There is an increasing interest in the Eustigmatophyceae, a class of stramenopile microalgae, because they offer a variety of high-value health-beneficial compounds, e.g. polyunsaturated fatty acids (PUFAs), while concomitantly producing high biomass. Clarification of the taxonomy of these organisms at the species level is important in order to achieve reproducible results and constant yields of valuable compounds in their exploitation. Here the distinction of the, so far exclusively, morphologically defined species of the genera Eustigmatos and Vischeria was tested. Distinctions inferred from almost full 18S and ITS2 rRNA as well as plastid-encoded rbcL gene sequences were evaluated following a morphological investigation. The ITS2 secondary-structure-based phylogenies separated independent lineages (species) with long internal branches. This recommends ITS2 as a promising marker for a DNA metabarcoding approach (culture-independent biodiversity assessment). In contrast, the 18S V4 region which is commonly used in metabarcoding was almost invariant, whereas the almost full length sequences distinguished eight groups/types of strains. Monophyly of the species was supported by shared ITS2 secondary structure features, making them distinct from other eustigmatophyte lineages in concordance with phylogenetic analyses. No groups of strains were congruently supported by all three markers. Consequently, the previous distinction of two genera on the basis of morphology cannot be retained and the species should be accommodated in a single genus, Vischeria. Taxonomic changes among the species with the definition of epitypes, on the basis of cryopreserved strains, are recommended. Two findings point to a more complex evolutionary history of the species. The rbcL and nuclear markers resulted in disparate groupings of strains. In three species divergent intragenomic ITS2 paralogues were revealed. Therefore, a still broader taxon sampling, in conjunction with a deep sequencing approach, is needed for a more comprehensive understanding of the complex evolution of eustigmatophyte species.

Background: Heterococcus is a microalgal genus of Xanthophyceae (Stramenopiles) that is common and widespread in soils, especially from cold regions. Species are characterized by extensively branched filaments produced when grown on agarized culture medium. Despite the large number of species described exclusively using light microscopic morphology, the assessment of species diversity is hampered by extensive morphological plasticity. Results: Two independent types of molecular data, the chloroplast-encoded psbA/rbcL spacer complemented by rbcL gene and the internal transcribed spacer 2 of the nuclear rDNA cistron (ITS2), congruently recovered a robust phylogenetic structure. With ITS2 considerable sequence and secondary structure divergence existed among the eight species, but a combined sequence and secondary structure phylogenetic analysis confined to helix II of ITS2 corroborated relationships as inferred from the rbcL gene phylogeny. Intra-genomic divergence of ITS2 sequences was revealed in many strains. The ‘monophyletic species concept’, appropriate for microalgae without known sexual reproduction, revealed eight different species. Species boundaries established using the molecular-based monophyletic species concept were more conservative than the traditional morphological species concept. Within a species, almost identical chloroplast marker sequences (genotypes) were repeatedly recovered from strains of different origins. At least two species had widespread geographical distributions; however, within a given species, genotypes recovered from Antarctic strains were distinct from those in temperate habitats. Furthermore, the sequence diversity may correspond to adaptation to different types of habitats or climates. Conclusions: We established a method and a reference data base for the unambiguous identification of species of the common soil microalgal genus Heterococcus which uses DNA sequence variation in markers from plastid and nuclear genomes. The molecular data were more reliable and more conservative than morphological data.

A prerequisite for the growth of microalgae on an industrial scale is the optimization of cultivation conditions and the reduction of production costs. The nutrient elements, heat and carbon dioxide provided by flue gas can be used to increase biomass production. The aim of this study was to investigate the effects of different CO₂ concentrations on algal strains with regard to biomass production, α-tocopherol content, fatty acid composition and fatty acid content. For this, we grew the three microalgae Coccomyxa sp., Desmodesmus sp. and Muriella terrestris that were isolated from two tufa-forming karstwater creeks characterized by high CO₂ partial pressures. No significant differences in biomass production rate were observed when the cultures were gassed with air or 15% (v/v) CO₂. However, cell growth increased substantially when 5% (v/v) CO₂ was used. The content of α-tocopherol increased in the stationary phase compared to the logarithmic phase in all investigated strains. In contrast, it decreased significantly when the cultures were aerated with either 5% (v/v) CO₂ or 15% (v/v) CO₂. The saturation level of fatty acids was significantly higher in cultures aerated with CO₂ in the stationary phase compared to the logarithmic phase. In contrast, it decreased in the stationary phase when the cultures were gassed with air. Fatty acid contents increased in the stationary phase compared to the logarithmic phase. It was also higher by gassing with 5% (v/v) CO₂ and decreased when the cultures were aerated with air or 15% (v/v) CO₂. When the biomass production rates were taken into consideration together with contents of α-tocopherol and total fatty acid, the productivity of both increased considerably when the cultures experienced gassing by a 5% (v/v) CO₂.