Abstract: Examining the pollination biology of plant species is not only crucial for enhancing our understanding of their reproductive biology, but also essential for elucidating their adaptation and evolutionary history. Here, we investigated the breeding system and pollination biology of two closely related species in Actaea. The flower of A. purpurea is unique in the genus with purple and chartaceous (paper-like) sepals, fewer stamens with yellow anthers and purple filaments. Through three seasons of field observation and exclusion experiments, we determined that A. purpurea was primarily pollinated by a hornet species, Vespa bicolor, which also served as the most efficient pollinator. In contrast, A. japonica was primarily pollinated by large flies. Actaea purpurea exhibited a significantly higher cumulative nectar volume than A. japonica, which could be a crucial factor in attracting V. bicolor. A control experiment further demonstrated that removing the nectar leaf (petal) significantly decreased the visiting frequency of V. bicolor. Breeding system studies revealed that both species were self-compatible, yet they primarily underwent outcrossing in natural habitats. Our study presents a compelling case of possible pollinator shift in A. purpurea accompanied by morphological divergence. A more in-depth investigation of this system would offer crucial insights into the extent to which pollinators are involved in the plant speciation process and whether they contribute to reproductive isolation between closely related species.

Key words: Actaea purpurea,  Actaea japonica,  Breeding system, Pollinator shift, Speciation, Vespa bicolor