Revisiting the Species Delimitation Within Amolops mantzorum (David, 1872), with a Description of a New Subspecies (Anura, Ranidae)

TL;DR

This study identifies a new subspecies of the Sichuan torrent frog, clarifying its taxonomy and emphasizing the importance of accurate species delimitation for conservation.

Contribution

The discovery and description of a new subspecies of Amolops mantzorum, supported by mitochondrial phylogeny and morphological analysis.

Findings

A new subspecies, Amolops mantzorum feiye, was identified based on mitochondrial and morphological data.

Four distinct lineages within A. mantzorum were clearly divided for the first time using phylogenetic analysis.

Morphometric differences confirmed the uniqueness of the Yalong River population compared to existing subspecies.

Abstract

Currently, controversy about the intraspecific taxonomy of Amolops mantzorum remains unresolved. The Litang population was overlooked for several years in recent phylogenetic studies. We revisited this population and described the third subspecies of A. mantzorum, clearly dividing the four recognized lineages within A. mantzorum for the first time based on mitochondrial phylogeny. Our findings highlighted the importance of clear delimitation and naming of subspecies for biological conservation and taxonomic description, and provided insights for future investigations into the taxonomy of the Amolops mantzorum species group.

Accurate species delimitation benefits detailed biological descriptions and conservation practices. In this study, we focus on a frog species distributed throughout the western edge of Sichuan Basin, China, the Sichuan torrent frog—Amolops mantzorum. Currently, A. mantzorum is recognized as comprising two subspecies—the nominate subspecies A. m. mantzorum and A. m. xinduqiao. We investigated a population from along the Yalong River, Sichuan Province, China which was previously overlooked in phylogenetic studies. Combining multiple mitochondrial genes (16S, CO1, cytb), we identified four distinct lineages within A. mantzorum using Bayesian inference and maximum likelihood phylogeny. Principal Component Analysis based on morphometrics revealed significant differences between the population from along the Yalong River and the other two subspecies for which morphological data were available. Combined with detailed morphological descriptions and comparisons, here we describe this population as the third subspecies of A. mantzorum—Amolops mantzorum feiye ssp. nov.

Figures

(A) Geographical locations of the type localities of species within the A. mantzorum group. (B) Distribution of specimens (or sequences) used in this study of A. m. ssp., A. m. xinduqiao, A. m. mantzorum, and “A. m ssp. (northern lineage)”. The numbers (B) beside the distribution points correspond to the IDs in Table 1.

Tables

Table 1. Sequences used in this study.

ID	Species	Voucher Number	Locality	16S	COI	Cytb
1	Amolops mantzorum ssp.	CIB QZ2021130	Bajiaolou Village, Bajiaolou Town, Yajiang, Sichuan	PX599057	PX599084	PX620422
2	A. m. ssp.	CIB QZ2021126	Bajiaolou Village, Bajiaolou Town, Yajiang, Sichuan	PX599068	PX599095	PX620433
3	A. m. ssp.	CIB QZ2021131	Bajiaolou Village, Bajiaolou Town, Yajiang, Sichuan	PX599075	PX599102	PX620438
4	A. m. ssp.	CIB QZ2021128	Bajiaolou Village, Bajiaolou Town, Yajiang, Sichuan	PX599077	PX599104	PX620440
5	A. m. ssp.	CIB QZ2021127	Bajiaolou Village, Bajiaolou Town, Yajiang, Sichuan	PX599079	PX599106	PX620442
6	A. m. ssp.	CIB YJ2019080602	Bajiaolou Village, Bajiaolou Town, Yajiang, Sichuan	PX599080	PX599107	PX620443
7	A. m. ssp.	CIB YJ2019080601	Bajiaolou Village, Bajiaolou Town, Yajiang, Sichuan	PX599081	PX599108	PX620444
8	A. m. ssp.	CIB YJ2019080603	Bajiaolou Village, Bajiaolou Town, Yajiang, Sichuan	PX599082	PX599109	PX620445
9	A. m. ssp.	CIB YJ202012	Nimazong Village, Zhusang Town, Yajiang, Sichuan	PX599083	PX599110	PX620446
10	A. m. ssp.	CIB YJ202011	Nimazong Village, Zhusang Town, Yajiang, Sichuan	PX599058	PX599085	PX620423
11	A. m. ssp.	CIB YJ202013	Nimazong Village, Zhusang Town, Yajiang, Sichuan	PX599059	PX599086	PX620424
12	A. m. ssp.	CIB LT20200712-4	Dewu Town, Litang, Sichuan	PX599060	PX599087	PX620425
13	A. m. ssp.	CIB LT20200712-5	Dewu Town, Litang, Sichuan	PX599061	PX599088	PX620426
14	A. m. ssp.	CIB LT20200712-6	Dewu Town, Litang, Sichuan	PX599062	PX599089	PX620427
15	A. m. ssp.	CIB LT20200712-8	Dewu Town, Litang, Sichuan	PX599063	PX599090	PX620428
16	A. m. ssp.	CIB DC20190905-89	Rihuo Village, Shengmu Town, Daocheng, Sichuan	PX599064	PX599091	PX620429
17	A. m. ssp.	CIB DC20190905-88	Rihuo Village, Shengmu Town, Daocheng, Sichuan	PX599065	PX599092	PX620430
18	A. m. ssp.	CIB DC20190905-91	Rihuo Village, Shengmu Town, Daocheng, Sichuan	PX599066	PX599093	PX620431
19	A. m. ssp.	CIB DC20190906-94	Maize Village, Mula Town, Daocheng, Sichuan	PX599067	PX599094	PX620432
20	A. m. ssp.	CIB DC20190906-95	Maize Village, Mula Town, Daocheng, Sichuan	PX599069	PX599096	PX620434
21	A. m. ssp.	CIB 2019ml0002	Xianggelila Village, Shuiluo Town, Muli, Sichuan	PX599070	PX599097	PX620435
22	A. m. ssp.	CIB 2019ml0001	Xianggelila Village, Shuiluo Town, Muli, Sichuan	PX599071	PX599098	PX620436
23	A. m. xinduqiao	CIB YJ202014	Wangxia Village, Bajiaolou Town, Yajiang, Sichuan	PX599072	PX599099	PX620437
24	A. m. xinduqiao	KIZ 041127	Xinduqiao Town, Kangding, Sichuan	MN953764	MN961465	-
25	A. m. xinduqiao	KIZ 041129	Xinduqiao Town, Kangding, Sichuan	MN953765	MN961466	-
26	A. m. xinduqiao	CIB GGS-PBX4-2	Geridi Village, Jiagenba Town, Kangding, Sichuan	PX599073	PX599100	-
27	A. m. xinduqiao	CIB GGS-SDX1-6	Laha Village, Shade Town, Kangding, Sichuan	PX599074	PX599101	-
28	A. m. xinduqiao	700307	Yajiang, Sichuan	-	-	KJ008410
29	A. m. xinduqiao	LCLH017	Luhuo, Sichuan	-	-	KJ008392
30	A. m. mantzorum	SYS a005360	Kangding, Sichuan	MK573807	MK568322	-
31	A. m. mantzorum	SYS a005336	Mt. Wawu, Hongya, Sichuan	MK573804	MK568319	-
32	A. m. mantzorum	SYS a005337	Mt. Wawu, Hongya, Sichuan	MK604853	MK605611	-
33	A. m. mantzorum	700114	Longdong Town, Baoxing, Sichuan	-	-	KJ008297
34	A. m. mantzorum	CIB 2020061501	Dongsheng Village, Wulong Town, Baoxing, Sichuan	PX599076	PX599103	PX620439
35	A. m. mantzorum	CIB 2020061502	Dongsheng Village, Wulong Town, Baoxing, Sichuan	PX599078	PX599105	PX620441
36	A. m. mantzorum	SYS a005370	Fengtongzhai, Baoxing, Sichuan	MK604865	MK605623	-
37	A. m. mantzorum	SYS a005365	Fengtongzhai, Baoxing, Sichuan	MK573808	MK568323	-
38	A. m. mantzorum	SCUM045825HX	Mt. Xiling Snow, Dayi, Sichuan	MN953707	MN961409	-
39	A. m. mantzorum	-	Mt. Xiling Snow, Dayi, Sichuan	KJ546429	KJ546429	KJ546429
40	A. m. mantzorum	700229	Chongzhou, Sichuan	-	-	KJ008339
41	A. m. mantzorum	SCUM045817HX	Wolong, Wenchuan, Sichuan	MN953706	MN961408	-
42	A. m. ssp. (northern lineage)	700040	Maoxian, Sichuan	-	-	KJ008277
43	A. m. ssp. (northern lineage)	700267	Wenxian, Gansu	-	-	KJ008360
44	A. tuberodepressus	SYS a003932	Mt. Wuliang, Jingdong, Yunnan	MK573800	MG991934	-
45	A. tuberodepressus	CIB-XM3125	Jingdong, Yunnan	KR559270	KR559270	KR559270
46	A. ailao	GXNU YU000004	Mt. Ailao, Xinping, Yunnan	MN650754	MN650740	MN650746
47	A. shuichengicus	SYS a004956	Shuicheng, Guizhou	MK604845	MK605603	-
48	A. granulosus	SCUM060911HX	Anxian, Sichuan	MN953681	MN961381	-
49	A. granulosus	20130258	Mt. Wawu, Sichuan, China	MH922934	MH922934	MH922934
50	A. loloensis	SYS a005346	Zhaojue, Sichuan	MK604854	MK605612	-
51	A. loloensis	SM-ZDTW-01	Shimian, Sichuan	KT750963	KT750963	KT750963
52	A. jinjiangensis	SYS a004571	Mt. Gaoligong, Yunnan	MK573801	MK568316	-
53	A. jinjiangensis	SCUM050435CHX	Deqing, Yunnan	EF453741	MN961403	-
54	A. jinjiangensis	KIZ047095	Chuxiong, Yunnan	MN953701	MN961404	-
55	A. sangzhiensis	CSUFT 907	Mt. Doupeng, Sangzhi, Hunan	OQ079540	OQ078905	-
56	A. chayuensis	SYS a007509	Baxoi, Xizang	MK573820	MK568333	-
57	A. lifanensis	SYS a005374	Lixian, Sichuan	MK573809	MK568324	-
58	A. dafangensis dafangensis	MT DF20230601002	Dafang, Guizhou	OR936315	OR924345	-
59	A. d. dafangensis	MT DF20230601001	Dafang, Guizhou	OR936314	OR924344	-
60	A. d. wumengmontis	SWU 0009561	Mt. Wumeng, Zhaotong, Yunnan, China	PX411479	PX410826	-
61	A. d. wumengmontis	SWU 0009562	Mt. Wumeng, Zhaotong, Yunnan, China	PX411478	PX410827	-
62	A. minutus	IEBR 4342	Muong La, Son La, Vietnam	-	-	MK941135
63	A. minutus	TBU 06	Muong La, Son La, Vietnam	-	-	MK941136
64	A. chunganensis	SYS a004212	Mt. Jinggang, Jinggang, Jiangxi	MK263263	MG991914	-
65	A. nyingchiensis	SYS a006679	Lhunze, Xizang	MK573814	MK568329	-
66	A. cremnobatus	ROM 14528	Khe Moi, Nghe An, Vietnam	DQ204477	-	-
67	A. daiyunensis	SYS a001739	Mt. Daiyun, Fujian, China	MK263243	KX507328	-
68	A. hainanensis	SYS a005281	Mt. Wuzhi, Hainan, China	MK263281	MG991916	-
69	A. ricketti	WUSTW01	Mt. Wugong, Jiangxi, China	KF956111	KF956111	KF956111
70	A. marmoratus	KUHE 19089	Chiang Mai, Thailand	AB211486	-	AB259738
71	A. spinapectoralis	ROM 37375	Ngoc Linh vicinity, Kon Tum, Vietnam	MN953726	MN961428	-
72	A. cremnobatus	KIZ 011621	Puhu National Reserve, Thanh Hoa, Vietnam	MN953672	MN961368	-
73	A. wangyufani	KIZ 014067	Zayü, Tibet, China	MN953740	MN961440	-
74	A. cuongi	IEBR A.5140	Tam Duong, Lai Chau, Vietnam	PX113529	-	PX119668
75	A. cuongi	IEBR A.5141	Tam Duong, Lai Chau, Vietnam	PX113530	-	PX119669
76	Odorrana wuchuanensis	LBML 5230	Libo, Guizhou, China	KU680791	KU680791	KU680791
77	Odorrana margaretae	HNNU 1207003	-	KJ815050	KJ815050	KJ815050

Full text

1. Introduction

The intraspecific taxonomy of Amolops mantzorum has been debated for several decades. Cai & Zhao [1] identified four genetic populations within the “A. mantzorum & A. kangtingensis complex” based on genetic distance. Two of these, exhibiting large morphometric divergence, are currently recognized as subspecies of A. mantzorum. Amolops m. xinduqiao (A. kangtingensis Kangding population sensu Cai & Zhao [1]) represents the lineage with smaller body size and a high-altitude distribution range (above 3000 m), distributed along the western slopes of Mt. Zheduo in the Yalong River Basin, whereas A. m. mantzorum (A. mantzorum Hongya and Dayi population sensu Cai & Zhao [1]) comprises larger-bodied, mid-to-high elevation (1200–2400 m) populations from the Dadu River Basin [2]. The two subspecies are also distinguished by different dorsal coloration patterns [2]. However, the coloration patterns can vary within a species and are thus insufficient for taxonomic diagnosis in the A. mantzorum group under current views. Given the small genetic divergence revealed by molecular phylogeny (i.e., Wu et al. [3]; Zeng et al. [4]), the subspecies-level classification proposed by Dufresnes & Litvinchuk [5] has been accepted in recent taxonomic systems (e.g., Tang et al. [6]; Frost [7]; Li et al. [8]).

Obviously, subspecies delimitation within A. mantzorum remains unresolved. Unfortunately, the CO1 and Cytb sequences used by Cai & Zhao [1] were not available in their original publication. However, some evidence could be traced. Lu et al. [9] recognized an undescribed lineage from Maoxian, Sichuan Province and Wenxian, Gansu Province, designating it as “A. mantzorum (northern lineage)”. This population is geographically close to the Lixian and Wolong populations of A. mantzorum in Cai & Zhao [1]. However, the Litang population—one of the four distinct genetic populations identified by Cai & Zhao [1]—was overlooked for several years in molecular phylogeny studies [3,4,5,10].

In this study, we revisited the Litang population of A. mantzorum, combining specimens collected from multiple localities along the Yalong River, Sichuan Province, China. Molecular phylogeny based on mitochondrial genes suggests that this population represents a monophyletic lineage. A comprehensive morphological examination was also conducted among the recognized subspecies of A. mantzorum. Based on this evidence, we describe this previously overlooked lineage as a new subspecies of A. mantzorum.

2. Material and Methods

2.1. Samples

From 2019 to 2021, 20 specimens and 2 tissue samples of the putative new subspecies were collected from Litang County, Yajiang County, Muli County, and Daocheng County in Sichuan Province (Figure 1). Additionally, 3 specimens of A. m. xinduqiao were collected from Yajiang County and Kangding City in Sichuan Province, and 14 specimens of A. m. mantzorum were collected from Baoxing County, Shimian County, Luding County and Kangding City in Sichuan Province. From the collected specimens and samples, we extracted tissues from 22 samples of the putative new subspecies, 2 of A. m. mantzorum, and 3 of A. m. xinduqiao. Muscle and/or liver tissues were preserved in 95% ethanol for molecular analyses. Specimens were fixed in formaldehyde solution and then transferred to 75% ethanol for long-term storage. All specimens were deposited in the Chengdu Institute of Biology (CIB) of Chinese Academy of Sciences (CAS). Museum specimens were also examined for morphological analyses (detailed in Appendix A).

(A) Geographical locations of the type localities of species within the A. mantzorum group. (B) Distribution of specimens (or sequences) used in this study of A. m. ssp., A. m. xinduqiao, A. m. mantzorum, and “A. m ssp. (northern lineage)”. The numbers (B) beside the distribution points correspond to the IDs in Table 1.

2.2. Molecular Analysis

We extracted genomic DNA from muscle or liver tissues using a DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd., Beijing, China). Segments of three mitochondrial genes, 16S ribosomal RNA (16S), cytochrome C oxidase subunit I (COI), and partial cytochrome b (Cytb) were amplified. The 16S primers were L3975 and H4551 [11], and the COI primers were Chmf4 and Chmr4 [12], and Cytb primers were Cytb-L and Cytb-H [13]. Amplification of the 16S, COI, and Cytb fragments was performed in a 25 μL volume reaction: initial denaturation at 94 °C for 5 min (95 °C for Cytb); 34 cycles (31 cycles for Cytb): denaturation for 30 s at 94 °C (95 °C for Cytb), annealing for 30 s at 55 °C (40 s for Cytb), extending for 30 s at 72 °C (1 min for Cytb); a final extension: 7 min at 72 °C (10 min for Cytb). Sequencing was conducted using a 3730xl DNA sequencer by Beijing Tsingke Biotech Co., Ltd. (Beijing, China).

In addition to the 27 sequences newly obtained in this study, 50 sequences were retrieved from GenBank, including two samples of “A. mantzorum (northern lineage)” from Lu et al. [9], currently recognized as “A. m. ssp. (northern lineage)” by Tang et al. [6]. The species Odorrana wuchuanensis and O. margaretae were selected as outgroups following Tang et al. [6]. Sequences of the species in the A. mantorum group were selected mostly following Lyu et al. [14] and supplemented with newly described species and subspecies. To reconstruct more stable trees, we also included one or two sequences from each species group of Amolops. In total, 77 sequences were used for phylogenetic analysis (Table 1).

The MAFFT algorithm [15] with its default parameters in MAFFT online service (https://mafft.cbrc.jp/alignment/server/index.html [accessed on 17 December 2025]) [16] was used to align the 16S (440 bp), COI (562 bp), and Cytb (756 bp) sequences, respectively, and AMAS [17] was used to concatenate the 16S, COI, and Cytb sequences of each specimen. Maximum-likelihood (ML) analysis was conducted using IQ-TREE [18,19]. The support values of the phylogenetic tree were assessed using 1000 bootstrap replicates (bootstrap support value, BSV). Bayesian inference (BI) was performed in MRBAYES 3.2.1 [20] based on the best partition scheme and substitution models: GTR + G for 16S, CO1 first codon, and Cytb third codon, HKY + I for COI second codon and Cytb first codon; GTR + I + G for CO1 third codon and Cytb second Codon, which were selected using ModelFinder [21] with Bayesian information criterion (BIC). Two independent runs were conducted during the BI analyses with 10,000,000 generations each, sampled every 1000 generations, with the first 25% of samples discarded as burn-in. We used a convergence diagnostic threshold of stopval = 0.01 to automatically terminate the MCMC run once convergence was achieved.

2.3. Morphological Analysis

Morphological characters and measurements followed Fei et al. [22], Jiang et al. [23], and Lyu et al. [14]. In total, 13 measurements were taken using slide calipers to the nearest 0.1 mm: (1) SVL, snout-vent length, from tip of snout to posterior margin of vent; (2) HL, head length, from tip of snout to the articulation of the jaw; (3) HW, head width, at the commissure of the jaws; (4) SL, snout length, from tip of snout to the anterior corner of the eye; (5) INS, internasal space, the distance between the nostrils; (6) IOS, interorbital space, the shortest distance between the upper eyelids; (7) UEW, upper eyelid width, maximum width of upper eyelid; (8) ED, eye diameter, horizontal distance from the anterior corner to the posterior corner of the eye; (9) LAHL, length of forearm and hand, from the tip of digit III to elbow joint; (10) HAL, hand length, from the proximal border of the outer palmar tubercle to the tip of digit III; (11) TL, tibia length, from the outer surface of the flexed knee to the heel; (12) TFL, length of tarsus and foot, from proximal end of tarsus to tip of toe IV; (13) FL, foot length, from proximal end of inner metatarsal tubercle to tip of toe IV.

Morphometric analyses were conducted for the putative new subspecies, A. m. xinduqiao and A. m. mantzorum. We measured specimens for numerical taxonomic analyses, including 10 adult males of the putative new subspecies, 10 adult males of A. m. mantzorum, and 15 adult males of A. m. xinduqiao. To avoid errors due to different measurement personnel, all 11 measurements were conducted by Y.-J.G. To reduce the impact of allometry, the logarithm (base 10) of the ratio of each measured value to SVL was used in the analyses. Mann–Whitney U test in SPSS (statistics 25.0) (In the “Two Independent Samples” option of the “Nonparametric Tests” option of the software, we ticked the “Mann–Whitney U” option box) was used to test for significant differences between the three subspecies of A. mantzorum, with significance level set at p < 0.05 (significant) and p < 0.01 (highly significant). Principal component analysis (PCA) in Origin (Pro) (Version 2024) (the “Correlation Matrix” option box in the “Analyze” option was ticked) was used to assess whether the subspecies were separated in morphometric space.

For morphological comparisons, we directly examined specimens of six species within the Amolops mantzorum group (detailed in Appendix A). For the remaining 12 species for which specimens were not examined, morphological data were obtained from the literature.

3. Results

3.1. Molecular Phylogeny

The reconstructed phylogenies are presented in Figure 2. Both the ML and BI trees show relatively weak support values for several major clades of previously identified species groups (e.g., A. lifanensis of A. mantrorum group clustered out of its clade), indicating that the complex evolutionary history of the A. mantzorum group remains unresolved. A total of 22 newly collected samples (A. mantzorum ssp., the new lineage) from along the Yalong River clustered together as one lineage with well support (BSV 99, BPP 1.00). Samples of A. m. xinduqiao, A. m. mantzorum, and “A. m. ssp. (northern lineage)” each formed distinct monophyletic lineages: the A. m. xinduqiao lineage with strong support in both the ML and BI tree (BSV 99, BPP 1.00), the A. m. mantzorum lineage with strong support in both the ML and BI trees (BSV 99, BPP 0.99), and the “A. m. ssp. (northern lineage)” with strong support in both the BI and ML trees (BSV 100, BPP 1.00) (Figure 2). These four monophyletic lineages formed a clade with strong support in both the ML and BI trees (BSV 100), BPP 1.00) (Figure 2).

3.2. Morphological Comparisons Within Subspecies of A. mantzorum

Comparisons among the new lineage from along the Yalong River, A. m. xinduqiao, and A. m. mantzorum are presented herein. Morphological data on “A. m. ssp. (northern lineage)” are missing.

The new lineage can be distinguished from A. m. xinduqiao by the absence of a tympanum (vs. small but distinct) (Figure 3), distinct and strong vomerine teeth (vs. small, two tiny rows) (Figure 4), and larger body size in adult females (SVL 56.7–63.7 mm [n = 10] vs. 48.5–56.6 mm [n = 15]) (Table 2); and from A. m. mantzorum by the absence of a tympanum (vs. small but distinct) (Figure 3), and smaller body size in adult males (SVL 42.3–51.2 mm [n = 10] vs. SVL 51.4–57.8 mm [n = 10]) (Table 2).

Amolops m. xinduqiao is distinguished from A. m. mantzorum by the smaller body size in adult males (SVL 40.3–46.7 mm [n = 15] vs. 51.4–57.8 mm [n = 10]) and adult females (SVL 48.5–56.6 mm [n = 15] vs. 59.0–72.0 mm [n = 10]) (Table 2).

3.3. Morphometric Analysis

Based on the measurements of 13 characters of the three lineages (the new lineage, A. m. xinduqiao, and A. m. mantzorum) (Table 3), the results of comparisons among them (Table 4) showed that: (1) two characters were significantly different between the new lineage and A. m. xinduqiao: SVL and SL/SVL; four characters were highly significantly different between them*:* UEW/SVL, ED/SVL, HAL/SVL, and TFL/SVL; (2) three characters were significantly different between the new lineage and A. m. mantzorum: HW/SVL, INS/SVL, and IOS/SVL, and four characters were highly significantly different between them: SVL, HL/SVL, SL/SVL, and TL/SVL; (3) one character was significantly different between A. m. xinduqiao and A. m. mantzorum: TL/SVL, and ten characters were highly significantly different between them*:* SVL, HL/SVL, HW/SVL, SL/SVL, INS/SVL, IOS/SVL, UEW/SVL, ED/SVL, HAL/SVL, and TFL/SVL.

In the PCA of males, the eigenvalues of the first three principal components (PC1, PC2, and PC3) were more than 1.0, and their total variation accounted for 69.5% (Table 5); therefore, the first three PCs could be used for elucidating their taxonomic significance. In the score plot of PCA (Figure 5), the plot of PC1 plus PC2 showed that the new lineage was almost separated from A. m. xinduqiao and A. m. mantzorum, though with some overlap, while the two lineages A. m. xinduqiao and A. m. mantzorum were well-separated. These differences were largely contributed by PC1 (Figure 5), to which SL and ED had higher contributions (Table 5). The plot of PC1 versus PC3 showed that the three lineages were distinctly separated from each other. These differences were contributed by combinations of PC1 and PC3 (Figure 5), of which HAL and FL had higher contributions to PC3 (Table 5). These results demonstrate that the three lineages exhibit distinct morphological differentiation.

3.4. Comparisons Between the New Lineage and Other Species Within the A. mantzorum Group

The morphological differences within the A. mantzorum group are summarized in Table 2. The new lineage can be clearly distinguished from A. tuberodepressus, A. granulosus, A. ailao, A. minutus, and A. dafangensis by the absence of a tympanum; differs from A. tuberodepressus by head width slightly larger than head length (vs. head slightly longer than broad); differs from A. granulosus by head width slightly larger than the head length (vs. head length slightly larger than head width), and the absence of vocal sacs (vs. present); differs from A. ailao by larger body size, SVL 42.3–51.2 mm in adult males and 56.7–63.7 mm in adult females (vs. 33.0–35.1 mm in males and 41.3 mm in female), head width slightly larger than head length (vs. head slightly longer than wide), and distinct and strong vomerine teeth (vs. absent); differs from A. minutus by larger body size, SVL 42.3–51.2 mm in adult males and 56.7–63.7 mm in adult females (vs. 29.70–36.42 mm in males and 38.47–50.22 mm in females), head width slightly larger than head length (vs. head length larger than head width), and the absence of dorsolateral folds (vs. very poorly developed); differs from A. shuichengicus by head width slightly larger than head length (vs. head length slightly larger than head width), and the absence of dorsolateral folds (vs. present); differs from A. jinjiangensis by head width slightly larger than head length (vs. head length slightly larger than head width), and the absence of dorsolateral folds (vs. present); differs from A. loloensis by smaller body size, SVL 42.3–51.2 mm in adult males and 56.7–63.7 mm in adult females (vs. 54.5–62.0 mm in males and 69.5–77.5 mm in females), head width slightly larger than head length (vs. head length almost equal to head width), distinct and strong vomerine teeth (vs. absent), and distinct and blunt canthus rostralis (vs. indistinct); differs from A. sangzhiensis by head width slightly larger than head length (vs. head length about equal to or larger than head width); differs from A. lifanensis by smaller body size, SVL 42.3–51.2 mm in adult males and 56.7–63.7 mm in adult females (vs. 52.0–56.0 mm in males and 61.0–79.0 mm in females), head width slightly larger than head length (vs. head length almost equal to width), and distinct and blunt canthus rostralis (vs. indistinct); differs from A. dafangensis by head width slightly larger than head length (vs. head length larger than head width slightly).

3.5. Taxonomic Account

Amolops mantzorum feiye ssp. nov. (Figure 6 and Figure 7; Figures S1 and S2)

Holotype. CIB QZ2021131, adult male, collected in Bajiaolou Village (30.0729° N, 101.1398° E; alt. 2841 m), Bajiaolou Town, Yajiang County, Sichuan Province, China by Shengchao Shi, Peng Yan, and Shun Ma on 20 August 2021.

Paratypes. Nineteen adult specimens (Appendix A (1)). One male (CIB QZ2021130) and three females (CIB QZ2021126, CIB QZ2021127, and CIB QZ2021128) were collected at the same time and locality as the holotype; two males (CIB YJ2019080601, CIB YJ2019080602) and one female (CIB YJ2019080603) were collected at the same locality as the holotype on 6 August 2019; two males (CIB YJ202012 and CIB YJ202013) and one female (CIB YJ202011) were collected in Nimazong Village, Zhusang Town, Yajiang County, Sichuan Province, China on 8 July 2020; four females (CIB LT20200712-4, CIB LT20200712-5, CIB LT20200712-6, CIB LT20200712-8) were collected in Dewu Town, Litang County, Sichuan Province, China on 12 July 2020; four males (CIB DC20190905-89, CIB DC20190905-91, CIB DC20190906-94, CIB DC20190906-95) and one female (CIB DC20190905-88) were collected in Daocheng County, Sichuan Province, China on 5–6 September 2019.

Diagnosis. Amolops mantzorum feiye ssp. nov. is distinguished from all other congeners by the following combination of characters: (1) moderate body size, SVL 42.3–51.2 mm in males (n = 10), and 56.7–63.7 mm in females (n = 10); (2) tympanum absent; (3) head width slightly larger than head length; (4) vomerine teeth distinct and strong; (5) male forearm relatively strong, inner side of first finger with a developed nuptial pad; (6) supernumerary tubercles below the base of fingers II, III, and IV distinct; (7) circummarginal grooves present on tips of outer three fingers, absent on finger I; (8) dorsolateral fold absent but dorsolateral fold-like glands thick and flat; (9) vocal sac absent in males.

Description of Holotype. Medium body size, SVL 47.4 mm; flat head, head width slightly larger than head length (HL/HW ratio 0.98); snout rounded, canthus rostralis distinct and blunt, loreal region concave and oblique; internasal space greater than interorbital space (INS/IOS ratio 1.28), interorbital space greater than the maximum width of upper eyelid (IOS/UEW ratio 1.52); eyes convex in dorsal view, moderate size (ED/HL ratio 0.34); tympanum absent; vomerine teeth distinct and strong; tongue deeply notched posteriorly; vocal sac absent.

Forelimbs: forearm robust, the length of forearm and hand approximately half of body length (LAHL/SVL ratio 0.47); fingers slender, all four fingers’ tips expanded into disks, and the order of finger length is I < II < IV < III; circummarginal grooves present on tips of outer three fingers, absent on finger I; subarticular tubercles distinct; supernumerary tubercles below the base of fingers II, III, and IV distinct; inner metacarpal tubercle oval, two outer metacarpal tubercles present; fringe on fingers absent; a developed nuptial pad on the inner side of the finger I.

Hindlimbs: long and thin, heels overlapping when hind limbs flexed and held perpendicular to body; all five toe tips expanded into disks; the order of toe length I < II < III < V < IV, the subarticular tubercles well-developed; entirely webbed on all toes except toe IV; inner metatarsal tubercle, outer metatarsal tubercle absent.

Skin: dorsal surface relatively smooth; supratympanic fold absent; dorsolateral fold absent but dorsolateral fold-like glands thick and flat; ventral surface smooth except for slightly flattened tubercles on basal ventral surface of thigh.

Color in life. Entire dorsal surface grayish brown with dense green stripes and black spots in the middle of the dorsal surface; upper lip grayish black, lower lip grayish white; a large area of green stripes on the lateral body, black stripes and brownish green stripes on the dorsal surface of the forelimbs, and reddish brown patches on the dorsal surface of hindlimbs; ventral surface of body opaque milky white; ventral surface of limbs red with a small amount of pale yellow spots; ventral surface of hands reddish gray; toe webs brown.

Color in preservative. Entire dorsal surface dark gray with black patches scattered; sides light grayish brown; ventral surface milky white without mottling; crotch slightly dark; dorsal surface of limbs grayish brown, with black stripes scattered, and ventral surface light gray.

Secondary sexual characters. Males obviously smaller than females; the forearm of males is relatively strong, and males have a gray nuptial pad on the inner side of finger I (Figure 7).

Variation. The dorsal surfaces of two individuals (CIB QZ2021126 and CIB YJ2019080602) from Yajiang County are light brown rather than grayish brown as in the holotype; in two individuals (CIB QZ2021127 and CIB YJ2019080603), ventral surfaces of hands are milky white rather than reddish gray as in the holotype; in three individuals (CIB QZ2021126, CIB QZ2021128 and CIB YJ2019080603), the supernumerary tubercle below the base of finger III and the lower subarticular tubercle on finger III are separate, rather than linked together as in the holotype.

In terms of body size (mean SVL), among males, the individuals from Yajiang County are larger than individuals from Daocheng County (SVL 47.8 mm [n = 6] vs. 45.2 mm [n = 4]). Among females, individuals from Yajiang County are larger than individuals from Litang County and Daocheng County (SVL 61.0 mm [n = 5] vs. 60.5 mm [n = 1] vs. 56.7 mm [n = 4]). All measurements of type series specimens are summarized in Table 6.

Etymology. The specific epithet “feiye” is named after Prof. Liang Fei and his wife Prof. Changyuan Ye, combining their first names “Fei” and “Ye”. Prof. Fei and Prof. Ye have made significant contributions to Chinese herpetology. We suggest the English common name “Feiye’s torrent frog” and the Chinese common name “费叶湍蛙 (in Chinese Pinyin: fèi yè tuān wā)”.

Distribution and ecology. At present, A. m. feiye ssp. nov. is known from Yajiang County, Daocheng County, Muli County, and Litang County in Sichuan Province (Figure 1). Specimens were found on rocks exposed to water or along the river (2800–2900 m a.s.l.), surrounded by shrubs or tussocks. The rivers crossed villages and roads where we collected these specimens.

4. Discussion

Amolops m. xinduqiao was previously recognized as “A. kangtingensis” in Cai & Zhao [1] (Kangding population), Lu et al. [9], and Zhang et al. [10]. However, “A. kangtingensis” was described based on specimens from multiple localities. Fei et al. [2] re-examined the holotype of “A. kangtingensis” and classified it as A. mantzorum (currently A. m. mantzorum), rendering “A. kangtintensis” an invalid name that should be synonymized with A. mantzorum (currently A. m. mantzorum). They proposed a new name, A. xinduqiao (currently A. m. xinduqiao) for the population studied by the above authors as distinct from A. m. mantzorum.

Amolops m. mantzorum represents the population along the Dadu River Basin according to Fei et al. [2], which also corresponds to the “A. mantzorum central population” of Lu et al. [9]. Samples from Hongya and Dayi represent one of the four distinct genetic populations of A. mantzorum identified by Cai & Zhao [1]. In our study, molecular phylogeny including samples from these two localities indicates that this population should be recognized as the nominate A. m. mantzorum. According to Zhang et al. [10], the distribution of A. m. mantzorum also includes Kangding, Luding, Yingjing, Hanyuan, Baoxing, Tianquan, and Chongzhou.

Geographically, A. mantzorum has a continuous distribution west of the Sichuan Basin, but four lineages can be separated based on their distribution patterns The northern lineage (“A. m. ssp. [northern lineage]”) is restricted to the east of the Min River. Amolops m. mantzorum is distributed from the Min River to the Dadu River. The new subspecies has nearly overlapping distribution with A. m. xinduqiao near site 23 (Figure 1) at the Yalong River, but samples were collected from different villages, and samples of A. m. xinduqiao were collected above 3000 m elevation, whereas samples of the new subspecies were collected at approximately 2800–2900 m. Geographical barriers formed by large rivers and different elevational preferences may have contributed to this separation of genetic lineages.

The description of the new subspecies has clarified species delimitation within Amolops mantzorum. The four monophyletic lineages recognized in this study are consistent with the four genetic populations revealed by Cai & Zhao [1]. Except for “A. m. ssp. (northern lineage)”, three of them have been described and exhibit morphological divergence. However, the delimitation of the four lineages relies solely on mitochondrial markers ([1]; this sudy). Separation in mitochondrial phylogeny is sometimes not supported by nuclear markers (e.g., Lu et al. [9]), but it can be confirmed after deeper genomic investigations [3,4]. Morphologicaly, the new subspecies can be clearly separated from the other two recognized subspecies by its distinct and strong vomerine teeth (vs. small in both A. m. mantzorum and A. m. xinduqiao), and the invisible tympanum (vs. distinct in both). The differences between A. m. mantrozum and A. m. xinduqiao are also recognized in body size.

Currently, controversy persists regarding whether these subspecies represent independent species (i.e., [28,31,32]). Given the limitations of mitochondrial genes in DNA barcoding and phylogenetic estimation [33,34,35], further studies—including a detailed description of “A. m. ssp. (northern lineage)”, and a more robust phylogeny based on SNP data—would benefit efforts to resolve this controversy.

5. Conclusions

Based on mitochondrial phylogeny and morphology, we clearly identified and described the third subspecies of Amolops mantzorum. The new subspecies, together with A. m. mantzorum, A. m. xinduqiao, and “A. m. ssp. (northern lineage)” forms four monophyletic lineages within A. mantzorum. Given that taxonomists do not fully agree on the placement of subspecies in these lineages, which remains controversial, future studies based on genome data and further surveys—especially on the “A. m. ssp. (northern lineage)”—should be prioritized.