Divergence in flowering time is a major component contributing to reproductive isolation between two wild rice species (Oryza rufipogon and O. nivara).

It is of critical importance for our understanding of speciation process to determine the forms of reproductive isolation and their relative importance in species divergence. Oryza nivara and O. rufipogon are direct ancestors of Asian cultivated rice and a progenitor-daughter species pair. Investigating the reproductive isolation between them provides insights into plant speciation and helps understanding of the rice domestication. Here, we quantitatively measured the major components of reproductive isolation between the two species based on common garden and crossing experiments for three pairs of sympatric populations in Nepal, Cambodia and Laos. We revealed significant differences in the flowering times between species pairs, with O. nivara flowering much earlier than O. rufipogon. A very weak reduction in seed set but no reduction in F1 viability and fertility were detected for the crosses between species relative to those within species. Moreover, we detected asymmetrical compatibility between species and found that emasculation significantly decreased pollination success in O. nivara but not in O. rufipogon. Our study demonstrates that the divergence between O. nivara and O. rufipogon is maintained almost entirely by the difference in flowering times and suggests that differential flowering times contribute to both habitat preferences and reproductive isolation between species.