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This study confirms the prolonged germination and fertilization capacities of orchid pollinaria, and to our knowledge is the first report demonstrating a clear relationship between pollen viability and pollination system. It is proposed that this relationship is attributed to the different types of reproductive barriers, pre- or post-zygotic, that characterize Ophrys and Serapias and the food-deceptive species, respectively.

Plant species diversification entails the action of reproductive barriers, which are severely challenged when related species grow in contact and form hybrid progeny. Orchis italica and O. anthropophora are two related orchid species that produce a known hybrid form, O. xbivonae. Here, we analysed a hybrid zone of these two orchids using molecular analysis and experimental crosses. As molecular tools, we employed both real-time PCR and PCR amplification of nuclear markers to evaluate the occurrence of backcross recombination. With these approaches, we demonstrated that all examined hybrids belong to the F(1) generation. Chloroplast DNA analysis showed that O. anthropophora was the maternal species of most of hybrid specimens and that cytoplasmic introgression was lacking in both parental species. Pollination experiments showed that the two orchid species were strictly out-crossing, although self-compatible, and have comparable levels of reproductive fitness in all crossing treatments. Conversely, hybrids demonstrated low reproductive success in all intra- and back-crossing treatments. The absence of any backcross generations and plastid introgression suggest that O. xbivonae does not represent a bridge to gene flow between O. italica and O. anthropophora. Indeed, the low hybrid fitness testifies to the effectiveness of late post-zygotic barriers occurring between the parental species.

We quantified inbreeding depression for fruit production, embryo vitality and seed germination in three deceptive orchids, Serapias vomeracea, S. cordigera and S. parviflora, which do not provide any reward to their pollinators, and are predicted to experience high outcrossing. Of the three species examined only S. parviflora was autonomously selfing. Both S. vomeracea and S. cordigera showed highly significant differences in fitness between selfed and outcrossed progenies, resulting in high levels of inbreeding depression, which increased in magnitude from seed set to seed germination. Inbreeding depression may promote outcrossing in Serapias by acting as a post-pollination barrier to selfing. Cumulative inbreeding depression across three stages in S. parviflora was lower that in both outcrossing species. The large difference in germination between selfed and outcrossed seeds is an important issue in conservation biology.

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