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.

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.

Differences in the Cyanobacterial Community Composition of Biocrusts From the Drylands of Central Mexico. Are There Endemic Species?

In drylands worldwide, biocrusts, topsoil microbial communities, are prevalent, contributing to the biostabilization of soils and allowing the subsequent establishment and growth of vascular plants. In early successional biocrusts, cyanobacteria are the first dominant colonizers of bare ground, largely determining their functioning. However, there are large gaps in our knowledge of the cyanobacterial diversity in biocrusts, particularly in understudied geographic regions, such as the tropical latitudes. We analyzed the diversity of the cyanobacteria inhabiting the biocrusts of semideserts from Central Mexico in two localities belonging to the same desert system (Chihuahuan Desert) that are separated by a cordillera that crosses the center of Mexico. Morphological identification of the cyanobacteria was carried out after cultivation in parallel with the direct observation of the environmental samples and was supported by genetic characterization through analysis of the 16S rRNA gene of the isolated strains and by next-generation sequencing of the soil samples. Taxonomic assignment revealed a clear dominance of heterocystous cyanobacteria at one of the studied locations (Actopan, Hidalgo state). Although heterocystous forms were abundant at the other location (Atexcac, Puebla state), almost a third of the cyanobacterial phylotypes were represented by unicellular/colonial cyanobacteria, mostly Chroococcidiopsis spp. Only 28.4% of the phylotypes were found to be common to both soils. Most of the other taxa, however, were biocrust-type specific, and approximately 35% of the phylotypes were found to be unique to the soil they were collected in. In addition, differences in the abundances of the shared cyanobacteria between the locations were also found. These differences in the cyanobacterial distribution were supported by the distinct responses of the isolated strains representative of the sites to extreme heat and desiccation in bioassays. Some cyanobacteria with high abundance or only present at the hottest Actopan site, such as Scytonema hyalinum, Scytonema crispum, Nostoc commune, Nostoc sp., and Calothrix parietina, survived extreme heat and desiccation. However, Tolypothrix distorta and Chroococcidiopsis spp. were clearly sensitive to these extreme conditions in relation to their lower abundances at Actopan as opposed to Atexcac. Since novel biocrust-associated phylotypes were also found, the emergence of endemic cyanobacterial taxa is 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.

Intracoronary tenecteplase versus abciximab as adjunctive treatment during primary percutaneous coronary intervention in patients with anterior myocardial infarction.

AIMS: We sought to compare the effects of intracoronary administration of a fibrinolytic drug (tenecteplase) to those of a glycoprotein IIb/IIIa inhibitor (abciximab) in patients with ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PPCI). METHODS AND RESULTS: In this pilot trial, 76 patients (59 male) with anterior STEMI were randomised to intracoronary infusion of reduced-dose tenecteplase or abciximab during PPCI. Angiography was repeated at 48 hours to assess corrected TIMI frame count (cTFC) and TIMI myocardial perfusion grade (TMPG). The primary endpoint was infarct size as assessed by cardiac MRI. The abciximab group showed lower cTFC (median 14.1 [IQR 9.4-17.1]) than the tenecteplase group (18.2 [10.0-28.2]) (p=0.02), and the proportion of patients with TMPG grade 2/3 was higher in the abciximab group (90.3% vs. 67.7%; p=0.03). Major cardiac and cerebrovascular event rates did not differ; however, notably, 2/38 patients in the tenecteplase group experienced subacute stent thrombosis. At four months, there were no significant differences in infarct size between the tenecteplase and abciximab groups (17.0 g [9.6-27.5] vs. 21.1 g [11.3-35.0], p=0.33). CONCLUSIONS: Intracoronary administration of tenecteplase did not reduce infarct size compared to abciximab in STEMI patients undergoing PPCI. Tenecteplase exhibited poorer myocardial reperfusion and might be associated with increased subacute stent thrombosis.

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.

Diversity of biocrust-forming cyanobacteria in a semiarid gypsiferous site from Central Spain.

Cyanobacteria are a key constituent of biocrusts, communities dominated by lichens, mosses and associated microorganisms, which are prevalent in drylands worldwide and that largely determine their functioning. Despite their importance, there are large gaps in our knowledge of the composition and diversity of cyanobacteria associated with biocrusts, particularly in areas such as the Mediterranean Basin. We studied the diversity of these cyanobacteria in a gypsiferous grassland from Central Spain using both morphological identification after cultivation and genetic analyses with the 16S rRNA gene. Nine different morphotypes were observed, eight corresponding to filamentous, and one to unicellular cyanobacteria. We found cyanobacterial genera typical of biocrust communities, such as Microcoleus and Trichocoleus, and N-fixing cyanobacteria such as Scytonema and Nostoc. Genetic information allowed us to identify cultures belonging to recently described genera such as Roholtiella, Nodosilinea and Mojavia. We also describe two new phylotypes of Microcoleus and Scytonema, which are key genera contributing to ecosystem functioning in biocrust-dominated ecosystems worldwide.

Validación de la Encuesta de Percepción de Enfermedad Revisada (Illness Perception Questionnaire) entre pacientes con patologías crónicas médicas y psiquiátricas / Validation of Illness Perception Survey Revised (Illness Perception Questionnaire) among patients with medical and psychiatric chronic diseases

MARCO TEÓRICO: La Percepción de Enfermedad es una variable importante en la evolución de los pacientes. Existen métodos de evaluación cualitativa de éste parámetro, sin embargo, no son aplicables a la población chilena por no estar adaptados al lenguaje local y cotidiano. OBJETIVO: Realizar una traducción del Illness Perception Questionnaire Revised, conservando la validez y fiabilidad original. MATERIALES Y MÉTODOS: Traducción y retraducción de la escala por experto. Piloteo de evaluación a 33 pacientes con enfermedades crónicas. Encuesta a 112 pacientes (49 diabéticos, 37 hipertensos y 33 esquizofrénicos) todos pertenecientes al CRS Hospital El Pino. CONCLUSIONES: En base al porcentaje de concordancia obtenido (95%) entre la versión original y la traducida a la lengua española y al reporte de los entrevistadores con respecto a la comprensibilidad manifestada por los pacientes, podemos concluir que la traducción por nosotros aplicada cumple con los criterios de validez.

THEORETICAL FRAMEWORK: The perception of disease is an important variable in the patient evolution. There are methods of qualitative evaluation of this parameter, however, are not applicable to the Chilean people for not being adapted to the local and everyday language. OBJECTIVE: A translation of the Revised Illness Perception Questionnaire, preserving the original set reliability and validity. MATERIALS AND METHODS: Translation and back translation of the scale by an expert. Piloting assessment to 33 patients with chronic diseases. Survey of 112 patients (49 diabetic and 33 hypertensive schizophrenics 37) all belonging to the CRS Hospital Pino. CONCLUSIONS: Based on the percentage of concordance obtained (95%) between the original version and the translated version in the Spanish language and the interviewers' report regarding the comprehensibility expressed by the patients, we can conclude that the translation applied by us meets the criteria of validity

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.

Valores de referencia en plasma de osmolalidad, electrolitos, calcio, magnesio y urea en una población del centro de España / Reference values in plasma for osmolality, electrolytes, calcium, magnesium and urea, in a spanish population

Introducción: El establecimiento de los valores de referencia es una tarea esencial de los laboratorios clínicos, ya que pueden variar según los instrumentos empleados y la población considerada. En el presente trabajo pretendemos definir el intervalo de referencia en nuestra población para las siguientes magnitudes biológicas en plasma: osmolalidad, sodio, potasio, calcio, urea, y magnesio con el analizador Unicell DXC 800. Material y métodos: Se seleccionó una muestra de referencia formada por 146 individuos sanos entre los donantes que acudieron a nuestro banco de sangre y se les extrajo sangre en un tubo de heparina litio que fue centrifugado a 1.700 g durante 5 minutos para la obtención de plasma. En el plasma resultante se analizaron urea, sodio, potasio, calcio y magnesio con el analizador Unicell DXC 800 (Beckman Coulter®), midiéndose la osmolalidad con el osmómetro 3300 (Advanced Instruments®). Se aplicó el método no paramétrico del CLSI C28-A3 para la obtención de los valores de referencia. Resultados: Se obtuvieron los siguientes intervalos de referencia: urea (13,67-48 mg/dL), sodio: 136,7-145 mmol/L), potasio: (3,37-4,6 mmol/L), osmolalidad: (290-305,42 mosm/kg), calcio (8-9,3 mg/dL) y magnesio (1,9-2,2 mg/dL). Estos intervalos fueron diferentes a los propuestos por el fabricante, lo que refuerza la necesidad de producir valores de referencia propios (AU)

Introduction: The establishing of reference values is an essential task of clinical laboratories. In this work the reference intervals for the following parameters in plasma were determined: osmolality, sodium, potassium, calcium, magnesium and urea, using a Unicell DXC 800 analyzer (Beckman Coulter®). Material and methods: A sample of 146 healthy individuals was selected from health blood donors of our institution. Plasma was obtained by centrifugation of lithium heparin tubes at 1700 g for five minutes. Sodium, potassium, calcium, magnesium and urea were measured on a Unicell DXC 800 analyzer (Beckman Coulter®), whereas osmolality was measured with an osmometer (Advanced Instruments® 3300). The CLSI C28 A-3 non-parametric method was used to determine the reference intervals. Results: The reference intervals obtained were: osmolality: (290-305.42 mosm/kg), sodium: (136.7-145 mmol/L), potassium: (3.37-4.6 mmol/L), calcium (8-9.3 mg/dL), magnesium (1.9- 2.2 mg/dL) and urea (13.67-48 mg/dL). These values are different from those claimed by the manufacturer, which reinforces the need for determining individual reference values in each laboratory (AU)

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.

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.

A battery of bioreporters of nitrogen bioavailability in aquatic ecosystems based on cyanobacteria.

Cyanobacteria are the main primary producers and are responsible for the blooms and eutrophication processes caused by excess nutrients in surface waters. The aim of this paper is to use cyanobacteria to monitor the presence and bioavailability of different chemical species of nitrogen in freshwater. Cyanobacteria have mechanisms which can detect the presence of nutrients in their environment and can activate or repress specific genes, or operons, depending on nutrient bioavailability. Therefore, monitoring the expression of these genes can facilitate measurement of the availability of nutrients. To achieve this we have constructed self-bioluminescent reporter strains of the filamentous nitrogen-fixing cyanobacterium Nostoc sp. PCC 7120 expressing promoters of genes responsive to nitrogen fused to the luxCDABE operon. Three promoters were selected to direct the expression of luxCDABE: The first, the glnA promoter, is activated in the absence of combined nitrogen. We found that it responded linearly to the addition of known amounts of combined N in the range 50-500 µM NH4(+) or NO3(-). The second, the nirA operon promoter, turns on in the presence of nitrate being inhibited by ammonium. The bioreporter responded linearly in the range of 10-100 µM NO3. The third, the gifA promoter, is activated in the presence of ammonium, responding linearly in the range 100-600 µM NH4(+). We also used a previously described strain of Synechococcus elongatus PCC 7942 expressing glnN (glutamine synthetase type III) fused to luxAB. We found that the glnN promoter responded linearly to the addition of known amounts of N in the range 50-500 µM NH4(+) or NO3(-). These cyanobacterial bioreporters were tested in real environmental samples (i.e. river waters) which confirmed their validity and showed a broad spectrum response. They are therefore useful in the detection of both total N-bioavailability and specific nitrogen species.

Temperature drives the continental-scale distribution of key microbes in topsoil communities.

Global warming will likely force terrestrial plant and animal species to migrate toward cooler areas or sustain range losses; whether this is also true for microorganisms remains unknown. Through continental-scale compositional surveys of soil crust microbial communities across arid North America, we observed a latitudinal replacement in dominance between two key topsoil cyanobacteria that was driven largely by temperature. The responses to temperature of enrichment cultures and cultivated strains support this contention, with one cyanobacterium (Microcoleus vaginatus) being more psychrotolerant and less thermotolerant than the other (M. steenstrupii). In view of our data and regional climate predictions, the latter cyanobacterium may replace the former in much of the studied area within the next few decades, with unknown ecological consequences for soil fertility and erodibility.

Structure of the O-antigen of the lipopolysaccharide isolated from Pantoea ananatis AEP17, a rhizobacterium associated with rice.

The lipopolysaccharide of a Gram-negative bacterium having a putative plant-growth promoting activity (Pantoea ananatis AEP17) has been isolated and subjected to partial hydrolysis. The O-antigen has been studied by mass spectrometry and NMR experiments. On the basis of these experiments it is concluded that the following repeating unit is present in the polysaccharide: →3)-ß-d-GlcpNAc-(1→3)[α-d-GalpAN-(1→2)]-α-l-Rhap-(1→2)-α-l-Rhap-(1→3)-α-l-Rhap-(1→2)-α-l-Rhap-(1→ The occurrence of d-galacturonamide (GalAN) is unusual in bacterial O-polysaccharides. It has only been reported in Escherichia coli O65 [Perry, M. B.; MacLean, L. L. Carbohydr. Res.1999, 322, 57-66].

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.

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.