Tatyana Darienko

Chloroidium, a common terrestrial coccoid green alga previously assigned to Chlorella (Trebouxiophyceae, Chlorophyta)

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.).

Evaluating the Species Boundaries of Green Microalgae (Coccomyxa, Trebouxiophyceae, Chlorophyta) Using Integrative Taxonomy and DNA Barcoding with Further Implications for the Species Identification in Environmental Samples

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.

Cyanobacteria and Algae of Biological Soil Crusts

Filamentous cyanobacteria are the key organisms in biological soil crust formation in all biomes of the world. However, especially in temperate, arctic, and high alpine regions, as well as in few dry Savannah ecosystems, filamentous green algae may act in a similar role. Here, we give an overview on the role, diversity, and biogeography of cyanobacteria and eukaryotic algae in biocrusts from all climatic regions and continents of the Earth. We refer to the species level wherever this is possible. Currently, there have been 320 species of cyanobacteria and more than 350 species of eukaryotic algae described from biocrusts. Despite this high diversity, only a minority of the cyanobacterial and algal species found is responsible for the bulk of biocrust formation. Others likely are opportunistic, utilizing the habitat created by biocrusts in the harsh regions of the Earth where habitable space is rare. We also discuss methods for the sampling and identification of biocrust algae and cyanobacteria.

Genetic variability and taxonomic revision of the genus Auxenochlorella (Shihira et Krauss) Kalina et Puncocharova (Trebouxiophyceae, Chlorophyta).

The monotypic genus Auxenochlorella with its type species A. protothecoides is so far only known from specific habitats such as the sap of several tree species. Several varieties were described according to physiological performances in culture on different organic substrates. However, two strains designated as Auxenochlorella were isolated from other habitats (an endosymbiont of Hydra viridis and an aquatic strain from an acidic volcano stream). We studied those isolates and compared them with six strains of Auxenochlorella belonging to different varieties. The integrative approach used in this study revealed that all strains showed similar morphology but differed in their SSU and ITS rDNA sequences. The Hydra endosymbiont formed a sister taxon to A. protothecoides, which included the varieties protothecoides, galactophila, and communis. The variety acidicola is not closely related to Auxenochlorella and represented its own lineage within the Trebouxiophyceae. In view of these results, we propose a new species of Auxenochlorella, A. symbiontica, for the Hydra symbiont, and a new genus Pumiliosphaera, with its type species, P. acidophila, for acidophilic strain. These results are supported by several compensatory base changes in the conserved region of ITS‐2 and ITS‐2 DNA barcodes.

Terrestrial algae of hypersaline environments of the Central Syvash islands (Kherson Region, Ukraine)

In hypersaline environments of the Churiuk and Kuyuk-Tuk islands located in Central Syvash lagoon (Ukraine) 93 species of oxygenic phototrophs (49 Cyanoprokaryota, 29 Chlorophyta, 12 Bacillariophyta, 2 Xanthophyta, 1 Streptophyta, 1 Eustigmatophyta) are recorded. The sites studied represent four basic types of habitats: semi-terrestrial ecotone (littoral heavy-loam solonchak free of vascular plants), wet gleyic solonchak covered by sparce halophytic vegetation, gleyic solonetz under saline meadow vegetation, and chestnut solonetzic soil with Steppa salsuginosa formation. They differ in the taxonomic composition and species diversity of algae. The highest species diversity is observed at sites of wet gleyic solonchaks (71 species of five divisions), the lowest diversity (23 species belonging to three divisions) in the harsh littoral ecotone. The distribution and abundance of species in the four habitats are discussed with reference to their ecology. Descriptions and original drawings of noteworthy taxa of Chlorophyta and Xanthophyta are presented.

Species concept and nomenclatural changes within the genera Elliptochloris and Pseudochlorella (Trebouxiophyceae) based on an integrative approach.

The genera Elliptochloris and Pseudochlorella were erected for Chlorella‐like green algae producing two types of autospores and cell packages, respectively. Both genera are widely distributed in different soil habitats, either as free living or as photobionts of lichens. The species of these genera are often difficult to identify because of the high phenotypic plasticity and occasional lack of characteristic features. The taxonomic and nomenclatural status of these species, therefore, remains unclear. In this study, 34 strains were investigated using an integrative approach. Phylogenetic analyses demonstrated that the isolates belong to two independent lineages of the Trebouxiophyceae (Elliptochloris and Prasiola clades) and confirmed that the genera are not closely related. The comparison of morphology, molecular phylogeny, and analyses of secondary structures of SSU and ITS rDNA sequences revealed that all of the strains belong to three genera: Elliptochloris, Pseudochlorella, and Edaphochlorella. As a consequence of the taxonomic revisions, we propose two new combinations (Elliptochloris antarctica and Pseudochlorella signiensis) and validate Elliptochloris reniformis, which is invalidly described according to the International Code for Nomenclature (ICN), by designating a holotype. To reflect the high phenotypic plasticity of P. signiensis, two new varieties were described: P. signiensis var. magna and P. signiensis var. communis. Chlorella mirabilis was not closely related to any of these genera and was, therefore, transferred to the new genus Edaphochlorella. All of the taxonomic changes were highly supported by all phylogenetic analyses and were confirmed by the ITS‐2 Barcodes using the ITS‐2/CBC approach.

ATP Content and Cell Viability as Indicators for Cryostress Across the Diversity of Life

In many natural environments, organisms get exposed to low temperature and/or to strong temperature shifts. Also, standard preservation protocols for live cells or tissues involve ultradeep freezing in or above liquid nitrogen (-196°C or -150°C, respectively). To which extent these conditions cause cold- or cryostress has rarely been investigated systematically. Using ATP content as an indicator of the physiological state of cells, we found that representatives of bacteria, fungi, algae, plant tissue, as well as plant and human cell lines exhibited similar responses during freezing and thawing. Compared to optimum growth conditions, the cellular ATP content of most model organisms decreased significantly upon treatment with cryoprotectant and cooling to up to -196°C. After thawing and a longer period of regeneration, the initial ATP content was restored or even exceeded the initial ATP levels. To assess the implications of cellular ATP concentration for the physiology of cryostress, cell viability was determined in parallel using independent approaches. A significantly positive correlation of ATP content and viability was detected only in the cryosensitive algae Chlamydomonas reinhardtii SAG 11-32b and Chlorella variabilis NC64A, and in plant cell lines of Solanum tuberosum. When comparing mesophilic with psychrophilic bacteria of the same genera, and cryosensitive with cryotolerant algae, ATP levels of actively growing cells were generally higher in the psychrophilic and cryotolerant representatives. During exposure to ultralow temperatures, however, psychrophilic and cryotolerant species showed a decline in ATP content similar to their mesophilic or cryosensitive counterparts. Nevertheless, psychrophilic and cryotolerant species attained better culturability after freezing. Cellular ATP concentrations and viability measurements thus monitor different features of live cells during their exposure to ultralow temperatures and cryostress.