and 4 Institute of Botany, Academy of Sciences of the Czech Republic, Pr ů honice 1, CZ-252 43 Pr ů honice, Czech Republic• Premise of the study : Genome duplication and interspecifi c hybridization are important evolutionary processes that significantly infl uence phenotypic variation, ecological behavior, and reproductive biology of land plants. These processes played a major role in the evolution of the Dryopteris carthusiana complex. This taxonomically intricate group composed of one diploid ( D. expansa ) and two allotetraploid ( D. carthusiana and D. dilatata ) species in Central Europe. Overall phenotypic similarity, great plasticity, and the incidence of interspecifi c hybrids have led to a continuous dispute concerning species circumscription and delimitation.• Methods : We used fl ow cytometry and multivariate morphometrics to assess the level of phenotypic variation and the frequency of hybridization in a representative set covering all recognized species and hybrids.• Key results : Flow cytometric measurements revealed unique genome sizes in all species and hybrids, allowing their easy and reliable identifi cation for subsequent morphometric analyses. Different species often formed mixed populations, providing the opportunity for interspecifi c hybridization. Different frequencies of particular hybrid combinations depended primarily on evolutionary relationships, reproductive biology, and co-occurrence of progenitors.• Conclusions : Our study shows that genome size is a powerful marker for taxonomic decisions about the D. carthusiana complex and that genome size data may help to resolve taxonomic complexities in this important component of the temperate fern fl ora.