## 3 Results
### Results from PCR Analysis for Microcystin-Producing Strains  
After the application of HEPF \(\times\) HEPR synthetase-gene-specific pair of primers, isolation of DNA fragments with expected length of 470 bp and their cloning into plasmid vector, we constructed eight _mcyDNA_-clone libraries. They reflect data on all 81 _mcyE_ sequences obtained from eight of the ten studied sites: Izvornik 2, Durankulak, Poroy, Mandra (with sites in its both parts--Mandra East and Mandra West), Uzunegren, Vaya and Sinyata Reka (Figure 7). PCR products were not obtained from the reservoirs Krapets and Kriva Reka. According to the BLAST search [54], 43 (53%) of all 81 obtained sequences had 100% identity with the NCBI [55]_mcyE_ sequences of known strains and could be affiliated to them. Another 31 sequences (38%) showed high level homology (>99% identity) and only seven strains, namely Blu4, Blu7, Blu8, Tol3, Uz9, Uz3, and Dur2 (Figure 7), were more distant, showing lower identity (>98%) to the known _mcyE_ sequences. All the analyzed 81 _mcyE_ sequences and their corresponding high homology NCBI sequences were used in the phylogenetic assay. In the constructed phylogenetic tree, four main clusters were formed (Figure 7). According to the combined results from the NCBI search and phylogenetic analysis, it was not possible to affiliate correctly to specific genera the seven strains mentioned above with lower identity, but 74 from all 81 obtained sequences clearly belonged to the genus _Microcystis_. Most of these 74 sequences (41, or 55%) were affiliated to uncultured or unidentified to species level strains of the genus and only 11 of them (15%) could be referred to two distinct species, with named sequences in NCBI--_M. aeruginosa_ and _M. wesenbergii_ (Figure 7). On the basis of _Microcystis_ strains, submitted by us to NCBI, sequenced in 2018 and described morphologically in [9], we can suppose that in the processed samples we found seven more sequences coinciding with _M. wesenbergii_ (with 100% homology). They are grouped together in cluster IV with NCBI clone _Microcystis_ sp. Brat 12/07-2 (KF219519.1) and are allied to the species _M. wesenbergii_ on the basis of the submitted by us strain Uncultured _Microcystis_ clone Blu1. MN417090.1 (Figure 7). In this way, during this study, we identified _M. wesenbergii_ as present in the reservoirs Sinyata Reka and Izvornik 2 (cluster IV), and in the reservoir Poroy, the last according to the sequences in cluster I of Figure 7. It is notable that the affiliation to the species in cluster I is based on the same strain _Microcystis wesenbergii_ NIES-107, NCBI:tvid315483, which was involved in the construction of the phylogenetic tree from June 2018 and is similar to the strain _Microcystis wesenbergii_ submitted by us (Vai1-2_11, NCBI:tvid2703880 with number MN417095.1) [9]. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Waterbody** & **Sampling Date** & **MC-RR, -YR, -LR** \\ \hline Reservoir Krapets & 14 August 2019 & \textless{}LOD \\ Reservoir Kriva Reka & 16 August 2020 & \textless{}LOD \\ Reservoir Izvornik 2 & 16 August 2019 & \textless{}LOD \\ Lake Durankulak & 16 August 2019 & LR 0.15 \(\mu\)g L\({}^{-1}\) \\ Reservoir Poroy & 17 August 2019 & \textless{}LOD \\ Reservoir Mandra (site West) & 18 August 2019 & LR 0.24 \(\mu\)g L\({}^{-1}\) \\ Reservoir Mandra (site East) & 19 August 2019 & LR 0.46 \(\mu\)g L\({}^{-1}\) \\ Lake Uzunegren & 19 August 2019 & \textless{}LOD \\ Lake Vaya & 19 August 2019 & \textless{}LOD \\ Reservoir Sinyata Reka & 20 August 2019 & \textless{}LOD \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Microcystis (MC–RR, -YR and -LR) in water samples from nine shallow Bulgarian waterbodies in August 2019. Limit of detection (LOD) of the applied method is 0.08–0.15 \(\mu\)g L\({}^{-1}\). Figure 7: Neighbor-joining phylogenetic tree constructed using nucleotides sequences from eight library samples and closest sequences retrieved after BLAST search in NCBI database with indication of their accession number in NCBI.