Genetic integrity of sympatric hybridizing plant species. The case of O. italica and O. anthropophora

Plant species diversification entails the action of reproductive barriers,which are severely challenged when related species grow in contact and formhybrid progeny. Orchis italica and O. anthropophora are two related orchidspecies that produce a known hybrid form, O. xbivonae. Here, we analyseda hybrid zone of these two orchids using molecular analysis and experimentalcrosses. As molecular tools, we employed both real-time PCR and PCRamplification of nuclear markers to evaluate the occurrence of backcrossrecombination. With these approaches, we demonstrated that all examinedhybrids belong to the F1 generation. Chloroplast DNA analysis showed thatO. anthropophora was the maternal species of most of hybrid specimens andthat cytoplasmic introgression was lacking in both parental species. Pollinationexperiments showed that the two orchid species were strictly out-crossing,although self-compatible, and have comparable levels of reproductivefitness in all crossing treatments. Conversely, hybrids demonstrated lowreproductive success in all intra- and back-crossing treatments. The absenceof any backcross generations and plastid introgression suggest that O. xbivonaedoes not represent a bridge to gene flow between O. italica andO. anthropophora. Indeed, the low hybrid fitness testifies to the effectivenessof late post-zygotic barriers occurring between the parental species.