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

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.