Incomplete reproductive isolation and low genetic differentiation despite floral divergence across varying geographic scales in Castilleja.

PREMISE: 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.

Adaptive generalization in pollination systems: Hawkmoths increase fitness to long-tubed flowers, but secondary pollinators remain important.

Selection on floral traits by animal pollinators is important in the evolution of flowering plants, yet whether floral divergence requires specialized pollination remains uncertain. Longer floral tubes, a trait associated with long-tongued pollinators, can also exclude other pollinators from accessing rewards, a potential mechanism for specialization. Across most of its range, Castilleja sessiliflora displays much longer corollas than most Castilleja species, though tube length varies geographically and correlates partially with hawkmoth visitation. To assess whether long corolla tubes reflect adaptation to hawkmoth pollinators, we performed a day/night pollinator exclusion experiment in nine natural populations that varied in corolla length across the range of C. sessiliflora and short-tubed members of the parapatric C. purpurea complex. We compared the fitness contributions of nocturnal and diurnal visitors, revealing that long-tubed populations visited predominantly by hawkmoths experienced greater fruit set at night, in contrast with short-tubed populations or those visited mainly by diurnal pollinators. Next, leveraging a range-wide multiyear dataset of pollinator visitation to these species, we identify that hawkmoth visitation is associated with increased fitness in long-tubed populations overall, and that long tubes are associated with less diverse visitor assemblages. Thus, long corollas represent an adaptation to hawkmoth pollination at the exclusion of diverse pollinators. Nonetheless, while hawkmoths were scarce in the northern range, secondary diurnal pollinators contributed to fruit set across the range, providing reproductive assurance despite possible trait mismatch. This study illustrates adaptive generalization in pollination systems and that floral divergence may proceed along a continuum of generalized and specialized pollinator interactions.