A new species of the genus Tylototriton (Caudata, Salamandridae) from Guangdong, southern China, with discussion on the subgenera and species groups within the genus

Zhi-Tong Lyu; Jian Wang; Zhao-Chi Zeng; Jia-Jun Zhou; Shuo Qi; Han Wan; You-Yu Li; Ying-Yong Wang

doi:10.3897/vz.71.e73563

Abstract

In this work, a new species of the genus Tylototriton is described from Guangdong, southern China. Tylototriton sini sp. nov. was recorded as T. asperrimus for decades, and was indicated to represent an independent lineage based on recent molecular phylogenetic analyses. After detailed molecular analysis and morphological comparisons, Tylototriton sini sp. nov. is recognized as a distinct species which can be clearly distinguished from all known congeners by a combination of morphological characteristics and the significant divergence in the mitochondrial gene. Because the genus Tylototriton is of high conservation concern and all formally described members are protected by law, we also provide first data on the conservation status and recommendations for IUCN categorization for Tylototriton sini sp. nov. A suggestion on the species groups division of the genus Tylototriton is also provided based on their morphological differences and phylogenetic relationships.

Key words

Chresonymy, conservation, morphology, phylogeny, Tylototriton sini sp. nov., Yunkai Mountains

Introduction

The newt genus Tylototriton Anderson, 1871 contains 32 known species distributed in the mountain areas of southern and eastern Himalaya, southern and central China, and northern Indochina Peninsula (Frost 2021). T. asperrimus Unterstein, 1930, the second described species within this genus, was originally nominated based on two specimens collected by Prof. Shu-Szi Sin (= Shu-Zhi Xin) from Loshiang (= Luoxiang Town), Yao Shan (= Mt Dayao, Jiuxiu Yao Autonomous County), Kwangsi (= Guangxi Zhuang Autonomous Region), China (Unterstein 1930; Fan 1931; Bauer et al. 1993). Subsequently, this species was widely recorded from multiple localities of China and Vietnam (Liu et al. 1973; Zhao and Adler 1993; Bain and Nguyen 2004; Fei et al. 1990, 2006, 2012; Nguyen et al. 2009; Fei and Ye 2016).

Liu et al. (1973) firstly noticed the morphological variations among different recorded populations of T. asperrimus in China, and suggested that detailed studies on this wide-spreading species are required. Afterward, based on the morphological differences, the populations in Hainan Island were proposed as an identical species T. hainanensis Fei, Ye & Yang, 1984, the populations in central China (Gansu, Sichuan, Guizhou, Hunan, and Anhui provinces) were assigned to T. wenxianensis Fei, Ye & Yang, 1984, and the populations in southern China (Guangxi Zhuang Autonomous Region, and Guangdong and Guizhou provinces) and northern Vietnam were kept as T. asperrimus (Fei et al. 1984, 1990, 2006). In recent years, the approach of integrative taxonomy combining morphological and molecular data has revealed that the recognition of T. asperrimus should be a species complex with multiple paraphyletic lineages, and the populations from northern Vietnam have been described as different new species, T. ziegleri Nishikawa, Matsui & Nguyen, 2013, T. pasmansi Bernardes, Le, Nguyen, Pham, Pham, Nguyen & Ziegler, 2020, and T. sparreboomi Bernardes, Le, Nguyen, Pham, Pham, Nguyen & Ziegler, 2020, respectively (Nishikawa et al. 2013a; Wang et al. 2018; Bernardes et al. 2020). After these taxonomic revisions, T. asperrimus is currently known only from southern China (Frost 2021). Nonetheless, the population in Mt Yunkai, Xinyi, Guangdong has been suggested to represent an independent lineage based on phylogenetic analyses using multi-locus of mitochondrial and nuclear data but without morphological comparisons (Wang et al. 2018; Poyarkov et al. 2021). Besides, this population was surprisingly reported as T. ziegleri after a rough phylogenetic analysis without including any data of T. hainanensis and other recently-described congeners from Vietnam (Li et al. 2020).

In this work, we performed detailed morphological comparisons and molecular analyses on the “T. asperrimus” population from Mt Yunkai, Xinyi, Guangdong, China (Fig. 1, site 1), to clarify its exact taxonomic status. The results substantiate that the Tylototriton population from Guangdong should be a distinct species and can be distinguished reliably from all known congeners in morphology and phylogeny, especially from T. asperrimus from Guangxi, China (Fig. 1, sites 2–4) and T. ziegleri from northern Vietnam (Fig. 1, sites 5–6). Therefore, we describe this Tylototriton population from Guangdong as a new species below.

Figure 1.

Map showing the localities of Tylototriton sini sp. nov., T. asperrimus, T. ziegleri, and T. hainanensis. Tylototriton sini sp. nov.: 1 Mt Yunkai, Xinyi, Guangdong, China (type locality); T. asperrimus: 2 Mt Dayao, Jiuxin, Guangxi, China (type locality) 3 Guiping, Guangxi, China 4 Longsheng, Guangxi, China; T. ziegleri: 5 Quan Ba, Ha Giang, Vietnam (type locality) 6 Bao Lac, Cao Bang, Vietnam; T. hainanensis: 7 Mt Jianfengling, Hainan, China 8 Mt Diaoluo, Hainan, China.

Materials and methods

Specimens and morphological analyses

Four specimens of the genus Tylototriton were collected from Mt Yunkai, Xinyi, Guangdong. All specimens were fixed in 10% buffered formalin, 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), PR China. External measurements were made for the unnamed specimens with digital calipers (Neiko 01407A Stainless Steel 6-Inch Digital Caliper) to the nearest 0.1 mm. These measurements are as follows: total length (TOL) from tip of snout to tip of tail; snout–vent length (SVL) from tip of snout to posterior edge of vent; head length (HL) from jugular fold to snout tip; maximum head width (HW); eye diameter (ED) from the anterior corner to the posterior corner of the eye; snout length (SL) from tip of snout to the anterior corner of eye; minimum interorbital (IOD) distance between the eyes; minimum internasal distance (IND) between the external nares; trunk length (TRL) from gular fold of throat to anterior tip of vent; tail length (TAL) from posterior edge of vent to tip of tail; maximum tail height (TH); lower arm length (LLA) from elbow to wrist; hand length (HL) from elbow to the tip of finger III; the third finger length (F3L) from base to tip of finger III; thigh length (TLH) from groin to knee; tibia length (TIB) from knee to tarsi; the third toe length (T3L) from base to tip of toe III.

The morphological comparisons for recognized congeners were attained from their original descriptions and latest revisions based on topotypic specimens (Anderson 1871; Unterstein 1930; Fang and Chang 1932; Liu 1950; Fei et al. 1984; Nussbaum et al. 1995; Böhme et al. 2005; Chen et al. 2010; Stuart et al. 2010; Hou et al. 2012; Nishikawa et al. 2013a, b, 2014; Yang et al. 2014; Le et al. 2015; Phimmachak et al. 2015; Fei and Ye 2016; Qian et al. 2017; Grismer et al. 2018, 2019; Zaw et al. 2019; Bernardes et al. 2020; Li et al. 2020; Pomchote et al. 2020; Poyarkov et al. 2021) and from the examination of museum specimens listed in Appendix.

Phylogenetic sampling and analyses

Totally 11 liver samples of the genus Tylototriton were used in this study, encompassing four samples of the undescribed Tylototriton specimens from Guangdong, four samples of T. asperrimus from Guangxi, one sample of T. broadoridgus Shen, Jiang & Mo, 2012, one sample of T. kweichowensis Fang & Chang, 1932, and one sample of T. maolanensis Li, Wei, Cheng, Zhang & Wang, 2020. All samples were attained from previously anesthetized and subsequently euthanized specimens and then preserved in 95% ethanol and stored at –40 °C.

Genomic DNA was extracted, using a DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd. One mitochondrion gene, namely NADH dehydrogenase subunit 2 (ND2), were amplified using the primers ND2-4F (5′-TATGAGTACGAGCATCATACCC-3′) and ND2-4R (5′-CTTCTGCTTAAGACTTTGAAGGTC-3′). PCR amplifications were processed with the cycling conditions that initial denaturing step at 95°C for 4 min, 35 cycles of denaturing at 95°C for 40 s, annealing at 53°C for 34 s and extending at 72°C for 60 s, and a final extending step at 72°C for 10 min. PCR products were purified with spin columns and then sequenced with both forward and reverse primers using BigDye Terminator Cycle Sequencing Kit from Applied Biosystems, on an ABI Prism 3730 automated DNA sequencer by Shanghai Majorbio Bio-pharm Technology Co., Ltd. All sequences were deposited in GenBank (Table 1).

For phylogenetic analyses, 35 sequences from additional Tylototriton congeners and 2 sequences of the out-group Echinotriton Nussbaum and Brodie, 1982, were obtained from GenBank and incorporated into our dataset. Detailed information is provided in Table 1. DNA sequences were aligned by the Clustal W algorithm with default parameters (Thompson et al. 1997). PartitionFinder2 was used to test the best partitioning scheme and jModelTest v2.1.2 was used to test the best fitting nucleotide substitution model. 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 10,000,000 generations and sampled every 1000 generations with the first 25% samples discarded as burn-in, resulting in a potential scale reduction factor (PSRF) of <0.005. In ML analysis, the bootstrap consensus tree inferred from 1000 replicates was used to represent the evolutionary history of the taxa analyzed. Genetic distances among samples were calculated in MEGA 6 using the uncorrected p-distance model.

Table 1.

Download as

CSV

XLSX

Localities, voucher information, and GenBank accession numbers for all ingroup Tylototriton and outgroup Echinotriton samples used in this study.

ID	Species	Locality	Voucher	ND2
1	Tylototriton sp. nov.	China: Guangdong: Mt Yunkai	SYS a004679	OK539834
2	Tylototriton sp. nov.	China: Guangdong: Mt Yunkai	SYS a008353	OK539835
3	Tylototriton sp. nov.	China: Guangdong: Mt Yunkai	SYS a008354	OK539836
4	Tylototriton sp. nov.	China: Guangdong: Mt Yunkai	SYS a008355	OK539837
5	Tylototriton sp. nov.	China: Guangdong: Xinyi	CIB XZ20091201	KY800876
6	Tylototriton sp. nov.	China: Guangdong: Mt Yunkai	GIABR 20187231	MH664279
7	Tylototriton sp. nov.	China: Guangdong: Mt Yunkai	GIABR 20187232	MH664280
8	T. asperrimus	China: Guangxi: Guiping	SYS a006890	OK539838
9	T. asperrimus	China: Guangxi: Guiping	SYS a006891	OK539839
10	T. asperrimus	China: Guangxi: Guiping	SYS a008200	OK539840
11	T. asperrimus	China: Guangxi: Guiping	SYS a008201	OK539841
12	T. asperrimus	China: Guangxi: Longsheng	CIB 70063	KC147816
13	T. asperrimus	China: Guangxi: Jinxiu	CIB 200807055	KC147815
14	T. asperrimus	China: Guangxi: Mt. Dayao	KIZ YPX9918	KT304303
15	T. ziegleri	Vietnam: Cao Bang: Bao Lac	VNMN 3389	KY800888
16	T. ziegleri	Vietnam: Ha Giang: Quan Ba	VNMN 3390	AB769539
17	T. hainanensis	China: Hainan: Mt Diaoluo	CIB 20081048	KC147817
18	T. hainanensis	China: Hainan: Mt Jianfengling	MVZ 236632	DQ517850
19	T. anguliceps	Laos: Luang Namtha: Viengphoukha	NCSM 82952	KT304300
20	T. anhuiensis	China: Anhui: Yuexi	AHU-16-EE-007	KY321413
21	T. broadoridgus	China: Hunan: Mt Huping	SYS a008391	OK539842
22	T. dabienicus	China: Henan: Shangcheng	HNNU10042015	KC147811
23	T. himalayanus	Nepal: Mechi: Illam	CIB 201406287	KT765210
24	T. kachinorum	Myanmar: Kachin: Indawgyi	ZMMU A5953	MK097273
25	T. kweichowensis	China: Guizhou: Shuicheng District	SYS a004967	OK539843
26	T. liuyangensis	China: Hunan: Liuyang	CSUFT 20100108	KJ205598
27	T. lizhengchangi	China: Hunan: Yizhang	KUHE 42317	AB769533
28	T. maolanensis	China: Guizhou: Libo County	SYS a002212	OK539844
29	T. ngarsuensis	Myanmar: Shan: Taunggyi	LSUHC 13763	MH836584
30	T. notialis	Vietnam: Nghe An: Pu Hoat	VNMN TAO1235	AB769536
31	T. panhai	Thailand: Loei: Phu Hin Rong Kla NP	KUHE PH019	AB830735
32	T. panwaensis	Myanmar: Kachin: Myitkyina	CAS 245418	KT304279
33	T. pasmansi	Vietnam: Phu Tho: Tan Son	IEBR 4467	MT210167
34	T. phukhaensis	Thailand: Nan: Doi Phu Kha NP	CUMZ A-7718	MN912574
35	T. podichthys	Laos: Luang Phabang: Phoukhoun	NCSM 77725	KT304295
36	T. pseudoverrucosus	China: Sichuan: Ningnan	CIB WCG2012003	KY800861
37	T. pulcherrima	China: Yunnan: Lyuchun	CIB TY040	KY800890
38	T. shanjing	China: Yunnan: Jingdong	MVZ219763	DQ517852
39	T. shanorum	Myanmar: Shan: Taunggyi	CAS 230933	AB922822
40	T. sparreboomi	Sin Ho, Lai Chau, Vietnam	IEBR 4476	MT210162
41	T. taliangensis	China: Sichuan: Liangshan	CAS 195126	DQ517853
42	T. uyenoi	Thailand: Chiang Mai: Doi Suthep	KUHE 19147	AB830733
43	T. verrucosus	China: Yunnan: Longchuan	CIB TSHS2	KY800848
44	T. vietnamensis	Vietnam: Bac Giang: Son Dong	IEBR 3243	HM770088
45	T. wenxianensis	China: Gansu: Wenxian	CIB 20090527	KC147813
46	T. yangi	China: Yunnan: Pingbian	KIZ RDQ201203001	LC017829
47	E. chinhaiensis	China: Zhejiang: Ningbo	TP26195	EU880315
48	E. maxiquadratus	China	SYNU SY20131101ENT	KM926344

Results

The BI and ML analyses resulted in identical topologies (Fig. 2). Most nodes were well supported with the Bayesian posterior probabilities (BPP) > 0.95 and the bootstrap supports (BS) > 70. The p-distances based on the ND2 gene among all samples used in this study are presented in Supplementary Table S1. As shown in the tree, five clades of Tylototriton were revealed. Within the clade IV (BPP 1.00, BS 100), the samples from Mt Yunkai, Xinyi, Guangdong, China clustered into a monophyletic lineage with small divergence (p-distances 0.0–0.7%) and strong support (BPP 1.00, BS 100). This lineage is phylogenetically distant from T. asperrimus from Guangxi, China and T. ziegleri from northern Vietnam, but forms the sister taxon to T. hainanensis from Hainan, China (BPP 1.00, BS 97).

Figure 2.

Bayesian inference and maximum-likelihood phylogenies based on mitochondrial ND2 gene. Bayesian posterior probabilities (BPP) > 0.95 and the bootstrap supports (BS) > 70 are shown. For the species with more than one samples used, number at the terminal of the lineage corresponds to the ID in Table 1 and the collecting locality is labelled.

Morphologically, the Tylototriton specimens from this unnamed lineage can be distinguished from all known congeners reliably (details in the Taxonomic account below). Combining the results of the morphological examination presented below and the phylogenetic analyses in this and previous studies, the Tylototriton population from Mt Yunkai, Xinyi, Guangdong is regarded as a new species and described herein.

Taxonomic account

Tylototriton sini Lyu, Wang, Zeng, Zhou, Qi, Wan & Wang, sp. nov.

http://zoobank.org/B9E2ABFD-4251-4B8A-8E99-902AFC57A02A

Tylototriton asperrimus — Fei et al. 1990 (Xinyi, Guangdong); Fei et al. 2006 (Xinyi, Guangdong); Li et al. 2011 (Mt Yunkai, Xinyi, Guangdong)

Echinotriton asperrimus asperrimus — Zhao and Adler 1993 (Guangdong)

Yaotriton asperrimus — Fei et al. 2012 (Xinyi, Guangdong); Fei and Ye 2016 (Xinyi, Guangdong)

Tylototriton asperrimus lineage 2 — Wang et al. 2018 (Xinyi, Guangdong)

Tylototriton ziegleri — Li et al. 2020 (Mt Yunkai, Xinyi, Guangdong)

Tylototriton sp. 3 — Poyarkov et al. 2021 (Xinyi, Guangdong)

Holotype

SYS a008354 (Figs 3, 4A), adult male, collected by Jian Wang, Shuo Qi, and Hong-Hui Chen on 14 June 2020 from Mt Yunkai (22°16’32.90”N, 111°11’42.87”E; ca 1500 m a.s.l.), Xinyi, Guangdong, China.

Paratypes

Two adult males and one adult female (Figs 4B, C, D, 5A, B). Male SYS a008353 and female SYS a008355, the same collection data as the holotype; male SYS a004679 /CIB 116083, collected by Jian Wang, Zhi-Tong Lyu, and Zhao-Chi Zeng on 16 April 2016 from the same locality as the holotype.

Figure 3.

Holotype SYS a008354 of Tylototriton sini sp. nov. in life., showing the features in dorsal full body, ventral full body, dorsal head, ventral head, ventral left hand, and ventral left foot.

Figure 4.

Type series of Tylototriton sini sp. nov. in preservative. A dorsal view B ventral view 1 male holotype SYS a008354 2 male paratype SYS a004679 3 male paratype SYS a008353 4 female paratype SYS a008355.

Etymology

The specific name sini refers to the outstanding biologist Prof. Shu-Szi Sin (= Shu-Zhi Xin, 辛树帜, 1894–1977). During his position at Sun Yat-sen University (1927–1931), Prof. Shu-Szi Sin organized repeated biology surveys throughout Guangxi, Guangdong, Guizhou, Hunan, and Hainan in southern China, pochally promoting the developments of zoological and botanic studies in this region. He collected specimens of T. asperrimus for the first time, as well as other amphibians and reptiles such as Quasipaa shini (Ahl, 1930) and the famous Shinisaurus crocodilurus Ahl, 1930. His family name “Sin” was mispronounced as “Shin” by the German researchers (Beolens et al. 2011), and we decided to use the correct spelling for this new species as Tylototriton sini sp. nov. in honor of Prof. Sin and his contributions.

Common name

Sin’s Knobby Newt (in English) / xīn shì yóu yuán (辛氏疣螈 in Chinese).

Diagnosis

(1) Dorsolateral bony ridges on head low; (2) quadrate spines absent; (3) medium body size, TOL 118.4–124.5 mm in males, 144.5 mm in a single female; (3) snout obtusely rounded in dorsal view and rounded in lateral profile; (4) head longer than wide, HW/HL ratio 0.87–0.95; (5) supratemporal bony ridges and the sagittal ridge on head distinctly visible; (6) limbs slender, tips of forelimb and hindlimb overlapping when adpressed along the body; (7) vertebral ridge distinct, relatively smooth, not segmented; (8) rib nodules 12–13, relatively small, distinctly isolated from each other; (9) ground coloration dark brown; (10) digits orange with irregular dark brown mottling; (11) in breeding season, rib nodules mottled with orange coloration, much brighter in the first two rib nodules; (12) in breeding season, lateral tail dark brown, fin with dorsal orange margin, ventral tail ridge orange.

Description of the holotype

SYS a008354 (Figs 3, 4A), adult male with a stout body, medium in size (SVL 62.0 mm, TAL 56.4 mm). Head longer than wide (HW/HL ratio 0.93); maximum head width slightly larger than the maximum trunk width; head nearly rounded hexagonal in shape in dorsal view, depressed, gently sloping in profile. Snout obtusely rounded in dorsal view, rounded in profile view, projecting beyond lower jaw. Nostril on anterior margin of snout, located notably closer to snout tip than to eye, with anterolateral orientation, not visible from dorsal view. Tongue oval, not notched distally; vomerine tooth series in an inverted ‘V’ shape, converging anteriorly but not reaching choanae. Parotoids distinct, large, crescentshaped, slightly projecting posteriorly. Dorsolateral supratemporal bony ridges on head wide, distinctly protruding, beginning at the anterior corner of orbit continuing to anterior end of parotoid, posterior ends slightly curved inside; sagittal bony ridge on head strong.

Vertebral middorsal ridge distinct, wide, not segmented, running from occiput region to sacrum and the base of tail. Rib nodules distinct, relatively small, distinctly isolated from each other but arranged in two longitudinal series on dorsolateral surfaces of dorsum from shoulder to base of tail, counting 13 nodules on each side of body.

Limbs slender, forelimb and hindlimb overlapping when adpressed towards each other along body; fingers and toes well developed, lacking webbing or fringes; relative finger lengths I < IV < III = II, relative toe lengths I = V < II < III = IV. Tail long, TAL/SVL ratio 0.91; laterally compressed along entire length, tapering posteriorly, lateral grooves on tail distinctly visible in dorsal view.

Skin of dorsum, flanks, and lateral sides of tail very rough with small granules and larger warts. Skin of head ridges and middorsal vertebral ridge relatively smooth. Skin of limbs with numerous tiny tubercles. Ventral surfaces relatively smoother, corrugated, with smaller granules arranged in transverse striations; throat with numerous tiny flat tubercles; weak gular fold present. Cloacal region slightly swollen, vent as a longitudinal slit, vent edges with numerous small transverse folds.

Coloration of holotype

In life (Fig. 3), ground color of head, dorsum, lateral tail, limbs, and venter uniform dark brown. Rib nodules mottled with orange coloration, the coloration of the first two rib nodules much brighter. Dorsal skin of hands and feet excluding the digits dark brown. Digits orange, with irregular dark brown mottling. Tail fin with dorsal orange margin, ventral tail ridge orange.

In preservative (Fig. 4A), ground color of head, dorsum, tail, limbs, and venter uniform dark. Orange coloration on all rib nodules fade. Orange coloration on the digits change into light brown. Ventral tail ridge pale yellow. Vent region pale yellow.

Variations

Measurements of the type series are given in Table 2. The single female paratype SYS a008355 (Figs 4D) possesses a larger and more robust body, however, its tail is relatively shorter, TAL/SVL ratio 0.82 vs 0.87–0.91 in males. Both the male holotype SYS a008354 (Figs 3, 4A) and the male paratype SYS a004679 (Figs 4B, 5A) bear 13 rib nodules on each side of body, while only 12 rib nodules in the male paratype SYS a008353 (Figs 4C, 5B) and the female paratype SYS a008355 (Figs 4D). In life, all rib nodules mottled with orange coloration, and much brighter in the first two rib nodules in several individuals (Figs 3, 5A, 5C).

Table 2.

Download as

CSV

XLSX

Measurements (in mm) of Tylototriton sini sp. nov., * for the holotype.

	SYS a008354 *	SYS a004679 /CIB 116083	SYS a008353	SYS a008355
Sex	M	M	M	F
TOL	118.4	124.5	118.6	144.5
SVL	62.0	66.5	63.2	79.3
HL	18.1	18.5	19.1	20.1
HW	16.8	16.8	16.7	19.0
ED	4.0	4.1	4.1	4.6
SL	5.8	5.7	5.9	6.5
IOD	7.3	7.6	7.1	6.7
IND	5.1	5.5	5.4	6.4
TRL	42.5	46.4	44.3	57.0
TAL	56.4	58.0	55.4	65.2
TH	8.1	8	7.1	7
LLA	7.3	7.3	6.9	8.9
HL	6.8	7.7	7.1	8.6
F3L	4.4	4.5	4.5	5.3
TLH	4.7	5.8	5.8	6.7
TIB	4.9	5.6	5.8	7.1
T3L	4.6	5.1	5.1	6.3

Comparisons

Tylototriton sini sp. nov. was recorded as T. asperrimus for a long time, but can be distinguished by the head longer than wide (vs head wider than long in T. asperrimus), the snout obtusely rounded in dorsal view (vs truncate), the distal tip of limbs greatly overlapping when the fore and hind limbs pressed along the trunk (vs slightly overlapping or just meeting), rib nodules small and distinctly isolated from each other (vs rib nodules large, knob-like, and nearly in contact with each other), rib nodules with orange coloration (vs rib nodules completely black or brown), and tail fin with dorsal orange margin (vs completely brownish black).

Tylototriton sini sp. nov. further differs from other congeners within the clade IV (Fig. 2) by the head longer than wide (vs head wider than long in T. hainanensis, T. thaiorum Poyarkov, Nguyen & Arkhipov, 2021, and T. ziegleri), snout obtusely rounded in dorsal view (vs truncate in T. pasmansi, T. sparreboomi, T. notialis Stuart, Phimmachak, Sivongxay & Robichaud, 2010, and T. ziegleri), snout rounded in profile view (vs slightly angular in T. pasmansi and T. notialis), sagittal bony ridge on head strong (vs obscure in T. hainanensis), vertebral ridge not segmented (vs segmented, forming a row of tubercles in T. ziegleri), absence of orange markings on the parotoid (vs present in T. pasmansi and T. notialis), and the presence of orange coloration on all rib nodules (vs absent in T. hainanensis, T. pasmansi, T. sparreboomi, T. thaiorum, and T. ziegleri).

For the species within clades III and IV (Fig. 2), Tylototriton sini sp. nov. can be distinguished from T. panhai Nishikawa, Khonsue, Pomchote & Matsui, 2013 by the absence of orange markings on the parotoid (vs present); from T. vietnamensis Böhme, Schöttler, Nguyen & Köhler, 2005 by the presence of gular fold (vs absent); from T. maolanensis by the smaller body size with TOL 118.4–124.5 mm in adult males (vs larger body size with TOL 151.0–172.0 mm in adult males); and from T. anhuiensis Qian, Sun, Li, Guo, Pan, Kang, Wang, Jiang, Wu & Zhang, 2017, T. broadoridgus, T. dabienicus Chen, Wang & Tao, 2010, T. liuyangensis Yang, Jiang, Shen & Fei, 2014, T. lizhengchangi Hou, Zhang, Jiang, Li & Lu, 2012, and T. wenxianensis by the rib nodules swollen and distinctly isolated from each other (vs rib nodules not separated, almost in continuous longitudinal rows).

For the remaining species within clades I and II (Fig. 2), Tylototriton sini sp. nov. can be distinguished from T. taliangensis Liu, 1950 by the absence of orange markings on the parotoid (vs present); from T. pseudoverrucosus Hou, Gu, Zhang, Zeng & Lu, 2012, T. anguliceps Le, Nguyen, Nishikawa, Nguyen, Pham, Matsui, Bernardes & Nguyen, 2015, T. himalayanus Khatiwada, Wang, Ghimire, Vasudevan, Paudel & Jiang, 2015, T. kachinorum Zaw, Lay, Pawangkhanant, Gorin & Poyarkov, 2019, T. ngarsuensis Grismer, Wood, Quah, Thura, Espinoza, Grismer, Murdoch & Lin, 2018, T. panwaensis Grismer, Wood, Quah, Thura, Espinoza & Murdoch, 2019, T. phukhaensis Pomchote, Khonsue, Thammachoti, Hernandez, Peerachidacho, Suwannapoom, Onishi & Nishikawa, 2020, T. podichthys Phimmachak, Aowphol & Stuart, 2015, T. pulcherrima Hou, Zhang, Li & Lu, 2012, T. shangjing Nussbaum, Brodie & Yang, 1995, T. shanorum Nishikawa, Matsui & Rao, 2014, T. uyenoi Nishikawa, Khonsue, Pomchote & Matsui, 2013, and T. verrucosus Anderson, 1871 by its limbs dark brown except for the orange digits, palms, and soles (vs limbs uniformly orange or light brown); and from T. kweichowensis and T. yangi Hou, Zhang, Zhou, Li & Lu, 2012 by its tail dark brown, except fin with dorsal orange margin and ventral tail ridge orange (vs tail uniformly orange).

Distribution

Tylototriton sini sp. nov. is currently known only from its type locality Mt Yunkai and the neighboring Mt Ehuangzhang (this study; Hernandez 2018), both situated in the Yunkai Mountains of western Guangdong.

Natural history

This newt is terrestrial and inhabits leaf litters in well-preserved montane evergreen broad-leaf forest. During its breeding season from April to July, adult individuals can be observed in small ponds with muddy bottoms, small marshes, and vernal pools. Larvae can be found from June to August. On 15 August 2017, different stages of larvae were observed in the same vernal pool near the road (ca 2 m long and ca 3 m wide of the pool with water depth ca 4 cm), without adults observed (Fig. 6).

Conservation recommendation

The extent of occurrence of Tylototriton sini sp. nov. is estimated to be less than 100 km², and the area of occupancy is estimated to be less than 10 km². Habitat degradation due to tourism development and illegal capture are the major threats. We recommend Tylototriton sini sp. nov. to be listed as Critically Endangered (CR) [IUCN Red List criteria B1ab(iii)+2ab(iii)]. This species must be added in the Appendix II of CITES, as the Tylototriton spp. are collectively included (CITES 2021). National Forestry and Grassland Administration of China (2021) has declared that the species T. asperrimus (as Y. asperrimus) is one of the Class II protected species of China, which was before the description of Tylototriton sini sp. nov. in this work. Therefore, the “T. asperrimus (as Y. asperrimus)” being listed as protected species of China should include the Guangdong population (now Tylototriton sini sp. nov.). Thus, we suggest Tylototriton sini sp. nov. should be also regarded as protected species of China with at least Class II.

Figure 5.

Tylototriton sini sp. nov. in life. A male paratype SYS a004679 B male paratype SYS a008353 in situ C an uncaptured individual in situ.

Figure 6.

Different stages of larvae were observed in the same vernal pool without adults observed on 15 August 2017 from the type locality of Tylototriton sini sp. nov. in Mt Yunkai.

Discussion

The phylogeny of genus Tylototriton has been well studied on the basis of multi-locus of mitochondrial and nuclear data (Wang et al. 2018). Subsequently, Poyarkov et al. (2021) employed two mitochondrial segments and got a unanimous result. In this study, our phylogenetic result from the sole mitochondrial ND2 segment is consistent with the topology revealed in the above studies, suggesting that the ND2 gene is adequate for settling the phylogeny in the genus Tylototriton.

After the taxonomic revisions in this work and previous studies (Nishikawa et al. 2013a; Wang et al. 2018; Bernardes et al. 2020), the species T. asperrimus can be confirmed to be present in Jiuxiu and Longsheng counties in northeastern Guangxi based on molecular data, while the taxonomic status for the populations from other localities such as northern and southwestern Guangxi (Mo et al. 2014) remain unknown and further studies are required. As the Tylototriton population from Guangdong is substantiated to be a new species Tylototriton sini sp. nov., the records of T. asperrimus and T. ziegleri should be removed from the herpetofauna of Guangdong. Given our recent findings, another record of T. ziegleri from China (Malipo County, Yunnan), recently published by Ye et al. (2017), should be re-examined whether it in fact represents the species that originally was described from northern Vietnam.

The taxonomy for interspecific relationships in the genus Tylototriton is controversial for decades (Zhao and Adler 1993; Dubois and Raffaëlli 2009; Fei et al. 2012; Nishikawa et al. 2013a; Fei and Ye 2016; Dubois et al. 2021; Poyarkov et al. 2021), and several nomenclatures were proposed to accommodate different species, such as Yaotriton Dubois & Raffaëlli, 2009, Qiantriton Fei, Ye & Jiang, 2012, and Liangshantriton Fei, Ye & Jiang, 2012. In the nearest study, the genus Tylototriton was partitioned into two subgenera Tylototriton and Yaotriton, and further into five species groups, based on the phylogenetic topology (Poyarkov et al. 2021). Despite the phylogenetic separation of Tylototriton and Yaotriton, the morphological characters for these two subgenera/genera remain unclear (Nishikawa et al. 2013a). The five species groups are corresponding to the five robust clades in phylogeny (Wang et al. 2018; Poyarkov et al. 2021; this work), while the morphological definitions for these five groups were not yet provided. Indeed, the morphological differences are unclear among some of these groups, for instance, T. maolanensis is morphologically similar to T. asperrimus but phylogenetically close to T. wenxianensis, and T. pasmansi, T. thaiorum, and T. ziegleri used to be morphologically identified as T. vietnamensis but phylogenetically close to T. asperrimus. Furthermore, as a genus including 33 recognized species, we consider it is too overstaffed to partition Tylototriton into both subgenera and species groups.

Thus, after a comprehensive review on these species, we suggest to divide the genus Tylototriton into three species groups, which is most reasonable with their morphological differences and phylogenetic relationships. Below we provide a key for the three species groups with their morphological definitions. We further provide a key for the T. asperrimus group recognized in this work, which includes the former T. asperrimus, T. wenxianensis, and T. vietnamensis groups in Poyarkov et al. (2021).

Key to species groups within genus Tylototriton

1a	Body relatively robust, TAL smaller, equal, or slightly larger than SVL, TAL/SVL < 120% in adult males	2
1b	Body slender, TAL distinctly larger than SVL, TAL/SVL > 125% in adult males	T. taliangensis group (2 species)
2a	Tail with different colorations, the lateral tail with the same color as the ground coloration of body, the dorsal tail fin or/and ventral tail ridge with orange margin	T. asperrimus group (17 species)
2b	Tail with uniform coloration, orange or light brown, much brighter and lighter than the ground coloration of body and head	T. verrucosus groups (14 species)

Key to species within Tylototriton asperrimus group

1a	Vertebral ridge segmented, tuberculate	*T. ziegleri*
1b	Vertebral ridge not segmented	2
2a	Rib nodules flatten, indistinct, not separated, and almost in continuous longitudinal rows forming a dorsolateral fold	3
2b	Rib nodules swollen, distinct, clearly separated or slightly contacted at the base	8
3a	Orange markings on the parotoid present in males	*T. lizhengchangi*
3b	Orange markings on the parotoid absent in males	4
4a	Head as wide as long	5
4b	Head longer than wide	6
5a	Peripheral area of cloaca brownish black	*T. wenxianensis*
5b	Peripheral area of cloaca orange	*T. liuyangensis*
6a	Vertebral ridge rather wide, its width approximately equal to the eye diameter	*T. broadoridgus*
6b	Vertebral ridge narrow, its width smaller than the eye diameter	7
7a	The orange coloration at the ventral edge of tail contacted with the orange coloration around the cloaca	*T. anhuiensis*
7b	The orange coloration at the ventral edge of tail isolated from the orange coloration around the cloaca	*T. dabienicus*
8a	Orange markings on the parotoid present	9
8b	Orange markings on the parotoid absent	10
9a	Anterior half of head and vertebral ridge yellow to reddish brown	*T. panhai*
9b	Anterior half of head and vertebral ridge dark brown, with the same color as the ground coloration of body	*T. notialis*
10a	Head wider than long	11
10b	Head longer than wide	12
11a	Sagittal bony ridge on head strong	*T. asperrimus*
11b	Sagittal bony ridge on head obscure	*T. hainanensis*
12a	Orange coloration of the rib nodules present	13
12b	Orange coloration of the rib nodules absent	14
13a	Smaller body size with TOL 118.4–124.5 mm in adult males	T. sini sp. nov.
13b	Larger body size with TOL 151.0–172.0 mm in adult males	*T. maolanensis*
14a	Gular fold absent	*T. vietnamensis*
14b	Gular fold present	15
15a	Snout obtusely rounded in dorsal view; rib nodules light brown	*T. thaiorum*
15b	Snout truncate in dorsal view; rib nodules uniformly black	16
16a	Tips of fingers reaching nostril, rib nodules slightly enlarged round-like	*T. sparreboomi*
16b	Tips of fingers reaching eye, rib nodules slightly smaller, pointy to rounded	*T. pasmansi*

Acknowledgments

We thank Hai-Long He, Hong-Hui Chen, Shun Ma, Zhi-Fei Ma, Yao Li, and Guangdong Ehuangzhang Provincial Nature Reserve for their help in the fieldwork. We are grateful to the editor Raffael Ernst and reviewers Thomas Ziegler and Peter Mikulicek for their helpful suggestions on our work. We thank Yinpeng Zhang from Michigan State University for polishing the language. This work was supported by DFGP Project of Fauna of Guangdong-202115, and the National Animal Collection Resource Center, China.

References

Anderson J (1871) Description of a new genus of newts from western Yunnan. Proceedings of the Zoological Society of London 1871: 423–425.

Bain RH, Nguyen TQ (2004) Herpetofauna diversity of Ha Giang Province in northeastern Vietnam, with descriptions of two new species. American Museum Novitates 3453: 1–42.

Bauer AM, Good DA, Günther R (1993) An annotated type catalogue of the caecilians and salamanders (Amphibia: Gymnophiona and Caudata) in the Zoological Museum, Berlin. Mitteilungen aus dem Zoologischen Museum in Berlin 69: 285–306.

Beolens B, Watkins M, Grayson M (2011) The Eponym Dictionary of Reptiles. The Johns Hopkins University Press, Baltimore, Maryland.

Bernardes M, Le MD, Nguyen TQ, Pham CT, Pham AV, Nguyen TT, Rödder D, Bonkowski M, Ziegler T (2020) Integrative taxonomy reveals three new taxa within the Tylototriton asperrimus complex (Caudata, Salamandridae) from Vietnam. ZooKeys 935: 121–164. https://doi.org/10.3897/zookeys.935.37138

Böhme W, Schöttler T, Nguyen TQ, Köhler J (2005) A new species of salamander, genus Tylototriton (Urodela: Salamandridae) from northern Vietnam. Salamandra 41: 215–220.

Chen XH, Wang XW, Tao J (2010) A new subspecies of genus Tylototriton from China (Caudata, Salamandridae). Acta Zootaxonomica Sinica 35: 666–670.

CITES (2021) Convention on international trade in endangered species of wild fauna and flora Appendices I, II and III valid from 22 June 2021. https://cites.org/eng/app/appendices.php

Dubois A, Raffaëlli J (2009) A new ergotaxonomy of the family Salamandridae Goldfuss, 1820 (Amphibia, Urodela). Alytes 26: 1–85.

Dubois A, Ohler A, Pyron RA (2021) New concepts and methods for phylogenetic taxonomy and nomenclature in zoology, exemplified by a new ranked cladonomy of recent amphibians (Lissamphibia). Megataxa 5: 1–738. https://doi.org/10.11646/megataxa.5.1.1

Fan TH (1931) Preliminary report of reptiles from Yaoshan, Kwangsi, China. Bulletin of the Biological Department, College of Science, Sun Yat-sen University 11: 1–154.

Fang PW, Chang MLY (1932) Notes on Tylototriton kweichowensis sp nov. and asperrimus Unterstein. Sinensia 2: 111–122.

Fei L, Hu SQ, Ye CY, Huang YZ (2006) Fauna Sinica. Amphibia. Volume 1. General Accounts of Gymnophiona and Urodela. Science Press, Beijing.

Fei L, Ye CY (2016) Amphibians of China (Vol. 1). Science Press, Beijing.

Fei L, Ye CY, Huang YZ (1990) Key to Chinese Amphibians. Publishing House for Scientific and Technological Literature, Chongqing.

Fei L, Ye CY, Jiang JP (2012) Colored Atlas of Chinese Amphibians and Their Distributions. Sichuan Publishing House of Science & Technology, Chengdu.

Fei L, Ye CY, Yang RS (1984) A new species and subspecies of the genus Tylototriton (Caudata: Salamandridae). Acta Zoologica Sinica 30: 85–91.

Frost DR (2021) Amphibian Species of the World 6.1, an Online Reference. American Museum of Natural History, New York. https://amphibiansoftheworld.amnh.org [accessed 20 August 2021]

Grismer LL, Wood PL Jr, Quah ESH, Thura MK, Espinoza RE, Grismer MS, Murdoch ML, Lin A (2018) A new species of Crocodile Newt Tylototriton (Caudata: Salamandridae) from Shan State, Myanmar (Burma). Zootaxa 4500: 442–573.

Grismer LL, Wood PL Jr, Quah ESH, Thura MK, Espinoza RE, Murdoch ML (2019) A new species of crocodile newt Tylototriton (Caudata: Salamandridae) from northern Myanmar (Burma). Journal of Natural History 53: 475–495. https://doi.org/10.1080/00222933.2019.1587534

Hernandez A (2018) Ecological affinities of Tylototriton asperrimus Unterstein, 1930 (Amphibia, Caudata: Salamandridae) at Bainiu, Dayaoshan, Guangxi Province, China with an emphasis on its cryptic diversity. Bulletin de la Société Herpétologique de France 166: 79–82.

Hou M, Li PP, Lü SQ (2012) Morphological research development of genus Tylototriton and primary confirmation of the status of four cryptic populations. Journal of Huangshan University 14: 61–65.

Le DT, Nguyen TT, Nishikawa K, Nguyen SHL, Pham AV, Matsui M, Bernardes M, Nguyen TQ (2015) A new species of Tylototriton Anderson, 1871 (Amphibia: Salamandridae) from northern Indochina. Current Herpetology 34: 38–50.

Li H, Yang B, Chi H, Lai R, Liu C, Xu Z, Zhu X, Gong S, Chen J (2020) Distribution and Supplementary Description of Tylototriton ziegleri in China. Chinese Journal of Wildlife 41: 791–795. https://doi.org/10.19711/j.cnki.issn2310-1490.2020.03.032

Li S, Wei G, Cheng Y, Zhang B, Wang B (2020) Description of a new species of the Asian newt genus Tylototriton sensu lato (Amphibia: Urodela: Salamandridae) from southwest China. Asian Herpetological Research 11: 282–296. https://doi.org/10.16373/j.cnki.ahr.200026

Li ZC, Xiao Z, Liu SR (2011) Amphibians and reptiles of Guangdong. Guangdong Science and Technology Press, Guangzhou.

Liu CC (1950) Amphibians of western China. Fieldiana. Zoology Memoires 2: 1–397.

Liu CC, Hu SQ, Fei L, Huang CC (1973) On collections of amphibians from Hainan Island. Acta Zoologica Sinica 19: 385–404.

Mo YM, Wei ZY, Chen WC (2014) Colored atlas of Guangxi amphibians. Guangxi Science & Technology Publishing House, Nanning.

National Forestry and Grassland Administration of China (2021) Official release of the updated List of Wild Animals under Special State Protection in China. https://www.forestry.gov.cn/main/586/20210208/095403793167571.html

Nguyen TQ, Nguyen VS, Ho LT, Le QK, Nguyen TT (2009) Phylogenetic relationships and taxonomic review of the family Salamandridae (Amphibia: Caudata) from Vietnam (in Vietnamese). Vietnam Journal of Biotechnology 7: 325–333.

Nishikawa K, Khonsue W, Pomchote P, Matsui M (2013a) Two new species of Tylototriton from Thailand (Amphibia: Urodela: Salamandridae). Zootaxa 3737(3): 261–279. https://doi.org/10.11646/zootaxa.3737.3.5

Nishikawa K, Matsui M, Nguyen TT (2013b) A New Species of Tylototriton from northern Vietnam (Amphibia: Urodela: Salamandridae). Current Herpetology 32: 34–49.

Nishikawa K, Matsui M, Rao DQ (2014) A new species of Tylototriton (Amphibia: Urodela: Salamandridae) from central Myanmar. Natural History Bulletin of the Siam Society 60: 9–22.

Nussbaum RA, Brodie ED Jr, Yang DT (1995) A taxonomic review of Tylototriton verrucosus Anderson (Amphibia: Caudata: Salamandridae). Herpetologica 51: 257–268.

Phimmachak S, Aowphol A, Stuart BL (2015) Morphological and molecular variation in Tylototriton (Caudata: Salamandridae) in Laos, with description of a new species. Zootaxa 4006: 285–310.

Pomchote P, Khonsue W, Thammachoti P, Hernandez A, Peerachidacho P, Suwannapoom C, Onishi Y, Nishikawa K (2020) A new species of Tylototriton (Urodela: Salamandridae) from Nan Province, northern Thailand. Tropical Natural History 20: 144–161.

Poyarkov NA Jr, Nguyen TV, Arkhipov DV (2021) A new species of the genus Tylototriton (Amphibia, Caudata, Salamandridae) from central Vietnam. Taprobanica 10: 4–22.

Qian L, Sun X, Li J, Guo W, Pan T, Kang X, Wang H, Jiang J, Wu J, Zhang BW (2017) A new species of the genus Tylototriton (Amphibia: Urodela: Salamandridae) from the southern Dabie Mountains in Anhui Province. Asian Herpetological Research 8: 151–164.

Ronquist F, Teslenko M, Van Der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61: 539–542. https://doi.org/10.1093/sysbio/sys029

Silvestro D, Michalak I (2012) RaxmlGUI: a graphical front-end for RAxML. Organisms Diversity and Evolution 12: 335–337. https://doi.org/10.1007/s13127-011-0056-0

Stuart BL, Phimmachak S, Sivongxay N, Robichaud WG (2010) A new species in the Tylototriton asperrimus group (Caudata: Salamandridae) from central Laos. Zootaxa 2650: 19–32.

Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25: 4876–4882. https://doi.org/10.1093/nar/25.24.4876

Unterstein W (1930) Beiträge zur Lurch- und Kriechtierfauna Kwangsi’s. 2. Schwanzlurche. Sitzungsberichte der Gesellschaft Naturforschender Freunde zu Berlin 1930: 313–315.

Wang B, Nishikawa K, Matsui M, Nguyen TQ, Xie F, Li C, Khatiwada JR, Zhang B, Gong D, Mo Y, Wei G, Chen X, Shen Y, Yang D, Xiong R, Jiang J (2018) Phylogenetic surveys on the newt genus Tylototriton sensu lato (Salamandridae, Caudata) reveal cryptic diversity and novel diversification promoted by historical climatic shifts. PeerJ 6: e4384. https://doi.org/10.7717/peerj.4384

Yang DD, Jiang JP, Shen YH, Fei DB (2014) A new species of the genus Tylototriton (Urodela: Salamandridae) from northeastern Hunan Province, China. Asian Herpetological Research 5: 1–11.

Ye J, Wei Z, Han F, Ni Q, Yao Y, Xu H, Li Y, Rao D, Zhang M (2017) The complete mitogenome sequence of Tylototriton ziegleri (Amphibia: Caudata). Conservation Genetics Resources 9: 503–506. https://doi.org/10.1007/s12686-017-0710-8

Zaw T, Lay P, Pawangkhanant P, Gorin VA, Poyarkov NA Jr (2019) A new species of Crocodile Newt, genus Tylototriton (Amphibia, Caudata, Salamandridae) from the mountains of Kachin State, northern Myanmar. Zoological Research 40: 151–174.

Zhao EM, Adler KA (1993) Herpetology of China. Contributions to Herpetology 10. Society for the Study of Amphibians and Reptiles, Oxford, Ohio.

Appendix 1

Examined specimens

Tylototriton asperrimus (N=4): China: Guangxi: Guiping: SYS a006890–6891, 8200–8201.

Tylototriton kweichowensis (N=6): China: Guizhou: Shuicheng: SYS a004967; Qixingguan: SYS a007119–7120, 7287, 7307–7308.

Tylototriton maolansis (N=5): China: Guizhou: Libo: SYS a000950–0953, 2212.

Tylototriton pulcherrima (N=3): China: Yunnan: Mojiang: SYS a003438–3440.

Tylototriton shanjing (N=3): China: Yunnan: Zhenyuan: SYS a001924–1925, 3371.

Tylototriton verrucosus (N=3): China: Yunnan: Tengchong: SYS a003768–3769; Longyang: SYS a006607.

Supplementary material

Supplementary material 1

Table S1

Lyu Z-T, Wang J, Zeng Z-C, Zhou J-J, Qi S, Wan H, Li Y-Y, Wang Y-Y (2021)

Data type: .xlsx

Explanation note: Pairwise distances among all Tylototriton samples used in this study.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Download file (26.96 kb)