The reproductive isolation barriers and the mating patterns among Pinus pumila, P. parviflora var. pentaphylla and their hybrids were examined by flowering phenology and genetic assays of three life stages: airborne-pollen grains, adults and seeds, in a hybrid zone on Mount Apoi, Hokkaido, Japan. Chloroplast DNA composition of the airborne-pollen was determined by single-pollen polymerase chain reaction. Mating patterns were analysed by estimating the molecular hybrid index of the seed parent, their seed embryos and pollen parents. The observation of flowering phenology showed that the flowering of P. pumila precedes that of P. parviflora var. pentaphylla by about 6 to 10 days within the same altitudinal ranges. Although this prezygotic isolation barrier is effective, the genetic assay of airborne-pollen showed that the two pine species, particularly P. pumila, still have chances to form F1 hybrid seeds. Both parental species showed a strong assortative mating pattern; F1 seeds were found in only 1.4% of seeds from P. pumila mother trees and not at all in P. parviflora var. pentaphylla. The assortative mating was concluded as the combined result of flowering time differentiation and cross-incompatibility. In contrast to the parental species, hybrids were fertilized evenly by the two parental species and themselves. The breakdown of prezygotic barriers (intermediate flowering phenology) and cross-incompatibility may account for the unselective mating. It is suggested that introgression is ongoing on Mount Apoi through backcrossing between hybrids and parental species, despite strong isolation barriers between the parental species.
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