Pre-zygotic reproductive isolation effectively isolates two Actae a species with different flower colour and pollinators

Speciation has long been a central topic in evolutionary biology, with reproductive isolation (RI) playing a fundamental role in this process by limiting gene flow between diverging populations. However, the relative contributions and temporal sequence of establishment of different reproductive barriers remain poorly understood, particularly in plants. In this study, we quantify RI between Actaea purpurea and A. japonica—two species exhibiting significant differences in floral morphology and pollinator preferences—with particular emphasis on pollinator isolation. We measured five reproductive barriers: ecogeographic isolation, phenological isolation, pollinator isolation, pollen-stigma incompatibility, and post-zygotic isolation, using hybridization experiments and ecological niche modelling with varying grid size. Pollinator isolation was assessed by establishing artificial sympatric populations. Our results demonstrate strong total RI between the two species (>0.99), supporting their status as distinct biological species with minimal gene flow. Pre-zygotic barriers contributed more significantly to RI than post-zygotic isolation. Ecogeographic isolation was the largest contributor to total RI, followed by pollinator isolation. This study underscores the importance of both pollinator isolation and ecogeographic factors in speciation and species maintenance in Actaea, and suggests that pre-zygotic reproductive barriers may arise earlier and play a dominant role in speciation: a pattern that may be widespread in flowering plants.