Strong post-pollination pre-zygotic isolation between sympatric, food-deceptive Mediterranean orchids

Mediterranean orchids that grow in admixed,co-flowering populations, and frequently show hybridprogenies are interesting to use to study the nature and thestrength of post-zygotic barriers. However, examination ofpre- and post-pollination pre-zygotic isolating mechanismsrequires sympatric, co-flowering species pairs that do notproduce hybrid swarms. In this study, we analyzed a contactzone between Orchis italica and O. papilionacea, inwhich hybrid forms have never been reported, althoughhybridization between members of their groups of appurtenancehas been signaled. We investigated pre-pollinationbarriers observing the floral phenology of both species andidentified pollinators by means of molecular analysis ofpollinaria collected on the insects captured in the study site.Post-pollination barriers were tested performing manualcrosses in order to evaluate pollen germination/pollen tubegrowth in vivo and fruit and seed formation. Floral phenologiesof O. italica and O. papilionacea display nearlyoverlapping trends, and two common pollinators have beenidentified by molecular analysis of pollinaria. Thus,pre-pollination barriers are very weak or nonexistent.Bidirectional crosses have shown that the growth ofheterospecific pollen tubes is fully blocked in stigmatic celllayers. Since no fruit formation was detected in bidirectionalinterspecific crosses, we assume that reproductiveisolation between the examined species is fully guaranteedby post-pollination pre-zygotic mechanisms acting at stigmatic level. Such condition has been rarely describedand may mask the potential action of post-zygoticmechanisms