Molecular Phylogeography And Genetic Structure Of Microtus Fortis(Arvicolinae,Rodentia) In China

Microtus fortis has a unique natural Schistosoma anti-infection characteristics which makes it become a special promising experimental animal model resource in China.However,the genome sequence,phylogenetic relationships and genetic structure of the M.fortis were not systematic reported before.The first complete mitochondrial genome sequence for M.f.fortis,a subspecies of the Microtus fortis(Arvicolinae,Rodentia),was reported in this study.Designed 16 pairs of primers,we amplified and sequenced the mitochondrial genome of the M.fortis from six regions(Hunan,Ningxia,Fujian,Heilongjiang,Jilin,Guangxi)in China.The mitochondrial genome length of the M.fortis samples was from 16303~16312 bp.The mitochondrial genome sequence of the M.f.fortis(Gen Bank:JF261174)showed a typical vertebrate pattern with 13 protein coding genes,2 ribosomal RNAs,22 transfer RNAs and one major noncoding region(Control region,CR).The CSB-1?ETAS-1?ETAS-2 and a Poly(C)n sequence sections were found in the CR region.The Poly(C)n structure was only found in the mitochondrial genome control region of Asian lineage genus Microtus.The Poly(C)n structure may be unique to the Asian lineage voles,which further supports the existence of the Asian lineage genus Microtus and the characteristics of its monophyletic lineage.It is found that a 3 base(CCC?TTT)mutation on the putative origin of replication for the light strand OL cervical loop of mitochondrial DNA between the south samples(Guangxi,Hunan,Fujian)and the northern samples(Ningxia,Heilongjiang,Jilin).This mutation can be used as an important molecular genetic marker for the differentiation of Microtus fortis from different sources in South and North china.After comparing the whole genome sequences of the 6 mitochondrial genomes,157 Parsimony sites(informative)and 450 variable sites(Singleton)were detected.The mitochondrial genome diversity of the southern samples was significantly higher than that of the northern samples.To examine the phylogeographic relationships of the M.fortis in China,we investigated 84 individuals collected from these six regions.The mitochondrial cytochrome b gene(cyt b)and control region(CR)were sequenced(Gen Bank accession number: KJ081871-KJ081954 for cyt b and KJ207290~KJ207373 for CR)and 49 haplotypes were observed from the combine data(cyt b+CR).No shared haplotype was found among different geographic populations.High FST values among the populations suggested that fragmentation of habitat has resulted in genetically distinct populations.The phylogenetic trees,inferred from maximum likelihood(ML)and Bayesian inference analysis(BI),highly supported all the M.fortis individuals clustering into one monophyletic lineage.Three main clades are recovered within M.fortis in China:(1)North population(Ningxia?Heilongjiang?Jilin samples);(2)South population(Hunan and Fujian samples);(3)GX population.It also showed that Microtus fortis formed the closest monophyletic relationship with M.middendorffii,M.mongolicus and M.maximowiczii in the phylogenetic tree.They should be differentiated from a common ancestor.The North Group distributed on the north side of Qinling Mountains-Huaihe river line as well as the South Group was on the south.It suggests this geographic barrier played an important role in differentiation of M.fortis in China.Furthermore,the samples all from Southwest China in the GX group may be an example of ‘refuge within refugia' in glacial period.According to our molecular clock analysis,the main clades of the M.fortis divergence and separated time at around 0.77~0.64 million years ago(Mya)located in the Penultimate Glaciation.Divergence time within the three clades taken place during the interglacial period between the Penultimate Glaciation and the Last Glaciation.Bayesian skyline plot(BSP)indicates the effective population size of the M.fortis had been experiencing a downward trend during the past 30-40 years,which may due to the habitats loss and environmental degradation.Genetic structure and differentiation of four Microtus fortis populations were detected by using twenty microsatellites DNA makers with multi-fluorescent STR-PCR analysis technology.19 STR loci exhibited high polymorphism(PIC = 0.83> 0.5).The average number of alleles,genetic heterozygosis also displayed rich genetic diversity of the four regions of M.fortis.According to the genetic distance and UPGMA phylogenetic analysis,it showed that the largest degree of genetic differentiation between the populations of Guangxi and Ningxia and the smallest degree of genetic differentiation between the populations of Hunan and Guangxi.The STRUCTURE program analysis(K=2)also given same illustration.The close geographical distance may result in the lowest genetic differentiation in the nuclear genome level between these two populations(Hunan and Guangxi).Through the comparisons,a number of interspecific and intraspecific single nucleotide polymorphisms(SNP)were counted in the mitochondrial cytochrome b(cyt b)gene and the control region(CR)of Microtus fortis.13 and 12 unique M.fortis SNP sites were found on the sequence cyt b gene and control region(CR)sequences,respectively.These specific SNP loci can be used as important molecular genetic markers for the identification of the M.fortis samples from the other voles.On the other hand,a certain number of intraspecific SNP loci were found in different geographical populations,which could provide important molecular marker for distinguishing the geographical origin of the Microtus fortis.These important mitochondrial genetic SNP loci and the microsatellite DNA genetic markers will provide an important basis for the study of subspecies classification,sample identification and genetic diversity analysis of Microtus fortis in the future.