## 2 Materials and Methods
### Molecular-Genetic Studies  
The sequence analysis of PCR amplified fragments of _mcyB_ and _mcyE_ genes from the _mcyA-J_ gene cluster involved in the biosynthesis of MCs [45; 46] was performed. The single copy genes _mcyA_, _mcyB_, _mcyD_ and _mcyE_ are the most commonly used target _mcy_ genes for detection of MC-producers (even when the toxin concentrations are too low to be detected) and primers for different regions of these genes can be generalists for any MC producer, or can be specific for _Microcytsis_[47; 46]. Considering the lack of universal primers (e.g., [48; 49]) we decided to combine primers for two genes from both different operons of the _mcyA-J_ gene cluster, namely _mcyA-C_ and _mcyD-J_[47]. In our previous study we pointed out the reliable MC-detective biomarker character of the _mcyE_ gene from the _mcyD-J_ operon and its strong indicator properties for identifying of potential risk from MCs in water bodies comprising mixed assemblages of toxic and non-toxic species (for details see [9]) and in samples with low cyanoprokaryote biomass [50]. The recent choice for additional application of primers for _mcyB_ gene from the _mcyA-C_ operon was based on the reported results of its successful application [51]. A few hours after collection, the samples for molecular-genetic analysis were filtered through 0.45 \(\upmu\)m cellulose filters Whatman NC45 ST/Sterile EO (Merck KGaA, Darmstadt, Germany). In the lab, the total DNA was isolated from the filters following the protocol of GeneJET(tm) Plant Genomic DNA Purification Mini Kit (Thermo Scientific, Waltham, MA, USA). For PCR amplification of the _mcyE_ gene the following steps were used: the DNA amplification was achieved by application of HEPF (5\({}^{\prime}\)TTTGGGGTTAACTTTTTTGGGCATAGTC-3\({}^{\prime}\)) \(\times\) HEPR (5\({}^{\prime}\)AAATTCTTGAGGCTGTAATCGGGTTT-3\({}^{\prime}\)) synthetase-gene-specific pair of primers [52]. Then PCR amplification was performed in a 25 \(\upmu\)L volume containing 10 pmol primers; 5 \(\upmu\)L of 5\(\times\) HF PCR buffer (2 mM MgCl\({}_{2}\)), 2.5 \(\upmu\)M deoxynucleoside triphosphates and 0.2 U/\(\upmu\)L Phusion(tm) High-Fidelity DNA Polymerase, supplied by Thermo Scientific, Waltham, MA, USA). The amplification of DNA was done in a thermal cycler QB-96 (Qianta Biotech) under the following PCR conditions: denaturation at 98 \({}^{\circ}\)C for 3 min, 35 cycles of denaturation (10 s at 98 \({}^{\circ}\)C), annealing at 57 \({}^{\circ}\)C for 30 s, extension at 72 \({}^{\circ}\)C for 15 s, and a final extension at 72 \({}^{\circ}\)C for 3 min. The resulting PCR products were purified using GeneJET(tm) Gel Extraction Kit (Thermo Fisher Scientific, Waltham, MA, USA). CloneJET(tm)PCR Cloning Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used for obtaining of clones of the _mcyE_ amplification products, and the Sigma Plasmid Miniprep Kit (Sigma, Tauftkirchen, Germany) was used for isolation of recombinant plasmids. For each site or waterbody between nine and fourteen clones were selected and sequenced by Macrogen Inc. (Seoul, South Korea). The fragments of _mcyB_ gene were selectively amplified from the purified metagenomics DNA by PCR using the following pair of primers: MIF GCAGCGAAACTTTGAAGGGTTATG and Mir GGCGATTCTGTGCAGCTTGTTCTC [53] in the above-described reaction mixtures. The amplification program included denaturation at 98 \({}^{\circ}\)C for 3 min, 35 cycles of denaturation (10 s at 98 \({}^{\circ}\)C), annealing at 60 \({}^{\circ}\)C for 30 s, extension 72 \({}^{\circ}\)C for 40 s, and a final extension at 72 \({}^{\circ}\)C for 3 min. The resulting PCR products were purified using the GeneJET(tm) Gel Extraction Kit (Thermo Scientific), following the manufacturer's instructions and directly sequenced by Macrogen Inc. (Seoul, South Korea). The obtained sequences from both _mcyE_ and _mcyB_ genes were processed with Vector NTI 11.5 software and used for BLAST search [54] in the NCBI database [55]. The phylogenetic tree, based on neighbor-joining method, was constructed by Mega 6.06 software [56].