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Two new species of Tylototriton from Thailand (Amphibia: Urodela: Salamandridae)

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Three morphological groups are found in a salamandrid newt Tylototriton shanjing from Thailand. We describe two of them as new species, one from northern and the other from northeastern Thailand, based on molecular and morphological data, however we could not make a taxonomic decision on the remaining one group because of the lack of voucher specimens and sufficient genetic data. The northern species differs morphologically from all known congeners by having the combination of orange to reddish brown markings, narrow and sharply protruding dorsolateral bony ridges on head, weakly segmented vertebral ridge, and long and high tail. The northeastern species is characterized by having the combination of yellow, orange, or reddish brown markings, wide and moderately protruding dorsolateral bony ridges on head, smooth vertebral ridge, black limbs, and black tail except for edges. Validity of taxonomic subdivision of the genus Tylototriton is discussed.
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Accepted by M. Vences: 29 Oct. 2013; published: 20 Nov. 2013
ZOOTAXA
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ISSN 1175-5334 (online edition)
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http://dx.doi.org/10.11646/zootaxa.3737.3.5
http://zoobank.org/urn:lsid:zoobank.org:pub:5AC9E7FB-1835-438C-962B-528986FEFAC3
Two new species of Tylototriton from Thailand
(Amphibia: Urodela: Salamandridae)
KANTO NISHIKAWA1,4, WICHASE KHONSUE2,4, PORRAWEE POMCHOTE2,3 & MASAFUMI MATSUI1
1Graduate School of Human and Environmental Studies, Kyoto University, Yoshida Nihonmatsu-cho, Sakyo-ku, Kyoto 606–8501,
Japan
2Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
3Primate Research Institute, Kyoto University, Kanrin 41-2, Inuyama, Aichi 484-8506, Japan
4Corresponding authors. E-mail: hynobius@zoo.zool.kyoto-u.ac.jp (Kanto Nishikawa) and Wichase.K@chula.ac.th (Wichase Khon-
sue)
Abstract
Three morphological groups are found in a salamandrid newt Tylototriton shanjing from Thailand. We describe two of
them as new species, one from northern and the other from northeastern Thailand, based on molecular and morphological
data, however we could not make a taxonomic decision on the remaining one group because of the lack of voucher spec-
imens and sufficient genetic data. The northern species differs morphologically from all known congeners by having the
combination of orange to reddish brown markings, narrow and sharply protruding dorsolateral bony ridges on head, weak-
ly segmented vertebral ridge, and long and high tail. The northeastern species is characterized by having the combination
of yellow, orange, or reddish brown markings, wide and moderately protruding dorsolateral bony ridges on head, smooth
vertebral ridge, black limbs, and black tail except for edges. Validity of taxonomic subdivision of the genus Tylototriton
is discussed.
Key words: Tylototriton, Molecular phylogeny, Morphology, New species, Thailand
Introduction
The salamandrid genus Tylototriton is widely distributed from eastern Himalaya, Indochina, to central and southern
China, and includes a total of 17 species/subspecies (after Zhao et al. 2012, Hou et al. 2012, and Nishikawa et al.,
2013): T. asperrimus Unterstein, 1930; T. broadoridgus Shen, Jiang, et Mo, 2012; T. daweishanensis Zhao, Rao,
Liu, Li, et Yuan, 2012; T. hainanensis Fei, Ye, et Yang, 1984; T. kweichowensis Fang et Chang, 1932; T.
lizhenchangi Hou, Zhang, Jiang, Li et Lu, 2012; T. notialis Stuart, Phimmachak, Sivongxay, et Robichaud, 2010; T.
pseudoverrucosus Hou, Gu, Zhang, Zeng, Li, et Lu, 2012; T. shanjing Nussbaum, Brodie, et Yang, 1995; T.
taliangensis Liu, 1950; T. verrucosus verrucosus Anderson, 1871; T. verrucosus pulcherrima Hou, Zhang, Li, et
Lu, 2012; T. vietnamensis Böhme, Schöttler, Nguyen, et Köhler, 2005; T. wenxianensis wenxianensis Fei, Ye, et
Yang, 1984; T. wenxianensis dabienicus Chen, Wang, et Tao, 2010; T. yangi Hou, Zhang, Zhou, Li, et Lu, 2012;
and T. ziegleri Nishikawa, Matsui, et Nguyen, 2013. Following Stuart et al. (2010), we here treated T. shanjing as a
valid species, in contrast to Zhang et al. (2007) who relegated it to a junior synonym of T. verrucosus.
These species are divided into two subgenera, Tylototriton Dubois et Raffaëlli, 2009 (= the T. verrucosus group
[Fei et al. 2005]) and Yaotriton Dubois et Raffaëlli, 2009 (= the T. asperrimus group [Fei et al. 2005]). The
subgenus Tylototriton includes T. daweishanensis, T. kweichowensis, T. pseudoverrucosus, T. shanjing, T.
taliangensis, T. verrucosus (also v. pulcherrima), and T. yangi, while Yaotriton includes T. asperrimus, T.
broadoridgus, T. hainanensis, T. lizhenchangi, T. notialis, T. vietnamensis, T. wenxianensis (also w. dabienicus),
and T. ziegleri (Zhao et al. 2012, Hou et al. 2012, Nishikawa et al., 2013).
Within this decade, a total of 10 new species/subspecies were described in the genus Tylototriton, which
greatly increased the number of taxa nearly two and half folds. However, still more cryptic lineages are suggested
NISHIKAWA ET AL.
262 · Zootaxa 3737 (3) © 2013 Magnolia Press
to be present in the genus (Stuart et al. 2010). In Thailand, Pomchote et al. (2008) reported two distinct color
morphs, Types I and II, in T. shanjing (“verrucosus” in their paper). Because T. verrucosus is described as having
black body except for ventral edge of tail (Anderson 1871; also see Nussbaum et al. 1995), we here identified the
Thai populations of Tylototriton possessing color markings as T. shanjing, and not T. verrucosus. The distribution
of the two types is geographically separated into two areas, Type I in northern and Type II in northeastern Thailand
(Pomchote et al. 2008). Our preliminary examination on morphology of the Thai populations revealed that the
Type I could be further separated into two groups based on coloration. Thus, a total of three groups (Groups 1–3)
are recognized. The Group 1 from Doi (=Mountain in Thai language) Lahnga, Chiang Rai Province, northern
Thailand, is black in the ground color, but has bright orange color on head, vertebral ridge, rib nodules, limbs, vent
region, and whole tail (part of Type I in Pomchote et al. 2008). This group is most similar in coloration to typical T.
shanjing. The Group 2 from Doi Inthanon, Doi Suthep, and Doi Ang Khang, Chinag Mai Province, northern
Thailand, has the ground color of dark brown, with orange to reddish brown color on head, vertebral ridge, rib
nodules, limbs, vent region, and whole tail (part of Type I in Pomchote et al. 2008). The Group 3 from Loei and
Phitsanulok provinces, northeastern Thailand, has also dark brown ground color, but is yellow, orange, or reddish
brown on head, vertebral ridge, rib nodules, tips of fingers and toes, margin of vent slit, and dorsal and ventral
edges of tail (Type II in Pomchote et al. 2008). Based in the coloration, these three groups can be assigned to
subgenus Tylototriton Dubois et Raffaëlli, 2009.
In this study, we examine the taxonomic status of these three groups of T. shanjing from Thailand, using both
genetic and morphological data.
FIGURE 1. Photos of the three groups of Tylototriton shanjing from Thailand. A: a subadult of unknown sex of Group 1 (T.
shanjing Lineage 5 in molecular phylogeny: see Fig. 3) from Doi Lahnga, Chiang Rai Province; B: an adult male of Group 2 (T.
shanjing Lineage 4) from Doi Inthanon, Chinag Mai Province; C: an adult female of Group 3 (T. shanjing Lineage 6) from Phu
Luang, Loei Province. Tips of tails of these individuals except for the one from Doi Inthanon were used in the molecular
analysis. All of these individuals were released without being deposited as voucher specimens.
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TWO NEW NEWTS FROM THAILA ND
Materials and methods
Molecular phylogenetic analyses: Because the species of Tylototriton have been protected in many areas in
Thailand, we could not collect voucher specimens for six individuals observed between 2001 and 2006 (each one
individual from Doi Ang Kang, Doi Lahnga, Doi Suthep, Phu Luang, Phu Suan Sai, and Phu Hin Rong Kla), but
we were permitted to get their tail tips for molecular analysis (Table 1). Identification of the six individuals was
mainly based on their body coloration. We added tissues of seven specimens collected from Doi Inthanon and Doi
Suthep in 1994.
We first obtained sequence data of NADH dehydrogenase subunit 2 region (ND2) of mitochondrial DNA
(mtDNA) from tissue samples preserved in 99 % ethanol. Methods for DNA extraction and amplification and
sequencing of the DNA fragment are the same as those reported by Nishikawa et al. (2013). The PCR primers were
Sal_ND2_F1 (forward: 5’-AAGCTTTTGGGCCCATACC-3’) and Sal_ND2_R2 (reverse: 5’-
GGTTGCATTCAGAAGATGTG-3’), and the cycle sequencing primers, Sal_ND2_R1 (reverse: 5’-
GTTATAAATATGGAKGARGTTA-3’) and Sal_ND2_F2 (forward: 5’-ATAGCATAYTCRTCYATTGC-3’), were
designed in the middle of the ND2 region, in addition to the two PCR primers shown in Nishikawa et al. (2013).
The present ND2 region of mtDNA has been proven to be useful for delineating species in the genus Tylototriton
(Nishikawa et al. 2013).
FIGURE 2. Map showing localities of the samples of Tylototriton and Echinotriton used for molecular analyses. For locality of
the sample numbers, refer to Table 1. The samples from unknown localities are shown in squares and positioned at supposed
localities. (Sample No. 18 corresponds to the Group 1 [T. shanjing Lineage 5], Nos. 19–24 to the Group 2 [T. shanjing Lineage
4: T. uyenoi sp. nov. described herein], and Nos. 25–27 to the Group 3 [T. shanjing Lineage 6: T. panhai sp. nov.])
NISHIKAWA ET AL.
264 · Zootaxa 3737 (3) © 2013 Magnolia Press
Sam-
ple
no. Species or subspecies Voucher Locality
GenBank
accession no. Source
GenBank
accession
no. Source
GenBank
accession
no. Source
Ingroup
1Tylototriton asperrimus CIB 200807055 Jinxiu, Guangxi, China KC147815 Shen et al. (2012)
2T. broadoridgus CIB 200085 Sangzhi, Hunan, China KC147814 Shen et al. (2012)
3T. hainanensis CIB 20081048 Diaoluoshan, Hainan, China KC147817 Shen et al. (2012)
4T. kweichowensis MVZ 230371 Daguan, Yunnan, China DQ517851 Weisrock et al. (2006)
5T. lizhenchangi KUHE 42316 Yizhang, Hunan, China AB769532 Nishikawa et al. (2013) AB856873 This study AB856886 This study
6T. notialis FMNH 271120 Boualapha, Khammouan, Laos HM462061 Stuart et al. (2010)
7T. shanjin
g
NMNS 3682 Jingdong, Yunnan, China AB830721 This study AB856875 This study AB856888 This study
8T. shanjing* MVZ 219763 Jingdong, Yunnan, China DQ517852 Weisrock et al. (2006)
9T. shanjing* CAS 234471 Nu Jiang, Yunnan, China HM462054 Stuart et al . (2010)
10 T. shanjing* CAS 234479 Nu Jiang, Yunnan, China HM462055 Stuart et al . (2010)
11 T. shanjin
g
KUHE 42342 Myanmar (Pet trade) AB830722 This study
12 T. shanjin
g
KUHE 42348 Myanmar (Pet trade) AB769544 Nishikawa et al . (2013)
13 T. shanjin
g
KUHE 43489 Myanmar (Pet trade) AB830723 This study
14 T. shanjin
g
KUHE 43578 Myanmar (Pet trade) AB830724 This study
15 T. shanjin
g
KUHE 43579 Myanmar (Pet trade) AB830725 This study
16 T. shanjin
g
KUHE no number Unknown (Pet trade) AB830726 This study
17 T. shanjin
g
KUHE 34399 Phu Pan, Xam Neua, Laos AB830727 This study AB856877 This study AB856890 This study
18 T. shanjin
g
(Group 1) - Doi Lahnga, Chiang Rai, Thailand AB830728 This study AB856867 This study AB856880 This study
19 T. shanjin
g
(Group 2) - Doi Ang Khang, Chiang Mai, Thailand AB830729 This study AB856868 This study AB856881 This study
20 T. shanjin
g
(Group 2) KUHE 19037 Doi Inthanon, Chiang Mai, Thailand AB830730 This study
21 T. shanjin
g
(Group 2) KUHE 19038 Doi Inthanon, Chiang Mai, Thailand AB830731 This study
22 T. shanjin
g
(Group 2) KUHE 19146 Doi Suthep, Chiang Mai, Thailand AB830732 This study
23 T. shanjin
g
(Group 2) KUHE 19147 Doi Suthep, Chiang Mai, Thailand AB830733 This study AB856869 This study AB856882 This study
24 T. shanjin
g
(Group 2) - Doi Suthep, Chiang Mai, Thailand AB830734 This study
25 T. shanjin
g
(Group 3) - Phu Hin Rong Kla NP, Phitsanulok, Thailand AB830735 This study AB856876 This study AB856889 This study
26 T. shanjin
g
(Group 3) - Phu Luang WS, Loei, Thailand AB830736 This study
27 T. shanjin
g
(Group 3) - Phu Suan Sai NP, Loei, Thailand AB830737 This study
28 T. taliangensis KUHE 43361 Unknown (Pet trade) AB769543 Nishikawa et al. (2013) AB856878 This study AB856891 This study
29 T. verrucosus verrucosus* CAS 230899 Taunggyi, Shan, Myanmar HM770087 Stuart et al . (2010)
30 T. verrucosus verrucosus* MVZ no number Nepal DQ517854 Weisrock et al. (2006)
31 T. verrucosus pulcherrima KUHE 46406 Yunnan, China (Pet trade) AB830738 This study AB856871 This study AB856884 This study
32 T. vietnamensis KUHE 55171 Yen Tu, Bac Giang, Vietnam AB769537 Nishikawa et al . (2013) AB856870 This study AB856883 This study
33 T. wenxianensis wenxianensis CIB 20090527 Wenxian, Gansu, China KC147813 Shen et al. (2012)
34 T. wenxianensis dabienicus HNNU 1004II015 Shangcheng, Anhui, China KC147811 Shen et al . (2012)
35 T. yangi NMNS 3114 Pingbian, Yunnan, China AB830739 This study
36 T. yangi KUHE 42282 China (Pet trade) AB769546 Nishikawa et al. (2013) AB856872 This study AB856885 This study
37 T. ziegleri VNMN 3390 Quan Ba, Ha Giang, Vietnam AB769539 Nishikawa et al. ( 2013) AB856874 This study AB856887 This study
Outgroup
38
E
chinotriton andersoni KUHE no number Nago, Okinawa, Japan AB769545 Nishikawa et al. (2013) AB856879 This study AB856892 This study
39
E
. chinhaiensis MVZ 230536 Ningbo, Zhejiang, China DQ517775 Weisrock et al . (2006)
40
P
leurodeles walt
l
MVZ 162384 Rabat, Morocco DQ517813 Weisrock et al. (2006)
41 Notophthalmus viridescens MVZ 230959 St.Charles, Missouri, USA DQ517795 Weisrock et al. (2006)
N
P: National Park; WS: Wildlife Sanctuary
mtDNA POMC Rag1
TABLE 1. Spec
i
mens o
f
Ty
l
ototriton spec
i
es an
d
re
l
ate
d
spec
i
es use
d
f
or mo
l
ecu
l
ar ana
l
yses. CAS = Ca
lif
orn
i
a Aca
d
emy o
f
Sc
i
ences; CIB = C
h
eng
d
u Inst
i
tute o
f
B
i
o
l
ogy; FMNH = F
i
e
ld
Museum o
f
Natura
l
H
i
story; HNNU =
Henan Normal University; KUHE = Graduate School of Human and Environmental Studies, Kyoto University; MVZ = Museum of Vertebrate Zoology, University of California, Berkeley; NMNS = National Museum of Natural
Science, Taiwan; VNMN = Vietnam National Museum of Nature. *Identification followed to Stuart et al. (2010). (Sample No. 18 correponds to T. shanjing Lineage 5, Nos. 19–24 to T. shanjing Lineage 4 [T. uyenoi sp. nov.],
and Nos. 25–27 to T. shanjing Lineage 6 [T. panhai sp. nov.]).
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TWO NEW NEWTS FROM THAILA ND
We reconstructed phylogenetic trees using our own samples of 19 specimens of Tylototriton including the Thai
sample. For comparison, DNA sequences of 18 sequences of Tylototriton and one sequence each of Echinotriton
andersoni, E. chinhaiensis, Pleurodeles waltl, and Notophthalmus viridescens were obtained from GenBank (Table
1, Fig. 2). Outgroup species (sample numbers 38–41 in Table 1) were selected based on Weisrock et al. (2006).
The optimum substitution models were selected by Kakusan4 (Tanabe 2011). We then constructed
phylogenetic trees by Bayesian inference (BI) and maximum likelihood (ML) methods using MrBayes v3.1.2
(Huelsenbeck & Ronquist 2001) and TREEFINDER ver. Mar. 2011 (Jobb 2011), respectively. The criterion used
for model selection was AIC.
The best substitution models for BI and ML were the general time reversible model (GTR: Tavaré 1986) + G
and the J3 (Jobb 2011) with gamma shape parameter (G), respectively. Two independent runs of four Markov
chains were conducted for ten million generations. We sampled one tree every 100 generations and calculated a
consensus topology for 70,000 trees after discarding the first 30,001 trees (burn-in=3,000,000).
Next, we analysed two nuclear (nu) DNA genes, prooptiomelanocortin (POMC) and Rag1 (recombination
activating protein 1), for 13 selected samples, representing main genetic groups recognized in the mtDNA
genealogical tree (Table 1). The PCR primers were POMC_DRV_F1 (forward: 5'-
ATATGTCATGASCCAYTTYCGCTGGAA-3'; Vieites et al. 2007) and POMC_DRV_R1 (reverse: 5'-
GGCRTTYTTGAAWAGAGTCATTAGWGG-3'; Vieites et al. 2007) for the POMC region, and RAG1_F_N1
(forward: 5’-RCAAGCCRAAYTCAGAGYTRTGCTGCAAGC-3’; this study) and RAG1_R_N3 (reverse: 5’-
TTCATTCTCATTATGGGCYTCARRTTCATCTTC-3’; this study) for the Rag1 region. We constructed a
Neighbor-joining tree in Splits Tree4 (Huson & Bryant 2006) with uncorrected p-distance and ambiguous (=
heterozygous) sites treated with the ‘‘Average States” option. This option calculates pairwise distance between
given two individuals by averaging distances among all possible combinations of homozygous and/or
heterozygous sites between the two individuals.
For the Bayesian analysis, we considered posterior probabilities (bpp) 95 % or greater as significant support
(Leaché & Reeder 2002). The robustness of the ML tree and NJ network was tested using bootstrap analysis
(Felsenstein 1985) with 1000 replicates. We regarded tree topologies with bootstrap values (bs) of 70 % or greater
as sufficiently supported (Huelsenbeck & Hillis 1993). Pairwise comparisons of uncorrected sequence divergences
(p-distance) were calculated using MEGA5 (Tamura et al. 2011).
Morphological comparisons. We compared morphology among the Groups 2 and 3 identified by molecular
analyses (see below), and the topotypic specimens of T. shanjing, which is most similar to the Thailand specimens.
Because we have no voucher specimens of the Group 3 used for molecular analyses (Table 1), we used a specimen
from Phu Luang Wildlife Sanctuary, Loei Province deposited in Thailand Natural History Museum (THNHM). The
specimen was identified as the Group 3 based on the characteristics noted above. We also used specimens of the
other congeners deposited in California Academy of Sciences (CAS), Chengdu Institute of Biology, Chinese
Academy of Sciences (CIB), Graduate School of Human and Environmental Studies, Kyoto University (KUHE),
Herpetological collection of the National Museum of Nature and Science, Tokyo (NSMT-H), National Museum of
Natural Science, Taiwan (NMNS), and Vietnam National Museum of Nature (VNMN) for comparisons (Appendix
1).
The following 29 measurements were taken for morphological comparisons (character definitions not
mentioned below are given in Nishikawa et al. [2011]): SVL (snout-vent length) from tip of snout to anterior tip of
vent; HL (head length); HW (head width); MXHW (maximum head width); SL (snout length); LJL (lower jaw
length); ENL (eyelid-nostril length); IND (internarial distance); IOD (interorbital distance); UEW (upper eyelid
width); UEL (upper eyelid length); OL (orbit length); AGD (axilla-groin distance); TRL (trunk length); TAL (tail
length) from anterior tip of vent to tail tip; VL (vent length); BTAW (basal tail width); MTAW (medial tail width);
BTAH (basal tail height); MXTAH (maximum tail height); MTAH (medial tail height); FLL (forelimb length);
HLL (hindlimb length); 2FL (second finger length); 3FL (third finger length); 3TL (third toe length); and 5TL
(fifth toe length) ; VTW (vomerine teeth series width): the greatest width of vomerine teeth series; VTL (vomerine
teeth series length): the greatest length of vomerine teeth series. All measurements were taken to the nearest 0.1
mm with a dial caliper. We used a stereoscopic binocular microscope when necessary. The sex and maturity of the
specimens and number of eggs were checked and counted by minor dissections.
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We compared SVL, 28 ratio values to SVL (R, %), and VTL/VTW ratio value among the Group 2, Group 3,
and topotypic specimens of T. shanjing. Differences in SVL and ratio values were tested between the two samples
by Student's t-test and Mann-Whitney's U test, respectively. The significance level was 95 % in all these statistical
tests.
We conducted multivariate analysis to examine overall morphological variation between male samples of the
Group 2 and topotypic Tylototriton shanjing from Jingdong, Yunnan, China. Using a total of 29 loge-transformed
values of the metric characters noted above, we conducted Principal Component Analysis (PCA) by SAS (1990).
We also examined skull morphology and counted the number of trunk vertebrae of specimens of all the voucher
specimens examined by us (see Appendix 1). These characters were examined in soft X-ray photographs using Fuji
Medical X-Ray Film.
Results
Phylogenetic relationships and genetic divergence: We obtained 988 bp sequences of partial ND2 region for 41
specimens, including outgroup (Table 1). Of 988 nucleotide sites, 420 were variable and 308 were parsimony
informative (sequence statistics available upon request from the senior author). The mean likelihood score of the
Bayesian analyses for all trees sampled at stationarity was -7448.217. The likelihood value of the ML tree was -
7399.447.
Phylogenetic analyses employing two different optimality criteria (BI and ML) yielded nearly identical
topologies. We therefore present only the ML tree in Fig. 3. Monophyly of Echinotriton was fully supported in the
BI and ML trees (bs=98 % and bpp=100 %), but that of Tylototriton was not supported in the BI tree (bs=74 % and
bpp=89 %). Within Tylototriton, two major clades (Clades I and II) were recognized: Clade I mainly consisted of
species with orange or yellow markings on the body (the subgenus Tylototriton), and Clade II mainly consisted of
species with blackish body (the subgenus Yaotriton). Each of these two major clades was highly supported (I:
bs=98 % and bpp=100 %; II: bs=93 % and bpp=100 %).
Clade I included 11 lineages: T. kweichowensis, T. shanjing Lineages 1–5 (including the Groups 1 and 2), T.
taliangensis, T. v. verrucosus Lineages 1 and 2, T. v. pulcherrima, and T. yangi (Fig. 3). Among them, T.
taliangensis was first separated from the clade including the remaining lineages. The latter clade was further
divided into three subclades: the subclade A, B, and C. The subclade C was separated into three groups of C-1, C-
2, and C-3 (T. shanjing Lineage 1 [the topotypic samples of T. shanjing], T. v. pulcherrima, and T. shanjing
Lineages 2 and 3 [hereafter, the T. shanjing complex]).
Clade II included 10 lineages: T. asperrimus, T. broadoridgus, T. hainanensis, T. lizhenchangi, T. notialis, T.
shanjing Lineage 6 (Group 3), T. vietnamensis, T. w. wenxianensis, T. w. dabienicus, and T. ziegleri. These 10
lineages were separated into three subclades of D, E, and F, whose relationships were not solved.
The p-distances between each pair of a total 21 lineages recognized above ranged from 2.6% (between T.
shanjing Lineage 1 and T. v. pulcherrima) to 14.7% (between the Group 2 and T. vietnamensis) in averaged values
(Table 2). The distances between a pair of the four lineages in the T. shanjing complex ranged from 2.5 to 4.3%
(average 3.2%), which were nearly same as the minimum values between other species in the subgenus Yaotriton
(>3.3 %: Nishikawa et al. 2013; Stuart et al. 2010; Shen et al. 2012) but tended to be smaller than some Asian
salamandrids (>4.0 % in Paramesotriton: Wu et al. 2010a; >6.6 % in Cynops: Wu et al. 2010b). Excluding the T.
shanjing complex, the distances between each pair of the remaining 17 lineages ranged from 3.3 % (between T. w.
dabienicus and T. broadoridgus) to 14.7 %. In contrast, the Groups 1, 2, and 3 differed from the topotypic T.
shanjing by as large as 5.2 %, 7.5 %, and 12.5 % difference, respectively. The averaged distance between these
groups and their sister lineages were also as large as 7.1 % between the Groups 1 and 2 and 10.6 % between the
Group 3 and T. vietnamensis.
For nuDNA, we obtained 475 bp of POMC, of which 13 were variable and five were parsimony informative,
and number of heterozygous sites was six and number of heterogenous specimens was four. We obtained 567 bp of
Rag1, of which 20 were variable and four were parsimony informative, and number of heterogenous sites was 17
and number of heterozygous specimens was seven. The obtained haplotypes (heterozygous sequences treated as
different haplotypes) of the topotype of T. shanjing were not shared with the Groups 1–3 in POMC, although the
Group 1 and 2 shared some haplotypes, while the haplotypes of topotypic T. shanjing in Rag1 were shared with the
Groups 1 and 2, but not with the Group 3. The tree based on concatenated nuDNA genes resulted in low resolution
for almost all relationships, except for a clade consisting of T. lizhenchangi and T. shanjing Lineage 3 (Fig. 4).
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FIGURE 3. Maximum likelihood tree based on the partial ND2 gene for Tylototriton and related species (see Table 1 and Fig.
1). Numbers at the end of braches represent sample numbers shown in Table 1. Numbers above branches represent bootstrap
supports for ML inference and Bayesian posterior probability (bs/bpp). Asterisks indicate nodes with bs>70 % and bpp>95 %.
Scale bar=0.02 substitutions/site. (Sample No. 18 correponds to the Group 1 [T. shanjing Lineage 5], Nos. 19–24 to the Group
2 [T. shanjing Lineage 4: T. uyenoi sp. nov. described herein], and Nos. 25–27 to the Group 3 [T. shanjing Lineage 6: T. panhai
sp. nov.])
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FIGURE 4. Neighbor-joining tree based on POMC and Rag1 genes for selected species of Tylototriton and related species (see
Table 1). Number above branch represents significant (>70 %) bootstrap support for the tree. Scale bar=0.001 substitutions/site.
(Sample No. 18 correponds to the Group 1 [T. shanjing Lineage 5], Nos. 19 and 23 to the Group 2 [T. shanjing Lineage 4: T.
uyenoi sp. nov. described herein], and No. 25 to the Group 3 [T. shanjing Lineage 6: T. panhai sp. nov.])
Unlike the relationships recovered by mtDNA, neither of the subgenus, Tylototriton or Yaotriton, was supported,
but the Group 1 was first grouped with the Group 2, supporting their monophyly in the mtDNA phylogeny.
Additionally, all of the Groups 1–3 were clearly differentiated from the topotypic T. shanjing (T. shanjing Lineage
1).
Morphological comparisons: A total of 16 adult males and six adult females of the Group 2 and 3 and the
topotypic specimens of T. shanjing were used for morphometric comparisons (Table 3, Appendix 1). Because of
the paucity of specimens, we could statistically compare only males of the Group 2 and the topotypic T. shanjing.
In SVL, the Group 2 was significantly larger than T. shanjing, although variation ranges largely overlapped
between them and the level of significance was low (t=2.178, p=0.047). In character ratios, the Group 2 was
significantly larger than the topotypic T. shanjing in RHW, RMXHW, RTAL, RBTAH, RMXTAH, RVTW, and
RVTL, and smaller in RMTAW (U<12.00, p<0.050). For females, the topotypic T. shanjing tended to show smaller
values than the specimens of the Group 2 and 3 in RHL, RHW, RMXHW, RLJL, RTAL, and RBTAH, and the
specimens of the Group 3 tended to show smaller values than those of the Group 2 and T. sha njing in RENL,
RHLL, and R3FL. As a whole, these ratio characters tended to separate the Groups 2 and 3, and the topotypic
specimens of T. shanjing, although we could not test significance of these differences because of paucity of
samples.
Then, we examined overall morphological differences by the use of PCA. In PCA, a total of eight males of the
Group 2 (holotype and all paratypes of T. uyenoi sp. nov. except for one paratype with a regenerated tail) and seven
males of topotypic T. shanjing (T. shanjing Lineage 1) were used. The first three principal components accounted
for 73.1 % of the total variation. On the two dimensional plot of PC 2 versus PC 1 (Fig. 5, left), the Group 2 and
topotypic T. shanjing were separated, and similar tendency was recognized in the plot of PC 3 versus PC 1 (Fig. 5,
right).
Color pattern varied among the Groups 1–3 and the topotypic T. shanjing. The Group 1 and 2 and the topotypic
T. shanjing had bright to dark orange, to reddish brown on anterior half of the head, dorsolateral bony ridges on
head, vertebral ridge, rib nodules, whole limbs, vent region, and whole tail. The color pattern of the Group 1 and 2
was similar, but the Group 1 had more bright markings and more blackish ground color than the Group 2 (Fig. 1).
The Group 3 differed from the Groups 1 and 2 and the topotypic T. shanjing by having black limbs and tail except
for edges (Fig. 1). The coloration of markings of the Group 3 was most variable among the groups examined, from
yellow, orange, to reddish brown.
Dorsal granules were more prominent and denser in the Groups 1 and 2 and the topotypic T. shanjing than the
Group 3. Bony ridges on the head were steeper and narrower in the Group 1 and 2 and the topotypic T. shanjing than
the Group 3. The surface of the bony ridges tended to be smoother in the Group 2 and 3 and the topotypic T. shanjing
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than the Group 1. Rib nodules were more prominent and larger in the Group 3 than the remaining groups and the
topotypic T. shanjing. The vertebral ridge was most tubercular in the Group 1 and smoothest in the Group 3.
FIGURE 5. Two dimensional plots of the first principal component (PC 1) versus the second (PC 2) (left), and PC 1 versus the
third (PC 3) (right) of the male specimens of the Group 2 (T. shanjing Lineage 4: T. u y e noi sp. nov. described herein) and
topotypic male specimens of T. shanjing (T. shanjing Lineage 1). Asterisk shows the holotype of T. uyenoi sp. nov.
The Groups 1, 2 and 3 of T. shanjing from northern and northeastern Thailand are distinguishable from the
remaining species of Tylototriton in both molecular and morphological properties. We consider the Group 2 (T.
shanjing Lineage 4) as distinct species because it differs strongly and consistently from topotypic specimens (T.
shanjing Lineage 1) in mitochondrial DNA, has a distinct POMC haplotype, and is also distinguished from
topotypic specimens by morphometry. We consider the Group 3 (T. shanjing Lineage 6) as distinct species because
it differs strongly and consistently from topotypic specimens (T. shanjing Lineage 1) and the Group 2 specimens in
mitochondrial DNA. The Group 3 is not placed sister to neither topotypic specimens nor to the Group 2 by the
mtDNA genealogy, and does not share POMC nor RAG1 haplotypes with these other two species.
We therefore describe the Groups 2 and 3 as two new species, but do not make a taxonomic decision on the
Group 1 because we have at present neither voucher specimens nor concrete data separating it from the Group 2 in
nuDNA.
Tylototriton uyenoi sp. nov.
(Figs. 1B, 6A, B, G, and J)
Tylototriton verrucosus: Smith (1924) : 309; Taylor (1962): 279;
Tylototriton verrucosus Type I: Pomchote et al. (2008): 39 (part).
Identity: This species corresponds to the Group 2 (T. shanjing Lineage 4 in molecular analyses).
Holotype: KUHE 19147, an adult male from Phuping Rajanives Palace, Doi Suthep, Chiang Mai Province,
Thailand (18 48’16’’ N, 98 54’9’’ E, 1436 m asl), collected on 10 August 1994 by Masafumi Matsui and Jarujin
Nabhitabhata.
Paratypes: A total of eight males: KUHE 19037 and 19038 from Royal Garden Siribhume, Doi Inthanon,
Chiang Mai Province, Thailand (18 32’46’’ N, 98 31’14’’ E, 1313 m asl), collected on 1 August 1994 by
Masafumi Matsui and Jarujin Nabhitabhata, KUHE 19146, and 19148–50 (data same as the holotype), and NSMT-
H 1076 and 1077 from Doi Suthep, Chiang Mai Province, Thailand, collected on 8 September 1967 by Shun-ichi
Uéno; two females: NSMT-H 1073 and 1075 (data same as NSMT-H 1076 and 1077); a larva: NSMT-H 1074 (data
same as NSMT-H 1076 and 1077).
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Etymology: The specific epithet is dedicated to Dr. Shun-ichi Uéno, who collected part of the paratypes of the
new species.
Diagnosis: The new species is placed in the genus Tylototriton by having the combination of: dorsal warts
present; dorsolateral bony ridges on head present; knob-like warts on dorsolateral body present; quadrate spine
absent. A medium-sized newt of Tylototriton; skin rough with fine granules; dorsolateral bony ridges on head
prominent and narrow; vertebral ridge distinct and slightly segmented; rib nodules prominent; limbs long and thin;
tips of forelimbs and hindlimbs greatly overlapping when adpressed along body; tail thin; dorsal head, upper and
lower lips, vertebral ridge, rib nodules, limbs, vent region, and whole tail orange to reddish brown.
FIGURE 6. The male holotype (KUHE 19147) of Tylototriton uyenoi (the Group 2 [T. shanjing Lineage 4]: A, B, G, and J), the
female holotype (THNHM 2800) of T. panhai (the Group 3 [T. shanjing Lineage 6]: C, D, H, and K), and male topotype
(NMNS 3682-38) of T. shanjing (T. shanjing Lineage 1: E, F, I, and L). Dorsal (A, C, and E) and ventral view (B, D, and F)
of the body, left lateral view of the tail (G, H, and I), and dorsal view of the head (J, K, and L). Scale bar=20 mm in body and
tail, 10 mm in head.
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Description of holotype: Body moderately stout; skin rough; fine granules dense on dorsum, arranged in
transverse striations on mid ventrum, and small and sparse on throat; head wider than long, hexagonal in shape,
depressed and slightly oblique in profile; snout short, truncate, slightly beyond lower jaw; nostril close to snout tip;
dorsolateral bony ridges on head prominent, narrow, sharply protruding, and slightly rough, from above eye to
above anterior end of parotoid, posterior ends scrolled proximally; middorsal ridge on head short, weak; labial fold
absent; skull wide and hexagonal, dorsolateral crests steep and narrow; middorsal crest prominent; maxillary
connecting with quadrate, but slightly separated from pterygoid; front-squamosal arch robust and wide; vomerine
tooth series in an inverted V-shape, converging anteriorly and reaching choana; tongue oval and attached to mouth
floor but free laterally and posteriorly; parotoid distinct but small, and projecting posteriorly; gular fold present;
costal folds absent; vertebral ridge prominent and slightly segmented, from neck to base of tail, separated from
middorsal ridge on head with a small gap; rib nodules distinct but small, forming knob-like warts, 15 on right side
and 14 on left side of body from axilla to base of tail; rib nodules slightly increasing in size from most anterior to
fourth nodule, then decreasing posteriorly; tips of fore- and hindlimbs greatly overlapping when adpressed along
body (longest toe reaching at elbow); number of trunk vertebrae (presacral vertebrae including atlas) 14; fingers
and toes free of webbing; tail laterally compressed, dorsal fin more distinct posteriorly, ventral edge smooth, tip
pointed; numbers of upper jaw teeth 107, lower jaw teeth 119, and vomerine teeth 102.
Measurements of holotype (in mm): SVL 68.6; HL 15.9; HW 16.5; MXHW 17.7; SL 6.4; LJL 14.0; ENL
3.9; IND 4.8; IOD 7.4; UEW 2.3; UEL 5.0; OL 3.3; AGD 34.8; TRL 52.7; TAL 87.6; VL 6.2; BTAW 5.5; MTAW
3.4; BTAH 8.5; MXTAH 9.0; MTAH 7.5; FLL 24.8; HLL 28.9; 2FL 4.6; 3FL 5.0; 3TL 6.8; 5TL 3.3; VTW 5.1;
VTL 6.9.
Color: In life, dorsal ground color dark brown to black; venter slightly lighter than dorsum; anterior half of
head, parotoids, vertebral ridge, rib nodules, limbs, vent region, and whole tail orange to reddish brown. In
preservative, dorsal coloration tending to fade, becoming light brown and orange to brown markings fading to
cream.
Variation: Males have more robust limbs than females. Males have a relatively longer tail (RTAL: 101.4–
127.7, median=115.0 %SVL in males vs. 88.0 and 97.0%SVL in females) and vent slit (RVL: 6.0–9.0, median=7.4
%SVL in males vs. 1.7 and 1.9%SVL in females) than females. Male specimens of the type series are basically
similar in morphology, but some specimens have more distinctly segmented vertebral ridges, larger rib nodules,
and rougher dorsolateral bony ridges on head than the holotype. Length of vomerine tooth rows is variable among
specimens, but reaching/exceeding posterior end of choana in all specimens except for one (KUHE 19148).
Number of trunk vertebrae is always 14. Specimens are generally uniform in color pattern, but markings slightly
vary in color from orange to reddish brown. One specimen (KUHE 19148) lacked distal half of right forelimb.
Egg: The mean diameter of ripe ova in ovaries of the paratype (NSMT-H 1075) ranged from 1.9 to 2.8 mm
(n=6, mean=2.4 mm). The clutch size is unknown. The animal pole is dark brown and the remaining area is dark
cream in color.
Larva: A paratype larva (NSMT-H 1074) was collected in Doi Suthep in early September. It is fully-grown
and has a total length of 66.1 mm. The gills started to shrink. The following description of larva is based on this
preserved individual.
Head rounded triangle; depressed and sloping in profile; snout short and rounded; labial fold distinct at
posterior half of upper jaw; caudal and dorsal fins nearly absorbed; tail tip rounded. Dorsum yellowish brown;
venter paler; anterior head, parotoids, vertebral ridge, rib nodules, limbs, and tail yellow.
Smith (1924) reported that the larvae at early developmental stages possessed a pair of balancers.
Comparisons: Tylototriton uyenoi (the Group 2 [T. shanjing Lineage 4]) is a member of the subgenus
Tylototriton based on the molecular phylogeny. This species differs from members of the subgenus Yaotriton
except for T. panhai (the Group 3 [T. shanjing Lineage 6]), another new species described below, and T.
daweishanensis, T. taliangensis, and T. v. verrucosus [data of T. daweishanensis are taken from Zhao et al. 2012
and those of T. v. verrucosus are taken from Anderson (1871)] of the subgenus Tylototriton, by having distinct
orange to reddish brown markings on head, trunk, limbs, and tail (vs. black body except for ventral edge of tail in
the remaining species of the subgenus Yaotriton except for T. panhai, T. daweishanensis, and T. v. verrucosus, and
black body except for posterior parotoids in T. taliangensis). The new species differs from T. kweichowensis and T.
pseudoverrucosus by having isolated orange to reddish brown markings on rib nodules (vs. connected markings
forming dorsolateral lines in T. kweichowensis and T. pseudoverrucosus [data of T. pseudoverrucosus are taken
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from Hou et al. 2012]), from T. sh a njing by having darker markings, relatively wider head, longer and higher tail,
and wider and longer vomerine teeth series (vs. bright markings, narrower head, shorter and lower tail, and
narrower and shorter vomerine teeth series in T. shanjing), from T. panhai by having orange to reddish brown limbs
and tail, and narrow dorsolateral ridges on head (vs. black limbs and tail except for edges, and wide dorsolateral
ridges on head in T. panhai), from T. v. pulcherrima by having black ground color of the body (vs. light brown
ground color in T. v. pulcherrima), from T. yangi by having orange anterior half of head (vs. black in T. yangi).
Range: Doi Ang Khang, Doi Chang Kien, Doi Inthanon, Doi Pui, and Doi Suthep, Chiang Mai Province,
Thailand.
Natural history: The holotype and the paratypes deposited in KUHE were collected in artificial pools. The
species is also found in natural or artificial ponds, ditches along farms, and in slow streams, with an average depth
of 38.2 cm (range 9–120 cm). Breeding occurs from May to July. Larvae are found in the water bodies from August
to December. The adults are probably semiaquatic, which are found in water even out of breeding season.
Tylototriton panhai sp. nov.
(Figs. 1C, 6C, D, H, and K)
Tylototriton verrucosus: Wongratana (1984): 107;
Tylototriton verrucosus Type II: Pomchote et al. (2008): 39.
Identity: This species corresponds to the Group 3 (T. shanjing Lineage 6 in molecular analyses).
Holotype: THNHM 2800, an adult female from Phu Ruea, Phu Luang Wildlife Sanctuary, Loei Province,
Thailand (17 29’59’’ N, 101 20’30’’ E, 1183 m asl), collected on 25 May 1998 by Tanya Chan-ard.
Etymology: The specific epithet is dedicated to Prof. Somsak Panha (Chulalongkorn University), who is an
active naturalist and helped our field surveys.
Diagnosis: The new species is placed in the genus Tylototriton by having the combination of: dorsal warts
present; dorsolateral bony ridges on head present; knob-like warts on dorsolateral body present,; quadrate spine
absent. A medium-sized Tylototriton; skin rough with fine granules; dorsolateral bony ridges on head wide;
vertebral ridge distinct and not segmented; rib nodules prominent and large; limbs long and thin; tips of forelimbs
and hindlimbs greatly overlapping when adpressed along body; tail thin; dorsal head, upper and lower lips,
parotoids, vertebral ridge, rib nodules, tips of fingers and toes, margin of vent slit, and dorsal and ventral edges of
tail yellow, orange, to reddish brown.
Description of holotype: Body moderately stout; skin rough; fine granules dense on dorsum, arranged in
transverse striations on mid ventrum, and small and sparse on throat; head longer than wide, hexagonal in shape,
depressed and slightly oblique in profile; snout short, truncate, slightly beyond lower jaw; nostril close to snout tip;
dorsolateral bony ridges on head prominent, wide, moderately protruding, and slightly rough, from above eye to
above anterior end of parotoid, posterior ends slightly scrolled proximally; middorsal ridge on head absent; labial
fold absent; skull wide and hexagonal, dorsolateral crests moderate and wide, middorsal crest absent; maxillary
connecting with quadrate, but slightly separated from pterygoid; front-squamosal arch robust and wide; vomerine
tooth series in an inverted V-shape, converging anteriorly and not reaching posterior end of choana; tongue oval
and attached to mouth floor but free laterally and posteriorly; parotoid large, protruding dorsolaterally and
posteriorly; gular fold present; costal folds absent; vertebral ridge prominent and smooth, from neck to base of tail;
rib nodules distinct and large except for the anteriormost one on right side, forming knob-like warts, 14 on both
sides of body from axilla to base of tail; rib nodules slightly increasing in size from most anterior to fourth or fifth
nodule, then decreasing posteriorly; tips of fore- and hindlimbs overlapping when adpressed along body (longest
toe reaching distal part of palm); number of trunk vertebrae 14; fingers and toes free of webbing; tail laterally
compressed, dorsal fin more distinct posteriorly, ventral edge smooth, tip pointed; numbers of upper jaw teeth 80,
lower jaw teeth 81, and vomerine teeth 66.
Measurements of holotype (in mm): SVL 71.6; HL 17.6; HW 16.9; MXHW 18.9; SL 7.4; LJL 13.8; ENL
3.9; IND 5.4; IOD 7.6; UEW 2.5; UEL 4.1; OL 2.9; AGD 36.5; TRL 54.0; TAL 63.3; VL 3.0; BTAW 5.9; MTAW
3.9; BTAH 8.9; MXTAH 9.0; MTAH 7.6; FLL 20.2; HLL 22.4; 2FL 3.2; 3FL 3.5; 3TL 6.0; 5TL 2.7; VTW 4.0;
VTL 6.8.
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Color: In life, dorsum ground color dark brown; venter slightly lighter than dorsum; anterior half of head,
parotoids, vertebral ridge, rib nodules, tips of fingers and toes, margin of vent slit, and dorsal and ventral edges of
tail yellow, orange, to reddish brown (based on the unvouchered samples whose tail tips were used for DNA
analysis). In preservative, dorsal coloration tending to fade, becoming light brown and orange to brown markings
fading to cream (the holotype).
Variation: Our field observation found a variation in color of markings, from yellow, dark orange, to reddish
brown, and in size of rib nodules. Individuals from Phu Hin Rong Kla National Park, Phitsanulok Province have
dark reddish brown markings, and those from Phu Suan Sai National Park, Loei Province have smaller nodules
than the holotype.
Egg: The mean diameter of ripe ova in right ovary that could be measured by a minor dissection of the
holotype ranged from 1.5 to 2.7 mm (n=7, gross mean=2.1 mm). The clutch size is unknown. The animal pole is
dark brown and the remaining area is dark cream in color.
Comparisons: Tylototriton panhai (the Group 3 [T. shanjing Lineage 6]) is a member of the subgenus
Yaotriton based on the molecular phylogeny, and differs from all other species of the subgenus by having distinct
yellow to reddish brown markings on head, trunk, and tail edges (vs. having black body except for tips of fingers
and toes, margin of vent, and ventral edge of tail in all other species of Yaotriton). The species looks similar to most
of the species of the subgenus Tylototriton, especially T. pseudoverrucosus, T. shanjing, T. uyenoi (the Group 2 [T.
shanjing Lineage 4]), and T. v. pulcherrima, however differs by having widely developed dorsolateral bony ridges
on head, black limbs and tail except for ridges (vs. less developed dorsolateral bony ridges on head, yellow or
orange limbs and whole tail in T. pseudoverrucosus [data of T. pseudoverrucosus are taken from Hou et al. 2012],
T. shanjing, T. uyenoi, and T. v. pulcherrima). The species differs from the remaining species of the subgenus
Tylototriton in the following way: from T. daweishanensis by having distinct yellow to reddish brown markings on
head, trunk, and tail edges (vs. having black body except for tips of fingers and toes, margin of vent, and ventral
edge of tail in T. daweishanensis [data are taken from Zhao et al. 2012]), from T. kweichowensis and T. taliangensis
by having isolated rib nodules (vs. connected nodules forming dorsolateral rides on body in T. kweichowensis and
T. taliangensis), from T. kweichowensis and T. yangi by having colored anterior half of head (vs. black head except
for posterior parotoids and jaw angles in T. kweichowensis and T. yangi), and from T. v. verrucosus by having
colored markings (vs. black body in T. v. verrucosus [data of T. v. verrucosus are taken from Anderson 1871]).
Range: Phu Hin Rong Kla National Park, Phitsanulok Province, and Phu Luang Wildlife Sanctuary and Phu
Suan Sai National Park, Loei Province, Thailand. The locality in Phu Hin Rong Kla National Park is the
southernmost record in the genus Tylototriton.
Natural history: The species was first reported by Wongratana (1984), which was collected walking on an
elephant trail at foggy morning. The habitat is moist forest with bamboo bushes and variety of water bodies like
temporal pools by rain, and streams. Eggs are found as attached on wet grass or plant overhanging the water
surface or under wood debris on land near small streams or ponds. In the breeding season from May to July, the
adults were found in the water bodies. Out of the breeding season, the species is probably terrestrial and difficult to
encounter.
Discussion
Although the genus Tylototriton has been reported to be monophyletic (e.g. Weisrock et al. 2006, but see Pyron &
Wiens 2011), monophyly of the genus was supported only in the maximum likelihood analysis in our results.
Partial ND2 region examined in this study has been proven to be useful for delimiting species in this genus
(Nishikawa et al. 2013), but seems to be too short (=not sufficiently informative) to resolve supraspecific
relationships.
The two clades recognized in the genus Tylototriton could be correspond to the two subgenera Tylototriton
(type species=T. verrucosus) and Yaotriton (type species=T. asperrimus) proposed by Dubois & Raffaëlli (2009).
These two subgenera are mainly defined by body color: presence of yellow to orange markings in Tylototriton and
almost black body without such markings in Yaotriton (Dubois & Raffaëlli 2009). However, this subgeneric
classification has been questioned, because “colored” Tylototriton includes T. daweishanensis and T. v. verrucosus
with black body and T. taliangensis with nearly black body (except for the posterior end of parotoid). Furthermore,
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the “black” Yaotriton includes T. notialis with orange rib nodules, although Nishikawa et al. (2013) reported a
Vietnamese population of T. notialis without orange markings. The present study showed that T. panhai with
distinct markings was clustered with T. asperrimus in the clade of Yaotriton. Although the two subgenera were
clearly separated by molecular data, they are not clearly split morphologically.
Dubois & Raffaëlli (2009) also noted that the subgenus Tylototriton has a large body and semiaquatic habit,
while the subgenus Yaotriton has a smaller body and basically terrestrial in habit. These trends are roughly
applicable to characterize them, but body size largely overlaps between the two subgenera (e.g. almost no
difference between T. [Tylototriton] uyenoi and T. [Yaotriton] panhai) and semiaquatic habit is observed in some
individuals of T. (Yaotriton) vietnamensis (Nishikawa, personal observation).
Recently, Fei et al (2012) proposed new genus Liantriton for T. taliangensis and new subgenus Qiantriton for
T. kweichowensis. However, these new taxa are not clearly defined, without any comparisons to the related taxa.
Our molecular analyses showed that T. taliangensis and T. kweichowensis were included in the subgenus
Tylototriton with significant supports, and rejected new classification by Fei et al (2012). Supraspecific taxonomy
of Tylototriton (sensu lato) needs further studies.
The present study showed the presence of more cryptic taxa in the subgenus Tylototriton. The T. shanjing
Lineage 5 (Group 1) and T. v. verrucosus Lineages 1 and 2 are candidates for cryptic species. In order to determine
taxonomic status of T. v. verrucosus Lineages 1 and 2, we must define the true T. verrucosus. In the original
description of the species, Anderson (1871) noted that the species has a black body. However, Fei et al. (2012)
treated the individuals with dark reddish brown markings from the type locality, western Yunnan, China, as this
species. In the present study, T. shanjing Lineage 2 from western Yunnan and an unknown locality represented such
individuals as having dark reddish brown markings and were nested within the T. shanjing complex in the
molecular phylogeny. At present we do not know what the true T. verrucosus is. Because the type specimens of T.
verrucosus had been believed to be lost, Nussbaum et al. (1995) designated the neotype for the species. However,
the original type specimens were found later (Chanda et al. 2000). It is most important to examine the type
specimens discovered for solving the taxonomic confusions on T. verrucosus.
Tylototriton shanjing was separated from T. verrucosus and described as a distinct species based on individuals
from western Yunnan with distinctly colored body (Nussbaum et al. 1995). Zhang et al. (2007) did not find
significant genetic difference between topotypic specimens of T. shanjing and T. verrucosus and synonymized the
former with the latter. However, Stuart et al. (2010) did not follow this taxonomic conclusion because Zhang et al.
(2007) used only a single, unvouchered sample of T. verrucosus for their study (no information on body color of
the sample is available). In the present study, all the samples from Yunnan except for T. kweichowensis and T. yangi
were nested within the T. shanjing complex. Genetic distances within the complex are generally much smaller than
those obtained between different species of the genus except for T. broadoridgus and T. w. dabienicus, whose
relationship we suspect to be conspecific. Based on this small genetic difference, we tentatively treat the complex
as one species, T. shanjing, by synonymizing T. v. pulcherrima with this species. This taxonomic idea is consistent
with Yu et al. (2013), who treated four lineages they found in T. shanjing from Yunnan as conspecific.
In the present paper, all of the three Thai groups of T. shanjing (Groups 1–3) were suggested as undescribed
species, and the two of them were described as T. uyenoi and T. panhai. However, the Group 1 from Doi Lahnga,
Chiang Rai Province could not be described by the lack of voucher specimens. In order to clarify true species
diversity and work on conservation plan of the endangered Thai species of Tylototriton, we must be hurry to
describe the Group 1.
Acknowledgements
We would like to thank Somsak Panha and the late Jarujin Nabhitabhata for their assistance in collecting
specimens, Dr. Miguel Vences and one anonymous reviewer for improving the earlier version of manuscript, the
National Research Council of Thailand and The Royal Forest Department of Thailand for permitting our fieldwork
in Thailand. We also thank Koshiro Eto for laboratory assistance, Max Sparreboom for providing literature, and
Jens Vindum (CAS), Jianping Jiang and Yuezhao Wang (CIB), Shin-ichiro Kawada (NSMT), Tanya Chan-ard
(THNHM), Tao T. Nguyen (VNMN), Wenhao Chou (NMNS) for allowing us to examine or borrow specimens
under their care. This work was partly supported by grants from The Monbusho (Field Research, Nos. 04041068,
NISHIKAWA ET AL.
278 · Zootaxa 3737 (3) © 2013 Magnolia Press
06041066, 08041144, 20405013, 20770066, and 23770084), TJTTP-OECF, JSPS AA Science Platform Program,
and the Kyoto University Foundation to MM and KN.
References
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APPENDIX 1. Specimens examined.
Tylototriton asperrimus: CIB GX200807010–16 (seven males) from Mt. Dayao, Jinxiu Yao Autonomous County, Guangxi
Zhuang Autonomous Region, China.
T. hainanensis: CIB 83360–63 (four males) from Jianfengling, Hainan Province, China.
T. kweichowensis: KUHE 40314 (one female) from pet trade; KUHE 46534, 46535 (one male and one female) from Shuicheng
County, Guizhou Province, China.
T. lizhenchangi: KUHE 42316 and 42317 (two males) from Mt. Mang, Yizhang County, Hunan Province, China.
T. notialis: VNMN 1194 (one male) from Pu Hoat District, Nghe An Province, Vietnam.
T. shanjing: NMNS 3682, 3682-6, 13, 38, 42, 53, 56, 68, 69, 92 (seven males and three females) from Jingdong County,
Yunnan Province, China
T. taliangensis: KUHE 40180, and 43361–63 (four males) from pet trade.
T. verrucosus verrucosus: CAS 234478, 234480–81 (one male and two females) from Nu Jiang, Yunnan Province, China.
T. verrucosus pulcherrima: KUHE 46406 (one female) from pet trade.
T. vietnamensis: KUHE 55172, VNMN 3032, 3168, 3363, 3364, 3374 (six males) from Yen Tu District, Bac Giang Province,
Vietnam.
T. wenxianensis wenxianensis: CIB 750041 (one male) from Pingwu County, Sichuan Province, China.
T. y a n gi: KUHE 42282–83 (two females) from pet trade.
T. ziegleri: VNMN 3390 (one male) from Quan Ba District, Ha Giang Province, Vietnam, VNMN 907, 1310–16 (eight males)
from Bac Quang District, Ha Giang Province, Vietnam, VNMN 3389 (one male) from Bao Lac District, Cao Bang
Province, Vietnam, NSMT-H 05679–88 (nine males and one female) from Nguyen Binh District, Cao Bang Province,
Vietnam.
... D-loop and Cytb; Honda et al., 2012;Yu et al., 2013), and the rare attempts to provide a nuclear phylogenetic tree have proven unsuccessful (e.g. Nishikawa et al., 2013b;Hou et al., 2014, Phimmachak et al., 2015a or uninformative (Wang et al., 2018). Furthermore, so far no molecular studies have considered all known lineages, but only those sequenced at a given locus (often ND2), and typically based on few specimens for each (e.g. ...
... Because previous studies could not retrieve meaningful nuclear inferences from the few loci and subsets of species those authors separately analysed (e.g. Nishikawa et al., 2013b;Hou et al., 2014;Wang et al., 2018), we attempted to build nuclear trees by combining all loci sequenced among distinct studies. To this end, we first gathered partial sequences from the genes POMC (~500 bp) and RAG1 (500-1300 bp), which both have relatively high polymorphism among Echinotriton and Tylototriton species (Nishikawa et al., 2013b). ...
... Nishikawa et al., 2013b;Hou et al., 2014;Wang et al., 2018), we attempted to build nuclear trees by combining all loci sequenced among distinct studies. To this end, we first gathered partial sequences from the genes POMC (~500 bp) and RAG1 (500-1300 bp), which both have relatively high polymorphism among Echinotriton and Tylototriton species (Nishikawa et al., 2013b). Those originating from the same individuals were concatenated, and sequences from Pleurodeles poireti (Gervais, 1835) were included as outgroup. ...
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... Tylototriton uyenoi (Nishikawa et al., 2013) (Figure 1) was described from nine specimens from Doi Suthep, Chiang Mai Province, northern Thailand and two specimens from Doi Inthanon, Chiang Mai Province, Northern Thailand. This species was separated using molecular and morphological evidence (Nishikawa et al., 2013). The distribution of Tylototriton uyenoi was reported at Doi Ang Khang, Doi Chang Kien, Doi Suthep and Doi Inthanon Chiang Mai Province, Northern Thailand (Gerlach, 2012(Gerlach, , 2020Nishikawa et al., 2013;and Michaels, 2015), the Maesa-Kogma Biosphere Reserve, Chiang Mai Province, Northern Thailand (Dowwiangkan et al., 2018), Doi Mon Jong, Tak Province, northwestern Thailand and Doi Mak Lang, Mae Ai District, Chiang Mai Province, Northern Thailand (Hernandez et al., 2019) and Khao Laem National Park, Kanchanaburi Province, Western Thailand (Hernandez and Pomchote, 2020). ...
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The morphology and distribution of the Chiang Mai Crocodile Newt Tylototriton uyenoi (Urodela: Salamandridae) in Chiang Mai Province, Northern Thailand were studied. The morphological characters were compared and revealed that the male body size did not differ from the female body size. In the group from Doi Chiang Dao Wildlife Sanctuary and the group from Doi Suthep-Pui National Park showed that the female body size that is no different than the female type specimens; however, in the group from Doi Inthanon, the male was smaller than the female type specimens. In this report, a new Tylototriton uyenoi locality was reported at Doi Lang, Mae Ai District, Chiang Mai Province, Northern Thailand. This study provides information on morphology and distribution which can used to aid conservation, protection, and taxonomy.
... [ Hou et al., 2012;Hernandez, 2016;Wang et al., 2018]. [ Nishikawa et al., 2013b;Hernandez, 2016Hernandez, , 2017Wang et al., 2018]. [ Stuart et al., 2010b;Nishikawa et al., 2013a;Wang et al., 2018]. ...
... [ Stuart et al., 2010b;Nishikawa et al., 2013a;Wang et al., 2018]. [ Nishikawa et al., 2013b;Phimmachak et al., 2015;Hernandez, 2016Hernandez, , 2017Wang et al., 2018]. Pham, Pham, Nguyen et Ziegler, 2020 (Son La Prov.). ...
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... panhai), and western (T. uyenoi) regions of Thailand (Pomchote et al. 2008;Nishikawa et al. 2013a;Hernandez 2016;Hernandez et al. 2019;Hernandez and Pomchote 2020a, 2020cPomchote et al. 2020aPomchote et al. , 2020bPomchote et al. 2021). ...
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... However, to understand evolutionary processes future studies especially on the ecology of the enigmatic crocodile newts are needed. Numerous species were just recently identified based on molecular data leading to an enormous increase in species numbers over the last decade (Stuart et al., 2010, Shen et al., 2012, Nishikawa et al., 2013a, Nishikawa et al., 2013b, Hou et al., 2014, Nishikawa et al., 2014, Khatiwada et al., 2015, Le et al., 2015, Phimmachak et al., 2015a, Qian et al., 2017, Grismer et al., 2018, Grismer et al., 2019, Zaw et al., 2019, Bernardes et al., 2020, Pomchote et al., 2020, Poyarkov et al., 2021. However morphological distinct characters are hard to identify and often appear somehow descriptive summarising differences of particular body part proportions based on a few specimens without any accounting for SD. ...
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Sexual dimorphism (SD) is a main source of intraspecific morphological variation, however sexual shape dimorphism (SShD) was long time neglected in evolutionary research. Especially in cold-blooded animal groups only subtle shape differences are expressed between males and females and the selective forces behind it are poorly understood. Crocodile newts of the genera Echinotriton and Tylototriton are highly polymorphic in their reproductive ecology and hence, are a highly suitable model system to investigate potential evolutionary forces leading to SShD differences. We applied 3D geometric morphometrics to the cranial and humerus morphology of nine species of crocodile newts to investigate patterns of SShD in relation to the different mating modes. Trajectories of shape differences between males and females differ in both, cranium and humerus but mating mode does explain differences in SShD trajectories between species only in cranial morphology. Nevertheless, cranial morphology shape differed between the amplecting and circle dancing species. Hence, other selective forces must act here. Variable interspecific allometric trajectories are a potential source of shape differences whereas these trajectories are quite stable for the sexes irrespective of the species.
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