The Orchidaceae is the most diverse family of angiosperms with an estimated number of over 24,000 species. It has long been believed that co-evolution with pollinating animals has played an instrumental role in the unparalleled diversification of the Orchidaceae. Many orchid species, however, are generalists with respect to their pollinators. Because the availability of a suitable mycorrhizal fungus is crucial to orchid establishment, it has recently been suggested that diversification might also have been driven by irregular fungal distributions, combined with high mycorrhizal specificities.Evidence for this hypothesis is still lacking, and at present, very little is known about the nature and specificity of orchid-mycorrhizae associations. Most studies so far have reported high specificity and single-species associations. However, from an ecological point of view high specificity and single-species associations are puzzling and it is unclear whether the reported results might be biased by methodological issues.The general aim of the proposed study is to reveal the potential role of mycorrhizal associations in orchid diversification. More specifically, I want to elucidate the nature and specificity of orchid-mycorrhizae associations, to investigate the availability of orchid mycorrhizae in natural populations and to study the role of mycorrhizal associations in acting as a post-mating reproductive barrier. To this end, I will apply, for the first time, multiplex assays that can detect several fungi simultaneously.The study will be conducted across the whole distribution area of 21 species of the genus Orchis. I will test for mycorrhizal specificity and investigate evolutionary trajectories of orchid-mycorrhizae associations. Advanced spatial point pattern analyses combined with seed baiting techniques will be used to detect spatial variation in fungal availability in natural populations.