DNA extraction, PCR and sequencing

DNA extraction was performed using the DNeasy Blood & Tissue Kit (Quiagen, Hilden, Germany) based on the standard protocol provided by the manufacturer. Two DNA regions were amplified. The partial gene coding 18S rRNA and complete ITS1 region was amplified using the primers S1 (forward, 5′-ATTCCGATAACGAACGAGACT-3′) and Lig5.8R (reverse, 5′-GATACTCGAGCCGAGTGATCC-3′) (Šimková et al., 2003; Blasco-Costa et al., 2012). Each amplification reaction was performed in a final volume of 20 μL, the reaction mixture comprising 1.5 U Taq polymerase (Fermentas), 1× buffer, 1.5 mm MgCl₂, 0.2 mm of dNTPs, 0.1 mg mL⁻¹ BSA, 0.5 μm of each primer and 2 μL of pure DNA (20 ng μL⁻¹). PCR was carried out using the following steps: 3 min initial denaturation at 95 °C, followed by 40 cycles of 40 s at 94 °C, 30 s at 52 °C and 45 s at 72 °C, and 4 min of final elongation at 72°C. The second marker, a part of the gene coding 28S rRNA, was amplified using the primers C1 (forward, 5′-ACCCGCTGAATTTAAGCA-3′) and D2 (reverse, 5′-TGGTCCGTGTTTCAAGAC-3′) (Hassouna et al., 1984), following the PCR protocol described in Šimková et al. (2006a). The PCR products were purified prior to sequencing using the ExoSAP-IT kit (Ecoli, Bratislava, Slovakia), following the standard protocol, and directly sequenced using the PCR primers and the BigDye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA). Sequencing was carried out on an ABI 3130 Genetic Analyzer (Applied Biosystems). The newly generated sequences were deposited in GenBank (see Table 1 for accession numbers).