Abstract: Wild perennial sister species Medicago archiducis-nicolai (rhizomatous/alpine) and M. ruthenica (non-rhizomatous/xeric) constitute vital genetic resources for forage improvement. To decode the genomic basis of their contrasting trait and habitat adaptation, we generated chromosome-scale genome assemblies, resequenced 128 individuals, profiled transcriptomes under cold/heat stress, and functionally validated causal alleles. We demonstrate that structural variations (SVs)—particularly gene duplications—are primary drivers of rhizome formation and alpine/xeric adaptation. Further, pervasive presence–absence SVs (PAVs) in noncoding regulatory regions underpin divergent allele-specific expression governing rhizome development and stress responses. Crucially, these regulatory PAVs induce contrasting expression patterns during trait development and stress adaptation. Our findings reveal a dual mechanism whereby coding and regulatory SVs convergently orchestrate phenotypic innovation and ecological specialization in sister species, offering valuable genomic resources for legume evolution studies and alfalfa breeding.

Key words: adaptive divergence, comparative genomics, gene duplication, rhizome development, sister species, structure variations