Phylogeny And Evolution Of Primula Section Sikkimensis(Primulaceae)

The Himalayan and the adjacent Hengduan Mountains of southwestern China,known as the Himalaya-Hengduan Mountains(HHM)region,have been designated as two of the world's 34 most important biodiversity hotspots.The region is also the species richness center of genus Primula(Primulaceae).Primula section Sikkimensis Balf.f.is a wholly HHM distributed taxa with ca.8 species.Primula sikkimensis is wide-ranging,occurring throughout the distribution of the section,but others are narrowly distributed.The group provides us a model system to test rapid speciation and divergence patterns in the HHM region.Here,we studied the molecular phylogeny and phylogeography of P.sect.Sikkimensis by widely sampling.We aimed to illustrate the phylogenetic relationships of species in the section,and uncover its demographic history which might feedback the rapid uplift of the HHM and Quaternary climatic oscillations.In addition,we developed 17 novel microsatellites and described seven whole chloroplast genomes for four species of sect.Sikkimensis.Main findings are as follows: 1.Phylogenetic study of Primula section SikkimensisThe first complete phylogenetic relationships in P.sect.Sikkimensis were reconstructed using nine chloroplast regions and ITS seqeuences.We obtained some novel conclusions from this study,(1)the section is monophyletic,and Primula reticulate is a basal one and divergent largely from other species in the section.(2)The relationships among most species(excluding P.reticulata)is complicated.Extremely,the most widely distributed species P.sikkimensis has at least six main lineages,and each grouped with other species.(3)DEC analyses shown sect.Sikkimensis origined from the central Himalaya,and subsequently dispersed to the southeast Tibet and Hengduan Mountains.The main divergences in the section probabily occurred at about 2-2.5 Ma,which is coincided with the recent rapid uplift of the Qinghai-Tibet Plateau and high climatic oscillations in Quaternary.These two events might co-affect the rapid speciation of section Sikkimensis.2.Phylogeographic study of Primula section SikkimensisIn this study,we investigated the phylogeographic structure and population dynamics of P.sect.Sikkimensis.Two chloroplast markers(trnL-F and rps16)were sequenced in 199 samples from 41 sampling sites in its whole distribution range.The phylogeographic analyses not only confirmed the main findings in chapter two,but also offered more invaluable information on the evolutionary history of sect.Sikkimensis.Eight haplotype groups were firstly recovered,and provided us more lines of evidences of hotspots of sect.Sikkimensis in the central Himalaya and southeast Tibet.In addition,Hengduan Mountains had a different demography history of sect.Sikkimensis than others,in which population expansion event was detected and the large area was occupied one main haplotype.Our dating results shown the divergence between the Hengduan Mountains group and its relative in southeast Tibet occurred around 1 Ma.3.Development of microsatellites in Primula sikkimensisA microsatellite-containing DNA library was constructed by enrichment approach using microsatellite probes,then sequenced through the Roche 454 pyrosequencing technology.61,755 unique reads were identified from a total of 62,470 reads.5,377 unique reads that each contains at least one microsatellite were filtered out.Furthermore,3,112 unique reads were found out that are suitable for primer design.Twenty-nine microsatellite loci were selected from them for further PCR amplification and polymorphic analyses.Seventeen microsatellites loci of P.sikkimensis populations were successfully identified,most of which could be amplified in its relatives(P.florindae and P.alpicola).4.The complete chloroplast genome in Primula sect.SikkimensisIn this study,whole chloroplast genomes of seven individuals from four species of P.sect.Sikkimensis were obtained using genome skimming method.The chloroplast genomes have typical circular quadripartite structure with size varied from153,862 bp to 154,505 bp.A total of 111-112 unique genes were identified from these cp genomes.We also compared them with other 11 published cp genomes of Primula.Our result indicated that there are little variations in structure of cp genomes and nucleotide differences among Primula species.The variations in sequence lengths among different Primula species are mostly coursed by their variable Large Single-Copies(LSC).Phylogenetic relationship of sect.Sikkimensis estimates using a matrix of 77 protein-coding gene sequences showed the identical result that was consistent with the results in chapter two and chapter three.