Differences in flowering time maintain species boundaries in a continental radiation of Viburnum.

PREMISE: We take an integrative approach in assessing how introgression and Pleistocene climate fluctuations have shaped the diversification of the core Lentago clade of Viburnum, a group of five interfertile species with broad areas of sympatry. We specifically tested whether flowering time plays a role in maintaining species isolation.

METHODS: RAD-seq data for 103 individuals were used to infer the species relationships and the genetic structure within each species. Flowering times were compared among species on the basis of historical flowering dates documented by herbarium specimens.

RESULTS: Within each species, we found a strong relationship between flowering date and latitude, such that southern populations flower earlier than northern ones. In areas of sympatry, the species flower in sequence rather than simultaneously, with flowering dates offset by ≥9 d for all species pairs. In two cases it appears that the offset in flowering times is an incidental consequence of adaptation to differing climates, but in the recently diverged sister species V. prunifolium and V. rufidulum, we find evidence that reinforcement led to reproductive character displacement. Long-term trends suggest that the two northern-most species are flowering earlier in response to recent climate change.

CONCLUSIONS: We argue that speciation in the Lentago clade has primarily occurred through ecological divergence of allopatric populations, but differences in flowering time were essential to maintain separation of incipient species when they came into secondary contact. This combination of factors may underlie diversification in many other plant clades.