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A new species of the genus Tylototriton (Caudata, Salamandridae) from Guangdong, southern China, with discussion on the subgenera and species groups within the genus
expand article infoZhi-Tong Lyu, Jian Wang§, Zhao-Chi Zeng§, Jia-Jun Zhou|, Shuo Qi, Han Wan#, You-Yu Li¤, Ying-Yong Wang
‡ Sun Yat-sen University, Guangzhou, China
§ Shenzhen Shuanghuamu Biological Technology Co., Shenzhen, China
| Zhejiang Forest Resource Monitoring Center / Zhejiang Forestry Survey Planning and Design Company Limited, Hangzhou, China
¶ Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China
# University of the Chinese Academy of Sciences, Kunming, China
¤ Guangdong Yunkaishan National Nature Reserve, Maoming, China
Open Access

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.

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.

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

Echinotriton asperrimus asperrimusZhao and Adler 1993 (Guangdong)

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

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

Tylototriton ziegleriLi 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.

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.

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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.