digital calipers (Neiko 01407A Stainless Steel 6-Inch Digital Caliper, USA) to the nearest 0.1 mm. These measurements are as follows: SVL snout-vent length (from tip of snout to posterior margin of vent); HDL head length (from tip of snout to the articulation of the jaw); HDW head width (head width at the commissure of the jaws); SNT snout length (from tip of snout to the anterior corner of the eye); IND internasal distance (distance between nares); IOD interorbital distance (minimum distance between upper eyelids); ED eye diameter (from the anterior corner of the eye to posterior corner of the eye); TD tympanum diameter (horizontal diameter of tympanum); TED tympanum-eye distance (from anterior edge of tympanum to posterior corner of the eye); HND hand length (from distal end of radioulna to tip of distal phalanx III); RAD radioulna length (from the flexed elbow to the base of the outer palmar tubercle); FTL foot length (from distal end of tibia to tip of distal phalanx IV); TIB tibial length (from the outer surface of the flexed knee to the heel); F3W width of digital disc on finger III; T4W width of digital disc on toe IV. Sex was determined by observation of secondary sexual characters, i.e. the presence of nuptial spines in males, following Fei et al. (2009) andLyu et al. (2018).
All specimens were fixed in 10% buffered formalin and later transferred to 70% ethanol, and deposited in The Museum of Biology, Sun Yat-sen University (SYS) and Chengdu Institute of Biology, the Chinese Academy of Sciences (CIB), P.R. China.

Results
The phylogenetic trees strongly supported that the Amolops ricketti group was a monophyletic species group, which can be further divided into six well-supported clades with marked divergences ( Fig. 2; Table 2). The four known species of this group, A. wuyiensis, A. ricketti, A. yunkaiensis and A. albispinus, formed the four basal clades respectively. The unnamed specimens from Shimentai Nature Reserve and Mt. Nankun in Guangdong, Mt. Dupangling in Guangxi, and Mt. Yangming and Mt. Hengshan in Hunan clustered into a clade (clade V) with highly supported node values (BS = 100, BPP = 1.00) and small divergences (p-distance 0.0-0.6); the speci- mens from Zhongshan City, Mt. Gudou, Shangchuan Island, Ehuangzhang Nature Reserve and Yunkaishan Nature Reserve clustered into a clade (clade VI) with highly supported node values (BS = 100, BPP = 1.00) and small divergences (p-distance 0.0-0.8). These two clades are sister taxa to each other with significant divergences (p-distance 3.5-4.2).
Morphologically, the specimens clustered into clade V from central Guangdong, northeastern Guangxi and southwestern Hunan can be distinguished from all known Amolops species by having unique longitudinal glandular folds on the skin of the shoulders and other characters (see diagnosis below). Therefore this clade represents a distinct evolutionary lineage, and is described below as a new species, Amolops sinensis sp. n. The specimens grouped in clade VI from the coastal hills of west Guangdong differ from all known Amolops species by having unique dense Table 2. 0.0 tiny round translucent, or white, spines on the dorsal skin of the body, dorsal and dorsolateral skin of the limbs and other characters (see diagnosis below). Therefore, this clade represents a distinct evolutionary lineage, and is described below as a new species Amolops yatseni sp. n. Diagnosis. The new species was assigned to genus Amolops and further to the A. ricketti group morphologically based on the absence of dorsolateral folds, the presence of a circummarginal groove on the disk of the first finger, the absence of tarsal glands, and the presence of nuptial pads with conical nuptial spines on the first finger in males.

Amolops sinensis
Amolops sinensis sp. n. is distinguished from its congeners by a combination of the following morphological characteristics: (1) body stout and robust, SVL 40.2-46.5 (43.1±2.2, n=6) mm in adult males, 47.7-52.7 (50.5±2.0, n=5) mm in adult females; (2) dorsal body olive-brown to dark brown, with irregular light strip-shaped patches or not; (3) ventral surface creamy white or beige, with dark gray patches; (4) dorsal skin of body very rough, granular and scattered with conical tubercles and raised large warts in males; (5) vomerine teeth strong, tongue cordiform, deeply notched posteriorly; (6) absence of dorsolateral folds; (7) a longitudinal glandular fold on skin of shoulder on each side; (8) supernumerary tubercles below base of fingers III and IV indistinct; (9) heels overlapping; (10) absence of outer metatarsal tubercles and tarsal glands; (11) absence of vocal sacs; (12) nuptial pad on first finger prominent with beige spines in breeding males; and (13) white conical spines present on skin of temporal region (including tympanum in several individuals) and loreal region in breeding males.
Description of holotype. Body stout, SVL 43.3 mm. Head width slightly smaller than head length (HDW/HDL = 1.04); snout short (SNT/HDL = 0.45) and rounded in profile, projecting beyond lower jaw; nostril closer to tip of snout than eye; loreal region concave; top of head flat; eye large (ED/HDL = 0.32) and convex; canthus rostralis distinct; pineal body distinct; tympanum small, edge distinct; tympanumeye distance smaller than tympanum, TED/TD 0.90; supratympanic fold distinct, start from back of eye and extending to shoulder, a well-developed gland on end of supratympanic fold; choanae moderate; vomerine teeth present; tongue cordiform, deeply notched posteriorly.
Forelimbs moderately robust; hands moderately long (HND/SVL = 0.33); relative finger lengths I = II < IV < III; finger tips dilated to wide oval disks with circummarginal grooves, relative width of finger disks I < II < III = IV; subarticular tubercles prominent, rounded; supernumerary tubercles below base of fingers III and IV indistinct, below base of fingers I and II absent; inner metacarpal tubercle small, outer metacarpal tubercle prominent and slightly separated; absence of webbing and presence of weak lateral fringes on fingers.
Hindlimbs long and robust (TIB/SVL = 0.60); tibio-tarsal articulation reaching tip of snout when hindlimb stretched alongside of body; relative toe lengths I < II < III = V < IV; tips of all toes expanded to well-developed oval discs with circummarginal grooves; subarticular tubercles oval and distinct; inner metatarsal tubercle prominent, elongated; outer metatarsal tubercles absent; toes fully webbed; lateral fringes of toes I and V developed; tarsal glands absent; heels overlapping when hindlimbs flexed at right angles to axis of body.
Dorsal skin of body rough, granular and scattered with raised tubercles and warts; underdeveloped conical spines on skin of loreal region and temporal region except tympanum; flanks very rough and granular with raised warts; dorsal limbs rough with numerous tubercles; several longitudinal dermal ridges on dorsal surfaces of thigh, tibia and tarsus; dorsolateral fold absent; a longitudinal glandular fold on skin of shoulder; posterior part of upper lip swollen; rictal gland prominent and ellipsoidal, posterior to corner of mouth.
Ventral surface slightly wrinkled with granules; ventral surface of hand smooth, ventral surface of foot granular; large warts surrounding cloaca.
Color of holotype in life. Dorsal body olive-brown with irregular light yellow patches; longitudinal glands on occipital region light yellow; warts on flanks dark or grayish white; irregular dark patches on dorsal surface of forearms, distinct dark transverse bars on dorsal surface of lower arms and hindlimbs; dorsal discs of digits brown or white; nuptial pads and nuptial spines beige; posterior edge of upper lip and rictal gland light maize-yellow; throat and chest creamy white; belly beige; several dark gray mottling on surface of throat, chest and anterior part of abdomen; ventral surfaces of limbs gray pink grounding; ventral hands and feet dark grey; warts around cloaca yellowish white tubercles and olive-brown.
Color of holotype in preservative. Dorsal surface dark brown with irregular gray patches, transverse bars on limbs more distinct; longitudinal glands on occipital region more distinct; webs on toes gray, mottling with olive-brown; ventral surface grayish white, mottling on surface of throat, chest and anterior part of abdomen become more distinct; ventral surface of limbs beige. Variations. Measurements of type series specimens are given in Table 3. All specimens are very similar in morphology except that: dorsal skin dark brown without any patterns in SYS a007109 (Fig. 4B); skin of tympanum with white conical spines in SYS a005710 (Fig. 4C); nuptial spines are conical in SYS a005710; tibia-tarsal articulation reaching forward to the loreal region in SYS a004257, 5712, 7109 and 7268.
Sexual dimorphism. Amolops sinensis sp. n. possesses distinct sexual dimorphism: (1) larger body size in females with SVL 47.7-52.7 mm (vs. SVL 40.2-46.5 mm in males); (2) beige nuptial spines on beige nuptial pads in breeding males; (3) dense white conical spines present on skin of temporal region and loreal region in males during breeding season (vs. absent in females); and (4) females bearing light yellow oocytes.
Comparisons. The character of longitudinal glandular folds on skin of shoulders makes Amolops sinensis sp. n. unique when compared with all known congeners within the genus. The new species is further compared with the four recognized species of the A. ricketti species group below (Fig. 5).
Amolops sinensis sp. n. was previously reported as A. ricketti, but significantly differs from the topotype of A. ricketti by the presence of longitudinal glandular folds on the skin of the shoulders (vs. absent), large raised warts on the dorsal surface of body (vs. relatively smooth), the presence of white conical spines on skin of temporal region and loreal region in breeding males (vs. absent), and nuptial pad and nuptial spines beige (vs. white).
Amolops sinensis sp. n. is phylogenetically close to A. albispinus, but can be distinguished from the later by the presence of longitudinal glandular folds on the skin of the shoulders (vs. absent), the presence of supernumerary tubercles below the base of fingers III and IV (vs. absent), outer metacarpal tubercle slightly separated (vs. completely divided into two tubercles), pineal body distinct (vs. indistinct), ventral surface smooth (vs. presence of tiny, transparent and dispersive conical spines on surface of chest in males), and nuptial spines beige (vs. white).
Amolops sinensis sp. n. can be easily distinguished from A. wuyiensis by the presence of longitudinal glandular folds on skin of shoulders (vs. absent), vomerine teeth present (vs. absent), lacking vocal sacs (vs. present), and nuptial spines beige (vs. black).
Amolops sinensis sp. n. further differs from A. yunkaiensis by the presence of longitudinal glandular folds on the skin of the shoulders (vs. absent), larger body size, SVL 40.2-46.5 mm in adult males and 47.7-52.7 mm in adult females (vs. SVL 31.8-34.1 mm in males and 35.2-39.0 mm in females), vomerine teeth present (vs. absent), lacking vocal sacs (vs. present), and ventral surface smooth (vs. presence of tiny transparent spines on surface of chest).
Etymology. The specific name "sinensis" refers to "Chinese", for this new species takes a wide distribution in southern China. We suggest its English common name "Chinese Torrent Frog" and Chinese name "Zhong Hua Tuan Wa (中华湍蛙)".
Distribution and habits. Currently, the Chinese Torrent Frog is recognized from the Shimentai Nature Reserve and Mt. Nankun in Guangdong, Mt. Dupangling in Guangxi, and Mt. Yangming and Mt. Hengshan in Hunan, which indicates the potential distribution area of Amolops sinensis sp. n. is from central Guangdong, to northeastern Guangxi and southwestern Hunan.
Amolops sinensis sp. n. inhabits rocky, fast-flowing streams (ca 500-1300 m a.s.l.) surrounded by moist subtropical secondary evergreen broadleaved forests. All individuals were observed from April to August. Males bear nuptial spines from April to July; females bear mature light yellow oocytes from April to August. Nevertheless, much of the ecology and behavior of this species remains unknown. Holotype. SYS a006807 (Fig. 6)  Diagnosis. The new species was assigned to genus Amolops and further to the A. ricketti group morphologically based on the absence of dorsolateral folds, the presence of a circummarginal groove on the disk of the first finger, the absence of tarsal glands, and the presence of nuptial pads with conical nuptial spines on the first finger in males.

Amolops yatseni
Amolops yatseni sp. n. is distinguished from its congeners by a combination of the following morphological characteristics: (1) body stout and robust, SVL 39.3-44.7 (42.5±2.1, n=6) mm in adult males, 42.1-48.9 (46.4±2.0, n=11) mm in adult females; (2) dorsal body olive-brown or light brown, with irregular light strip-shaped patches or not; (3) ventral surface creamy white, with nebulous dark gray patches or not; (4) dorsal skin of body very rough, granular and scattered with tubercles and raised large warts, lacking warts on central back of trunk in females; (5) dense tiny round translucent, or white, spines present on dorsal skin of body, dorsal and dorsolateral skin of limbs in males, denser in females; (6) vomerine teeth strong, tongue cordiform, deeply notched posteriorly; (7) absence of the dorsolateral folds; (8) supernumerary tubercles below the base of fingers II, III and IV distinct and prominent; (9) heels just meeting; (10) absence of outer metatarsal tubercles and tarsal glands; (11) absence of vocal sacs; (12) nuptial pad on the first finger prominent with developed white conical spines in breeding males, tip of nuptial spines brown; and (13) dense white conical spines present on the skin of the temporal region (including the tympanum in several individuals), loreal region, snout, lips and chin in males during breeding season, and such spines less developed and rounded only on skin of temporal region except the tympanum and lower lips in females.
Description of holotype. Body stout, SVL 41.0 mm. Head width slightly smaller than head length (HDW/HDL = 1.05); snout short (SNT/HDL = 0.38) and rounded in profile, projecting beyond lower jaw; nostril closer to tip of snout than eye; loreal region concave; top of head flat; eye large (ED/HDL = 0.31) and convex; canthus rostralis distinct; pineal body distinct; tympanum small, edge faintly distinct, upper margin of tympanum in contact with supratympanic fold; tympanum-eye distance larger than tympanum, TED/TD 1.08; supratympanic fold distinct, start from back of eye and extending to shoulder; choanae moderate; vomerine teeth present; tongue cordiform, deeply notched posteriorly.
Forelimbs moderately robust; hands moderately long (HND/SVL = 0.31); relative finger lengths I < II < IV < III; finger tips dilated to wide oval disks with circummarginal grooves, relative width of finger disks I < II < III = IV; subarticular tubercles prominent, rounded; supernumerary tubercles below the base of fingers II, III and IV distinct and prominent, below base of fingers I absent; inner metacarpal tubercle elongated and prominent, outer metacarpal tubercle prominent and slightly separated; absence of webbing and presence of weak lateral fringes on fingers.
Hindlimbs long and robust (TIB/SVL = 0.55) ; tibio-tarsal articulation reaching tip of snout when hindlimb stretched alongside of body; relative toe lengths I < II < III = V < IV; tips of all toes expanded to well-developed oval discs with circummarginal grooves; subarticular tubercles oval and distinct; inner metatarsal tubercle prominent, elongated; outer metatarsal tubercles absent; toes fully webbed; lateral fringes of toes I and V developed; tarsal glands absent; heels just meeting when hindlimbs flexed at right angles to axis of body.
Dorsal skin of body very rough, granular and scattered with raised large warts; dense rounded spines present on dorsal body, dorsal limbs, and many well developed and denser ones on sacral region; dense conical spines present on skin of temporal region except tympanum, loreal region, snout, lips and chin, conical spines on skin of lower lips much smaller; flanks very rough and granular with raised warts; dorsal limbs rough with numerous tubercles; several longitudinal dermal ridges on dorsal surfaces of thigh, tibia and tarsus; dorsolateral fold absent; posterior part of upper lip swollen; rictal gland prominent and ellipsoidal, posterior to corner of mouth.
Ventral surface slightly wrinkled with round spines on chest; ventral surface of hand and foot granular; large warts surrounding the vent.
Color of holotype in life. Dorsal body dark green; faint dark transverse bars on dorsal surface of limbs; dorsal discs of digits yellowish brown; posterior edge of upper lip and rictal gland light maize-yellow; all round spines and conical spines on skin grayish white; throat, chest, and belly creamy white; several dark gray nebulous mottling on surface of throat, chest and anterior part of abdomen; ventral surfaces of limbs gray pink grounding; creamy white blotches on ventral thighs; rear of thighs mottled with dark brown; ventral hands and feet dark grey; yellowish white tubercles and olivebrown warts around cloaca.
Color of holotype in preservative. Dorsal surface dark brown, irregular light strip-shaped patches present, transverse bars indistinct; ventral surface grayish white, mottling on surface of throat, chest and anterior part of abdomen become more distinct; ventral surface of limbs light brown.
Variations. Measurements of type series specimens are given in Table 3. All specimens are very similar in morphology except that: dorsal skin without any patterns in the specimens from Zhongshan City (vs. dorsal skin with irregular light strip-shaped patches in the remaining specimens); skin of tympanum with white conical spines in SYS a004643 (Fig. 7A) and 4676; tibia-tarsal articulation reaching anterior corner of eye in SYS a003633,3634,3678,3680,4994,6806 and 6809).
Sexual dimorphism. Amolops yatseni sp. n. possesses significantly-distinct sexual dimorphism: (1) larger body size in females with SVL 42.  in males); (2) white nuptial spines with brown tips on white nuptial pads in breeding males; (3) rounded spines on dorsal skin denser and more distinct in females (Fig. 7B); (4) skin of central back bearing raised large warts in males (vs. such warts absent in females); (5) dense white conical spines on skin of temporal region (including the tympanum in several individuals), loreal region, snout, lips and chin in males during breeding season (vs. spines underdeveloped and rounded only on skin of temporal region and lower lips in females); and (6) females bearing light yellow oocytes.
Comparisons. The dense tiny round translucent, or white, spines on dorsal skin of body, dorsal and dorsolateral skin of the limbs makes Amolops yatseni sp. n. unique when compared with all known congeners within the genus. Amolops yatseni sp. n. is further compared with Amolops sinensis sp. n. and other four recognized species within the A. ricketti species group below (Fig. 5).
Amolops yatseni sp. n. is a sister taxon to A. sinensis sp. n. in our phylogenetic trees (Fig. 2), and differs from the later by a significant genetic divergence of 3.5-4.2%. Mor- 6.0-7.5 (6.6±0.6) 6.6-7.9 (7.1±0.5) 5.6-7.4 (6.4±0.6) 6.2-7.2 (6.7±0.4) IND 5.3-6.9 (5.9±0.5) 5.6-7.4 (6.6±0.8) 5.7-6.4 (6.1±0.3) 5.7-6.6 (6.3±0.3) IOD 4.  phologically, A. yatseni sp. n. differs from A. sinensis sp. n. by the presence of dense tiny round translucent, or white, spines on the dorsal skin of the body, dorsal and dorsolateral skin of limbs (vs. absent), the presence of rounded spines on the skin of the temporal region and lower-lips in females (vs. absent), the absence of longitudinal glandular folds on the skin of the shoulders (vs. present), supernumerary tubercles below the base of fingers II, III and IV distinct and prominent (vs. indistinct below the base of fingers III and IV, absent below the base of finger II), and heels just meeting (vs. overlapping). Amolops yatseni sp. n. was previously reported as A. ricketti, but significantly differs from the topotype A. ricketti by the presence of dense tiny round translucent or white spines on the dorsal skin of the body, dorsal and dorsolateral skin of the limbs (vs. absent), large raised warts on dorsal surface of body (vs. relatively smooth), supernumerary tubercles below the base of fingers II, III and IV distinct and prominent (vs. indistinct below the base of fingers III and IV, absent below the base of finger II), and the presence of white conical spines on skin of temporal region and loreal region in breeding males (vs. absent).
Amolops yatseni sp. n. differs from A. albispinus by the presence of dense tiny round translucent, or white, spines on the dorsal skin of the body, dorsal and dorsolateral skin of the limbs (vs. absent), pineal body distinct (vs. indistinct), the presence of conical spines on skin of the tympanum (vs. absent), the presence of rounded spines on skin of temporal region and lower-lips in females (vs. absent), the presence of supernumerary tubercles below the base of fingers II, III and IV (vs. absent), and ventral surface smooth (vs. presence of tiny, transparent and dispersive conical spines on surface of chest in males).
Amolops yatseni sp. n. can be easily distinguished from A. wuyiensis by the presence of dense tiny round translucent or white spines on dorsal skin of body, dorsal and dorsolateral skin of limbs (vs. absent), vomerine teeth present (vs. absent), lacking vocal sacs (vs. present), and conical nuptial spines white (vs. black). Amolops yatseni sp. n. further differs from A. yunkaiensis by the presence of dense tiny round translucent or white spines on dorsal skin of body, dorsal and dorsolateral skin of limbs (vs. absent), a significantly larger body size, SVL 39.3-44.7 mm in adult males and 42.1-48.9 mm in adult females (vs. SVL 31.8-34.1 mm in males and 35.2-39.0 mm in females), vomerine teeth present (vs. absent), lacking vocal sacs (vs. present), and ventral surface smooth (vs. presence of tiny transparent spines on surface of chest).
Etymology. The specific name "yatseni" refers to the founder of Sun Yat-sen University, Dr. Sun Yat-sen, who was born in Cuiheng Village, Zhongshan City, about five kilometers from the type locality, Mt. Wugui. We suggest its English common name "Yat-sen's Torrent Frog" and Chinese name "Yi Xian Tuan Wa (逸仙湍蛙)".
Distribution and habits. Currently, the Yat-sen's Torrent Frog is known from the Zhongshan City, as well as from Mt. Gudou, Shangchuan Island, Ehuangzhang Nature Reserve, and Yunkaishan Nature Reserve. All these localities are situated in the coastal hills of west Guangdong, indicating the potential distribution area of Amolops yatseni sp. n. is from the west border of Pearl River Delta to the Yunkai Mountains. However, the five known localities of the new species are being threatened by hydropower station construction and tourism development respectively, and surveys are needed in western Guangdong to investigate the accurate population status and the distribution of this species.
Amolops yatseni sp. n. inhabits rocky, fast-flowing streams (ca 250-1000 m a.s.l.) surrounded by moist subtropical secondary evergreen broadleaved forests (Fig. 7C). All individuals were observed from March to August when males bear nuptial spines and females bear mature oocytes. Nevertheless, much of the ecology and behavior of this species remains unknown.

Discussion
The species Amolops ricketti was originally described based on two specimens from Mt. Wuyi, Fujian (Boulenger 1899), and was recorded subsequently over wide area from southern China to northern and central Indochina (Bourret 1942;Liu 1950;Fei et al. 2012;Frost 2018). In this work, we have found that the recorded population of A. ricketti from central Guangdong, northeastern Guangxi and southwestern Hunan (now recognized as A. sinensis sp. n.) and from coastal hills of west Guangdong (now recognized as A. yatseni sp. n.), are markedly different from the topotype of A. ricketti from Fujian, both morphologically and genetically. This indicates that the current records of A. ricketti might be a species complex (designated here as A. ricketti sensu lato) composed of multiple species. Further surveys and studies are required to clarify the concept of A. ricketti, especially for the reported populations from southwestern China and Indochina and to determine the accurate distribution of A. sinensis sp. n. and A. yatseni sp. n. Southwestern China has been considered as hotspot area with highest species diversity over time, while southeastern China, which suffers from more human activities, is considered as much less diverse, which may reflect the lack of biodiversity surveys over time. Recently, a number of new amphibian species were described from southeastern China Wang et al. 2017;Yuan et al. 2017;Zeng et al. 2017;Lyu et al. 2018;Wang et al. 2018a;Wang et al. 2018b;this study), to be the greatest number of new amphibian species in China in recent times. These discoveries indicate that the species diversity in southeastern China is highly underestimated. Comprehensive and careful surveys are urgently demanded to investigate the biodiversity status in this area, especially for herpetological species which are sensitive to rapid environmental changes.