# Multiple lines of evidence for a new cryptic species of *Tylototriton* (Amphibia, Salamandridae) from northern Vietnam

Nikolay A. POYARKOV<fnref n="1" />,<fnref n="2" />,*, Tan Van NGUYEN<fnref n="3" />,<fnref n="4" />, Son Xuan LE<fnref n="2" />, Dac Xuan LE<fnref n="2" />, Dmitriy V. ARKHIPOV<fnref n="1" />, Vladislav A. GORIN<fnref n="1" />, Axel HERNANDEZ<fnref n="5" /> & Christophe DUFRESNES<fnref n="6" />,<fnref n="7" />,*

Using molecular (mitochondrial DNA, multilocus genomic loci) and phenotypic (morphology, coloration, osteology) analyses, we reveal a new cryptic species of crocodile newt of the *Tylototriton asperrimus* species group from Viet Bac Mountain Range, northern Vietnam. The new species was previously confounded with *T. asperrimus*, *T. vietnamensis* and more recently *T. ziegleri*, its closest relative, from which it can be distinguished by a combination of diagnostic characters at phenotypic traits including rib nodules, cranial bony ridges, skin granulation, orange spotting patterns, vomerine teeth shape and relative limb size. General differences in body shape between the new species and *T. ziegleri* s. str. are further corroborated by multivariate morphological analyses. Phylogenetic analysis of the *ND2* and 16S rRNA mtDNA gene fragments (1665 bp) confirm the placement of the new species in the subgenus *Yaotriton* as the sister species of *T. ziegleri* s. str. (*p*-distance 2.7 % in the *ND2* gene). Furthermore, the differentiation

<footnote n="1">¹ Department of Vertebrate Zoology, Lomonosov Moscow State University, Leninskiye Gory, Moscow 119234, Russia.</footnote>

<footnote n="2">² Joint Vietnam - Russia Tropical Science and Technology Research Center, 63 Nguyen Van Huyen Road, Nghia Do, Cau Giay, Hanoi 122000, Vietnam.</footnote>

<footnote n="3">³ Institute for Research and Training in Medicine, Biology and Pharmacy, Duy Tan University, Da Nang 550000, Vietnam.</footnote>

<footnote n="4">⁴ College of Medicine and Pharmacy, Duy Tan University, 120 Hoang Minh Thao, Lien Chieu, Da Nang 550000, Vietnam.</footnote>

<footnote n="5">⁵ Department of Environmental Sciences, Faculty of Sciences and Technics, University Pasquale Paoli of Corsica, Corte, 20250, France.</footnote>

<footnote n="6">⁶ Laboratory of Amphibian Systematics and Evolutionary Research, College of Biology and Environment, Nanjing Forestry University, Nanjing 210037, People's Republic of China.</footnote>

<footnote n="7">⁷ Institut de Systématique, Evolution, Biodiversité (ISYEB), Muséum national d'Histoire naturelle, CNRS, Sorbonne Université, EPHE-PSL, Université des Antilles, 45 rue Buffon, CP 50, 75005 Paris, France.</footnote>

<footnote>*Corresponding authors: Nikolay A. Poyarkov <n.poyarkov@gmail.com>; Christophe Dufresnes <christophe.dufresnes@hotmail.fr>.</footnote>
of the two species is fully supported by the analysis of 3901 genomic loci (556,209 bp) obtained by double digest RAD-sequencing. The new species is presently isolated in the northeastern parts of the Viet Bac Massif (Ngan Son Mountain Range) while *T. ziegleri* s. str. is restricted to its western part (Song Gam Mountain Range); their respective ranges are presumably separated by the Gam River Valley. The new species is probably threatened by extinction on the short term, being currently known only from montane evergreen broadleaf forests (~1000–1400 m of elevation) in the Nguyen Binh and Bao Lac districts of Cao Bang Province of Vietnam, but potentially extending to Jingxi County in the adjacent Guangxi Province of China. Our study emphasizes the benefits of relatively new technologies such as high-throughput sequencing and X-ray microtomography for the integrative taxonomy of cryptic, closely related amphibian species, and brings research on the iconic genus *Tylototriton* into the genomic era.

**Keywords:** 16S rRNA, Cao Bang Province, ddRAD-seq, endemism, Gam River, micro-CT scanning, mtDNA genealogy, *ND2*, taxonomy, *Tylototriton ziegleri*

urn:lsid:zoobank.org:pub:983AB200-7C21-4F5C-B80A-566BBD820A8C

INTRODUCTION

The genus *Tylototriton* Anderson, 1871, commonly known as crocodile newts, is the largest genus in the family Salamandridae and currently includes 41 recognized species (Frost 2024). Crocodile newts inhabit montane forest areas throughout the Asian monsoon climate zone from the southern and eastern Himalaya, eastern Nepal, northern India, Bhutan, Myanmar, central to southern China, and southwards across Laos, Thailand, and Vietnam (Poyarkov *et al.* 2021a; Dufresnes & Hernandez 2022; Decemson *et al.* 2023; Phung *et al.* 2023; Frost 2024). All known *Tylototriton* species are terrestrial and discrete, most having restricted distributions and being regarded as niche specialists typically found from middle to high elevations in subtropical moist forested environments, which provide a relatively narrow thermal range (15–24.0°C) and high annual rainfall, especially during the monsoon season (Hernandez *et al.* 2018). The genus is generally subdivided into two subgenera: the subgenus *Tylototriton* is characterized by the presence of orange spots on the head, dorsum, tail, or sides of the body, while the subgenus *Yaotriton* Dubois & Raffaëlli, 2009 is characterized by generally much darker, almost blackish coloration and fewer light markings (Yang *et al.* 2014; Li *et al.* 2020b; Poyarkov *et al.* 2021a; Lyu *et al.* 2021; Dufresnes & Hernandez 2022); the recognition of these subgenera was, however, questioned by several recent studies (Than Zaw *et al.* 2019; Poyarkov *et al.* 2021a; Lyu *et al.* 2021). With over 80 % of species described in the 21st century (Frost 2024), most of the known diversity of the genus *Tylototriton* was documented only recently, in no small part due to integrative taxonomic approaches that implement biometric together with molecular data, assisted by new technologies such as X-ray microtomography (micro-CT scanning) (e.g., Le *et al.* 2015). Such approaches have allowed to detect many new species even among superficially resembling populations, such as the *T. asperrimus* Unterstein, 1930 and *T. wenxianensis* Fei, Ye & Yang, 1984 species groups of the subgenus *Yaotriton*
(Bernardes *et al.* 2020; Li *et al.* 2020b; Poyarkov *et al.* 2021a; Lyu *et al.* 2021; Li *et al.* 2022; Phung *et al.* 2023).

Beside China, Vietnam currently hosts the largest proportion of *Tylototriton* diversity, with nine species, including six endemics, although a single one was known for the country in the late 2000s (Poyarkov *et al.* 2021b; Phung *et al.* 2023). Among them, Ziegler’s crocodile newt, *Tylototriton ziegleri* Nishikawa, Matsui & Nguyen, 2013, was described based on 20 adult specimens collected from Viet Bac Massif of northern Vietnam. Originally, this species was confounded with *T. asperrimus* (currently considered to be restricted to Guangxi Province, China) or with *T. vietnamensis* Böhme, Schöttler, Nguyen & Köhler, 2005 (currently restricted to northeastern Vietnam) (see Böhme *et al.* 2005; Weisrock *et al.* 2006; Nguyen *et al.* 2009; Stuart *et al.* 2010; Gu *et al.* 2012; Shen *et al.* 2012). The molecular study of Yuan *et al.* (2011) subsequently reported that the population from Bac Quang District (Ha Giang Province of Vietnam) identified as ‘*T. asperrimus*’, and the population from Quang Thanh District (Cao Bang Province of Vietnam) identified as ‘*T. cf. vietnamensis*,’ are in fact not closely related to neither *T. asperrimus* sensu stricto (hereafter, **s. str.**) or to *T. vietnamensis s. str.*, but instead form a separate clade with a substantial level of divergence from any other known *Tylototriton* taxon. These authors refrained from any taxonomic conclusions, and Nishikawa *et al.* (2013a) eventually described the Ha Giang and Cao Bang populations as *T. ziegleri*, based on divergence at the mitochondrial DNA (hereafter, mtDNA) gene *ND2*. The type locality of *T. ziegleri* is given as Ta Boc Mountain (hereafter, Mt.), Ban Thang Village, Tung Vai Municipality, Quan Ba District, Ha Giang Province, and Nishikawa *et al.* (2013a) further listed Vi Xuyen District of Ha Giang Province and Bao Lac and Nguyen Binh districts of Cao Bang Province as other localities of this species. The *ND2* analyses of Nishikawa *et al.* (2013a) also suggested molecular differentiation among the populations of *T. ziegleri*, namely between the Ha Giang and Cao Bang provinces, but these were based only on a few samples, and no further characterization was conducted. Since then, *T. ziegleri* has been documented in nearby China, namely the Malipo County of Yunnan Province (Ye *et al.* 2017; Liu *et al.* 2022) and perhaps the Jingxi County of Guangxi Province (Hernandez 2016). Li *et al.* (2020a) also mentioned the presence of *T. ziegleri* in Yunkai Mt., Xinyi County, Guangdong Province, but a recent reassessment has updated this record as *T. sini* Lyu, Wang, Zeng, Zhou, Qi, Wan, Li & Wang, 2021 (Lyu *et al.* 2021).

In Ha Giang Province (where its type locality is located), *T. ziegleri* inhabits primary evergreen forests on limestone parent rock characterized mainly by bamboo vegetation, while in Cao Bang Province it is known from montane evergreen forests on granite rocks (our observations). Rare and geographically restricted, the species has been evaluated as Vulnerable (VU) by the IUCN Red List (Anonymous 2017). Recent phylogenetic reconstructions confirmed the earlier results of Yuan *et al.* (2011) and Nishikawa *et al.* (2013a) that *T. ziegleri* comprises at least two divergent mtDNA lineages with levels of *ND2* sequence divergence (2.6 %–2.7 %) comparable to presently recognized species (Wang *et al.* 2018; Liu *et al.* 2022; Dufresnes & Hernandez 2022). One lineage is found in Ha Giang Province (Vietnam) and Yunnan Province (China) and corresponds to *T. ziegleri s. str.* The other lineage, which is so far reliably known from three localities in Cao Bang Province (Vietnam) and likely occurs
in the adjacent Guangxi Province (China; pending a molecular confirmation) thus potentially represents an unnamed candidate species.

In the present study, we implement an integrative taxonomic approach to test whether the Cao Bang populations of *T. ziegleri* correspond to a different species. Using newly-collected specimens and samples, we conduct phylogenetic analyses of mitochondrial sequences and nuclear loci obtained with double digest Restriction-Site Associated DNA sequencing (ddRAD-seq), and quantify differences in body shape. We further compare topotypical *T. ziegleri* *s. str.* specimens with specimens from Cao Bang to document stable character state differences in coloration and external morphology. The results call to implement taxonomic revisions, and we describe the Cao Bang population as a new species of *Tylototriton*. Finally, our new DNA barcoding data refine the respective ranges of *T. ziegleri* *s. str.* and the new species, and provides a new record for another rare species of Vietnam, *T. thaiorum* Poyarkov, Nguyen & Arkhipov, 2021.

## MATERIAL AND METHODS

### Sample collection

Specimens of *T. ziegleri* sensu lato (hereafter, *s. l.*) were collected from Ha Giang and Cao Bang provinces, northern Vietnam, during several field surveys in 2017–2022. In addition, specimens provisionally identified on the field as *T. thaiorum* were collected from a new population, namely in Thanh Hoa Province, northern Vietnam (Fig. 1; Table 1). Permissions to conduct fieldwork and collect specimens were granted by the Department of Forestry, Ministry of Agriculture and Rural Development of Vietnam (permit numbers #712/TCLN-BTTN of 17.05.2017 and #1539/TCLN-DDPH of 19.09.2018) and by the Forest Protection Departments of the Peoples’ Committees of Cao Bang Province (permit number #1659/UBND-NC of 02.06.2017), Ha Giang Province (permit number #109/SNgV-LS of 15.03.2022), and Thanh Hoa Province (permit number #562/GP of 01.06.2022) of Vietnam.

Specimens of *Tylototriton* spp. were collected by hand in swamps of forest clearings surrounded by montane evergreen subtropical forest. Geographic coordinates and altitude were obtained with a Garmin GPSMAP 60CSx GPS receiver (Garmin Ltd., USA) and recorded in datum WGS 84. Specimens were euthanized by 20 % benzocaine, and tissue samples (liver) were taken for genetic analyses and stored in 96 % ethanol prior to preservation in 70 % ethanol and curation in the herpetological collections of the Zoological Museum of Moscow State University (ZMMU), Moscow, Russia, and of the Joint Vietnam-Russia Tropical Science and Technology Research Center (VRTC), Hanoi, Vietnam.
<table><caption>Table 1. List of the localities of *Tylototriton koliaensis* sp. nov., *T. ziegleri* s. str. and *T. thaiorum* appearing on Fig. 1. Symbols and abbreviations as follows. (#): Locality number on the map in Fig. 1; (Mor.): Verified by morphology data (yes/no); (DNA): Verified by molecular data (yes/no).</caption><thead><tr><th>#</th><th>Species</th><th>(Mor.)</th><th>(DNA)</th><th>Locality</th><th>Source(s)</th></tr></thead><tbody><tr><td>1</td><td><i>T. koliaensis</i> sp. nov.</td><td>yes</td><td>yes</td><td>Kolia Mountain Pass and Kolia Organic Farm within the Phia Oac-Phia Den NP, Thanh Cong Commune, Nguyen Binh District, Cao Bang Province, Vietnam (type locality)</td><td>Nishikawa et al. 2013a; our data</td></tr><tr><td>2</td><td><i>T. koliaensis</i> sp. nov.</td><td>yes</td><td>yes</td><td>Quang Thanh Commune, Nguyen Binh District, Cao Bang Province, Vietnam</td><td>Nishikawa et al. 2013a; our data</td></tr><tr><td>3</td><td><i>T. koliaensis</i> sp. nov.</td><td>yes</td><td>yes</td><td>Bao Lac District, Cao Bang Province, Vietnam</td><td>Nishikawa et al. 2013a</td></tr><tr><td>4</td><td><i>T. cf. koliaensis</i> sp. nov.</td><td>yes</td><td>no</td><td>Jingxi, Guangxi Province, China</td><td>Hernandez 2016</td></tr><tr><td>5</td><td><i>T. ziegleri</i> s. str.</td><td>yes</td><td>yes</td><td>Tung Vai Commune, Quan Ba District, Ha Giang Province, Vietnam (type locality)</td><td>Nishikawa et al. 2013a; our data</td></tr><tr><td>6</td><td><i>T. ziegleri</i> s. str.</td><td>yes</td><td>yes</td><td>Bat Dai Son NR, Thanh Van Commune, Quan Ba District, Ha Giang Province, Vietnam</td><td>our data</td></tr><tr><td>7</td><td><i>T. ziegleri</i> s. str.</td><td>yes</td><td>yes</td><td>Bac Quang District, Ha Giang Province, Vietnam</td><td>Nishikawa et al. 2013a</td></tr><tr><td>8</td><td><i>T. ziegleri</i> s. str.</td><td>yes</td><td>no</td><td>Chieu Lau Thi Mt., Xin Man District, Ha Giang Province, Vietnam</td><td>our data</td></tr><tr><td>9</td><td><i>T. ziegleri</i> s. str.</td><td>yes</td><td>yes</td><td>Malipo, Yunnan Province, China</td><td>Liu et al. 2022</td></tr><tr><td>10</td><td><i>T. ziegleri</i> s. str.</td><td>yes</td><td>no</td><td>Wenshan, Yunnan Province, China</td><td>https://www.inaturalist.org/observations/153349883</td></tr><tr><td>11</td><td><i>T. thaiorum</i></td><td>yes</td><td>yes</td><td>Xuan Lien NR, Bat Mot Commune, Thuong Xuan District, Thanh Hoa Province, Vietnam</td><td>our data</td></tr><tr><td>12</td><td><i>T. thaiorum</i></td><td>yes</td><td>yes</td><td>Pu Hoat N.R., Thong Tu Commune, Que Phong District, Nghe An Province, Vietnam (type locality)</td><td>Poyarkov et al. 2021a</td></tr></tbody></table>

<figure><img src="image_1.png" /><figcaption>Figure 1. Distribution of *Tylototriton koliaensis* sp. nov. (red), *T. ziegleri* s. str. (blue) and *T. thaiorum* (green) in northern Indochina. A, map of Indochina, the inset shows the area of the studied species. B, localities of the studied species; see Table 1 for locality details. Dots show type localities. Locality 4 was not confirmed with molecular data and should be regarded as *Tylototriton cf. koliaensis* sp. nov.</figcaption></figure>
*Molecular laboratory methods*

Total genomic DNA was extracted from ethanol-preserved muscle tissues using standard phenol-chloroform extraction protocols (Hillis *et al.* 1996). Total DNA concentration was estimated from 1 µL drops with a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA) and consequently adjusted to 100 ng/µL. We amplified two mtDNA fragments, including the partial sequences of the *ND2* and 16S rRNA mtDNA genes. These markers were chosen as they have been extensively used in phylogenetic and taxonomic studies of *Tylototriton* (e.g., Wang *et al.* 2018; Poyarkov *et al.* 2021a; Dufresnes & Hernandez 2022). We used the 16L-1 (forward) (5'-CTGACCGTGCAAAGGTAGCGTAATCACT-3') and 16H-1 (reverse) (5'-CTCCGGTCTGAACTCAGATCACGTAGG-3') primers to amplify the 16S rRNA fragments following Hedges (1994). For amplification and sequencing of the *ND2* gene, we used the SL-1 (forward) (5'-ATAGAGGTTCAAACCCTCTC-3') and SL-2 (reverse) (5'-TTAAAGTGTCTGGGTTGCATTCAG-3') primers of Wang *et al.* (2018). Polymerase chain reaction (PCR) conditions followed Poyarkov *et al.* (2021a). PCRs were performed in 20 µL using 50 ng genomic DNA, 10 nmol of each primer, 15 nmol of each dNTP, 50 nmol additional MgCl2, Taq PCR buffer (10 mmol / L Tris-HCl, pH 8.3, 50 mmol/L KCl, 1.1 mmol/L MgCl2, and 0.01 % gelatin), and 1 U of Taq DNA polymerase. PCR cycles included an initial denaturation step of 4 min at 94°C and 35 cycles of denaturation for 30 s at 94°C, primer annealing for 30 s at 48–58°C, and extension for 1 min 30 s at 72°C. PCR products were visualized by agarose gel electrophoresis (in the presence of ethidium bromide) and subsequently purified using 2 µL of a 1:4 dilution of ExoSapIt (Amersham, UK) per 5 µL of PCR product prior to cycle sequencing. Sequencing was performed in both directions using the same primers on an ABI3730xl automated sequencer (Applied Biosystems, USA) at Evrogen Inc., Moscow (Russia). The newly obtained sequences of *T. ziegleri s. l.* and *T. thaiorum* were aligned and deposited in GenBank under the accession numbers PQ530356–PQ530364 and PQ530959–PQ530967, as summarized in Table 2. For analysis, 44 sequences representing the remaining *Tylototriton* species available, as well as two sequences representing two species of the genus *Echinotriton* Nussbaum & Brodie, 1982 as outgroups, were obtained from GenBank (see Table 2).

To assess the whole-genome nuclear differentiation of the candidate species, we considered ddRAD-seq data obtained from nine *Tylototriton* specimens, representing the two *T. ziegleri* s. l. populations (three specimens from Cao Bang, two specimens from Ha Giang), as well as *T. thaiorum* (two topotypical samples) and *T. vietnamensis* (two samples) as outgroups. To this end, DNA samples from these specimens were extracted with the DNeasy Blood & Tissue kit (Qiagen) and included in a genomic library of 96 *Tylototriton* samples prepared with a ddRAD-seq protocol adapted from Brelsford *et al.* (2016). Library preparation involves *MseI/SbfI* enzyme restriction, ligation of barcoded adapters, and size selection of 400–500 bp fragments. The full procedures are detailed in a freely available online protocol<fnref n="1" />. The library was sequenced paired end on an DNBSEQ-G400 sequencer (PE150 kit).

<footnote n="1">¹ See: http://dx.doi.org/10.17504/protocols.io.kxygx3nzwg8j/v1.</footnote>
<table><caption>Table 2. Sequences and voucher specimens of the genus *Tylototriton* and outgroup taxa used in this study.</caption><thead><tr><th>No.</th><th>Species</th><th>Voucher Number</th><th>Locality</th><th>16S rRNA</th><th>ND2</th></tr></thead><tbody><tr><td colspan="6">Ingroup:</td></tr><tr><td>1</td><td><i>T. anguliceps</i></td><td>TBU PAE671</td><td>Thuan Chau, Son La, Vietnam</td><td>—</td><td>LC017833</td></tr><tr><td>2</td><td><i>T. anhuiensis</i></td><td>CIB 08042905-2</td><td>Yuexi Co. Anhui, China</td><td>KY800587</td><td>KY800853</td></tr><tr><td>3</td><td><i>T. asperrimus</i></td><td>CIB 70063</td><td>Longsheng Co., Guangxi, China</td><td>KY800549</td><td>KC147816</td></tr><tr><td>4</td><td><i>T. broadoridgus</i></td><td>CIB 200084</td><td>Sangzhi Co., Hunan, China</td><td>KY800570</td><td>KY800837</td></tr><tr><td>5</td><td><i>T. dabienicus</i></td><td>HNNU 1004-015</td><td>Shangcheng Co., Anhui, China</td><td>KY800607</td><td>KC147811</td></tr><tr><td>6</td><td><i>T. daloushanensis</i></td><td>CIB WG200600019</td><td>Suiyang Co., Zunyi, Guizhou, China</td><td>KY800544</td><td>KY800817</td></tr><tr><td>7</td><td><i>T. gaowangjienensis</i></td><td>JWS20210100</td><td>Guzhang Co., Hunan, China</td><td>ON764431</td><td>ON764431</td></tr><tr><td>8</td><td><i>T. hainanensis</i></td><td>CIB 20081048</td><td>Mt. Diaoluo, Hainan, China</td><td>KY800553</td><td>KC147817</td></tr><tr><td>9</td><td><i>T. himalayanus</i></td><td>CIB 201406246</td><td>Mai Pokhari, Illam, Mechi, Nepal</td><td>KY800590</td><td>KT765173</td></tr><tr><td>10</td><td><i>T. houi</i></td><td>MZL46960</td><td>Yulong, Lijiang, Yunnan, China</td><td>ON008211</td><td>—</td></tr><tr><td>11</td><td><i>T. kachinorum</i></td><td>ZMMU A5953</td><td>In Gyi Taung Mt., Indawgyi, Kachin, Myanmar</td><td>MK095618</td><td>MK097273</td></tr><tr><td>12</td><td><i>T. koliaensis sp. nov.</i></td><td>ZMMU A-8157</td><td>Kolia, Thanh Cong, Nguyen Binh, Cao Bang, Vietnam</td><td>PQ530962</td><td>PQ530359</td></tr><tr><td>13</td><td><i>T. koliaensis sp. nov.</i></td><td>ZMMU A-8158</td><td>Kolia, Thanh Cong, Nguyen Binh, Cao Bang, Vietnam</td><td>PQ530963</td><td>PQ530360</td></tr><tr><td>14</td><td><i>T. koliaensis sp. nov.</i></td><td>ZMMU A-8159</td><td>Kolia, Thanh Cong, Nguyen Binh, Cao Bang, Vietnam</td><td>PQ530964</td><td>PQ530361</td></tr><tr><td>15</td><td><i>T. koliaensis sp. nov.</i></td><td>ZMMU A-8160</td><td>Kolia, Thanh Cong, Nguyen Binh, Cao Bang, Vietnam</td><td>PQ530965</td><td>PQ530362</td></tr><tr><td>16</td><td><i>T. koliaensis sp. nov.</i></td><td>ROM26520</td><td>Quang Thanh, Nguyen Binh, Cao Bang, Vietnam</td><td>—</td><td>JQ046351</td></tr><tr><td>17</td><td><i>T. koliaensis sp. nov.</i></td><td>ROM35364</td><td>Quang Thanh, Nguyen Binh, Cao Bang, Vietnam</td><td>—</td><td>HM462056</td></tr><tr><td>18</td><td><i>T. koliaensis sp. nov.</i></td><td>ROM35330</td><td>Quang Thanh, Nguyen Binh, Cao Bang, Vietnam</td><td>—</td><td>DQ517856</td></tr><tr><td>19</td><td><i>T. koliaensis sp. nov.</i></td><td>ROM26519</td><td>Quang Thanh, Nguyen Binh, Cao Bang, Vietnam</td><td>—</td><td>JQ046337</td></tr><tr><td>20</td><td><i>T. kweichowensis</i></td><td>CIB Wg20080818014</td><td>Bijie City, Guizhou, China</td><td>KY800551</td><td>KY800823</td></tr><tr><td>21</td><td><i>T. liuyangensis</i></td><td>CSUFT 20100108</td><td>Liuyang City, Hunan, China</td><td>KY800606</td><td>KJ205598</td></tr><tr><td>22</td><td><i>T. lizhenchangi</i></td><td>KUHE 42316</td><td>Yizhang Co., Hunan, China</td><td>KY800621</td><td>KY800881</td></tr><tr><td>23</td><td><i>T. maolanensis</i></td><td>CIB Wg20090730001</td><td>Libo Co., Guizhou, China</td><td>KY800575</td><td>KY800842</td></tr><tr><td>24</td><td><i>T. ngarsuensis</i></td><td>LSUHC 13763</td><td>Ngar Su, Taunggyi Dist., Shan, Myanmar</td><td>—</td><td>MH836585</td></tr><tr><td>25</td><td><i>T. notialis</i></td><td>FMNH 271120</td><td>Boualapha Dist., Khammouan, Laos</td><td>—</td><td>HM462061</td></tr><tr><td>26</td><td><i>T. obsti</i></td><td>IEBR 4471</td><td>Xuan Nha NR, Van Ho, Son La, Vietnam</td><td>—</td><td>MT210168</td></tr><tr><td>27</td><td><i>T. panhai</i></td><td>PH019</td><td>Phu Hin Rong Kla NP, Phitsanulok, Thailand</td><td>—</td><td>AB830735</td></tr><tr><td>28</td><td><i>T. panwaensis</i></td><td>CAS 245418</td><td>Panwa, Myitkyina Dist., Kachin, Myanmar</td><td>—</td><td>KT304279</td></tr><tr><td>29</td><td><i>T. pasmansi</i></td><td>IEBR 4466</td><td>Phu Canh NR, Da Bac, Hoa Binh, Vietnam</td><td>—</td><td>MT201166</td></tr><tr><td>30</td><td><i>T. phukhaensis</i></td><td>CUMZ A-7717</td><td>Doi Phu Kha NP, Nan, Thailand</td><td>—</td><td>MN912573</td></tr><tr><td>31</td><td><i>T. podichthys</i></td><td>IEBR A.2014.1</td><td>Xam Neua, Huaphanh, Laos</td><td>—</td><td>LC017835</td></tr></tbody></table>
<table><thead><tr><th>No.</th><th>Species</th><th>Voucher Number</th><th>Locality</th><th>16S rRNA</th><th>ND2</th></tr></thead><tbody><tr><td>32</td><td>T. pseudoverrucosus</td><td>CIB WCG2012012</td><td>Ningnan Co., Liangshanyizu, Sichuan, China</td><td>KY800599</td><td>KY800860</td></tr><tr><td>33</td><td>T. pulcherrimus</td><td>CIB TY040</td><td>Lüchun Co., Yunnan, China</td><td>KY800626</td><td>KY800890</td></tr><tr><td>34</td><td>T. shanjing</td><td>KIZ 201306081</td><td>Yongde Co., Yunnan, China</td><td>KY800593</td><td>KY800856</td></tr><tr><td>35</td><td>T. shanorum (lineage 1)</td><td>CAS 230940</td><td>Taunggyi Township, Shan, Myanmar</td><td>—</td><td>AB922823</td></tr><tr><td>36</td><td>T. shanorum (lineage 2)</td><td>KUHE 42348</td><td>Pinluang Township, Shan, Myanmar</td><td>—</td><td>AB769544</td></tr><tr><td>37</td><td>T. sini</td><td>CIB XZ20091201</td><td>Xinyi City, Guangdong, China</td><td>KY800616</td><td>KY800876</td></tr><tr><td>38</td><td>T. sparreboomii</td><td>IEBR 4476</td><td>Sin Ho, Lai Chau, Vietnam</td><td>—</td><td>MT210162</td></tr><tr><td>39</td><td>T. taliangensis</td><td>CIB GG200110183</td><td>Shimian Co., Yan'an City, Sichuan, China</td><td>KY800559</td><td>KC147819</td></tr><tr><td>40</td><td>T. thaiorum</td><td>ZMMU A-7577</td><td>Pu Hoat NR, Nghe An, Vietnam</td><td>MW883482</td><td>MW883478</td></tr><tr><td>41</td><td>T. thaiorum</td><td>ZMMU NAP-13138</td><td>Ban Vin, Thuong Xuan, Thanh Hoa, Vietnam</td><td>PQ530966</td><td>PQ530363</td></tr><tr><td>42</td><td>T. thaiorum</td><td>ZMMU NAP-13139</td><td>Ban Vin, Thuong Xuan, Thanh Hoa, Vietnam</td><td>PQ530967</td><td>PQ530364</td></tr><tr><td>43</td><td>T. tongziensis</td><td>CIB WH10001</td><td>Wufeng Co., Hubei, China</td><td>KY800600</td><td>KY800863</td></tr><tr><td>44</td><td>T. umphangensis</td><td>CUMZ-A-8243</td><td>Umphang WS., Tak, Thailand</td><td>—</td><td>OK092618</td></tr><tr><td>45</td><td>T. uyenoi</td><td>KUHE 19037</td><td>Doi Inthanon, Chiang Mai, Thailand</td><td>—</td><td>AB830730</td></tr><tr><td>46</td><td>T. verrucosus</td><td>CIB TSHS1</td><td>Longchuan Co., Dehong, Yunnan, China</td><td>KY800581</td><td>KY800847</td></tr><tr><td>47</td><td>T. vietnamensis</td><td>IEBR A.3674</td><td>Tay Yen Tu, Bac Giang, Vietnam</td><td>KY800614</td><td>KY800874</td></tr><tr><td>48</td><td>T. wenxianensis (lineage 1)</td><td>CIB 20090527</td><td>Wenxian Co., Gansu, China</td><td>KY800579</td><td>KC147813</td></tr><tr><td>49</td><td>T. wenxianensis (lineage 2)</td><td>CIB 20070638</td><td>Qingchuan Co., Sichuan, China</td><td>KY800543</td><td>KY800816</td></tr><tr><td>50</td><td>T. wenxianensis (lineage 3)</td><td>CIB 20090601</td><td>Wangcang Co., Sichuan, China</td><td>KY800571</td><td>KY800838</td></tr><tr><td>51</td><td>T. wenxianensis (lineage 4)</td><td>CIB 20080002</td><td>Yunyang Co., Chongqing, China</td><td>KY800540</td><td>KY800813</td></tr><tr><td>52</td><td>T. wenxianensis (lineage 5)</td><td>CIB 20080003</td><td>Yunyang Co., Chongqing, China</td><td>KY800541</td><td>KY800814</td></tr><tr><td>53</td><td>T. yangi</td><td>KUHE 42282</td><td>Pingbian Co., Yunnan, China</td><td>KY800624</td><td>KY800887</td></tr><tr><td>54</td><td>T. zaimeng</td><td>MZMU-2947</td><td>Zaimeng Lake, Kangpokpi Dist., Manipur, India</td><td>OQ996133</td><td>OR039162</td></tr><tr><td>55</td><td>T. ziegleri</td><td>VNMN1200A</td><td>Quan Ba, Ha Giang, Vietnam</td><td>—</td><td>JQ046350</td></tr><tr><td>56</td><td>T. ziegleri</td><td>KUHE55078</td><td>Quan Ba, Ha Giang, Vietnam</td><td>—</td><td>AB769541</td></tr><tr><td>57</td><td>T. ziegleri</td><td>KUHE55077</td><td>Quan Ba, Ha Giang, Vietnam</td><td>—</td><td>AB769540</td></tr><tr><td>58</td><td>T. ziegleri</td><td>ZMMU A-8166</td><td>Bat Dai Son NR, Thanh Van, Quan Ba, Ha Giang, Vietnam</td><td>PQ530959</td><td>PQ530356</td></tr><tr><td>59</td><td>T. ziegleri</td><td>ZMMU A-8167</td><td>Bat Dai Son NR, Thanh Van, Quan Ba, Ha Giang, Vietnam</td><td>PQ530960</td><td>PQ530357</td></tr><tr><td>60</td><td>T. ziegleri</td><td>ZMMU A-8168</td><td>Bat Dai Son NR, Thanh Van, Quan Ba, Ha Giang, Vietnam</td><td>PQ530961</td><td>PQ530358</td></tr><tr><td>61</td><td>T. ziegleri</td><td>VNUH HG.081</td><td>Quan Ba, Ha Giang, Vietnam</td><td>KY800610</td><td>KY800870</td></tr><tr><td>62</td><td>T. ziegleri</td><td>VNUH HG.082</td><td>Quan Ba, Ha Giang, Vietnam</td><td>KY800611</td><td>KY800871</td></tr><tr><td>63</td><td>T. ziegleri</td><td>VNMN3390</td><td>Quan Ba, Ha Giang, Vietnam</td><td>KY800625</td><td>KY800889</td></tr><tr><td>64</td><td>T. ziegleri</td><td>KIZ20210504</td><td>Malipo, Yunnan, China</td><td>—</td><td>ON159334</td></tr><tr><td>65</td><td>T. ziegleri</td><td>KIZ20210505</td><td>Malipo, Yunnan, China</td><td>—</td><td>ON159333</td></tr><tr><td>66</td><td>T. ziegleri</td><td>KIZ2015000082</td><td>Malipo, Yunnan, China</td><td>KY398015</td><td>KY398015</td></tr><tr><td colspan="6"><strong>Outgroup:</strong></td></tr><tr><td>67</td><td>Echinotriton chinhaiensis</td><td>CIB ZHJY1</td><td>Zhenhai Co., Zhejiang, China</td><td>KY800627</td><td>KY800891</td></tr><tr><td>68</td><td>Echinotriton raffaellii</td><td>MVZ 232187</td><td>Tokunoshima, Kagoshima, Japan</td><td>EU880314</td><td>EU880314</td></tr></tbody></table>
Phylogenetic analyses of mtDNA data

Mitochondrial analyses covered partial fragments of the genes *ND2* and 16S rRNA (total length of up to 1665 bp) sequenced in 68 specimens, including 66 *Tylototriton* (representing 39 species prior to our study), and two closely related outgroup species, namely *E. chinhaiensis* (Chang, 1932) and *E. raffaellii* Hernandez & Dufresnes, 2022 *in* Dufresnes & Hernandez (2022). Information on voucher specimens and GenBank accession numbers is provided in Table 2. Nucleotide sequences were initially aligned in MAFFT v.6 (Katoh *et al.* 2002) with default parameters and then manually checked and adjusted in BioEdit 7.0.5.2 (Hall 1999). The dataset was divided into four partitions: three codon partitions for the *ND2* gene and a single partition for 16S rRNA; their optimal evolutionary models were determined with MODELTEST v. 3.06 (Posada & Crandall 1998) according to the Akaike information criterion (AIC). For the *ND2* gene, the HKY+G model was considered the best fit for the first and second codon partitions, and the J2+G model was considered the best fit for the third codon partition, while the TVM+G model was considered the best fit for the 16S rRNA partition. Mean uncorrected genetic distances (*p*-distances) between sequences were calculated in MEGA 7.0 (Kumar *et al.* 2016), excluding missing data and gaps.

The matrilineal genealogy was reconstructed from the *ND2*-16S partitioned alignment through Bayesian inference (BI) and maximum likelihood (ML) methods. The BI analyses were conducted in MrBayes v3.1.2 (Ronquist & Huelsenbeck 2003). Metropolis-coupled Markov chain Monte Carlo (MCMCMC) analyses were run with one cold chain and three heated chains for twenty million generations, sampled every 2000 generations. Five independent MCMCMC runs were performed, each ran for 100 million generations, sampled every 10,000 generations, with the first 10 % of the trees discarded as burn-in. We checked the convergence of the runs and that the effective sample sizes (ESS) of all parameters were above 200 with TRACER v1.6 (Rambaut *et al.* 2014). Posterior probabilities (BI PP) were considered to assess confidence in node support (Huelsenbeck & Ronquist 2001). Nodes with PP over 0.95 were regarded as significantly supported; nodes between 0.95 and 0.90 were regarded as tendencies; nodes below 0.90 were regarded as unsupported. The ML analysis was conducted with IQ-TREE (Trifinopoulos *et al.* 2016) using an online webserver. For node support, we computed 1000 bootstrap pseudoreplicates with the ultrafast bootstrap approximation (UFBS; Hoang *et al.* 2018). Nodes with UFBS over 95 were regarded as significantly supported; nodes with UFBS between 94 and 90 were regarded as well supported, and nodes with UFBS between 89 and 70 were regarded as tendencies (Bui *et al.* 2013).

ddRAD-seq analyses

Single-end reads were demultiplexed with STACKS 2.63 (Catchen *et al.* 2013) and the denovo_map.pl pipeline was applied for RAD loci construction, assembly, and cataloguing (default -m, -n, and -M values). A supermatrix alignment (phylip format, including variable and invariable sites) was obtained with the module population of STACKS by considering the loci sequenced in all 9 samples (-p 9 -phylip-var-all). The obtained alignment featured 3901 concatenated loci for a total length of 556,209 bp, and was converted to fasta format using an online tool<fnref n="2" />. A maximum-likelihood tree was built with IQtree 1.6

<footnote n="2">² See: https://www.hiv.lanl.gov/content/sequence/FORMAT_CONVERSION/form.html.</footnote>
(Trifinopoulos *et al.* 2016) using the model finder tool to determine and apply the optimal model of sequence evolution, and computing 1000 UFBS to assess node support.

## External morphology

Specimens of *Tylototriton* spp. were photographed in life and after preservation. The sex and maturity of the specimens were confirmed by minor dissections. Measurements were taken with a digital caliper to the nearest 0.01 mm, subsequently rounded to 0.1 mm, and using an Olympus stereoscopic light binocular microscope when necessary. Morphometric measurements followed Poyarkov *et al.* (2021a) and included the following 24 characters measured in 17 *T. ziegleri s. l.* specimens for descriptive and statistical comparisons: (1) SVL (snout-vent length) from tip of snout to anterior tip of vent; (2) HL (head length); (3) HW (head width); (4) MXHW (maximum head width); (5) IND (internarial distance); (6) AGD (axilla-groin distance); (7) TRL (trunk length); (8) TAL (tail length) from anterior tip of vent to tail tip; (9) VL (vent length); (10) FLL (forelimb length); (11) HLL (hindlimb length); (12) VTW (vomerine tooth series width): greatest width of vomerine tooth series; (13) VTL (vomerine tooth series length): greatest length of vomerine tooth series; (14) LJL (lower jaw length from tip of lower jaw to articulation of upper and lower jaws); (15) SL (snout length from tip of snout to anterior tip of upper eyelid); (16) IOD (minimum interorbital distance); (17) UEW (maximum upper eyelid width); (18) UEL (upper eyelid length, distance between anterior and posterior angles); (19) OL (orbit length); (20) BTAW (basal tail width at level of anterior tip of cloaca); (21) MTAW (tail width at mid-level of tail); (22) MXTAH (maximum tail height); (23) MTAH (tail height at mid-level of tail); and (24) ON (orbitonarial distance). To characterize the holotype of the new species (see Results), we additionally measured the following 12 characters following Poyarkov *et al.* (2012; 2021a): (25) ICD (intercanthal distance); (26) CW (chest width); (27) NSD (nostril-snout distance); (28) 1FL (first finger length from base to tip); (29) 2FL (second finger length from base to tip); (30) 3FL (third finger length from base to tip); (31) 4FL (fourth finger length from base to tip); (32) 1TL (first toe length from base to tip); (33) 2TL (second toe length from base to tip); (34) 3TL (third toe length from base to tip); (35) 4TL (fourth toe length from base to tip); and (36) 5TL (fifth toe length from base to tip).

To find statistically significant morphological differences between the Ha Giang and Cao Bang specimens of *T. ziegleri s. l.*, an analysis of variance (ANOVA) was performed on SVL, as well as on each morphometric characters separately, after correction by body size (character/SVL, thereafter body ratios). To assess how specimens from the two *T. ziegleri s. l.* populations segregate in the morphospace, we performed a Principal Component Analysis (PCA) on the 24 variables following Nishikawa *et al.* (2007). Specifically, we considered body ratios for characters 2–24 and searched for colinear variables by pairwise correlations, removing one variable for each correlated pair to exclude possible overweighting effects. Retained variables were scaled (log-transformed) prior to analysis. Statistical analyses were carried out using Statistica 8.0 (StatSoft, Tulsa, OK, USA).

The diagnosis of the subgenus *Yaotriton* and external morphological characters used for interspecific comparisons were taken from original descriptions and taxonomic reviews of the genus, including Bernardes *et al.* (2020), Böhme *et al.* (2005), Chen *et al.*
(2010), Fei & Ye (2016), Hou *et al.* (2012), Li *et al.* (2020b), Lyu *et al.* (2021), Liu *et al.* (2022), Nishikawa *et al.* (2013a, 2013b, 2020), Phimmachak *et al.* (2015), Phung *et al.* (2023), Poyarkov *et al.* (2021a), Qian *et al.* (2017), Shen *et al.* (2012), Stuart *et al.* (2010) and Yang *et al.* (2014).

Skeletal morphology

To study the skeletal morphology of *T. ziegleri* s. l., notably to compare the two
candidate species, we examined X-ray projections for seven specimens from Cao Bang
(ZMMU A-8157–8163) and eight specimens from Ha Giang (*T. ziegleri* s. str., ZMMU
A-8166–8173), and performed detailed micro-CT scanning for one specimen of each
(ZMMU A-8157 and ZMMU A-8169). The scans were obtained with a SkyScan 1272
microtomograph (Bruker, Billerica, USA) equipped with a Hamamatsu L10101-67
source (Hamamatsu Photonics, Hamamatsu, Japan) and a Ximea xiRAY16 camera
(Ximea GmbH, Münster, Germany) at the Biological Faculty of Moscow University.
The specimens were scanned at a source voltage of 100 kV and a source current of 100
µA, without an X-ray filter. The samples were rotated 360° around the vertical axis with
a rotation step of 0.1, with 4 frames averaging. From the obtained X-ray projections,
stacks of virtual cross sections through the specimens’ skeletal structures were
reconstructed with the software NRecon® (Bruker micro-CT, Kontich, Belgium). The
resulting stack consisted of 3599 images of 4,504×4,504 resolution and a pixel size of
5.42 µm. This stack of images was imported into the three-dimensional visualization
software package Avizo 8.1. for subsequent processing and examination of osteological
traits. The obtained scans were deposited in MorphoSource<fnref n="3" />.

RESULTS

Characteristics of the genetic datasets

The final alignment of the *ND2* gene contained 1157 nucleotides, of which 1002 sites were conserved and 155 sites were variable, including 94 parsimony-informative sites. The transition-transversion bias (R) was estimated to 4.77. Nucleotide frequencies were 37.59 % (A), 23.73 % (T), 28.06 % (C), and 10.61 % (G). The final alignment of the 16S rRNA gene contained 508 nucleotides, of which 484 sites were conserved and 22 sites were variable, including 13 parsimony-informative sites. The transition-transversion bias (R) was estimated to 2.05. Nucleotide frequencies were 37.64 % (A), 24.89 % (T), 20.31 % (C), and 17.16 % (G) (all data given for the ingroup only).

The ddRAD-seq catalog contained 801,425 loci with a mean effective per-sample coverage of 10.0×. Among them, 3901 loci of ~142 bp (totaling 556,209 bp) were sequenced in all nine individuals.

<footnote n="3">³ See: https://www.morphosource.org/concern/media/000665372 and
https://www.morphosource.org/concern/media/000665321.</footnote>
## MtDNA genealogy

Our mtDNA-based genealogy of *Tylototriton* included sequences of 39 out of the 41 species recognized in the genus prior to our study (see Table 2). The two exceptions are the recently described *T. ngoclinhensis* Phung, Pham, Nguyen, Ninh, Nguyen, Bernardes, Le, Ziegler & Nguyen, 2023 and *T. soimailai* Pomchote, Peerachidacho, Khonsue, Sapewisut, Hernandez, Phalaraksh, Siriput & Nishikawa, 2024, for the following reasons. The GenBank accession numbers LC575221–LC575223 given by Phung *et al.* (2023) as ND2 sequences of *T. ngoclinhensis* correspond to D-loop sequences of the chicken *Gallus gallus* (Linnaeus, 1758). In parallel, *T. soimailai* was published while this manuscript was under review and its associated sequences had not yet been released.

BI and ML phylogenetic analyses resulted in almost identical tree topologies, in which the monophyly of the genus *Tylototriton* in respect to its sister genus *Echinotriton* is always strongly supported (ML UFBS/BI PP: 100/1.0, hereafter reported in this format; see Fig. 2). Our tree is largely consistent with the *Tylototriton* mitochondrial trees presented in earlier works based on various markers and sample sets (Wang *et al.* 2018; Poyarkov *et al.* 2021a; Lyu *et al.* 2021; Dufresnes & Hernandez 2022; Decemson *et al.* 2023). In particular, we recovered five main clades grouped into two major groups that correspond to the subgenera *Tylototriton* and *Yaotriton* (Fig. 2).

The monophyly of the subgenus *Yaotriton* is poorly supported by the ML analysis but got high support in the BI analysis (Fig. 2, 88/1.0). This group includes three main clades, all highly supported:

1) Clade I (100/1.0) regroups the members of the *T. asperrimus* species group from China, northern Vietnam, and Laos, and includes nine described species: *T. asperrimus*; *T. hainanensis* Fei, Ye & Yang, 1984; *T. notialis* Stuart, Phimmachak, Sivongxay & Robichaud, 2010; *T. pasmansi* Bernardes, Le, Nguyen, Pham, Pham, Nguyen & Ziegler in Bernardes *et al.* (2020); *T. obsti* Bernardes, Le, Nguyen, Pham, Pham, Nguyen & Ziegler in Bernardes *et al.* (2020); *T. sini*; *T. sparreboomi* Bernardes, Le, Nguyen, Pham, Pham, Nguyen & Ziegler in Bernardes *et al.* (2020); *T. thaiorum*; *T. ziegleri s. str.* from Ha Giang Province. It further distinguishes the population from Cao Bang Province, Vietnam, previously assigned to *T. ziegleri* (noted *Tylototriton* sp.).

2) Clade II (98/1.0) regroups the members of the *T. wenxianensis* species group from China, and includes ten species: *T. anhuiensis* Qian, Sun, Li, Guo, Pan, Kang, Wang, Jiang, Wu & Zhang, 2017; *T. broadoridgus* Shen, Jiang & Mo, 2012; *T. dabienicus* Chen, Wang & Tao, 2010; *T. daloushanensis* Zhou, Xiao & Luo *in Luo* *et al.* (2022); *T. gaowangjienensis* Huang, Xiang, Wu, Zhang, Zhang, Wang, Lan, Huang, Jiang & Jiang, 2024; *T. liuyangensis* Yang, Jiang, Shen & Fei, 2014; *T. lizhenchangi* Hou, Zhang, Jiang, Li & Lu *in Hou* *et al.* (2012); *T. maolanensis* Li, Wei, Cheng, Zhang & Wang, 2020; *T. tongziensis* Li, Liu, Shi, Wei & Wang, 2022; and *T. wenxianensis*. The analysis confirms the deep divergence within *T. wenxianensis s. l.*, which includes five distinct mtDNA lineages (noted *T. wenxianensis* lineages 1–5, see Fig. 2).

3) Clade III (95/1.0), to which we refer herein as the *T. vietnamensis* species group, includes two species from northern Vietnam and north-eastern Thailand and
adjacent Laos: *T. panhai* Nishikawa, Khonsue, Pomchote & Matsui, 2013 and *T. vietnamensis*.

The monophyly of the subgenus *Tylototriton* is strongly supported by both ML and BI analyses (100/1.0, Fig. 2). This group includes two strongly supported clades:

4) Clade IV (100/1.0) regroups the members of the *T. verrucosus* Anderson, 1871 species group from Nepal, Myanmar, China, and northern Indochina, and includes 17 species: *T. anguliceps* Le, Nguyen, Nishikawa, Nguyen, Pham, Matsui, Bernardes & Nguyen, 2015; *T. himalayanus* Khatiwada, Wang, Ghimire, Vasudevan, Paudel & Jiang, 2015; *T. houi* Hernandez & Dufresnes *in* Dufresnes & Hernandez, 2022; *T. kachinorum* Than Zaw, Lay, Pawangkhanant, Gorin & Poyarkov, 2019; *T. kweichowensis* Fang & Chang, 1932; *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. pulcherrimus* Hou, Zhang, Li & Lu, 2012; *T. shanjing* Nussbaum, Brodie & Yang, 1995; *T. shanorum* Nishikawa, Matsui & Rao, 2014; *T. umphangensis* Pomchote, Peerachidacho, Hernandez, Sapewisut, Khonsue, Thammachoti & Nishikawa, 2021; *T. uyenoi* Nishikawa, Khonsue, Pomchote & Matsui, 2013; *T. verrucosus* Anderson, 1871; *T. yangi* Hou, Zhang, Zhou, Li & Lu *in* Hou *et al.* (2012); and *T. zaimeng* Decemson, Lalremsanga, Elangbam, Mathipi, Shinde, Purkayastha, Arkhipov, Bragin & Poyarkov, 2023. In our tree, *T. shanorum* is paraphyletic with respect to *T. ngarsuensis* (Fig. 2).

5) Clade V (100/1.0), to which we refer herein as the *T. taliangensis* Liu, 1950 species group, includes two species from China: *T. pseudoverrucosus* Hou, Gu, Zhang, Zeng & Lu *in* Hu *et al.* (2012) and *T. taliangensis*.

Genealogical relationships within the *T. asperrimus* species group (Clade I) are not entirely resolved. The sister relationship of *T. hainanensis* from Hainan Island and *T. sini* from the mainland of southern China is robustly supported (97/1.0) but the clade regrouping the remaining members of the group is not (84/0.96; Fig. 2). *Tylototriton pasmansi* and *T. obsti* from northern Vietnam are robustly retrieved as sister taxa (98/1.0) and branches with *T. asperrimus* from southern China (98/1.0; Fig. 2). The sister relationship of *T. notialis* from central Laos and *T. thaiorum* from northern and central Vietnam is strongly supported (99/1.0), while the phylogenetic position of *T. sparreboomi* remains unresolved (Fig. 2). Our new samples from a previously unstudied population in Thanh Hoa Province (locality 11; Fig. 1–2) are grouped with the holotype specimen of *T. thaiorum* from Nghe An Province (locality 11; Fig. 1–2), with full support (100/1.0).

The populations previously attributed to *T. ziegleri* form a well-supported clade (100/1.0) that contains two divergent fully supported mtDNA lineages (100/1.0; Fig. 2). The first lineage includes our new samples from Ha Giang Province (locality 6; Fig. 1–2), which share identical mtDNA-haplotypes with the type specimens of *T. ziegleri* (locality 5; Fig. 1–2), as well as three specimens of *T. ziegleri* from Malipo, Yunnan Province of China (locality 9; Fig. 1–2). The second lineage includes our newly-collected specimens from Cao Bang Province (locality 1; Fig. 1–2) and published sequences from a nearby locality of that province (locality 2; Fig. 1–2).
<figure><img src="image_2.png" /><figcaption>Figure 2. Mitochondrial tree of *Tylototriton* obtained from the ML phylogenetic analysis of the concatenated *ND2* and 16S rRNA gene fragments (1665 bp). See Table 2 for voucher specimen information and GenBank accession numbers. Red, blue and green colors denote the new species *Tylototriton koliaensis* sp. nov., *T. ziegleri* s. str., and *T. thaiorum*, respectively (color codes as in Fig. 1). Numbers in brackets correspond to locality numbers (Fig. 1, Table 1). Numbers at tree nodes correspond to UFBS/PP support values. Photographs by Nikolay A. Poyarkov.</figcaption></figure>
*Genetic distances in mtDNA genes*

The pairwise uncorrected genetic *p*-distances at the *ND2* mtDNA sequences between *Tylototriton* sp. from Cao Bang and other members of the *T. asperrimus* species group varied from 2.67 % (between *Tylototriton* sp. and its sister species *T. ziegleri s. str.*) to 5.13 % (between *Tylototriton* sp. and *T. sini* and *T. thaiorum*) (see Table 3). The pairwise uncorrected genetic *p*-distances in the 16S rRNA gene varied from 1.12 % (between *Tylototriton* sp. and its sister species *T. ziegleri s. str.*) to 2.02 % (between *Tylototriton* sp. and *T. asperrimus*) (see Table 3).

There was no variation in the 16S rRNA gene among the Cao Bang specimens. *ND2* sequence variation was much lower (0.02 % of substitutions) within *Tylototriton* sp. from Cao Bang than within *T. ziegleri s. str.* (0.16 %) (Table 3).

*ddRAD-seq phylogenomics*

The ML tree constructed from the ddRAD-seq loci retrieved the four examined *Tylototriton* species/populations included in the analysis as fully supported divergent nuclear lineages (UFBS: 100; Fig. 3). Specifically, the tree distinguishes *T. vietnamensis*, *T. thaiorum*, and a clade regrouping the *T. ziegleri s. l.* specimens in two diverged groups: those from Ha Giang (*T. ziegleri s. str.*) vs. those from Cao Bang (*Tylototriton* sp.).

*Morphological differentiation*

As only a single female could be measured, morphological comparisons of the Ha Giang (*T. ziegleri s. str.*) and Cao Bang (*Tylototriton* sp.) specimens of *T. ziegleri s. l.* were performed only for males. The ANOVA retrieved a statistically significant difference in body size, with the Ha Giang specimens being larger (SVL: 62.4 ± 4.5 mm, n = 9; hereafter mean values ± standard deviation are given, see Table 4 for ranges) than the Cao Bang specimens (58.5 ± 2.0 mm, n = 8). Several body ratios also significantly differ (see Table 4). Specifically, the Cao Bang specimens have proportionally wider heads (MXHW/SVL: 0.29 ± 0.01 vs. 0.27 ± 0.01), greater distance between nostrils (IND/SVL: 0.10 ± 0.006 vs. 0.08 ± 0.004), shorter axilla-groin distance (AGD/SVL: 0.50 ± 0.01 vs. 0.52 ± 0.02), longer forelimbs (FLL/SVL: 0.36 ± 0.03 vs. 0.34 ± 0.02), longer hindlimbs (HLL/SVL: 0.38 ± 0.03 vs. 0.35 ± 0.01), shorter upper eyelids (UEL/SVL: 0.068 ± 0.003 vs. 0.072 ± 0.003), as well as longer (TAL/SVL: 1.22 ± 0.07 vs. 1.12 ± 0.02), wider (BTAW/SVL: 0.16 ± 0.008 vs. 1.13 ± 0.01; MTAW/SVL: 0.05 ± 0.004 vs. 0.04 ± 0.001) and higher (MTAH/SVL: 0.13 ± 0.008 vs. 0.10 ± 0.013) tails than in the Ha Giang specimens. In *Tylototriton* newts, tail length and width may fluctuate due to changes in tail shape during the breeding season and tail regeneration after injuries (our observations), so the latter variation should be interpreted with caution. Nevertheless, both the Cao Bang and Ha Giang specimens were collected at the peak of the breeding season, which should mitigate some of these effects.
<table><caption>Table 3. Genetic differentiation among the *Tylototriton asperrimus* species group. Pairwise uncorrected *p*-distance (percentage) are given for the *ND2* (below diagonal) and the 16S rRNA (above diagonal) sequences; intraspecific pairwise *ND2* *p*-distances are given on the diagonal in bold when computable.</caption><thead><tr><th>Species</th><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th><th>6</th><th>7</th><th>8</th><th>9</th><th>10</th></tr></thead><tbody><tr><td>1 <i>T. koliaensis sp. nov.</i></td><td><strong>0.02</strong></td><td>1.12</td><td>1.13</td><td>1.57</td><td>1.57</td><td>—</td><td>2.02</td><td>1.57</td><td>—</td><td>—</td></tr><tr><td>2 <i>T. ziegleri s. str.</i></td><td>2.67</td><td><strong>0.16</strong></td><td>0.68</td><td>0.9</td><td>1.35</td><td>—</td><td>1.35</td><td>1.35</td><td>—</td><td>—</td></tr><tr><td>3 <i>T. hainanensis</i></td><td>4.26</td><td>4.35</td><td>—</td><td>0.9</td><td>1.35</td><td>—</td><td>1.35</td><td>1.8</td><td>—</td><td>—</td></tr><tr><td>4 <i>T. sini</i></td><td>5.13</td><td>5.60</td><td>3.57</td><td>—</td><td>1.35</td><td>—</td><td>1.35</td><td>1.8</td><td>—</td><td>—</td></tr><tr><td>5 <i>T. thaiorum</i></td><td>5.13</td><td>4.76</td><td>4.64</td><td>6.09</td><td><strong>0.00</strong></td><td>—</td><td>1.8</td><td>2.25</td><td>—</td><td>—</td></tr><tr><td>6 <i>T. notialis</i></td><td>4.75</td><td>4.61</td><td>4.64</td><td>5.51</td><td>3.00</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>7 <i>T. asperrimus</i></td><td>4.55</td><td>4.47</td><td>5.02</td><td>5.60</td><td>5.31</td><td>4.73</td><td>—</td><td>1.35</td><td>—</td><td>—</td></tr><tr><td>8 <i>T. obsti</i></td><td>4.65</td><td>4.45</td><td>4.93</td><td>5.89</td><td>4.73</td><td>4.35</td><td>3.38</td><td>—</td><td>—</td><td>—</td></tr><tr><td>9 <i>T. pasmansi</i></td><td>4.46</td><td>4.88</td><td>4.93</td><td>5.51</td><td>4.73</td><td>4.54</td><td>3.29</td><td>3.09</td><td>—</td><td>—</td></tr><tr><td>10 <i>T. sparreboomi</i></td><td>4.26</td><td>4.40</td><td>4.44</td><td>5.22</td><td>4.64</td><td>4.54</td><td>4.15</td><td>4.06</td><td>4.25</td><td>—</td></tr></tbody></table>

In parallel, the PCA suggests that the Cao Bang and Ha Giang specimens do not overlap in the morphospace (Fig. 4). PC1, which accounts for 32.59 % of the total variance, loads most heavily on FLL and HLL. PC2, which accounts for 18.44 % of the total variance, loads most heavily on SVL, AGD, IND, and MXHW. Overall, the morphometric data was distributed in two clusters that correspond to the two studied populations, with the Ha Giang populations showing generally more variation (Fig. 4). The relatively low sample sizes might underestimate the actual variation (and potential morphometric overlap) of these populations, so these results should also be interpreted with caution.

<figure><img src="image_3.png" /><figcaption>Figure 3. Nuclear tree obtained from the ML phylogenetic analysis of 3901 genomic loci (556,209 bp) for the two candidate species of *T. ziegleri* s. l., as well as *T. thaiorum* and *T. vietnamensis* as outgroups. Color codes as in Fig. 1–2; node labels are UFBS support values.</figcaption></figure>
<table><caption>Table 4. Measurements (in mm) of the *Tylototriton koliaensis* sp. nov. type series and topotypes of *T. ziegleri* s. str. Abbreviations are defined in the Methods. Characters which body ratios significantly differed between species are noted with an asterisk (*) with their values in bold font.</caption><thead><tr><th rowspan="3">Character</th><th colspan="2">Tylototriton koliaensis sp. nov</th><th colspan="2">Tylototriton ziegleri s. str.</th></tr><tr><th>Phia Oac-Phia Den NP, Cao Bang Province, Vietnam</th><th>Quan Ba, Ha Giang Province, Vietnam</th><th>Phia Oac-Phia Den NP, Cao Bang Province, Vietnam</th><th>Quan Ba, Ha Giang Province, Vietnam</th></tr><tr><th>ZMMU A-8157<br>Holotype female (n = 1)</th><th>All paratype males<br>Range; Mean ± SD (n = 8)</th><th>All topotype males<br>Range; Mean ± SD (n = 9)</th><th>All topotype males<br>Range; Mean ± SD (n = 9)</th></tr></thead><tbody><tr><td>SVL (mm)*</td><td>73.0</td><td><strong>56.0-61.4</strong></td><td><strong>58.5±2.0</strong></td><td><strong>52.7-67.4</strong></td><td><strong>62.4±4.5</strong></td></tr><tr><td>HL (mm)</td><td>14.5</td><td>12.0-14.2</td><td>13.1±0.6</td><td>12.3-15.9</td><td>14.0±1.0</td></tr><tr><td>HW (mm)</td><td>14.6</td><td>12.1-13.0</td><td>12.6±0.3</td><td>11.8-14.6</td><td>13.2±1.0</td></tr><tr><td>MXHW (mm)*</td><td>17.8</td><td><strong>15.8-18.5</strong></td><td><strong>16.8±0.8</strong></td><td><strong>15.1-18.3</strong></td><td><strong>16.8±1.1</strong></td></tr><tr><td>IND (mm)*</td><td>6.0</td><td><strong>5.0-5.9</strong></td><td><strong>5.5±0.3</strong></td><td><strong>4.0-5.7</strong></td><td><strong>5.0±0.5</strong></td></tr><tr><td>AGD (mm)*</td><td>39.3</td><td><strong>27.8-31.1</strong></td><td><strong>29.5±1.1</strong></td><td><strong>26.5-35.6</strong></td><td><strong>32.5±2.8</strong></td></tr><tr><td>TRL (mm)</td><td>60.5</td><td>45.5-52.2</td><td>48.3±2.5</td><td>44.3-55.2</td><td>51.0±3.3</td></tr><tr><td>TAL (mm)*</td><td>71.1</td><td><strong>66.2-76.8</strong></td><td><strong>71.1±4.1</strong></td><td><strong>57.3-76.6</strong></td><td><strong>69.5±5.7</strong></td></tr><tr><td>VL (mm)</td><td>6.1</td><td>5.2-6.7</td><td>6.1±0.5</td><td>5.5-17.2</td><td>8.2±3.8</td></tr><tr><td>FLL (mm)*</td><td>22.8</td><td><strong>19.3-22.9</strong></td><td><strong>21.2±1.4</strong></td><td><strong>18.4-23.0</strong></td><td><strong>21.2±1.6</strong></td></tr><tr><td>HLL (mm)*</td><td>24.6</td><td><strong>20.7-24.0</strong></td><td><strong>22.0±1.3</strong></td><td><strong>19.3-24.3</strong></td><td><strong>22.1±1.4</strong></td></tr><tr><td>LJL (mm)</td><td>13.7</td><td>12.0-13.8</td><td>12.6±0.5</td><td>11.7-15.4</td><td>13.6±1.2</td></tr><tr><td>SL (mm)</td><td>6.6</td><td>5.6-6.5</td><td>6.1±0.3</td><td>5.8-7.2</td><td>6.5±0.5</td></tr><tr><td>IOD (mm)</td><td>8.3</td><td>7.0-7.5</td><td>7.3±0.2</td><td>6.4-8.3</td><td>7.3±0.7</td></tr><tr><td>UEW (mm)</td><td>2.5</td><td>1.9-2.2</td><td>2.0±0.1</td><td>2.0-2.5</td><td>2.2±0.2</td></tr><tr><td>UEL (mm)*</td><td>4.5</td><td><strong>3.7-4.2</strong></td><td><strong>4.0±0.2</strong></td><td><strong>3.6-5.0</strong></td><td><strong>4.4±0.4</strong></td></tr><tr><td>OL (mm)</td><td>3.0</td><td>2.7-3.2</td><td>2.9±0.1</td><td>2.9-3.8</td><td>3.3±0.3</td></tr><tr><td>BTAW (mm)*</td><td>7.9</td><td><strong>8.2-9.8</strong></td><td><strong>9.2±0.5</strong></td><td><strong>6.4-9.3</strong></td><td><strong>7.9±1.0</strong></td></tr><tr><td>MTAW (mm)*</td><td>2.9</td><td><strong>2.4-3.0</strong></td><td><strong>2.7±0.2</strong></td><td><strong>2.1-2.6</strong></td><td><strong>2.4±0.2</strong></td></tr><tr><td>MXTAH (mm)*</td><td>9.5</td><td><strong>8.6-9.9</strong></td><td><strong>9.3±0.5</strong></td><td><strong>7.5-10.2</strong></td><td><strong>9.2±1.0</strong></td></tr><tr><td>MTAH (mm)*</td><td>7.0</td><td><strong>6.8-7.7</strong></td><td><strong>7.3±0.4</strong></td><td><strong>5.3-7.5</strong></td><td><strong>6.5±0.9</strong></td></tr><tr><td>ON (mm)</td><td>4.1</td><td>3.6-3.8</td><td>3.7±0.1</td><td>3.5-5.1</td><td>4.2±0.5</td></tr><tr><td>HW/HL</td><td>1.01</td><td>0.92-1.05</td><td>0.96±0.05</td><td>0.89-1.01</td><td>0.94±0.44</td></tr><tr><td>FLL/SVL*</td><td>0.31</td><td><strong>0.33-0.40</strong></td><td><strong>0.36±0.03</strong></td><td><strong>0.32-0.37</strong></td><td><strong>0.34±0.01</strong></td></tr><tr><td>HLL/SVL*</td><td>0.34</td><td><strong>0.34-0.41</strong></td><td><strong>0.38±0.02</strong></td><td><strong>0.33-0.37</strong></td><td><strong>0.35±0.01</strong></td></tr></tbody></table>

<figure><img src="image_4.png" /><figcaption>Figure 4. Scatterplot of the first and second PCA factors built from SVL and 23 body ratios measured in adult male newt specimens of *Tylototriton koliaensis* sp. nov. and *T. ziegleri* s. str. Color-code as in Fig. 1–3.</figcaption></figure>
Examination of the cranial morphology of specimens from both populations suggest several qualitative osteological characters that appear to be diagnostic between them (see Fig. 5). Specimens from Cao Bang (*Tylototriton* sp.) generally have wider vomerine tooth series, in the form of two gently curving diverging lines with the anterior corner of the series forming a sharp angle (Fig. 5B), while in most Ha Giang specimens (*T. ziegleri* s. str.), the anterior corner of the vomerine tooth series is rounded, the anterior parts of their branches are located close to each other, are almost parallel, and diverge in the posterior one-third of their length, forming a blunt angle (Fig. 5E). Other important differences in skull morphology include an excavation pattern on the processus oticum of pterygoid, the shape of the dorsal grove on the processus palatinum of pterygoid, the shape of the lateral margin of the processus palatinum of pterygoid, and the degree of ossification of saccular otoconia and the stapedial footplate (Fig. 5). These differences are detailed in the “Comparisons” section below.

## Systematic revisions

Our phylogenetic analyses retrieve the Cao Bang population as a divergent mitochondrial and nuclear lineage placed as the sister lineage of *T. ziegleri* s. str. from Ha Giang Province, in agreement with previous phylogenies (Yuan *et al.* 2011; Nishikawa *et al.* 2013a; Wang *et al.* 2018; Liu *et al.* 2022; Dufresnes & Hernandez 2022). Although their degree of *ND2* divergence appears moderate (*p* = 2.7 %), it is comparable or even exceeds the divergence observed between many recognized species of *Tylototriton*, both within the subgenus *Tylototriton* (*T. verrucosus* and *T. shanjing* [1.3 %]; *T. verrucosus* and *T. pulcherrimus* [2.2 %]; *T. verrucosus* and *T. panwaensis* [2.5 %]; *T. pulcherrimus* and *T. panwaensis* [2.7 %]; *T. taliangensis* and *T. pseudoverrucosus* [2.4 %]; *T. ngarsuensis* and *T. shanorum* [1.1 %]; see Decemson *et al.* 2023) and the subgenus *Yaotriton* (*T. anhuiensis* and *T. broadoridgus* [2.6 %]; *T. anhuiensis* and *T. maolanensis* [2.6 %]; *T. thaiorum* and *T. notialis* [3.0 %]; *T. obsti* and *T. pasmansi* [3.1 %]; see Table 3). This degree of molecular differentiation suggests that the Cao Bang population (*Tylototriton* sp.) represents a distinct species from *T. ziegleri* s. str., which is further supported by their differentiation at phenotypic traits. Specifically, the external morphology features a number of statistically significant differences between males, including non-overlapping body shapes (Fig. 4), as well as diagnostic characters in skull morphology and coloration that readily distinguish the Cao Bang population (*Tylototriton* sp.) from *T. ziegleri* s. str. and all other congeners (see the “Morphological differentiation” section above and the “Comparisons” section below). From a biogeographic perspective, the Cao Bang and Ha Giang lineages are probably allopatric, being isolated by the Gam River valley, which separates the two main mountain ranges of the Viet Bac Massif (the subtropical montane forests of Ngan Son Mountain Range in the east from the evergreen montane forests of Song Gam Mountain Range in the west, Fig. 1), and thus potentially acts as a dispersal barrier (Bain & Hurley 2011; Abramov & Tran 2017; Poyarkov *et al.* 2021b).

Altogether, these elements support that the Cao Bang *Tylototriton* sp. newts represent an independently evolving population distinct from *T. ziegleri* s. str., which we formally describe below as a new species.
<figure><img src="image_5.png" /><figcaption>Figure 5. Cranial morphology of *Tylototriton koliaensis* sp. nov. (ZMMU A-8157, holotype; A–C) and *T. ziegleri* s. str. (ZMMU A-8169; D–F) visualized via micro-CT scanning. Skulls are shown in dorsal (A, D), ventral (B, E) and lateral (C, F) aspects. **Abbreviations:** a.f.sq. – fronto-squamosal arc; art. – articular; c.int. – internasal fontanelle; c.occ. – occipital condyle; den. – dentary; exoc.+prot. – prootic–exoccipital; f.a.m. – antero-medial fenestra; f.o. – foramen opticum; fr. – frontal; mx. – maxilla; nas. – nasal; oper. – operculum; osph. – orbitosphenoid; p.cor. – coronoid process; par. – parietal; pfr. – prefrontal; pmx. – premaxilla; prart. – prearticular; psph. – parasphenoid; pter. – pterygoid; q. – quadrate; sq. – squamosal; vom. – vomer.</figcaption></figure>
Family *SALAMANDRIDAE* Goldfuss, 1820

Subfamily *PLEURODELINAE* Tschudi, 1838

Genus *Tylototriton* Anderson, 1871

Subgenus *Yaotriton* Dubois & Raffaëlli, 2009

*Tylototriton koliaensis* sp. nov.

urn:lsid:zoobank.org:act:DB001E6F-71F6-4964-929E-7E2308EF0FB5

(Fig. 2, Fig. 5–8; Tables 4–5)

*Chresonymy*

*Tylototriton vietnamensis* (non *Tylototriton vietnamensis* Böhme, Schöttler, Nguyen & Köhler, 2005) — Böhme *et al.* (2005, in part); Weisrock *et al.* (2006); Nguyen *et al.* (2009, in part); Shen *et al.* (2012). *Tylototriton* cf. *vietnamensis* — Stuart *et al.* (2010); Gu *et al.* (2012). *Tylototriton asperrimus* (non *Tylototriton asperrimus* Unterstein, 1930) — Yuan *et al.* (2011, in part). *Tylototriton ziegleri* (non *Tylototriton ziegleri* Nishikawa, Matsui & Nguyen, 2013) — Nishikawa *et al.* (2013a, in part); Phimmachak *et al.* (2015, in part); Hernandez (2016, in part); Jiang *et al.* (2017, in part); Wang *et al.* (2018, in part); Than Zaw *et al.* (2019, in part); Lyu *et al.* (2021, in part); Liu *et al.* (2022, in part); Poyarkov *et al.* (2021a, in part); Poyarkov *et al.* (2021b, in part); Dufresnes & Hernandez (2022, in part); Nguyen *et al.* (2022, in part).

*Holotype*

Adult female ZMMU A-8157 (field number NAP-07305), collected from Kolia Organic Farm within the Phia Oac-Phia Den National Park, Thanh Cong Commune, Nguyen Binh District, Cao Bang Province, Vietnam (22.562423°N, 105.862566°E; 1025 m a.s.l.) by Nikolay A. Poyarkov and Son Xuan Le on June 9, 2017.

*Paratypes (n=8)*

Eight adult males: ZMMU A-8158–8165 (field numbers NAP-07306–07313), all collected from Kolia Mountain Pass within the Phia Oac-Phia Den National Park, Thanh Cong Commune, Nguyen Binh District, Cao Bang Province, Vietnam (22.59938°N, 105.88328°E; 1341 m a.s.l.) by Nikolay A. Poyarkov on June 9, 2017.

*Diagnosis*

The new species is assigned to the genus *Tylototriton* and to the subgenus *Yaotriton* based on its phylogenetic placement and the following combination of morphological attributes: granules on dorsal skin present, dorsolateral bony ridges on head present, dorsolateral series of knob-like warts (rib nodules) present, and quadrate spine absent (Fig. 5). *Tylototriton koliaensis* sp. nov. is differentiated from all other members of the subgenus *Yaotriton* by the following combination of morphological characters: medium body size (SVL: 56.0–61.4 mm in males; 70.8–73.0 in females); snout bluntly truncate in dorsal view and rounded in lateral profile; head large, as wide as long; gular fold weak; skin rough with fine granules laterally, granules getting smaller at mid-dorsum; supratemporal bony ridges on head wide, protruding, beginning at the snout; sagittal ridge on head indistinct; limbs very long and thin; tips of forelimbs and hindlimbs greatly overlapping when adpressed along body; vertebral ridge prominent, glandular and not segmented or slightly segmented; tail base widened; rib nodules 10–12, small, knob-like, rounded, protruding, arranged in two irregular dorsolateral series intermittent with smaller warts; dorsal background coloration black, ventral coloration dark-gray to black,
limbs black; finger and toe tips, parts of soles and palms, and vent bright orange, orange stripe continuing along the ventral edge of tail to the tail tip; bright orange markings on the parotoids or rib nodules absent; vomerine teeth arranged in two gently curving diverging ^-shaped series, converging anteriorly with their anterior corner forming a sharp angle. At the molecular level, it differs by private nucleotides at the mitochondrial genes *ND2* and 16S, as well as in multiple loci across the nuclear genome.

*Description of holotype*

A medium-sized female specimen (SVL: 73.0 mm) in a good state of preservation (Fig. 6). Head as wide as long (HW/HL: 0.99), large (HL/SVL: 0.20), notably wider than body; rounded hexagonal in shape in dorsal view (Fig. 6C), slightly depressed, gently sloping in profile (Fig. 6E); snout comparatively long and wide, three times longer than eye (UEW/SL: 0.38), bluntly truncated in dorsal and ventral views (Fig. 6C), obtusely rounded in lateral profile (Fig. 6E), almost not projecting beyond lower jaw (Fig. 6E); nostrils located on lateral surfaces of the anterior margin of snout, located notably closer to snout tip than to eye (NSD/ON: 0.68), with antero-lateral orientation, not visible from dorsal view (Fig. 6C); labial fold weakly developed in the posterior one third of jaw length (Fig. 6C); tongue oval, attached to anterior floor of mouth, free posteriorly and laterally; vomerine teeth arranged in two gently curving diverging ^-shaped series, converging anteriorly but not reaching choanae, with their anterior corner forming a sharp angle, each vomer bearing 34 vomerine teeth (Fig. 7); parotoids distinct, large, bean-shaped, slightly projecting posteriorly (Fig. 6C); dorsolateral supratemporal bony ridges on head wide, slightly protruding, beginning at the snout, continuing to the anterior corner of orbit and further posteriorly to the anterior end of parotoid, posterior ends of supratemporal ridges bulge-shaped, slightly curved inside (Fig. 6C); sagittal bony ridge on head indistinct (Fig. 6C); gular fold weak and almost indistinct (Fig. 6D).

Body habitus moderately stout (Fig. 6A–B); costal folds absent; vertebral middorsal ridge distinct, wide, rough, and glandular, not segmented, running from occiput region to sacrum and further continuing on the anterior part of tail (Fig. 6A). Rib nodules distinct, small, rounded, forming knoblike glandular warts, arranged in two regular longitudinal series on dorsolateral surfaces of dorsum, with 12 nodules placed on each side of body from the area posterior to axilla to the level of the posterior vent margin (tail base) (Fig. 6A). Rib nodules not in contact with each other, their size varies from rounded, distinctly knob-like anteriorly to smaller and of more irregular shape posteriorly; rib nodules intermittent with smaller irregular granules, which makes the dorsolateral series slightly ragged in shape (Fig. 6A; Fig. 8A).

Limbs comparatively long and thin (Fig. 6A–B); forelimbs slightly shorter than hindlimbs; relative lengths of limbs: FLL/SVL: 0.31, HLL/SVL: 0.34; fore- and hindlimbs overlapping when adpressed towards each other along body; fingers and toes well developed (Fig. 6F–G), free of webbing or folds; fingers four, comparative finger lengths: 4FL<1FL=2FL<3FL; toes five, comparative toe lengths: 5TL<1TL<2TL<4TL<3TL. Tail comparatively short, subequal to body length (TAL/SVL: 0.97); laterally compressed along entire length, sharply tapering posteriorly; lateral grooves on tail indistinct; dorsal tail fin comparatively low, starting at anterior one fourth of tail length, becoming higher and more distinct posteriorly, with maximal tail height at the middle of tail length, dorsal tail fin smooth; ventral tail ridge smooth; tail tip pointed.
<figure><img src="image_6.png" /><figcaption>Figure 6. Holotype of *Tylototriton koliaensis* sp. nov. (ZMMU A-8157, female) in preservative. A, dorsal view; B, ventral view; C, head, dorsal view; D, head, ventral view; E, head, lateral view; F, palmar view of right hand; G, plantar view of left foot; H, ventral view of cloacal area.
Photographs by Nikolay A. Poyarkov.</figcaption></figure>
Dorsally skin rough, granulated, with numerous small granules and larger warts present on lateral surfaces of head, body, and tail (Fig. 6E, 8A); granules getting less dense and notably smaller on dorsum between the dorsolateral rows of rib nodules and at the head basis (Fig. 6A, Fig. 8A); ventral surfaces smoother with smaller granules arranged in transverse striations (Fig. 6B); throat with numerous tiny flat tubercles (Fig. 6D); surfaces of head ridges and middorsal vertebral ridge rough; limbs dorsally with numerous tiny tubercles, volar and plantar surfaces of hands and feet with tiny grooves forming reticulated pattern (Fig. 6F–G). Flattened outer metacarpal and inner metacarpal tubercles on palms (Fig. 6F); flattened inner metatarsal tubercle distinct on soles (Fig. 6G). Cloacal region slightly swollen, vent as a shirt longitudinal slit, vent edges with numerous small transverse folds (Fig. 6H).

*Coloration of holotype*

In life, dorsum uniformly black (Fig. 8A); venter slate-black to dark gray, slightly lighter than dorsum; limbs black; rib nodules and vertebral ridge dark brownish-black; fingers and toes brownish-orange (ventrally on all of their lengths, dorsally only tips of the digits); parts of soles and palms corresponding to outer and inner metacarpal and inner metatarsal tubercles reddish-orange, vent and ventral tail ridge bright orange, separated by a dark patch at the beginning of the ventral tail fin. After seven years in preservative, dorsal coloration slightly faded, becoming dark brown, while orange markings on limbs and tail faded to cream (Fig. 6).

<figure><img src="image_7.png" /><figcaption>Figure 7. Schematic drawing of vomers and vomerine tooth series of *Tylototriton koliaensis* sp. nov. holotype (ZMMU A-8157). Drawing by Dmitriy V. Arkhipov.</figcaption></figure>
*Measurements of the holotype*

Measurements of the holotype are given in Table 4. Additional morphometric characters (all in mm) as follows. ICD: 9.4; CW: 15.7; NSD: 2.8; 1FL: 4.6; 2FL: 4.6; 3FL: 5.1; 4FL: 2.8; 1TL: 3.0; 2TL: 4.4; 3TL: 5.6; 4TL: 5.4; 5TL: 2.9.

*Variation*

All males are generally similar in morphology and coloration and agree with the holotype description in coloration; variation of morphometric characters among the type series is given in Table 4. Variation of the dorsal coloration in the type series in life is presented in Fig. 8: all specimens show dark black to slate-black dorsal coloration without any light markings. The number of rib nodules in males varies from 10 to 12 (mean: 11.1 ± 1.1; n = 8). All paratype males have a notably smaller body size than the single female holotype (SVL: 56.0–61.4 mm vs. 73.0 mm); their body habitus is more slender, tails are notably longer than body (TAL/SVL: 1.22 ± 0.07 vs. 0.97) and the head is relatively bigger and wider than in the female specimen (MXHW/SVL: 0.29 ± 0.01 vs. 0.24). Male ZMMU A-8158 has a more segmented middorsal ridge (Fig. 8B) than other specimens, in which the middorsal ridge was continuous and almost not segmented. Some noticeable variation was also observed in the shape of the vomerine tooth series: while most paratypes show gently curving, diverging ^-shaped vomerine tooth series, the paratype male ZMMU A-8158 has a ∩-shaped anterior part of the series with a blunt angle in the middle of the series length.

*Osteological description*

The following description of skull morphology is based on the tomographic data obtained for the holotype ZMMU A-8157, female (Fig. 5); the associated metadata are provided in the MorphoSource project<fnref n="4" />.

*Cranium*

Overall, the cranium of *Tylototriton koliaensis* sp. nov. is heavy and strongly ossified; some elements on the dorsal surfaces of the cranium show traces of hyperossification; the number of cartilaginous elements is comparatively low; the skull shape is acute trapezoidal in dorsal (Fig. 5A) and ventral views (Fig. 5B) and close to right-angled trapezoidal in lateral view (Fig. 5C); snout distinctly truncate in dorsal and ventral views.

*Premaxilla*

The premaxilla is a paired bone, slightly arched dorsally, bearing teeth; the right premaxilla bears 18, and the left maxilla bears 16 teeth (Fig. 5B). The premaxilla dorsally contributes to the anterodorsal internasal fontanelle (cavum internasale), ventrally contributes to the anteromedial fenestra, and anteriorly forms a ventromedial part of the external nares. Anterior and dorsal surfaces of premaxillary bones are granular with numerous small ossifications (Fig. 5A). The premaxilla contacts the prefrontal, nasal, and maxillary bones posteriorly and contributes to the anterior part of the nasal cavity.

<footnote n="4">⁴ See https://www.morphosource.org/concern/media/000665372</footnote>
<figure><img src="image_8.png" /><figcaption>Figure 8. Specimens of *Tylototriton koliaensis* sp. nov. in life from Kolia Mountain Pass, Phia Oac-Phia Den National Park, Nguyen Binh, Cao Bang Province, Vietnam. A, holotype ZMMU A-8157, female, in situ; B, paratype ZMMU A-8158, male, in situ.
Photographs by Nikolay A. Poyarkov.</figcaption></figure>
### Maxilla

The paired maxilla runs laterally from the nasal capsule to the level of the otic region, bearing teeth on its anterior half; the right maxilla bears 21, and the left maxilla bears 19 teeth (Fig. 5B). Dorsal and lateral surfaces of the maxillary bone are rough and tuberous. The maxilla contacts premaxillar, prefrontal, and vomerine bones anteriorly, and pterygoid and quadrate posteriorly. Anteriorly maxilla forms an anterolateral part of the external nares and dorsomedially contributes to the orbit cavity.

### Prefrontal

The prefrontal (sensu Huxley 1875) is a paired bone located between nasal anteriorly, maxillary laterally, prefrontal posteriorly, and frontal medially. The dorsal surface of the prefrontal is densely tuberous, forming the medial part of the supratemporal cranial crests (Fig. 5A). The prefrontal contributes posteriorly to the orbit cavity and ventrally to the nasal cavity.

### Nasal

The paired nasals are located anteriorly; they contact premaxillary anteromedially, maxillary anterolaterally, prefrontal posterolaterally, and frontal posteromedially (Fig. 5A). The nasal forms the dorsal part of external nares and contributes ventrally to the nasal cavity. Dorsal surfaces of nasal bones granular, forming the anterior part of the supratemporal cranial crests. A pair of nasals meet anteriorly and form internasal fontanelle medially.

### Frontal

The frontal is a paired bone, forming the large portion of the cranial roof. The frontal is located between the nasal anteriorly, prefrontal anterolaterally, orbitosphenoid ventrally, squamosal posterolaterally, and parietal posteromedially. A pair of frontals meet along the midline forming a suture (Fig. 5A), anteromedially contribute to the internasal fontanelle, and ventromedially to the orbit cavity. A broad and massive posterolateral process of each frontal contacts squamosal forming fronto-squamosal arc (Fig. 5A). The frontal bones' dorsal surface is tubercular, with ossified granules becoming denser posteriorly. The sagittal cranial crest is not pronounced; posterolaterally frontal highly tuberous, contributing to the medial part of supratemporal cranial crest; anteromedially frontal is almost smooth.

### Parietal

The paired parietals meet each other along their length, forming a suture (Fig. 5A); they contact frontal anteriorly, orbitosphenoid anteroventrally, occipito-otic complex laterally and posteriorly, and touch squamosal dorsolaterally. The dorsal surface of the parietal is depressed and smooth posterolaterally, elevated, and granular anteromedially; however, no sagittal crest is formed.

### Vomer

The vomer is a paired bone, covering the cranial base between the internal nares and bearing vomerine teeth forming two gently curving, diverging ^-shaped series (Fig. 5B); each vomer bears 34 teeth. The vomer is located between prefrontal anteriorly, maxillary anterolaterally, orbitosphenoid dorsomedially, and parasphenoid posteriorly, covering the latter with its posterior process ventrally. A pair of vomers forms an
anteromedial fenestra, meets medially behind this fenestra, and contributes to the nasal cavity anterodorsally and orbit cavity laterally.

### Parasphenoid

The single parasphenoid is a large sword-shaped bone plate, covered with the posterior portion of the vomers (Fig. 5B). The parasphenoid contacts vomers anterolaterally, orbitosphenoid laterally, occipito-otic complex laterally and posteriorly, and the posteromedial portion of the parasphenoid contributes to the ventral edge of the foramen magnum.

### Squamosal

The paired squamosals exhibit a T-shaped structure with massive anterior process and stem portion, which is located at the right angle to the jaw arc (Fig. 5A, C). The anterior process of the squamosal has a rough and tuberous dorsal surface; it contacts the posterolateral process of the frontal anteriorly, forming the fronto-squamosal arc. Squamosal is barely attached to parietal with a small dorsomedial portion, while mostly being in contact medially with occipito-otic complex. The stem portion of the squamosal contacts quadrate ventrally and pterygoid ventromedially. The squamosal is densely tuberous dorsally, with a well-developed posterior process, forming the posterior part of the supratemporal cranial crest (Fig. 5C).

### Quadrate

The quadrate is a small paired bone contacting squamosal dorsally, pterygoid medially, maxillary anteriorly, and forming the jaw joint with the mandible ventrally (Fig. 5B). The lateral surface of the quadrate is granular.

### Pterygoid

The paired pterygoids are triangular in shape. The processus oticum is directed dorsomedially, with a prominent excavation in lateral view (Fig. 5B). The processus palatinum is directed anterolaterally with its ventral margin rounded, bearing a bulb-shaped pterygoid groove in lateral view (Fig. 5C). The processus quadratum of the pterygoid is directed posteriorly.

### Orbitosphenoid

The orbitosphenoid is a single bone that forms the anterior portion of the neurocranium, bearing on each side a large foramen opticum that opens in its posterior part (Fig. 5C). The orbitosphenoid contacts prefrontal dorsally, parietal posterodorsally, occipito-otic complex posteriorly, parasphenoid posteroventrally, and vomer ventrally and contributes to the nasal cavity anteriorly and orbit cavity laterally.

### Occipito-otic complex

The occipito-otic complex is a paired structure comprised of fused prootic and exoccipital bones. This complex contacts squamosal dorsolaterally, pterygoid laterally, parasphenoid ventrally, parietal anteromedially, and narrowly touches orbitosphenoid anterolaterally. A pair of these structures meet dorsally, behind the parietals (Fig. 5A). These bones form the lateral part of the otic capsule, fenestra ovalis, foramen magnum, and bear paired occipital condyles.
*Stapes*

The paired stapes encompass a disc-like footplate and stylus. The footplate was barely seen in our stacks, which indicates its poor or partial ossification; the stylus was completely invisible, likely due to it being cartilaginous. Examination of cleared and stained specimens is needed to clarify the morphology of this structure.

*Operculum*

The paired oval operculum covers the posterior part of the fenestra ovalis and is attached to the lateral side of the occipito-otic complex (Fig. 5B). It was likely cartilaginous in all the specimens examined and, thus, is invisible in our reconstructions. Examination of cleared and stained specimens is needed to clarify the morphology of this structure.

*Saccular otoconia*

The otic capsule in *Tylototriton* is filled with calcium carbonate, which was visible in our reconstructions. This structure was notably less dense than the other bones, which indicates poor or partial mineralization. The saccule was close to a hemisphere or ellipsoid in shape.

*Mandible*

The lower jaw has a shape of solid bony arch and comprises three bone elements, namely, paired dentaries, prearticulars, and the Meckel's cartilage (Fig. 5C), which is poorly seen in our reconstructions. Only a trace of Meckel's cartilage on the proximal surface, anterior of the prearticular and in between the two portions of the dentary is visible; the poorly-ossified posterior edge of the Meckel's cartilage corresponds to the location of the articular, which forms the jaw joint with the quadrate.

*Dentary*

The paired dentaries comprise most of the mandible and bear 38–41 teeth across the two-thirds of their length (Fig. 5C). The dentary contacts articular posterolaterally and prearticular posteromedially, contributing to the coronoid process. A pair of dentaries meet anteriorly with no trace of mentomeckelian between them.

*Prearticular*

The paired prearticulars cover the posterior part of the medial surface of the mandible (Fig. 5C). The prearticular, together with the dentary, forms the coronoid process.

*Hyobranchial apparatus*

The hyobranchial apparatus consists of a few predominantly cartilaginous elements. In our scans, we were able to observe only three bony elements: ceratobranchial, ceratohyal, and hypobranchial I.

*Etymology*

The specific epithet 'koliaensis' is a Latinized toponymic adjective in nominative singular and is given in reference to the Kolia Mountain Pass and Kolia Farm, the type locality of the new species. The exploration of the Ngan Son Mountains in Cao Bang Province dates back to the early 20th century, when, starting in 1901, French colonialists built a road and camps for gold and tin mining in the area of Phia
Oac-Phia Den NP. The local legend says that a female French engineer named “Kolia” (also spelled as “Cô Lê A”) participated in construction of the road to the mountain pass and of a small French settlement on the slopes of the Phia Oac Mountain, built as a mountain retreat during hot summer months (presently known as “Nhà Pháp,” or “French houses”). Local people believe that this French lady was eaten by a tiger during her work in Phia Oac, and the place was named “Kolia” for her memory.

*Suggested common names*

We suggest the following common names: *Cao Bang* (*Crocodile Newt*) (English), *Cá cóc Cao Bằng* (Vietnamese), *Kaobangskiy krokodilovyi triton* (Russian), *Tylototriton de Cao Bang* (French).

*Comparisons*

According to mitochondrial phylogeny, *Tylototriton koliaensis sp. nov.* falls into Clade I, which corresponds to the *T. asperrimus* species group of the subgenus *Yaotriton*. Differences between the new species and all other members of the subgenus *Yaotriton* are summarized in Table 5. The new species can be easily distinguished from all members of the subgenus *Tylototriton* (clades 4–5 in Fig. 2), as well as from *T. panhai* and *T. ngoclinhensis*, by a dark body coloration, except for bright orange palms and soles, vent region, and ventral ridge of tail (vs. light color markings present on parotoids, lips, vertebral ridge, rib nodules, limbs, and ventral tail ridge).

*Tylototriton koliaensis sp. nov.* can be distinguished from *T. anhuiensis*, *T. asperrimus*, *T. broadoridgus*, *T. dabienicus*, *T. daloushanensis*, *T. hainanensis*, *T. liuyangensis*, *T. lizhenchangi*, *T. tongziensis*, *T. vietnamensis*, and *T. wenxianensis* by having prominent bony ridges on the head, denser granules on the body, large and small warts surrounding the rib nodules, and distinct interspaces between the rib nodules (vs. comparatively less developed ridges on head, smoother skin, slightly flattened to nearly indistinct rib nodules with no distinct interspaces between them). The new species differs from *T. maolanensis*, *T. notialis*, and *T. thaiorum* by a blackish coloration of dorsum (vs. dark brown); from *T. asperrimus* by comparatively larger eyes and a thinner and lower tail, as well as a weak, almost indistinct gular fold (vs. gular fold distinct); from *T. pasmansi* by a smaller male body size (SVL: 56.0–61.4 mm vs. 61.6–70.7 mm), the head wider than long (vs. slightly longer than wide); from *T. sini* by a smaller body size in both sexes (SVL: 56.0–61.4 mm in males, 70.8–73.0 mm in females vs. 62.0–66.5 mm in males, 79.3 mm in a single female), the snout truncated in dorsal view (vs. rounded), and the head wider than long (vs. slightly longer than wide); from *T. sparreboomi* by a smaller male body size (SVL: 56.0–61.4 mm vs. 63.2–68.7 mm) and the head wider than long (vs. longer than wide).

Although *Tylototriton koliaensis sp. nov.* was previously confounded with its sister species *T. ziegleri*, it can be readily distinguished by several coloration, morphological and cranial specificities.
<caption>Table 5. Morphological comparisons between *Tylototriton koliaensis* sp. nov. and the 20 currently recognized species of the subgenus *Yaootriton*. **Character definitions:** (1) SVL in mm (males); (2) SVL in mm (females); (3) Snout in dorsal view; (4) Snout in profile view; (5) Head; (6) Gular fold; (7) Rib nodules shape; (8) Vertebral ridge; (9) Ground color; (10) Orange markings on the parotoid; (11) Orange coloration of the rib nodules.</caption>

Sources as follows. 1: Böhme *et al.* (2005); 2: Chen *et al.* (2010); 3: Stuart *et al.* (2010); 4: Yuan *et al.* (2011); 5: Shen *et al.* (2012); 6: Hou *et al.* (2012); 7: Nishikawa *et al.* (2013a);

8: Nishikawa *et al.* (2013b); 9: Yang *et al.* (2014); 10: Phimmachak *et al.* (2015); 11: Fei & Ye (2016); 12: Hernandez (2016); 13: Qian *et al.* (2017); 14: Wang *et al.* (2018); 15: Bernardes *et al.* (2020); 16: Li *et al.* (2020a); 17: Li *et al.* (2020b); 18: Poyarkov *et al.* (2021a); 19: Lyu *et al.* (2021); 20: Li *et al.* (2022); 21: Luo *et al.* (2022); 22: Liu *et al.* (2022); 23: Phung *et al.* (2023); 24: this study.

<table><thead><tr><th>No.</th><th>Species</th><th>(1)</th><th>(2)</th><th>(3)</th><th>(4)</th></tr></thead><tbody><tr><td>1</td><td><i>T. koliaensis sp. nov.</i></td><td>56.0–61.4</td><td>70.8–73.0</td><td>truncate</td><td>rounded</td></tr><tr><td>2</td><td><i>T. anhuiensis</i></td><td>59.4–74.8</td><td>51.0–96.1</td><td>rounded</td><td>rounded</td></tr><tr><td>3</td><td><i>T. asperrimus</i></td><td>55.8–63.3</td><td>73.5</td><td>truncate</td><td>rounded</td></tr><tr><td>4</td><td><i>T. broadoridgus</i></td><td>66.1–68.7</td><td>76.3–87.4</td><td>truncate</td><td>rounded</td></tr><tr><td>5</td><td><i>T. dabienicus</i></td><td>67.4–74.0</td><td>66.1</td><td>rounded</td><td>rounded</td></tr><tr><td>6</td><td><i>T. daloushanensis</i></td><td>64.7–83.6</td><td>70.5–100.3</td><td>rounded</td><td>rounded</td></tr><tr><td>7</td><td><i>T. hainanensis</i></td><td>55.7–57.7</td><td>—</td><td>rounded</td><td>rounded</td></tr><tr><td>8</td><td><i>T. liuyangensis</i></td><td>64.2–82.0</td><td>80.8–88.0</td><td>truncate</td><td>slightly angular</td></tr><tr><td>9</td><td><i>T. lizhenchangi</i></td><td>—</td><td>—</td><td>truncate</td><td>slightly angular</td></tr><tr><td>10</td><td><i>T. maolanensis</i></td><td>76.8–85.2</td><td>76.3–87.4</td><td>truncate</td><td>slightly angular</td></tr><tr><td>11</td><td><i>T. notialis</i></td><td>56.5–60.2</td><td>73.4–78.3</td><td>truncate</td><td>slightly angular</td></tr><tr><td>12</td><td><i>T. ngoclinhensis</i></td><td>60.8–66.5</td><td>72.5–75.6</td><td>truncate</td><td>rounded</td></tr><tr><td>13</td><td><i>T. panhai</i></td><td>—</td><td>71.6</td><td>truncate</td><td>rounded</td></tr><tr><td>14</td><td><i>T. obsti</i></td><td>62.0–68.0</td><td>—</td><td>truncate</td><td>slightly angular</td></tr><tr><td>15</td><td><i>T. pasmansi</i></td><td>61.6–70.7</td><td>—</td><td>truncate</td><td>slightly angular</td></tr><tr><td>16</td><td><i>T. sini</i></td><td>62.0–66.5</td><td>79.3</td><td>rounded</td><td>rounded</td></tr><tr><td>17</td><td><i>T. sparreboomi</i></td><td>63.2–68.7</td><td>—</td><td>truncate</td><td>rounded</td></tr><tr><td>18</td><td><i>T. thaiorum</i></td><td>59.3–68.6</td><td>—</td><td>obtusely rounded</td><td>rounded</td></tr><tr><td>19</td><td><i>T. tongziensis</i></td><td>61.1–65.9</td><td>66.7–69.2</td><td>truncate</td><td>slightly angular</td></tr><tr><td>20</td><td><i>T. vietnamensis</i></td><td>48.3–53.6</td><td>—</td><td>truncate</td><td>slightly angular</td></tr><tr><td>21</td><td><i>T. wenxianensis</i></td><td>61.0–71.0</td><td>70.1–77.2</td><td>rounded</td><td>rounded</td></tr><tr><td>22</td><td><i>T. ziegleri s. str.</i></td><td>52.7–67.4</td><td>—</td><td>truncate</td><td>rounded</td></tr></tbody></table>

<table><thead><tr><th>No.</th><th>Species</th><th>(5)</th><th>(6)</th><th>(7)</th></tr></thead><tbody><tr><td>1</td><td><i>T. koliaensis sp. nov.</i></td><td>wider than long</td><td>weak</td><td>knob-like, rounded</td></tr><tr><td>2</td><td><i>T. anhuiensis</i></td><td>longer than wide</td><td>present</td><td>slightly flattened</td></tr><tr><td>3</td><td><i>T. asperrimus</i></td><td>wider than long</td><td>present</td><td>knob-like</td></tr><tr><td>4</td><td><i>T. broadoridgus</i></td><td>longer than wide</td><td>absent</td><td>slightly flattened</td></tr><tr><td>5</td><td><i>T. dabienicus</i></td><td>longer than wide</td><td>present</td><td>slightly flattened</td></tr><tr><td>6</td><td><i>T. daloushanensis</i></td><td>slightly longer than wide</td><td>present</td><td>slightly flattened</td></tr><tr><td>7</td><td><i>T. hainanensis</i></td><td>wider than long</td><td>present</td><td>slightly flattened</td></tr><tr><td>8</td><td><i>T. liuyangensis</i></td><td>equally long and wide</td><td>present</td><td>slightly flattened</td></tr><tr><td>9</td><td><i>T. lizhenchangi</i></td><td>longer than wide</td><td>present</td><td>slightly flattened</td></tr><tr><td>10</td><td><i>T. maolanensis</i></td><td>longer than wide</td><td>present</td><td>knob-like</td></tr><tr><td>11</td><td><i>T. notialis</i></td><td>slightly longer than wide</td><td>present</td><td>knob-like</td></tr><tr><td>12</td><td><i>T. ngoclinhensis</i></td><td>longer than wide</td><td>present</td><td>knob-like</td></tr><tr><td>13</td><td><i>T. panhai</i></td><td>longer than wide</td><td>present</td><td>knob-like</td></tr><tr><td>14</td><td><i>T. obsti</i></td><td>slightly longer than wide</td><td>present</td><td>knob-like</td></tr><tr><td>15</td><td><i>T. pasmansi</i></td><td>slightly longer than wide</td><td>present</td><td>knob-like</td></tr><tr><td>16</td><td><i>T. sini</i></td><td>slightly longer than wide</td><td>present</td><td>knob-like</td></tr><tr><td>17</td><td><i>T. sparreboomi</i></td><td>longer than wide</td><td>present</td><td>knob-like</td></tr><tr><td>18</td><td><i>T. thaiorum</i></td><td>wider than long</td><td>present</td><td>knob-like in irregular series</td></tr><tr><td>19</td><td><i>T. tongziensis</i></td><td>slightly longer than wide</td><td>present</td><td>slightly flattened</td></tr><tr><td>20</td><td><i>T. vietnamensis</i></td><td>longer than wide</td><td>absent</td><td>slightly flattened</td></tr><tr><td>21</td><td><i>T. wenxianensis</i></td><td>longer than wide</td><td>absent</td><td>slightly flattened</td></tr><tr><td>22</td><td><i>T. ziegleri s. str.</i></td><td>wider than long</td><td>present</td><td>knob-like</td></tr></tbody></table>

(Continues on the next page).
<table><caption>Table 5 (continued).</caption><thead><tr><th>No.</th><th>Species</th><th>(8)</th><th>(9)</th><th>(10)</th><th>(11)</th></tr></thead><tbody><tr><td>1</td><td><i>T. koliaensis</i> sp. nov.</td><td>not or slightly segmented</td><td>black</td><td>absent</td><td>absent</td></tr><tr><td>2</td><td><i>T. anhuiensis</i></td><td>not segmented</td><td>blackish</td><td>absent</td><td>absent</td></tr><tr><td>3</td><td><i>T. asperrimus</i></td><td>not segmented</td><td>brownish black</td><td>absent</td><td>absent</td></tr><tr><td>4</td><td><i>T. broadoridgus</i></td><td>not segmented</td><td>brownish black</td><td>absent</td><td>absent</td></tr><tr><td>5</td><td><i>T. dabienicus</i></td><td>not segmented</td><td>brown</td><td>absent</td><td>absent</td></tr><tr><td>6</td><td><i>T. daloushanensis</i></td><td>not segmented</td><td>blackish</td><td>absent</td><td>absent</td></tr><tr><td>7</td><td><i>T. hainanensis</i></td><td>not segmented</td><td>blackish or dark brown</td><td>absent</td><td>absent</td></tr><tr><td>8</td><td><i>T. liuyangensis</i></td><td>not segmented</td><td>blackish</td><td>absent</td><td>absent</td></tr><tr><td>9</td><td><i>T. lizhenchangi</i></td><td>not segmented</td><td>blackish</td><td>present</td><td>absent</td></tr><tr><td>10</td><td><i>T. maolanensis</i></td><td>not segmented</td><td>dark brown</td><td>absent</td><td>absent</td></tr><tr><td>11</td><td><i>T. notialis</i></td><td>not segmented</td><td>brownish black to black</td><td>present</td><td>present</td></tr><tr><td>12</td><td><i>T. ngoclinhensis</i></td><td>segmented, tuberculate</td><td>blackish</td><td>present</td><td>present</td></tr><tr><td>13</td><td><i>T. panhai</i></td><td>not segmented</td><td>blackish</td><td>present</td><td>present</td></tr><tr><td>14</td><td><i>T. obsti</i></td><td>not segmented</td><td>blackish</td><td>absent</td><td>absent</td></tr><tr><td>15</td><td><i>T. pasmansi</i></td><td>not segmented</td><td>blackish</td><td>absent</td><td>absent</td></tr><tr><td>16</td><td><i>T. sini</i></td><td>not segmented</td><td>blackish</td><td>absent</td><td>absent</td></tr><tr><td>17</td><td><i>T. sparreboomi</i></td><td>not segmented</td><td>blackish</td><td>absent</td><td>absent</td></tr><tr><td>18</td><td><i>T. thaiorum</i></td><td>not segmented</td><td>dark brown</td><td>absent</td><td>absent</td></tr><tr><td>19</td><td><i>T. tongziensis</i></td><td>not segmented</td><td>blackish</td><td>absent</td><td>absent</td></tr><tr><td>20</td><td><i>T. vietnamensis</i></td><td>not segmented</td><td>gray to tan</td><td>absent</td><td>absent</td></tr><tr><td>21</td><td><i>T. wenxianensis</i></td><td>not segmented</td><td>blackish</td><td>absent</td><td>absent</td></tr><tr><td>22</td><td><i>T. ziegleri</i> s. str.</td><td>segmented, tuberculate</td><td>blackish</td><td>absent</td><td>absent or present</td></tr></tbody></table>

<table><thead><tr><th>No.</th><th>Species</th><th>Distribution (* = type locality)</th><th>Sources</th></tr></thead><tbody><tr><td>1</td><td><i>T. koliaensis</i> sp. nov.</td><td>Vietnam (Cao Bang: Nguyen Binh, Bao Lac), China (Guangxi: Jingxi?)</td><td>7, 12, 24</td></tr><tr><td>2</td><td><i>T. anhuiensis</i></td><td>China (Anhui: Yaoluoping NNR*)</td><td>13</td></tr><tr><td>3</td><td><i>T. asperrimus</i></td><td>China (Guangxi: Yaoshan Mt.*, Dayao Mt., Jinxiu, Longsheng &amp; Guiping; Guizhou: Libo)</td><td>4, 11, 14, 19</td></tr><tr><td>4</td><td><i>T. broadoridgus</i></td><td>China (Hunnan: Tianping Mt.*, Huping Mt.)</td><td>5, 19</td></tr><tr><td>5</td><td><i>T. dabienicus</i></td><td>China (Anhui: Dabie Mt.*)</td><td>2, 11, 17</td></tr><tr><td>6</td><td><i>T. daloushanensis</i></td><td>China (Guizhou: Huoqiuba NR*, Kuankuoshui NNR)</td><td>21</td></tr><tr><td>7</td><td><i>T. hainanensis</i></td><td>China (Hainam: Wuzhishan NNR*, Jianfengling, Diaoluo Mt.)</td><td>11, 14</td></tr><tr><td>8</td><td><i>T. liuyangensis</i></td><td>China (Hunan: Daweishan NFP*)</td><td>9, 14</td></tr><tr><td>9</td><td><i>T. lizhenchangi</i></td><td>China (Hunan: Mangshan NNR*, Yizhang)</td><td>6, 11</td></tr><tr><td>10</td><td><i>T. maolanensis</i></td><td>China (Guizhou: Maolan NR*, Leigong Mt.)</td><td>14, 17</td></tr><tr><td>11</td><td><i>T. notialis</i></td><td>Laos (Khammouan: Nakai-Nam Theun NP*)</td><td>3, 10, 18</td></tr><tr><td>12</td><td><i>T. ngoclinhensis</i></td><td>Vietnam (Kon Tum: Ngoc Linh Mt); possibly Laos (Xekong)</td><td>23, 24</td></tr><tr><td>13</td><td><i>T. panhai</i></td><td>Thailand (Loei: Phu Luang WS*); Phitsanulok: Phu Hin Rong Kla NP; Phetchabun: Phu Thap Boek Mt.), Laos (Xaignabouli: Botene)</td><td>8, 10</td></tr><tr><td>14</td><td><i>T. obsti</i></td><td>Vietnam (Son La: Xuan Nha NR*); Thanh Hoa: Xuan Lien NR &amp; Quan Son)</td><td>15, 24</td></tr><tr><td>15</td><td><i>T. pasmansi</i></td><td>Vietnam (Hoa Binh: Phu Canh NR*; Lao Cai: Van Ban; Phu Tho: Xuan Son NP)</td><td>15</td></tr><tr><td>16</td><td><i>T. sini</i></td><td>China (Guangdong: Yunkai Mt*; Guangxi: Baise)</td><td>4, 16, 19</td></tr><tr><td>17</td><td><i>T. sparreboomi</i></td><td>Vietnam (Lai Chau: Sin Ho*)</td><td>15</td></tr><tr><td>18</td><td><i>T. thaiorum</i></td><td>Vietnam (Nghe An: Pu Hoat NR*)</td><td>18, 24</td></tr><tr><td>19</td><td><i>T. tongziensis</i></td><td>China (Guizho: Tongziu*; Hubei: Yichang)</td><td>14, 20</td></tr><tr><td>20</td><td><i>T. vietnamensis</i></td><td>Vietnam (Bac Giang: Tay Yen Tu NR*; Lang Son: Mau Son Mt.; Quang Ninh: Dong Son-Ky Thuong NR)</td><td>1, 15, 24</td></tr><tr><td>21</td><td><i>T. wenxianensis</i></td><td>China (Gansu: Wenxian*; Chongqing: Yunyang; Sichuan: Wangcang &amp; Qingchuan)</td><td>11, 14</td></tr><tr><td>22</td><td><i>T. ziegleri</i> s. str.</td><td>Vietnam (Ha Giang: Quan Ba*, Vi Xuyen &amp; Bac Quang); China (Yunnan: Malipo)</td><td>7, 22, 24</td></tr></tbody></table>
First, the dorsal coloration varies from black to slate-black in *Tylototriton koliaensis sp. nov.* (Fig. 2, 8), while it is generally lighter, varying from blackish-brown (Fig. 9A, C) to dark-brown (Fig. 2, 9B) or orange-brown in *T. ziegleri*; the tail fin is dorsally completely black in *Tylototriton koliaensis sp. nov.*, while it presents an orange-brown dorsal margin in *T. ziegleri* (Fig. 2, 10); the dorsum is homogeneous in *Tylototriton koliaensis sp. nov*, while it shows weak orange markings on rib nodules or head in some specimens of *T. ziegleri* (Fig. 2, 9C).

<figure><img src="image_9.png" /><figcaption>Figure 9. Specimens of *Tylototriton ziegleri s. str.* in life. A, male ZMMU A-8167 from Thanh Van Village, Quang Ba, Bat Dai Son Nature Reserve, Ha Giang Province, Vietnam, in situ; B, male from Chieu Lau Thi Mt., Ha Giang Province, Vietnam, in situ (not collected); C, male from Wenshan, Yunnan Province, China, in situ (not collected).</figcaption></figure>

Photographs by Nikolay A. Poyarkov (A), Loi Phuoc Tran (B), and Yinpeng Zhang (C).
Second, *Tylototriton koliaensis* sp. nov, differs from *T. ziegleri* in body size and for several body ratios (see Table 4). Specifically, the males of *Tylototriton koliaensis* sp. nov. are generally smaller than the males of *T. ziegleri*, although with wide overlap (SVL: 58.5 ± 2.0 mm, range 56.0–61.4 mm vs. 62.4 ± 4.5 mm, range 52.7–67.4 mm); the males of *Tylototriton koliaensis* sp. nov. have wider heads (MXHW/SVL: 0.29 ± 0.01 vs. 0.27 ± 0.01), greater distance between nostrils (IND/SVL: 0.10 ± 0.006 vs. 0.08 ± 0.004), shorter axilla-groin distance (AGD/SVL: 0.50 ± 0.01 vs. 0.52 ± 0.02), longer forelimbs (FLL/SVL: 0.36 ± 0.03 vs. 0.34 ± 0.02), longer hindlimbs (HLL/SVL: 0.38 ± 0.03 vs. 0.35 ± 0.01), and shorter upper eyelids (UEL/SVL: 0.068 ± 0.003 vs. 0.072 ± 0.003) than the males of *T. ziegleri*.

Third, *Tylototriton koliaensis* sp. nov. shows unique cranial characters compared to *T. ziegleri*. Specifically, *Tylototriton koliaensis* sp. nov. often has wider vomerine tooth series, in the form of two gently curving diverging ^-shaped lines with the anterior corner of the series forming a sharp angle (Fig. 5B), while in *T. ziegleri*, the anterior part of the vomerine tooth series is ∩-shaped, their anterior corner is rounded, and the anterior parts of their branches are almost parallel, diverging in the posterior one-third of their length, forming a blunt angle (Fig. 5E); but note that the shape of the vomerine tooth series is subjected to individual variation. Moreover, *Tylototriton koliaensis* sp. nov. shows an excavation on the processus oticum of pterygoid (Fig. 5C) (vs. absent in *T. ziegleri*, Fig. 5F), the dorsal grove on the processus palatinum of the pterygoid is bulb-shaped (Fig. 5C) (vs. slit-like in *T. ziegleri*, Fig. 5F), the lateral margin of the processus palatinum of pterygoid is rounded (Fig. 5B) (vs. flattened in *T. ziegleri*, Fig. 5E), the saccular otoconia is poorly mineralized (vs. completely mineralized in *T. ziegleri*), and the stapedial footplate is partially ossified (vs. completely ossified in *T. ziegleri*).

## Distribution

To date, *Tylototriton koliaensis* sp. nov. has been recorded from the Ngan Son Mountain Range in Bao Lac and Nguyen Binh districts of Cao Bang Province, at elevations between 1025 and 1425 m a.s.l. (Bernades *et al.* 2017; this study), and is reliably known from only three localities (localities 1–3 in Fig. 1). Hernandez (2016) reported this species (as *T. ziegleri*) from 920 m. a.s.l. elevation in Jingxi County of Guangxi Province, China, but based on personal field observations and without voucher specimen collection that could have allowed subsequent reassessments. Given the overall morphological similarity within the *T. asperrimus* species group, species identification based on photographs or personal observations, i.e., without molecular or quantitative morphological data, should be taken with caution. Accordingly, and acknowledging that the Guangxi population is geographically proximate to the confirmed range of the new species, we herein treat this population as *Tylototriton* cf. *koliaensis* sp. nov.
<figure><img src="image_10.png" /><figcaption>Figure 10. Topotype of *Tylototriton ziegleri* (ZMMU A-8167, male) Thanh Van Village, Quang Ba, Bat Dai Son Nature Reserve, Ha Giang Province, Vietnam, in life. A, dorsal view; B, ventral view. Photographs by Nikolay A. Poyarkov.</figcaption></figure>
*Natural history notes*

At the type locality in Phia Oac-Phia Den NP, *Tylototriton koliaensis sp. nov.* is known from primary subtropical broadleaf montane forests on granite substrates. Phia Oac Mt. (1935 m a.s.l.) is the highest peak of the Ngan Son-Yen Lac Mountain Range, the major mountain system of northeastern Vietnam (Cao Bang, Bac Kan, and Thai Nguyen provinces). The occurrence of *Tylototriton koliaensis sp. nov.* in other montane forest areas of the Ngan Son-Yen Lac Mountain Ridge at similar elevations is thus considered likely. The polydominant evergreen subtropical forests in Phia Oac-Phia Den NP at elevations of 1200-1500 m a.s.l. are characterized by thick bamboo undergrowth and are dominated by trees from the families Fagaceae (genera *Lithocarpus* Blume and *Castanopsis* (D. Don) Spach), Sapindaceae (genus *Acer* L.), Platanaceae (genus *Platanus* L.), Elaeocarpaceae (genus *Elaeocarpus* L.), Ericaceae (genus *Rhododendron* L.), Lauraceae (genus *Cinnamomum* Schaeff.), and Theaceae (genus *Schima* Reinw. ex Blume), with thick layers of moss and numerous epiphytic plants (Orchidaceae, Ericaceae, Pteridophyta). At higher elevations, they are characterized mainly by bamboo vegetation, patches of grasses, and woody shrubs (Fig. 11A; Hernandez 2016; Bernardes *et al.* 2017; Poyarkov *et al.* 2018; this study).

The Phia Oac-Phia Den NP, where *Tylototriton koliaensis sp. nov.* has also been recorded, experiences a comparatively colder climate. Under the influence of the monsoon tropical climate of northeast Vietnam, which comprises cold winters and summer rains, the area has a mean annual temperature of 20.6°C, a mean annual precipitation of 1718 mm, and an average humidity rate of 83.4 % (Averyanov *et al.* 2003; Le 2005; Le *et al.* 2021). Unusually for northern Vietnam, the temperature can fall below zero, and snow is not rare in December and January. The dry season extends from November to April, with a mean precipitation of 295 mm (17.2 % of total annual rainfall); the rainy season runs from May to November, with the peak of precipitation in

<figure><img src="image_11.png" /><figcaption>Figure 11. The type locality of *Tylototriton koliaensis sp. nov.* in Phia Oac-Phia Den National Park, Cao Bang Province, Vietnam. A, macrohabitat. B, microhabitat. Photographs by Son Xuan Le.</figcaption></figure>
July and August, which average 1423 mm (82.8 % of total annual rainfall; Le 2005). These conditions support a variety of forest types, particularly low to high montane broadleaf evergreen forests (Tran *et al.* 2014). Though vegetation covers approximately 84 % of the total area of Phia Oac-Phia Den NP, it mostly consists of secondary forests or plantations, while the primary and undisturbed forests are found only above 1,000 m a.s.l. (Tran *et al.* 2014).

*Tylototriton koliaensis* sp. nov. breeds in the summer, when rainfall is abundant. During the breeding season, from April to July, it occurs in slow-flowing streams and temporary pools of 30–50 cm of depth (Fig. 11B). It hides under rocks and cavities during its winter terrestrial phase. In Cao Bang Province, *Paramesotriton guangxiensis* (Huang, Tang & Tang, 1983) is sympatric with *Tylototriton koliaensis* sp. nov., but it presumably occupies cooler streams at higher elevations (Hernandez 2016). Other species of amphibians recorded at the type locality of *Tylototriton koliaensis* sp. nov. include *Vietnamophryne orlovi* Poyarkov, Suwannapoom, Pawangkhanant, Aksornneam, Duong, Korost & Che, 2018, *Raorchestes* cf. *malipoensis* Huang, Liu, Du, Bernstein, Liu, Yang, Yu & Wu, 2023, *Kurixalus* cf. *hainanus* (Zhao, Wang & Shi in Zhao *et al.* 2005), *Gracixalus gracilipes* (Bourret, 1937), *Gracixalus* cf. *jinxiuensis* (Hu in Hu *et al.* 1978), *Polypedates mutus* (Smith, 1940), and *Ophryophryne microstoma* Boulenger, 1903.

Several natural history features that pertain to *Tylototriton koliaensis* sp. nov. were documented by Bernardes *et al.* (2017). These authors reported that females are supposadly larger than males and have distended abdomens during the breeding season, whereas males tend to have more robust-looking limbs than females. Females deposit their eggs on the ground (terrestrial oviposition) from April to July, and tadpoles undergo metamorphosis in July. However, in June 2017, we also observed egg deposition directly in the water at the type locality, in the context of large aggregations of males and females in the slow-moving parts of partially flooded mountain streams. In Bao Lac District, Cao Bang Province, Bernardes *et al.* (2017) observed eggs and larvae in July, and reported egg diameters (from randomly sampled eggs) of 7.2 mm to 8.9 mm (mean: 8.1 ± 0.8 mm, n = 28). On average, the egg clutches of *Tylototriton koliaensis* sp. nov. appear to be bigger than other *Yaotriton* members, with a mean size of 67 ± 32 eggs, deposited on rock and soil substrates at a distance of 50 ± 28 cm from the water (Bernardes *et al.* 2017). Bernardes *et al.* (2017) also found clutches of *Tylototriton koliaensis* sp. nov. on the slopes of a slow-flowing forest stream, suggesting some flexibility in the breeding habitat.

According to Bernardes *et al.* (2017), larvae of the new species have relatively broad head, the interorbital distance being wider than the internarial distance, a pointed tail tip, the dorsal tail fin being higher than the ventral fin, and the tail being shorter than the body. The early larval stages of *Tylototriton koliaensis* sp. nov. lack balancers, while the more advanced larval stages have dark ground color with the exception of yellow digits and ventral fin (Bernardes *et al.* 2017).

*Conservation status*

Habitat loss due to destructive anthropogenic activities, such as road construction, agriculture, forest destruction caused by logging, hydroelectric development, and rock excavation, are the main threats faced by the new species (Hernandez 2016). During the past French colonial era, mining and farming activities
resulted in the clearing of large areas of evergreen montane forest in Cao Bang Province, and possibly caused local population declines. At present, *Tylototriton koliaensis sp. nov.* is only distributed in one protected area, Phia Oac-Phia Den NP, which covers 663,798 ha. Given its presumably small range that potentially continues to be subjected to many threats, we suggest to list *Tylototriton koliaensis sp. nov.* under the IUCN's Red List category of Endangered (EN) (Anonymous 2019).

DISCUSSION

New *Tylototriton* species are being described on a yearly basis, and most recent descriptions stemmed from taxonomic revisions of previously known populations regrouped under species complexes. This is particularly true for the subgenus *Yaotriton*, where the external morphology remains overall similar even between distantly related species (Hernandez 2016; Bernardes *et al.* 2020; Poyarkov *et al.* 2021a). There is thus little doubt that an intensification of survey efforts, especially in the unexplored remote montane areas of East and Southeast Asia, combined with the implementation of new methods, will continue to uncover cryptic diversity in crocodile newts (Dufresnes & Hernandez 2022). Accordingly, twenty species, i.e., about half of the presently recognized diversity of *Tylototriton*, were described during the last six years (Decemson *et al.* 2023; Frost 2024). Our description of *Tylototriton koliaensis sp. nov.* now brings the total number of species in the genus to 42, including 10 in Vietnam alone. Vietnam is predominantly inhabited by members of the subgenus *Yaotriton* (8 out of 10), namely *Tylototriton koliaensis sp. nov.*, *T. ngoclinhensis*, *T. obsti*, *T. pasmansi*, *T. sparreboomi*, *T. thaiorum*, *T. vietnamensis*, and *T. ziegleri* (Bernardes *et al.* 2020; Poyarkov *et al.* 2021a; Phung *et al.* 2023; this study), and this diversity emphasizes well the country as an important center of amphibian species richness and endemism (see Poyarkov *et al.* 2021b).

Beyond the characterization of *Tylototriton koliaensis sp. nov.*, our updated mtDNA-based genealogy of *Tylototriton* (which is largely concordant with previous studies, e.g., Yuan *et al.* 2011; Nishikawa *et al.* 2013a; Wang *et al.* 2018; Liu *et al.* 2022; Dufresnes & Hernandez 2022) also provides new insights on the distribution of the recently described *T. thaiorum*. Until recently, this species was only known from Nghe An Province in the northern part of central Vietnam. Here, we provide DNA barcoding evidence for its occurrence in Thanh Hoa Province of northern Vietnam (Fig. 2, Table 1). This range extension is not necessarily surprising as the new locality (Vin Village, Bat Mot Commune within Xuan Lien NR, Thuong Xuan District, Thanh Hoa Province; see Table 1) is situated only 34 km away (direct distance) from the type locality (Pu Hoat Nature Reserve in Nghe An Province). Based on this updated distribution, we predict that *T. thaiorum* might also be discovered in the adjacent parts of Laos (Houaphan Province).

An important step forward to study the diversity of *Tylototriton* is the application of high-throughput sequencing for the first time in these newts. Using ddRAD-seq, we could robustly establish the nuclear differentiation between *T. ziegleri* and *Tylototriton koliaensis sp. nov.* based on thousands of loci, whereas traditional nuclear sequence markers were shown to be largely uninformative in the genus, due to slow evolution and incomplete lineage sorting (Wang *et al.* 2018; Dufresnes &
Hernandez 2022). Extending the application of genomic methods to taxonomy (“genomic” taxonomy), notably to ascertain that the genetic divergence of candidate species is not only limited to the mitochondrion, has been recently advocated (see Dufresnes et al. 2023; Trofimets et al. 2024; Vences et al. 2024). Although it remains cumbersome to implement without proper laboratory and bioinformatic resources and training, the ambition to include genomics among the toolkit of taxonomists is becoming increasingly realistic, thanks to simplified, freely-accessible protocols (see Methods) and most importantly, more affordable sequencing costs. These aspects offer exciting prospects to revisit the entire diversification of crocodile newts with genome-wide loci, especially since most species were described based on mitochondrial DNA as the sole molecular evidence (Dufresnes & Hernandez 2022).

It is notoriously difficult to find steady morphological differences among closely related species of *Tylototriton* (many of which are considered ‘cryptic’), and in this respect, our study shows that detailed scrutiny of skull osteology can provide informative insights. Micro-CT scanning, which in *Tylototriton* was so far only applied in the *T. verrucosus* species complex (Le *et al.* 2015), revealed species-distinctive bony features in *T. ziegleri* s. l. The pterygoid is consistently different in shape and sculpture between the two examined series of *T. ziegleri* and *Tylototriton koliaensis* sp. nov., notably the shape of the processi palatini and otici of the pterygoid and the shape of the pterygoid groove. Reciprocally, the examinations demonstrated that candidate diagnostic characters, such as the shape of the vomerine tooth series (∧ or ∩-shaped), as well as the degree of development of sagittal and supratemporal cranial crests, may show substantially individual variation. These observations emphasize the need to include representative series of specimens for morphological descriptions, in order to carefully establish interspecific differences when intraspecific polymorphism is high.

Crocodile newts rise among the most threatened amphibians in the world, and all *Tylototriton* species are listed in the Appendices of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES; Anonymous 2022). In Vietnam, these species fall under the Governmental Decree No. 84/2021/ND-CP, which automatically confers *Tylototriton koliaensis* sp. nov. a protected status. Its conservation at the global scale should benefit from further survey efforts to clarify the extent of its distribution in northern Vietnam, as well as in the adjacent Guangxi Province of China. The species should also be under watch to limit illegal poaching for the pet trade, an important threat to crocodile newts that should not be underestimated (Rowley *et al.* 2016; Bernardes *et al.* 2017, 2020; Grismer *et al.* 2018). The description of *Tylototriton koliaensis* sp. nov. thus emphasizes the unique but fragile natural heritage of Vietnam, where the rapid economic development, even in rural areas deemed hardly accessible just a few years ago, is putting an unprecedented pressure on wildlife. Such pressure, which takes many forms, including deforestation, agriculture, water pollution, and the construction of tourist infrastructure, does not spare the mountain forests of Vietnam. Given that these mountains host a large number of rare and endemic amphibian species (Poyarkov *et al.* 2021b), immediate efforts to document their biodiversity are imperative to ensure their conservation.
ACKNOWLEDGEMENTS

The fieldwork in Vietnam was completed within the frameworks and with partial
financial support from the research project E-1.2 of the Joint Vietnam - Russia Tropical
Science and Technology Research Center for 2024 (Task No. 3). Permission to conduct
fieldwork in Vietnam was granted by the Bureau of Forestry, the Ministry of
Agriculture and Rural Development of Vietnam, and local administrations. Permissions
to conduct fieldwork and collect specimens were granted by the Department of Forestry,
Ministry of Agriculture and Rural Development of Vietnam (permit numbers
#712/TCLN-BTTN of 17.05.2017 and #1539/TCLN-DDPH of 19.09.2018) and by the
Forest Protection Departments of the Peoples' Committees of Cao Bang Province
(permit number #1659/UBND-NC of 02.06.2017), Ha Giang Province (permit number
#109/SNgV-LS of 15.03.2022), and Thanh Hoa Province (permit number #562/GP of
01.06.2022) of Vietnam. The authors are grateful to Andrey N. Kuznetsov (VRTC,
Hanoi), Hoi Dang Nguyen (VRTC, Hanoi), Leonid P. Korzoun (MSU, Moscow), Bao
Nguyen Le (DTU, Da Nang), Andrey M. Bragin (VRTC, Hanoi), as well as the Ban Vin
Ranger Station staff (Thanh Hoa, Vietnam) for support, organization and assistance of
the fieldwork; to Loi Phuoc Tran (Vietnam) and Yinpeng Zhang (China) for providing
photos and information of *T. ziegleri*; to Alexey A. Polilov (MSU, Moscow) and
Evgeny Scherbakov (MSU, Moscow) for the opportunity to conduct osteological study
using the Bruker Skyscan 1272 tomograph of the Biological Faculty of Moscow State
University; and to the MSU HerpLab members including Alexei V. Trofimets, Sabira S.
Idiiatullina, and Nikita S. Kliukin for assistance in the lab. We also thank Spartak N.
Litvinchuk and Anton O. Svinin for their feedback on our manuscript. This work was
supported by the Russian Science Foundation (Grant Number 22-14-00037) for
molecular and phylogenetic analyses to NAP, as well as by the Research Found for
International Scientists (RFIS) of the National Natural Science Foundation of China
(NSFC) (N°3211101356) for genomic analyses to CD.

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Submitted: 28 September 2024.

Accepted: 30 October 2024.

Published: 12 November 2024.

*Corresponding Editor:* Spartak N. LITVINCHUK