Molecular and cytomorphological characterization of Pseudanabaena vesniana sp. nov. and Pseudanabaena suomiensis var. macedonica var. nov. (Pseudanabaenales, Cyanobacteriota) with evidence of microcystin-producing Pseudanabaena taxa.

Detailed molecular and cytomorphological characterization of two toxic Pseudanabaena strains (NMCCC 011 and NMCCC 012), isolated from highly polluted shallow areas of Monospitovo Marsh (North Macedonia), was performed using a "polyphasic" approach. This involved morphological and ultrastructural characterization, phylogenetic assessment of the 16S rRNA gene, analyses of the 16S-23S internal transcribed spacer (ITS) rRNA region, and investigations of the mcyB gene. The 16S rRNA gene phylogenetic analysis and the ITS rRNA region analyses confirmed that strain NMCCC 011 was a novel species for which we have proposed the name Pseudanabaena vesniana sp. nov., whereas NMCCC 012 represented a novel variety of Pseudanabaena suomiensis, here designated as P. suomiensis var. macedonica. The overall 16S rRNA gene phylogenetic analysis grouped Pseudanabaena into four large clades. Two of them comprised the primary lineages of diversification, hosting most Pseudanabaena sensu stricto infrageneric units. Most of these units formed distinctly recognizable subclades corresponding to different Pseudanabaena species (Cyanobacteriota); however, some strains were misidentified, leading to several scattered species through different subclades within the genus. Additionally, the genus Pseudanabaena was determined to be polyphyletic, with some strains clustering within the genera Tumidithrix and Thalassoporum (Cyanobacteriota). The 16S rRNA gene sequence identity within Pseudanabaena sensu stricto ranged from 95.7% to 99.9%, while the identity with Tumidithrix, Thalassoporum, and Thermostichus (Cyanobacteriota) ranged from 87.6% to 92.0%. Furthermore, both strains NMCCC 011 and NMCCC 012 produced microcystins and 2-methylisoborneol. The production of microcystins was confirmed by genetic and enzyme-linked immunosorbent assay (ELISA) analyses. Through this study, we report the presence of the mcyB gene in Pseudanabaena.