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. INTRODUCTIONHeterocystous cyanobacteria correspond to a monophyletic lineage (Wilmotte & Herdman, 2001) that contains the orders Nostocales and Stigonematales (subsections IV and V) (Rippka et al., 1979;Castenholz, 2001). The genera Rivularia and Calothrix belong to the order Nostocales and the family Rivulariaceae according to traditional classification (Geitler, 1932; Komárek & Anagnostidis, 1989;Whitton, 2002) and to subsection IV by bacteriological classification (Rippka et al., 2001a). The Rivulariaceae are among the most morphologically complex cyanobacteria (Whitton, 1987). They are characterized by tapered trichomes, apart from short phases of hormogonium formation. The mature trichome has a terminal heterocyst, although some species also have intercalary heterocysts, and cell division is largely localized to a region near the heterocyst (Whitton, 2002). Traditional taxonomy (Geitler, 1932) included 12 genera, some of which are widespread and others that have rarely been recorded (Whitton, 1987). Thus, Calothrix, Rivularia and Gloeotrichia can be considered the most representative genera of this group and are the most thoroughly studied. The genus Rivularia Agardh is easily distinguishable in the field by its characteristic development as gelatinous, hemispherical or subspherical colonies containing a large number of filaments, arranged radially or sometimes parallel ...

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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. INTRODUCTIONCyanobacteria are the simplest and oldest oxyphototrophic micro-organisms, comprising a single taxonomic and phylogenetic group. They are important since they can be used as experimental and model strains for studying the diversification of prokaryotic cells and the physiological processes occurring within the cell. The correct determination of cyanobacteria species or strains is one of the main challenges of experimental work. However, the extreme phenotypic flexibility of many species of cyanobacteria in culture produces atypical or anomalous morphologies that differ from those that are characteristic in nature. As a result, identification and taxonomy based on morphology are often difficult. A serious problem in experimental studies is the arbitrarily selected names for strains. The number of incorrectly identified strains in collections is surprisingly high (Komárek, 1994;Wilmotte, 1994). Attempts to identify cyanobacteria in culture by using a field-based system of classification, combined with the placement of cyanobacteria under the rules of the Bacteriological Code (Stanier et al., 1978), have added more ambiguities, but the establishment of two parallel and competing systems has been avoided. Oren (2004) stressed the need for botanical and bacteriological taxonomists to use unified rules to describe new taxa and to adopt a sin...

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.