ABSTRACT: The strains LEGE 06306, LEGE 06307, LEGE 11436, and LEGE 10375, isolated from northern Portugal, were characterized using a polyphasic taxonomic approach that included 16S rRNA gene phylogenetic analyses (ML and BI), 16S-23S ITS secondary structures, p-distance calculations, phylogenomics, morphological and ultrastructural (TEM) observations, MALDI-TOF MS profiling, as well as ecological and biochemical characterization. Although LEGE 06306, LEGE 06307, and LEGE 11436 share high 16S rRNA gene sequence similarity and display very similar morphology, the three ML and BI phylogenetic analyses revealed that these strains form three distinct lineages within the family Prochlorococcaceae, without consistent affiliation to any previously described genera. The phylogenomic analysis, ITS comparisons, and the biochemical statistical comparisons further supported the separation of these lineages. MALDI-TOF analysis revealed unique spectral profiles for each strain, while their pigment compositions were similar but varied in pigment concentrations, supporting their distinction. Based on these combined results, the three strains are described as representatives of three new cyanobacterial genera. Strain LEGE 10375 showed an uncertain placement in both the 16S rRNA and whole-genome phylogenies and therefore could not be taxonomically assigned. The high content of bioactive metabolites of the studied strains, including pigments, phenolic compounds, and sugars, positions them as sustainable sources for functional ingredients with potential applications in the food, feed, and pharmaceutical industries.
