Description of a species of the genus Paramesotriton (Caudata, Salamandridae) from Guizhou, China, based on morphological and genomic evidence

Desc ip ion o a species o he genus Pa ameso i on (Cauda a, Salamand idae) om Guizhou, China, based on mo phological and genomic e idence Tao Luo1,2,3*, Jia-Jia Wang2*, Mei Liao2, Ming-Yuan Xiao4, Huai-Qing Deng4, Ning Xiao5, Jiang Zhou2 1 School o Li e Sciences, Yunnan Uni e si y, Kunming, China 2 School o Ka s Science, Guizhou No mal Uni e si y, Guiyang, China 3 Sou hwes Uni ed G adua e School, Kunming, China 4 School o Li e Sciences, Guizhou No mal Uni e si y, Guiyang, China 5 Guiyang Heal hca e Voca ional Uni e si y, Guiyang, China h ps://zoobank.o g/8D40ED2F-A2AB-463F-97D3-C97884C8BE1E Co esponding au ho : Jiang Zhou ([email p o ec ed]) * These au ho s con ibu ed equally o his pape . Academic edi o : Umilaela A i in ♦ Recei ed 11 Feb ua y 2025 ♦ Accep ed 4 Sep embe 2025 ♦ Published 1 Oc obe 2025 Abs ac Species iden i ica ion and delimi a ion a e c i ical o biodi e si y conse a ion. Recen mi ochond ial-based phylogene ic s udies o he genus Pa ameso i on, a g oup o small salamande s inhabi ing moun ain s eams in sou he n China and no he n Vie nam, sugges ha i s di e si y may be unde es ima ed. In his s udy, we con i m and cha ac e ize a new species, Pa ameso i on wumengshanensis sp. no ., using mo phological, mi ochond ial NADH dehyd ogenase subuni 2 (ND2), mi ogenome, and genomic e idence. This new species can be dis inguished om o he Pa ameso i on species by cha ac e is ics such as ough and la ge body wa s, la ge eyes, he absence o es igial gills and gill ilamen s in adul s, and dis inc i e ome ine ee h. S a is ical analysis o he mo phological da a u he showed ha he new species is signi ican ly di e en om i s close ela i es. Phylogene ic econs uc ion based on conca ena ed and coalescen app oaches, using bo h mi ochond ial DNA and nuclea single nucleo ide polymo phisms (SNPs) gene a ed by es ic ion si e-associa ed DNA sequencing, e ealed ha he new species o ms a dis inc lineage wi hin he genus Pa ameso i on, exhibi ing a minimum gene ic dis ance o 0.63% in mi ochond ial ND2 compa ed o congene s. The obse ed cy onuclea disco dance and mino mi ochond ial di e - ences among species may s em om his o ical gene low and/o incomple e lineage so ing, and u u e s udies using genomic e idence a e needed o in es iga e hei espec i e con ibu ions. Based on his disco e y, we ecommend ha independen e idence, especially ge- nomic e idence, be in eg a ed in o he classi ica ion o closely ela ed species o a oid alling in o he ap o mi ochond ial in og ession. Key Wo ds Guizhou, mo phology, mi ochond ial in og ession, phylogenomics In oduc ion The Asian new s o he genus Pa ameso i on Chang, 1935 (Cauda a, Salamand idae) a e among he mos spe- cies-di e se cauda e amphibians in China, second only o he genus Tylo o i on (AmphibiaChina 2025). Cu en ly, 15 species ha e been eco ded, which a e di ided in o wo species g oups: he P. caudopunc a us g oup, which in- cludes P. caudopunc a us (Liu & Hu, 1973), P. longliensis Li, Tian, Gu & Xiong, 2008, P. malipoensis Rao, Liu, Zhu Zoosys . E ol. 101 (4) 2025, 1933–1947|DOI 10.3897/zse.101.149144 Copy igh Luo, T. e al. This is an open access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (CC BY 4.0), which pe mi s un es ic ed use, dis i- bu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. zse.penso .ne Luo, T. e al.: Genus Pa ameso i on1934 & Ma, 2022, P. maolanensis Gu, Chen, Tian, Li & Ran, 2012, P. wulingensis Wang, Tian & Gu, 2013, and P. zhi- jinensis Li, Tian & Gu, 2008, and he P. chinensis g oup, which includes P. au an ius Yuan, Wu, Zhou & Che, 2016, P. chinensis (G ay, 1859), P. delous ali (Bou e , 1934), P. uzhongensis Wen, 1989, P. guangxiensis (Huang, Tang & Tang, 1983), P. hongkongensis (Mye s & Le i- on, 1962), P. labia us (Un e s ein, 1930), P. qixilingen- sis Yan, Zhao, Jiang, Hou, He, Mu phy & Che, 2014, and P. yunwuensis Wu, Jiang & Hanken, 2010 (Fei e al. 2006; Fei and Ye 2016; Rao 2022). These species a e p ima ily dis ibu ed in sou he n China and no he n Vie nam (Fei e al. 2006; Fei and Ye 2016; AmphibiaChina 2025). The mi ochond ial gene-based phylogeny iden i ied wo o h ee c yp ic species wi hin he P. caudopunc a us g oup (Luo e al. 2021; Luo e al. 2022). Luo e al. (2021) iden i ied a Pa ameso i on popula ion in Da ang Coun y, Guizhou P o ince, China, as a po en ial c yp ic species, ini ially e e ed o as Pa ameso i on sp2 (DF) and la e as P. longliensis (DF) (Luo e al. 2022). Howe e , his spe- cies was no o mally desc ibed in ei he s udy. This c yp- ic species exhibi s minimal mi ochond ial a ia ion (less han 2%) compa ed o cu en ly ecognized species, ais- ing ques ions ega ding i s alidi y. This ela i ely small mi ochond ial a ia ion may be a ibu ed o his o ical mi- ochond ial in og ession. Fo ins ance, mi ogenome in o- g ession be ween Odo ana g ahami and O. junlianensis in sou he n China led o minimal di e ences in mi ochon- d ial cy och ome oxidase subuni I bu signi ican nuclea gene di e gence (Yuan e al. 2022). This highligh s he impo ance o geno yping-by-sequencing o ob ain hou- sands o nuclea single nucleo ide polymo phisms (SNPs) o accu a e species delimi a ion. Simila ly, whole-ge- nome esequencing has been employed o gene a e la ge SNP da ase s, esol ing he axonomy o closely ela ed species and iden i ying c yp ic di e si y (Hu e al. 2020; Yang e al. 2022; Gu e al. 2023). Thus, genome-le el SNP da a p o ided c i ical insigh s o esol ing axonomic unce ain ies among closely ela ed species, pa icula ly hose a ec ed by mi ochond ial in og ession, and o e ed obus suppo o species delimi a ion. Al hough ou p e ious wo k using mi ochond ial ND2 and combining phylogene ic and species delimi a- ion me hods iden i ied he c yp ic species Pa ameso i- on sp2 (DF) o P. longliensis (DF) (Luo e al. 2021; Luo e al. 2022), he smalle mi ochond ial di e ences wi h closely ela ed species make hei alidi y no s ongly suppo ed. An addi ional p oblem is ha while whole-ge- nome esequencing can yield a la ge numbe o SNPs, he P. caudopunc a us g oup cu en ly has no close e e ence genome ha can be used. The e o e, we pe o med e- s ic ion si e-associa ed DNA sequencing (RADseq), a e- duced- ep esen a ion genome sequencing echnique (Mill- e e al. 2007; And ews e al. 2016) ha is e sa ile and does no equi e a e e ence genome, on 46 samples, he e- by ob aining a la ge amoun o SNP da a. In his s udy, ou aim is o con i m he alidi y o his c yp ic species and o mally desc ibe i in combina ion wi h mo phology. Ma e ials and me hods Sampling and mo phological analyses Among he 46 samples collec ed, nine we e om P. cau- dopunc a us, 10 om P. longliensis, wo om P. maolan- ensis, 10 om P. zhijinensis, eigh om Pa ameso i on sp2 (DF), and se en om he ou g oup (Fig. 1, Suppl. ma e ial 1). Tissue samples and specimens o he e- maining species we e collec ed be o e 2018, excep o Pa ameso i on sp2 (DF), which was collec ed in July 2018. Muscle samples we e collec ed om he ail ip and immedia ely p ese ed in 95% anhyd ous e hanol, and specimens we e ixed using 75% alcohol imme sion and s o ed indoo s in a e ige a o a −80 °C. The ield su - ey ollowed he ules o he Wildli e P o ec ion Law o he People’s Republic o China. All muscle samples and specimens we e p ese ed in he Animal Ecology Lab- o a o y, Guizhou No mal Uni e si y, Guiyang, Guizhou P o ince, China. All animal expe imen s we e app o ed by he Resea ch E hics Commi ee o Guizhou No mal Uni e si y (app o al numbe : 202504001). In his s udy, we measu ed 15 mo phological cha - ac e s o 29 specimens using digi al calipe s accu a e o 0.1 mm (Gu e al. 2012; Suppl. ma e ial 1), including new species ( ou males and h ee emales), P. longliensis (eigh males and nine emales), and P. zhijinensis ( i e e- males). The measu ed cha ac e s we e as ollows: TOL = o al leng h ( om ip o snou o ip o ail); SVL = snou – en leng h ( om ip o snou o pos e io edge o en ); HL = head leng h ( om pos e io edge o le pa o oid o snou ip); HW = head wid h (maximum head wid h); SL = snou leng h ( om ip o snou o he an e io co ne o eye); TKL = unk leng h (dis ance be ween axillae along igh body side); IOD = in e o bi al dis ance (minimum dis ance be ween he eyes); ED = eye diame e ( om he an e io co ne o he pos e io co ne o he eye); TAL = ail leng h ( om an e io ip o cloaca o ip o ail); TH = ail heigh (maximum ail heigh ); TW = ail wid h (max- imum ail wid h); FLL = o elimb leng h ( om he base o he o elimb o he ip o he longes inge ); HLL = hindlimb leng h ( om he base o he hindlimb o he ip o he longes oe); DAG = dis ance be ween axilla and g oin ( om he pos e io ma gin o he base o he o e- limb o he an e io ma gin o he base o he hindlimb); and SW = snou wid h (maximum snou wid h). P incipal componen analysis (PCA) wi h eigen alues g ea e han 1, he maximum a iance me hod, and simple bi a ia e sca e plo s we e used o explo e and cha ac e ize he mo phome ic di e ences be ween he new species and closely ela ed species. A canonical disc iminan analy- sis (CDA) was employed o dis inguish be ween he new species and closely ela ed species, as well as o p edic g oup membe ship, ollowing me hods used in p e ious s udies (Pa sons and Jones 2000; Polaszek e al. 2004; Xiong e al. 2015; Shen e al. 2020). To educe he e ec o allome y, each mo phome ic measu emen was co - ec ed by di iding i by he TOL be o e conduc ing he Zoosys . E ol. 101 (4) 2025, 1933–1947 zse.penso .ne 1935 PCA and CDA analyses. One-way analysis o a iance (ANOVA) was conduc ed o de e mine he signi icance o di e ences in mo phome ic cha ac e s be ween he new species and he a o emen ioned closely ela ed spe- cies. All s a is ical analyses we e pe o med using SPSS .21.0 (SPSS, Inc., Chicago, IL, USA), wi h di e ences conside ed s a is ically signi ican a p < 0.05. Mo pho- logical compa isons o he new species wi h congene s we e based on specimen examina ion and li e a u e. Sex iden i ica ion was pe o med based on Fei e al. (2006) and Fei and Ye (2016)—speci ically, males pos- sess a la ge and low cloaca, wi h a ela i ely long longi- udinal anal issu e and inge -like papillae on he cloacal wall. In con as , emales ha e a small and high cloaca, a sho o al-shaped anal issu e, and lack inge -like papillae on he cloacal wall. Labo a o y p o ocols, sequencing, and bioin o ma ic me hods The genomic DNA o 46 samples was ex ac ed om 95% e hanol-p ese ed issue using a s anda d CTAB p o ocol (Hanania e al. 2004). Genomic DNA om each sample was diges ed using he es ic ion enzyme EcoRI (GAATTC) ollowing p e iously published p ocedu es. DNA lib a ies o each sample we e cons uc ed acco d- ing o he RAD p o ocol (Bai d e al. 2008). PCR p oduc s we e pu i ied o eco e 350–550 bp DNA, and pai ed- end sequencing was conduc ed on he MGISEQ-2000. Raw eads we e deposi ed in he NCBI Sequence Read A chi e da abase (h ps://www.ncbi.nlm.nih.go /s a) un- de he BioP ojec accession numbe PRJNA1016311. Raw eads o samples we e il e ed using as p .0.23.4 (Chen 2023) wi h de aul pa ame e s. Nex , we pe o med u he quali y con ol on he cleaned eads using STACKS .2.61 (Roche e e al. 2019). The p o- cess_ ad ags module was used o clean he ags by dis- ca ding low-quali y eads, emo ing eads wi h uncalled bases, and escuing ba codes and ad ags. Addi ionally, we used he clone_ il e module in STACKS o iden i- y and emo e PCR duplica es om he RADseq da a. Single nucleo ide polymo phism (SNP) calling o each sample was pe o med using he deno o_map.pl sc ip in STACKS. We used he us acks, cs acks, ss acks, s 2bam, and popula ions modules om he STACKS pipeline o comple e he de no o assembly o cleaned eads, gene a e consensus sequences, and pe o m SNP calling o each sample. To de e mine he op imal assembly pa ame e s, we ollowed p e ious me hods o e alua e wo key pa- ame e s (Pa is e al. 2017; Gundappa e al. 2022): (−M), he maximum numbe o nucleo ide misma ches allowed be ween s acks, and (−m), he minimum dep h o co e - age equi ed o c ea e a s ack. Fo ou da ase , M anged om 3 o 8 (in in e als o 1), while m anged om 4 o 14 ( alues: 4, 6, 8, 9, 10, 12, 14). Fo he pa ame e n Figu e 1. Dis ibu ion and sampling o he P. caudopunc a us g oup and ou g oup Pachy i on inexpec a us in Guizhou, China. zse.penso .ne Luo, T. e al.: Genus Pa ameso i on1936 ( he numbe o misma ches allowed be ween sample loci when building he ca alog) in he cs acks module, we ol- lowed he ecommenda ion o Pa is e al. (2017) and se n = M. Based on he 80 ule (Pa is e al. 2017), he inal op imal pa ame e combina ion was M = 6, m = 10, and n = 6. In o al, he STACKS p og am iden i ied 15,581,665 biallelic SNPs ac oss 46 indi iduals. In his s udy, we geno yped and il e ed he samples o ob ain eliable SNPs using he ollowing p ocess: (1) ini ial geno ype il e ing was pe o med based on geno ype miss- ing a e and minimum allele equency using VCF ools .0.1.16 (Danecek e al. 2011) wi h he ollowing unning pa ame e s: ---ma 0.01 ---max-missing 0.5 ---min-alleles 2 -- ecode -- ecode-INFO-all –ou . (2) PLINK .1.90 (Pu - cell e al. 2007) was hen used o il e ou samples wi h a missing geno ype a e g ea e han 50%. (3) Missing gen- o ypes we e impu ed using Beagle (B owning and B own- ing 2016). (4) Finally, he SNPs gene a ed in he p e i- ous s ep we e u he il e ed using he R package bigsnp .1.9.11 (P i é e al. 2018) based on linkage disequilib i- um-based SNP clumping. In his s udy, only one SNP pe locus was e ained in he inal SNP da ase . Phylogene ic econs uc ion and gene ic di e en ia ion based on SNP da a We used IQ-TREE .1.6.12 (Nguyen e al. 2015) o econ- s uc phylogene ic ees based on maximum likelihood o conca ena ed sequences om all loci o in e phylog- enomic ela ionships. The analysis was pe o med wi h he ollowing pa ame e s: −s −keep-iden −s DNA −n −m GTR+ASC −bb 1000 −p e. Node suppo alues we e assessed using he ul a as boo s ap app oxima ion (UF- Boo ) me hod (Hoang e al. 2018). Species ee in e ence was pe o med using SVDqua e s (Chi man and Kuba - ko 2014) implemen ed in PAUP* 4.0a (Wilgenbusch and Swo o d 2003). SVDqua e s es ima es ela ionships be- ween axa unde he coalescen model by in e ing spli s among qua e s o andomly sampled axa. All possible axon qua e s we e e alua ed, and node suppo was es- ima ed using 100 s anda d boo s ap eplica es. In cases whe e node suppo was low, he SVDqua e s analysis helped iden i y po en ial incomple e lineage so ing o in- og ession among species. Bo h analyses used 1,475,246 unlinked SNPs. The ixa ion index (Fs ) be ween he new species and i s closely ela ed species was assessed using VCF ools .0.1.16 (Danecek e al. 2011) o quan i y he deg ee o gene ic a ia ion among di e en species. Phylogene ic econs uc ion, gene ic dis ance, and haplo ype ne wo k based on mi ochond ial sequences The 48 mi ochond ial ND2 sequences and 19 mi ochon- d ial genomes used o phylogene ic econs uc ion we e p ima ily sou ced om ou p e iously published da a (Table 1) (Luo e al. 2021; Luo e al. 2022). Mul iple se- quence alignmen was pe o med using MUSCLE (Edga 2004) wi hin MEGA .7.0 (Kuma e al. 2016), wi h de- aul se ings. Based on he Bayesian in o ma ion c i e- ion, we e alua ed he bes - i pa i ioning schemes and co esponding nucleo ide subs i u ion models o ND2 and mi ochond ial genomes using Pa i ionFinde .2.1.1 (Lan ea e al. 2017). Phylogene ic ees we e cons uc ed using bo h maximum likelihood (ML) and Bayesian in- e ence (BI) me hods. ML analysis was un in IQ-TREE .1.6.1 (Nguyen e al. 2015) wi h he bes model and 20,000 ul a as boo s ap (UBP) eplica es. The BI ee was econs uc ed using M Bayes .3.2.1 (Ronquis e al. 2012). Two independen uns we e conduc ed in he BI analysis, each o which was pe o med o 5 million gen- e a ions and sampled e e y 1000 gene a ions. The i s 25% o he samples we e disca ded as a bu n-in. Nodes we e conside ed well suppo ed when he Bayesian pos- e io p obabili y (BPP) was g ea e han 0.95 and he ML ul a as boo s ap alue (UBP) was g ea e han 95%. Unco ec ed p-dis ances (1000 eplica es) based on he ND2 gene we e calcula ed using MEGA .7.0 (Kuma e al. 2016). Addi ionally, we econs uc ed he mi ochon- d ial ND2 gene haplo ype ne wo k using PopART .1.7 (Leigh e al. 2015) based on he median-joining me hod (Bandel e al. 1999). Da a a ailabili y s a emen The da ase s con aining he accession numbe s o he P. caudopunc a us g oup gene a ed in his s udy a e a ailable as supplemen al in o ma ion. O iginal sequence eads o genome da a a e a ailable a he Na ional Cen- e o Bio echnology In o ma ion (accession numbe s SAMN37379588–SAMN37379633). Resul s Mo phological di e ences based on measu ed da a We measu ed 15 mo phological cha ac e s in 29 speci- mens (Suppl. ma e ial 1) and pe o med PCA, CDA, and ANOVA analyses o assess di e ences be ween P. wu- mengshanensis sp. no . and closely ela ed species. Fo males and emales, h ee and h ee p incipal componen s we e ex ac ed, espec i ely. The i s wo componen s accoun ed o 72.87% and 73.84% o he o al a ia- ion (Suppl. ma e ial 2). In he PC1 e sus PC2 sca e - plo , P. wumengshanensis sp. no ., P. longliensis, and P. zhijinensis o med nea ly dis inc clus e s, wi h sligh sepa a ion along he PC1 axis (Fig. 2A, B). Fo males, ai s ha loaded on he PC1 axis included o al leng h, snou – en leng h, head leng h, head wid h, eye diam- e e , ail wid h, o elimb leng h, and dis ance be ween axilla and g oin (Suppl. ma e ial 2). Fo emales, ai s Zoosys . E ol. 101 (4) 2025, 1933–1947 zse.penso .ne 1937 ha loaded on he PC1 axis included o al leng h, snou – en leng h, snou leng h, unk leng h, in e o bi al dis- ance, ail leng h, o elimb leng h, hindlimb leng h, and dis ance be ween axilla and g oin (Suppl. ma e ial 2). The CDA based on he 15 mo phological ai s clea ly classi ied all indi iduals in o speci ic axa, wi h CAN1 and CAN2 explaining 87.2% and 12.8% o he o al a i- ance, espec i ely. Dis inc clus e s we e o med along he CAN1 axis (Fig. 2C; Suppl. ma e ial 2). Uni a ia e ANOVA e ealed signi ican mo phological di e en ia ion be ween he new species and i s congene s (Table 2). Among males, P. wumengshanensis sp. no . and P. longliensis showed s a is ically signi ican di e - ences (p < 0.05) in snou leng h, ail wid h, and snou wid h (Table 2). In con as , P. wumengshanensis sp. no . and P. zhijinensis exhibi ed signi ican mo phological di- e gence ac oss nine measu ed ai s: o al leng h, unk leng h, in e o bi al dis ance, ail leng h, ail heigh , ail wid h, o elimb leng h, hindlimb leng h, and snou wid h (p < 0.05). Among emales, P. wumengshanensis sp. no . and P. longliensis showed signi ican di e en ia ion in eigh mo phological cha ac e s (Table 2). Table 1. Locali ies, ouche in o ma ion, and GenBank numbe s o all samples used. Fo he ouche numbe and locali y o he mi ogenome, see Luo e al. (2022b). ID Species Vouche numbe Locali y (* ype locali ies) ND2 Mi ogenome 1P. longliensis GZNU 20070421001 Longli, Guizhou, China* FJ169608 ON357921 2P. longliensis KIZ-GZH 081025 Longli, Guizhou, China* GU980576 3P. longliensis GZNU 20070421004 Longli, Guizhou, China* JF438975 4P. longliensis GZNU 20070421003 Longli, Guizhou, China* JF438974 5P. longliensis GZNU 20070421002 Longli, Guizhou, China* JF438973 6P. longliensis GZNU 2018061813 Huishui, Guizhou, China MW147285 ON357922 7P. longliensis GZNU 2018061814 Huishui, Guizhou, China MW147283 MW524141 8P. longliensis GZNU 20180618016 Huishui, Guizhou, China MW147286 9P. longliensis GZNU 20180618017 Huishui, Guizhou, China MW147284 10 P. maolanensis GZNU 2006030003 Libo, Guizhou, China* FJ169607 ON357924 11 P. maolanensis GZNU 2006030006 Libo, Guizhou, China* JF438972 12 P. maolanensis GZNU 2006030004 Libo, Guizhou, China* JF438993 13 P. maolanensis GZNU 2006030005 Libo, Guizhou, China* JF438994 14 P. wumengshanensis sp. no . GZNU 20180709024 Da ang, Guizhou, China* MT811029 MW524142 15 P. wumengshanensis sp. no . GZNU 2018070904 Da ang, Guizhou, China* MT811030 ON357926 16 P. wumengshanensis sp. no . GZNU 2018070905 Da ang, Guizhou, China* MT811031 17 P. wumengshanensis sp. no . GZNU 2018070903 Da ang, Guizhou, China* MT811028 18 P. zhijinensis GZNU 20070415002 Zhijin, Guizhou, China* JF438976 ON357925 19 P. zhijinensis GZNU 20070415003 Zhijin, Guizhou, China* JF438977 20 P. zhijinensis GZNU 20070415004 Zhijin, Guizhou, China* JF438978 21 P. zhijinensis GZNU 20070415001 Zhijin, Guizhou, China* FJ169609 22 P. zhijinensis KIZ-GZH 081026 Zhijin, Guizhou, China* GU980575 23 P. wulingensis GZNU 07072001 Jiangkou, Guizhou, China FJ938040 ON357928 24 P. wulingensis GZNU 2007071002 Jiangkou, Guizhou, China JF438987 25 P. wulingensis GZNU 2007071004 Jiangkou, Guizhou, China JF438989 26 P. wulingensis GZNU 08072603 Youyang, Chongqing, China* JF438991 27 P. wulingensis GZNU 08072602 Youyang, Chongqing, China* JF438990 28 P. wulingensis GZNU 2007071003 Youyang, Chongqing, China* JF438988 29 P. wulingensis GZNU 08072604 Youyang, Chongqing, China* JF438992 30 P. wulingensis KIZ 21898 Fanjingshan, Guizhou, China KJ650055 31 P. wulingensis KIZ 21899 Fanjingshan, Guizhou, China KJ650056 32 P. caudopunc a us GZNU 2007071001 Leishan, Guizhou, China* FJ169606 ON357930 33 P. caudopunc a us GZNU 200904251 Leishan, Guizhou, China* MW147288 34 P. caudopunc a us GZNU 200904252 Leishan, Guizhou, China* MW147287 35 P. caudopunc a us GZNU 2007072005 Leishan, Guizhou, China* JF438986 36 P. caudopunc a us GZNU 2009042501 Leishan, Guizhou, China* JF438983 37 P. caudopunc a us GZNU 20050727001 Leishan, Guizhou, China* JF438985 38 P. caudopunc a us GZNU 20050727002 Leishan, Guizhou, China* JF438984 39 P. delous ali Tissue ID: GZNU35 Hekou Coun y, Yunnan, China ON357931 ON357931 40 P. guangxiensis Tissue ID: GZNU36 Shiwandashan, Guangxi, China* ON357932 ON357932 41 P. yunwuensis GZNU20201018003 Xinyi Ci y, Guangdong, China* ON357933 ON357933 42 P. labia us GZNU20210531001 Jinxiu Coun y, Guangxi, China* ON357934 ON357934 43 P. uzhongensis GZNU20070520002 Zhongshan, Guangxi, China* ON357935 ON357935 44 P. hongkongensis Tissue ID: GZNU37 Hong Kong, China* ON357937 ON357937 45 P. qixilingensis Tissue ID: GZNU38 Yongxin Coun y, Jiangxi, China* ON357938 ON357938 46 P. au an ius GZNU20201018001 Jingning Coun y, Zhejiang, China* ON357939 ON357939 47 P. chinensis Tissue ID: GZNU39 Ningbo Ci y, Zhejiang, China* ON357940 ON357940 48 Pachy i on inexpec a us GZNU20180706001 Danzhai Coun y, Guizhou, China ON422325 ON422325 zse.penso .ne Luo, T. e al.: Genus Pa ameso i on1938 Gene ic da a in o ma ion In his s udy, he ull leng h o he mi ochond ial ND2 used o phylogene ic econs uc ion was 1407 base pai s (bp), o which 409 we e a iable and 277 we e pa simony-in o - ma i e. The model selec ion analysis sugges ed a di ision in o h ee pa i ions, i.e., he i s codon, he second codon, and he hi d codon o ND2, co esponding o he mod- els HKY+G, HKY+I, and HKY+I. Fo he mi ochond ial genome o 15,467 bp, he ollowing e olu iona y mod- els we e iden i ied as op imal o he co esponding gene segmen s: TVM+I+G o 12S RNA and 16S RNA, HKY+I+G o RNAs, GTR+G o p o ein-coding genes (COX2, COX1, COX3, Cy b, ATP8, ND2, ND3, ND4, ATP6, ND1, ND5, and ND4L), and HKY+G o ND6. A o al o 46 samples gene a ed app oxima e- ly 2948.5 Gb o aw da a and 1004 million pai ed-end eads, wi h an a e age o 64.09 Gb o da a and 21.8 mil- lion pai ed-end eads pe sample. A o al o 15,581,665 biallelic SNPs we e gene a ed om he STACKS-cleaned eads. A e il e ing o low-quali y indi iduals using PLINK, 1,475,250 SNPs emained. Table 2. Mo phome ic s a is ics and esul s o analysis o a iance om P. wumengshanensis sp. no . (PW), P. longliensis (PL), and P. zhijinensis (PZ). All uni s a e in mm. P- alues a e a 95% signi icance. Measu emen s P. wumengshanensis sp. no . P. longliensis P. zhijinensis P- alue om ANOVA Male (n = 4) Female (n = 3) Male (n = 8) Female (n = 9) Male (n = 5) Male Female Range (Mean ± SD) Range (Mean ± SD) Range (Mean ± SD) Range (Mean ± SD) Range (Mean ± SD) PW s. PL PW s. PZ PW s. PL TOL 95.5–123.6 (110.2 ± 13.6) 150.1–160.0 (155.8 ± 5.1) 97.2–143.6 (117.0 ± 15.5) 114.6–136.3 (126.2 ± 8.4) 128.1–142.4 (136.7 ± 5.4) 0.610 0.014 0.013 SVL 53.0–72.6 (63.4 ± 8.9) 82.3–87.8 (85.6 ± 2.9) 47.7–78.3 (60.7 ± 9.9) 65.4–91.8 (71.5 ± 8.3) 63.7–68.3 (66.3 ± 1.8) 0.610 1.000 0.052 HL 16.5–23.9 (20.8 ± 3.3) 23.5–25.2 (24.6 ± 0.9) 16.3–22.1 (19.4 ± 1.8) 18.5–43.1 (22.7 ± 7.7) 20.9–23.9 (22.3 ± 1.1) 0.308 0.806 0.052 HW 11.8–18.9 (16.0 ± 3.3) 18.3–19.2 (18.7 ± 0.5) 12.5–16.6 (13.9 ± 1.5) 13.2–37.5 (17.8 ± 7.6) 15.1–17.9 (17.1 ± 1.1) 0.308 0.902 0.052 SL 6.3–8.6 (7.1 ± 1.1) 7.1–8.3 (7.9 ± 0.7) 5.7–7.6 (6.2 ± 0.6) 5.8–7.3 (6.5 ± 0.5) 7.0–8.5 (7.6 ± 0.6) 0.042 0.327 0.033 TKL 37.5–52.5 (45.6 ± 6.5) 61.0–66.5 (63.6 ± 2.7) 36.2–57.9 (46.7 ± 6.7) 46.9–59.8 (51.6 ± 4.3) 52.2–56.8 (54.6 ± 1.7) 0.865 0.027 0.013 IOD 4.5–6.8 (5.8 ± 1.1) 6.6–7.7 (7.3 ± 0.6) 4.5–7.7 (6.5 ± 0.9) 3.6–5.3 (4.4 ± 0.6) 8.3–9.5 (8.7 ± 0.6) 0.308 0.014 0.013 ED 2.9–3.9 (3.4 ± 0.5) 3.6–4.8 (4.1 ± 0.6) 2.3–3.8 (2.9 ± 0.5) 2.9–4.5 (3.6 ± 0.5) 2.7–3.6 (2.9 ± 0.4) 0.126 0.050 0.116 TL 40.8–55.1 (48.6 ± 6.1) 66.6–74.4 (70.0 ± 4.0) 45.8–63.8 (55.8 ± 6.1) 48.3–60.2 (54.7 ± 4.4) 63.8–71.9 (69.0 ± 3.5) 0.089 0.014 0.012 TH 6.3–9.9 (7.6 ± 1.6) 6.6–11.2 (9.1 ± 2.3) 6.7–10.0 (8.1 ± 1.0) 7.6–11.2 (9.0 ± 1.4) 8.4–20.0 (12.5 ± 4.5) 0.234 0.027 0.926 TW 2.4–3.3 (2.9 ± 0.4) 3.5–5.8 (4.7 ± 1.1) 3.4–5.1 (4.3 ± 0.5) 3.5–4.4 (3.9 ± 0.3) 4.5–7.3 (5.8 ± 1.1) 0.007 0.014 0.309 FLL 13.9–24.5 (18.4 ± 5.1) 26.0–28.2 (27.2 ± 1.1) 17.5–23.0 (20.4 ± 1.8) 19.4–23.6 (20.8 ± 1.3) 22.7–31.5 (27.5 ± 3.2) 0.497 0.027 0.013 HLL 14.8–26.2 (21.1 ± 4.8) 26.2–31.0 (28.0 ± 2.6) 17.5–27.1 (21.1 ± 2.9) 21.5–24.9 (23.2 ± 1.3) 24.2–30.9 (27.4 ± 2.4) 0.734 0.027 0.013 DAG 23.1–35.4 (29.9 ± 5.2) 41.3–41.9 (41.6 ± 0.3) 23.2–38.2 (28.7 ± 4.9) 23.0–40.5 (31.6 ± 5.7) 30.1–35.2 (32.2 ± 2.1) 0.865 0.462 0.013 SW 4.4–4.7 (4.6 ± 0.2) 4.1–5.8 (4.9 ± 0.9) 4.4–7.3 (5.5 ± 0.8) 4.1–5.8 (4.8 ± 0.7) 5.5–6.1 (5.8 ± 0.2) 0.027 0.014 0.926 Figu e 2. Plo s o p incipal componen analysis (A, B) and canonical disc iminan analysis (C) sco es o P. wumengshanensis sp. no ., P. longliensis, and P. zhijinensis based on mo phological cha ac e s. A. Male; B. Female; C. Male and emale. Zoosys . E ol. 101 (4) 2025, 1933–1947 zse.penso .ne 1939 Phylogene ic econs uc ion, gene ic dis ance, and haplo ype Phylogene ic econs uc ion based on he mi ochond ial ND2 gene and mi ogenome e ealed ha Pa ameso i on can be di ided in o Clade I and Clade II (Fig. 3A, B). Clade I co esponds o he P. chinensis g oup, wi h a la ge gene ic di e gence be ween he species, and he in e speci ic ela- ionships a e ini ially esol ed. Clade II co esponds o he P. caudopunc a us g oup, which exhibi s ela i ely low mi- ochond ial di e gence ye encompasses six dis inc e o- lu iona y lineages (Fig. 3A). The ou samples om Da- ang, Guizhou, o m a dis inc e olu iona y lineage wi hin Clade II, which is close o P. longliensis and P. maolan- ensis (BPP/UFB = 0.84/72; Fig. 3A). Howe e , he phy- logene ic ee econs uc ed om he mi ogenome showed ha he wo Da ang samples om Guizhou clus e ed close o P. longliensis wi h low suppo (BPP/UFB = 0.47/42; Fig. 3B). The new species had small mi ochond ial gene - ic dis ances om ela i es, anging om 0.63 o 9.39% (Table 3). The haplo ype ne wo k based on ND2 showed no sha ed haplo ypes wi hin he P. caudopunc a us g oup, wi h he new species o ming a unique haplo ype (Fig. 3C). Using bo h conca ena ion and coalescen -based app oaches, we econs uc ed maximum-likelihood (ML) (Fig. 4A) and species ees (Fig. 4B) based on SNP da a h ough IQ-TREE and SVDqua e s analyses, espec- i ely. Bo h analyses s ongly suppo ed he samples om Da ang, Guizhou, as a dis inc e olu iona y lin- eage. Howe e , we obse ed phylogene ic disco dance be ween he ML and species ee opologies ega ding he placemen o he new species and P. zhijinensis, sug- ges ing po en ial in luences o incomple e lineage so - ing and gene low e en s on phylogene ic econs uc ion. The new species exhibi s mode a e gene ic di e en ia ion Table 3. Unco ec ed p-dis ance (%) be ween six species o he P. caudopunc a us g oup based on mi ochond ial ND2. ID Species 1 2 3 4 5 1P. wumengshanensis sp. no . 2P. caudopunc a us 9.39 3P. longliensis 1.03 9.68 4P. maolanensis 0.63 9.49 0.61 5P. wulingensis 9.39 2.70 9.69 9.50 6P. zhijinensis 1.84 9.64 1.82 1.42 9.65 Figu e 3. Recons uc ed phylogeny and haplo ype ne wo k. A. Phylogene ic ee econs uc ed based on he mi ochond ial ND2 gene; B. Phylogene ic ee econs uc ed based on he mi ochond ial genome. In phylogene ic ees, Bayesian pos e io p obabili ies (BPP) om BI analyses/ul a- as boo s ap suppo s (UFB) om ML analyses we e no ed beside nodes. The scale ba ep esen s 0.02 nucleo ide subs i u ions pe si e; C. ND2-based mi ochond ial haplo ype ne wo k. 0.02 Pa ameso i on qixilingensis Pa ameso i on yunwuensis Pa ameso i on hongkongensis Pa ameso i on au an ius Pa ameso i on guanxiensis Pa ameso i on chinensis Pachy i on inexpec a us Pa ameso i on delous ali Pa ameso i on labia us Pa ameso i on uzhongensis 1.00/99 100/1.00 1.00/94 0.71/65 0.99/89 0.52/71 0.97/96 1.00/100 1.00/100 1.00/100 1.00/100 0.41/77 1.00/98 0.84/72 P. longliensis Pa ameso i on maolanensis Pa ameso i on wumengshanensis sp. no . Pa ameso i on zhijinensis Pa ameso i on wulingensis Pa ameso i on caudopunc a us Pa ameso i on Ou g oup BPP/UFB A I II P. caudopunc a us P. wulingensis P. wumengshanensis sp. no . P. maolanensis P. longliensis P. zhijinensis 1 6 C B Pa ameso i on Ou g oup I II 0.02 Pa ameso i on labia us Pachy i on inexpec a us Pa ameso i on hongkongensis Pa ameso i on delous ali Pa ameso i on uzhongensis Pa ameso i on qixilingensis Pa ameso i on guanxiensis Pa ameso i on yunwuensis Pa ameso i on caudopunc a us Pa ameso i on chinensis Pa ameso i on zhijinensis Pa ameso i on maolanensis Pa ameso i on wulingensis Pa ameso i on au an ius 0.47/42 1.00/100 1.00/100 1.00/100 1.00/98 0.65/46 1.00/100 1.00/100 1.00/100 1.00/74 1.00/100 1.00/100 1.00/98 Pa ameso i on longliensis Pa ameso i on wumengshanensis sp. no . BPP/UFB 0.99/99 1.00/99 1.00/100 1.00/100 zse.penso .ne Luo, T. e al.: Genus Pa ameso i on1940 om i s closely ela ed congene s, wi h Fs alues ang- ing om 0.16 o 0.19 (Fig. 4C), sugges ing pa ial bu signi ican species di e gence. Based on mo phological, mi ochond ial, and nuclea gene ic di e ences, he e is clea suppo ha he Pa ame- so i on geog aphic popula ion om Da ang, Guizhou, China, should be ea ed as a new species. Taxonomic accoun Pa ameso i on wumengshanensis Luo & Zhou, sp. no . h ps://zoobank.o g/72A9ACFC-B93C-4727-BA3F-ACA804B7B041 Figs 5, 6, Suppl. ma e ial 1 Ch esonymy. Pa ameso i on sp2 (DF): Luo e al. 2021 (Da ang, Guizhou, China); Pa ameso i on longliensis (DF): Luo e al. 2022 (Da ang Coun y, Guizhou, China). Holo ype. Adul male, GZNU20180711, collec ed by Tao Luo on 11 July 2018, in Yuchong Township, Da- ang Coun y, Guizhou P o ince, China, 27.44933332°N, 105.95403671°E, ele a ion 1137 m a.s.l. Pa a ypes. Th ee adul males: GZNU2018070903, GZNU2018070904, and GZNU2018070905. Th ee adul emales: GZNU2018070901, GZNU2018070902, and GZNU20180709011. The locali y and da e o all pa a- ypes a e same as holo ype. E ymology. The speci ic epi he wumengshanensis e e s o he ype locali y, loca ed wi hin he Wumeng Moun ains, Guizhou, China. The sugges ed English name is he Wumeng Moun ains Wa y New , and he Chinese name is Wū Mēng Shān Luǒ Yuán (乌蒙山瘰螈). Diagnosis. (1) la ge body size, TOL 95.5–123.6 mm in adul males, TOL 150.1–160.0 mm in adul emales; (2) absence o dis inc colo ed s ipes on do sola e al idges, consis en wi h body colo a ion; (3) absence o es igial gills and gill ilamen s; (4) ex e nal eyes no mal, eye diame e 14–20% o head leng h; (5) absence o colo ed spo s on he ails o males, a single ed-o ange s ipe om he pos e io ma gin o he cloaca o he 4/5 pa s o ail on en al side; (6) ough skin, body wa s ough and la ge, and longi udinal g oo es dis inc on he body lanks; (7) yellow spo below he pos e io ma gin o he eye absen ; (8) ome ine ee h dis inc , middle pa dep essed ou wa d, ha ing a sligh ly wide and shallow g oo e a middle base o each choana, and a dis inc ly p ominen bony pla e is p esen pos e io o he choana; (9) an e io end o he epib anchial bone o he hyoid appa a us is nea T-shaped. Desc ip ion o he holo ype. Adul male, TOL 123.6 mm, SVL 72.6 mm. Measu emen s a e p esen ed in Suppl. ma e- ial 1. Body ela i ely slende . Head la , dis inc ly longe han wide (leng h/wid h = 1.4), dis inc scen glands on side o head; knob-like (Fig. 6D), wi h a leshy p o ube ance p es- en in he b anchial egion. Snou sho ; snou a is e iden , unca ed, ex ending beyond lowe lip; snou leng h dis inc - ly longe han eye diame e ; nos ils sho , close o snou ip; eyes la ge; labial old de eloped, ex ending om lowe eye o ip o snou ; gula old de eloped; glandula idge on each side o he head, sligh ly de eloped; ongue sho , ellip i- cal, bo h la e al sides dissocia ed; one p emaxilla wi h iny ee h on he uppe and lowe jaws. Vome ine ee h dis inc , Ʌ-shaped, middle pa dep essed ou wa d; den i ion slow- ly comp essed om pos e io o an e io - o-an e io ma gin 0.02 D5 A8 A5 Y2 C2 C9 H9 A6 C4 A2 H3 Y1 H10 C10 Y5 A7 A4 H7 C6 C5 C8 D2 A1 Y7 D3 A3 H6 H5 H2 D6 Y4 A10 Y6 H4 D1 D4 A9 Y3 C7 D7 C1 H8 H1 M1 M2 C3 100 100 100 100 100 100 100 100 98 100 99 100 100 97 100 98 100 100 100 96 100 100 100 98 100 P. longliensis P. caudopunc a us UFBoo = 100 95≤UFBoo <100 UFBoo <95 P. maolanensis P. zhijinensis P. wumengshanensis sp. no . 0.6 100 100 100 Ou g oup Clade I Clade II A B PLPC PW PZ PM 0.16 0.17 0.19 0.16 C Pachy i on inexpec a us Figu e 4. Phylogeny and in e speci ic di e en ia ion. Maximum likelihood ee econs uc ed (A) and species ee gene a ed om SVDqua e s (B) analysis based on 1,475,246 SNPs. Suppo alues a e displayed on nodes, and dashed lines indica e opology-con- lic ing species. Tip labels: b anch ip numbe s a e sample numbe s o he co esponding species. No e: Sample C1 ep esen s he new species bu e ains i s o iginal label due o ini ial misiden i ica ion and o consis ency wi h NCBI eco ds. (C) Species di e - en ia ion be ween new species and o he species. The numbe s abo e and below he dashed line ep esen Fs alues. Abb e ia ion: PC = P. caudopunc a us, pho o modi ied om Fei e al. (2006); PW = P. wumengshanensis sp. no .; PZ = P. zhijinensis; PL = P. longliensis; PM = P. maolanensis, pho o modi ied om Gu e al. (2012). Zoosys . E ol. 101 (4) 2025, 1933–1947 zse.penso .ne 1941 mee ing be ween wo na es; ha ing a sligh ly wide and shallow g oo e a middle base o each choana; a dis inc - ly p ominen bony pla e is p esen pos e io o he choana (Fig. 7A). An e io end o he epib anchial bone o he hyoid appa a us is nea T-shaped, connec ed wi h he i s and sec- ond ce a ob anchials o he bone; ce a ohyal kni e-like, wi h he an e io wo- hi ds in ca ilage, le and igh unconnec - ed (Fig. 7D). Ve eb al idge de eloped, ligh an, conspic- uous, unin e mi en , om occipu o ail. Fo elimbs sho , eaching o ip o snou when adp essed o wa d; hind limbs longe and s u die han o elimbs; palm and sole o e lap when adp essed o elimb backwa d and hind limb o wa d along body lank; wi hou me aca pal o me a a sal ube - cles; ou inge s and i e oes wi hou webbing o inge; ela i e inge leng h 1 < 4 < 2 < 3; ela i e oe leng h 1 < 5 < 2 < 4 < 3. Tail ela i ely long; do sal and en al caudal ins e iden , expanding on pos e io hal o ail; ail- ip ound- ed. Cloaca a la ge sli , swollen in o a small mound, illous mas oid a ound cloaca, many pubes on gaps o cloaca. Skin ough, especially on he back o he body; many wa s and g anula glands on do sal skin, do sal su ace o he limbs, and la e al sides o ail. Longi udinal lank g oo es dis inc on he body lanks, app oxima ely 17 (Fig. 6E). Ven al skin w inkled, densely co e ed wi h small wa s (Fig. 6C, H). Sexual dimo phism. Apophysis o cloaca o he male la ge and low, and he coun e pa o he emale small and high (Fig. 5C, F). Female cloaca small and o al wi hou gland in inside and lack inge -like papillae on he cloacal wall. Colo a ion. In li e, do sal su ace o body black- b own and ligh oli e colo . Head, body, and en al su aces o limbs da k b own o e all wi h i egula ed- dish-o ange ma kings. Absence o dis inc colo ed s ipes on do sola e al idges, consis en wi h body colo a ion. A single ed-o ange o ange ine yellow s ipe ex ends om he pos e io ma gin o cloaca o he 4/5 pa s o he ail on he en al side (Fig. 6H). Finge and oe ips g ay- ish b own. Colo ligh ens in 75% e hanol p ese a ion. Compa ison. The new species can be assigned o he P. caudopunc a us g oup on he basis o phylogeny and can be dis inguished om o he species wi hin ha g oup by he ollowing mo phological cha ac e is ics (Table 4). Pa ameso i on wumengshanensis sp. no . di e s om P. caudopunc a us and P. wulingensis by absence o colo ed spo s on he ails o males and colo ed s ipes on do sola e al idges ( s. p esence o pu plish ed spo s on he ails o males and h ee yellowish b own longi udinal s ipes e y dis inc on do sola e al idges). Pa ameso i on wumengshanensis sp. no . di e s om P. malipoensis by absence o es igial gills and gill ilamen s in adul s ( s. p esence), snou - en leng h g ea e han ail leng h ( s. almos equal), and absence o dis inc colo ed s ipes on do sola e al idges, consis en wi h body colo a ion ( s. do sola e al idges sca e ed wi h dis inc i e o ange- ed o b ownish-yellow spo s). Pa ameso i on wumengshanensis sp. no . di e s om P. zhijinensis by absence o dis inc colo ed s ipes on do sola e al idges, consis en wi h body colo a ion ( s. wo o h ee nea -con inuous yellow s ipes on he do sola e al idge), yellow spo below he pos e io ma - gin o he eye absen ( s. p esen ), absence o es igial gills and gill ilamen s in adul s ( s. neo eny is common wi h mos adul specimens ha ing es igial gills and gill ilamen s), ome ine ee h dis inc , middle p o uding ou wa d, den i ion slowly comp essed om pos e io o an e io o an e io ma gin mee ing be ween wo na es, ha ing a sligh ly wide and shallow g oo e a middle base Figu e 5. Holo ype GZNU20180711 (A–C) and emale pa a ype GZNU20180709011 (D–F) o Pa ameso i on wumengshanensis sp. no . in p ese a i e. (A, D) La e al iew. (B, E) Do sola e al iew. (C, F) Ven al iew.