Phylogeny Utility Of Nuclear And Chloroplast Genomes Of Populus

The species of the genus Populus(Salicaceae)are one of the most important plantation trees,which are well known for its rapid growth,profuse vegetative propagation,adaptability to various ecological conditions and the numerous uses of its wood.Futhermore,the genus has become an excellent research model due to its small genome size.Populus were classified into six sections(Populus,Tacamahaca,Leucoides,Aigeiros,Turanga and Abaso)based on morphological characteristics.However,the extensive interspecific hybridization and high levels of morphological variation among poplars have made it difficult to classify individual species for systematic and comparative evolutionary studies.For the accessment of genetic relationships among Populus species,different methods have been applied,the results showed that the relationship have not been well resolved or have proved inconsistent based on different methods.In addition,the relationships among species and taxonomic status of purported hybrids have remained controversial because of the relatively low phylogenetic resolution of the genes considered as well as the limited number of samples or insufficient characteristic information used in these studies.In recent years,due to the rapid development of next-generation sequencing technology,the maternally inherited chloroplast genomes,because of its relatively conserved size,gene content and gene structure,which have been used to make significant contributions concerning evolutionary mechanisms for species and phylogenetic reconstruction.In addition,the completion of the whole genome of P.trichocarpa provides a means to find highly variable single copy nuclear DNA sequences to assess the evolutionary relationships of Populus.Here,we reconstructed the phylogenetic relationship of Populus based on the complete chloroplast genome and the single copy nuclear data.The results were as follows:(1)Plastome sequences help to resolve deep-level relationships of Populus: total DNA of 25 Populus species was used to generate libraries and sequence using the Illumina HiSeqX platform.Approximately 15.0 GB raw data were generated.Then,the raw data were used to assemble the complete chloroplast genome using GetOrganelle software with P.trichocarpa as the reference.The ten most dissimilar regions of the 40 Populus plastomes were trnK intron + trnK-psbK,rpoB-petN,psbM-trnD,psbZ-trnfM,trnL-ndhJ and ndhC-trnV,ycf1,ndhF-ccsA,ccsA + ccsA-ndhD and rps15-ycf1.We present phylogenetic analyses of 25 newly sequenced Populus plastomes and 35 plastomes from GenBank,which represent seven genera of Salicaceae,in combination with a matrix of eighteen morphological characters of 40 Populus taxa to reconstruct highly supported relationships of genus Populus.Relationships among the 60 taxa of Salicaceae strongly supported two monophyletic genera: Populus and Salix and five clades within Populus were divided.Clade I included the three taxa P.euphratica,P.pruinosa,and P.ilicifolia.Clade II contained thirteen taxa [P.adenopoda,P.alba,P.bolleana,P.davidiana,P.hopeiensis,P.nigra,P.qiongdaoensis,P.rotundifolia,P.rotundifolia var.duclouxiana,P.tremula,P.tremula × alba,P.tomentosa,and P.tomentosa(NC)].Clade III included the ten taxa P.haoana,P.kangdingensis,P.lasiocarpa,P.pseudoglauca,P.qamdoensis,P.schneideri,P.simonii,P.szechuanica,P.szechuanica var.tibetica,and P.yunnanensis.Clade IV included P.cathayana,P.gonggaensis,P.koreana,P.laurifolia,P.trinervis,P.wilsonii,and P.xiangchengensis.The last clade comprised P.angustifolia,P.balsamifera,P.deltoides,P.deltoides × nigra,P.fremontii,P.mexicana,and P.trichocarpa.This phylogeny is also supported by six morphological traits,including bark smoothness,bud size,petiole shape,leaf inflorescence,male anther length and male anther tip.(2)Phylogenetic relationship of Populus based on single copy nuclear genes: the phylogenetic relationship of 39 Populus was reconstructed based on the combined data of 16 single copy nuclear genes and one ITS fragment.The phylogenetic tree showed that monophyly of Populus was strongly supported,and four clades within Populus were divided.P.mexicana diverged first and formed a single clade with high support,followed by the nine species in section Populus.Clade III included three samples of section Turanga.The remaining species of section Tacamahaca,Leucoides and Aigeiros were in the last clade.(3)Phylogenetic relationship of Populus based on multiple nuclear genes and chloroplast genomes: with a combined data set of 17 nuclear DNA sequences and chloroplast genome sequences,all species of Populus clustered together as one clade and separated from outgroup species.The section of Turanga and Populus were all formed a robust monophyly by the comparison of the cpDNA tree and the nuclear gene tree.However,the relationships among some species were inconsistent between cpDNA tree and the nuclear gene tree:(1)In the cp genome phylogeny,P.mexicana of section Abaso was clustered with all the North American species in section Aigeiros and Tacamahaca,while in the nuclear phylogeny,P.mexicana of section Abaso diverged first with high support value in a basal position.(2)In P.nigra,cp genome analysis showed similarity to species of the section Populus,but nuclear gene analysis had a genetic affinity to species of section Tacamahaca.(3)In the cp genome phylogeny,the species in section Tacamahaca,Leucoides and Aigeiros were divided into three clades,and clade IV with clade V have obvious geographical distribution.However,in the nuclear gene phylogeny,the species in section Tacamahaca,Leucoides and Aigeiros clustered together as one clade with high resolution.(4)Alternative parents of P.tomentosa: We speculate that P.tomentosa was a nature hybrid between P.alba as female parent and P.tremula as male parent based on the nuclear genome and chloroplast genome data.(5)Phylogenetic relationship of Populus in southwest China: in the cp genome phylogeny,the species in southwest China were divided into Clade III and Clade IV.Clade III includes P.gonggaensis and P.wilsonii of section Leucoides,P.xiangchengensis,P.trinervis,P.koreana,P.cathayana and P.laurifolia of section Tacamahaca,and P.xiangchengensis is closely related to P.trinervis.In Clade IV,P.haoana and P.kangdingensis cluster together sister to P.pseudoglauca and P.schneideri,P.yunnanensis and P.szechuanica var.tibetica clustered together separating from other species in Clade IV.In the phylogenetic tree generated using nuclear data,the species in southwest China was divided into subclades D1 and D2.The first subclade consisted of two groups: P.szechuanica var.tibetica,P.xiangchengensis and P.haoana in the first group,two species of section Tacahamaca(P.trinervis and P.cathayana)and three species of section Leucoides(P.lasiocarpa,P.gonggaensis and P.pseudoglauca)were in the second group.In the second subclade(D2),P.yunnanensis,P.qamdoensis,P.szechuanica,P.ciliata cluster with P.kangdingensis and P.simonii,and was sister to P.schneideri.In addition,phylogenetic relationship of five species P.kangdingensis,P.xiangchengensis,P.cathayana,P.pseudoglauca and P.schneideri based on nuclear and cp genomes both supported that P.kangdingensis and P.schneideri,had a close genetic affinity.