*4.1. Plant Material, DNA Extraction, Library Preparation and Illumina Sequencing*

For both preparation of the DNA libraries and cytogenetic experiments, plants of the diploid species *C. acuminatum*, *C. bryoniifolium*, *C. ficifolium*, *C. iljinii*, *C. pamiricum*, *C. suecicum* and *C. vulvaria*, which represent the main lineages of the *C. album* aggregate as described in Mandák et al. [27], were used (Table 1). For our research, we sampled genotypes that, according to our previous data, have average parameters for the lineage [27]. All plants were cultivated at the experimental garden of the Institute of Botany, Czech Academy of Sciences, Pr*u*˚honice, Czech Republic (49.9917◦ N, 14.5667◦ E, ca. 320 m above sea level). Leaves were collected, and DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Venlo, The Netherlands) according to the manufacturer's instructions. For in situ hybridization experiments, root tips of young, fine roots were collected and fixed as described in Mandák et al. [26] and stored until use. For all analyzed accessions, the ploidy level was verified by flow cytometry as described in Vít et al. [52].

One individual per species was used for library preparation and NGS. One microgram of extracted DNA was sheared to fragments of approximately 500 to 600 bp using a Bioruptor Pico sonication device (Diagenode, Liège, Belgium). The NEBNext adaptors for Illumina were ligated to the resulting fragments using the NEBNext Ultra DNA Library Prep Kit for Illumina (New England BioLabs, Ipswich, MA, USA), following the manufacturer's instructions. The QIAquick PCR Purification Kit (Qiagen) was used to clean the samples from unbound adapters and to concentrate the samples to a total volume of 30 µL. Afterwards, the samples were loaded onto a 1% agarose gel in low EDTA/TAE buffer. Fragments with sizes ranging from 500 to 750 bp were excised and purified using the Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, CA, USA) and eluted into 20 µL of ddH2O Concentration was estimated with a Qubit fluorometer using the Qubit HS Assay kit (Thermo Scientific, Waltham, MA, USA). The individual libraries (corresponding to individual species) were enriched and indexed by unique barcodes using PCR with NEBNext Q5 HotStart HiFi PCR Master Mix and NEBNext Multiplex Oligos for Illumina (New England BioLabs) according to the manufacturer's instructions. The enriched libraries were purified twice using AMPure magnetic beads (Beckman Coulter, Pasadena, CA, USA). The bead:library ratio was 0.7:1 in the first purification and 1:1 in the second purification. The libraries were verified on 1% agarose gels after each purification step. Concentration was measured using the Qubit HS Assay kit (Thermo Scientific) after the final purification step. Libraries of all seven species were pooled and sequenced on an Illumina MiSeq system at Macrogen Inc., to obtain 2*x* 300 bp paired-end reads.
