Phylogenetic Analysis And Taxonomic Study Of Medicago Sensu Lato In China

Medicago sensu lato(including Medicago,Trigonella and Melilotus;Leguminosae)is mainly distributed in temperate regions of the world and widely existed in grassland ecosystem,and all of them are important forage resource.Among them,alfalfa has the reputation of being the king of forages and is widely cultivated worldwide.However,the taxonomic status of Medicago sensu lato is chaotic,leading to different taxonomic opinions on some taxa.Here,we focus on species of the three genera Medicago,Trigonella and Melilotus that are distributed in China.We used DNA barcodes,chloroplast genome sequences and whole genome resequencing data,and combined morphological traits to investigate the phylogenetic relationships and species delimitation among these Chinese species.Wer then based on the results to carry out the taxonomic revisions.The main results are as follows:1.We collected samples representing 21 Chinese naturally distributed species of these three genera,and applied an integrative approach by combining evidences from population-based morphological clusters and molecular data to investigate phylogenetic relationships and species boundaries.A total of 186 individuals selected from 156 populations and 454 individuals from 124 populations were collected for genetic and morphological analyses,respectively.The results showed that 16 out of 21 species could be well delimited based on phylogenetic analyses and morphological clusters.The four "Medicagoid" species is more closely to Medicago edgeworthii than the other Trigonella species,and nested within the Medicago clade in nuclear DNA(nr DNA)phylogenetic tree,supporting the previous finding that the "Medicagoid" species should be transfer to the genus Medicago.Trigonella emodi and Trigonella fimbriata cannot be delimited based on either phylogenetic analyses or morphological clusters,which suggest that the two species should be treated as one species.Medicago sativa,Medicago falcata and Medicago × varia cannot be separated either in phylogenetic tree or in morphological principal component analysis,indicating that these three species should be treated as subspecies of the Medicago sativa complex.The relationships among the four species of Medicago section Platycarpae are incongruent in the chloroplast tree and nr DNA tree,especially Medicago edgeworthii,which is clustered with the four "Medicagoid" species.The incongruent relastionships between the two types of markers may be resulted from hybrization/introgression and incomplete lineage sorting.2.In order to further clarify the phylogenetic relationships of these Chinese Medicago sensu lato species,we assembled the chloroplast genomes of 75 individuals representing 21 species and downloaded the chloroplast genome sequences of Medicago truncatula available in Gen Bank database.After including Trifolium subterraneum(NC?011828.1)as the out group,a matrix containing 311 alignment sequences(113 coding regions and 198 non-coding regions)of 21 species was used to reconstruct a phylogenetic tree.The results showed that 18 species are well delimeted except for Medicago sativa,Medicago falcata and Melilotus officinalis.The chloroplast genomic tree also supports two large clades: Medicago clade and Melilotus/Trigonella clade.The four "Medicagoid" species are nested in the Medicago clade and grouped with Medicago edgeworthii.Melilotus and Trigonella are reciprocally monophyletic to each other.3.We used whole-genome resequencing data of 80 individuals representing 21 species of the three genera to reconstruct a phylogenomic tree.The main clades inferred from this phylogenomic tree is similar to that inferred from the chloroplast genomic tree.Furthermore,Melilotus officinalis is grouped as monophyletic clade,and Medicago sativa and Medicago falcata can be separated in the nuclear genomic tree.However,the basal position of the Medicago clade between the two trees is different:three species of Medicago section Platycarpae is basal in the nuclear genomic tree,while in the chloroplast genomic tree,the six species clade(i.e.,M.lupulina,M.minima,M.polymorpha,M.truncatula,M.sativa,M.falcata and M.× varia)is basal.Hybrization/introgression,incomplete lineage sorting and incomplete samping may account for this incongruent relastionship.Finally,the phylogenetic relationships based on genomic level data both support that the four "Medicagoid" species should be transfered into the genus Medicago,and Trigonella emodi and Trigonella fimbriata should be combined as one species.4.Based on the morphological and molecular evidence,we reveals the phylogenetic relationship of Medicago sensu lato species in China and carry on an appropriate taxonomical revision: the four "Medicagoid" species(Trigonella cancellata,T.arcuata,Medicago orthoceras and M.monantha)should be transfer to the genus Medicago,and Trigonella emodi and Trigonella fimbriata should be treated as one species.Therefore,there are 15 Medicago species in China(Medicago sativa complex,M.lupulina,M.platycarpos,M.archiducis-nicolai,M.ruthenica,M.edgeworthii,M.minima,M.polymorpha,M.monantha,M.orthoceras,M.connivens,M.arcuata,M.arabica,M.praecox and M.arborea)and three subspecies(Medicago sativa L.subsp.sativa,Medicago sativa L.subsp.falcata L.and Medicago sativa L.subsp.× varia Martyn),four Melilotus species(Melilotus officinalis,M.albus,M.dentatus,M.indicus),and four Trigonella species(Trigonella foenum-graecum,T.coerulea,T.emodi,T.cachemiriana).