Changing environmental conditions underpin long-term patterns of phytoplankton in a freshwater reservoir.

Environmental changes can exert strong pressure on freshwater biota and lead to unwanted alterations of local communities and deterioration of ecosystem services. Disentangling the links between environmental and community changes is, therefore, essential to understand and predict the impact of human activities on freshwater ecosystems. This is particularly relevant for man-made freshwater reservoirs that represent a nexus between anthropogenic, environmental, and biotic effects. Reservoir food webs depend strongly on phytoplankton dynamics, which are affected by abiotic conditions, nutrient availability and grazing pressure by zooplankton. We studied the effects of relevant environmental drivers (hydrochemistry, hydrodynamics and zooplankton) on the composition, diversity and community stability of main morpho-functional phytoplankton groups over 32 years in the Rímov Reservoir (Czech Republic). Environmental conditions in the reservoir are characterised by three distinct periods (1983-89, 1990-99, and 2000-14) defined by shifts and breakpoints in temporal trends in reservoir hydrochemistry and hydraulic conditions, and we examined if and how phytoplankton responded to these abrupt changes. We found significant differences in phytoplankton composition among the three periods. Phytoplankton underwent a substantial compositional shift towards a dominance of pennate diatoms. Time-lag analysis of dissimilarity in phytoplankton composition revealed higher and stochastic annual variations until 1999, followed by a lower variability and divergence in phytoplankton composition in subsequent years. Changes in overall phytoplankton assemblage and most abundant morpho-functional phytoplankton groups were driven mainly by hydrochemical (total nitrogen) and hydrodynamic variables (inflow rate, surface level and mixing depth) and less by zooplankton dynamics. These results suggest that phytoplankton are driven primarily by nutrient input and water regime, both of which can be appropriately managed to support valuable ecosystem services provided by phytoplankton in freshwater reservoirs.

POLYPHASIC CHARACTERIZATION OF DOLICHOSPERMUM SPP. AND SPHAEROSPERMOPSIS SPP. (NOSTOCALES, CYANOBACTERIA): MORPHOLOGY, 16S rRNA GENE SEQUENCES AND FATTY ACID AND SECONDARY METABOLITE PROFILES(1).

The genera Dolichospermum (Ralfs ex Bornet et Flahault) Wacklin, L. Hoffm. et Komárek and Sphaerospermopsis Zapomelová, Jezberová, Hrouzek, Hisem, K. Reháková et Komárk.-Legn. represent a highly diversified group of planktonic cyanobacteria that have been recently separated from the traditional genus Anabaena Bory ex Bornet et Flahault. In this study, morphological diversity, phylogeny of the 16S rRNA gene, production of fatty acids, and secondary metabolite profiles were evaluated in 33 strains of 14 morphospecies isolated from the Czech Republic. Clustering of the strains based on 16S rRNA gene sequences corresponded to wider groups of species in terms of morphology. The overall secondary metabolite and fatty acid profiles, however, were not correlated to each other and neither were they correlated to the 16S rRNA phylogeny nor the morphology of the strains. Nevertheless, a minor part of the detected secondary metabolites (19% of all compounds) was present only in close relatives and can be thus considered as autapomorphic features.

POLYPHASIC CHARACTERIZATION OF THREE STRAINS OF ANABAENA RENIFORMIS AND APHANIZOMENON APHANIZOMENOIDES (CYANOBACTERIA) AND THEIR RECLASSIFICATION TO SPHAEROSPERMUM GEN. NOV. (INCL. ANABAENA KISSELEVIANA)(1).

Occurrences of rare cyanobacteria Anabaena reniformis Lemmerm. and Aphanizomenon aphanizomenoides (Forti) Horecká et Komárek were recently detected at several localities in the Czech Republic. Two monoclonal strains of An. reniformis and one strain of Aph. aphanizomenoides were isolated from distant localities and different sampling years. They were characterized by a combination of morphological, genetic, and biochemical approaches. For the first time, partial 16S rRNA gene sequences were obtained for these morphospecies. Based on this gene, all of these strains clustered separately from other planktonic Anabaena and Aphanizomenon strains. They appeared in a cluster with Cylindrospermopsis Seenaya et Subba Raju and Raphidiopsis F. E. Fritsch et M. F. Rich, clustered closely together with two An. kisseleviana Elenkin strains available from GenBank. A new generic entity was defined (Sphaerospermum gen. nov., with the type species S. reniforme, based on the traditional species An. reniformis). These results contribute significantly to the knowledge base about genetic heterogeneity among planktonic Anabaena-like and Aphanizomenon-like morphospecies. Accordingly, the subgenus Dolichospermum, previously proposed for the group of planktonic Anabaena, should be revaluated. Secondary metabolite profiles of the An. reniformis and Aph. aphanizomenoides strains differed considerably from 17 other planktonic Anabaena strains of eight morphospecies isolated from Czech water bodies. Production of puwainaphycin A was found in both of the An. reniformis strains. Despite the relatively short phylogenetic distance from Cylidrospermopsis, the production of cylindrospermopsin was not detected in any of our strains.

Morphological transformation in a freshwater Cyanobium sp. induced by grazers.

We document a remarkable morphological transformation, attributable to grazing by nanoflagellate Ochromonas sp. DS, of a phycocyanin-rich freshwater Cyanobium sp. (10-NR 98.2% similar 16S rRNA gene sequence to the type species Cyanobium gracile). The single cells aggregated into microcolonies (average size 40 microm) in the presence of the protist. Colonies were characterized by hundreds of tubes (spinae), 100 nm to 1 microm long and 63 +/- 6 nm wide, on the surfaces of the Cyanobium cells co-cultured with Ochromonas. Spinae production, previously unknown for the freshwater Cyanobium species, suggests that picocyanobacterial life strategies are more flexible than previously thought.

Summer changes in cyanobacterial bloom composition and microcystin concentration in eutrophic Czech reservoirs.

In mid-July and August 2003 and 2004, 18 reservoirs in the Czech Republic were sampled for phytoplankton species composition and concentration of intracellular microcystins (MCs). As a consequence of high nutrient loading, most of the reservoirs experienced cyanobacterial blooms of various intensities, with the prevalence of cyanobacteria increasing markedly in August, along with a conspicuous shift in species composition toward dominance of Microcystis spp. Microcystins were detected in 90% of the samples, and their amount also increased considerably in August, reflecting the cyanobacterial biomass. In Microcystis-dominated samples, a significantly higher amount of MCs (p < 0.001) occurred than in samples in which other taxa prevailed. Microcystins were positively correlated with chlorophyll a and cyanobacterial biovolume (p < 0.05, R2 = 0.61 and 0.66, respectively), with the strongest correlation found for Microcystis spp. biovolume (p < 0.001, R2 = 0.87). This taxon was the most important producer of MCs in Czech reservoirs. The main structural variants of MCs were MC-LR, MC-RR, and MC-YR. This study's data also indicate that the relative share of MC variants (MC-LR and MC-RR) varies considerably with time, most likely as a consequence of different species and strain compositions during the summer. This study clearly demonstrates a high prevalence of MC-producing cyanobacteria in Czech reservoirs. Therefore, regular monitoring of these reservoirs is highly desirable in an effort to minimize potential health risks to the human population.

Extracellular phosphatase activity of natural plankton studied with ELF97 phosphate: fluorescence quantification and labelling kinetics.

ELF(R)97 phosphate (ELFP) is a phosphatase substrate which produces ELF(R)97 alcohol (ELFA), a fluorescent water-insoluble product, upon hydrolysis. We studied the kinetics of ELFA precipitation in freshwater samples at levels of total plankton and single phytoplankton cells, and tested the suitability of ELFP for measurement of surface-bound algal extracellular phosphatases. Samples from acidic Plesné Lake (pH approximately 5; high phosphatase activity) and eutrophic Rímov reservoir (pH approximately 7-10; moderate phosphatase activity) were incubated with ELFP for 5-300 min, fixed with HgCl2 and filtered through polycarbonate filters. Relative fluorescence of filter-retained ELFA precipitates was quantified with image analysis. Time-courses of ELFA formation exhibited lag periods followed by finite periods of linear increase. In Plesné Lake, lag-times were shorter (1-18 min) and rates of increase in ELFA fluorescence higher (by approximately 2 orders of magnitude) than in Rímov reservoir (lag-times 30-200 min). Similar patterns of ELFA formation kinetics were also observed in Plesné Lake samples in cuvette spectrofluorometer measurements (which failed in Rímov reservoir). Linear regression of seasonal data on rates of increase in ELFA fluorescence from image cytometry and spectrofluorometry (r2 = 0.65, n = 10) allowed for calibration of image cytometry in terms of amount of cell-associated ELFA. Preliminary measurements of extracellular phosphatase activities of several algae resulted in rates (10-2260 fmol cell-1 h-1) which are comparable to data reported in the literature for algal cultures.