Plastid Phylogenomics Of Fabaceae

Fabaceae,also named as Leguminosae,is the third largest and second economically important angiosperm family,consisting of ca.770 genera and more than 19,500 species.Legumes distribute worldwide,vary extremely in life forms and habitat,and are found to be major components of most of the world's vegetation types with great ecological value.Traditionally,Fabaceae is divided into three subfamilies based on flower structure,i.e.,Caesalpinioideae,Mimosoideae,and Papilionoideae.With the development of molecular phylogenetics,however,the subfamily Caesalpinioideae was found non-monophyletic,a revised classification system for legumes is gradually recognized urgent by legume researchers.Based on a phylogenetic framework reconstructed from a near-complete sampling of genera and the chloroplast matK gene,the Legume Phylogeny Working Group proposed a six-subfamily classification at the beginning of 2017,i.e.,Caesalpinioideae,Cercidoideae,Detarioideae,Dialioideae,Duparquetioideae,and Papilionoideae.However,the phylogenetic relationships of subfamilies remain unresolved.In addition,taxonomic and phylogenetic issues of major clades within subfamilies remain confused.Plastid phylogenomics has been successfully used in resolving difficult relationships of many plant lineages,and thus provides better frameworks for researching on the feature,variation,and evolution of plastomes.Nevertheless,few legume plastomes have been reported of which most are from Papilionoideae.Extreme variations have been found in papilionoid plastomes,and only a few other variations have been reported in other subfamilies recently.Then,could phylogenomics based on plastomes resolve the phylogenetic relationships of major lineages of Fabaceae?Are non-papilionoid legumes also have major variation?To explore these issues,this study conducted a plastid phylogenomics of Fabaceae based on a wide sampling for the first time,by sequencing and assembling plastomes of 148 legume species(representing 135 genera and all six subfamilies),and integrating previously published plastome data.Maximum likelihood phylogenetic trees were reconstructed using different combination of plastid loci and different partitioning methods.And the plastome feature,variation and evolution of two lineages were analyzed in detail on plastome phylogeny frameworks.Main findings are summarized as following:1.Legume phylogenetic reconstruction based on plastomesRelatively constant topology and high support values were obtained from the maximum likelihood phylogenetic trees of Fabaceae based on different plastomic loci and partitioning methods.All six subfamilies were supported as monophyly.Phylogenetic relationships of subfamilies were resolved as(Cercidoideae,Detarioideae),(Duparquetioideae,(Dialioideae,(Caesalpinioideae,Papilionoideae))).Major clades within subfamilies were strongly supported as monophyly,and phylogenetic relationships among them were mostly resolved.Different loci with different evolutionary rate have different ability in resolving phylogenetic relationships at different taxonomic level.This study found that plastid introns are the best choice for resolving deep phylogenetic relationships of legumes,while inter-genic spacers(IGS)are better for tip taxa.Different partitioning methods got similar topologies and bootstrap values(BS),implying the results based on both partitioning methods are reliable.In the phylogenies reconstructed from GBLOCKS-treated IGS,BS are less,genera relationships are worse resolved,and conflicts among loci are higher.2.Character and variation of mimosoid plastomesThe plastomes of all sampled species from the mimosoid clade displayed the typical quadripartite structure.Part of them also have conserved size,gene content and order.While these within a clade comprising Ingeae and Acacia s.s.all experienced ca.13-kb IR(inverted repeat region)expansion into SSC,resulting in the largest known legume plastomes.This 13-kb IR expansion into SSC was traced as the synapomorphy of this clade,which was then named as Inverted repeat-expanding clade(IREC).Plastomes of a few individuals in this clade also experienced some other structural variations,such as IR-LSC junction shift,intron loss,gene duplication,and sequence variation.Plastome size was influenced by not only IR expansion and contraction,but also composition of repeats.In addition,repeats may also contribute to the high variation of plastome.Employing sequence divergence analysis,this study calculated the number and percentage of variable sites and parsimony informative sites for every gene,intron,and IGS,this could supply information of valuable markers for further phylogenetic and evolutionary studies.Additionally,nucleotide substitution rate of plastome coding genes has been proved to be decreased after translocating from single-copy region to IR following the expansion and contraction of IR.3.Character and variation of Cercidoideae plastomes and isomeric plastomesThe plastomes of all sampled Cercidoideae species also presented typical quadripartite structure and conserved gene content,except one intron loss in one species and the truncated accD and matK at their 5'-ends in several species.While plastomes of some other species experienced unusual rearrangements like IR expansion/contraction and sequence inversion.IR expansion/contraction were discovered common in species from Bauhinia s.1.except Bauhinia.Four different inversions were found in four species,a 24-kb and a 1.3-kb inversions were found in Griffonia simplicifolia and Piliostigma thonningii,respectively,a 7.5-kb inversion was found in the two Tylosema species,and a 38-kb inversion was found in the newly assembled plastome of Tylosema fassoglensis,locating between a pair of 29 bp inverted repeats located at the 3'-ends of tmSGCU and trnSGGA genes.Read-mapping and PCR results both indicated the plastomes with this inversion coexist with canonical-type plastomes in all four sampled Tylosema individuals,with one type is major while another is minor,and the abundance of the two arrangements shift among different individuals.Isomeric plastomes are firstly found in legumes.