Fluorescent oligonucleotide probes can be used to identify bacterial cells by hybridizing to specific rRNA sequences that are unique to a taxon. It has been suggested that single-cell RNA content could be estimated from the resulhng cell fluorescence, which is directly proportional to rRNA content However, this possibility was never assessed quantitatively for use in field studies, where most natural bacteria, due to their small size and slow growth, do not have enough rRNA to yield sufficient fluorescence for detection. One approach for increasing the fluorescence per rRNA molecule IS the use of multiple probes targeted to independent sites in the rRNA molecule. In this study, we hybridized n~ultiple probes (3 universal and 4 bacterial) to cultured marine bacterial isolates, and to natural bacterial assemblages from coastal water. For S of the 7 probes (2 probes did not hybridize to some of the isolates), cell fluorescence increased linearly with the number of probes hybridized. The natural bacterial assemblages showed an asymptotic increase of the probe-labeled fraction from ca 20% (1 probe) to 75% (5 probes) of the total population. The estimated detection limit was 0.3 fg RNA cell-' with S probes. RNA contents estimated from the fluorescence of probe-labeled cells agreed well to those determined separately by ethidium bromide fluorometry (differed by average 16 %, n = 3). Calibration of the photometer against fluorescence standards was critical for accurate measurements and comparisons.

The genus Leptolyngbya Anagnostidis & Komá rek (1988) was described from a set of strains identified as 'LPP-group B'. The morphology within this group is not particularly informative and underestimates the group's genetic diversity. In the present study, two new pseudanabaenacean genera related to Leptolyngbya morphotypes, Pantanalinema gen. nov. and Alkalinema gen. nov., are described under the provisions of the International Code of Nomenclature for Algae, Fungi and Plants, based on a polyphasic approach. Pantanalinema gen. nov. (type species Pantanalinema rosaneae sp. nov.) has sheaths and trichomes with slight gliding motility, which distinguish this genus from Alkalinema gen. nov. (type species Alkalinema pantanalense sp. nov.), which possesses trichomes arranged in an ornate (interwoven) pattern. 16S rRNA gene sequences of strains of Pantanalinema and Alkalinema exhibited low identity to each other (¡91.6 %) and to other sequences from known pseudanabaenacean genera (¡94.3 and 93.7 %, respectively). In a phylogenetic reconstruction, six sequences from strains of Pantanalinema and four from strains of Alkalinema formed two separate and robust clades (99 % bootstrap value), with the genera Oculatella and Phormidesmis, respectively, as the closest related groups. 16S-23S rRNA intergenic spacer sequences and secondary structures of strains of Pantanalinema and Alkalinema did not correspond to any previous descriptions. The strains of Pantanalinema and Alkalinema were able to survive and produce biomass at a range of pH (pH 4-11) and were also able to alter the culture medium to pH values ranging from pH 8.4 to 9.9. These data indicate that cyanobacterial communities in underexplored environments, such as the Pantanal wetlands, are promising sources of novel taxa.The cyanobacteria constitute a bacterial phylum with great morphological and metabolic diversity and are ubiquitous on Earth, including extreme environments (Castenholz & Waterbury, 1989). The classification of this microbial group has long been based on morphological traits, which are currently insufficient to delimit genera and species (Taton et al., 2003(Taton et al., , 2006Turicchia et al., 2009;Zammit et al., 2012; Genuário et al., 2013;Silva et al., 2014). In attempting to clarify cyanobacterial classification, many studies have applied a combination of morphological, ecological and molecular data (Perkerson et al., 2011; Hašler et al., 2012;Zammit et al., 2012;Andreote et al., 2014;Silva et al., 2014), and phylogenies based on the 16S rRNA gene have been widely used for generic definitions (Fox et al., 1992; Johansen & Casamata, 2005;Perkerson et al., 2011). Phylogenies based on this gene have demonstrated that some morphologically described genera are well defined in terms of evolutionary relationships (Komárek & Kaštovský, 2003; Willame et al., 2006; Komárek, 2010). However, data that have been obtained from 16S rRNA gene phylogenies have also led to the separation and definition of new genera, such as Desmonostoc The GenBank/EMBL/DDBJ acce...

RESUMOAs lagoas alcalinas do Pantanal da Nhecolândia, denominadas "salinas", são exemplos típicos de ambientes extremos por apresentarem elevados valores de pH (˃9). Este estudo mostrou as particularidades da microflora das "salinas" que é composta por espécies alcalíficas tais como Anabaenopsis elenkinii, Anomoeoneis sphaerophora var. sphaerophora e Nitzschia intermedia. Além disso, foi observado que alterações naturais e/ou antrópicas, que permitem a entrada de água doce de sistemas adjacentes às "salinas", descaracterizam a flora típica desses ambientes. Assim, o presente estudo enfatiza que as características naturais de isolamento das "salinas" devem ser preservadas e que a composição da sua microflora é uma importante ferramenta para avaliar mudanças nestes sistemas. Palavras-chave: Pantanal; lagoas alcalinas; biodiversidade; microflora. ABSTRACTALGAE AND CYANOBACTERIA FROM EXTREME ENVIRONMENT OF THE BRAZILIAN PANTANAL. The alkaline lakes, known as "salinas", in Pantanal are typical examples of extreme environments due to their high pH (>9) values. The present study showed the particularities of the microflora in these alkaline lakes that is composed of alkaliphic species such as Anabaenopsis elenkinii, Anomoeoneis sphaerophora var. sphaerophora and Nitzschia intermedia. Besides, it was detected that natural or anthropogenic disturbances that allowed freshwater from adjacent lakes go into the "salinas", totally modified the typical flora of these systems. Thus, our study indicates that the natural condition of isolation of the alkaline lakes should be preserved and that the composition of its microflora is a very important tool to evaluate the changes in these systems. RESUMEN ALGAS Y CIANOBACTERIAS DE AMBIENTES EXTREMOS DEL PANTANAL BRASILEÑO.Los lagos alcalinos del Pantanal, conocidos como "salinas", son ejemplos típicos de ambientes extremos debido a sus elevados valores de pH (>9). El presente estudio mostró las particularidades de la microflora de estos lagos alcalinos que está compuesta por especies alcalófilas tales como Anabaenopsis elenkinii, Anomoeoneis sphaerophora var. sphaerophora y Nitzschia intermedia. Además, se observó que los disturbios naturales y/o antrópicos, que permiten el ingreso de agua dulce de los sistemas adyacentes a las "salinas", modifican totalmente la flora típica de los estos ambientes. De esta manera, este estudio enfatiza en que las condiciones naturales de aislamiento de las "salinas" debe ser preservado y que la composición de su microflora es una herramienta importante para evaluar los cambios en estos sistemas. Palabras-clave: Pantanal; lagos alcalinos; biodiversidad; microflora.

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