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Research Article
Southbound – the southernmost record of Tylototriton (Amphibia, Caudata, Salamandridae) from the Central Highlands of Vietnam represents a new species
expand article infoTrung My Phung‡, Cuong The Pham§|, Truong Quang Nguyen|§, Hoa Thi Ninh, Huy Quoc Nguyen#, Marta Bernardes¤«, Son Thanh Le», Thomas Ziegler«¤, Tao Thien Nguyen
‡ Unaffiliated, Bien Hoa, Vietnam
§ Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology, Hanoi, Vietnam
| Graduate University of Science and Technology, Hanoi, Vietnam
¶ Institute of Genome Research, Vietnam Academy of Science and Technology, Hanoi, Vietnam
# Vietnam National Museum of Nature, Vietnam Academy of Science and Technology, Hanoi, Vietnam
¤ University of Cologne, Cologne, Germany
« Cologne Zoo, Cologne, Germany
» National Institute of Medicinal Materials, Ho Chi Minh City, Vietnam

Corresponding author: Thomas Ziegler ( ziegler@koelnerzoo.de )

Academic editor: Johannes Penner
© 2023 Trung My Phung, Cuong The Pham, Truong Quang Nguyen, Hoa Thi Ninh, Huy Quoc Nguyen, Marta Bernardes, Son Thanh Le, Thomas Ziegler, Tao Thien Nguyen.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Phung TM, Pham CT, Nguyen TQ, Ninh HT, Nguyen HQ, Bernardes M, Le ST, Ziegler T, Nguyen TT (2023) Southbound – the southernmost record of Tylototriton (Amphibia, Caudata, Salamandridae) from the Central Highlands of Vietnam represents a new species. ZooKeys 1168: 193-218. https://doi.org/10.3897/zookeys.1168.96091
ZooBank: urn:lsid:zoobank.org:pub:0100D617-C563-4A6F-B57C-1528B0F11446
Open Access

Abstract

A new species of the genus Tylototriton is described from Ngoc Linh Mountain, Kon Tum Province, in the Central Highlands of Vietnam based on integrative taxonomy, namely by combining molecular and morphological evidence. Tylototriton ngoclinhensis sp. nov. differs from all other congeners based on morphological data, allopatric distribution, and molecular divergence. In terms of genetic divergence, Tylototriton ngoclinhensis sp. nov. distinctly differs from the sister species T. panhai (6.77%) and from T. ngarsuensis (12.36%) based on the mitochondrial NADH dehydrogenase subunit 2 (ND2) gene. Tylototriton ngoclinhensis sp. nov. is a moderate sized and robust salamander species with large cephalic edges, parotoids, and vertebral ridge orange in coloration. The new taxon differs from its congeners by a combination of the following morphological characteristics: size medium (SVL 60.8–66.5 mm, TL 57.6–61.8 mm in males, and SVL 72.5–75.6 mm, TL 62.9–67.9 mm in females); head longer than wide; parotoids very prominent and enlarged, projecting backwards; tail length shorter than snout-vent length; vertebral ridge large, high and glandular in appearance; 14 large and distinct dorsolateral glandular warts; gular fold present; tips of fore and hind limbs overlapping when adpressed along the body; tips of fingers reaching between eye and nostril when foreleg is laid forward; dorsal surface and lateral sides of the head, upper and lower lips, dorsolateral glandular warts, vertebral ridge, the peripheral area of the cloaca and the ventral edge of the tail orange in coloration; the presence of a distinct black line extending from the posterior end of the eye towards the shoulder. Tylototriton ngoclinhensis sp. nov. is restricted to evergreen montane forests near water bodies on Ngoc Linh Mountain. We suggest that the new species should be classified as Endangered (EN) in the IUCN Red List. This new important discovery represents the eighth Tylototriton taxon described from Vietnam, and at the same time constitutes the southernmost distributional record for the whole genus in Asia.

Key words

Crocodile newt, ND2 gene, Ngoc Linh Mountain, Salamandridae, taxonomy, Tylototriton ngoclinhensis sp. nov.

Introduction

The salamandrid genus Tylototriton Anderson, 1871, commonly known as crocodile newts, currently contains 38 species inhabiting montane forest areas throughout the Asian monsoon climate zone and is distributed across Asia, from eastern Himalayas, eastern Nepal, northern India, Bhutan, Myanmar, central to southern China (including Hainan Island), and southwards through Laos, Thailand, and Vietnam (Dufresnes and Hernandez 2022; Li et al. 2022; Wang et al. 2022; Frost 2023).

Remarkably, 15 new species have been described in the past five years (Hernandez 2016; Dufresnes and Hernandez 2022; Frost 2023). The genus was subdivided into three subgenera, Tylototriton, Yaotriton, and Liangshantriton (e.g., Dubois and Raffaëlli 2009; Nishikawa et al. 2013a; Bernardes et al. 2020; Pomchote et al. 2021) and includes several, as yet, unnamed taxa, which contain cryptic species that are morphologically difficult to distinguish, especially in their southern range of distribution (Hernandez 2016; Pomchote et al. 2021; Poyarkov et al. 2021b). Most of the new discoveries derived from the splitting of widely distributed Tylototriton taxa through the efforts of integrative taxonomy, namely the combination of morphological and phylogenetic analyses.

In Vietnam, six species and seven taxa are currently known, Tylototriton anguliceps Le, Nguyen, Nishikawa, Nguyen, Pham, Matsui, Bernardes & Nguyen, 2015; T. pasmansi pasmansi Bernardes, Le, Nguyen, Pham, Pham, Nguyen & Ziegler, 2020; T. pasmansi obsti Bernardes, Le, Nguyen, Pham, Pham, Nguyen & Ziegler, 2020; T. sparreboomi Bernardes, Le, Nguyen, Pham, Pham, Nguyen & Ziegler, 2020; T. thaiorum Poyarkov, Nguyen & Arkhipov, 2021; T. vietnamensis Böhme, Schöttler, Nguyen & Köhler, 2005; and T. ziegleri Nishikawa, Matsui & Nguyen, 2013 (Bernardes et al. 2017a; 2017b; Poyarkov et al. 2021b; Raffaëlli 2022; Frost 2023). All afore mentioned species are known from northern Vietnam, from Ha Giang Province southwards to Nghe An Province.

During recent fieldwork in May 2022 a new Tylototriton population was discovered in Ngoc Linh Mountain, Kon Tum Province, Central Vietnam resembling the T. panhai phenotype I from Phu Luang Wildlife Sanctuary and Phu Ruea National Park, Loei Province, northeastern Thailand (see Hernandez 2016; Hernandez and Pomchote 2020). However, morphological comparisons and molecular phylogenetic analyses revealed this southernmost Tylototriton population to be distinct from all related species of the genus. We herein describe the newly discovered Tylototriton population from the Central Highlands of Vietnam as a new species.

Materials and methods

Sampling

A field survey was conducted in Ngoc Linh Nature Reserve, Kon Tum Province of the Central Highlands, Vietnam, on 22nd of May 2022. Crocodile newts were found on the forest floor between 9:00 and 16:00. After having been photographed in life, six specimens were anaesthetized and euthanized in a closed vessel with a piece of cotton wool containing ethyl acetate (Simmons 2002), fixed in 80% ethanol for five hours, and then later transferred to 70% ethanol for permanent storage. Muscle tissue from the central upper part of the tail was taken for tissue samples, which were preserved separately in 70% ethanol prior to fixation. Voucher specimens referred to in this paper were deposited in the collections of the Institute of Ecology and Biological Resources (IEBR), Hanoi, Vietnam.

Molecular analyses

DNA from tissue samples of the preserved specimens were extracted using the Dneasy blood and tissue kit, Qiagen (California, USA). A fragment of a mitochondrial gene, the NADH dehydrogenase subunit 2 (ND2), was amplified by PCR master mix (Fermentas, Burlington, ON, Canada) using the primer pair, Sal_Nd2_F1 (5’- AAGCTTTTGGGCCCATACC-3’), Sal_Nd2_R1 (5’-GTTATAAATATGGAKLARGTTA-3’) (Nishikawa et al. 2013b).

For the phylogenetic analyses, 53 sequences of species of the genus Tylototriton were used in combination with a sequence of Pleurodeles waltl and Echinotriton chinhaiensis as outgroups according to Li et al. (2022) (Table 1).

Table 1.
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Samples of the Tylototriton species and other species used for DNA analyses in this study.

No. Scientific name Voucher number Locality GenBank number Reference
1. Tylototriton ngoclinhensis sp. nov. IEBR A.5131 Kon Tum Prov., Vietnam LC575223 This study
2. Tylototriton ngoclinhensis sp. nov. IEBR A.5130 Kon Tum Prov., Vietnam LC575221 This study
3. Tylototriton ngoclinhensis sp. nov. IEBR A.5133 Kon Tum Prov., Vietnam LC575222 This study
4. T. anguliceps NUOL 00420 Viengphoukha, Luang Namtha, Laos KT304301 Phimmachak et al. (2015)
5. T. anhuiensis CIB 08042905-3 Yuexi Co. Anhui Prov., China KY800854 Wang et al. (2018)
6. T. anhuiensis CIB 08042905-4 Yuexi Co. Anhui Prov., China KY800855 Wang et al. (2018)
7. T. asperrimus CIB GX20080714 Jinxiu Co., Guangxi Prov., China KY800819 Wang et al. (2018)
8. T. broadoridgus CIB 200084 Sangzhi Co., Hunan Prov., China KY800837 Wang et al. (2018)
9. T. dabienicus HNNU1004-024 Shangcheng Co., Henan Prov., China KC147812 Nishikawa et al. (2014)
10. T. dabienicus HNNU 1004-015 Shangcheng Co., Henan Prov., China KC147811 Nishikawa et al. (2014)
11. T. dabienicus HNNU 1004-026 Shangcheng Co., Henan Prov., China KY800869 Wang et al. (2018)
12. T. daloushanensis GZNU 20060626002 Suiyang Co., Guizhou Prov., China JF825872 Li et al. (2022)
13. T. daloushanensis GZNU 20060626001 Suiyang Co., Guizhou Prov., China FJ415600 Wang and Gu (2008)
14. T. hainanensis CIB 20081048 Mt. Diaoluo, Hainan Prov., China KC147817 Nishikawa et al. (2013b)
15. T. himalayanus CIB 201406246 Mai Pokhari, Illam, Mechi, Nepal KT765173 Khatiwada et al. (2015)
16. T. kweichowensis CIB Wg20080818014 Bijie City, Guizhou Prov., China KY800823 Wang et al. (2018)
17. T. liuyangensis CIB 110601F06 Liuyang City, Hunan Prov., China KY800875 Wang et al. (2018)
18. T. maolanensis CIB ML20180427003 Libo Co., Guizhou Prov., China MK820701 Li et al. (2020)
19. T. maolanensis CIB ML20180427004 Libo Co., Guizhou Prov., China MK820702 Li et al. (2020)
20. T. maolanensis GZNU 200706050101 Leishan Co., Guizhou Prov., China FJ415596 Li et al. (2020)
21. T. maolanensis GZNU 200706050102 Leishan Co., Guizhou Prov., China JF825868 Li et al. (2020)
22. T. ngarsuensis LSUHC13762 Shan State, Myanmar MH836585 Grismer et al. (2018)
23. T. notialis FMNH: HERP:271120 Boualapha, Khammouan, Laos HM462061 Stuart et al. (2010)
24. T. panhai NUOL 00424 Botene, Xaignabouli, Laos KT304309 Phimmachak et al. (2015)
25. T. panhai NUOL 00425 Botene, Xaignabouli, Laos KT304311 Phimmachak et al. (2015)
26. T. panhai NUOL 00421 Botene, Xaignabouli, Laos KT304310 Phimmachak et al. (2015)
27. T. pasmansi IEBR 4466 (Holotype) Da Bac, Hoa Binh Prov., Vietnam MT210166 Bernardes et al. (2020)
28. T. pasmansi IEBR:4467 Da Bac, Hoa Binh Prov., Vietnam MT210167 Bernardes et al. (2020)
29. T. podichthys NCSM 77725 Phoukhoun, Luang Phabang, Laos KT304295 Phimmachak et al. (2015)
30. T. pseudoverrucosus CIB WCG2012003 Ningnan Co., Liangshanyizu State, Sichuan Prov., China KY800861 Wang et al. (2018)
31. T. pulcherrimus KUHE:46406 Pet Trade KY800880 Wang et al. (2018)
32. T. shanjing CIB 980004 Baoshan City, Yunnan Prov., China KY800831 Wang et al. (2018)
33. T. shanorum KUHE 42348 Shan State, Myanmar AB769544 Nishikawa et al. (2013b)
34. T. sini SYS a008354 Mt Yunkai, Guangdong Prov., China OK539836 Lyu et al. (2021)
35. T. sparreboomi IEBR 4477 Sin Ho, Lai Chau Prov., Vietnam MT210163 Bernardes et al. (2020)
36. T. taliangensis CIB GG200110183 Shimian Co., Yan’an City, Sichuan Prov., China KC147819 Yang et al. (2014)
37. T. thaiorum ZMMU A-7577 Pu Hoat NR, Nghe An Prov., Vietnam MW883478 Poyarkov et al. (2021a)
38. T. tongziensis CIB WB2020081511 Tongzi Co., Guizhou Prov., China OK349411 Li et al. (2022)
39. T. tongziensis CIB TZ20160714002 Tongzi Co., Guizhou Prov., China OK349413 Li et al. (2022)
40. T. tongziensis TZ20160714010 Tongzi Co., Guizhou Prov., China OK349414 v
41. T. tongziensis CIB WB2020202 Tongzi Co., Guizhou Prov., China OK349415 Li et al. (2022)
42. T. uyenoi KUHE:19147 Doi Suthep, Chiang Mai Prov., Thailand AB830733 Nishikawa et al. (2013a)
43. T. verrucosus CIB-TSHS1 Longchuan Co., Dehong State, Yunnan Prov., China KY800847 Wang et al. (2018)
44. T. wenxianensis CIB 2010123101 Pingwu Co., Gansu Prov., China KY800867 Wang et al. (2018)
45. T. wenxianensis CIB 2010123102 Pingwu Co., Gansu Prov., China KY800868 Wang et al. (2018)
46. T. yangi KUHE:42282 Pet Trade KY800887 Nishikawa et al. (2014)
47. T. ziegleri VNMN 3390 Quan Ba, Ha Giang Prov., Vietnam KY800889 Wang et al. (2018)
48. T. liuyangensis CSUFT20100108 Liuyang City, Hunan Prov., China KJ205598 Yang et al. (2014)
49. T. phukhaensis CUMZ-A-7717 DPKNP, Nan Prov., Thailand MN912573 Pomchote et al. (2020)
50. T. sparreboomi IEBR 4477 Sin Ho, Lai Chau Prov., Vietnam MT210163 Bernardes et al. (2020)
51. T. vietnamensis IEBR A.2014.44 Mau Son, Loc Binh, Lang Son Prov., Vietnam KX609962 Bernardes et al. (2017a)
52. T. vietnamensis IEBR A.2014.43 Hoanh Bo, Quang Ninh Prov., Vietnam KX609961 Bernardes et al. (2017a)
53. T. vietnamensis IEBR A.0701 Mau Son, Lang Son Prov., Vietnam KY800873 Wang et al. (2018)
54. Echinotriton chinhaiensis CIB ZHJY2 Zhenhai Co., Zhejiang Prov., China KY800892 Wang et al. (2018)
55. Pleurodeles waltl - Cadiz, Andalusia, Spain EU880330 Zhang et al. (2008)

CHROMAS PRO software (Technelysium Pty Ltd., Tewantin, Australia) was used to edit the sequences, which were aligned using MAFFT v. 7 (Katoh and Standley 2013) with default settings. We then checked the initial alignments by eye and adjusted them slightly. Phylogenetic trees were constructed by using maximum likelihood ML) and Bayesian inference (BI). Prior to ML and Bayesian analyses, we chose the optimum substitution models for entire sequences using KAKUSAN 4 (Tanabe 2011) based on the Akaike information criterion (AIC). The best model selected for ML and BI was the general time-reversible model (GTR: Tavaré 1986) with a gamma shape parameter (G: 0.337 in ML and 0.376 in BI). For ML analysis, the TREEFINDER software (Jobb et al. 2004) was used while BI analysis was conducted using MRBAYES v. 3.2.7a (Ronquist et al. 2012). The strength of nodal support in the ML tree was analyzed using non-parametric bootstrapping (MLBS) with 1,000 replicates. We regarded tree nodes in the ML tree with bootstrap values of 75% or greater as sufficiently resolved (Hillis and Bull 1993), and nodes with a BPP of 95% or greater as significant in the BI analysis (Leaché and Reeder 2002). The BI summarized two independent runs of four Markov Chains for 10,000,000 generations. A tree was sampled every 100 generations and a consensus topology was calculated for 70,000 trees after discarding the first 30,001 trees (burn-in 1,000,000). We checked parameter estimates and convergence using TRACER v. 1.6 (Rambaut and Drummond 2013). Pairwise comparisons of uncorrected sequence divergences (p-distance) were calculated with MEGA 7 (Kumar et al. 2016) for ND2 fragments only between species of the genus Tylototriton. Variance was estimated using bootstrap method with 1,000 replicates using nucleotide substitution while gap/missing data were treated via pairwise deletion.

Morphological characters

A total of 27 morphological characters were measured following Bernardes et al. (2020) to the nearest 0.01 mm with a digital caliper as follows: anterior snout-vent length (SVL) from tip of snout to anterior tip of vent ; head length (HL) ; head width (HW) measured behind the eyes and before the beginning of the parotoids ; maximum head width (MHW); parotoid length (PL) ; parotoid width (PW) ; maximum parotoid height (PH) ; eye length (EL) ; eye-narial distance (EN) ; inter-narial distance (IN) ; inter-eye distance (IE) ; lower jaw length (LJL) from tip of lower jaw to jaw angle ; maximum upper eyelid length (UEL) ; humerus length (HUM) ; radius length (RAD) ; femur length (FEM) ; tibia length (TIB) ; total forelimb length (FORE) ; total hindlimb length (HIND) ; tail length (TL) from anterior of vent to tail tip ; tail height (TH) ; cloaca length (CIL): length of cloaca muscle ; cloaca width (CIW) ; width of vertebral cord measured at the height of the 5th nodule (WVr) ; length of the 5th anterior dorsal nodule (L5W) ; axilla to groin (AG) ; trunk length from wrinkle of throat to anterior tip of vent (TkL) ; total length (TOL).

Morphological comparisons between the new taxon and its congeners were based on the specimens examination and the following literature: Fei et al. (1984), Nussbaum et al. (1995), Böhme et al. (2005), Chen et al. (2010), Stuart et al. (2010), Hou et al. (2012), Shen et al. (2012), Nishikawa et al. (2013a,b), Nishikawa et al. (2014), Yang et al. (2014), Khatiwada et al. (2015), Le et al. (2015), Phimmachak et al. (2015), Fei and Ye (2016), Hernandez (2016), Qian et al. (2017), Grismer et al. (2018, 2019), Zaw et al. (2019), Bernardes et al. (2017a, 2020), Pomchote et al. (2020, 2021), Lyu et al. (2021), Poyarkov et al. (2021a), Li et al. (2022), and Luo et al. (2022).

Results

Phylogenetic analyses

Aligned, combined sequences yielded a total of 1,035 characters. Of 1,035 nucleotide sites, 416 were variable and 336 were parsimony informative within the in-group. The ML and Bayesian analyses produced topologies with -lnL = 7442.0236 and 7521.862, respectively. Phylogenetic analyses employing ML and BI methods yielded slightly different topologies only among referenced species, and only the ML tree is presented in Fig. 1.

Figure 1. 

BI tree from a 1035 bp sequence of the mitochondrial ND2 gene for Tylototriton and outgroup species; ML inferences bootstrap support value (ML-BS) and Bayesian posterior probabilities (BPP) are shown near the node. For GenBank accession numbers, refer to Table 1.

Monophyly of Tylototriton with respect to the outgroup species was fully supported (each 100% support in ML bootstrap value and Bayesian posterior probability) and samples were split into four major clades named A, B, C, and D.

Monophyly of clade A, including T. maolanensis Li, Wei, Cheng, Zhang & Wang, 2020; T. wenxianensis Fei, Ye & Yang, 1984; T. anhuiensis Qian, Sun, Li, Guo, Pan, Kang, Wang, Jiang, Wu & Zhang, 2017; T. dabienicus Chen, Wang & Tao, 2010; T. tongziensis Li, Liu, Shi, Wei & Wang, 2022; T. broadoridgus Shen, Jiang & Mo, 2012 T. daloushanensis Zhou, Xiao, and Luo, 2022; and T. liuyangensis Yang, Jiang, Shen, and Fei, 2014, was well supported (100% and 92% support).

The undescribed species of Tylototriton from Kon Tum Province, Vietnam was clustered in clade B with T. panhai Nishikawa, Khonsue, Pomchote & Matsui, 2013 from Laos and T. vietnamensis from Vietnam, but the support was not significant, particularly for Bayesian posterior probability (0.83 and 60%) (Table 2).

Table 2.
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Mean uncorrected (p) distance (%) among 1,035 bp fragments of ND2 of the genus Tylototriton and related taxa.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
1. Tylototriton ngoclinhensis
sp. nov.		 0.00–0.31
2. T. anguliceps 10.08–11.55 -
3. T. anhuiensis 10.19–10.64 10.82 -
4. T. asperrimus 9.88–10.64 11.30 8.31 -
5. T. broadoridgus 9.54–9.73 10.92 3.67 8.50 -
6. T. dabienicus 9.23–10.03 11.88–12.08 3.86–3.96 8.99–9.08 3.38–3.48 0.10–0.29
7. T. daloushanensis 9.54–9.73 11.69 4.64 9.28 4.25 4.44–4.45 -
8. T. hainanensis 7.72–8.21 9.95 7.83 5.02 7.63 8.79–8.89 9.18 -
9. T. himalayanus 8.64–9.12 6.86 10.82 11.50 10.43 11.01–11.21 11.01 9.76 -
10. T. kweichowensis 8.64–9.12 6.18 9.86 10.14 8.99 10.24–10.34 10.63 8.60 5.31 -
11. T. liuyangensis 8.62–9.12 10.14–10.24 7.25–7.34 8.70–8.79 7.05–7.15 7.25–7.44 7.44–7.54 7.73–7.83 10.05–10.14 9.47–9.57 0.10
12. T. maolanensis 9.57–11.55 10.72–11.40 3.48–3.57 8.60 2.80–3.00 3.38–3.67 4.35–4.44 8.21–8.31 10.63–11.11 9.66–9.95 7.05–7.25 0.00–2.13
13. T. ngarsuensis 12.04–12.36 7.74 12.05 12.34 11.56 12.34–12.54 12.44 10.97 6.46 6.95 11.36–11.46 11.46–11.85 -
14. T. notialis 9.26–9.73 10.63 8.50 4.93 8.41 9.18–9.28 9.47 4.83 10.63 9.86 8.70–8.79 8.60–8.70 11.75 -
15. T. panhai 6.77–6.99 12.95 9.57 11.11 8.37 9.47–9.57 10.24 9.66 12.27 10.72 9.37–9.47 9.86–10.14 13.22 10.34 0.00
16. T. pasmansi 9.57–10.33 10.40–10.69 8.41–8.50 3.38–3.48 8.31–8.41 9.28–9.37 9.57–9.66 4.83 11.21–11.69 9.95–10.24 8.31–8.41 8.89–9.08 12.44–12.93 4.64–4.93 10.43–10.53 0.48
17. T. phukhaensis 11.11–11.38 4.29 11.40 11.99 11.11 11.89–12.09 12.28 10.62 7.02 6.34 10.33–10.43 11.11–11.89 9.01 11.01 12.87 12.09–12.38
18. T. podichthys 9.88–10.15 5.31 9.86 10.82 9.95 10.63–10.82 10.14 10.14 6.76 6.09 10.05–10.14 10.05–10.43 8.33 10.72 12.27 11.21–11.50 6.24
19. T. pseudoverrucosus 9.26–9.54 8.50 9.47 9.66 9.28 9.47–9.66 9.66 8.50 7.05 5.99 9.80–9.18 9.28–9.86 8.72 9.28 10.43 9.37–9.86 8.38 7.25
20. T. pulcherrimus 9.73–10.15 3.96 9.55–9.95 10.92 9.86 10.34–10.53 12.24 9.47 6.18 5.41 9.76–9.86 9.76–10.14 6.95 10.24 12.08 10.50–11.20 4.39 3.67 6.67
21. T. shanjing 11.25–11.69 4.35 10.43 11.40 10.24 10.82–11.01 10.63 10.24 6.28 5.99 10.53–10.63 9.95–10.72 7.35 11.01 12.66 12.08–11.79 5.36 4.25 7.73 2.51
22. T. shanorum 9.88–10.33 6.67 10.82 11.30 10.14 10.92–11.11 11.40 9.76 5.02 5.89 10.24–10.34 10.24–10.63 1.76 10.63 12.37 11.21–11.69 7.60 7.44 7.92 6.18 6.57
23. T. sini 7.72–8.81 9.57 8.21 5.22 8.60 8.99–9.08 8.79 3.19 9.47 8.89 7.83–7.92 8.31–8.6 10.77 5.51 10.34 5.12–5.60 10.62 9.66 8.50 9.28 10.24 9.57
24. T. sparreboomi 8.64–10.03 10.72–10.92 7.92–8.12 4.15 8.02–8.21 8.50–8.70 9.28–9.47 4.44–4.64 10.53–10.72 9.57–9.76 8.60–8.79 8.50–8.79 11.95–12.14 4.73 9.95–10.14 3.96–4.25 11.31–11.50 10.92–11.11 9.08–9.28 10.34–10.53 11.40–11.59 10.53–10.72 4.83–5.02 0.19
25. T. taliangensis 9.42–9.85 8.41 8.99 9.28 9.28 9.08–9.28 9.47 8.60 7.54 6.47 8.99–9.08 8.89–9.57 9.11 9.47 10.53 9.76–10.05 8.58 7.73 2.71 7.25 7.34 8.12 8.79 9.28–9.47
26. T. thaiorum 8.64–9.12 10.72 8.21 5.31 7.92 8.70–8.79 8.99 4.64 10.53 9.95 8.50–8.60 8.12–8.21 12.05 2.80 10.24 4.64–4.93 10.82 10.72 8.89 10.24 11.01 10.92 5.89 4.64–4.83 9.08
27. T. tongziensis 8.33–9.12 10.05–10.14 2.80–2.90 7.83–7.92 2.61–2.71 2.61–2.80 3.67 7.44–7.54 9.95–10.05 8.99–9.08 6.47–6.67 2.42–2.80 10.87–10.97 7.92–8.02 9.18–9.28 8.12–8.21 10.43–10.53 9.37–9.47 8.12–8.21 9.08–9.18 9.86–9.95 9.66–9.76 7.44–7.54 7.73–8.02 8.02–8.12 7.44–7.54 0.00–0.10
28. T. uyenoi 12.96–13.23 7.25 12.75 12.85 12.75 13.04–13.14 13.24 12.46 8.50 8.02 12.56–12.66 12.56–12.75 10.09 13.04 14.40 13.04–13.33 7.21 8.21 9.95 6.76 7.25 9.08 12.27 13.62–13.82 9.95 12.66 11.59–11.69
29. T. verrucosus 10.94–11.08 4.35 10.63 11.50 10.24 10.82–11.01 10.82 10.24 6.47 5.70 10.24–10.34 9.95–10.72 6.95 11.01 12.56 11.79–12.08 4.97 4.06 7.54 2.13 1.16 6.18 10.05 11.40–11.59 7.44 11.01 9.86–9.95 7.25
30. T. vietnamensis 11.11–11.38 13.04–13.17 10.64–10.84 10.82–11.04 10.84–10.92 10.44–10.92 11.14–11.21 10.72–10.94 12.36–12.75 11.65–11.98 10.64–10.94 10.53–11.14 12.97–13.17 11.35–11.55 10.54–11.24 11.59–12.75 13.48–13.74 12.95–13.07 11.45–11.65 11.79–12.06 12.57–12.66 11.85–12.06 11.01–11.45 10.54–10.82 10.94–11.14 11.55–11.75 9.83–10.14 14.78–14.99 12.08–12.06 0.00
31. T. wenxianensis 9.88–10.33 10.72 4.15 8.70 4.25 4.73–4.83 3.77 8.70 10.53 9.76 6.96–7.05 4.15–4.25 12.05 8.89 10.14 9.47–9.57 11.11 9.57 9.28 9.08 9.86 11.11 9.08 9.08–9.28 8.60 8.60 3.57–3.67 12.46 9.66 10.23–10.24 0.00
32. T. yangi 11.73–12.00 4.15 10.14 10.14 10.05 10.82–11.01 10.43 9.57 6.57 6.18 9.37–9.47 9.57–10.34 7.35 10.24 12.46 10.53–10.82 5.56 5.12 7.44 3.77 4.35 6.38 8.79 9.95–10.14 7.83 10.34 9.18–9.28 7.54 3.96 11.96–12.16 9.66
33. T. ziegleri 8.95–9.73 10.82 8.12 4.44 7.92 8.79–8.89 9.28 4.35 10.92 9.18 8.41–8.50 8.41–8.50 12.05 4.93 9.95 4.83–5.12 11.21 10.72 9.28 10.43 11.21 10.92 5.22 4.35 9.47 4.83 7.54–7.63 13.33 11.21 10.94–11.21 8.79 10.05

Monophyly of clade C, including T. pasmansi; T. sparreboomi; T. asperrimus Unterstein, 1930; T. thaiorum; T. notialis Stuart, Phimmachak, Sivongxay & Robichaud, 2010; T. sini Lyu, Wang, Zeng, Zhou, Qi, Wan & Wang, 2021; T. hainanensis Fei, Ye & Yang, 1984 and T. ziegleri, was well supported (100% and 98% support).

Monophyly of clade D, including T. verrucosus Anderson, 1871; T. shanjing Nussbaum, Brodie & Yang, 1995; T. podichthys Phimmachak, Aowphol & Stuart, 2015; T. pulcherrimus Hou, Zhang, Li & Lu, 2012; T. uyenoi Nishikawa, Khonsue, Pomchote & Matsui, 2013; T. phukhaensis Pomchote, Khonsue, Thammachoti, Hernandez, Peerachidacho, Suwannapoom, Onishi & Nishikawa, 2020; T. anguliceps; T. yangi Hou, Zhang, Zhou, Li & Lu, 2012; T. shanorum Nishikawa, Matsui & Rao, 2014; T. ngarsuensis Grismer, Wood, Quah, Thura, Espinoza, Grismer, Murdoch & Lin, 2018; T. himalayanus Khatiwada, Wang, Ghimire, Vasudevan, Paudel & Jiang, 2015; T. kweichowensis Fang & Chang, 1932; T. taliangensis Liu, 1950 and T. pseudoverrucosus Hou, Gu, Zhang, Zeng & Lu, 2012, was also strongly supported (100% and 97% support).

There is a clear genetic distance between clade A and the remaining clades B, C, and D: from 8.33% (Tylototriton sp. from Kon Tum Province, Vietnam and T. tongziensis) to 11.55% (Tylototriton sp. from Kon Tum Province, Vietnam and T. maolanensis); from 7.44% (T. sini and T. tongziensis) to 9.66% (T. pasmansi and T. daloushanensis); from 8.02 (T. daloushanensis and T. uyenoi) to 13.24% (T. tongziensis and T. taliangensis), respectively. The genetic distance between clade B and the two clades C and D ranges from 7.72% (Tylototriton sp. from Kon Tum Province, Vietnam and T. thaiorum) to 11.79% (T. vietnamensis and T. thaiorum); from 8.64% (Tylototriton sp. from Kon Tum Province, Vietnam and T. pseudoverrucosus) to 14.99% (T. vietnamensis and T. uyenoi) and clear genetic distances between clades C and D: from 8.5% (T. hainanensis and T. pseudoverrucosus) to 13.82 (T. sparreboomi and T. uyenoi).

The Tylototriton population from Kon Tum Provice, Vietnam exhibited distinct genetic distances from the thirty-three examined species of Tylototriton, with uncorrected p-distance of 6.77% (compared to T. panhai from Laos) to 12.36% (compared to T. ngarsuensis), being higher than that between some pairs of sister species, for example, T. verrucosus vs T. shanjing (1.16%), and T. maolanensis vs T. tongziensis (2.42%).

Furthermore, the population of Tylototriton sp. from Kon Tum Province, Vietnam was also clearly separated morphologically from all the other congeners, including its sister species T. panhai, which is in congruence with the genetic separation. Thus, we describe the new Tylototriton population from Kon Tum Province, Vietnam as a new species.

Taxonomy

Tylototriton ngoclinhensis sp. nov.

https://zoobank.org/CBC407D7-E692-4363-A2BA-EECA307C7AC9
Figs 2, 3, 4, 5, 6, 7, 8 Proposed common name: Ngoc Linh Crocodile Newt

Material examined

Holotype. IEBR A.5130 (Field No KT 2022.02), an adult male collected by T. M. Phung on 22 May 2022 in the montane evergreen forests of Ngoc Linh Natural Reserve, Dak Glei District, Kon Tum Province, Central Vietnam at 1.854 m a.s.l. Paratypes. IEBR A.5131, A.5132 (Field No KT 2022.01, KT 2022.5), two adult males and IEBR A.5133, A.5134 (Field No KT2022.03, KT 2022.6), two adult females, collected by T. M. Phung; IEBR A.5135 (Field No KT 2022.4), an adult female, collected by S. T. Le on 20 May 2022, the same collection data as the holotype.

Figure 2. 

Dorsolateral view of Tylototriton ngoclinhensis sp. nov., holotype male (IEBR A.5130), in life.

Etymology

The specific epithet ngoclinhensis refers to the type locality of the new species, Ngoc Linh Mountain in the Central Highlands of Vietnam. As common names, we suggest Ngoc Linh Crocodile Newt (English), Cá cóc sần ngọc linh (Vietnamese).

Figure 3. 

Dorsal views of the holotype B of Tylototriton ngoclinhensis sp. nov. (IEBR A.5130, male) and two paratypes A (IEBR A.5133, female) and C (IEBR A.5131, male) in preservative.

Diagnosis

The new species is assigned to the genus Tylototriton and the subgenus Yaotriton based on the results of the molecular phylogenetic analyses and the following combination of morphological attributes: rough skin covered with fine warts, the presence of dorsolateral bony ridges on the head; the presence of dorsolateral glandular warts on the body; quadrate spine absent (Nussbaum and Brodie 1982; Nishikawa et al. 2013a; Le et al. 2015). Furthermore, the species is diagnosed from its congeners by the following combination of morphological characters: (1) size medium (SVL 60.8–66.5 mm, TL 57.6–61.8 mm in males, and SVL 72.5–75.6 mm, TL 62.9–67.9 mm in females); (2) head longer than wide; (3) parotoids very prominent and enlarged, projecting backwards; (4) tail length shorter than the snout-vent length; (5) vertebral ridge large, high, and glandular in appearance (6) 14 distinct dorsolateral glandular warts; (7) gular fold present; (8) tips of fore- and hind limbs overlapping when adpressed along the body; (9) tips of fingers reaching between eye and nostril when foreleg is laid forward; (10) dorsal surface and lateral sides of the head, upper and lower lips, rib nodules, vertebral ridge, peripheral area of the cloaca, and the ventral edge of tail with orange coloration; (11) presence of a distinct black line extending from the posterior end of the eye towards the shoulder.

Figure 4. 

Ventral views of the holotype B of Tylototriton ngoclinhensis sp. nov. (IEBR A.5130, male) and two paratypes A (IEBR A.5133, female) and C (IEBR A.5131, male) in preservative.

Description of holotype

A medium-sized male (SVL 66.5 mm, TL 59.5 mm). Head longer than wide (HW/HL 81.6%); head slightly concave on the top; snout short, truncate in dorsal view, slightly angular shaped in profile and protruding beyond lower jaw; nostril closer to the snout tip than to the eye; upper lip thick, fleshy and overlapping lower lip under the eye region; dorsolateral bony ridges on head prominent, moderately protruding, from above eye to above anterior end of parotoid, posterior ends relatively thick and scrolled inside; mid-dorsal ridge on head distinct and thin; parotoids enlarged, projecting backwards; tongue oval, attached to anterior floor of mouth, free laterally and posteriorly; vomerine teeth series in an inverted V-shape, converging anteriorly and reaching choanae; glandular vertebral ridge large, high, segmented tuberculate, extending from top of head to base of tail; rib nodules large, forming knob-like warts, distinctly isolated from each other, 14 on each side of body from axilla to base of tail; gular fold present.

Limbs comparatively long, and slender; length of forelimbs approximately equal to hind limbs; relative length of forelimb FORE/SVL ratio 39.0%, relative length of hind limb HIND/SVL ratio 38.1%; tips of forelimb and hind limb overlapping when adpressed along the body; tips of fingers reaching between eye and nostril when foreleg is laid forward; fingers and toes well developed, free of webbing; fingers four, comparative finger lengths 1FL<4FL<2FL<3FL; toes five, comparative toe lengths 1TL<5TL<2TL<4TL<3TL.

Tail length shorter than the snout-vent length (TL/SVL 89.5%); tail compressed laterally, the base relatively broad, tapering posteriorly, tail tip pointed; tail height less than the width at the tail base; dorsal fin fold relatively high; ventral side smooth. In general, the appearance of the tail is relatively low and flat.

Dorsal skin very rough, with small granules and larger warts on dorsal surfaces of head and dorsum, lateral sides of body and tail; ventral skin with tubercles shaped like transverse wrinkles; throat with numerous tiny flat tubercles; 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; flattened outer metacarpal and metatarsal tubercles distinct on palms and soles, respectively. Cloacal region slightly swollen, vent as a longitudinal slit, vent edges with numerous small transverse folds.

Coloration in life

In life, ground color of dorsal and ventral surfaces black; dorsal surface and lateral sides of head and lower lips to jaw angles, rib nodules, vertebral ridge, the peripheral area of the cloaca and the ventral edge of the tail orange; tips of fingers, toes and elbow orange-brown.

Coloration in preservative

The specimen in preservative is blackish brown. The orange coloration in life has faded to pale yellow.

Secondary sexual characteristics

Males are probably smaller than females but sample size was small (n = 3) and thus needs confirmation based on further records in the future. The female cloacal slit is short and its inner cloacal walls have no papilla. The male has papilla on its inner cloacal wall and its cloaca presents a long slit.

Distribution

The new species is currently known only from the Ngoc Linh Nature Reserve, Kon Tum Province, in the Central Highlands of Vietnam (Fig. 5).

Figure 5. 

Type locality of Tylototriton ngoclinhensis sp. nov. (yellow star) and congeners; elevations (from 0-1200+ m) increasing from green to brown (Tran Anh Tuan, after Sterling et al. 2006).

Ecological notes

All specimens were collected during the day on the forest floor, under rotten trees or under moss, near a small rocky stream (Figs 6, 7). The surrounding habitat at the type locality of the new species in Ngoc Linh Mountain was primary montane evergreen broadleaf forest, at elevations between 1,800 and 2,300 m asl.

Figure 6. 

Habitat at the type locality of Tylototriton ngoclinhensis sp. nov. on Ngoc Linh Mountain.

Figure 7. 

Tylototriton ngoclinhensis sp. nov. in its microhabitat at the type locality.

Morphological measurements

Morphometric measurements of Tylototriton ngoclinhensis sp. nov. examined in this study are given in Table 3.

Table 3.
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Morphometric measurements (mm) of Tylototriton ngoclinhensis sp. nov. examined in this study.

Voucher IEBR A.5130 Holotype IEBR A.5131 Paratype IEBR A.5132 Paratype IEBR A.5133 Paratype IEBR A.5134 Paratype IEBR A.5135 Paratype
Sex ♂ ♂ ♂ ♀ ♀ ♀
SVL 66.5 65.7 60.8 75.6 74.8 72.5
HL 20.6 19.9 18.9 23.8 22.2 21.5
HW 16.8 15.6 16.3 18.6 17.5 17.4
MHW 18.5 18.4 17.9 20.1 19.5 19.4
PL 11.5 11.4 11.2 13.5 12.1 12.4
PW 5.7 5.8 5.3 5.9 5.9 5.8
PH 5.9 6.3 5.2 6.5 6.2 6.1
EL 4.8 4.4 4.4 5.2 4.9 4.9
EN 4.2 4 3.9 4.8 4.6 4.5
IN 5.5 5.7 5.4 6.4 6.6 6.2
IE 8.4 8.2 8.5 9.8 9.5 9.4
LJL 14.1 13.6 13.7 17.1 16.1 16.6
UEL 2.5 2.6 2.2 2.8 2.5 2.6
HUM 9.2 8.4 8.7 9.5 9.4 9.3
RAD 16.7 15.6 15.8 17.6 17.3 17.2
FEM 8.7 8.2 8.3 8.8 8.9 8.6
TIB 16.6 15.9 16.8 17.8 18.1 17.2
FORE 25.9 24 24.5 27.1 26.7 26.5
HIND 25.3 24.1 25.1 26.6 27 26.2
TL 59.5 57.6 61.8 67.9 62.9 66.2
TH 7.2 7 7.5 8.2 7.2 7.6
CIL 7.4 6.9 6.7 5.9 5.5 5.8
CIW 4.9 4.6 4.3 3.7 3.5 3.5
WVr 4.2 4.5 3.8 4.4 4.2 4.1
L5W 3.2 3.1 2.7 3.5 3.3 3.4
AG 35.4 34.5 31.7 41.4 39.4 37.8
TkL 49.7 49.3 45.5 58.7 55.7 53.8
ToL 126 123.3 122.6 143.5 137.7 138.7

Morphological comparisons

We compared the new species with other members of the genus Tylototriton based on data obtained from the literature (Table 4).

Table 4.
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Morphological comparisons between Tylototriton ngoclinhensis sp. nov. with other members of the subgenus Yaotriton (morphological data obtained from Fei et al. (1984), Nussbaum et al. (1995), Böhme et al. (2005), Chen et al. (2010), Stuart et al. (2010), Hou et al. (2012), Shen et al. (2012), Nishikawa et al. (2013a,b), Yang et al. (2014), Phimmachak et al. (2015), Fei and Ye (2016), Hernandez (2016), Qian et al. (2017), Bernardes et al. (2017a, 2020), Li et al. (2020), Lyu et al. (2021), Poyarkov et al. (2021a), Li et al. (2022), Luo et al. (2022). Abbreviations are as follows: TOL = total length, / = characters unobtainable from literature.

Species TOL of males TOL of females Gular fold Reach of finger tips when forelimbs stretched forward Reach of tips of fore-and hind limbs when adpressed along body Rib nodules shape Vertebral ridge Orange markings on the parotoid Orange coloration on the rib nodules
Tylototriton ngoclinhensis sp. nov. 122.6–126.0 137.7–143.5 present between the eye and nostril overlapping knob-like segmented tuberculate present present
T. anhuiensis 118.9–145.7 103.8–165.4 present / meeting slightly flattened not segmented absent absent
T. asperrimus 118.0-138.0 149.0–202.0 present reaching to the nostril or eye meeting or overlapping knob-like not segmented absent absent
T. broadoridgus 110.4–140.3 138.9–162.5 absent anterior corner of eye not touched slightly flattened not segmented absent absent
T. dabienicus 120.3–135.3 134.9–155.5 present anterior corner of eye not touched slightly flattened not segmented absent absent
T. daloushanensis / / present eyes to nostrils overlapping slightly flattened not segmented absent absent
T. hainanensis 137.0–148.0 125.0–140.0 present eye meeting or overlapping slightly flattened not segmented absent absent
T. liuyangensis 110.1–146.5 138.6–154.2 present eye not touched slightly flattened not segmented absent absent
T. lizhenchangi 145.6–173.0 150.0–156.5 present Tip of snout overlapping slightly flattened not segmented absent absent
T. maolanensis 151.0–172.0 142.7–170.5 present beyond the snout overlapping knob-like not segmented absent absent
T. notialis 109.1–130.4 141.8 present / / knob-like not segmented absent present
T. panhai 129.9–1603 160.0–166.8 present / / knob-like not segmented present present
T. pasmansi / 160.0 present eye / knob-like not segmented absent absent
T. sini 118.4–124.5 144.5 present / overlapping knob-like not segmented absent absent
T. sparreboomi / / present nostril / knob-like not segmented absent absent
T. thaiorum 116.1–138.0 / present / overlapping knob-like, in irregular series not segmented absent absent
T. tongziensis 120.5–135.1 123.5–127.6 present beyond the snout overlapping slightly flattened not segmented absent absent
T. vietnamensis 113.0–121.8 / absent / / slightly flattened not segmented absent absent
T. wenxianensis 126.0–133.0 / present nostril meeting or overlapping slightly flattened not segmented absent absent
T. ziegleri / / present / overlapping knob-like segmented tuberculate absent absent

Tylototriton ngoclinhensis sp. nov. differs from T. anhuiensis by different shape of dorsolateral glandular warts (knob-like vs slightly flattened), the presence of orange markings on the parotoids (vs absent), the presence of orange coloration on the dorsolateral glandular warts (vs absent), and tips of fore-and hind limbs overlapping when adpressed along the body (vs meeting); from T. asperrimus by having a smaller size in females (TOL 137.7–143.5 mm vs 149.0–202.0 mm), head longer than wide (vs head wider than long), the presence of orange markings on the parotoids (vs absent), and the presence of orange coloration on the dorsolateral glandular warts (vs absent); from T. broadoridgus by different shape of dorsolateral glandular warts (knob-like vs slightly flattened), the presence of orange markings on the parotoids (vs absent), the presence of orange coloration on the dorsolateral glandular warts (vs absent), and tips of fore and hind limbs overlapping when adpressed along the body (vs separated from each other); from T. dabienicus by different shape of dorsolateral glandular warts (knob-like vs slightly flattened), the presence of orange markings on the parotoids (vs absent), the presence of orange coloration on the dorsolateral glandular warts (vs absent), tips of fore-and hind limbs overlapping when adpressed along the body (vs separated from each other), and finger tips reaching between eye and nostril when foreleg is laid forward (vs reaching anterior corner of eye); from T. daloushanensis by different shape of dorsolateral glandular warts (knob-like vs slightly flattened), the presence of orange markings on the parotoids (vs absent), and the presence of orange coloration on dorsolateral glandular warts (vs absent); from T. hainanensis by having a smaller size in males (TOL 122.6–126.0 mm vs 137.4–148.0 mm), head longer than wide (vs head wider than long), different shape of dorsolateral glandular warts (knob-like vs slightly flattened), the presence of orange marking on the parotoids (vs absent), the presence of orange coloration on the dorsolateral glandular warts (vs absent), and finger tips reaching between eye and nostril when foreleg laid forward (vs reaching eye); from T. joe by having larger size (TOL 122.6–126.0 mm vs 108–115 mm in males and 137.7–143.5 mm vs 121–128 mm in females), ventral edge of the tail orange, and tip of fingers, toes and elbow orange-brown (vs whole body dark brown but brownish tip of toes and tip of tail in some individuals); from T. liuyangensis by different shape of dorsolateral glandular warts (knob-like vs slightly flattened), the presence of orange markings on the parotoids (vs absent), the presence of orange coloration on the dorsolateral glandular warts (vs absent), tips of fore-and hind limbs overlapping when adpressed along body (vs separated from each other), and finger tips reaching between eye and nostril when foreleg laid forward (vs reaching eye); from T. lizhenchangi by having a smaller size (TOL 122.6–126.0 mm vs 145.6–173.0 mm in males, TOL 137.7–143.5 mm vs 150.0–156.5 mm in females), different shape of dorsolateral glandular warts (knob-like vs slightly flattened), the presence of orange coloration on the dorsolateral glandular warts (vs absent), tips of fingers reaching between eyes and nostrils when foreleg laid forward (vs reaching to tip of snout), and the presence of orange markings on the parotoids (vs absent); from T. maolanensis by having a smaller size (TOL 122.6–126.0 mm vs 151.0–172.0 mm in males, TOL 137.7–143.5 mm vs 142.7–170.5 mm in females), the presence of orange markings on the parotoids (vs absent), and the presence of orange coloration on the dorsolateral glandular warts (vs absent); from T. notialis by different color pattern on head and vertebral ridge (orange vs dark brown), lower lip with orange marking (vs brown), and dorsolateral glandular warts and vertebral ridge distinctively large (vs small); from T. panhai by having a different ground color (black vs dark brown to brown), the presence of a distinct black line extending from the back of the eye towards the shoulder (vs less evident brownish line to absent line), dorsal edges of tail black (vs yellow, orange to reddish brown). Since T. panhai is the closest known taxon to Tylototriton ngoclinhensis sp. nov., additional morphological comparisons were made between the two species, especially between topotypic T. panhai type I and type II from Laos. Tail height was the only character (with n ≥ 3) that showed variation, presenting higher values both in males and in females (Fig. 8) of T. panhai, in relation to the new species.

Figure 8. 

Comparison of average tail heights (mm) between Tylototriton ngoclinhensis sp. nov and T. panhai. The left side shows data for males (A) of Tylototriton ngoclinhensis sp. nov. (n = 3; own data) and T. panhai (n = 15; taken from Phimmachak et al. 2015), showing 2 standard deviation (2SD) bars, which represent 95% of the population. The right side shows data for females (B) of Tylototriton ngoclinhensis sp. nov. (n = 3; own data) and T. panhai (n = 3) including two data points from Phimmachak et al. (2015), and one from Nishikawa et al. (2013a); averages marked by green horizontal lines, and ranges as vertical lines.

The new species is distinguished from T. pasmansi by having rib nodules distinctively large (vs small), the presence of orange markings on the parotoid (vs absent), and the presence of orange colorations of the dorsolateral glandular warts (vs absent); from T. sini by different color pattern on head and vertebral ridge (orange vs dark brown), the presence of orange markings on the parotoids (vs absent), the presence of orange coloration on the dorsolateral glandular warts (vs absent), and dorsolateral glandular warts distinct and large (vs small); from T. sparreboomi by different color pattern on head and vertebral ridge (orange vs dark brown), the presence of orange markings on the parotoids (vs absent), the presence of orange coloration on the dorsolateral glandular warts (vs absent), and tips of fingers reaching between eye and nostril when foreleg laid forward (vs reaching nostril); from T. thaiorum by having head longer than wide (vs head wider than long), the presence of orange markings on the parotoids (vs absent), and the presence of orange coloration on the dorsolateral glandular warts (vs absent); from T. tongziensis by different shape of dorsolateral glandular warts (knob-like vs slightly flattened), the presence of orange markings on the parotoids (vs absent), and the presence of orange coloration on the dorsolateral glandular warts (vs absent); from T. vietnamensis by different shape of dorsolateral glandular warts (knob-like vs slightly flattened), the presence of orange markings on the parotoids (vs absent), the presence of orange coloration on the dorsolateral glandular warts (vs absent), and the presence of gular fold (vs absent); from T. wenxianensis by different shape of dorsolateral glandular warts (knob-like vs slightly flattened), the presence of orange markings on the parotoids (vs absent), the presence of orange coloration on the dorsolateral glandular warts (vs absent), and finger tips reaching to between eyes and nostrils (vs reaching nostril); from T. ziegleri by having head longer than wide (vs head wider than long), different color pattern on head and vertebral ridge (orange vs dark brown), the presence of orange markings on the parotoids (vs absent), and the presence of orange coloration on the dorsolateral glandular warts (vs absent); from T. anguliceps, T. himalayanus, T. kachinorum, T. ngarsuensis, T. panwaensis, T. phukhaensis, T. podichthys, T. pulcherrimus, T. pseudoverrucosus, T. shanorum, T. shanjing, T. uyenoi, T. umphangensis, and T. verrucosus by having limbs and tail edges dark brown except for the orange digits, palms, and soles (vs limbs and tail edges uniformly orange or pale brown in the latter), the presence of a black line extending from the back of the eye towards the shoulder (vs absent); and from T. kweichowensis and T. yangi by different color pattern on tail (black vs uniformly orange in the latter), and ventral side dark brown (vs ventrolateral sides yellow in the latter).

Discussion

New species are being continuously described within the genus Tylototriton. A total of 12 new species has been recorded during the last three years alone, from China, Thailand and northern Vietnam (Bernardes et al. 2020; Li et al. 2020; Li et al. 2022; Luo et al. 2022; Lyu et al. 2021; Pomchote et al. 2021; Poyarkov et al. 2021a; Dufresnes and Hernandez 2022; Rao 2022; Wang et al. 2022). Most recent descriptions were due to the separation of species complexes, that were previously masked by phenotypic similarities (Bernardes et al. 2020; Li et al. 2020; Lyu et al. 2021; Pomchote et al. 2021; Poyarkov et al. 2021a; Luo et al. 2022). There certainly is high potential to continue uncovering new species by applying integrative taxonomic analyses (Dufresnes and Hernandez 2022). However, some novel species descriptions occur by conducting field work in previously unexplored areas. In our case, a new species was discovered in a region where several field surveys to assess the herpetological diversity had been conducted in the past (exp. Orlov 2005, 2009; Jenkins et al. 2007; David et al. 2011). However, individuals of the new species have been only found in a recent field survey in 2022.

This is also the first time that a crocodile newt species is recorded from the Central Highlands of Vietnam. Occurring at an elevation more than 1,800 m, this discovery sets an altitudinal record for the genus in the country, with former ranges distributed between 250 m (for T. vietnamensis) and 1,740 m (for T. anguliceps). Furthermore, this discovery represents the southernmost distribution range of the genus known to date. The new species is located approximately 370 air km distant from the nearest T. notialis population from Khammouan Province, Laos. Ngoc Linh Mountain is located on the northwestern border of the Kon Tum Massif and is the highest peak in Central Vietnam with 2,598 m (Sterling et al. 2006). The new species is also expected to be found in other localities on the Kon Tum Plateau. The climate in Ngoc Linh Mountain is relatively cool, as it is part of a tropical mountain system (averages of 15–18.5 °C throughout the year). Humidity is high, with strong amounts of rainfall (ranging from 2,600 to 3,200 mm per year) and distinct cloud coverage. Due to this unique climate and geographical position, Ngoc Linh has become a hotspot of amphibian diversity, with numerous endemic species. The area is also the type locality for some recently discovered species, namely Leptobrachium ngoclinhense (Orlov 2005), Theloderma nebulosum Rowley, Le, Hoang, Dau & Cao, 2011, Leptobrachella firthi (Rowley, Hoang, Dau, Le & Cao, 2012), Gracixalus lumarius Rowley, Le, Dau, Hoang & Cao, 2014, and G. trieng Rowley, Le, Hoang, Cao & Dau, 2020 (Orlov 2005; Rowley et al. 2011, 2012, 2014, 2020). Most recently Krzikowski et al. (2022) highlighted the extraordinary endemism rate of amphibians in the Central Highlands of Vietnam, where the highest species diversity, with 130 species, was recorded among the eight regions of Vietnam while also containing the highest number of regionally occurring, micro-endemic amphibians, amounting for 26 species, for example: Leptobrachella crocea (Rowley, Hoang, Le, Dau & Cao, 2010) and Microhyla darevskii Poyarkov, Vassilieva, Orlov, Galoyan, Tran, Le, Kretova & Geissler, 2014, next to the afore-mentioned species (Krzikowski et al. 2022; Frost 2023). Thus, the discovery dealt with herein is another and very remarkable case, demonstrating that the Central Highlands play a special role in Vietnamese amphibian diversification and evolution.

Ngoc Linh Nature Reserve has been established in 1986 with an initial protected area of 41,424 hectares and represents a key biodiversity area for the threatened Golden-winged Laughingthrush, Trochalopteron ngoclinhense, listed as Endangered in the IUCN Red List, as well as for other rare species like the Truong Son Muntjac, Muntiacus truongsonensis, Rhesus Macaque, Macata mulatta, and the Stump-tailed Macaque, Macaca arctoides (Le et al. 1998; BirdLife International 2023). The Ngoc Linh Crocodile Newt certainly will represent another flagship species of this protected area and its surroundings, as individuals have been found both inside and outside of the Ngoc Linh Nature Reserve.

Together with T. panhai, T. ngoclinhensis is the only known species within the subgenus Yaotriton to present characteristic colorful markings on the body. Although there is a high phenotypic variation recorded for T. panhai (types I, II, and III from Hernandez 2016), T. ngoclinhensis is clearly distinguishable both morphologically and phylogenetically from the former. Nevertheless, further studies should be conducted to obtain additional data on population characteristics, including further biogeographical analyses.

Tylototriton species so far have been documented as having little dispersal abilities due to limitations in vagility and their specific habitat requirements (Zamudio and Wieczorek 2007). Therefore, the discovery of this new species geographically separated by more than 300 km from all known congeners, and bearing a particularly unique colorful pattern represents not only an important discovery in terms of evolution and zoogeography, but also reveals to be of high conservation relevance.

Conservation recommendation

Due to the aforementioned reasoning and given that Tylototriton ngoclinhensis sp. nov. is currently known only from Ngoc Linh Mountain, implying a limited distribution range composed of a single small isolated mountain population, is distinct evidence for a high threat potential. In addition to its special zoogeographic situation and rarity, the particular colorful appearance of the new crocodile newt species is very likely to draw the interest of illegal collectors. Therefore, this species should be provisionally considered to be listed as Endangered (EN) under IUCN Red List criteria B1ab(i,iii), as it is known only from Ngoc Linh Mountain in Kon Tum Province; the estimated extent of occurrence (EOO) is less than 500 km2; and the species' habitat is currently being degraded due to human impacts, for example forest product exploitation, tourism development and agricultural cultivation. All species of the genus Tylototriton are listed in the Appendices of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES 2022) and also in the Governmental Decree No. 84/2021/ND-CP of Vietnam and therefore the new species is automatically protected under these regulations.

Acknowledgements

We are grateful to the directorates of the Ngoc Linh Nature Reserve and the Forest Protection Department of Kon Tum Province for support of our field work and issuing relevant permits. We thank Duy D.T. (Kon Tum Forest Ranger), Mai N.T.T, Anh D.N.H, Lam P.N.T (Ho Chi Minh City) for their assistance in the field. For constructive comments on a previous version of the manuscript we thank L. Lee Grismer, Axel Hernandez and Bryan Stuart. For the fruitful cooperation within joint research projects, we cordially thank Nguyen S.V. (IEBR, Hanoi), as well as T. Pagel and C. Landsberg (Cologne Zoo).

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This research was supported by the Vietnam Academy of Science and Technology (Project Code: ĐLSĐ00.01/20-23).

Author contributions

Field survey: Trung My Phung and Son Thanh Le; conceptualization: Tao Thien Nguyen, Truong Quang Nguyen, Thomas Ziegler; methodology: Marta Bernardes, Tao Thien Nguyen; data analysis: Cuong The Pham, Hoa Thi Ninh, Huy Quoc Nguyen, Marta Bernardes; writing, review and editing: all authors.

Author ORCIDs

Trung My Phung https://orcid.org/0000-0001-7086-8110

Cuong The Pham https://orcid.org/0000-0001-5158-4526

Truong Quang Nguyen https://orcid.org/0000-0002-6601-0880

Huy Quoc Nguyen https://orcid.org/0000-0003-3171-1561

Marta Bernardes https://orcid.org/0009-0008-2847-8862

Thomas Ziegler https://orcid.org/0000-0002-4797-609X

Tao Thien Nguyen https://orcid.org/0000-0002-5640-4536

Data availability

All of the data that support the findings of this study are available in the main text or Supplementary Information.

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