The presence and strength of post-pollination barriers in an orchid lineage that lacks pollinator specialization

Background and aims: Post-pollination pre-zygotic barriers involve interactions between pollen, pistil, and ovules that impede fertilization by heterospecific pollen. These barriers act sequentially when pollen from different donors simultaneously pollinates the same flower, by inhibiting the development of foreign pollen or conferring a fecundation advantage to the specific pollen tube. Here, we investigated the presence and strength of post-pollination barriers in an orchid lineage that lacks pollinator specialization. We then evaluated the relationship between early and late post-pollination pre-zygotic barriers and their chronology of insurgence. Methods: We conducted hand pollinations by adding conspecific and/or heterospecific pollen to the stigma of the receiving species. From all crosses, we investigated the formation of fruits, the development of the pollen tubes (using scanning and epifluorescence microscopy), the percentage of embryonated seeds, and the seed paternity by molecular analyses of in vitro germinated seeds (protocorms) produced in double pollinations. Key results: Post-pollination pre-zygotic barrier related to the interaction between pollen tube and pistil or between pollen tubes and ovules were found to be variable in strength and with strong asymmetry in species pairs. Conspecific pollen precedence (CPP) was found to be strong in most cross combinations: when both conspecific and heterospecific pollen were added simultaneously, conspecific pollen fertilized most of the ovules. Conclusions: We observed that CPP is stronger than early gametic incompatibility as pollen-pistil incompatibility, despite the latter occurring earlier than CPP. We did not find that species with stronger early gametic incompatibility have less CPP, as expected from a sequential development of these post-pollination barriers. CPP likely existed in the lineage before species separation and has progressively strengthened over the time of species divergence. Instead pollen-pistil incompatibilities may have evolved several times and asymmetrically after species divergence, likely prompted by ecological interactions between species during secondary contact.