The Medicago sativa species complex includes tetraploid cultivated alfalfa and several other diploid and tetraploid taxa that are recognized either as subspecies of M. sativa or as separate species. The two principal diploid taxa are “caerulea,” with purple
flowers and coiled pods, and “falcata” with yellow flowers and falcate pods. To understand the evolutionary relationships among taxa in the complex, sequence variation in two noncoding regions of cpDNA (rpl20–rps12 and trnS–trnG spacers)
and three regions of mitochondrial DNA (mtDNA: nad4 intron, nad 7 intron, and rpS14–cob spacer) were surveyed from 48 (37 for mtDNA) individuals representing these and other diploid taxa in the complex. These sequences afforded independent perspectives on
the evolutionary history of the group, because mtDNA is maternally inherited in Medicago whereas cpDNA is biparentally inherited with strong paternal bias. Twenty and 21 haplotypes were identified for cpDNA and mtDNA, respectively. Haplotype networks were constructed and tests of differentiation
were conducted. Results from cpDNA sequences supported the recognition of “caerulea” and “falcata” as differentiated taxa, despite the presence of some shared haplotypes, in agreement with morphological characters. In contrast, no significant evidence of mtDNA haplotype
differentiation was observed. Incongruence between cpDNA and mtDNA is more likely explained by introgression of the mitochondrial genome than by incomplete lineage sorting of mtDNA haplotypes, given the expected smaller effective population size for uniparentally inherited mtDNA than for biparentally
inherited cpDNA. Moreover, the two taxa are readily crossable, making natural hybridization possible. The long-time disagreement on whether to recognize “falcata” as a separate species or a subspecies of M. sativa s. l. is due to the common problem of unequal rates of differentiation
for different characters during speciation.