Confirmation of Tylototritonziegleri Nishikawa, Matsui & Nguyen, 2013 in China, with discussion on the relationship between T.verrucosus Anderson, 1871 and T.panwaensis Grismer, Wood, Quah, Thura, Espinoza & Murdoch, 2019 (Caudata, Salamandridae)

Abstract Background The distribution of the Ziegler’s Crocodile Newt Tylototritonziegleri Nishikawa, Matsui & Nguyen, 2013 in China has been controversial. This species was originally recorded uncertainly from Guangxi Autonomous Region, China. Subsequently, this species was recorded from Yunnan and Guangdong provinces, China. Thereafter, the record from Guangdong was denied and the record from Yunnan was questioned. New information Two specimens of Tylototriton Anderson, 1871 were collected from Wenshan Prefecture, Yunnan Province, China, in 2020. Phylogenetically, the sequences of these two specimens clustered with the sequences of T.ziegleri (including the holotype) from its type locality with strong support, and morphologically agree well with the original description of T.ziegleri. We confirm the record of T.ziegleri in China and present detailed collection site and morphological description of the specimens from China. In addition, we found that T.panwaensis Grismer, Wood, Quah, Thura, Espinoza & Murdoch, 2019 may be the synonym of T.verrucosus Anderson, 1871. We discussed the relationship between T.verrucosus and T.panwaensis.


Introduction
Tylototriton ziegleri Nishikawa, Matsui & Nguyen, 2013 is a member of the T. asperrimus species group, that was described from northern Vietnam (Nishikawa et al. 2013). This species was previously reported in China by Hernandez (2016) from Jingxi County, Guangxi Autonomous Region; however, he also mentioned that the population in Jingxi may not be the true T. ziegleri. Subsequently, this species was reported from Malipo County, Yunnan Province, based on only molecular data of a single specimen without morphological description and detailed collection information (Ye et al. 2017). Thereafter, this species was reported from Guangdong Province, China by Li et al. 2020. Lyu et al. (2021 found that the record of T. ziegleri from Guangdong by Li et al. (2020) actually represented a new species at that time and described it as T. sini Lyu, Wang, Zeng, Zhou, Qi, Wan & Wang, 2021. Therefore, Lyu et al. (2021 removed the record of T. ziegleri from the herpetofauna of Guangdong. In addition, Lyu et al. (2021) questioned the record of T. ziegleri from Yunnan. Anderson, 1871 is the type species of the genus Tylototriton. However, the current taxonomy of the true T. verrucosus is problematic because Anderson (1871) neither specified an exact type locality nor mentioned which specimens the original description of T. verrucosus was based on. Tylototriton panwaensis Grismer, Wood, Quah, Thura, Espinoza & Murdoch, 2019 is another species of Tylototriton closely resembling T. verrucosus and this species was described from Kachin State, north-eastern Myanmar, a location close to the border between Myanmar and western Yunnan of China.

Tylototriton verrucosus
During our field surveys in south-eastern Yunnan, China, in 2020, two specimens of Tylototriton were collected from Malipo County, Wenshan Prefecture. Detailed morphological comparisons and molecular analysis indicated that these specimens belong to T. ziegleri. Therefore, we confirm the distribution of T. ziegleri in China and provide a detailed description of these specimens herein. In addition, we collected some specimens of T. panwaensis in 2019 from western Yunnan, China, which is probably the type locality of T. verrucosus. Therefore, we also discuss the relationship between T. verrucosus and T. panwaensis herein.

Sampling
Specimens were collected, humanely euthanised and then fixed in 75% ethanol for permanent storage. Photographs were taken to document the colour pattern in life prior to euthanasia. Liver tissue samples were preserved in 99% ethanol for molecular analysis. All specimens were deposited at Kunming Natural History Museum of Zoology, Kunming Institute of Zoology, Chinese Academy of Sciences (KIZ).

Morphological characteristics
Morphological terminology followed Nishikawa et al. (2013). Measurements were taken with an electronic vernier caliper to the nearest 0.1 mm. Morphometric characters included: SVL: snout-vent length, from tip of snout to anterior tip of vent; HL: head length, from tip of snout to wrinkle of throat; HW: head width, measured at the angle anterior to the parotid gland; MXHW: maximum head width, measured at widest point; SL: snout length, from tip of snout to anterior tip of upper eyelid; LJL: lower jaw length, from tip of lower jaw to jaw angle; ENL: eyelid-nostril length, minimum distance between eyelid and nostril; IND: internarial distance, minimum distance between the external nares; IOD: interorbital distance, minimum distance between upper eyelids; UEW: upper eyelid width, greatest width of upper eyelid; UEL: upper eyelid length, greatest length of upper eyelid; OL: orbit length, maximum length of orbit; AGD: axilla-groin distance, minimum distance between axilla and groin; TRL: trunk length, from wrinkle of throat to anterior tip of vent; TAL: tail length, from anterior tip of vent to tail tip; VL: vent length, from anterior to posterior tip of vent; BTAW: basal tail width, tail width measured at root of tail; MTAW: medial tail width, tail width measured at middle; BTAH: basal tail height, tail height measured at base of tail; MXTAH: maximum tail height, tail height measured at highest point; MTAH: medial tail height, tail height measured at middle; FLL: fore-limb length, distance from axilla to tip of longest finger; HLL: hind-limb length, 2FL: second finger length; 3FL: third finger length; 3TL: third toe length; and 5TL: fifth toe length.

Molecular analysis
Genomic DNA was extracted from liver tissue samples preserved in 99% ethanol using a standard phenol-chloroform extraction protocol (Sambrook et al. 1989). A fragment of the NADH dehydrogenase subunit two (ND2) was amplified for the newly-collected specimens. Methods for amplification and sequencing of the DNA fragment are the same as Lyu et al. (2021). All new sequences were deposited in GenBank. Homologous and outgroup sequences were obtained from GenBank (Table 1).

Phylogenetic analyses
Sequences were aligned using MUSCLE 3.6 (Edgar 2004) with default parameters. Average genetic distances were calculated in MEGA 11 (Tamura et al. 2021) using the uncorrected p-distance model. The best substitution model GTR+F+I+G4 was selected using the Akaike Information Criterion (AIC) in ModelFinder (Kalyaanamoorthy et al. 2017). Bayesian Inference was performed in MrBayes 3.2.7 (Ronquist et al. 2012). Two runs were performed simultaneously with four Markov chains, the chains were run for 10,000,000 generations and sampled every 1,000 generations, the first 25% of the initial samples was discarded as burn-in after the standard deviation of split frequencies of the two runs was less than a value of 0.01. Maximum Likelihood analysis was performed in IQ-TREE 1.6.12 (Nguyen et al. 2015). The ultrafast bootstrap approximation algorithm was used via 1,000 bootstrap pseudoreplicates. Description the specimens from China: Morphometric data are provided in Table 2. Body moderately stout, medium in size (SVL 58.2-60.8 mm, TAL 68.0-69.7 mm). Head width almost equal to head length (HW/HL 0.96-1.05); head nearly hexagonal in shape in dorsal view, depressed, gently sloping in profile. Snout short, truncate, slightly beyond lower jaw. Nostril on anterior margin of snout, located notably closer to snout tip than to eye. Tongue oval, not notched distally, attached to mouth floor, but free laterally; vomerine tooth series in an inverted V-shape, converging anteriorly, but not reaching choanae. Labial fold absent; gular fold present, but weak; parotoids distinct, projecting posteriorly; costal folds absent. Dorsolateral supratemporal bony ridges on head protruding, beginning at the anterior corner of orbit continuing to anterior end of parotoid, posterior ends curved inside; mid-dorsal bony ridge on head short. Vertebral mid-dorsal ridge distinctly protruding, segmented, forming a row of tubercles, running from occiput region to the base of tail, separated from mid-dorsal bony ridge on head by a small gap. Rib nodules distinct, forming knob-like warts, relatively small, arranged in two longitudinal series on dorsolateral surfaces of dorsum from axilla to base of tail, counting 15-17 nodules on each side of body; rib nodules in the middle largest and decreasing anteriorly and posteriorly (Fig. 1 Limbs slender, tips of fore-limbs and hind-limbs overlapping when adpressed towards each other along body; fingers and toes free of webbing; relative finger lengths III ＞ II ＞ I ≥ IV, relative toe lengths III ＞ IV ＞ II ＞ I ≥ V. Tail long, TAL/SVL 1.12-1.20; laterally compressed along entire length, tapering posteriorly, tip pointed, dorsal fin more distinct posteriorly, ventral ridge smooth.

Taxon treatment
Skin rough with fine granules, dense on dorsum and ventrum, but small and sparse on throat. Cloacal region slightly swollen, vent as a longitudinal slit, vent edges with numerous small transverse folds.
Colouration in life: Dorsum almost uniformly black; venter slightly lighter than dorsum; bony ridges on head and vertebral ridge black, rib nodules black or reddish, only tips or most regions of fingers and toes, vent and ventral ridge of tail orange (Fig. 2).  Ecological notes: The specimens were collected in a small stream in the forest at night, the water in the stream was shallow, and both sides of the stream were covered with vegetation. No eggs or larvae were found.
Distribution: This species was recorded from Ha Giang and Cao Bang provinces, northern Vietnam; Malipo County, Yunnan Province and Jingxi County, Guangxi Autonomous Region, China (Fig. 3). However, the populations in Cao Bang and Jingxi may not represent the true Tylototriton ziegleri (see Discussion section).

Analysis
Morphologically, the specimens from Malipo County, Wenshan Prefecture, Yunnan, China, agree well with the original description of Tylototriton ziegleri: medium body size; skin rough with fine granules; bony ridges on head distinct; vertebral ridge prominent and segmented; rib nodules prominent; tips of fore-limbs and hind-limbs overlapping when adpressed along body; tail thin; dorsum uniform blackish; finger and toe tips, vent and ventral ridge of tail orange. Map showing the type locality of Tylototriton ziegleri in Ha Giang, Vietnam (1); the collection site of T. ziegleri in Malipo, Yunnan, China (2); the distributions of T. cf. ziegleri in Cao Bang, Vietnam (3 and 4); the distribution of T. cf. ziegleri in Jingxi, Guangxi, China (5); the type locality of T. verrucosus designated by Nussbaum et al. (1995) in Longchuan, Yunnan, China (6); the type locality of T. panwaensis in Kachin, Myanmar (7); the collection site of T. panwaensis in Tengchong, Yunnan, China (8); and the collection site of T. panwaensis in Yingjiang, Yunnan, China (9).
The obtained sequence alignment is 1044 bp in length. BI and ML analyses showed basically consistent topology (Fig. 4). The two specimens collected from Malipo County, Wenshan Prefecture, Yunnan, China, were homogeneous and clustered with Tylototriton ziegleri (including the holotype) from Ha Giang Province, Vietnam, with strong support. The genetic distance (uncorrected p-distance) between the specimens from China and T. ziegleri (including the holotype) from Ha Giang, Vietnam was only 0.1% (Suppl. material 1). Combining the results of morphological and molecular analysis, we determined that the specimens from Malipo County, Wenshan Prefecture, Yunnan, China, belong to Tylototriton ziegleri.

Discussion
In the phylogenetic analyses in Nishikawa et al. (2013) and in this study, Tylototriton ziegleri formed two strongly-supported lineages, one (including the holotype) from Ha Giang Province, Vietnam and the other from Cao Bang Province, Vietnam and there is a relatively large genetic divergence (2.6%) between the two lineages, more than that (2.1%) between T. verrucosus and T. pulcherrima and close to that (2.7%) between T. taliangensis and T. pseudoverrucosus. Therefore, we regard the lineage containing the holotype as T. ziegleri sensu stricto and the other lineage as Tylototriton cf. ziegleri. The lineage of Tylototriton cf. ziegleri may, therefore, represent a cryptic new species of Tylototriton. Our newly-collected specimens from China clustered with the lineage of T. ziegleri sensu stricto and showed negligible genetic divergence (0.1%) with this lineage, confirming T. ziegleri sensu stricto. Ziegler et al. (2018) reported the longevity of T. ziegleri and extended the diagnosis of this species, based on specimens collected from Cao Bang, Vietnam; according to their result of molecular identification, these specimens from Cao Bang should be assigned to to Tylototriton cf. ziegleri rather than T. ziegleri sensu stricto. As for the population in Jingxi, Guangxi, China, record by Hernandez (2016), judging from the geographical location, we consider that this population should also belong to Tylototriton cf. ziegleri.
In the original description of Tylototriton verrucosus, Anderson (1871) described this species as uniform blackish-brown, but not orange-patterned. Usually, the uniform blackish-brown colour of these salamanders in life remains constant in preservative and the orange colour of these salamanders in life fades to light yellow or white, but the pattern remains distinct in preservative. However, according to Nussbaum et al. (1995) and Chanda et al. (2000), all the syntypes of T. verrucosus are orange-patterned. This has always made people wonder why the syntypes were inconsistent with the original description by Anderson (1871). Anderson (1871) did not specify a precise type locality, but merely mentioned some valleys in western Yunnan, including Nantin, Momien, Hotha, Ponsee and Nampoung. That is to say, any of these valleys may be the type locality of T. verrucosus. "Momien" generally refers to Tengchong City, Yunnan, China, nowadays; "Hotha" generally refers to Husa Township, Longchuan County, Yunnan, China, nowadays; "Ponsee" generally refers to Xueli Village, Yingjiang County, Yunnan, China, nowadays; "Nampoung" generally refers to Nabang Town, Yingjiang County, Yunnan, China, nowadays; and "Nantin" is uncertain. Nussbaum et al. (1995) (Sclater 1892, Chanda et al. 2000, one of the syntypes should be selected as the lectotype instead of designating a neotype and this designation contravenes the requirements of Art. 74.7 of the Code (ICZN, 1999). Therefore, we agree with Fei et al. (2006) that the designation of the neotype (KIZ 74II0061 VI.16) is unvalid.
We collected some specimens from western Tengchong City and neighbouring northwestern Yingjiang County, Yunnan, China, in 2019; these specimens were uniform blackish-brown or uniform brown in life, that is to say, not only the specimens from Husa agree with the original description of T. verrucosus, but also the specimens from western Tengchong and north-western Yingjiang agree with the original description of T. verrucosus . Therefore, Tengchong or Yingjiang cannot be ruled out as the type locality of T. verrucosus. Interestingly, the black surface skins of the ones who underwent poor preservation fell off and the body colour turned to orange-patterned, while others are still uniform blackish-brown or uniform brown (Fig. 5). The colour of some of these specimens in preservative became inconsistent with that in life; this seems to be similar to the case of the syntypes of T. verrucosus. Nevertheless, as we do not know the previous preservation status of the syntypes of T. verrucosus, we cannot determine whether the colours of these syntypes have changed. More interestingly, the specimens from western Tengchong and north-western Yingjiang were assigned to T. panwaensis in the molecular analysis.
Although the population in Husa and the population in western Tengchong are both in agreement with the original description of Tylototriton verrucosus, the two populations The specimens of Tylototriton panwaensis from western Yunnan, China, in preservative.