Phylogeographic Study Of Tethys Relic Smilax Aspera L.,An Intercontinental Disjunct Species

Smilax aspera is one of the most widely distributed species in Smilacaceae.It distributed in the Mediterranean region,the East African highland forest and the India-China Himalaya seasonal rain forest,showing a disjuncted distribution patten in Mediterranean-Eastern African-Southern Asia.S.aspera is one of components of the evergreen broad-leaved sclerophyllous forest and originated from the warm and humid Tertiary Paleo-Tethys subtropical flora.The previous studies indicated that S.aspera is a basal taxon on the old continental branch and is a key link between the Old World and the New World of Smilacaceae.It is of great significance to elucidate the phylogenetic evolution of the Smilacaceae by studying speciation and phylogeographics of S.aspera.In this study,we used microsatellite markers,SNP loci and ecological niche modeling to reveal the genealogical divergenece,population dynamic history and origin and evolution of S.aspera with the theory and methods of phylogeograpy and population genetics.The major results were listed below:1)Microsatellite markers.10754 microsatellite loci were identified by scanning the transcriptome of S.bona-nox,a related specice of S.aspera and 4862 pairs of SSR markers were designed.Moer than 150 pairs of primers were synthesized randomly for PCR amplification and35 markers were amplificated efficiently and highly polymorphic.These 35 microsatellite markers were employed in the population study of S.aspera in 34 populations.The population genentic diversity based on 35 SSR markers showed that the range of total genetic diversity(H_S)is from0.372 to 0.608 with the average is 0.537;the range of observed heterozygosity(H_O)is from 0.349to 0.738 with the average is 0.650;the range of expected heterozygosity(H_E)is from 0.354 to0.588 with the average is 0.518.PCoA analysis based on Nei distance showed that there were two distinct cluster of S.aspera,that is,Mediterranean lineage and the East African-South Asian lineage.Further more,the STRUCTURE analysis based on Bayes algorithm of all populations indicated that the best grouping value(K)is two and also revealed the lineage of Mediterranean and East Africa-South Asia.In the Mediterranean lineage,the STRUCTURE analyse indicated two gene pools:the west Mediterranean and east Mediterranean.And in Eastern African-Southern Asian lineage,the groups shared gene pools with each other.IBD test showed that S.aspera isolated significantly by distance.2)SNP loci based on RAD-tags.We chose the restriction enzymes MluCI and AvaII for DNA library construction of S.aspera after experimental and insilico digestion.With experimental process optimized and promoted,40 populations of S.aspera(250 individuals)were invoveled for double digest RAD library construction and libraries were sequenced on Illumina HiSeq X Ten platform.First,the next generation sequencing data were processed by the software Stacks version1.47.the block process_radtags in Stacks was used to take apart the data set into single sample.Detailly,the number of rad tags ranged from 338,582 to 5670,635.And then,the workflow of ustacks,cstacks and sstacks was employed to identify the SNPs among popualtions and indviduals.Finally,49,138 SNPs were identified.DAPC(Discriminant analysis of principal components)based on SNP loci indicated that S.aspera populations were grouped into 3 clusters,which is,western Mediterranean cluster(containing Iberian Peninsula populations and Apennine Peninsula populations),eastern Mediterranean cluster(containing Balkan Peninsula populations and Asia Minor populations)and East African-Southern Asian cluster.The phylogenetic analysis based on maximum likelihood method revealed that two clades were covered in Mediterranean and East African-Southern Asian region and divergenced deeply with 100%supported value.According to the toplogical relation,it indicated Asia Minor was the basal taxon of Mediterranean lineage and the Eastern African was the basal taxon of EA-SA lineage.BEAST result based on 49318 SNPs suggested that the widespread distributed S.aspera divergenced in middle Miocene(11.71 Ma,95%HPD:8.8866-14.24 Ma).Of two major lineages,Mediterranean lineage divergenced in late Miocene(5.62 Ma,95%HPD:0.95-11.03 Ma)while EA-SA lineage divergenced in late Pliocene(4.09 Ma,95%HPD:0.32-9.71 Ma).The reconstruction of ancestral areas using Bayesian Binary BBM method indicated that the vicariance event in middle Miocence resulted in the Mediterranean lineage and EA-SA lineage with originated from Himalaya region.3)Ecological niche modeling.The ENM analysis based on MAXENT showed that the potential distribution of S.aspera was equal to the actual distribution at present.During Last Glacial Maximum,S.aspera disjuncted in Mediterranean region,Eatern Africa and Southern Asia.The distributions moved southward in Mediterranean region,expanded in Eastern Africa and Southern Asia.The habitat declined dramatically while the climate becoming warmer by greenhouse effect in future(the year of 2070).In Mediterranean region,the suitable habitat will move northward arriving in northwestern France.In East Africa,the habitat will be fragmented and in Southern Aisa,S.aspera will still distribute in Himalay and Hengduan mountain.