Divergence in basic chromosome numbers among closely related species is widespread in plants, yet a fundamental question regarding the evolutionary direction of karyotype—whether descending (from higher to lower numbers) or ascending (from lower to higher)—remains contentious. Alfalfa (Medicago sativa L.), a key forage crop, displays two basic chromosome numbers (x = 8 and x = 7) within the genus, and whether this divergence arose through descending evolution from 8 to 7 or the reverse remains unclear. Here, we developed a set of chromosome-specific painting markers capable of tracing chromosomal evolutionary trajectories among Medicago species. Comparative cytological analysis of seven accessions (x = 8) from the M. sativa L. complex revealed conserved chromosomal synteny in both diploid and autotetraploid species, with no detectable interchromosomal rearrangements. In Medicago polymorpha (x = 7), we discovered that the divergence in basic chromosome numbers (x = 7 vs. x = 8) resulted from large-scale fission–fusion events involving chromosomes 3, 5, and 6, rather than the simple fusion of chromosomes 3 and 7 as previously published genomic hypotheses. Further supporting evidence from rDNA remodeling and phylogenetic analysis indicates a descending evolutionary pathway, with the ancestral x = 8 transitioning to x = 7 approximately Mid-Miocene (~12 million years ago). Our results offer new insights into Medicago speciation and evolutionary origins, and instantiate a strategy for studying karyotypic evolutionary direction in other plant taxa with similar chromosomal dynamics.

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