Closely related species occurring in sympatry may experience negative consequences from interspecific pollen transfer. Pre-pollination reproductive isolation is a crucial mechanism that minimizes the opportunity for interspecific pollen transfer and facilitates the coexistence of closely related species. It is widely accepted that multiple barriers contribute to pre-pollination reproductive isolation between closely related species; however, few studies have quantified the relative importance of these factors. Here, we present a detailed analysis of isolation barriers among three species of the genus Tricyrtis: T. hirta, T. affinis, and T. ohsumiensis. These species coexist sympatrically within the same mountainous area in Kagoshima Prefecture. We quantified the roles of (1) spatial isolation, (2) temporal isolation, (3) differentiation of reproductive traits, (4) differentiation in pollinator assemblages, and (5) behavioral isolation of pollinators.

The results showed that: (1) T. hirta primarily occurs in low-elevation areas, while T. affinis and T. ohsumiensis co-occur at relatively higher elevations; (2) the peak blooming times of the three species do not overlap; (3) the flowers of each species are distinguishable from the perspective of bumblebees; (4) Bombus diversus is the primary pollinator, and pollinator assemblages overlap significantly among the species; and (5) in areas where all three species co-occur, heterospecific visitation events are limited by the pollinator constancy of B. diversus.

Our results suggest that the cumulative effects of multiple factors establish nearly complete pre-pollination reproductive isolation among three species. In particular, in species with highly overlapping spatial distributions, the synergistic effects of differing flowering phenology and pollinator constancy are likely to contribute significantly to reproductive isolation.