Cyanobacterial Harmful Algal Mats (CyanoHAMs) in tropical rivers of central Mexico and their potential risks through toxin production.

Cyanobacteria inhabiting lotic environments have been poorly studied and characterized in Mexico, despite their potential risks from cyanotoxin production. This article aims to fill this knowledge gap by assessing the importance of benthic cyanobacteria as potential cyanotoxin producers in central Mexican rivers through: (i) the taxonomic identification of cyanobacteria found in these rivers, (ii) the environmental characterization of their habitats, and (iii) testing for the presence of toxin producing genes in the encountered taxa. Additionally, we introduce and discuss the use of the term "CyanoHAMs" for lotic water environments. Populations of cyanobacteria were collected from ten mountain rivers and identified using molecular techniques. Subsequently, these taxa were evaluated for genes producing anatoxins and microcystins via PCR. Through RDA analyses, the collected cyanobacteria were grouped into one of three categories based on their environmental preferences for the following: (1) waters with high ionic concentrations, (2) cold-temperate waters, or (3) waters with high nutrient enrichment. Populations from six locations were identified to genus level: Ancylothrix sp., Cyanoplacoma sp., and Oxynema sp. The latter was found to contain the gene that produces anatoxins and microcystins in siliceous rivers, while Oxynema tested positive for the gene that produces microcystins in calcareous rivers. Our results suggest that eutrophic environments are not necessarily required for toxin-producing cyanobacteria. Our records of Compactonostoc, Oxynema, and Ancylothrix represent the first for Mexico. Four taxa were identified to species level: Wilmottia aff. murrayi, Nostoc tlalocii, Nostoc montejanii, and Dichothrix aff. willei, with only the first testing positive using PCR for anatoxin and microcystin-producing genes in siliceous rivers. Due to the differences between benthic growths with respect to planktonic ones, we propose the adoption of the term Cyanobacterial Harmful Algal Mats (CyanoHAMs) as a more precise descriptor for future studies.

Lithic cyanobacterial communities in the polyextreme Sahara Desert: implications for the search for the limits of life.

The hyperarid Sahara Desert presents extreme and persistent dry conditions with a limited number of hours during which the moisture availability, temperature and light allow phototrophic growth. Some cyanobacteria can live in these hostile conditions by seeking refuge under (hypolithic) or inside (endolithic) rocks, by colonizing porous spaces (cryptoendoliths) or fissures in stones (chasmoendoliths). Chroococcidiopsis spp. have been reported as the dominant or even the only phototrophs in these hot desert lithic communities. However, the results of this study reveal the high diversity of and variability in cyanobacteria among the sampled habitats in the Sahara Desert. The chasmoendolithic samples presented high coccoid cyanobacteria abundances, although the dominant cyanobacteria were distinct among different locations. A high predominance of a newly described cyanobacterium, Pseudoacaryochloris sahariense, was found in hard, compact, and more opaque stones with cryptoendolithic colonization. On the other hand, the hypolithic samples were dominated by filamentous, non-heterocystous cyanobacteria. Thermophysiological bioassays confirmed desiccation and extreme temperature tolerance as drivers in the cyanobacterial community composition of these lithic niches. The results of the present study provide key factors for understanding life strategies under polyextreme environmental conditions. The isolated strains, especially the newly described cyanobacterium P. sahariense, might represent suitable microorganisms in astrobiology studies aimed at investigating the limits of life.

Microenvironmental Conditions Drive the Differential Cyanobacterial Community Composition of Biocrusts from the Sahara Desert.

The Sahara Desert is characterized by extreme environmental conditions, which are a unique challenge for life. Cyanobacteria are key players in the colonization of bare soils and form assemblages with other microorganisms in the top millimetres, establishing biological soil crusts (biocrusts) that cover most soil surfaces in deserts, which have important roles in the functioning of drylands. However, knowledge of biocrusts from these extreme environments is limited. Therefore, to study cyanobacterial community composition in biocrusts from the Sahara Desert, we utilized a combination of methodologies in which taxonomic assignation, for next-generation sequencing of soil samples, was based on phylogenetic analysis (16S rRNA gene) in parallel with morphological identification of cyanobacteria in natural samples and isolates from certain locations. Two close locations that differed in microenvironmental conditions were analysed. One was a dry salt lake (a "chott"), and the other was an extension of sandy, slightly saline soil. Differences in cyanobacterial composition between the sites were found, with a clear dominance of Microcoleus spp. in the less saline site, while the chott presented a high abundance of heterocystous cyanobacteria as well as the filamentous non-heterocystous Pseudophormidium sp. and the unicellular cf. Acaryochloris. The cyanobacteria found in our study area, such as Microcoleus steenstrupii, Microcoleus vaginatus, Scytonema hyalinum, Tolypothrix distorta, and Calothrix sp., are also widely distributed in other geographic locations around the world, where the conditions are less severe. Our results, therefore, indicated that some cyanobacteria can cope with polyextreme conditions, as confirmed by bioassays, and can be considered extremotolerant, being able to live in a wide range of conditions.

Overview of toxic cyanobacteria and cyanotoxins in Ibero-American freshwaters: Challenges for risk management and opportunities for removal by advanced technologies.

The increasing occurrence of cyanobacterial blooms worldwide represents an important threat for both the environment and public health. In this context, the development of risk analysis and management tools as well as sustainable and cost-effective treatment processes is essential. The research project TALGENTOX, funded by the Ibero-American Science and Technology Program for Development (CYTED-2019), aims to address this ambitious challenge in countries with different environmental and social conditions within the Ibero-American context. It is based on a multidisciplinary approach that combines ecology, water management and technology fields, and includes research groups from Chile, Colombia, Mexico, Peru and Spain. In this review, the occurrence of toxic cyanobacteria and cyanotoxins in freshwaters from these countries are summarized. The presence of cyanotoxins has been confirmed in all countries but the information is still scarce and further monitoring is required. In this regard, remote sensing or metagenomics are good alternatives at reasonable cost. The risk management of freshwaters from those countries considering the most frequent uses (consumption and recreation) has been also evaluated. Only Spain and Peru include cyanotoxins in its drinking water legislation (only MC-LR) and thus, there is a need for regulatory improvements. The development of preventive strategies like diminishing nutrient loads to aquatic systems is also required. In the same line, corrective measures are urgently needed especially in drinking waters. Advanced Oxidation Processes (AOPs) have the potential to play a major role in this scenario as they are effective for the elimination of most cyanotoxins classes. The research on the field of AOPs is herein summarized considering the cost-effectiveness, environmental character and technical applicability of such technologies. Fenton-based processes and photocatalysis using solar irradiation or LED light represent very promising alternatives given their high cost-efficiency. Further research should focus on developing stable long-term operation systems, addressing their scale-up.

Analysis of molecular diversity within single cyanobacterial colonies from environmental samples.

Attached or floating macroscopic cyanobacteria can be found in shallow waters and can be easily hand-collected, but their identification is often challenging due to their high morphological variability. In addition, many members of environmental samples lose their morphological adaptations under controlled conditions, making the integration of analyses of field populations and derived isolated cultures necessary in order to evaluate phenotypic plasticity for identification purposes. Therefore, in this study, twenty-nine macroscopic field samples were analyzed by Illumina sequencing and parallel optical microscopy. Some colonies showed the typical morphological characteristics of Rivularia biasolettiana, and others showed those of Rivularia haematites. However, other Rivularia-like colonies showed ambiguous morphologies, and some of them showed the phenotypic features of the new genus Cyanomargarita, which is virtually indistinguishable from Rivularia in the field. In all of the colonies, phylotype composition was highly heterogeneous, with abundances varying depending on the analyzed sample. Some colonies were dominated (97-99%) by a single phylotype, while in others, the percentage of the dominant phylotype decreased to approximately 50-60%. Surprisingly, the same dominant phylotype was found in R. biasolettiana and R. haematites colonies. The relationships between environmental and/or biological factors and morphological variability in these colonies are discussed.

Polyphasic evaluation of key cyanobacteria in biocrusts from the most arid region in Europe.

Cyanobacteria are key microbes in topsoil communities that have important roles in preventing soil erosion, carbon and nitrogen fixation, and influencing soil hydrology. However, little is known regarding the identity and distribution of the microbial components in the photosynthetic assemblages that form a cohesive biological soil crust (biocrust) in drylands of Europe. In this study, we investigated the cyanobacterial species colonizing biocrusts in three representative dryland ecosystems from the most arid region in Europe (SE Spain) that are characterized by different soil conditions. Isolated cyanobacterial cultures were identified by a polyphasic approach, including 16S rRNA gene sequencing, phylogenetic relationship determination, and morphological and ecological habitat assessments. Three well-differentiated groups were identified: heterocystous-cyanobacteria (Nostoc commune, Nostoc calcicola, Tolypothrix distorta and Scytonema hyalinum), which play an important role in N and C cycling in soil; nonheterocystous bundle-forming cyanobacteria (Microcoleus steenstrupii, Trichocoleus desertorum, and Schizothrix cf. calcicola); and narrow filamentous cyanobacteria (Leptolyngbya frigida and Oculatella kazantipica), all of which are essential genera for initial biocrust formation. The results of this study contribute to our understanding of cyanobacterial species composition in biocrusts from important and understudied European habitats, such as the Mediterranean Basin, a hotspot of biodiversity, where these species are keystone pioneer organisms.

Cyanobacterial biocrust diversity in Mediterranean ecosystems along a latitudinal and climatic gradient.

Cyanobacteria are a key biotic component as primary producers in biocrusts, topsoil communities that have important roles in the functioning of drylands. Yet, major knowledge gaps exist regarding the composition of biocrust cyanobacterial diversity and distribution in Mediterranean ecosystems. We describe cyanobacterial diversity in Mediterranean semiarid soil crusts along an aridity gradient by using next-generation sequencing and bioinformatics analyses, and detect clear shifts along it in cyanobacterial dominance. Statistical analyses show that temperature and precipitation were major parameters determining cyanobacterial composition, suggesting the presence of differentiated climatic niches for distinct cyanobacteria. The responses to temperature of a set of cultivated, pedigreed strains representative of the field populations lend direct support to that contention, with psychrotolerant vs thermotolerant physiology being strain dependent, and consistent with their dominance along the natural gradient. Our results suggest a possible replacement, as global warming proceeds, of cool-adapted by warm-adapted nitrogen-fixing cyanobacteria (such as Scytonema) and a switch in the dominance of Microcoleus vaginatus by thermotolerant, novel phylotypes of bundle-forming cyanobacteria. These differential sensitivities of cyanobacteria to rising temperatures and decreasing precipitation, their ubiquity, and their low generation time point to their potential as bioindicators of global change.

Nitrogen fixation in a non-heterocystous cyanobacterial mat from a mountain river.

In situ nitrogen fixation was investigated in a cyanobacterial mat growing on the bed of rocks of the Muga River, Spain. The filamentous non-heterocystous cyanobacterium Schizothrix dominated the mat, showing nitrogenase activity in the light at similar rates to those found in nearby heterocystous Rivularia colonies. N2 fixation in the light was significantly increased by an inhibitor of PSII and oxygen evolution, DCMU (3-[3,4-dichlorophenyl]-1,1-dimethylurea), and anaerobic conditions. However, no nitrogenase activity was found in the dark. Addition of fructose as a respiratory substrate induced nitrogenase activity in samples incubated under aerobic conditions in the dark but not in anaerobic conditions. Microelectrode oxygen profiles showed internal microaerobic microzones where nitrogen fixation might concentrate. Analyses of the 16S rRNA gene revealed only the presence of sequences belonging to filamentous non-heterocystous cyanobacteria. nifH gene diversity showed that the major phylotypes also belonged to this group. One of the three strains isolated from the Schizothrix mat was capable of fixing N2 and growing in the absence of combined N. This was consistent with the nifH gene analysis. These results suggest a relevant contribution of non-heterocystous cyanobacteria to nitrogen fixation in these mats.

Monitoring bioavailable phosphorus in lotic systems: a polyphasic approach based on cyanobacteria.

Conventional assays to measure phosphorus in freshwater systems are sometimes not sufficient to quantify the actual bioavailable P for aquatic biota since some inorganic or organic P species may not be detected by chemical methods, and their bioavailability can be affected by a range of environmental factors. This situation could lead regulatory agencies to be unable to detect imminent ecosystem-degrading phenomena such as cyanobacterial blooms. It could also be an obstacle in studying the ecophysiological requirements of freshwater communities. P bioavailability in five rivers located in central Spain was analysed by a polyphasic approach (combinations of different marker types) based on cyanobacteria. This approach included a parallel study with the use of a self-luminescent P-cyanobacterial bioreporter based on a phosphatase alkaline promoter, determination of in situ alkaline phosphatase activities from cyanobacteria found at sampling sites, and the characterisation of cyanobacterial morphological features related to P bioavailability (hairs, polyphosphate granules and calyptras). An inverse relationship was found between values of bioavailable P, measured by the bioreporter and phosphatase activities. Cyanobacteria from sampling sites with low bioavailable P showed high phosphatase activity and vice versa, although some differences in values of this activity were observed in different cyanobacteria found at the same place, in relation to different growth strategies. Morphological characteristics associated with P limitation or P enrichment also varied between sampling locations. Cyanobacteria collected from sampling sites with reduced P bioavailability, measured by bioreporter and phosphatase activity, had a lower abundance of polyphosphate granules; those cyanobacteria capable of developing hairs or calyptras showed a greater abundance of these structures. Conversely, polyphosphate granules in cyanobacteria increased as P bioavailability increased as measured by the bioreporter and phosphatase activity. The study shows that the results of genetic, physiological and microscopic analyses based on these methods complement each other, implying that combining their findings would provide a more complete analysis of the nutrient status of running waters.

Specific responses to nitrogen and phosphorus enrichment in cyanobacteria: factors influencing changes in species dominance along eutrophic gradients.

Anthropogenic eutrophication is a worldwide problem, causing proliferation of cyanobacterial masses, some of which may be toxic. However, little is known about whether the response to nutrient enrichment differs among cyanobacterial species. To address this issue, distinct patterns in growth and competitive response of benthic cyanobacteria under N and P nutrient regimes were studied. Nine cyanobacterial species, collected from Guadarrama river biofilms at several locations with different nutrient concentrations, were isolated and used for a series of N and P enrichment bioassays. In competition experiments with a mixture of all nine species, a great predominance of certain cyanobacteria over others was noted at high nutrient conditions, while under low nutrient conditions some others dominated. On the basis of these results four selected strains were subjected to a gradient of different concentrations of phosphate, nitrate and ammonium, in independent bioassays, both in monocultures and mixed cultures. Depending on the concentration of N and P, stimulation or inhibition of growth was observed. Some species grew better, dominating at high nutrient concentrations, while higher yields were recorded for others under low nutrient regimes, dominating in these conditions. Results from this study clarify previously published field observations, whereby a group of species occurred mostly in downstream nutrient-rich locations, while other was typical of upstream oligotrophic conditions. Our findings concerning differential growth in relation to nutrient concentrations may be useful for environmental management, because they help us predict which cyanobacteria may be expected to occur under certain conditions.

Polyphasic characterization of benthic cyanobacterial diversity from biofilms of the Guadarrama river (Spain): morphological, molecular, and ecological approaches(1).

The occurrence and environmental factors responsible for the distribution of benthic cyanobacteria in running waters remain largely unexplored in comparison with those of other aquatic ecosystems. In this study, combined data of ecological characteristics, molecular analysis (based on 16S rRNA gene), and direct microscopic inspection of environmental samples were analyzed in parallel with the morphological characterization of the isolated strains to investigate benthic cyanobacterial diversity in the Guadarrama river (Spain). A total of 17 species were identified that belonged to the genera Aphanocapsa, Pleurocapsa, Chroococcus, Chamaesiphon, Cyanobium, Pseudan-abaena, Leptolyngbya, Phormidium, Nostoc, and Tolypothrix. Phenotypic features were associated with the results of 16S rRNA gene sequencing, complementing existing morphological and genetic databases. A decrease in the cyanobacterial diversity was observed along a pollution gradient in the river. Water quality differed among the sampling sites, and variation in nutrient content was the principal difference among locations. These characteristics were closely associated with an upstream-downstream eutrophic gradient. Canonical correspondence analysis distinguished three groups of species with respect to the eutrophication gradient. The first group (Tolypothrix cf. tenuis, Nostoc punctiforme, Nostoc piscinale, Chamaesiphon investiens, Chroococcus minor, Leptolyngbya nostocorum, and Leptolyngbya tenuis) was characteristic of waters with low levels of nutrients. The second group (Cyanobium sp., Chamaesiphon polymorphus, Leptolyngbya boryana, Phormidium autumnale, Phormidium sp., and Aphanocapsa cf. rivularis) was characteristic of polluted waters, its members appearing mainly in great abundance under eutrophic-hypertrophic conditions. The third group of species (Pseudanabaena catenata, Aphanocapsa muscicola, and Nostoc carneum) was present at upstream and downstream sites.

Molecular fingerprinting of cyanobacteria from river biofilms as a water quality monitoring tool.

Benthic cyanobacterial communities from Guadarrama River (Spain) biofilms were examined using temperature gradient gel electrophoresis (TGGE), comparing the results with microscopic analyses of field-fixed samples and the genetic characterization of cultured isolates from the river. Changes in the structure and composition of cyanobacterial communities and their possible association with eutrophication in the river downstream were studied by examining complex TGGE patterns, band extraction, and subsequent sequencing of 16S rRNA gene fragments. Band profiles differed among sampling sites depending on differences in water quality. The results showed that TGGE band richness decreased in a downstream direction, and there was a clear clustering of phylotypes on the basis of their origins from different locations according to their ecological requirements. Multivariate analyses (cluster analysis and canonical correspondence analysis) corroborated these differences. Results were consistent with those obtained from microscopic observations of field-fixed samples. According to the phylogenetic analysis, morphotypes observed in natural samples were the most common phylotypes in the TGGE sequences. These phylotypes were closely related to Chamaesiphon, Aphanocapsa, Pleurocapsa, Cyanobium, Pseudanabaena, Phormidium, and Leptolyngbya. Differences in the populations in response to environmental variables, principally nutrient concentrations (dissolved inorganic nitrogen and soluble reactive phosphorus), were found. Some phylotypes were associated with low nutrient concentrations and high levels of dissolved oxygen, while other phylotypes were associated with eutrophic-hypertrophic conditions. These results support the view that once a community has been characterized and its genetic fingerprint obtained, this technique could be used for the purpose of monitoring rivers.

Phenotypic variability and phylogenetic relationships of the genera Tolypothrix and Calothrix (Nostocales, Cyanobacteria) from running water.

The taxonomy of heterocystous cyanobacteria belonging to the genera Calothrix and Tolypothrix has long been a matter of debate, but their phylogenetic relationships are still not well understood. Our aim was to compare the phylogeny and morphology of members of these genera, which exhibit basal-apical polarity. A phylogeny was reconstructed on the basis of 16S rRNA gene sequences and compared with the morphological characterization of new isolates and environmental samples. Strains isolated from several rivers and streams showed a high degree of tapering when they were cultured in a nutrient-rich medium. However, clear differences were apparent when they were transferred to a nutrient-poor medium. Some strains showed a low degree of tapering and other morphological features corresponding to the genus Tolypothrix, such as false branching, whereas others maintained the morphological characteristics of the genus Calothrix. Phylogenetic analysis was congruent with the phenotypic characterization, in which the strains and environmental samples of the Tolypothrix and Calothrix morphotypes could be clearly separated. Isolates with a low degree of tapering and natural samples of Tolypothrix distorta were grouped in the same cluster, but strains of the genus Calothrix fell into well separated clades. Results from this study showed that representatives of the genus Tolypothrix share most morphological and developmental properties and a high degree of 16S rRNA gene sequence similarity. However, although similar and sometimes overlapping morphologies may occur in isolates of the genus Calothrix, these morphotypes may be distinguished on the basis of their clear genetic divergence.

Life cycle as a stable trait in the evaluation of diversity of Nostoc from biofilms in rivers.

The diversity within the genus Nostoc is still controversial and more studies are needed to clarify its heterogeneity. Macroscopic species have been extensively studied and discussed; however, the microscopic forms of the genus, especially those from running waters, are poorly known and likely represented by many more species than currently described. Nostoc isolates from biofilms of two Spanish calcareous rivers were characterized comparing the morphology and life cycle in two culture media with different levels of nutrients and also comparing the 16S rRNA gene sequences. The results showed that trichome shape and cellular dimensions varied considerably depending on the culture media used, whereas the characteristics expressed in the course of the life cycle remained stable for each strain independent of the culture conditions. Molecular phylogenetic analysis confirmed the distinction between the studied strains established on morphological grounds. A balanced approach to the evaluation of diversity of Nostoc in the service of autecological studies requires both genotypic information and the evaluation of stable traits. The results of this study show that 16S rRNA gene sequence similarity serves as an important criterion for characterizing Nostoc strains and is consistent with stable attributes, such as the life cycle.

Genetic and morphological characterization of Rivularia and Calothrix (Nostocales, Cyanobacteria) from running water.

In this study, a polyphasic approach was adopted to investigate natural freshwater (river and stream) samples of Rivularia colonies and isolated strains of cyanobacteria with a high degree of trichome tapering (genera Rivularia and Calothrix). Analysis of the phycocyanin (PC) operon and the intervening intergenic spacer (cpcBA-IGS) and 16S rRNA gene sequences were used for genetic characterization. In addition, a molecular fingerprinting method, temperature-gradient gel electrophoresis, which allows sequence-dependent separation of PCR products, was used to assess genotypic diversity in environmental samples and isolated strains. The results showed a high variability of the PC-IGS among the genotypes that was not associated with the morphologies observed. This study underlines the importance of choosing a low-nutrient-content culture medium, especially one with a low phosphorus concentration, for studying typical morphological features of Rivularia for taxonomic purposes. Molecular fingerprinting methods and morphological analyses confirmed the diversity in Rivularia colonial structure and trichome features corresponding to genetic diversity within a single colony. Phylogenetic analysis of cpcBA-IGS was largely consistent with that obtained from 16S rRNA gene sequence analysis and confirmed the high level of divergence between genotypes. The sequences of Rivularia and Calothrix from this study and database sequences showed great heterogeneity and were clearly not monophyletic. The results of this genetic and morphological study of field samples and fresh isolates indicated that the current classification of these genera needs to be revised.

A molecular fingerprint technique to detect pollution-related changes in river cyanobacterial diversity.

Humans now have a strong influence on almost every major aquatic ecosystem, and our activities have dramatically altered the quality of receiving waters worldwide. Thus, there is a continuous need to develop and apply novel and effective technologies to detect, manage, and correct human-induced degradation of aquatic systems. In the present work, we evaluated the molecular approach using polymerase chain reaction (PCR)-temperature gradient gel electrophoresis (TGGE) to measure changes in cyanobacterial diversity along a pollution gradient in a river and compared it with that of using microscopic observations of field-fixed and cultured samples. The different 16S rDNA genes present in the cyanobacterial community of each sampling point of the river were separated by TGGE, giving a characteristic pattern of bands for each site. This pattern represents a "fingerprint" of the community, allowing direct comparisons of the different samples. The TGGE results revealed that the structure of the cyanobacterial community differed along the pollution gradient of the river. Microscopic and molecular approaches showed that cyanobacterial diversity decreased in a downstream direction. Similar results were obtained by the two methods, as indicated by the high correlation between them. We suggest PCR-TGGE could be a useful and rapidly applied technique for the routine analysis of changes in cyanobacterial diversity in response to pollution, which would allow us to monitor rivers in surveillance networks of watercourse quality.