The acquisition of £oral nectar spurs is correlated with increased species diversity across multiple clades. We tested whether variation in nectar spurs in£uences reproductive isolation and, thus, can potentially promote species diversity using two species of Aquilegia, Aquilegia formosa and Aquilegia pubescens, which form narrow hybrid zones. Floral visitors strongly discriminated between the two species both in natural populations and at mixed-species arrays of individual £owers. Bees and hummingbirds visited £owers of A. formosa at a much greater rate than £owers of A. pubescens. Hawkmoths, however, nearly exclusively visited £owers of A. pubescens. We found that altering the orientation of A. pubescens £owers from upright to pendent, like the £owers of A. formosa, reduced hawkmoth visitation by an order of magnitude. In contrast, shortening the length of the nectar spurs of A. pubescens £owers to a length similar to A. formosa £owers did not a¡ect hawkmoth visitation. However, pollen removal was signi¢cantly reduced in £owers with shortened nectar spurs. These data indicate that £oral traits promote £oral isolation between these species and that speci¢c £oral traits a¡ect £oral isolation via ethological isolation while others a¡ect £oral isolation via mechanical isolation.

SummaryOne of Verne Grant's lasting contributions to plant evolutionary biology has been the recognition that differences between plants in floral characters can have a dramatic impact on both pollinator visitation and pollen transfer and thus affect reproductive isolation between nascent plant species (collectively, floral isolation). Here we review some of the concepts and findings from Grant's work on floral isolation, particularly with respect to the genus Aquilegia (Ranunculaceae). It has now been over 50 yr since Grant first published on the role of floral isolation on reproductive isolation and speciation in Aquilegia and we compare and contrast his findings with our own work on this genus. We find that the data largely support Grant's findings and that Aquilegia will continue to offer great opportunities to learn about the processes of adaptation and speciation.

Reproductive isolation between Aquilegia formosa and Aquilegia pubescens is influenced by differences in their flowers through their effects on pollinator visitation and pollen transfer. Here, we investigate the genetic basis of floral characters differentiating these species. We found that in addition to the effects of flower orientation and the length of nectar spurs previously described, other characters such as flower color or odor affect hawkmoth visitation. Repeatability of measurements in an F2 population ranged from 0.53 to 0.83 among five floral traits, indicating that using the means of multiple measures per plant will substantially increase the power of a quantitative trait locus (QTL) analysis. Integration of floral traits was indicated by significant correlations among traits in an F2 population. In a separate F2 population we found that QTL for different floral traits were often closely associated, indicating that linkage or pleiotropy cause at least some of this integration. In addition, we found QTL for all floral traits examined. Because Aquilegia species are largely interfertile and vary extensively in both floral morphology and ecology, they offer the opportunity for QTL studies of a wide range of characters affecting reproductive isolation.