Five different ploidy levels (from 2x to 6x) were found, with triploids of K. arvensis being recorded for the first time. The species also exhibited variation in the monoploid genome size, corresponding to the types of habitats occupied (grassland diploid populations had larger genome sizes than relict and subalpine diploid populations). Disregarding relict populations, the distribution of 2x and 4x cytotypes was largely parapatric, with a diffuse secondary contact zone running along the north-west margin of the Pannonian basin. Spatial segregation of the cytotypes was also observed on regional and microgeographic scales. The newly detected sympatric growth of diploids and tetraploids in isolated relict habitats most likely represents the primary zone of cytotype contact. Ploidy level was found to be a major determinant of the strength of inter-cytotype reproductive barriers. While mixed 2x + 4x populations virtually lacked the intermediate ploidy level at any ontogenetic stage, pentaploid hybrids were common in 4x +6x populations, despite the cytotypes representing different taxonomic entities.

Nuclear genome size is an inherited quantitative trait of eukaryotic organisms with both practical and biological consequences. A detailed analysis of major families is a promising approach to fully understand the biological meaning of the extensive variation in genome size in plants. Although Orchidaceae accounts for $10% of the angiosperm diversity, the knowledge of patterns and dynamics of their genome size is limited, in part due to difficulties in flow cytometric analyses. Cells in various somatic tissues of orchids undergo extensive endoreplication, either whole-genome or partial, and the G1-phase nuclei with 2C DNA amounts may be lacking, resulting in overestimated genome size values. Interpretation of DNA content histograms is particularly challenging in species with progressively partial endoreplication, in which the ratios between the positions of two neighboring DNA peaks are lower than two. In order to assess distributions of nuclear DNA amounts and identify tissue suitable for reliable estimation of nuclear DNA content, we analyzed six different tissue types in 48 orchid species belonging to all recognized subfamilies. Although traditionally used leaves may provide incorrect C-values, particularly in species with progressively partial endoreplication, young ovaries and pollinaria consistently yield 2C and 1C peaks of their G1-phase nuclei, respectively, and are, therefore, the most suitable parts for genome size studies in orchids. We also provide new DNA C-values for 22 orchid genera and 42 species. Adhering to the proposed methodology would allow for reliable genome size estimates in this largest plant family. Although our research was limited to orchids, the need to find a suitable tissue with dominant 2C peak of G1-phase nuclei applies to all endopolyploid species. V C 2015 International Society for Advancement of Cytometry Key terms

SummaryPolyploidy is widely recognized as a major mechanism of sympatric speciation in plants, yet little is known about its effects on interactions with other organisms. Mycorrhizal fungi are among the most common plant symbionts and play an important role in plant nutrient supply. It remains to be understood whether mycorrhizal associations of ploidy-variable plants can be ploidy-specific.We examined mycorrhizal associations in three cytotypes (2x, 3x, 4x) of the Gymnadenia conopsea group (Orchidaceae), involving G. conopsea s.s. and G. densiflora, at different spatial scales and during different ontogenetic stages. We analysed: adults from mixed-and single-ploidy populations at a regional scale; closely spaced adults within a mixed-ploidy site; and mycorrhizal seedlings.All Gymnadenia cytotypes associated mainly with saprotrophic Tulasnellaceae (Basidiomycota). Nonetheless, both adults and seedlings of diploids and their autotetraploid derivatives significantly differed in the identity of their mycorrhizal symbionts. Interploidy segregation of mycorrhizal symbionts was most pronounced within a site with closely spaced adults.This study provides the first evidence that polyploidization of a plant species can be associated with a shift in mycorrhizal symbionts. This divergence may contribute to niche partitioning and facilitate establishment and co-existence of different cytotypes.