Ellipsoidal Chlorella-like species are very common in all kinds of aquatic and terrestrial habitats, and often identified as Chlorella saccharophila or C. ellipsoidea. However, the taxonomic status of these species remains unclear, because they are not related to the type species of the genus, Chlorella vulgaris. In this study, 23 strains isolated from different habitats, were investigated using a polyphasic approach, i.e. morphology and reproduction, ecophysiology, and combined SSU and ITS rDNA sequences. Phylogenetic analyses clearly demonstrated that these isolates formed a monophyletic lineage within the green algal class Trebouxiophyceae. All strains were characterized by ellipsoidal cell shape, unequal autospores during reproduction, and parietal chloroplasts, as well as by the biochemical capability to synthesize and accumulate the rather unusual polyol, ribitol. Although ribitol is a typical stress metabolite involved in osmotic acclimation, it can also be used as a chemotaxonomic marker. Comparative growth measurements under different temperature regimes indicated similar optimum growth temperatures and maximum growth rates in all studied Chlorella-like species. However, these were different from those of C. vulgaris. We therefore propose to transfer all Chlorella-like strains related to Chlorella saccharophila and C. ellipsoidea to the genus Chloroidium Nadson and to emend its diagnosis. We propose four new combinations: Chloroidium saccharophilum comb. nov., Chloroidium ellipsoideum comb. nov., Chloroidium angusto-ellipsoideum comb. nov. and Chloroidium engadinensis comb. nov. In contrast, Chlorella ellipsoidea sensu Punčochárová, which has other morphological and ecophysiological characters, should be assigned to the genus Pseudochlorella (P. pringsheimii comb. nov.).

Integrative taxonomy is an approach for defining species and genera by taking phylogenetic, morphological, physiological, and ecological data into account. This approach is appropriate for microalgae, where morphological convergence and high levels of morphological plasticity complicate the application of the traditional classification. Although DNA barcode markers are well-established for animals, fungi, and higher plants, there is an ongoing discussion about suitable markers for microalgae and protists because these organisms are genetically more diverse compared to the former groups. To solve these problems, we assess the usage of a polyphasic approach combining phenotypic and genetic parameters for species and generic characterization. The application of barcode markers for database queries further allows conclusions about the ‘coverage’ of culture-based approaches in biodiversity studies and integrates additional aspects into modern taxonomic concepts. Although the culture-dependent approach revealed three new lineages, which are described as new species in this paper, the culture-independent analyses discovered additional putative new species. We evaluated three barcode markers (V4, V9 and ITS-2 regions, nuclear ribosomal operon) and studied the morphological and physiological plasticity of Coccomyxa, which became a model organism because its whole genome sequence has been published. In addition, several biotechnological patents have been registered for Coccomyxa. Coccomyxa representatives are distributed worldwide, are free-living or in symbioses, and colonize terrestrial and aquatic habitats. We investigated more than 40 strains and reviewed the biodiversity and biogeographical distribution of Coccomyxa species using DNA barcoding. The genus Coccomyxa formed a monophyletic group within the Trebouxiophyceae separated into seven independent phylogenetic lineages representing species. Summarizing, the combination of different characteristics in an integrative approach helps to evaluate environmental data and clearly identifies microalgae at generic and species levels.

Summary: Symbiosis of green algae with protozoa and invertebrates has been studied for more than 100 years. Endosymbiotic green algae are widely distributed in ciliates (e.g. Paramecium , Stentor, Climacostomum, Coleps, Euplotes ), heliozoa (e.g. Acanthocystis ) and invertebrates (e.g. Hydra , Spongilla ), and have traditionally been identified as named or unnamed species of Chlorella Beij. or Zoochlorella K. Brandt or referred to as Chlorella ‐like algae or zoochlorellae. We studied 17 strains of endosymbionts isolated from various hosts and geographical localities using an integrative approach (nuclear encoded small subunit and internal transcribed spacer regions of rRNA gene sequences including their secondary structures, morphology, physiology and virus sensitivity). Phylogenetic analyses have revealed them to be polyphyletic. The strains examined belong to five independent clades within the Trebouxiophyceae ( Choricystis ‐, Elliptochloris ‐, Auxenochlorella ‐ and Chlorella ‐clades) and Chlorophyceae ( Scenedesmus ‐clade). The most studied host organism, Paramecium bursaria , harbours endosymbionts representing at least five different species. On the basis of our results, we propose a taxonomic revision of endosymbiotic ‘ Chlorella ’‐like green algae. Zoochlorella conductrix K. Brandt is transferred to Micractinium Fresen. and Zoochlorella parasitica K. Brandt to Choricystis (Skuja) Fott. It was shown that Choricystis minor (Skuja) Fott, the generitype, is a later heterotypic synonym of Choricystis parasitica (K. Brandt) comb. nov. A new species, Chlorella heliozoae , is proposed to accommodate the endosymbiont of Acanthocystis turfacea .

The evolution of streptophytes had a profound impact on life on Earth. They brought forth those photosynthetic eukaryotes that today dominate the macroscopic flora: the land plants (Embryophyta).¹ There is convincing evidence that the unicellular/filamentous Zygnematophyceae—and not the morphologically more elaborate Coleochaetophyceae or Charophyceae—are the closest algal relatives of land plants.2, 3, 4, 5, 6 Despite the species richness (>4,000), wide distribution, and key evolutionary position of the zygnematophytes, their internal phylogeny remains largely unresolved.^7,8 There are also putative zygnematophytes with interesting body plan modifications (e.g., filamentous growth) whose phylogenetic affiliations remain unknown. Here, we studied a filamentous green alga (strain MZCH580) from an Austrian peat bog with central or parietal chloroplasts that lack discernible pyrenoids. It represents Mougeotiopsis calospora PALLA, an enigmatic alga that was described more than 120 years ago⁹ but never subjected to molecular analyses. We generated transcriptomic data of M. calospora strain MZCH580 and conducted comprehensive phylogenomic analyses (326 nuclear loci) for 46 taxonomically diverse zygnematophytes. Strain MZCH580 falls in a deep-branching zygnematophycean clade together with some unicellular species and thus represents a formerly unknown zygnematophycean lineage with filamentous growth. Our well-supported phylogenomic tree lets us propose a new five-order system for the Zygnematophyceae and provides evidence for at least five independent origins of true filamentous growth in the closest algal relatives of land plants. This phylogeny provides a robust and comprehensive framework for performing comparative analyses and inferring the evolution of cellular traits and body plans in the closest relatives of land plants.