Reproductive isolation emerges from coordinated barriers during mating system divergence in plants.

Understanding how reproductive barriers combine to restrict gene flow remains a central challenge in speciation research. Although reproductive isolation is inherently a composite process, most empirical studies have focused on individual barriers in isolation, limiting our ability to capture their joint effects, particularly in systems undergoing evolutionary transitions such as shifts in mating system. Here, we provide a comprehensive, life-cycle-wide quantification of reproductive isolation between two closely related species of the Erysimum incanum complex that differ markedly in mating system: the predominantly selfing E. incanum and the outcrossing E. wilczekianum. By integrating ecological, phenological, behavioural, and post-pollination components, we show that total reproductive isolation is nearly complete (T = 0.999) but largely driven by pre-pollination barriers. Ecogeographical differentiation and, most prominently, pollinator-mediated isolation dominates, with pollinators showing a strong bias towards E. wilczekianum. Floral traits associated with mating system divergence, particularly flower size, emerge as key drivers of assortative mating, supporting their role as 'magic traits' linking ecological divergence and reproductive isolation. In contrast, post-pollination barriers are weaker but strongly asymmetric. Hybrid seed formation is largely prevented when E. wilczekianum acts as the maternal parent, whereas reciprocal crosses are relatively successful. Despite reduced germination, hybrids show enhanced growth and no evidence of hybrid breakdown, suggesting that intrinsic incompatibilities remain incomplete. Overall, our results indicate that mating system divergence reshapes the entire architecture of reproductive isolation, highlighting the importance of integrative approaches to understand how multiple barriers interact during early stages of speciation.