A critical evaluation of reproductive barriers between closely related species using DNA markers - a case study in Pinus

Former controlled crosses between twelve Pinus montana var. rostrata (Pinus mugo complex) and eight P. sylvestris clones revealed that only two P. sylvestris had efficiently fertilised P. montana. Two species-diagnostic chloroplast DNA markers were applied to verify the species purity of the parental clones. All maternal P. montana were unambiguously confirmed to belong to the P. mugo complex at both chloroplast DNA marker loci. Six P. sylvestris clones carried the 'sylvestris' haplotypes. However, the same two P. sylvestris clones that had efficiently fertilised P. montana displayed the chloroplast haplotypes diagnostic to the P. mugo complex. The patterns of highly polymorphic cpDNA microsatellite markers in parents and offspring ruled out contamination by foreign pollen. We concluded that the two clones successful in the crosses represent fertile hybrids between the two species with P. mugo as the pollen donor. Consequently, DNA markers are proposed for verifying or falsifying the success of artificial fertilisation in general. The existence of crossing barriers between the two Pinus species, meaningful to the postulated natural hybridisation and the evolution of their populations in sympatric stands, was indicated and is newly discussed.