Medicago truncatula is a model legume that has been extensively investigated in diverse subdisciplines of plant science. Medicago littoralis can interbreed with M. truncatula and M. italica; these three closely related species form a clade, i.e. TLI clade. Genetic studies have indicated that M. truncatula accessions are heterogeneous, but their taxonomic identities have not been verified. To elucidate the phylogenetic position of diverse M. truncatula accessions within the genus, we assembled 54 plastid genomes (plastomes) using publicly available next-generation sequencing data and conducted phylogenetic analyses using maximum likelihood. Five accessions showed high levels of plastid DNA polymorphism. Three of these highly polymorphic accessions contained sequences from both M. truncatula and M. littoralis. Phylogenetic analyses of sequences placed some accessions closer to distantly related species suggesting misidentification of source material. Most accessions were placed within the TLI clade and maximally supported the interrelationships of three subclades. Two Medicago accessions were placed within a M. italica subclade of the TLI clade. Plastomes with a 45-kb (rpl20-ycf1) inversion were placed within the M. littoralis subclade. Our results suggest that the M. truncatula accession genome pool represents more than one species due to possible mistaken identities and gene flow among closely related species.

Plastid genomes were assembled using GetOrganelle based on publicly available next-generation sequencing data.

Sequences were aligned using MAFFT.

Phylogenetic analyses were conducted using IQ-TREE 2.

GenBank format files (.gb) of plastomes can be opened using SnapGene Viewer.

Tree files (.newick) and sequence alignments (.fasta) can be opened using MEGA.