A Transcriptomic Phylogenomic Study Of Primula L.(Primulaceae)

The Primula is one of the largest genera of the primrose family(Primulaceae).There are more than 500 species in the world,most of which are distributed in the northern hemisphere.China is the center of distribution of the genus Primula,with a total of more than 300 species.As a rapid radiation group with extremely rich species diversity,the phylogenetic relationship between primroses,especially between sections,has not been fully solved.In particular,primroses,as a model group for studying heterostyly,have led to bottlenecks in the study of evolutionary biology issues based on species relationships.In order to understand the relationships within the genus Primula,and to find the cause of the rapid differentiation and diversity of the genus Primula,this study selected representative samples under the genus Primula and reconstructed the phylogenetic relationships using transcriptomic data.Transcriptome sequencing was performed on 76 fresh samples and combining with the publicly available data to obtain a data set containing 81 samples.Nuclear gene transcripts and chloroplast transcripts were extracted from transcriptome data.The nuclear gene transcript uses two orthologous inferring methods,based on the expansion of the core orthologous gene and the All-by-All searching and clustering method.The expansion method includes 1364 orthologous nuclear genes with over 80% sample coverage,and the supermatrix total length is 1,296,993 bases.Based on the clustering tree method,8271 orthologous nuclear gene were obtained with over 60% sample coverage,and the supermatrix total length was 9,706,584 bases.Futhurmore,the isolated chloroplast transcripts were combined with protein coding sequences from 12 whole chloroplast genomes,resulting in 36 chloroplast homologous alignments by using “anchor and prune” method,including 93 samples,total length 29,337 bases.Using these three methods,the phylogenetic relationships were reconstructed for the genus Primula using a variety of phylogenetic methods.Almost all methods obtained phylogenetic trees that were nearly completely solved and reproduced five clades defined by previous studies.Our result supported the previous reports of the split of the subg.Aleuritia.However,the phylogenetic trees obtained from the nuclear supermatrix and the chloroplast supermatrix are very different.This difference is comfirmed by severel phylogenetic studies based on a small number of sequence fragments(ITS and chloroplast fragments).Unexpectedly,two nuclear supermatrix placed the outgroup Omphalogramma vinciflora within the clade of the subg.Auganthus in the genus Primula and those clades were highly supported.To address this problem,we examined the spatial distribution of gene tree topological distances that support this relative position hypothesis of Omphalogramma vinciflora in the nuclear and chloroplast datasets.It was found that the number of gene trees that met the two hypotheses was roughly the same size,and Omphalogramma vinciflora was the most influential factor inducing the difference in gene tree topologies.It is noticed that the gene trees supporting Omphalogramma vinciflora within the genus Primula are more concentrated.If we assume that ILS occurs at a uniform rate along the species tree.Then these nuclear genes,which support for the hypothesis of Omphalogramma vinciflora in the genus Primula,could be younger than the others,suggesting possible ancient lateral gene transfer events.Futhermore,a site-based approach was used to detect possible introgression and gene flow events in the genus Primula.And the gene trees were used to explore the possible evolutionary network of the genus Primula.The results of both analyses indicate that several major branches of the genus Primula have experienced extensive geneflow during rapid radiation speciation,especially in the early stage of differentiation,the genus Primula and Omphalogramma vinciflora exhibit a complex network of evolutionary structures.Finally,this study examined the ancient polyploidization events that may be experienced by the genus Primula and its closely related groups.The results of gene replication patterns indicated that there were multiple whole genome duplication(WGD)events in the early divergent stage of Primula,which were likely to lead subsequently rapid radiation of this genus Primula.At the same time,we used Ks density distribution calculated on the intra-species and inter-species,and it suggested that Primula and its related group may have experienced at least two ancient polyploidization events.By locating these two ancient WGDs,it can be determined that the older WGD occurred much earlier than the divergence of the family Primulaceae,could be happened around the age of the ancestor of angiosperms or before it.And the signal distribution suggested this event may also be generated from overlapping by multiple WGDs.The younger WGD event occurred later than the divergence of the common ancestors of Primulaceae,earlier than the divergence of the common ancestor of the genus Primula.More precise position of this WGD event remains further analysis.