A new species of Crocodile Newt, genus Tylototriton (Amphibia, Caudata, Salamandridae) from the mountains of Kachin State, northern Myanmar

2019·11.1 MB·

by  Zaw, Than

ZOOLOGICAL RESEARCH
A new species of Crocodile Newt, genus Tylototriton
(Amphibia, Caudata, Salamandridae) from the
mountains of Kachin State, northern Myanmar
ThanZaw1,PawLay2,ParinyaPawangkhanant3,VladislavA.Gorin4,NikolayA.Poyarkov,Jr.4,5,*
1ZoologyDepartment,MohnyinDegreeCollege,Mohnyin,KachinState,Myanmar
2FloraandFaunaInternational,LonTonVillage,Indawgyi,KachinState,Myanmar
3BansomdejchaoprayaRajabhatUniversity,HiranRuchi,ThonBuri,Bangkok10600,Thailand
4DepartmentofVertebrateZoology,BiologicalFaculty,LomonosovMoscowStateUniversity,Moscow119234,Russia
5JointRussian-VietnameseTropicalResearchandTechnologicalCenter,NghiaDo,CauGiay,Hanoi,Vietnam
ABSTRACT studies of the genus Tylototriton has indicated that the genus
ismonophyletic(Nishikawaetal.,2013a,2013b;Phimmachak
We describe a new species of the genus Tylototriton et al., 2015; Wang et al., 2018) and includes two major
from Ingyin Taung Mt., Mohnyin Township, Kachin groups,correspondingtothesubgeneraTylototritons.str.and
State, Myanmar, based on morphological and Yaotriton (Wang et al., 2018). Molecular taxonomy methods
molecular evidence. The new species is assigned to have proven to be useful for deciphering taxonomic diversity
the subgenus Tylototriton s. str. and is clearly distinct ofthegenusTylototriton,with13species(over50%)described
fromallknowncongenersbythefollowingcharacters: inthepastfiveyears(Grismeretal.,2018a;Houetal.,2012;
medium body size; thin, long tail, lacking lateral Khatiwadaetal.,2015;Nishikawaetal.,2013a,2013b,2014;
grooves; rough skin; truncate snout; wide, protruding Phimmachaketal.,2015;Shenetal.,2012;Yangetal.,2014;
supratemporal bony ridges on head, beginning at Zhaoetal.,2012).However,recentmolecularsurveysindicate
anteriorcorneroforbit;weak,almostindistinctsagittal that our knowledge on taxonomic diversity of the genus
ridge; long, thin limbs, broadly overlapping when Tylototriton is still far from complete, revealing several cryptic
adpressedalongbody;distinct,wide,non-segmented lineages likely corresponding to as yet undescribed species
vertebral ridge; 13 or 14 rib nodules; brown to dark- (Grismeretal.,2018a;Wangetal.,2018).
brown background coloration with dull orange-brown Myanmar,previouslyknownasBurma,isthelargestcountry
to yellowish-brown markings on labial regions, of mainland Southeast Asia. Despite this, its herpetofauna
parotoids, rib nodules, whole limbs, vent, and ventral remainsoneoftheleastexploredintheregion(Grismeretal.,
tail ridge. We also briefly discuss biogeography and 2018a). Members of the genus Tylototriton have long been
speciesdiversityofthegenusTylototritoninMyanmar. recorded from northern and eastern parts of Myanmar and
have been traditionally classified as T. verrucosus Anderson,
Keywords: Tylototritonkachinorumsp.nov.;mtDNA 1871 (Gyi, 1969). Nishikawa et al. (2014), based on the
genealogy; ND2; 16S rRNA; Shan; Biogeography; examinationofspecimensassignedtoT.verrucosuscollected
Endemism;Taxonomy from the Shan Plateau in eastern Myanmar and pet-trade
animalsassumedtooriginatefromMyanmar,recentlydescribed
a new species, T. shanorum Nishikawa, Matsui & Rao, 2014.
INTRODUCTION Soon after, Phimmachak et al. (2015) published sequence
The salamandrid genus Tylototriton Anderson, 1871, or
Received:30 October 2018;Accepted:21 February 2019;Online:28
Crocodile Newts, currently includes 24 recognized species,
March2019
inhabiting montane forest areas throughout the Asian
monsoon climate zone from eastern Himalaya, southern and Foundation items: The study was partially supported by the Russian
central China including Hainan Island, to northern Indochina ScienceFoundation(19-14-00050)
includingVietnam,Laos,Thailand,andMyanmar(Hernandez, *Correspondingauthor,E-mail:[email protected]
2016; Wang et al., 2018). Recent progress in phylogenetic DOI:10.24272/j.issn.2095-8137.2019.043
SciencePress ZoologicalResearch 40(3):151-174,2019 151
data for specimens collected from the Sagaing Region and In the present paper, we report on a new population of the
Kachin State in northern Myanmar, which were reported asT. genusTylototritonfromtheKachinHillsinthesouthernpartof
verrucosus.Morerecently,Grismeretal.(2018a)demonstrated Kachin State, northern Myanmar. We applied morphological
the presence of two morphologically and genetically distinct and molecular methods to evaluate its taxonomic status and
lineages of Tylototriton in the Shan Plateau and described a describe it as a new species. We also discuss biogeography
new species from its north-western edge, T. ngarsuensis andtaxonomyofthegenusTylototritoninMyanmar.
Grismer, Wood, Quah, Thura, Espinoza, Grismer, Murdoch &
MATERIALSANDMETHODS
Lin,2018.ThesameworkofGrismeretal.(2018a)reanalyzed
sequences of specimens from the Sagaing and Kachin
Samplecollection
regions of Myanmar and demonstrated that they belong to a
FieldworkwascarriedoutinnorthernMyanmar,KachinState,
distinctlineage–Tylototritonsp.1,distinctfromT.verrucosus
from 14 to 21 July 2018. Specimens of Tylototriton sp. were
s. str. Recent work also indicated the presence of T.
collectedbyhandinswampsinforestclearingssurroundedby
himalayanus Khatiwada, Wang, Ghimire, Vasudevan, Paudel
montane evergreen tropical forests of Ingyin Taung Mountain,
& Jiang, 2015 (a species described from Nepalese Himalaya)
IndawgyiLakearea,KachinState(Figure1;samples22–23).
in northern Myanmar, but without providing voucher specimen
Geographic coordinates and altitude were obtained using a
information or any other justification for this identification
Garmin GPSMAP 60CSx GPS receiver (Garmin Ltd., USA)
(Hernandez, 2016; Hernandez et al., 2018). The recent
and recorded in datum WGS 84. Specimens were euthanized
monographic review of the genus Tylototriton by Hernandez
by 20% benzocaine and tissue samples for genetic analysis
(2016) also indicated the possibility of the occurrence of T. weretakenandstoredin96%ethanol(femoralmuscles)prior
uyenoiNishikawa,Khonsue,Pomchote&Matsui,2013andT. to preservation. Specimens were subsequently preserved in
shanjingNussbaum,Brodie&Yang,1995inpartsofMyanmar 70% ethanol and deposited in the herpetological collection of
adjacent to northern Thailand and the southwestern Yunnan the Zoological Museum of Moscow State University (ZMMU)
Province of China; however, these records are not supported in Moscow, Russia; Zoological Institute of Russian Academy
byvoucherspecimens.Thus,ourknowledgeonthetaxonomic of Sciences (ZISP) in St. Petersburg, Russia; and Zoology
compositionanddiversityofthegenusTylototritoninMyanmar Department of University of Mandalay (ZDUM), Mandalay,
isstillfarfromcomplete. Myanmar.
Figure1 DistributionofTylototritonandsamplinglocalitiesexaminedinthisstudy
Colorsofdotscorrespondtoclades 1–5 denotedinFigure2.Samplenumbers 1–115 refertoTable1.TypelocalityofnewspeciesTylototriton
kachinorumsp.nov.ismarkedwithastar.PhotobyNikolayA.Poyarkov.
152 www.zoores.ac.cn
Morphologicaldescription et al. (2005); Chen et al. (2010); Fang & Chang (1932); Fei
Specimens of Tylototriton sp. were photographed in life and et al. (1984); Grismer et al. (2018a); Hernandez (2016); Hou
afterpreservation.Thesexandmaturityofthespecimensand et al. (2012); Khatiwada et al. (2015); Le et al. (2015); Liu
number of eggs were checked and counted by minor (1950); Nishikawa et al. (2013a; 2013b; 2014); Nussbaum et
dissections. Measurements were taken using a digital caliper al. (1995); Phimmachak et al. (2015); Qian et al. (2017);
tothenearest0.01mm,subsequentlyroundedto0.1mm.We Shen et al. (2012); Stuart et al. (2010); Unterstein (1930);
used a stereoscopic light binocular microscope when Yang et al. (2014).
necessary. Statistical analyses were performed with Statistica
8.0(Version8.0;StatSoft,Tulsa,OK,USA). Larvalmorphology
Description of larval morphology followed Okamiya et al.
Adultmorphology (2018). For larval specimens, we recorded nine morphometric
MorphometricsfollowedNishikawaetal.(2014),Khatiwadaet charactersincluding(1)SVL,(2)HL,(3)HW,(4)OL,(5)AGD,
al.(2015),andOkamiyaetal.(2018)andincludedthefollowing (6)TAL, (7) FLL, (8) HLL, and (9) MXTAH (definition same as
24 measurements: (1) SVL (snout-vent length) from tip of foradultmorphology).Developmentalstagesweredetermined
snout to anterior tip of vent; (2) HL (head length); (3) HW followingGrosse(2013).
(head width); (4) MXHW (maximum head width); (5) IND
(internarial distance); (6) AGD (axilla-groin distance); (7) TRL DNAisolation,PCR,andsequencing
(trunk length); (8) TAL (tail length) from anterior tip of vent to TotalgenomicDNAwasextractedfrom95%ethanol-preserved
tailtip;(9)VL(ventlength);(10)FLL(forelimblength); (11) HLL muscle tissues using standard phenol-chloroform extraction
(hindlimb length); (12) VTW (vomerine tooth series width): protocols (Hillis et al., 1996). Total DNA concentration was
greatest width of vomerine tooth series; (13) VTL (vomerine estimated in 1 μLusing a NanoDrop 2000 spectrophotometer
tooth series length): greatest length of vomerine tooth series; (Thermo Scientific, USA), and consequently adjusted to
(14) LJL(lower jaw length from tip of lower jaw to articulation 100ngDNA/μL.
of upper and lower jaws); (15) SL (snout length from tip of We amplified two mtDNA fragments consisting of partial
snout to anterior tip of upper eyelid); (16) IOD (minimum sequences of the ND2 and 16S rRNA mtDNA genes. These
interorbitaldistance);(17)UEW(maximumuppereyelidwidth); markerswerechosenastheyareusefulinstudiesofTylototriton
(18) UEL(upper eyelid length, distance between anterior and phylogeny and taxonomy (Nishikawa et al., 2013a, 2013b,
posteriorangles);(19)OL(orbitlength);(20)BTAW(basaltail 2014; Wang et al., 2018; Zhao et al., 2012 and references
widthatlevelofanteriortipofcloaca);(21)MTAW(tailwidthat therein).Weusedthe16L-1(forward)(5'-CTGACCGTGCAAA
mid-leveloftail);(22)MXTAH(maximumtailheight);(23)MTAH GGTAGCGTAATCACT-3') and 16H-1 (reverse) (5'-CTCCGG
(tailheightatmid-leveloftail);(24)ON(orbitonarialdistance). TCTGAACTCAGATCACGTAGG-3') primers to amplify the
For holotype description, we examined the following 12 16S rRNA fragments (Hedges, 1994). For amplification and
morphometricandthreemeristiccharactersfollowingPoyarkov sequencing of the ND2 gene, we used the SL-1 (forward) (5'-
etal.(2012)andOkamiyaetal.(2018):additionalmorphometric ATAGAGGTTCAAACCCTCTC-3') and SL-2 (reverse) (5'-
characters: (25) ICD (intercanthal distance); (26) CW (chest TTAAAGTGTCTGGGTTGCATTCAG-3') primers of Wang et
width); (27) NSD (nostril-snout distance); (28) 1FL(first finger al. (2018). Polymerase chain reaction (PCR) was performed
lengthfrombasetotip);(29)2FL(secondfingerlengthfrombase in 20 μL reactions using 50 ng genomic DNA, 10 nmol of
to tip); (30) 3FL(third finger length from base to tip); (31) 4FL each primer, 15 nmol of each dNTP, 50 nmol additional
(fourthfingerlengthfrombasetotip);(32)1TL(firsttoelength MgCl, Taq PCR buffer (10 mmol/L Tris-HCl, pH 8.3,
2
from base to tip); (33) 2TL (second toe length from base to 50 mmol/L KCl, 1.1 mmol/L MgCl, and 0.01% gelatin), and
2
tip);(34)3TL(thirdtoelengthfrombasetotip);(35)4TL(fourth 1 U of Taq DNA polymerase. PCR cycles included an initial
toe length from base to tip); (36) 5TL (fifth toe length from denaturation step of 4 min at 94 °C and 35 cycles of
base to tip); meristic characters: (37) UJTN (number of teeth denaturation for 30 s at 94 °C, primer annealing for 30 s at
onupperjaw);(38)LJTN(numberofteethonlowerjaw);(39)VTN, 48–58 °C, and extension for 1 min 30 s at 72 °C. PCR
number of teeth on vomer. We also recorded the following products were visualized by agarose gel electrophoresis in
characters as per Nishikawa et al. (2014) and Grismer et al. the presence of ethidium bromide and consequently purified
(2018a): shape of vomerine teeth and their positional using 2 μL from a 1:4 dilution of ExoSapIt (Amersham, UK)
relationship relative to choanae; skin texture; number and per 5 μL of PCR product prior to cycle sequencing.
shape of rib nodules counted from posterior margin of vent to Sequencingwasperformedinbothdirections using the same
axilla; width and prominence of vertebral ridge and head primersasusedinPCRonanABI3730xlautomatedsequencer
ridges; and coloration of dorsum, venter, head, labial region, (AppliedBiosystems,USA)atEvrogenInc.,Moscow(Russia).
parotoidglands,ribnodules,limbs,soles,palms,tailsurfaces, The newly obtained sequences were aligned and deposited
andventregion. in GenBank under the accession numbers MK095616–
The characters of adult morphology chosen for MK095619 and MK097271–MK097274 (Table 1). Sequences
comparison and data on other Tylototriton species were of 25 other Tylototriton species used for comparisons were
taken from the following sources: Anderson (1871); Böhme obtained from GenBank (Table 1).
www.zoores.ac.cn ZoologicalResearch 40(3):151-174,2019 153
Table1 SequencesandvoucherspecimensofTylototritonandoutgrouptaxausedinthisstudy
Sample
Speciesname Vouchernumber Locality 16SrRNA ND2
No.
Ingroup:
1 Tylototritontaliangensis CIBGG200110183 ShimianCo.,Ya'anCity,Sichuan,China KY800559 KC147819
2 Tylototritontaliangensis CIBGG200110185 ShimianCo.,Ya'anCity,Sichuan,China KY800560 KY800829
3 Tylototritontaliangensis CIBGG200110186 ShimianCo.Ya'anCity,Sichuan,China KY800561 KY800830
NingnanCo.,LiangshanYiAutonomousPrefec-
4 Tylototritonpseudoverrucosus CIBWCG2012003 KY800597 KY800861
ture,Sichuan,China
NingnanCo.,LiangshanYiAutonomousPrefec-
5 Tylototritonpseudoverrucosus CIBWCG2012007 KY800598 KY800862
ture,Sichuan,China
NingnanCo.,LiangshanYiAutonomousPrefec-
6 Tylototritonpseudoverrucosus CIBWCG2012012 KY800599 KY800860
ture,Sichuan,China
7 Tylototritonkweichowensis CIBWG20080818014 BijieCity,Guizhou,China KY800551 KY800823
8 Tylototritonkweichowensis CIBWG20080818018 BijieCity,Guizhou,China KY800552 KY800824
9 Tylototritonkweichowensis CIB20050213 ShuichengCo.,Guizhou,China KY800557 KY800827
10 Tylototritonkweichowensis CIB20050215 ShuichengCo.,Guizhou,China KY800558 KY800828
11 Tylototritonshanorumlineage1 CAS230933 TaunggyiTownship,Shan,Myanmar — AB922822
12 Tylototritonshanorumlineage1 CAS230940 TaunggyiTownship,Shan,Myanmar — AB922823
13 Tylototritonshanorumlineage1 CAS230899 TaunggyiTownship,Shan,Myanmar — HM770087
14 Tylototritonshanorumlineage2 KUHE42348 pettrade,presumablyfromMyanmar — AB769544
15 Tylototritonngarsuensis LSUHC13762 NgarSu,Shan,Myanmar — MH836585
16 Tylototritonngarsuensis LSUHC13763 NgarSu,Shan,Myanmar — MH836584
17 Tylototritonsp.1 CAS245418 ChipwiTownship,KachinState,Myanmar — KT304279
18 Tylototritonsp.1 CAS245290 LaheTownship,SagaingRegion,Myanmar — KT304278
19 Tylototritonhimalayanus CIB201406246 MaiPokhari,Illam,Mechi,Nepal KY800590 KT765173
20 Tylototritonhimalayanus CIB201406284 MaiPokhari,Illam,Mechi,Nepal KY800591 KT765207
21 Tylototritonhimalayanus CIB201406285 MaiPokhari,Illam,Mechi,Nepal KY800592 KT765208
22 Tylototritonkachinorumsp.nov. ZMMUA5953 IngyinTaungMt.,Indawgyi,Kachin,Myanmar MK095618 MK097273
23 Tylototritonkachinorumsp.nov. ZDUM-0103 IngyinTaungMt.,Indawgyi,Kachin,Myanmar MK095619 MK097274
24 Tylototritonyangi KUHE42282 PingbianCo.,Yunnan,China KY800624 KY800887
25 Tylototritonuyenoi ZMMUNAP-08220 DoiInthanon,ChiangMai,Thailand MK095617 MK097272
26 Tylototritonuyenoi KUHE19037 DoiInthanon,ChiangMai,Thailand — AB830730
27 Tylototritonuyenoi KUHE19147 DoiAngKhang,ChiangMai,Thailand — AB830729
28 Tylototritonuyenoi ITNT1 DoiSuthep,ChiangMai,Thailand — AB830733
29 Tylototritonanguliceps TBUPAE671 ThuanChau,SonLa,Vietnam — LC017833
30 Tylototritonanguliceps VNMNA20143 MuongNhe,DienBien,Vietnam — LC017832
31 Tylototritonanguliceps LK003 DoiLahnga,ChiangMai,Thailand — AB830728
32 Tylototritonpodichthys KUHE34399 PhuPan,XamNeua,Laos — AB830727
33 Tylototritonpodichthys IEBRA2014-1 XamNeua,Huaphanh,Laos — LC017835
34 Tylototritonpulcherrimus CIBTY040 LüchunCo.,Yunnan,China KY800626 KY800890
35 Tylototritonpulcherrimus KUHE46406 PetTrade KY800620 KY800880
36 Tylototritonverrucosus CIBTSHS1 LongchuanCo.,Dehong,Yunnan,China KY800581 KY800847
37 Tylototritonverrucosus CIB-TSHS2 LongchuanCo.,Dehong,Yunnan,China KY800582 KY800848
38 Tylototritonverrucosus CIBTSHS3 LongchuanCo.,Dehong,Yunnan,China KY800583 KY800849
154 www.zoores.ac.cn
Continued
Sample
Speciesname Vouchernumber Locality 16SrRNA ND2
No.
39 Tylototritonverrucosus CIBTSHS4 LongchuanCo.,Dehong,Yunnan,China KY800584 KY800850
40 Tylototritonverrucosus CIBTSHS5 LongchuanCo.,Dehong,Yunnan,China KY800585 KY800851
41 Tylototritonverrucosus CIBTSHS6 LongchuanCo.,Dehong,Yunnan,China KY800586 KY800852
42 Tylototritonshanjing KIZ201306081 YongdeCo.,Yunnan,China KY800593 KY800856
43 Tylototritonshanjing KIZ201306098 YunCo.,Yunnan,China KY800594 KY800857
44 Tylototritonshanjing KIZ201306102 JingdongCo.,Yunnan,China KY800595 KY800858
45 Tylototritonshanjing KIZ201306108 NanjianCo.,Yunnan,China KY800596 KY800859
46 Tylototritonshanjing CIB980004 BaoshanCity,Yunnan,China KY800562 KY800831
47 Tylototritonshanjing CIB980005 BaoshanCity,Yunnan,China KY800563 KY800832
48 Tylototritonshanjing CIB980006 BaoshanCity,Yunnan,China KY800564 KY800833
49 Tylototritonpanhai ZMMUNAP-08217 PhuHinRongKlaNP,Phitsanulok,Thailand MK095616 MK097271
50 Tylototritonpanhai PH019 PhuHinRongKlaNP,Phitsanulok,Thailand — AB830735
51 Tylototritonpanhai PL009 PhuLuangWS,Loei,Thailand — AB830736
52 Tylototritonvietnamensis IEBRA.3674 YenTu,BacGiang,Vietnam KY800614 KY800874
53 Tylototritonvietnamensis IEBRA.0702 Mauson,LangSon,Vietnam KY800612 KY800872
54 Tylototritonvietnamensis IEBRA.0701 Mauson,LangSon,Vietnam KY800613 KY800873
55 Tylototritonziegleri VNMN3389 BaoLac,CaoBang,Vietnam — KY800888
56 Tylototritonziegleri VNMN3390 QuanBa,HaGiang,Vietnam KY800625 KY800889
57 Tylototritonziegleri VNUHHG.082 QuanBa,HaGiang,Vietnam KY800610 KY800870
58 Tylototritonziegleri VNUHHG.081 QuanBa,HaGiang,Vietnam KY800611 KY800871
59 Tylototritonhainanensis CIB20081048 Mt.Diaoluo,Hainan,China KY800553 KC147817
60 Tylototritonhainanensis CIB20081049 Mt.Diaoluo,Hainan,China KY800554 KC147818
61 Tylototritonhainanensis CIB20081051 Mt.Diaoluo,Hainan,China KY800555 KY800825
62 Tylototritonhainanensis CIB20081052 Mt.Diaoluo,Hainan,China KY800556 KY800826
63 Tylototritonasperrimuslineage2 CIBXZ20091201 XinyiCity,Guangdong,China KY800616 KY800876
64 Tylototritonasperrimuslineage2 CIBXZ20091204 XinyiCity,Guangdong,China KY800619 KY800879
65 Tylototritonasperrimuslineage2 CIBXZ20091 XinyiCity,Guangdong,China KY800618 KY800878
66 Tylototritonasperrimuslineage2 CIBXZ20092 XinyiCity,Guangdong,China KY800617 KY800877
67 Tylototritonnotialis FMNHHERP271120 BoualaphaDist.,Khammouan,Laos — HM462061
68 Tylototritonnotialis FMNHHERP271121 BoualaphaDist.,Khammouan,Laos — HM462062
69 Tylototritonnotialis VNMNTAO1229 PuHoat,NgheAn,Vietnam — KY800883
70 Tylototritonnotialis VNMNTAO1235 PuHoat,NgheAn,Vietnam — KY800884
71 Tylototritonasperrimuslineage1 VNMNTAO1213 ThuongTien,HoaBinh,Vietnam KY800623 KY800885
72 Tylototritonasperrimuslineage1 VNMNTAO1214 ThuongTien,HoaBinh,Vietnam — KY800886
73 Tylototritonasperrimuslineage1 CIB70063 LongshengCo.,Guangxi,China KY800549 KC147816
74 Tylototritonasperrimuslineage1 CIBGX20080714 JinxiuCo.,Guangxi,China KY800546 KY800819
75 Tylototritonasperrimuslineage1 CIBGX200807010 JinxiuCo.,Guangxi,China KY800547 KY800820
76 Tylototritonasperrimuslineage1 CIBGX200807012 JinxiuCo.,Guangxi,China KY800548 KY800821
77 Tylototritonasperrimuslineage1 CIBGX200807016 JinxiuCo.,Guangxi,China KY800550 KY800822
78 Tylototritonasperrimuslineage1 CIB20070715 JinxiuCo.,Guangxi,China KY800565 KY800834
79 Tylototritonasperrimuslineage1 CIB200807055 JinxiuCo.,Guangxi,China KY800566 KC147815
www.zoores.ac.cn ZoologicalResearch 40(3):151-174,2019 155
Continued
Sample
Speciesname Vouchernumber Locality 16SrRNA ND2
No.
80 Tylototritonliuyangensis CSUFT20100108 LiuyangCity,Hunan,China KY800606 KJ205598
81 Tylototritonliuyangensis CIB110601F06 LiuyangCity,Hunan,China KY800615 KY800875
82 Tylototritonlizhenchangi KUHE42316 YizhangCo.,Hunan,China KY800621 KY800881
83 Tylototritonlizhenchangi KUHE42317 YizhangCo.,Hunan,China KY800622 KY800882
84 Tylototritondabienicuslineage2 CIB08042905-2 YuexiCo.Anhui,China KY800587 KY800853
85 Tylototritondabienicuslineage2 CIB08042905-3 YuexiCo.Anhui,China KY800588 KY800854
86 Tylototritondabienicuslineage2 CIB08042905-4 YuexiCo.Anhui,China KY800589 KY800855
87 Tylototritonbroadoridgus CIB200085 SangzhiCo.,Hunan,China KY800569 KC147814
88 Tylototritonbroadoridgus CIB200084 SangzhiCo.,Hunan,China KY800570 KY800837
89 Tylototritondabienicuslineage1 HNNU1004-015 ShangchengCo.,Anhui,China KY800607 KC147811
90 Tylototritondabienicuslineage1 HNNU1004-024 ShangchengCo.,Anhui,China KY800608 KC147812
91 Tylototritondabienicuslineage1 HNNU1004-026 ShangchengCo.,Anhui,China KY800609 KY800869
Tylototritonwenxianensis
92 CIBWH10001 WufengCo.,Hubei,China KY800600 KY800863
lineage3
Tylototritonwenxianensis
93 CIBWH10002 WufengCo.,Hubei,China KY800601 KY800864
lineage3
Tylototritonwenxianensis
94 CIBWH10003 WufengCo.,Hubei,China KY800602 KY800865
lineage3
Tylototritonwenxianensis
95 CIBWH10007 WufengCo.,Hubei,China KY800603 KY800866
lineage3
Tylototritonwenxianensis
96 CIBWg20090730001 LiboCo.,Guizhou,China KY800575 KY800842
lineage2
Tylototritonwenxianensis
97 CIBWg20090730002 LiboCo.,Guizhou,China KY800576 KY800843
lineage2
Tylototritonwenxianensis
98 CIBWg20090730003 LiboCo.,Guizhou,China KY800577 KY800844
lineage2
Tylototritonwenxianensis
99 CIBWg20090730005 LiboCo.,Guizhou,China KY800578 KY800845
lineage2
Tylototritonwenxianensis
100 CIBWg20090730004 LiboCo.,Guizhou,China KY800580 KY800846
lineage2
Tylototritonwenxianensis
101 CIBWG200600019 SuiyangCo.,Zunyi,Guizhou,China KY800544 KY800817
lineage1
Tylototritonwenxianensis
102 CIBWG20060007 SuiyangCo.,Zunyi,Guizhou,China KY800545 KY800818
lineage1
Tylototritonwenxianensis
103 CIBWG20090601002 SuiyangCo.,Zunyi,Guizhou,China KY800573 KY800840
lineage1
Tylototritonwenxianensis
104 CIBWG20090601001 SuiyangCo.,Zunyi,Guizhou,China KY800574 KY800841
lineage1
Tylototritonwenxianensis
105 CIB20090527 WenxianCo.,Gansu,China KY800579 KC147813
lineage1
Tylototritonwenxianensis
106 CIB2010123101 PingwuCo.,Gansu,China KY800604 KY800867
lineage1
Tylototritonwenxianensis
107 CIB2010123102 PingwuCo.,Gansu,China KY800605 KY800868
lineage1
Tylototritonwenxianensis
108 CIB20070639 QingchuanCo.,Sichuan,China KY800542 KY800815
lineage1
156 www.zoores.ac.cn
Continued
Sample
Speciesname Vouchernumber Locality 16SrRNA ND2
No.
Tylototritonwenxianensis
109 CIB20070638 QingchuanCo.,Sichuan,China KY800543 KY800816
lineage1
Tylototritonwenxianensis
110 CIB20080002 YunyangCo.,Chongqing,China KY800540 KY800813
lineage1
Tylototritonwenxianensis
111 CIB20080003 YunyangCo.,Chongqing,China KY800541 KY800814
lineage1
Tylototritonwenxianensis
112 CIB20081201 YunyangCo.,Chongqing,China KY800567 KY800835
lineage1
Tylototritonwenxianensis
113 CIB20081202 YunyangCo.,Chongqing,China KY800568 KY800836
lineage1
Tylototritonwenxianensis
114 CIBWA20090601 WangcangCo.,Sichuan,China KY800571 KY800838
lineage1
Tylototritonwenxianensis
115 CIBWA20090602 WangcangCo.,Sichuan,China KY800572 KY800839
lineage1
Outgroup:
116 Echinotritonchinhaiensis CIBZHJY1 ZhenhaiCo.,Zhejiang,China KY800627 KY800891
117 Echinotritonchinhaiensis CIBZHJY2 ZhenhaiCo.,Zhejiang,China KY800628 KY800892
118 Echinotritonandersoni MVZ232187 Tokunoshima,Kagoshima,Japan EU880314 EU880314
119 Pleurodeleswaltl MVZ231894 Cadiz,Andalusia,Spain EU880330 EU880330
For sampling localities see Figure 1. Institutional abbreviations: CAS: California Academy of Sciences, Department of Herpetology, USA; CIB:
ChengduInstituteofBiology,ChineseAcademyofSciences,China;CSUFT:CentralSouthUniversityofForestryandTechnology,China;FMNH:
FieldMuseum,USA;HNNU:HenanNormalUniversity,China;IEBR:InstituteofEcologyandBiologicalResources,Vietnam;KIZ:KunmingInstitute
of Zoology, Chinese Academy of Sciences, China; KUHE: Graduate School of Human and Environmental Studies of Kyoto University, Japan;
LSUHC:LaSierraUniversity,HerpetologicalCollection,USA;MVZ:MuseumofVertebrateZoology,UniversityofCalifornia,USA;TBU:ThuanChau
University,Vietnam;VNMN:VietnamNationalMuseumofNature,Vietnam;VNUH:VietnamNationalUniversityHanoi,Vietnam;ZDUM:Zoological
Department, University of Mandalay, Myanmar; ZISP: Zoological Institute, RussianAcademy of Sciences, Russia; ZMMU: Zoological Museum of
LomonosovMoscowStateUniversity,Russia.Co.:County.—:Notavailable.
Phylogeneticanalyses etal.,2016).
SequencesofpartialfragmentsofND2and16SrRNAmtDNA The matrilineal genealogy was inferred using Bayesian
for119Salamandridaespecimens,including115representatives inference (BI) and maximum likelihood (ML) algorithms. BI
of Tylototriton (26 species) and four sequences of outgroup analyses were conducted in MrBayes v3.1.2 (Huelsenbeck &
members of Salamandridae (Echinotriton and Pleurodeles) Ronquist, 2001; Ronquist & Huelsenbeck, 2003). Metropolis-
wereincludedinthefinalalignmentwithatotallengthofupto coupled Markov chain Monte Carlo (MCMCMC) analyses
1 665 bp. Information on voucher specimens and GenBank were run with one cold chain and three heated chains for
accession Nos. used in phylogenetic analyses is summarized twenty million generations and sampled every 2000
in Table 1. Nucleotide sequences were initially aligned in generations. Five independent MCMCMC runs were
MAFFT v.6 (Katoh et al., 2002) with default parameters, and performed and 1 000 trees were discarded as burn-in. We
then checked by eye in BioEdit 7.0.5.2 (Hall, 1999) and checked the convergence of the runs and that the effective
slightlyadjusted. sample sizes (ESS) were all above 200 by exploring the
The dataset was divided into four partitions: three codon- likelihood plots using TRACER v1.6 (Rambaut et al., 2014).
partitions for the ND2 gene and a single partition for 16S Confidenceintreetopologywastestedbyposteriorprobability
rRNA,withtheoptimalevolutionarymodelsforeachestimated (PP) for the BI trees (Huelsenbeck & Ronquist, 2001). Nodes
using MODELTEST v. 3.06 (Posada & Crandall, 1998). withPPvaluesover0.95werea-prioriregardedassufficiently
According to theAkaike information criterion (AIC), theTVM+ resolved, those between 0.95 and 0.90 were regarded as
G model was the best fit for the 16S rRNA partition; for the tendencies, and values below 0.90 were considered to be not
ND2 gene, however, the HKY+G model was considered the supported.
best fit for the first and second codon partitions, whereas the We conducted ML analyses using the RAxML web server
J2+G model was selected as the best fit for the third codon (http://embnet.vital-it.ch/raxml-bb/; Stamatakis et al., 2008)
partition. Mean uncorrected genetic distances (P-distances) and searched ML trees using the gamma model of rate
between sequences were determined with MEGA7.0 (Kumar heterogeneityoption.Confidenceinnodetopologywastested
www.zoores.ac.cn ZoologicalResearch 40(3):151-174,2019 157
by non-parametric bootstrapping with 1 000 replicates (ML group is incomplete and further taxonomic and phylogenetic
BS, see Felsenstein, 1985). We a-priori regarded tree nodes researchisrequired.
with bootstrap (ML BS) values of 70% or greater and BI PP The newly discovered population of Tylototriton sp. nov.
values over 0.95 as sufficiently resolved; ML BS values from the Indawgyi Lake area belongs to clade 1 (Figure 2),
between 70% and 50% (BI PP between 0.95 and 0.90) were which occurs in western and northern Indochina, Himalaya,
treated as tendencies, and nodes with ML BS values below andYunnanProvinceofChina(Figure1),andisgroupedwith
50% (BI PP below 0.90) were regarded as unresolved T. himalayanus from Nepal, though not with significant node
(Felsenstein,2004;Huelsenbeck&Hillis,1993). support (0.85/57, hereafter given for BI PP/ML BS,
respectively). Tylototriton sp. nov. from the Indawgyi Lake
area and T. himalayanus form a well-supported monophyletic
RESULTS
group with Tylototriton species from the Shan Plateau in
Phylogeneticanalyses Myanmar (1.0/96), whereas populations of Tylototriton sp. 1
Sequences and statistics: The final alignment of the ND2 from the Sagaing and Kachin states in northern Myanmar,
gene contained 1157 aligned characters, including 712 reported by Grismer et al. (2018a), are clustered with other
conserved sites and 445 variable sites, of which 405 were members of Tylototriton clade 1 from Yunnan and northern
parsimony-informative.Thetransition-transversionbias(R)was Indochina and belong to the T. verrucosus species complex
estimated to be 4.56 (all data for ingroup only). Nucleotide (Figure2).Ouranalysessuggestsisterrelationshipsofclosely
frequencies were 37.5% (A), 23.7% (T), 28.3% (C), and related T. verrucosus and T. shanjing but only with support
10.5% (G). The final alignment of the 16S rRNA gene from BI (1.0/-), suggesting that the taxonomic status of T.
contained 508 aligned characters, including 424 conserved shanjing might need to be reconsidered. Finally, in our tree,
sites and 82 variable sites, of which 69 were parsimony- the recently described Myanmar species T. ngarsuensis is
informative.Thetransition-transversionbias(R)wasestimated nested within differentiation of T. shanorum, rendering the
to be 5.84 (all data for ingroup only). Nucleotide frequencies latterparaphyletic.
were36.8%(A),24.9%(T),20.3%(C),and18.0%(G).
Sequence divergence: The uncorrected P-distances among
Position of Tylototriton sp. in matrilineal genealogy: BI and within the studied mtDNA fragments for the examined
and ML phylogenetic analyses resulted in essentially similar Tylototriton species are shown in Table 2 (data for ingroup
topologies (Figure 2). In general, the topology of the mtDNA- only).TheinterspecificuncorrectedgeneticP-distancesbetween
basedmatrilinealgenealogywasconsistentwiththephylogeny the Tylototriton sp. from Kachin State of Myanmar and other
of Tylototriton presented by Wang et al. (2018), suggesting congeners varied from 5.3% (between Tylototriton sp. and its
that the genus is divided into five clades (1–5) grouped into sister species T. himalayanus) to 14.6% (between Tylototriton
twomajorreciprocallymonophyleticgroups(Figure2): sp. and T. lizhenchangi) for the ND2 gene; and from 2.4%
(1) Thefirstgroupjoinedtwocladeswithsisterrelationships: (betweenTylototritonsp.anditssisterspeciesT.himalayanus)
clade1(includingT.verrucosus(typespeciesofTylototritons. to 5.9% (between Tylototriton sp. and T. liuyangensis and T.
str. Anderson, 1871), T. anguliceps, T. himalayanus, T. dabienicus, lineage 1) for the 16S rRNAgene (Table 2). This
kweichowensis (type species of Qiantriton Fei, Ye & Jiang, degree of pairwise divergence is quite high, notably greater
2012), T. ngarsuensis, T. podichthys, T. pulcherrimus, T. than the genetic divergence observed between many
shanjing,T. shanorum,T. uyenoi,T. yangi,andTylototriton sp. recognizedspeciesofTylototriton(seeTable2andGrismeret
1 from the western part of the Kachin and Sagaing States of al.,2018a;Wangetal.,2018).
Myanmar and the newly discovered population of Tylototriton
sp. nov. from the Indawgyi Lake area in the southern part of Taxonomy
KachinState)andclade2(includingT.pseudoverrucosusand Our mtDNA genealogy analyses based on the ND2 and 16S
T. taliangensis, the latter being the type species of rRNAgenes indicated that the newly discovered population of
LiangshantritonFei,Ye&Jiang,2012). Tylototriton sp. nov. from the Indawgyi Lake area belongs to
(2)Thesecondgroupjoinedclade3(includingT.broadoridgus, clade 1 of the subgenus Tylototriton s. str. and is clustered
T. dabienicus, T. liuyangensis, T. lizhenchangi, and T. with two other species of the genus known from Myanmar, T.
wenxianensis), clade 4 (including T. panhai and T. ngarsuensis and T. shanorum, and with T. himalayanus from
vietnamensis), and clade 5 (including T. asperrimus (type Nepal (Figure 2). The lineage of Tylototriton sp. nov. from
species ofYaotriton Dubois & Raffaëlli, 2009),T. hainanensis, IndawgyiLakeisclearlydistinctandnotablydivergentfromall
T. notialis, and T. ziegleri); the topological relationships other congeners with the uncorrected genetic distance for
betweenthesethreecladesareessentiallyunresolved. interspecificcomparisonsexceedingP=5.3%intheND2gene
In accordance with the results of Wang et al. (2018) our andP=2.4%inthe16SrRNAgene.Theobserveddifferences
analysisindicateddeepphylogeneticstructuringandparaphyly in mtDNA sequences are congruent with evidence from
ofT.asperrimus(consistingoftwonon-monophyleticlineages), diagnostic morphological characters (see“Comparisons”).
T. wenxianensis (consisting of three lineages, not forming a Theseresultssupportourhypothesisthatthenewlydiscovered
monophyly), and T. dabienicus (consisting of two non- populationofTylototritonsp.nov.fromIndawgyiLakerepresents
monophyletic lineages), suggesting that taxonomy of this apreviouslyunknownspecies,whichwedescribeherein.
158 www.zoores.ac.cn
Figure 2 Bayesian inference consensus tree of genus Tylototriton derived from analysis of 1 157 bp ND2 and 508 bp 16S rRNAgene
fragments
Specimennumbers(1–115),correspondingvoucherspecimeninformation,andGenBankaccessionNos.aregiveninTable1.Colordenotesfive
major clades revealed within genusTylototriton (clades 1–5). Information on type species of subgeneric-level taxaTylototriton s. str., Qiantriton,
Liangshantriton,andYaotritonisprovided.NumberattreenodescorrespondstoBIPP/MLBSsupportvalues,respectively.PhotobyNikolayA.
Poyarkov.
www.zoores.ac.cn ZoologicalResearch 40(3):151-174,2019 159
of
al) 31 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – ––
n
ago 30 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –– 6.8
edi 29 3.4 – – 4.2 2.5 4.8 – – 3.3 3.6 3.5 4.9 3.8 3.2 3.6 4.0 3.4 2.7 3.0 – 3.4 1.7 1.6 1.3 1.1 1.9 1.3 1.1 –– 11.9 10.1
v
o 6 0
ab 28 3.4 – – 4.5 2.8 5.3 – – 3.8 4.0 3.9 5.2 4.0 3.6 3.2 4.0 3.4 2.6 3.0 – 3.7 1.3 1.8 1.2 0.8 1.7 1.2 –– 4.0 11. 10.
(
ment 27 3.7 – – 3.9 2.3 5.0 – – 3.5 3.7 3.6 5.0 3.6 3.5 3.9 4.2 3.7 2.9 3.0 – 3.4 2.0 2.0 0.9 0.9 1.0 –– 2.9 4.0 11.5 10.2
enefrag 2526 3.84.7 –– –– 4.34.5 2.83.3 4.65.6 –– –– 3.64.5 3.84.7 3.74.5 5.05.9 4.24.7 3.64.5 4.04.9 3.84.3 3.84.7 3.03.9 3.03.9 –– 3.44.3 1.52.4 1.62.5 0.81.3 0.9–– 3.4–– 2.22.5 2.83.6 4.14.6 11.312.5 10.010.5
g 7 2
NA 24 3.8 – – 4.3 2.8 5.1 – – 3.6 4.2 4.0 5.0 4.2 4.0 4.4 4.2 3.8 3.4 3.4 – 3.9 1.9 2.0 –– 3.4 3.9 3.2 3.3 4.4 11. 10.
R 6 4
Sr 23 4.0 – – 4.3 3.2 5.5 – – 3.6 3.8 3.7 5.0 4.2 3.2 3.8 4.2 4.0 2.8 2.8 – 3.0 2.4 –– 8.3 8.1 8.8 7.8 8.0 8.3 12. 11.
6
1 3 2
bp 22 4.2 – – 5.2 3.2 5.3 – – 3.8 4.2 4.0 5.9 4.7 3.8 3.8 4.5 4.2 3.1 3.5 – 3.9 –– 7.1 7.3 7.0 7.4 7.0 7.2 7.3 11. 10.
508 21 4.4 – – 4.7 3.6 5.1 – – 4.0 4.2 4.1 5.5 4.7 4.0 3.8 3.8 4.4 1.4 1.4 – –– 8.6 10.4 8.1 8.0 9.0 8.2 8.5 9.0 11.9 10.9
d 8 8
an 20 – – – – – – – – – – – – – – – – – – – –– 4.8 8.6 9.7 8.2 7.9 8.9 7.8 8.2 8.9 11. 10.
onal) 19 3.8 – – 4.5 3.2 4.8 – – 3.6 3.8 3.7 5.2 4.8 3.6 3.6 4.0 3.8 0.8 –– 5.9 5.7 8.5 9.6 8.8 8.8 9.6 8.5 9.0 9.5 10.5 10.1
g 6 1
dia 18 3.4 – – 4.5 2.8 4.8 – – 3.4 3.6 3.5 5.2 4.4 3.2 3.2 4.0 3.4 –– 3.6 4.7 4.9 7.8 8.7 7.9 7.5 8.9 7.5 8.2 8.9 10. 10.
w 0 9 8
elo 17 0.0 – – 3.3 2.0 4.4 – – 2.8 2.4 2.7 2.5 4.0 2.4 2.8 3.8 –– 4.4 5.6 4.9 4.5 8.5 10. 8.3 8.0 9.1 7.8 8.6 9.3 11. 10.
b 3 8 6 6 9 9 7 8 7 9 7 0 7 1 1
nt( 16 3.8 – – 4.1 3.8 4.8 – – 4.2 4.4 4.3 4.7 4.4 4.2 4.0 –– 11. 10. 11. 11. 10. 10. 11. 10. 10. 10. 10. 11. 10. 13. 12.
gme 15 2.8 – – 4.5 2.8 4.8 – – 2.6 2.6 2.5 4.3 4.8 1.2 –– 11.7 9.3 8.4 9.1 8.9 9.5 9.0 9.8 9.4 9.0 9.6 8.9 9.2 9.4 8.6 7.3
a
efr 14 2.4 – – 4.3 2.4 4.4 – – 2.2 2.0 2.3 4.1 5.1 –– 2.7 11.2 9.7 8.6 9.3 9.2 9.4 9.1 9.5 9.0 9.1 9.2 8.8 9.0 9.1 9.1 7.5
n
e 5 3 0 3 4 5 4 3 5 5 5
2g 13 4.0 – – 3.8 3.5 6.2 – – 4.8 5.3 5.2 5.2 –– 10. 10. 11. 10. 9.6 11. 10. 11. 9.5 9.8 9.7 9.5 9.7 9.2 10. 10. 12. 12.
D 3 1 8 9 0 4 1 3 6 9 2 6 8 7 9
pN 12 2.5 – –– 2.4 3.2 5.2 – – 3.9 3.9 3.7 –– 13. 7.3 7.1 13. 11. 11. 13. 12. 13. 12. 14. 10. 11. 11. 10. 11. 11. 8.0 6.6
b 0 5 4 3 7 0 5 7 7 5 2 1 4 2 3
57 11 2.7 –– – 3.5 1.8 3.2 – – 1.0 0.7 –– 7.9 13. 7.5 7.7 12. 11. 10. 10. 11. 11. 10. 11. 10. 10. 11. 10. 10. 10. 7.0 3.2
1 7 1 3 2 5 9 4 3 5 6 1 9 3 0 1
of1 10 2.4 – – 3.7 2.0 3.1 – – 1.6 –– 1.1 7.3 12. 7.4 7.4 12. 11. 10. 10. 10. 11. 10. 11. 10. 10. 10. 10. 10. 10. 6.7 2.5
8 0 6 0 2 9 9 5
ces 9 2.8 – – 3.7 1.6 3.1 – – –– 2.1 2.6 7.7 11. 7.3 6.6 12. 10. 9.5 10. 10. 10. 9.9 10. 9.9 9.7 10. 9.6 9.7 9.6 6.8 2.8
n 4 6 9 1 7 8 5 9 9 0 1 0
que 8 – – – – – – – –– 3.3 3.6 4.0 8.0 12. 7.9 7.4 12. 10. 9.9 10. 10. 10. 10. 11. 9.9 9.9 10. 10. 10. 10. 7.6 3.5
e 3 4 2 3 0 9 4 4 6 1 0 2 2 0 4
ntage)betweens neticanalyses 4567 3.32.04.4– –––– –––– 2.34.8––– 6.52.6––– 8.57.5––– 7.04.57.6–– 6.75.08.75.4 6.33.86.84.2 6.43.97.24.6 6.24.57.24.8 5.38.211.68.3 11.912.714.313. 7.67.99.78.8 7.17.49.78.6 12.712.215.013. 11.210.213.411. 9.99.612.610. 10.09.313.210. 10.610.312.710. 11.410.213.411. 10.19.512.710. 11.511.113.212. 10.910.112.711. 10.59.912.311. 11.211.013.012. 10.39.912.011. 10.79.712.511. 10.910.012.711. 6.26.99.37.5 6.74.27.74.7
perce yloge 3 0.0 – –– 4.9 6.4 9.1 6.8 6.9 6.1 6.0 6.4 7.1 12.1 8.0 7.7 12.2 11.1 9.8 10.0 10.7 11.2 10.3 11.8 10.8 10.2 11.1 10.2 10.4 11.1 1.5 6.4
( h 2 5 1 2 7 2 3 9 8 3 0 2 3 0
Table2UncorrectedP-distance Tylototritonspeciesincludedinp Species12 1T.kweichowensis––– 2T.shanorum15.9–– 3T.shanorum25.60.6 4T.himalayanus5.25.1 5T.yangi6.16.6 6T.uyenoi8.19.2 7T.anguliceps6.37.0 8T.podichthys5.87.2 9T.pulcherrimus5.36.4 10T.verrucosus5.66.2 11T.shanjing5.96.6 12T.kachinorumsp.nov.6.57.6 13T.panhai10.712. 14T.taliangensis6.38.2 15T.pseudoverrucosus5.77.9 16T.vietnamensis11.812. 17T.ziegleri9.411. 18T.hainanensis8.59.9 19T.asperrimus29.310. 20T.notialis9.710. 21T.asperrimus110.011. 22T.liuyangensis9.410. 23T.lizhenchangi10.711. 24T.dabienicus29.710. 25T.broadoridgus9.110. 26T.dabienicus110.211. 27T.wenxianensis39.110. 28T.wenxianensis29.510. 29T.wenxianensis19.911. 30T.ngarsuensis6.61.8 31Tylototritonsp.15.76.7
160 www.zoores.ac.cn