A New Species Of The Genus Tylototriton From Dabie Mountains And The Preliminary Conservation Genetics Research Of It

From 2013 to 2016,a total of 32 individuals from genus Tylototriton were collected from Yaoluoping Nature Reserve in the Dabie Mountains,Anhui,China..This species is identified belonging to Tylototriton asperrimus group and shares a number of similarities with T.wenxianensis,T.broadoridgus and T.dabienicus.The diagnostic characters of the new species are as follow:head length is greater than width of head,bony ridges on the head are notable and necked-in,and distal digit ends,ventral digits,peripheral area of cloaca and tail lower margin are orange.Based on the tree of three mitochondrial genes(ND1,ND2 and Cyt b),these 32 species clustered an independent clade,which is close to T.wenxianensis,T.dabienicus and T.broadoridgus.This result was consistent with the morphological analysis above,which supported that the species distributed in southern of Dabie Mountains from Yuexi county,Anhui province is a new species,named Tyltotriton anuiensis sp.nov.,which has significant difference with other Tylototriton species.In this research,dinucleotide and trinucleotide and tetranucleotide microsatellite loci in Tylototriton anhuiensis sp.nov.were isolated successfully.Then,we used these microsatellite loci to do further research on Tylototriton anhuiensis sp.nov.,such as conservation genetics.The main experiment results as the following:we used the High-throughput sequencing protocol to isolate dinucleotide and trinucleotide and tetranucleotide microsatellite loci in Tylototriton anhuiensis sp.nov.After digestion-ligation,hybridization with biotinylated probe,magnetic field separation,DNA recovery,cloning and sequence,primer designing,PCR optimization,genotyping and data analysis,we get eight dinucleotide microsatellite loci and one trinucleotide microsatellite loci and eight tetranucleotide microsatellite loci.The expected heterozygosities of inucleotide microsatellite loci ranged from 0.538 to 0.881,and the observed heterozygosity ranged from 0.719 to 1.000,and the PIC values ranged form 0.419 to 0.85,while the number of alleles ranged form 3 to 13.We isolated only one trinucleotide microsatellite loci,its expected heterozygosities is 0.813,and the observed heterozygosity is 1.000,while the PIC value is 0.776.The expected heterozygosities of inucleotide microsatellite loci ranged from 0.593 to 0.843,and the observed heterozygosity ranged from 0.448 to 0.8,and the PIC values ranged form 0.558 to 0.811,the number of alleles ranged from 5 to 13.In this research,the mitochondrial genome of T.anhuiensis sp.nov.was sequenced,which was 16,259 bp in length and the overall base composition was A(33.6%),G(14.5%),C(26.3%)and T(25.6%).The arrangement of mitochondrial genome of T.anhuiensis sp.nov.was consistent with vertebrate,which was consist of 38 genes and included 22 Trna,2 rRNA(12S rRNA ? 16S rRNA),13 protein coding genes and lcontrol region(D-loop).The phylogenetic relationship was constructed using complete mitochondrial genomes.The result showed that the Tylototriton was consisting of T.verucosus group and T.asperrimus group.However,the phylogenetic relationship of T.verucosus group was not so good.The relationship of T.verucosus,T.yangi and T.shanjing was very close and the value of bootstrap was low,which indicate that the taxon of T.verucosus need to further test using more molecular markers.The T.asperrimus group was monophyly and had high bootstrap values.T.anhuiensis sp.nov.and T.wenxianensis were sibling species.The phylogenetic result pointed out the relationship of T.asperrimus,T.wenxianensis and T.anhuiensis sp.nov.was very close to each other,which could explained the change process of species name between this three species.