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