Divergence depends on the strength of selection and frequency of gene flow between taxa, while reproductive isolation relies on mating barriers and geographic distance. Less is known about how these processes interact at early stages of speciation. Here, we compared population-level differentiation in floral phenotype and genetic sequence variation among recently diverged Castilleja to explore patterns of diversification under different scenarios of reproductive isolation. METHODS:Using target enrichment enabled by the Angiosperms353 probe set, we assessed genetic distance among 50 populations of four Castilleja species. We investigated whether patterns of genetic divergence are explained by floral trait variation or geographic distance in two focal groups: the widespread C. sessiliflora and the more restricted C. purpurea species complex. RESULTS:We document that C. sessiliflora and the C. purpurea complex are characterized by high diversity in floral color across varying geographic scales. Despite phenotypic divergence, groups were not well supported in phylogenetic analyses, and little genetic differentiation was found across targeted Angiosperms353 loci. Nonetheless, a principal coordinate analysis of single nucleotide polymorphisms revealed differentiation within C. sessiliflora across floral morphs and geography and less differentiation among species of the C. purpurea complex. CONCLUSIONS:Patterns of genetic distance in C. sessiliflora suggest species cohesion maintained over long distances despite variation in floral traits. In the C. purpurea complex, divergence in floral color across narrow geographic clines may be driven by recent selection on floral color. These contrasting patterns of floral and genetic differentiation reveal that divergence can arise via multiple eco-evolutionary paths.

Floral trait evolution mediated by pollinators is important in the diversification of flowering plants, yet few studies have demonstrated the range-wide geographic variation in both floral traits and pollinators which represents a predicted precursor for pollinator-mediated speciation. This study explores whether geographic variation in pollinator interactions underlies the observed patterns of floral divergence both 1) among species of the Castilleja purpurea complex (C. purpurea, C. citrina and C. lindheimeri) and the congener C. sessiliflora, as well as 2) within C. sessiliflora, across its wide geographic range. We sampled floral visitors and floral traits (morphology and color) at 23 populations across a 1900 km-wide study area in 1-3 years, with reproductive fitness (fruit set) data for 18 of these populations. A wide diversity of pollinator functional groups visited the focal species, including bees, butterflies, hawkmoths and hummingbirds, and visitor assemblages varied among species and across geography. We identified relationships between floral traits and visitation by certain pollinator groups, which often aligned with predictions based on pollination syndromes. Despite visitor assemblages being largely generalized across most populations, we found that the observed changes in floral traits were associated with shifts in the relative frequencies of key pollinator functional groups. Hence this study demonstrates that variation in pollinator assemblages across the distributions of taxa may underlie divergence in floral traits and suggests that highly specialized relationships may not be required for early stages of pollinator-mediated floral divergence. Our extensive sampling of 23 populations over multiple years across a large geographic area highlights the value of range-wide studies for characterizing patterns of divergence mediated by ecological interactions.