Salinity matters the most: How environmental factors shape the diversity and structure of cyanobacterial mat communities in high altitude arid ecosystems.

Introduction: Microbial mats are complex communities of benthic microorganisms that occur at the soil-water interphase in lakes' shores, streams, and ponds. In the cold, mountainous desert of Eastern Pamir (Tajikistan), where scarce water bodies are influenced by extreme environmental conditions, photosynthetic cyanobacteria form diverse mats. The mats are characterized by different morphology and thickness. Their habitats exhibit a wide range of conditions; from oligosaline to hypersaline, oligotrophic to hypertrophic, and from cold ponds to hot springs. The aim of the present study was to reveal the taxonomic composition and structure of these mats and to examine which environmental factors influence them. Methods: Fifty-one mats were collected from small water bodies around Bulunkul, Karakul, and Rangkul Lakes in 2015 and 2017. The physical and chemical properties of the water were measured in situ, while the concentration of nutrients was analyzed ex-situ. To reveal the taxonomic composition of the mats, the hypervariable V3-V4 region of the 16S rRNA gene was examined using NGS technology. Results: The results of bioinformatic analyses were compared with microscopic observations. They showed that Cyanobacteria was the dominant phylum, constituting on average 35% of bacterial ASVs, followed by Proteobacteria (28%), Bacteroidota (11%), and Firmicutes (9%). Synechococcales, Oscillatoriales, and Nostocales orders prevailed in Oxyphotobacteria, with a low contribution of Chroococcales, Gloeobacterales, and Chroococcidiopsidales. Occasionally the non-photosynthetic Vampirivibrionia (Melainabacteria) and Sericytochromatia from sister clades to Oxyphotobacteria were noted in the samples. Moreover, there was a high percentage of unidentified cyanobacterial sequences, as well as the recently described Hillbrichtia pamiria gen. et sp. nov., present in one of the samples. Salinity, followed by Na and K concentrations, correlated positively with the composition and structure of Oxyphotobacteria on different taxonomic levels and the abundance of all bacterial ASVs. Discussion: The study suggests that the investigated communities possibly host more novel and endemic species. Among the environmental factors, salinity influenced the Oxyphotobacteria communities the most. Overall, the microenvironmental factors, i.e. the conditions in each of the reservoirs seemed to have a larger impact on the diversity of microbial mats in Pamir than the "subregional" factors, related to altitude, mean annual air temperature and distance between these subregions.

Cyanobacteria in hot pursuit: Characterization of cyanobacteria strains, including novel taxa, isolated from geothermal habitats from different ecoregions of the world.

Well-studied thermal spring microbial mat systems continue to serve as excellent models from which to make discoveries of general importance to microbial community ecology in order to address comprehensively the question of "who is there" in a microbial community. Cyanobacteria are highly adaptable and an integral part of many ecosystems including thermal springs. In this context, we sampled disparate thermal springs, spanning from Iceland and Poland to Greece and Tajikistan. Thirteen (13) strains were isolated and characterised with taxonomic indices and molecular markers (16S-23S rRNA region and cpcBA gene), whilst their thermotolerance was evaluated. Screening for the presence of genes encoding three heat shock proteins, as well as non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) was performed. This approach resulted in the description of two new genera (Hillbrichtia and Amphirytos) and their type species (Hillbrichtia pamiria and Amphirytos necridicus) representing Oscillatoriales and Synechococcales orders, respectively. We also found unique lineages inside the genus Thermoleptolyngbya, describing a novel species (T. hindakiae). We described the presence of sub-cosmopolitan taxa (such as Calothrix, Desertifilum, and Trichormus). Strains were diverse concerning their thermophilic ability with the strains well adapted to high temperatures possessing all three investigated genes encoding heat shock proteins as well as studied PKS and NRPS genes. In this work, we show novel cyanobacteria diversity from thermal springs from disparate environments, possible correlation of thermotolerance and their genetic background, which may have implications on strategic focusing of screening programs on underexploited taxa in these habitats.

Limited Microcystin, Anatoxin and Cylindrospermopsin Production by Cyanobacteria from Microbial Mats in Cold Deserts.

Toxic metabolites are produced by many cyanobacterial species. There are limited data on toxigenic benthic, mat-forming cyanobacteria, and information on toxic cyanobacteria from Central Asia is even more scarce. In the present study, we examined cyanobacterial diversity and community structure, the presence of genes involved in toxin production and the occurrence of cyanotoxins in cyanobacterial mats from small water bodies in a cold high-mountain desert of Eastern Pamir. Diversity was explored using amplicon-based sequencing targeting the V3-V4 region of the 16S rRNA gene, toxin potential using PCR-based methods (mcy, nda, ana, sxt), and toxins by enzyme-linked immunosorbent assays (ELISAs) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Molecular identification of cyanobacteria showed a high similarity of abundant taxa to Nostoc PCC-73102, Nostoc PCC-7524, Nodularia PCC-935 and Leptolyngbya CYN68. The PCRs revealed the presence of mcyE and/or ndaF genes in 11 samples and mcyD in six. The partial sequences of the mcyE gene showed high sequence similarity to Nostoc, Planktothrix and uncultured cyanobacteria. LC-MS/MS analysis identified six microcystin congeners in two samples and unknown peptides in one. These results suggest that, in this extreme environment, cyanobacteria do not commonly produce microcystins, anatoxins and cylindrospermopsins, despite the high diversity and widespread occurrence of potentially toxic taxa.

Predicting blooms of toxic cyanobacteria in eutrophic lakes with diverse cyanobacterial communities.

We investigated possibility of predicting whether blooms, if they occur, would be formed of microcystin-producing cyanobacteria. DGGE analysis of 16S-ITS and mcyA genes revealed that only Planktothrix and Microcystis possessed mcy-genes and Planktothrix was the main microcystin producer. qPCR analysis revealed that the proportion of cells with mcy-genes in Planktothrix populations was almost 100%. Microcystin concentration correlated with the number of potentially toxic and total Planktothrix cells and the proportion of Planktothrix within all cyanobacteria, but not with the proportion of cells with mcy-genes in total Planktothrix. The share of Microcystis cells with mcy-genes was low and variable in time. Neither the number of mcy-possessing cells, nor the proportion of these cells in total Microcystis, correlated with the concentration of microcystins. This suggests that it is possible to predict whether the bloom in the Masurian Lakes will be toxic based on Planktothrix occurrence. Two species of toxin producing Planktothrix, P. agardhii and P. rubescens, were identified by phylogenetic analysis of 16S-ITS. Based on morphological and ecological features, the toxic Planktothrix was identified as P. agardhii. However, the very high proportion of cells with mcy-genes suggests P. rubescens. Our study reveals the need of universal primers for mcyA genes from environment.

Reconstructing euglenoid evolutionary relationships using three genes: nuclear SSU and LSU, and chloroplast SSU rDNA sequences and the description of Euglenaria gen. nov. (Euglenophyta).

Using Maximum Likelihood and Bayesian analyses of three genes, nuclear SSU (nSSU) and LSU (nLSU) rDNA, and chloroplast SSU (cpSSU) rDNA, the relationships among 82 plastid-containing strains of euglenophytes were clarified. The resulting tree split into two major clades: clade one contained Euglena, Trachelomonas, Strombomonas, Colacium, Monomorphina, Cryptoglena and Euglenaria; clade two contained Lepocinclis, Phacus and Discoplastis. The majority of the members of Euglena were contained in clade A, but seven members were outside of this clade. Euglena limnophila grouped with, and was thus transferred to Phacus. Euglena proxima was a single taxon at the base of clade one and is unassociated with any subclade. Five members of Euglena grouped together within clade one and were transferred into the newly erected genus Euglenaria. The monophyly of the remaining genera was supported by Bayesian and Maximum Likelihood analyses. Combining datasets resolved the relationships among ten genera of photosynthetic euglenoids.

PHYLOGENY AND SYSTEMATICS OF EUGLENA (EUGLENACEAE) SPECIES WITH AXIAL, STELLATE CHLOROPLASTS BASED ON MORPHOLOGICAL AND MOLECULAR DATA-NEW TAXA, EMENDED DIAGNOSES, AND EPITYPIFICATIONS(1).

Morphological and molecular studies, as well as original literature reexamination, necessitate establishment of five Euglena species with a single axial, stellate chloroplast [Euglena viridis (O. F. Müller) Ehrenberg 1830, Euglena pseudoviridis Chadefaud 1937, Euglena stellata Mainx 1926, Euglena pseudostellata sp. nov., and Euglena cantabrica Pringsheim 1956], three species with two chloroplasts (Euglena geniculata Dujardin ex Schmitz 1884, Euglena chadefaudii Bourrelly 1951, and Euglena pseudochadefaudii sp. nov.), and one species with three chloroplasts (Euglena tristella Chu 1946). The primary morphological features, allowing distinction of the considered species are the presence and the shape of mucocysts, as well as the number of chloroplasts. Spherical mucocysts occur in E. cantabrica and E. geniculata, while spindle-shaped mucocysts are present in E. stellata, E. pseudostellata, E. chadefaudii, E. pseudochadefaudii, and E. tristella. No mucocysts are observed in E. viridis and E. pseudoviridis. Two new species (E. pseudochadefaudii sp. nov. and E. pseudostellata sp. nov.) differ from the respective species, E. chadefaudii and E. stellata, only at the molecular level. Molecular signatures and characteristic sequences are designated for nine distinguished species. Emended diagnoses for all and delimitation of epitypes for seven species (except E. viridis and E. tristella) are proposed.

Structural relationships among the ribosomal stalk proteins from the three domains of life.

The GTPase center of the large ribosomal subunit, being a landing platform for translation factors, and regarded as one of the oldest structures in the ribosome, is a universally conserved structure in all domains of life. It is thought that this structure could be responsible for the major breakthrough on the way to the RNA/protein world, because its appearance would have dramatically increased the rate and accuracy of protein synthesis. The major part of this center is recognized as a distinct structural entity, called the stalk. The main functional part of the stalk in all domains of life is composed of small L12/P proteins, which are believed to form an evolutionarily conserved group. However, some data indicate that the bacterial and archaeo/eukaryal proteins are not related to each other structurally, and only a functional relationship may be clearly recognized. To clarify this point, we performed a comprehensive comparative analysis of the L12/P proteins from the three domains of life. The results show that bacterial and archaeo/eukaryal L12/P-proteins are not structurally related and, therefore, might not be linked evolutionarily either. Consequently, these proteins should be regarded as analogous rather than homologous systems and probably appeared on the ribosomal particle in two independent events in the course of evolution.