Function and evolution of isolation genes in Ophrys orchids

Orchids of the European genus Ophrys imitate the mating signals of female insects and are pollinated by sexually aroused male insects, often on a species-specific basis. The specificity of pollinator attraction is determined by the ratios of different alkenes in the floral odour bouquet. Genes encoding proteins involved in the biosynthesis of these compounds may thus be "isolation genes" since they are directly linked to reproductive isolation among plant individuals. This project seeks to use powerful molecular tools that have already been shown to work in the study system, to identify the molecular basis of specific odour bouquets and their evolutionary patterns. The questions addressed are: (i) What is the molecular basis for differences in floral odour within and among species? (ii) What is the evolutionary pattern apparent in genes involved in the biosynthesis of floral odour in the study system? An Ophrys species-pair with distinct floral odours will be used to identify the genes responsible for the differences. Based upon promising preliminary investigations, the candidate gene approach will be followed to identify stearoyl-ACP desaturase genes. The enzymes encoded by these genes catalyse the first introduction of a double bond into fatty acids, the alkenes` precursor compounds, and are thus of key importance for the regulation of alkene production. The expression of these genes in different tissues in the two species will be investigated by quantitative PCR, since a change in the expression patterns among species may be a mechanism leading to a change in the alkene pattern. Desaturase enzymes may also change their substrate preference, or the position of double bond introduction in the fatty acyl chain. Therefore, the relevance of these mechanisms in the study system will be investigated by functional assays. Yeast mutants deficient of the target genes will be transformed with Ophrys desaturase genes and fed with saturated fatty acids. Unsaturated fatty acids produced by the transformed yeast will be analysed by gas chromatography. To test the hypothesis that different odour bouquets leading to reproductive isolation can evolve in sympatric populations, the evolutionary history of functional, orthologous genes underlying odour differences will be reconstructed using modern phylogenetic methods. This project, by investigating the molecular basis of floral traits responsible for reproductive isolation, will be highly novel and relevant for understanding the evolution of floral diversity in many plant-pollinator systems.