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.

Connectivity of Edaphic and Endolithic Microbial Niches in Cold Mountain Desert of Eastern Pamir (Tajikistan).

Microbial communities found in arid environments are commonly represented by biological soil crusts (BSCs) and endolithic assemblages. There is still limited knowledge concerning endoliths and BSCs occurring in the cold mountain desert of Pamir. The aim of the study was to investigate the composition and structure of endolithic bacterial communities in comparison to surrounding BSCs in three subregions of the Eastern Pamir (Tajikistan). The endolithic and BSC communities were studied using culture-independent and culture-dependent techniques. The structure of the endolithic bacterial communities can be characterized as Actinobacteria-Proteobacteria-Bacteroidetes-Chloroflexi-Cyanobacteria, while the BSCs' can be described as Proteobacteria-Actinobacteria-Bacteroidetes-Cyanobacteria assemblages with low representation of other bacteria. The endolithic cyanobacterial communities were characterized by the high percentage of Chroococcidiopsaceae, Nodosilineaceae, Nostocaceae and Thermosynechococcaceae, while in the BSCs were dominated by Nodosilineaceae, Phormidiaceae and Nostocaceae. The analysis of 16S rRNA genes of the cyanobacterial cultures revealed the presence of possibly novel species of Chroococcidiopsis, Gloeocapsopsis and Wilmottia. Despite the niches' specificity, which is related to the influence of microenvironment factors on the composition and structure of endolithic communities, our results illustrate the interrelation between the endoliths and the surrounding BSCs in some regions. The structure of cyanobacterial communities from BSC was the only one to demonstrate some subregional differences.

Diversity and Colonization Strategies of Endolithic Cyanobacteria in the Cold Mountain Desert of Pamir.

Microorganisms can survive in extreme environments and oligotrophic habitats thanks to their specific adaptive capacity. Due to its severe and contrasting climate conditions, the cold mountain desert in Eastern Pamir provides a unique environment for analyzing microbial adaptation mechanisms occurring within colonization of endolithic habitats. This study aims to investigate the composition and structure of endolithic microbial communities and analyze the interactions between microorganisms and colonized lithic substrates. Endolithic biofilms were examined using scanning electron microscopy in backscattered electron mode (SEM-BSE) and next-generation sequencing (NGS) applying amplicon sequence variants (ASVs) approach. The investigation of the V3-V4 region of 16S rRNA gene revealed that endolithic communities are dominated by Actinobacteria (26%), Proteobacteria (23%), and Cyanobacteria (11.4%). Cyanobacteria were represented by Oxyphotobacteria with a predominance of subclasses of Oscillatoriophycidae, Synechococcophycideae, and Nostocophycidae as well as the rarely occurring Sericytochromatia. The positive correlation between the contribution of the orders Synechococcales and Rhizobiales to community structure suggests that some functionally closed taxa of Cyanobacteria and Proteobacteria can complement each other, for example, in nitrogen fixation in endolithic communities. The endolithic communities occurring in Eastern Pamir were identified as complex systems whose composition and structure seem to be influenced by the architecture of microhabitats and related microenvironmental conditions.

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.

Distribution of invasive Cylindrospermopsis raciborskii in the East-Central Europe is driven by climatic and local environmental variables.

Mechanisms behind expansion of an invasive cyanobacterium Cylindrospermopsis raciborskii have not been fully resolved, and different hypotheses, such as global warming, are suggested. In the East-Central Europe, it is widely occurring in western part of Poland but only in single locations in the East due to some limiting factors. Therefore, broad-scale phytoplankton survey including 117 randomly selected lakes in Poland and Lithuania was conducted. The results showed that C. raciborskii occurred widely in western part of Poland but was absent from other regions and Lithuania except one lake. The regions in which C. raciborskii was present had higher annual mean air temperature, higher maximum air temperature of the warmest month and higher minimum temperature of the coldest month, demonstrating that average air temperature, and indirectly, the duration of growing season might be more important factor driving C. raciborskii distribution than measured in situ water temperature. In turn, the presence of C. raciborskii in single localities may be more related to physiological adaptations of separated ecotype. Collectively, these results provide novel evidence on the influence of temperature on C. raciborskii distribution in East-European regions but also indicate high ecological plasticity of this species.

Molecular (PCR-DGGE) versus morphological approach: analysis of taxonomic composition of potentially toxic cyanobacteria in freshwater lakes.

BACKGROUND: The microscopic Utermöhl method is commonly used for the recognition of the presence and taxonomic composition of potentially toxic cyanobacteria and is especially useful for monitoring reservoirs used as drinking water, recreation and fishery resources. However, this method is time-consuming and does not allow potentially toxic and nontoxic cyanobacterial strains to be distinguished. We have developed a method based on denaturing gradient gel electrophoresis (DGGE) of the marker gene ITS and the mcy-gene cluster, and DNA sequencing. We have attempted to calibrate the DGGE-method with a microscopic procedure, using water samples taken in 2011 from four lakes of the Great Mazurian Lakes system. RESULTS: Results showed that the classic microscopic method was much more precise and allowed the classification of the majority of cyanobacterial taxa to the species or genus. Using the molecular approach, most of the sequences could only be assigned to a genus or family. The results of DGGE and microscopic analyses overlapped in the detection of the filamentous cyanobacteria. For coccoid cyanobacteria, we only found two taxa using the molecular method, which represented 17% of the total taxa identified using microscopic observations. The DGGE method allowed the identification of two genera of cyanobacteria (Planktothrix and Microcystis) in the studied samples, which have the potential ability to produce toxins from the microcystins group. CONCLUSIONS: The results confirmed that the molecular approach is useful for the rapid detection and taxonomic distinction of potentially toxic cyanobacteria in lake-water samples, also in very diverse cyanobacterial communities. Such rapid detection is unattainable by other methods. However, with still limited nucleotide sequences deposited in the public databases, this method is currently not sufficient to evaluate the entire taxonomic composition of cyanobacteria in lakes.

Seasonal and spatial diversity of picocyanobacteria community in the Great Mazurian Lakes derived from DGGE analyses of ITS region of rDNA and cpcBAIGS [corrected] markers.

The seasonal and spatial diversity of picocyanobacteria (Pcy) in lakes of the Great Mazurian Lakes (GLM) system was examined by DGGE analysis of molecular markers derived from the 16S-23S internal transcribed spacer (ITS) of the ribosomal operon and the phycocyanin operon (cpcBA-IGS). The study of nine lakes, ranging from mesotrophy to hypereutrophy, demonstrated seasonal variance of Pcy. The richness and Shannon diversity index calculated on the basis of both markers were higher in spring and lower in early and late summer. No statistically significant relationships were found between the markers and trophic status of the studied lakes or Pcy abundance. There were, however, statistically significant relationships between the diversity indices and sampling time. The analysis pointed to a different distribution of the two markers. The ITS marker exhibited more unique sequences in time and space, whereas a greater role for common and ubiquitous sequences was indicated by the cpcBA-IGS data. Examination of the Pcy community structure demonstrated that communities were grouped in highly similar clusters according to sampling season/time rather than to the trophic status of the lake. Our results suggest that time is more important than trophic status in shaping the diversity and structure of Pcy communities. The seasonal changes in picocyanobacteria and differences in diversity and community structures are discussed in the context of well-established ecological hypotheses: the PEG model, intermediate disturbance hypothesis (IDH), and horizontal gene transfer (HGT).

A novel phylogenetic clade of picocyanobacteria from the Mazurian lakes (Poland) reflects the early ontogeny of glacial lakes.

The community of picocyanobacteria inhabiting the Great Mazurian Lakes system (comprising lakes ranging from mesotrophic to hypertrophic) is dominated by phycoerythrin-rich cells, which outnumber phycocyanin-rich cells, even in hypertrophic lakes. The genetic diversity and phylogeny of 43 strains of picocyanobacteria isolated from four Mazurian lakes were studied by analyzing the nucleotide sequences of the 16S rRNA gene and cpcBA-IGS operon. Phylogenetic analyses assigned some of the strains to several previously described clusters (Groups A, B, C, E and I) and revealed the existence of a novel clade, Group M (Mazurian), which exhibited a low level of similarity to the other clusters. Both phycocyanin and phycoerythrin picocyanobacteria were assigned to this clade based on an analysis of the 16S rRNA gene. The cpcBA sequence analysis assigned only phycocyanin strains to Group M, whereas the phycoerythrin strains from the M ribogroup were assigned to Groups B and E. We hypothesize that Group M originally contained only phycocyanin picocyanobacteria. The phycoerythrin found in strains belonging to ribogroup M seems to have been acquired through horizontal gene transfer as an adaptation to the changing environment early in the ontogeny of these glacial lakes.

Composition of picocyanobacteria community in the Great Mazurian Lakes: isolation of phycoerythrin-rich and phycocyanin-rich ecotypes from the system--comparison of two methods.

The study showed that the picocyanobacteria community of the Great Mazurian Lakes system (GML) was dominated by phycoerythrin-rich (PE) ecotypes and demonstrated a gradual decrease of the ratio between PE and phycocyanin-rich (PC) ecotypes. The Great Mazurian Lakes offer better conditions for the PE ecotype than for the PC one, despite the considerably high trophic status, probably thanks to low turbidity and attenuation of light in the water column. The successful isolation of PE and PC picocyanobacteria was achieved by two methods: the classic plate method and a modified flow-cytometry method. The modified flow-cytometry method proved to be superior: being more selective for PE picocyanobacteria as well as less time consuming and less laborious. The modifications introduced to the method, such us concentration of cyanobacterial cells by centrifugation to the density required by the flow cytometer, did not hinder the isolation while allowing to skip an intermediate phase of enrichment cultures that had been formerly proposed. The first phylogenetic analyses based on cpcBA operon and 16S rRNA gene demonstrated that picocyanobacteria isolates from GML could, with a high bootstrap support, be grouped into five and four clusters, respectively. Based on a cpcBA-IGS analysis and IGS length the study suggests that at least one of the clusters is new and has not been previously described.