Sample Collection
Samples were collected from a total of 398 localities across the Czech Republic. At each locality, samples were taken from five individual plants for every species present from the list. To minimize the risk of collecting genetically identical individuals (clones), the minimum distance between sampled plants was three meters. Each sample consisted of 1–30 leaves, depending on their size. The plant material was immediately stored and dried in silica gel to preserve DNA quality for subsequent analyses. With the help of 66 collectors, we successfully gathered 10,200 samples.
For the downstream analyses, up to 80 populations per studied species were selected, evenly distributed across the Czech Republic with a minimum distance of 10 km between sites. Priority was given to valuable localities monitored under the Czech Union for Nature Conservation (ČSOP) framework. For polyploid species, the selection was carried out separately for each ploidy level.
Flow Cytometry
For all samples originating from polyploid complexes (i.e., species with multiple ploidy levels), the ploidy level was reliably verified using flow cytometry (FCM). This method enabled the precise identification of individual cytotypes and the detection of potential intra-population variability, which is crucial for the correct taxonomic interpretation of the studied plants.
DNA analysis (ddRADseq)
DNA was isolated from each sample and analyzed using modern genomic markers (RADseq – Restriction Site-Associated DNA Sequencing). This high-throughput method allows for the acquisition of large amounts of genetic information across the entire genome, making it highly suitable for detailed studies of population structure and fine-scale differentiation among populations of common meadow species.
Population Genetic Data Analysis
Applied analytical approaches
The genetic data were processed using three key analytical approaches commonly used in population genetics:
- Multivariate analyses (e.g., PCA / PCoA) determined the degree of similarity and differentiation among populations within individual species.
- Genetic structure analyses (e.g., using the program STRUCTURE) identified the number and spatial distribution of genetically distinct groups (clusters).
- Isolation-by-distance testing verified whether genetic differentiation increases with geographical distance. This relationship was confirmed in all studied species, although its intensity varied among them.
Key methodological aspect: In all the analyses, we rigorously account for the ploidy levels of individual samples, ensuring high accuracy and biological relevance of the resulting genetic models.