Paternal apomixis was recently reported in the endangered Mediterranean cypress, Cupressus dupreziana. This species acts as a surrogate mother for the development of all-paternal embryos from pollen grains. C. dupreziana production of Cupressus sempervirens haploid or diploid seedlings from C. sempervirens pollen was also demonstrated. The haploid progeny was derived from the embryogenic development of haploid gametes, but the origin of the diploid progeny remained unknown. To determine the ontogenic origin of the diploid C. sempervirens progeny, we analyzed the heterozygozity of 63 diploid all-paternal C. sempervirens seedlings using highly variable co-dominant nuclear microsatellite markers. The bi-parental inheritance of the markers was checked in C. sempervirens controlled crosses. A high level of polymorphism was observed among the diploid all-paternal trees. All but three individuals exhibited single-band profiles as expected for homozygotes, which may originate from natural diploidization of a C. sempervirens haploid embryo or from the fusion of two male gametes produced by the same C. sempervirens microgametophyte. The three heterozygous seedlings must be derived from the fusion of male gametes produced by two different C. sempervirens microgametophytes. These findings offer a unique opportunity in conifers to produce homozygous lines, highly valuable for genetic analyses or breeding
