Speciation in sexually deceptive orchids: pollinator-driven selection maintains discrete odour phenotypes in hybridizing species

Ophrys orchids mimic the female sex pheromones of their pollinator species to attract males for pollination. Reproductive isolation in Ophrys is based on the selective attraction of only a single pollinator species. A change of floral odour can result in the attraction of a new pollinator species that acts as an isolation barrier towards other sympatrically occurring Ophrys species. Ophrys lupercalis, Ophrys bilunulata, and Ophrys fabrella grow sympatrically and bloom consecutively on Majorca and are pollinated by three species of Andrena. We investigated variation of phenotypic and genotypic flower traits, aiming to study the role of the floral odour for reproductive isolation and speciation. Using chemical and electrophysiology (gas chromatography coupled with an electroantennographic detector) methods, we show that the three Ophrys species use the same odour compounds for pollinator attraction, but in different proportions. A comparison of the floral odour bouquets in a multivariate analysis revealed a clear grouping of plants from the same species, although with an overlap between species. A comparison of the same plants using molecular markers gave a contrasting result. Although O. lupercalis and O. fabrella were genetically well separated, plants of O. bilunulata did not form a distinct group but were similar to either O. lupercalis or O. fabrella. Our data indicate gene flow and hybridization to occur between O. bilunulata and O. lupercalis as well as between O. bilunulata and O. fabrella. All plants of O. bilunulata, despite having different genotypes, showed a very similar floral odour. This reflects a strong selective pressure by the pollinating males. The overlap of genotypes of O. bilunulata and O. fabrella supports our hypothesis that O. fabrella diverged from O. bilunulata by scent variation and the attraction of a new pollinator species, Andrena fabrella