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

Background and AimsUnderstanding 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. MethodsHere, we provide a comprehensive, life-cycle-wide quantification of reproductive isolation between two closely related species of the Erysimum incanum complex that differ strikingly in mating system: the predominantly selfing E. incanum and the outcrossing E. wilczekianum. Key ResultsBy integrating ecological, phenological, behavioural, and post-pollination components, we show that total reproductive isolation is nearly complete (T{approx}0.999), but overwhelmingly driven by pre-pollination barriers. Ecogeographical differentiation and, most prominently, pollinator-mediated isolation dominate, with pollinators exhibiting a strong bias toward E. wilczekianum. Floral traits linked to mating system divergence, particularly flower size, emerge as key drivers of assortative mating, supporting their role as "magic traits" coupling ecological divergence with 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, consistent with expectations from mating system differences, whereas reciprocal crosses are relatively successful. Despite reduced germination, hybrids display enhanced growth and no evidence of hybrid breakdown, suggesting that intrinsic incompatibilities remain incomplete. ConclusionsThese findings reveal that mating system divergence restructures the entire architecture of reproductive isolation rather than acting as a single barrier. More broadly, our results highlight that early-stage speciation can be driven by coordinated shifts in ecological and reproductive traits, emphasizing the need for integrative approaches to fully understand how barriers interact to generate species boundaries.