Artificial crossing and pollen tracking reveal new evidence of hybridization between sympatric Platanthera species

Studies focusing on hybrid zones contribute to a better understanding of reproductive isolation and speciation processes. Reproductive isolation is achieved by various pre- and postmating barriers that act sequentially and together determine whether interspecific mating and hybridization can occur. In this study, we investigated various reproductive barriers acting between Platanthera bifolia and P. chlorantha. These species are known to hybridize in the field, suggesting that reproductive barriers are weak. We conducted a pollen tracking and crossing experiments within and between the species in natural populations in Estonia and germinated seeds in vitro for molecular analysis. We also combined morphological measurements with analysis of 233 AFLP loci to examine variation in P. chlorantha, P. bifolia and their putative hybrids in both sympatric and allopatric populations of these species. Pollen flow occurred from P. bifolia to P. chlorantha but not in the opposite direction. Crossing experiments revealed no postmating isolation between the Platanthera species. Out of nine measured morphological traits, three were intermediate for putative hybrids; for other traits, they resembled either of the parental species. Hybrids with known parents had intermediate molecular traits, as did the putative hybrids. Our results showed weak reproductive isolation between P. bifolia and P. chlorantha and may refer to the early stages of speciation of the species.