Genetic Andphylogenetic Structure Of Zanthoxylum Species

Zanthoxylum genus,comprising more than 250 species,belongs to the Rutaceae family.There are 39 species and 14 varieties in China.Z.bungeanum and Z.armatum have great economic importance because of their pericarps as source of culinary and medical applications with more than 2000 years.Z.bungeanum is native of China and widely cultivated throughout China.Recently,Z.armatum also became the second cultivars widely distributed in southwest regions.So China possessed the numerous germplasm resources of Z.bungeanum and Z.armatum.A large number of cultivars and ecotypes of Z.bungeanum and Z.armatum were formed by the long cultivation process.Therefore,their use for cultivar improvement is limited by the scarcity of genetic diversity,genetic relationships and proper phenotypy.We investigated the genetic diversity,phylogenetic relationships and phylogeography at 16 SRAP and 11 SSR markers and 3 cpDNA sequences matK,rbc L and trnL-F in Z.bungeanum and Z.armatum provenances.The main results are as follows:1.A total of 145 clear repetitive bands were generated by 16 primer pairs and the percentage of polymorphic bands were 100%,indicating a relatively high diversity among provenances.The abundant genetic variation was mainly caused by variation of intra-provenances based on AMOVA analysis.The gene differentiation of Z.armatum provenances(Gst=0.385)was higher than Z.bungeanum provenances(Gst=0.138),conversely,the gene flow of Z.bungeanum provenances(Nm=3.112)was relatively high.One hundred and seventy-five accessions were divided four groups according to NJ tree,a divergence between the accessions of Z.bungeanum and Z.armatum(Group?and?).Moreover,three accessions of Z.piperitum without prickles introduced from Japan gathered the Group ?.Group IV is composed of 11 wild species and 10 cultivated accessions of Z.bungeanum.The cluster analysis reflected a relatively close relationship between the geographical origins and the classification of accessions.2.A relatively unambiguous genetic relationship among 31 accessions of Z.bungeanum and Z.armatum were elucidated via 11 SSR primer pairs that were developed from the transcriptome dataset of Z.bungeanum.The genetic diversity of Z.bungeanum was relatively high than Z.armatum based on the 11 primer pairs detected allele 53 and 23 in these two species,respectively.In Z.bungeanum provenances,GMXHY(He=0.521)maintained the highest level of genetic diversity,and in Z.armatum provenances,ZZZYJ(He=0.277)possessed the highest genetic diversity.Z.bungeanum experienced the bottleneck along the long cultivation process.The coefficient of genetic differentiation(Fst)for Z.bungeanum and Z.armatum were 0.3961 and 0.2156,indicating a high level of genetic differentiation.Furthermore,the Mantel test displayed a significance of correlation between genetic distance and geographic distance in the sampled provenances(P<0.05).These results suggested that geographic isolation was the primary factor leading togenetic differentiation of Z.bungeanum and Z.armatum.AMOVA indicated that the most of the genetic variation of Z.bungeanum and Z.armatumexists within individuals.As a geographic barrier,Qinling Mountains closed the gene flow between the Z.bungeanum provenances of northern and southern China,which lead to the formation of strong population structure.However,we detected slightly gene introgression from northern provenances to Fengxian,Qin'an and Wu du located southern Qinling piedmont.A Mantel test was performed in order to test whether there is a geographic-genetic distances correlation.When all North and SouthChina provenaces are included in the analysis,there is a positive correlation between genetic and geographic distances of r2 = 0.074.However,this result is driven by isolation by distance within the North and South provenances.When we compared genetic and geographic distances focusing only on pairwise North vs.South provenances,no correlation between genetic and geographic distance is found,r2< 0.001(P>0.1),ruling out the hypothesis that gene flow between North and South provenances follows an isolation-by-distance model.Therefore,Shaanxi and Gansu provinces were formed a transitional provenance.As a boundary,Qinling Mountains dividiedZ.bungeanum provenancesinto two groups ‘Nanjiao' and ‘Beijiao',whereasall Z.armatum accessions gathered a single group.The SSR markers also supported that two cultivars ‘‘Qinghuajiao'' and “Dingtanhuajiao” belonged to Z.armatum.3.Nineteen haplotypes with 64 polymorphic sites in Zanthoxylum 182 accessions were identified(H1~H19).Z.bungeanumand Z.armatum harbored eight and three haplotypes,respectively.Other eights haplotypes were harbored by the other Zanthoxylum species.The total haplotype diversity(Hd)and nucleotide diversity(Pi)were 0.588 and 0.259×10-3,respectively.The highest haplotype diversity was found in provenanceLZ(Hd=0.833),BJX had the highest nucleotide diversity(Pi=0.881×10-3).The species had high level of chloroplast genetic variation(HT=0.600),whereas the low level of genetic variation whin populations was onlyHS=0.438,indicating a higher genetic differentiation.The coefficient of population genetic differentiation(Nst=0.373>Gst=0.271,P<0.05),indicating significant phylogeographic structure across the species' distribution range.All 19 haplotypes were divided into four groups based on phylogenetic tree and haplotype network.Ancestral area reconstruction indicated that the ancestors of Zanthoxylum first colonized Yunnan and Guizhou provinces.The current geographic distribution of cultivated species may represent long-distance dispersal and several vicariance events of ancestors of Z.bungeanum and Z.armatum.Additionally,Z.bungeanum and Z.armatum most probably diverged during the Late Miocene.Neutrality test and the mismatch distribution analysis showed Z.bungeanum experienced sudden and spatial expansion events in history,whereas Z.armatum experienced sudden expansion event.