Molecular Phylogeny Of The Genus Pleione(Orchidaceae)

The genus Pleione D.Don(Orchidaceae)has high ornamental and medicinal value.In this genus,P.bulbocodioides and P.yunnanensis are the source plants of Chinese traditional Chinese medicine “Shancigu”.The mountainous area of Southwest China,which is rich in germplasm resources of Pleione,is the diversity distribution center of this genus.Due to the lack of in-depth phylogenetic studies,the interspecific relationship of Pleione is unclear and species identification is difficult.This limits the conservation and utilization of species resources in the genus.In the present thesis,we greatly increased the number of population samples of Pleione,and constructed phylogenetic relationships of the entire genus by integrating chloroplast whole genome and nuclear gene data,which provided a reliable phylogenetic background for species identification and classification of the genus.The main results are as follows:1)Based on the genome skimming data from 115 populations of 24 species of Pleione,115 chloroplast whole genome(cp DNA)and ribosomal DNA(nr DNA)sequences were assembled.The phylogenetic maximum likelihood trees were constructed using these two datasets,respectively.Chloroplast genome structure was intact without inverted repeat sequence region(IR region)expansion or other structure variations.The phylogenetic trees constructed from plastid genome and ITS sequences all supported the monophyletic nature of this genus,but the interspecific relationships and topological structures were highly conflicted among different phylogenetic trees,which was likely caused by interspecific hybridization events.The genome size and chromosome ploidy of 100 samples from 24 species were further determined.It was found that there were great differences in genome size and chromosome ploidy between species and within species.This suggested that hybridization events occurred frequently in Pleione,which may be the main reason for the conflict of phylogenetic relationship between nuclear and plastome data.2)Based on ITS tree and plastome tree,the genus Pleione was divided into 7 clades.Among them,the clustering of Clade I,Clade II and Clade III was basically consistent with that of the previous study.This thesis increased the range and quantity of sampling,and subdivided the previously general division of Clade IV into four clades(Clade Ⅳ,Clade Ⅴ,Clade Ⅵ,Clade Ⅶ)to provide a clearer phylogenetic relationship among the species of Pleione.This study clarified the phylogenetic positions of P.arunachalensis and P.kaatiae which have never been systematically studied before.Plastome data supported the exclusion of P.Hui from P.formosana,but whether it was a new species still needed further study.Pleione dilamellata was closely related to P.limprichtii.These was very limited morphological differences between the two species.Thus,P.dilamellata was treated as a sysnonym of P.limprichtii.Both ITS and plastome phylogenetic trees supported the exclusion of P.formosana from the P.bulbocodioides complex.However,the interspecific relationships of P.limprichtii,P.bulbocodioides,and P.yunnanensis remained unclear due to the frequent gene exchanges and reticular evolution.3)Four species(52 individuals)in the P.bulbocodioides complex and seven species(9 individuals)closely related to this complex were selected for reduced-representation genome sequencing,and a large number of SNP mutation sites were obtained for further analysis of the evolutionary relationship within the P.bulbocodioides complex.The results of population genetic structure analysis further confirmed that interspecific hybridization frequently occurred in Pleione,and supported the exclusion of P.formosana from the P.bulbocodioides complex.The species of P.bulbocodioides complex were distributed in Southwest China,and there was a strong correlation between genetic composition and geographical distribution.Based on molecular evidence,the populations of the P.bulbocodioides complex(excluding P.formosana)was divided into three groups,namely CSB group,YGP group and WSP group.Each population of CSB group shared one set of genetic material,while the population of YGP group had another set of genetic material,and the WSP group was a hybridization group.This study speculated that different populations of same species(P.bulbocodioides,P.pleionoides,P.limprichtii)in CSB and YGP regions were identified as different species because of extensive morphological differentiation among populations of same species driven by the high diversity of topography and climate in Southwest China.This thesis studied chloroplast phylogenetic genomics of Pleione based on a large-scale sampling to clarify the phylogenetic relationships among species,and conducted reducedrepresentation genome sequencing of the P.bulbocodioides complex to understand the geographical distribution,speciation and evolutionary pattern of the complex.This thesis provided important data for understanding the adaptive evolution of Orchidaceae,and provide a theoretical basis for species identification as well as the conservation and utilization of germplasm resources of Pleione.