Pilar Mateo

Spatial and temporal changes in water quality in a Spanish river

Spatial and temporal variation in the water quality of a Spanish river (Alberche River) were examined during 2 consecutive years. Principal component analysis was used to analyse the environmental factors associated with the physico-chemical variability. The first principal component corresponded to the variation of water solute content along the course of the river. In general, the conservative parameters (alkalinity, sulphate, calcium, and chloride) gradually increased in a downstream direction. However, nutrient variables showed marked differences depending on the location and the season. Two sections can be distinguished in the river. The first in an upstream zone with little human occupation, a siliceous substrate produces waters with low ionic composition and few nutrients. However, the second, downstream zone, featured high levels of phosphate and dissolved inorganic nitrogen in summer and to a lesser extent in spring. The variation in nutrient content in this section of the river can be attributed to anthropogenic sources since in these seasons the presence of holiday-makers leads to a high population density in the residential buildings and recreation areas. Thus, in this area, seasonal increases in human activities at some locations cause high levels of nutrients and may therefore be considered to be classed subject to spatio-temporal eutrophication.

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.

Implications of cyanophyte toxicity on biological monitoring of calcareous streams in north-east Spain

There is increasing evidence that benthic cyanophytes in Mediterraneancalcareous streams can influence the macroinvertebrate community, probablydirectly due to toxicity. A study was therefore planned to establish whetherthis also influenced the results of indices used for the biological monitoringof stream water quality. Two indices (BMWPc based on macroinvertebrates; IBDbased on diatoms) widely used to assess water quality based on the diversity ofmacroinvertebrate and diatom assemblages were calculated for two unpollutedMediterranean calcareous streams in north-east Spain. The toxicity levelsof the cyanophyte communities were quantified by the Microtox test. There was aclear inverse relationship between the dominance of cyanophytes and the valuesobtained for the biological indices. It is recommended that cyanophyte biomassshould be incorporated in some way in the calibration of index values; the useof phycobiliprotein concentration and/or the chlorophyll a:phycobiliproteinratio is proposed.

Epilithic cyanobacterial communities and water quality: an alternative tool for monitoring eutrophication in the Alberche River (Spain)

Changes in epilithic cyanobacterial communities were determined in a river characterized by variations in nutrient content. The cyanobacterial community composition of the upstream sites was different from that of the downstream communities, where anthropogenic influences lead to an increase in nutrients (principally soluble reactive phosphate, SRP). There was a general trend in downstream sites towards a decrease in species richness, abundance, and diversity of cyanobacteria. The reduced cyanobacterial species richness in downstream locations was due largely to a marked decrease in the number of heterocystous species, although the number of non-heterocystous species also decreased. Epilithic phycobiliprotein content was positively correlated with the number of cyanobacterial cells, implying that this pigment provides information about the abundance of the cyanobacteria community in the epilithon. The lowest concentrations of phycobiliprotein in the epilithon were observed where concentrations of phosphate were highest. Similarly, the number of heterocystous and non-heterocystous species tended to decrease as the SRP increased, and as the DIN:SRP ratio decreased. However, no relation was found with dissolved inorganic nitrogen (DIN). The differences among cyanobacterial communities could be interpreted as being a consequence of variations in nutrient composition. Finally, the usefulness of cyanobacteria as an alternative tool for assessing changes in water quality is discussed.

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.

Molecular Fingerprinting of Cyanobacteria from River Biofilms as a Water Quality Monitoring Tool

ABSTRACT 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.

PHYSIOLOGICAL DIFFERENCES BETWEEN TWO SPECIES OF CYANOBACTERIA IN RELATION TO PHOSPHORUS LIMITATION1

In order to gain a better understanding of the factors responsible for changes in cyanobacterial species composition in the Alberche River (central Spain), which is characterized by spatiotemporal N and P variability, we undertook P‐limitation studies on two isolated N2‐fixing species that are well represented in the river: Calothrix elenkinii (Kosinskaja) UAM 225 and Nostoc punctiforme (Kützing) UAM 220. We examined differences in phosphorus‐storage capacity, phosphate uptake, and phosphatase activity between the two isolates grown under N2‐fixing conditions. Although they shared a similar pattern of phosphate uptake, C. elenkinii cultures had a greater phosphorus storage capacity and greater phosphatase activity than those of N. punctiforme. The former showed a stronger response when cultured under low P concentrations. The two species showed specific differences in the rates of intracellular polyphosphate granule utilization and phosphatase activity. Our results suggest that C. elenkinii might be particularly well suited to river environments in which nutrient concentrations are generally low, but which experience episodic pulses of high P supply (inorganic and/or organic), favoring “luxury uptake” and storage strategies.

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.

Novel cyanobacterial bioreporters of phosphorus bioavailability based on alkaline phosphatase and phosphate transporter genes of Anabaena sp. PCC 7120

AbstractThere is heterogeneity in the way cyanobacteria respond to P starvation and subsequently how they adapt to environments with low or fluctuating P concentrations. In this study, we have fused the promoterless lux operon luxCDABE to the promoter regions of Anabaena sp. PCC 7120 phoA genes putatively encoding alkaline phosphatases, phoA (all2843) and phoA-like (alr5291) and to the promoter region of one operon putatively encoding a high affinity phosphate transporter pst1 (all4575-4572). The self-bioluminescent strains constructed in this way, Anabaena AP (phoA promoter), Anabaena AP-L (phoA-like promoter), and Anabaena PST (pst1 promoter) have been used to study the expression of these genes in response to P starvation and P re-feeding with inorganic and organic phosphate sources. Our data showed that the pst1 promoter was activated at much higher level than the phoA-like promoter following P starvation; however, we did not observe activation of the phoA promoter. The P re-feeding experiments revealed that both strains, Anabaena (A.) PST and A. AP-L could be used as novel bioreporters of P availability in environmental samples. Both strains were used to estimate bioavailable P in environmental samples (fresh- and wastewaters) with a wide range of soluble P concentrations. The results indicated that most of the P in the water samples was in chemical forms available to the cyanobacterium; however there were some differences in the estimates given by both strains as A. PST appeared to be more adequate for the samples with the lowest P load while A. AP-L gave similar or even higher values of P concentrations than those chemically measured in samples with higher P load. FigureSchematic representation of the cellular elements involved in the Pho regulon in most cyanobacteria. The micrograph shows a filament of an Anabaena P-bioavailability bioreporter where P-responses are coupled to a luminescent signal

Polyphasic characterization of benthic cyanobacterial diversity from biofilms of the Guadarrama river (Spain): morphological, molecular, and ecological approaches1

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.