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

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.


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. 1990Fei et al. , 2006Fei et al. , 2012Nguyen 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(Fei et al. , 1990(Fei et al. , 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. 2013aWang 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 .
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.

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.

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 am plifications 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 additio nal Tylototriton congeners and 2 sequences of the outgroup 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). Partition-Finder2 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.

Results
The BI and ML analyses resulted in identical topologies (Fig. 2
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. Dorsolate ral 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 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).
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 broadleaf 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 km 2 , and the area of occupancy is estimated to be less than 10 km 2 . Habitat degradation due to tourism development and illegal capture are the major threats. We recommend Tylototriton sini sp. nov. to be listed

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).