Detection of reproductive interference between closely related Salvia species with small-scale separated distributions by multifaceted pollination and molecular analyses.

Reproductive interference, an interspecific interaction in reproductive process that exerts an adverse effect, has gained attention as a contributing factor in promoting exclusive distributions between closely related species. However, detailed studies on the possibility of reproductive interference between native plants are still lacking, presumably because strong reproductive interference can rapidly realize exclusive distributions, leaving the two species apparently independent. Salvia japonica and S. lutescens are found in separate localities at a small scale, although their distributions overlap at a large scale. We investigated the possibility of reproductive interference between them through field surveys, hand-pollination experiments, evaluation of hybrid fertility, cpDNA and nrDNA genotyping, and genome-wide DNA analysis. The field survey results did not reveal apparent negative interaction in competition for pollinator services. Mixed pollination with conspecific pollen and counterpart pollen reduced seed set in S. japonica, and hybrid progeny produced by mixed pollination were less than 20% as fertile compared to the pure species. The DNA genotyping results suggested the possibility of hybridization where their distributions overlap, and the genome-wide DNA analysis results showed clear genetic differentiation between the two species as well as the existence of hybrids. These results suggest that bi-directional reproductive interference between S. japonica and S. lutescens may have led to their present separated distributions at a small scale.