Minority cytotypes in European populations of the Gymnadenia conopsea complex (Orchidaceae) greatly increase intraspecific and intrapopulation diversity

Patterns of ploidy variation among and within populations can provide valuable insights into the evolutionary mechanisms shaping the dynamics of plant systems showing ploidy diversity. Whereas data on majority ploidies are, by definition, often sufficiently extensive, much less is known about the incidence and evolutionary role of minority cytotypes. Ploidy and proportions of endoreplicated genome were determined using DAPI (4,6-diamidino-2-phenylindole) flow cytometry in 6150 Gymnadenia plants (fragrant orchids) collected from 141 populations in 17 European countries. All widely recognized European species, and several taxa of less certain taxonomic status were sampled within Gymnadenia conopsea sensu lato. Most Gymnadenia populations were taxonomically and/or ploidy heterogeneous. Two majority (2x and 4x) and three minority (3x, 5x and 6x) cytotypes were identified. Evolution largely proceeded at the diploid level, whereas tetraploids were much more geographically and taxonomically restricted. Although minority ploidies constituted 2 of the individuals sampled, they were found in 35 of populations across the entire area investigated. The amount of nuclear DNA, together with the level of progressively partial endoreplication, separated all Gymnadenia species currently widely recognized in Europe. Despite their low frequency, minority cytotypes substantially increase intraspecific and intrapopulation ploidy diversity estimates for fragrant orchids. The cytogenetic structure of Gymnadenia populations is remarkably dynamic and shaped by multiple evolutionary mechanisms, including both the ongoing production of unreduced gametes and heteroploid hybridization. Overall, it is likely that the level of ploidy heterogeneity experienced by most plant species/populations is currently underestimated; intensive sampling is necessary to obtain a holistic picture.