The Study On Conservation Genetics Of The Endangered Emberiza Jankowskii

The Rufous-backed Bunting(Emberiza jankowskii)is a passerine bird(Passeriformes,Emberizidae)with a narrow distribution in the world.Historically,two E.jankowskii breeding populations have been recognized.The one occurred the junction among Russia,North Korea and China.The other one occurred in west Jilin Province and the adjacent areas of Inner Mongolia.At present,the survival of E.jankowskii has been subjected to human disturbance as well as habitat fragmentation.It has been partially disappeared in the historical breeding distributions and been listed as an endangered species by the International Union for Conservation of Nature and Natural Resources due to a sharp decline in population size.In order to estimate the genetic diversity,genetic differentiation and adaptive evolutionary potentials,we studies on five recently discovered breeding populations(Aolunhua(ALH),Gahaitu(GHT),Lubei557(LB557),Tongliao(TL)and Kundu(KD))of E.jankowskii in Inner Mongolia.The neutral and adaptive genetic markers were used to detect the genetic diversity,genetic structure,population dynamics and adaptive evolution of E.jankowskii.Our aims are to answer the following scientific questions:(1)if genetic diversity and genetic structure of E.jankowskii were effected by habitat fragmentation and human disturbances;(2)if the small E.jankowskii population size had led to the inbreading;(3)if the distribution of E.jankowskii populations was effected by cold climate during ice age;(4)how was the evolutionary machanism of MHC gene in E.jankowskii;and(5)how to define the conservation unit of the sharply declining E.jankowskii populations.Our results could provide the important scientific information for the conservation of the endangerd species,E.jankowskii.The main results are as follows:1.Sixty-one mitochondrial control region(CR)and cytochrome b(Cyt b)gene sequences of E.jankowskii were detected,respectively,with the length of 542/543 bp and 1143 bp.Among the 20 and 19 haplotypes identified based on the mitochondrial CR and Cyt b gene sequences,respectively,70% and 73.68% of the specific haplotypes were detected.In E.jankowskii,the overall haplotype diversity and nucleotide diversity were 0.94 and 0.0084 based on mitochondrial DNA(CR and Cyt b gene united sequences),and the overall expected heterozygosity and observed heterozygosity were 0.76 and 0.61 based on microsatellite genetic markers.These results indicated the relatively high level of genetic diversity in E.jankowskii.However,the relatively low genetic diversity was detected based on the adaptive genetic marker,MHC gene.On the one hand,among the 12 and 17 MHCIIB exon 2 alleles identified by the intron primers in E.jankowskii and its closely related species(Emberiza cioides),the nucleotide diversities were 0.1865 and 0.1667,respectively,and the at least 3 and 4 functional loci were detected,respectively.On the other hand,among 70 MHCIIB exon 2 alleles identified by the exon primers in E.jankowskii,the overall nucleotide diversity was 0.1900,and the at least 8 functional loci were detected.2.The relatively low genetic differentiation and frequent gene flow in E.jankowskii populations were detected by Fst and Nm which were estimated by neutral(mtDNA,microsatellite)and adaptive(MHC gene)genetic markers.The phylogenetic trees or the haplotype network diagrams constructed by mt DNA and MHCIIB exon 2 alleles sequences showed that the mixed clusters of the different E.jankowskii populations.These suggested that there was not an obvious genetic structure in E.jankowskii distributions.According to the individual neighbor-joining tree and the structure dendrogram based on microsatellite data,the non-significant genetic differentiation was detected within E.jankowskii.Based on the results of molecular variation(AMOVA)by neutral and adaptive genetic markers,the population genetic variations mainly produced within populaions,and the relatively low level of genetic differentiation was detected by microsatellite DNA and MHC gene.Furthermore,according to the Mantel tests based on neutral and adaptive markers,the genetic differentiation between populations did not conform to the pattern of isolation by distance.In addition,according to the bottleneck effect based on microsatellites,the GHT,ALH,LB557 and TL E.jankowskii populations might undergo the bottleneck effects during the recent period.Inbreeding phenomenon might exist in E.jankowskii,because the coefficient of inbreeding was larger than zero.3.According to the time to most recent common ancestor(TMRCA)estimated by mtDNA Cyt b gene,Emberiza jankowskii might originate from Tertiary pliocene to Pleistocene(2.90 Mya,95%CI: 2.09-3.81 Mya),and it occurred the differentiation subsequently(2.83 Mya,95%CI: 2.01-3.67 Mya).At that time,the global climate was cold,and it was dry and cold in Inner Mongolia without forming a glacier.Thus,a refuge of E.jankowskii might be in Inner Mongolia.During the Pleistocene,the second differentiation might occur in E.jankowskii(0.22 Mya,95%CI: 0.07-0.38 Mya);and the four specific haplotypes were formed in GHT population during the dagu-lushan interglacial period(200-300/380 Ka BP).It might be relevant to the frequent volcanic activities in the middle-east of Inner Mongolia during that period.According to neutrality tests and mismatch distribution analyses based on mitochondrial genes,population expansion events were detected in ALH,LB557,TL and KD E.jankowskii populations,and the estimated expansion time was from 17.53 to 51.46 Ka BP(95%CI: 0.00-89.64 Ka BP)corresponding to MIS3 interglacial period and last glacial maximum(MIS2)with the decreasing temperature finally to the lowest.4.Trans-species polymorphism was detected according to phylogenetic analyses based on the MHC alleles amplified by the intron and exon primers.The dN value was significantly higher than the dS value in the putative PBR based on the MHC gene amplified by the exon primers,suggesting positive selection play a role on E.jankowskii MHC gene.Whereas the dN value was significantly higher than the dS value in the putative non-PBR based on the MHC gene amplified by the intron and exon primers,it might be due to the substitution rate of these loci effected by the high codon usage bias.We detected the codons under positive selection using at least one test(PAML/SLAC/FEL)in E.jankowskii,which matched with homologous codons under positive selection found in other avian species.Furthermore,no recombination was detected in MHC gene in E.jankowskii.5.Due to the overall low level of genetic differentiation in the endangered E.jankowskii populations,we defined the five E.jankowskii populations as one conservation unit for major concernings and managment.Simultaneously,the different conservations and attentions based on the different results were as follows: according to the points of view of maternal inheritance and the high quality gene pools of MHC gene,the GHT and TL populations should be paid major attention,respectively;the GHT,ALH,LB557 and TL populations which might have undergone bottleneck effects should be protected and paid close attention.In addition,the knowledge of wildlife protection and the “Wildlife protection law of the People's Republic of China” should be increase publicity efforts,and the protective consciousness of people should be promoted to protect the endangered E.jankowskii species.