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Accepted by M. Vences: 6 Sept. 2018; published: 18 Oct. 2018
ZOOTAXA
ISSN 1175-5326 (print edition)
ISSN
1175-5334
(online edition)
Copyright © 2018 Magnolia Press
Zootaxa 4500 (4): 553
–
573
http://www.mapress.com/j/zt/
Article
553
https://doi.org/10.11646/zootaxa.4500.4.5
http://zoobank.org/urn:lsid:zoobank.org:pub:3518D551-CA4B-43A4-B6D3-8A159E2398E1
A new species of Crocodile Newt Tylototriton (Caudata: Salamandridae)
from Shan State, Myanmar (Burma)
L. LEE GRISMER
1,8
, PERRY L. WOOD JUNIOR
2
, EVAN S. H. QUAH
3
, MYINT KYAW THURA
4
,
ROBERT E. ESPINOZA
5
, MARTA S. GRISMER
1
, MATTHEW L. MURDOCH
6
& AUNG LIN
7
1
Herpetology Laboratory, Department of Biology, La Sierra University, 4500 Riverwalk Parkway, Riverside, California 92515, USA.
2
Department of Ecology and Evolutionary Biology and Biodiversity Institute, University of Kansas, Dyche Hall, 1345 Jayhawk Blvd,
Lawrence, Kansas 66045-7561, USA.
3
School of Biological Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia.
4
Myanmar Environment Sustainable Conservation, Yangon, Myanmar.
5
Department of Biology, California State University, Northridge, 18111 Nordhoff Street, Northrdge, California 91330-8303, USA.
6
Department of Biology, Villanova University, 800 Lancaster Avenue, Villanova, Pennsylvania 19085, USA.
7
Fauna and Flora International, No(35), 3rd Floor, Shan Gone Condo, Myay Ni Gone Market Street, Sanchaung Township, Yangon,
Myanmar.
8
Corresponding author. E-mail: lgrismer@lasierra.edu
Abstract
A phylogenetic taxonomic analysis of a monophyletic subgroup of the salamandrid genus Tylototriton revealed that a new-
ly discovered population from Ngar Su Village, 1 km south of Ywangan, Shan State, Myanmar is a new species and most
closely related to T. shanorum from approximately 80 km to the west in the vicinity of Taunggyi, Shan State. Tylototriton
ngarsuensis sp. nov. differs from other closely related species of Tylototriton on basis of varying combinations of charac-
teristics associated with it shorter head, larger size, rib nodule morphology, and overall drab, very dark, coloration, along
with its genetic differentiation. Tylototriton ngarsuensis sp. nov. also appears to breed later in the year than most other
species. Unfortunately, this species like many other Asian newts, is being harvested for the pet and medicinal trade and
given its restricted distribution, this could pose a serious threat to its long-term survival.
Key words: Integrative taxonomy, Tylototriton, Shan State, Ywangan, new species, Myanmar, conservation, pet trade
Introduction
The salamandrid genus Tylototriton contains 23 nominal species (Frost 2018) that range across the Asian monsoon
climatic zone from the eastern Himalayas through northern Myanmar and China (including Hainan Island) and
southward through Laos to central Thailand and Vietnam (Wang et al. 2018). The life history and ecology of a
number of species of Tylototriton has been very well-studied (e.g. Kuzmin et al. 1994; Dasgupta 1996; Tian et al.
1998; Roy & Mushahindunnabi 2001; Yu & Zhao 2005; Gong & Mu 2008; Ziegler et al. 2008; Seglie et al. 2003,
2010; Sun et al. 2011; Fe et al. 2012; Jun et al. 2012; Wangyal & Gurung 2012; Benardes et al. 2013, 2017;
Phimmachak et al. 2015). These studies have revealed that in general, Tylototriton are sexually dimorphic
terrestrial salamanders with low-dispersal capabilities that migrate short distances (50–200 m) during the early
monsoon seasons to ponds or slow-moving streams to reproduce. After mating, eggs are usually deposited in soil or
vegetation near the shore and the females return to land. Males usually remain in the water for longer periods of
time. Although widely distributed across much of Asia, their ecological requirements contribute to their allopatric,
fragmented distribution and restriction to relatively cool, mesic, upland forests in the vicinity of streams and
ephemeral ponds. This no doubt has contributed to a number of site-specific or nearly site-specific endemic species
and the generally narrow distributions of most others (see Wang et al. 2018:Fig. 1). Such demographics provide
excellent opportunities for addressing phylogenetic, taxonomic, and historical biogeographic issues as well as
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investigations into the correlation between current species boundaries and geography. Yet despite Tylototrition
being one of the most ecologically, taxonomically, and phylogenetically best studied reptile or amphibian genera in
Asia, the taxonomy of this group remains surprising unresolved—replete with potential synonymies and unnamed
lineages (Zang et al. 2007, 2013; Stuart et al. 2010; Nishikawa et al. 2013a,b, 2015; Hou et al., 2012; Yuan et al.
2011; Shen et al. 2012; Sparreboom 2014, Yang et al. 2014; Khatiwada et al. 2015; Le et al. 2015; Phimmacak et
al. 2015; Qian et al. 2017; Wang et al. 2018). This is due largely to relatively low uncorrected pairwise sequence
divergences (3.1–3.9%) of the mitochondrial gene NADH dehydrogenase subunit 2 (ND2) between even well-
established closely related species and subtle differences in morphology and coloration (Stuart et al. 2010).
FIGURE 1. Distribution of Tylototriton ngarsuensis sp. nov., T. shanorum, and Tylototriton sp. nov. from Shan State, Kachin
State, and Sagaing Region, Myanmar.
In Myanmar, Tylototriton is currently known from only two species—T. verrucossus and T. shanorum
(Nishsikawa et al. 2014; Phimmachak et al. 2015). Tylototriton shanorum, is a range-restricted endemic from the
vicinity of Taunggyi, Shan State that is known from at least four geographically proximate and genetically similar
populations (Fig. 1). Phimmachak et al. (2015) published sequence data on specimens from Sagaing Region and
Kachin State reported to be T. verrucosus. Curiously however, these populations were omitted from the most recent
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phylogenetic survey of the genus that was purported to reveal species biogeography and cryptic diversity (Wang et
al. 2018). We discovered a new population of Tylototriton from Baw Hto Chang, a small stream that courses
through Ngar Su Village, 1 km south of Ywangan, Shan State (Fig. 1). A molecular phylogeny based on the
mitochondrial gene ND2 indicates this population is most closely related to T. shanorum that occurs approximately
80 km to the west but differs from it in morphology and color pattern. Our phylogeny also suggests that the Sagaing
and Kachin populations are each other’s closest relatives and phylogenetically distinct from T. verrucosus—the
nomen under which they are currently recognized—as well as all other species of Tylototriton. Below we describe
the new population from Ngar Su Village and discuss the taxonomic implications of the phylogenetic relationships
of the Sagaing and Kachin populations.
Materials and methods
Species delimitation. The general lineage concept (GLC: de Queiroz 2007) adopted herein proposes that a species
constitutes a population of organisms independently evolving from other such populations owing to a lack of gene
flow. By “independently,” it is meant that new mutations arising in one species cannot spread readily into another
species (Barraclough et al. 2003 and de Queiroz 2007). Integrative studies on the nature and origins of species are
using an increasingly wider range of empirical data to delimit species boundaries (Coyne & Orr 1998; Fontaneto et
al. 2007; Knowles & Carstens 2007, Leaché et al. 2009; Abdala et al. 2016), rather than relying solely on
traditional taxonomic methods. Under the GLC herein, molecular phylogenies were used to define mitochondrial
lineages of individuals from the same populations in order to develop initial species hypotheses. Univariate and
multivariate analyses (Student’s t-tests, analysis of variance [ANOVA], and principal component analysis [PCA])
of morphological and color pattern characters were then used to search for concordance among those lineages to
further support their species-level differentiation. These species boundaries were subsequently cross-checked using
a Generalized Mixed Yule Coalescent (GMYC) approach (Pons et al. 2006), thus providing an independent
framework to complement the empirically based hypotheses of the morphological and molecular analyses.
Phylogenetic data and analyses. In a comprehensive phylogenetic analysis of the genus Tylototriton, Wang et
al. (2018) delimited five well-supported clades. Clade I, containing the sister species T. pseudoverrucosus and T.
taliangensis, was recovered as the sister lineage to clade II which contains T. shanorum from Myanmar as well as
T. kweichowensis, T. himalayanus, T. yangi, T. uyenoi, T. anguliceps, T. podichthys, T. pulcherrimus, T. verrucosus,
and T. shanjing from China, Laos, and Thailand. We follow the taxonomy of Wang et al. (2018) and augmented
their data set for clades I and II with additional specimens from Myanmar: two specimens from Ngar Village, Shan
State; one specimen from Panwa Village, Kachin State; and one specimen from the Lahe Township, Sagain Region.
A total of 38 specimens composed the ingroup (clade II) and six specimens composed the outgroup (clade I). All
specimens, their catalog numbers, locality, and GenBank accession numbers are listed in Table 1.
Genomic DNA was isolated from liver or skeletal muscle tissue stored in 95% ethanol using a Maxwell® RSC
Tissue DNA kit on the Promega Maxwell® RSC extraction robot. The mitochondrial gene ND2 was amplified
using a double-stranded Polymerase Chain Reaction (PCR) under the following conditions: 1.0 µl genomic DNA
(10–30 µg), 1.0 µl light strand primer (concentration 10 µM), 1.0 µl heavy strand primer (concentration 10 µM),
1.0 µl dinucleotide pairs (1.5 µM), 2.0 µl 5x buffer (1.5 µM), MgCl 10x buffer (1.5 µM), 0.1 µl Taq polymerase
(5u/µl), and 6.4 µl ultra-pure H
2
O. PCR reactions were executed on Bio-Rad gradient thermocycler under the
following conditions: initial denaturation at 95°C for 2 min, followed by a second denaturation at 95°C for 35 s,
annealing at 48–52°C for 35 s, followed by a cycle extension at 72°C for 35 s, for 31 cycles. All PCR products
were visualized using electrophoresis on a 1.0 % agarose gel. Successful PCR products were sent to GENEWIZ®
for PCR purification, cycle sequencing, sequencing purification, and sequencing using the same primers as in the
amplification step (Table 2). Sequences were analyzed from both the 3' and the 5' ends separately to confirm
congruence between the reads. Forward and reverse sequences were uploaded and edited in Geneious
TM
version
v6.1.8. Following sequence editing we aligned the protein-coding region and the flanking tRNAs using the
MAFTT v7.017 (Katoh & Kuma, 2002) plugin under the default settings in Geneious
TM
(Kearse et al. 2012).
Mesquite v3.04 (Maddison & Maddison 2015) was used to calculate the correct amino acid reading frame and to
confirm the lack of premature stop codons in the ND2 portion of the DNA fragment.
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TABLE 1. GenBank accession numbers, voucher number, and locality for the specimens used for the molecular phylogenetic analyses. / = voucher number
unavailable.
Species name Voucher Number Locality GenBank no.
Tylototriton taliangensis CIBGG 200110183 Shimian Co., Yan'an City, Sichuan Province, China KC147819
Tylototriton taliangensis CIBGG 200110185 Shimian Co., Yan'an City, Sichuan Province, China KY800829
Tylototriton taliangensis CIBGG 200110186 Shimian Co., Yan'an City, Sichuan Province, China KY800830
Tylototriton pseudoverrucosus CIBWCG 2012003 Ningnan Co., Liangshanyizu state, Sichuan Province, China KY800861
Tylototriton pseudoverrucosus CIBWCG 2012007 Ningnan Co., Liangshanyizu state, Sichuan Province, China KY800862
Tylototriton pseudoverrucosus CIBWCG 2012012 Ningnan Co., Liangshanyizu state, Sichuan Province, China KY800860
Tylototriton kweichowensis CIBWg 20080818014 Bijie City, Guizhou Province, China KY800823
Tylototriton kweichowensis CIBWg 20080818018 Bijie City, Guizhou Province, China KY800824
Tylototriton kweichowensis CIB 20050213 Shuicheng City, Guizhou Province, China KY800827
Tylototriton kweichowensis CIB 20050215 Shuicheng City, Guizhou Province, China KY800828
Tylototriton shanorum CAS 230933 Taunggyi Township, Shan State, Myanmar AB922822
Tylototriton shanorum CAS 230940 Taunggyi Township, Shan State, Myanmar AB922823
Tylototriton shanorum CAS 230899 Taunggyi Township, Shan State, Myanmar AB769544
Tylototriton himalayanus CIB 201406246 Mai Pokhari, Illam, Mechi,Nepal KT765173
Tylototriton himalayanus CIB 201406284 Bagh Khor, Illam, Mechi,Nepal KT765207
Tylototriton himalayanus CIB 201406285 Bagh Khor, Illam, Mechi,Nepal KT765208
Tylototriton yangi KUHE 42282 Pet Trade KY800887
Tylototriton uyenoi / Doi Ang Khang, Chiang Mai, Thailand AB830729
Tylototriton uyenoi KUHE 19147 Doi Inthanon, Chiang Mai, Thailand AB830730
Tylototriton uyenoi KUHE 19037 Doi Suthep, Chiang Mai, Thailand AB830733
Tylototriton anguliceps TBU PAE.671 Thuan Chau, Son La, Vietnam LC017833.1
Tylototriton anguliceps VNMN A 2014 3 LCO Muong Nhe, Dien Bien, Vietnam LC017832.1
Tylototriton anguliceps / Doi Lahnga, Chiang Mai, Thailand AB830728
Tylototriton podichthys / Phu Pan, Xam Neua, Laos AB830727
……continued on the next page
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TABLE 1 . (Continued)
Species name Voucher Number Locality GenBank no.
Tylototriton podichthys / Xam Neua, Huaphanh, Laos LC017835
Tylototriton pulcherrima CIBTY 040 Lƺchun Co., Yunnan Province, China KY800890
Tylototriton pulcherrima KUHE 46406 Pet Trade KY800880
Tylototriton verrucosus CIB TSHS1 Longchuan Co., Dehong state, Yunnan Province, China KY800847
Tylototriton verrucosus CIB TSHS2 Longchuan Co., Dehong state, Yunnan Province, China KY800848
Tylototriton verrucosus CIB TSHS3 Longchuan Co., Dehong state, Yunnan Province, China KY800849
Tylototriton verrucosus CIB TSHS4 Longchuan Co., Dehong state, Yunnan Province, China KY800850
Tylototriton verrucosus CIB TSHS5 Longchuan Co., Dehong state, Yunnan Province, China KY800851
Tylototriton verrucosus CIB TSHS6 Longchuan Co., Dehong state, Yunnan Province, China KY800852
Tylototriton shanjing KIZ 201306081 Yongde Co., Yunnan Province, China KY800856
Tylototriton shanjing KIZ 201306098 Yun Co., Yunnan Province, China KY800857
Tylototriton shanjing KIZ 201306102 Jingdong Co., Yunnan Province, China KY800858
Tylototriton shanjing KIZ 201306108 Nanjian Co., Yunnan Province, China KY800859
Tylototriton shanjing CIB 980004 Baoshan City, Yunnan Province, China KY800831
Tylototriton shanjing CIB 980005 Baoshan City, Yunnan Province, China KY800832
Tylototriton shanjing CIB 980006 Baoshan City, Yunnan Province, China KY800833
Tylototriton ngarsuensis sp. nov. LSUHC 13762 Ngar Su Village, Ywangan Township, Taunggyi District, Shan State, Myanmar MH836585
Tylototriton ngarsuensis sp. nov. LSUHC 13763 Ngar Su Village, Ywangan Township, Taunggyi District, Shan State, Myanmar MH836584
Tylototriton sp. nov. CAS 245418 Panwa Town, Panwa Township, Myitkyina District, Kachin State, Myanmar KT304279
Tylototriton sp. nov. CAS 245290 Lahe Township, Sagaing Region, Myanmar KT304278
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TABLE 2. Primer sequences used in this study for amplification and sequencing the ND2 gene and the flanking tRNAs.
Two model-based phylogenetic analyses, maximum likelihood (ML) and Bayesian Inference (BI) were
employed. The ML analysis was implemented in IQ-TREE (Nguyen et al. 2015) and used a Bayesian Information
Criterion (BIC) to calculate that HKY+F+G4 was the best-fit model of evolution for the first codon position,
TN+F+G4 was the best-fit model for the second codon positions, and TPM2u+F+G4 was the best-fit model for the
third codon position. Ultrafast Bootstrap Approximation (UFB; Hoang et al. 2017) using 1000 bootstrap replicates
were used to construct a final consensus tree. Nodes with UFB values of 95 and above were considered
significantly supported. A BI analysis was implemented in MrBayes 3.2.3. on XSEDE (Ronquist et al. 2012) using
CIPRES (Cyberinfrastructure for Phylogenetic Research; Miller et al. 2010) employing default priors and a GTR +
Gamma model of evolution for all codon positions. Two simultaneous runs were performed with four chains, three
hot and one cold. The simulation ran for 50,000,000 generations, was sampled every 5000 generations using the
Markov chain Monte Carlo (MCMC), and the first 25% of each run were discarded as burn-in. Stationarity and .p
files from each run were checked in Tracer v1.6 (Rambaut et al. 2014) to ensure effective sample sizes (ESS) were
above 200 for all parameters. Nodes with Bayesian posterior probabilities (BPP) of 0.95 and above were
considered well-supported (Huelsenbeck et al. 2001; Wilcox et al. 2002).
A time-calibrated Bayesian Inference analysis was constructed and used to perform a generalized mixed yule
coalescent (GMYC) approach to species delimitation (Pons et al. 2006), thus providing an independent framework
to complement the empirically based thresholds of the BI and ML analyses. The analysis was implemented in
BEAUti version 2.4.7 (Bayesian Evolutionary Analysis Utility) and run with BEAST version 2.4.6 (Bayesian
Evolutionary Analysis Sampling Trees; Drummond et al. 2012) on CIPRES employing a lognormal relaxed clock
with unlinked substitution and clock models and an HKY substitution model selected for each codon position.
MCMC chains were run using a coalescent exponential population prior for 50,000,000 million generations and
logged every 5,000 generations. A mean age of 10.4 million years based on the calibrated mtDNA tree of Wang et
al. (2018) was used herein as an internal constraint prior to date the node delimiting clade II with a standard
deviation of 1.4% as calculated from the highest posterior density (HPD). The BEAST log file was visualized and
checked in Tracer v. 1.6.0 (Rambaut et al. 2014) to ensure ESS values were above 200 for all parameters and a
maximum clade credibility tree using mean heights at the nodes was generated using TreeAnnotator v.1.8.0
(Rambaut & Drummond 2013) with a burnin of 1000 trees (10%). After removing outgroup taxa, MEGA7 (Kumar
et al. 2016) was used to calculate uncorrected pairwise sequence divergence among the 10 ingroup species.
The GMYC approach is a method for delimiting mitochondrial lineages of closely related individuals from
single-locus, time-calibrated ultrametric gene trees by detecting genetic clustering beyond the expected levels of a
null hypothesis which infers that all individuals of a population form a genetically, interacting nexus. In clades
where effective population sizes are relatively low and divergence times among the populations are relatively high,
the single-threshold version of the model (such as that used herein) out performs the multi-threshold version
(Fujisawa & Barrenclough 2013) so long as there are not multiple intermixing haplotypes in the same population.
The GMYC relies on the prediction that independent evolution leads to the appearance of distinct genetic clusters,
separated by relatively longer internal branches (Barraclough et al. 2003; Acinas et al. 2004). Such groups
therefore, diverge into discrete units of morphological and genetic variation that are recovered with surveys of
higher clades. The analysis was run on a web server at http://species.h-its.org/gmyc/ on 25 July 2018.
Morphological data and analyses. Color pattern notes were taken from living and preserved specimens and
digital images of living specimens. Measurements were taken on the right side of the body when possible to the
nearest 0.1 mm using dial calipers under a Leica Wild M 10 stereo dissecting microscope following Nishikawa et
al. (2011, 2014) and Phimmachak et al. (2015). Measurements taken were: snout-vent length (SVL), taken from
the tip of snout to anterior margin of the vent; head length (HL), measured from the tip of the snout to the gular
fold; (HW), measured across the widest distance across the parotoid glands; internarial distance (IND), measured
as the minimum distance between the internal nares; trunk length (TRL), measured from the gular fold to the
anterior margin of the vent; axilla to groin distance (AGD), taken from the posterior margin of the forelimb at its
Primer name Primer reference Sequence
L4437b (Macey et al., 1997) External 5’-AAGCAGTTGGGCCCATACC-3’
H5934 (Macey et al., 1997) External 5' -AGRGTGCCAATGTCTTTGTGRTT-3'
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insertion point on the body to the anterior margin of the hind limb at its insertion point on the body; forelimb length
(FLL), distance from the axilla to the tip of the longest finger; hind limb length (HLL), distance from the groin to
the tip of the longest toe; vent length (VL), measured from the anterior to the posterior end of the vent; and tail
length (TAL), taken from the anterior margin of the vent to the tip of the tail. All mensural data were converted to
ratiometric data by dividing each by SVL. Welch Two-sample t-tests were performed (because an F-test indicated
non-homogeneity of the variances) to search for significant mean differences among the ratiometric characters
between the sister species (see below) of the males of T. shanorum and the Ngar Su Village population. Females
were not tested because only one specimen was available for each species.
Other characters evaluated were the shape of the vomerine teeth and their positional relationship relative to the
choanae; numbers of and shape of the rib nodules counted from the posterior margin of the vent to the axilla; width
and prominence of the vertebral ridge; and coloration of the dorsum, venter, head, labial region, parotoid glands,
rib nodules, limbs, soles, palms, tail surfaces, and vent region.
A principal component analysis (PCA) of the ratiometric data (i.e. a mensural character divided by a different
mensural character, e.g. head length divided by snout-vent length) run on the platform R v 3.2.1 (R Core Team
2015) was used to determine if the new population of Tylototriton from Ngar Su Village was morphospacially
distinct from its sister species T. shanorum from Taunggyi and the degree to which their variation in morphospace
coincided with the putative species boundaries delimited by the molecular phylogenetic and GMYC analyses (see
Results). PCA, implemented by the prcomp() command in R, is an indiscriminate analysis plotting the overall
variation among individuals (i.e. data points) while treating each individual independently (i.e. not coercing data
points into pre-defined groups). Because of small, sexually asymmetric sample sizes, males and females from their
respective populations were pooled together even though they are sexually dimorphic. To remove allometric effects
due to body size, each character was adjusted using the following equation: Xadj=X-β(SVL-SVLmean), where
Xadj=adjusted value; X=measured value; β=unstandardized regression coefficient for each OTU; SVL=measured
snout-vent-length; SVLmean=overall average SVL of all OTU’s (Thorpe, 1975, 1983; Turan, 1999; Lleonart et al.,
2000). Adjusted data for males and females were then combined into a single PCA data matrix. The PCA data were
log-transformed and scaled to their standard deviation prior to analysis in order to normalize their distribution so as
to ensure characters with very large and very low values did not over-leverage the results owing to intervariable
nonlinearity and to insure the data were analyzed on the basis of correlation not covariance.
Results
Both the ML and BI analyses recovered the same topology as that recovered in Phimmachak et al. (2015) that was
based five concatenated mitochondrial genes and their associated tRNAs and the topologies herein bear the same
areas of week nodal support (Fig. 2). Both topologies differ from that of Wang et al. (2018) in that T.
kweichowensis is not recovered as the sister species of the T. himalayanus-T. shanorum lineage but the sister
species to a clade containing the remaining species of clade II. The analyses also recovered the Ngar Su Village
population as a phylogenetically distinct lineage and the well-supported sister lineage of T. shanorum and that these
two lineages share an uncorrected pairwise sequence divergence between them of 3.0–3.4% which is
commensurate with a number of other well-differentiated sister species (Stuart et al. 2010). Additionally, the
analyses recovered the Saigaing and Kachin populations as phylogenetically distinct within the verrucosus group
(sec. Phimmachak et al. 2015) and referred to here as T. cf. verrucosus (Fig. 2). Despite combining the sexes, the
PCA aligns itself well with the phylogenetic analyses in that the Ngar Su Village population and T. shanorum
occupy completely separate positions in morphospace with the first two components (PC1 and PC2) accounting for
74% of the variation in the data set (Fig. 3). PC1 accounts for 54% of the variation and loads most heavily for head
width, axilla-groin distance, and trunk length. PC2 accounts for an additional 24% of the variation and loads most
heavily for hind limb length (Table 3). PC2 is demonstrates the sexually dimorphic nature in limb length (shorter in
females) for each species in that males and females are completely separated along this axis.
The GMYC species delimitation analysis independently recovered nearly the same ingroup lineages as the
phylogenetic analyses (subsequently considered to be species based on corroborative morphological evidence)
with a highly significant likelihood ratio of 12.241 (p = 0022). The difference being that the GMYC recovered T.
verrucosos and T. shanjing as conspecific unlike the taxonomy of Wang et al. (2018). Bryan Stuart (in litt. 2018)
informed one of us (LLG) he believes these are distinct species and that mitochondrial introgression between them
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is responsible for the GMYC recognizing them as conspecific. The GMYC also recovered T. shanorum and the
Ngar Su Village population as potential species and it recognized the Lahe Township and the Panwa Village
populations from Sagaing Region and Kachin State, respectively as conspecific and unique from all other species
in clade II (Fig. 2). Sukumaran & Knowles (2017) demonstrated that species delimitation methods generally
overestimate species diversity by recovering clades not species and additional criteria such as morphology should
be used in conjunction with these analyses. Fujisawa & Barraclough (2013) specifically noted that the GMYC
approach should be used in conjunction with additional independent data. We agree with these recommendations
and believe the GMYC recovered noteworthy interspecific genetic structure within the phylogeny that corroborates
the species boundaries (Fig. 2) characterized by the morphological and color pattern analyses of each currently
recognized species (except T. sh a njing) in that each bears a unique and/or a unique suite of diagnostic characters
(see Stuart et al. 2010; Hou et al., 2012; Le et al. 2015; Nishikawa et al. 2013a,b, 2014; Khatiwada et al. 2015;
Phimmachak et al. 2015 and Table 4). Therefore, we base our species delimitations on this more integrative
approach.
FIGURE 2. Maximum likelihood consensus tree topology of Tylototriton clades I and II (sec. Wang et al. 2018) based on ND2
showing nodal support and the species delimited by the GMYC.
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TABLE 3. Summary statistics and principal component analysis scores for Tylototriton ngarsuensis sp. nov. and T. shanorum. Abbreviations are listed in the
Materials and Methods.
PC1 PC2 PC3 PC4 PC5 PC6 PC7
Standard deviation 2.194876269 1.471349011 1.296381418 0.436087397 0.36922865 0.102681331 6.97E-16
Proportion of variance 0.53528 0.24054 0.18673 0.02113 0.01515 0.00117 0
Cumulative proportion 0.53528 0.77582 0.96255 0.98368 0.99883 1 1
Eigenvalue 4.817481838 2.164867911 1.680604781 0.190172218 0.136329796 0.010543456 4.86E-31
HL 0.372772026 -0.253168368 0.281782489 -0.522564636 -0.128665798 -0.629781535 -0.128794349
HW 0.43270682 0.03684891 0.107092912 0.307141529 -0.650286583 0.135446949 0.421542651
IND 0.07405733 0.324590159 -0.628031417 -0.618379675 -0.265263736 0.170741673 0.077759525
AGD 0.427625552 0.222904611 0.038629879 0.082926646 0.234150951 -0.13876746 0.380131891
TRL 0.427173415 0.218832935 -0.088175765 0.127309192 0.074570035 0.180078357 -0.570682699
TAL 0.407332999 0.195593782 -0.238415376 0.266817999 0.249303916 -0.174403317 -0.242549004
VL -0.133939663 -0.326599236 -0.62089495 0.374477597 -0.200383291 -0.498645678 -0.023357724
FLL 0.270766971 -0.4810778 -0.251029576 -0.118737593 0.518592709 0.224046827 0.398971279
HLL 0.214022987 -0.596032723 -0.008412205 -0.039855614 -0.242024711 0.418247523 -0.333068436
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FIGURE 3. Left: box plot analysis showing the differences in relative head length between Tylototriton ngarsuensis sp. nov.
and T. shanorum. Right: principle component analysis (PCA) of the mensural characters of T. ngarsuensis sp. nov. and T.
shanorum from Shan State showing sexual dimorphism associated primarily with hind limb length along PC2; M = male and F
= female.
Taxonomy
Tylototriton ngarsuensis sp. nov.
Suggested common name: Ywangan Crocodile Newt
Figs. 4, 5
Holotype. Gravid female LSUHC 13762 collected on 24 October 2017 at 2330 hrs by Evan S. H. Quah, Robert E.
Espinoza, Myint Kwaw Thura, and L. Lee Grismer from Baw Hto Chang in Ngar Su Village, Ywangan Township,
Taunggyi District, Shan State, Myanmar (21.15364° N, 96.43660°E WGS84) at 1212 m in elevation.
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Paratypes. Adult male paratypes LUSHC 13763–64 bear the same collecting data as the holotype (Table 4).
Additional referred material. A lot of 15 gilled larvae (LSUHC 13761) ranging from 10–33 mm SVL (stage
44 of Grosse 2013) bearing the same locality data as the holotype were collected during the evening of 24 October
2017 and the morning of 25 October 2017 between 1000 and 1100 hrs.
Diagnosis. A member of the genus Tylototriton based on its dark dorsal ground color with light-colored
markings, well-developed cephalic and vertebral ridges, strong sexual dimorphism (Dubois & Raffaëlli 2009), and
its phylogenetic relationships (Wang et al. 2018). A large-sized newt (female SVL = 102.3 mm; male SVL 74.9–
76.4 mm); head relatively short (HL/SVL = 0.22–0.26), subtriangular in dorsal profile; skin rough, glandular,
bearing fine granules; dorsolateral boney ridges on head prominent; vertebral ridge low and wide; rib nodules
relatively indistinct, sometimes lacking medially; vomeropalatine tooth series form an inverted, narrow V-shape,
anteriorly projecting beyond the choanae but not in contact with them, teeth confined to palatine bone, not vomers;
dorsal ground color black; anterior of head, paratoid region, vertebral ridge, rib nodules, limbs, and side of tail
dark-brown, nearly black; labial regions, palms and soles, vent region, and subcaudal region light-brown.
FIGURE 4. Tylototriton ngarsuensis sp. nov. from Baw Hto Chang in Ngar Su Village, Ywangan Township, Taunggyi
District, Shan State, Myanmar (21.15364 °N, 96.43660 °E WGS84) at 1212 m in elevation. A. Gravid female holotype LUSHC
13762. B. Adult male paratype LSUHC 13764. C. Adult male paratype LSUHC 13763. D. Stage 44 larva (Grosse 2013) from
lot LSUHC 13761 (SVL = 30 mm).
Description of holotype. Habitus stout; head narrower than body, wider than long, subtriangularly shaped in
dorsal profile, slightly sloping in lateral profile; snout short, rounded in dorsal profile, truncate in lateral profile,
extending beyond lower jaw; nostrils on anterior margin of snout, facing forward, narrowly visible from above;
labial fold absent; vomeropalatine tooth series form an inverted, narrow V-shape, anteriorly projecting beyond the
choanae but not in contact with them, teeth confined to palatine bone, not vomers; tongue oval, attached to anterior
floor of mouth but free posteriorly and laterally; no glandular ridge on midline of crown but short ridge on snout;
dorsolateral bony ridges on head wide and slightly protruding, extending from lore to anterior end of parotoids,
posterior ends turned slight toward midline; parotoids enlarged, crescent-shaped, projecting posteriorly with
posterior margins curved medially; low, wide, weakly segmented, vertebral tubercular ridge extends from occiput
onto anterior one-half of dorsal margin of tail, separated from ridge on midline of snout; dorsolateral row of weak
glandular warts (rib nodules) on each side from level of axillae to level of posterior margin of vent (i.e. base of tail)
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on anterior margin of tail but not extending father down the tail; anterior and posterior nodules more prominent
than the barely discernable medial series; approximately 15 rib nodules on both sides; skin rough; small, glandular
warts on most dorsal and ventral surfaces; warts on crown, nape, and ventrolateral region in clusters of small
glands, those on throat, tail, and ventral surfaces of limbs granular, those on belly arranged in transverse striations;
pectoral region bearing a smooth, glandular, ovoid-shaped patch; gular fold prominent; adpressed limbs do not
overlap; four fingers, five toes, all without webbing; and tail laterally compressed, tapering posteriorly, bearing
sharp narrow dorsal fin most distinct posteriorly, a smooth ventral ridge, bluntly acuminate tip in lateral profile.
FIGURE 5. A. Dorsal view of the type series of Tylototriton ngarsuensis sp. nov. from Baw Hto Chang in Ngar Su Village,
Ywangan Township, Taunggyi District, Shan State, Myanmar (21.15364 °N, 96.43660 °E WGS84) at 1212 m in elevation. B.
Dorsal view of the type seires of T. shanorum from the vicinity of Taunggyi, Shan State. C. Stage 44 larva (Grosse 2013) of T.
ngarsuensis sp. nov. from lot LSUHC 13761 (SVL = 33 mm). E. Ventral view of the type series of T. ngarsuensis sp. nov. E.
Ventral view of the type series of T. shanorum. F. Adult male T. ngarsuensis sp. nov. LSUHC 13763.
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FIGURE 6. A. Baw Hto stream wherein adults and larvae of Tylototriton ngarsuensis sp. nov. were collected. B. Pond
associated with Baw Hto stream where some of the larvae were also collected.
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TABLE 4. Mensural and character state data for the type series of Tylototriton ngarsuensis sp. nov. and T. shanorum. See Methods for abbreviations. Mensural data are in mm.
shanorum shanorum shanorum shanorum ngarsuensis
s
p
. nov.
ngarsuensis
s
p
. nov.
ngarsuensis
s
p
. nov.
CAS 230940 CAS 230899 CAS 20814 CAS 230933 LSUHC 13763 LSUHC 13764 LSUHC 13762
holotype additional
specimen
paratype paratype paratype paratype holotype
Sex male male male female male male female
SVL 76 66.7 76.5 87.9 76.4 74.9 102.3
HL 22.4 19.7 24.3 25.7 18.3 19.5 22
HW 25.9 18.6 25.7 27.1 18.7 21.1 27.2
IND 6.7 4.14 6 6.3 6.7 6 7.9
AGG 49.6 33.3 49.4 51.9 38.1 36.1 52.4
TRL 77.6 49 74.3 75.7 57.7 55.6 79.3
TAL 111.2 64.8 97 97.8 79.1 73.4 107
VL 9.3 6.3 3.4 3.5 8.2 9.2 8.2
FLL 34.7 29.5 31.7 32.5 30.5 29.7 36.3
HLL 37.2 35 35.6 37 32 35.3 39.6
HL/SVL 0.29 0.30 0.32 0.29 0.24 0.26 0.22
HW/SVL 0.34 0.28 0.34 0.31 0.24 0.28 0.27
AGD/SVL 0.65 0.50 0.65 0.59 0.50 0.48 0.51
TRL/SVL 1.02 0.73 0.97 0.86 0.76 0.74 0. 78
TAL/SVL 1. 46 0.97 1.27 1.11 1. 04 0.98 1.05
VL 0.12 0.09 0.04 0.04 0.11 0.12 0.08
FLL/DVL 0.46 0. 44 0.41 0. 37 0.40 0.40 0.35
HLL/SVL 0.49 0.52 0.47 0.42 0.42 0.47 0.39
vomerine teeth contact
choanae
yes yes yes yes no no no
rib nodules 14 14 14 14 15 15 15
supratemporal ridge begins in loreal
region
begins in loreal
region
begins in loreal
region
begins in loreal
region
begins posterior to
orbit
begins posterior to
orbit
begins posterior to
orbit
vertebral ridge narrow narrow narrow narrow wide wide wide
top of head red-brown red-brown red-brown red-brown nearly black nearly black nearly black
vertebral ridge red-brown red-brown red-brown red-brown nearly black nearly black nearly black
rib nodules red-brown red-brown red-brown red-brown nearly black nearly black nearly black
limbs red-brown red-brown red-brown red-brown nearly black nearly black nearly black
side of tail red-brown red-brown red-brown red-brown nearly black nearly black nearly black
labial regions dark-yellow dark-yellow dark-yellow dark-yellow dark-yellow dark-yellow dark-yellow
palms and soles dark-yellow dark-yellow dark-yellow dark-yellow dark-yellow dark-yellow dark-yellow
vent region dark-yellow dark-yellow dark-yellow dark-yellow dark-yellow dark-yellow dark-yellow
subcaudal region dark-yellow dark-yellow dark-yellow dark-yellow dark-yellow dark-yellow dark-yellow
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Color of holotype in life (Fig. 4). Dorsal ground color of top of head and trunk black; dorsal surfaces of limbs,
and lateral surfaces of tail dark-brown; iris black; gular region, belly, and ventral surfaces of limbs dark-grey;
anterior of head and parotoids dull-brown; rib nodules and vertebral ridge dark-brown, barely discernable from
black trunk coloration; upper and lower lips, palms and soles, and subcaudal region light-brown.
Variation (Figs 4, 5). The male paratypes generally approximate the female holotype in coloration and pattern.
LSUHC 13764 is darker overall than the holotype in that it is essentially black on all dorsal surfaces except for the
upper lips, lores, and parotoids. The vertebral ridge is the same color as the trunk and the rib nodules are only
weakly discernable by being slightly lighter in color. In LSUHC 13763, these areas are only slightly lighter but
they are more discernable than in the holotype or LSUHC 13764. The middle series of rib nodules on the left side
in LSUHC 13763 are barely discernable as in the holotype. The adpressed limbs greatly overlap in the paratypes
but do not overlap in the holotype. The holotype measures 102.3 mm in SVL whereas the largest male (LSUHC
13763) measures 76.4 mm. Other mensural differences are presented in Table 2.
Distribution (Fig. 1). Tylototriton ngarsuensis sp. nov. is known only from Baw Hto Chang in Ngar Su
Village, Ywnagan Township, Taunggyi District, Shan State, Myanmar (21.15364° N, 96.43660°E WGS84)
although locals told us they can be found in other nearby streams and ponds.
Etymology. The specific epithet ngarsuensis is a toponym in reference to Ngar Su Village, the type locality.
Natural history. Baw Hto Chang is a shallow, slow-moving stream during the monsoon months (May-
September) that runs through a narrow corridor of forest (Fig. 6). The area beyond the corridor has been converted
to paddy fields and thus this narrow band of forest is likely to serve as the only place where newts can estivate
during the colder and drier winter months. The adult newts were found in the late evening walking along the sandy
bottom in clear water 1 m deep in the vicinity of underwater vegetation. Larvae were collected from shallower
areas near the shore. We returned the next morning and found more larvae in the water along the edges of the shore,
usually beneath leaves or other cover. The fact that we collected larvae as small as 10 mm SVL and a gravid female
indicates newts are still breeding during late October. Most other species breed from April through July (Bernardes
et al. 2013, 2017) or from June through September in the case of Tylototriton himalayanus (Dasgupta 1996).
Villagers from Ngar Su Village told us the newts are quite common on land during the months of May and June at
the beginning of monsoon season at which time they are presumably migrating to the water. Villagers also say it is
not uncommon for newts to wander into their houses during this period.
Comparisons. Tylototriton ngarsuensis sp. nov. is most closely related to T. shanorum but differs from it by
having much larger adult females (102.3 mm vs 87.9 mm); a significantly shorter head in males (HL/SVL 0.22–
0.26 vs. 0.29–0.32; p = 0.041 for males; 0.22 vs 0.29 for females; Fig. 3); having large, rounded rib nodules with
diameters equivalent to or greater than that of eye as opposed to having small, slightly elongated rib nodules with
diameters less than that of the eye; by having a thick, glandular, vertebral tubercular ridge as opposed to a narrow
and less glandular ridge; 15 vs 14 rib nodules; paratoid ridge beginning posterior to the orbits as opposed beginning
in the loreal region; top of head, vertebral ridge, rib nodules, limbs, and side of tail nearly black as opposed being
red-brown; labial regions, palms, soles, and subcaudal region dark-brown as opposed to being dull-yellow; and
venter and underside of limbs being dark-gray as opposed to dull-yellow. Tylototriton ngarsuensis sp. nov. and T.
shanorum share an uncorrected pairwise sequence divergence between them of 3.0–3.4% which is commensurate
with that among other well-established sister species (Stuart et al. 2010).
Tylototriton ngarsuensis Color of holotype in life (Fig. 4). Dorsal ground color of top of head and trunk black;
dorsal surfaces of limbs, and lateral surfaces of tail dark-brown; iris black; gular region, belly, and ventral surfaces
of limbs dark-grey; anterior of head and parotoids dull-brown; rib nodules and vertebral ridge dark-brown, barely
discernable from black trunk coloration; upper and lower lips, palms and soles, and subcaudal region light-brown.
Variation (Figs 4, 5). The male paratypes generally approximate the female holotype in coloration and pattern.
LSUHC 13764 is darker overall than the holotype in that it is essentially black on all dorsal surfaces except for the
upper lips, lores, and parotoids. The vertebral ridge is the same color as the trunk and the rib nodules are only
weakly discernable by being slightly lighter in color. In LSUHC 13763, these areas are only slightly lighter but
they are more discernable than in the holotype or LSUHC 13764. The middle series of rib nodules on the left side
in LSUHC 13763 are barely discernable as in the holotype. The adpressed limbs greatly overlap in the paratypes
but do not overlap in the holotype. The holotype measures 102.3 mm in SVL whereas the largest male (LSUHC
13763) measures 76.4 mm. Other mensural differences are presented in Table 2.
Distribution (Fig. 1). Tylototriton ngarsuensis sp. nov. is known only from Baw Hto Chang in Ngar Su
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Village, Ywnagan Township, Taunggyi District, Shan State, Myanmar (21.15364° N, 96.43660°E WGS84)
although locals told us they can be found in other nearby streams and ponds.
Etymology. The specific epithet ngarsuensis is a toponym in reference to Ngar Su Village, the type locality.
Natural history. Baw Hto Chang is a shallow, slow-moving stream during the monsoon months (May-
September) that runs through a narrow corridor of forest (Fig. 6). The area beyond the corridor has been converted
to paddy fields and thus this narrow band of forest is likely to serve as the only place where newts can estivate
during the colder and drier winter months. The adult newts were found in the late evening walking along the sandy
bottom in clear water 1 m deep in the vicinity of underwater vegetation. Larvae were collected from shallower
areas near the shore. We returned the next morning and found more larvae in the water along the edges of the shore,
usually beneath leaves or other cover. The fact that we collected larvae as small as 10 mm SVL and a gravid female
indicates newts are still breeding during late October. Most other species breed from April through July (Bernardes
et al. 2013, 2017) or from June through September in the case of Tylototriton himalayanus (Dasgupta 1996).
Villagers from Ngar Su Village told us the newts are quite common on land during the months of May and June at
the beginning of monsoon season at which time they are presumably migrating to the water. Villagers also say it is
not uncommon for newts to wander into their houses during this period.
Comparisons. Tylototriton ngarsuensis sp. nov. is most closely related to T. shanorum but differs from it by
having much larger adult females (102.3 mm vs 87.9 mm); a significantly shorter head in males (HL/SVL 0.22–
0.26 vs. 0.29–0.32; p = 0.041 for males; 0.22 vs 0.29 for females; Fig. 3); having large, rounded rib nodules with
diameters equivalent to or greater than that of eye as opposed to having small, slightly elongated rib nodules with
diameters less than that of the eye; by having a thick, glandular, vertebral tubercular ridge as opposed to a narrow
and less glandular ridge; 15 vs 14 rib nodules; paratoid ridge beginning posterior to the orbits as opposed beginning
in the loreal region; top of head, vertebral ridge, rib nodules, limbs, and side of tail nearly black as opposed being
red-brown; labial regions, palms, soles, and subcaudal region dark-brown as opposed to being dull-yellow; and
venter and underside of limbs being dark-gray as opposed to dull-yellow. Tylototriton ngarsuensis sp. nov. and T.
shanorum share an uncorrected pairwise sequence divergence between them of 3.0–3.4% which is commensurate
with that among other well-established sister species (Stuart et al. 2010).
Tylototriton ngarsuensis sp. nov. differs from all other species of Tylototriton as follows (data from Stuart et al.
2010; Hou et al., 2012; Nishikawa et al. 2013a,b, 2014; Khatiwada et al. 2015; Le et al. 2015; Phimmachak et al.
2015): from T. podicthys it differs by lacking orange coloration on the limbs and lateral surfaces of the tail and by
lacking a boney ridge on the midline of the crown as opposed to having these traits; from T. anguliceps and T.
shanjing it differs by lacking a boney ridge on the midline of the crown as opposed to the presence of a boney
ridge; from T. anguliceps it differs by lacking an orange head, limbs, tail, parotoids, rib nodules, and vertebral ridge
as opposed to having these structures being orange; from T. uyenoi and T. pulcherrima it differs by lacking orange-
colored limbs, tail, parotoids, rib nodules, and vertebral ridge as opposed to having these structures colored orange;
from T. kweichowensis and T. pseudoverrucosus it differs by having isolated rib nodules as opposed to the rib
nodules being connected and forming dorsolateral ridges; from T. shanjing it differs by having no sharp contrast
between the orange crown of the head and black nape as opposed to a sharp contrast, having dark coloration on
ventral surfaces of limbs versus orange coloration, and the tips of the digits being dark as opposed uniformly
orange; from T. taliangensis it differs by having as opposed to lacking rib nodules; from T. verrucosus it differs by
lacking a glandular ridge on the midline of the crown as opposed to having a ridge on the crown; from T. yangi it
differs by having uniformly orange parotoids as opposed to the light-colored parotoid regions being restricted to
the posterior margins of the glands; and from T. himalayanus it differs in having the head longer than wide, a
truncate snout in lateral profile as opposed to a rounded snout, and ventral surfaces being dark-gray as opposed to
cream-colored. differs from all other species of Tylototriton as follows (data from Stuart et al. 2010; Hou et al.,
2012; Nishikawa et al. 2013a,b, 2014; Khatiwada et al. 2015; Le et al. 2015; Phimmachak et al. 2015): from T.
podicthys it differs by lacking orange coloration on the limbs and lateral surfaces of the tail and by lacking a boney
ridge on the midline of the crown as opposed to having these traits; from T. anguliceps and T. shanjing it differs by
lacking a boney ridge on the midline of the crown as opposed to the presence of a boney ridge; from T. anguliceps
it differs by lacking an orange head, limbs, tail, parotoids, rib nodules, and vertebral ridge as opposed to having
these structures being orange; from T. uyenoi and T. pulcherrima it differs by lacking orange-colored limbs, tail,
parotoids, rib nodules, and vertebral ridge as opposed to having these structures colored orange; from T.
kweichowensis and T. pseudoverrucosus it differs by having isolated rib nodules as opposed to the rib nodules
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being connected and forming dorsolateral ridges; from T. shanjing it differs by having no sharp contrast between
the orange crown of the head and black nape as opposed to a sharp contrast, having dark coloration on ventral
surfaces of limbs versus orange coloration, and the tips of the digits being dark as opposed uniformly orange; from
T. taliangensis it differs by having as opposed to lacking rib nodules; from T. verrucosus it differs by lacking a
glandular ridge on the midline of the crown as opposed to having a ridge on the crown; from T. yangi it differs by
having uniformly orange parotoids as opposed to the light-colored parotoid regions being restricted to the posterior
margins of the glands; and from T. himalayanus it differs in having the head longer than wide, a truncate snout in
lateral profile as opposed to a rounded snout, and ventral surfaces being dark-gray as opposed to cream-colored.
Discussion
The BEAST analysis recovered the divergence between Tylototriton ngarsuensis sp. nov. and T. shanorum as
occurring no later than 2.4 million years ago. These species are currently separated by approximately 80 km that
encompasses three intervening basins and two mountain ranges (Fig. 1). Given these geographical barriers, their
sequence divergence, and poor dispersal capabilities we suspect that recent gene flow is highly unlikely. The
discovery of another new species of Tylototriton from the unexplored upland areas of northern Myanmar is more of
an expectation than it is a surprise and we are aware of other unreported populations from these regions as well.
The Shan State species and their phylogenetic affinities with species from southwestern China, northern Thailand,
and Laos represents the westernmost extension of this clade to the eastern edge of the Ayeyarwady Basin—a
phylogeographic pattern that has been reported for other new species found in this region (Grismer et al. 2017a,b,
2018a,b,c). However, the distribution of another potentially new species of Tylototrion from Sagaing Region (CAS
245290) and Kachin State (CAS 245418) expands the range of this clade (and this species) across the narrow
northern end of the Ayeyarwady Basin through a series of intervening hilly areas that are likely to harbor
unreported populations as well (Fig. 1).
The phylogenetic tree recovered herein is just another tree among many Tylototriton phylogenies (Zang et al.
2007, 2013; Stuart et al. 2010; Nishikawa et al. 2013a,b, 2015; Hou et al., 2012; Yuan et al. 2011; Shen et al. 2012;
Sparreboom 2014, Yang et al. 2014; Khatiwada et al. 2015; Le et al. 2015; Phimmacak et al. 2015; Qian et al.
2017; Wang et al. 2018) that does little to resolve the issues of species boundaries or taxonomy within this genus.
Even the latest comprehensive phylogenies of Phimmachak et al. (2015)—constructed from 13 mitochondrial
genes and tRNA fragments using 137 samples—and that of Wang et al. (2018)—constructed from two
mitochondrial genes and two nuclear genes and 108 ingroup samples—are incongruent in many places. The
phylogenetic and taxonomic issues within this genus would benefit greatly from a genomic data set which should
clearly be the next step in studying the evolutionary history of this widespread genus as opposed to generating
additional competing trees based on Sanger sequencing. The phylogeny herein extends the now well-established
trend of new species being discovered in the Shan Hills (Grismer et al. 2017a,b, 2018a,b,c) and that as more
remote, unstudied areas within this geographically complex region are surveyed, more of the unrealized
biodiversity from this area will be discovered and described.
Conservation
A villager from Ngar Su Village told us that people from Yangon come to his village and other nearby villages each
year and hire people to collect newts during their migration period (presumably during May and June). The newts
are used for indigenous medicines and taken back to Yangon for sale in pet stores. Tylototriton shanorum is also
harvested for the pet trade and sold in European markets (Nishsikawa et al. 2015). In a set of thoroughly researched
studies, Rowley et al. (2010, 2016) noted the ongoing crisis surrounding the illegal pet trade in Asian newts.
Rowley et al. (2016) documented the demand for several species noting that between the years of 2005 and 2014,
8702 individual newts from just four species—including T. verrucosus and T. shanjing—were imported live into
the USA alone. They went on to report that Southeast Asian newts were listed for sale in the pet trade from 15 other
countries throughout Europe and Asia.
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Acknowledgements
We wish to thank Mr. Win Naing Thaw of the Ministry of Natural Resources and Environmental Conservation
Forest Department for the collection and export permits. We thank the staff of the Shwe Gue Gu Hotel and Genious
Coffee for their hospitality. We thank our driver Ko Tun for logistic support an unending antics. LLG thanks the
College of Arts and Sciences of La Sierra University and Fauna & Flora International for partial funding. Research
by ESHQ was partially covered by the USM Postdoctoral Scheme. REE thanks the College of Science and
Mathematics at California State University, Northridge for partial funding.
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