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

Abstract Background and Aims Post-pollination pre-zygotic barriers involve interactions between the 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 by 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 barriers related to the interaction between the pollen tube and pistil or between the pollen tube and ovule 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 is likely to have existed in the lineage before species separation and has strengthened progressively over the time of species divergence. In contrast, pollen–pistil incompatibilities might have evolved several times and asymmetrically after species divergence, probably prompted by ecological interactions between species during secondary contact.