The Molecular Phylogeography Of Clintonia Udensis Revealed By CpDNA PCR-RFLP Markers And Sequencing

Clintonia udensis Trautv. et Mey., a pennial herb, was belonged to the genus Clintonia Rif. (Liliaceae). This genus is commonly regarded as containing five species: four species exist in North America and only one specie exists in East Asia. C. udensis is considered the primitive specie in the genus, due to the coexistence of two distinct cytotypes (2n=14 and 2n=28) and the overlapping of their distribution areas, information on population structure and genetic variability is of great significance to the understanding of the polyploid origin, dispersal and evolution of Clintonia.In this study, 19 populations (included 5 tetraploid populations) were analyzed to reveal the Molecular Phylogeography of C. udensis. We used chloroplast DNA markers to investigate the intraspecific phylogeographic patterns of C. udensis. PCR-RFLP results showed that among 96 bands produced, 36 were polymorphic (37.5% ). The results showed that Shannon index (H_st = 0.8739), Nei's index (G_st = 0.9688) and the gene differentiation coefficient (φ_st=0.9427) together revealed that the molecular variation mainly existed among populations. The level of genetic diversity was probably related with its wide geographical distribution and its evolutionary history; and the biological characteristics of this species, limited gene flow and genetic drift might account for the existence of relatively high inter-populational genetic variation. Moreover, the phylogeographic relationships of cpDNA variants can be uncovered by first studying the haplotype diversity and then mapping their geographic distribution. We distinguished 26 cpDNA haplotypes belonging to five distinct clades over the populations' range in Eastern Asia. The two chloroplast DNA PCR products for which size variation was found in restriction digests (trnL-F and rpl20-rps12) were sequenced to determine the nature of the observed variation. The results indicated that differentiations between diploid and tetraploid populations were significant, except three populations-LJ (4x), XZ (4x), ZD (2x). By summarizing the results of cytological, PCR-RFLP and cpDNA sequence analyses, it indicated that the origin of C. udensis polyploidy was autopolyploidy polytopic, and there existed at least three geographical origination regions or secondary differentiation centers. Under the influence of Quaternary climate, C. udensis later dispersed toward Southwest and Northeast China, and finally formed the present mosaic distribution pattern of distinct cytotypes. The results developped the genetic information of molecular Phylogeography of C. udensis, and provided supporting evidences for the further investigation into the origination and evolution of Clintonia.