Plastid phylogenomics uncovers multiple species in Medicago truncatula (Fabaceae) germplasm accessions

<p><em>Medicago</em> <em>truncatula</em> is a model legume that has been extensively investigated in diverse subdisciplines of plant science. <em>Medicago</em> <em>littoralis</em> can interbreed with <em>M</em>. <em>truncatula</em> and <em>M</em>. <em>italica</em>; these three closely related species form a clade, i.e. TLI clade. Genetic studies have indicated that <em>M</em>. <em>truncatula</em> accessions are heterogeneous, but their taxonomic identities have not been verified. To elucidate the phylogenetic position of diverse <em>M</em>. <em>truncatula</em> 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 <em>M</em>. <em>truncatula</em> and <em>M</em>. <em>littoralis</em>. 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 <em>M</em>. <em>italica</em> subclade of the TLI clade. Plastomes with a 45-kb (rpl20-ycf1) inversion were placed within the <em>M</em>. <em>littoralis</em> subclade. Our results suggest that the <em>M</em>. <em>truncatula</em> accession genome pool represents more than one species due to possible mistaken identities and gene flow among closely related species.</p>