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Biofilms microscopically dominated by cyanobacteria and diatoms of two CO 2 degassing karst-water creeks in Germany were investigated for their diversities along a gradient of calcification using SSU rRNA gene cloning and sequencing from environmental samples. The recovered totals of 731/413 cyanobacteria/diatom clones were grouped at 97/98% similarity levels into 28/29 molecular OTUs widely spread over their corresponding sequence phylogenies forming mostly monophyletic subclades. Sequence comparisons with named reference strains from NCBI/GenBank as well as newly determined references from the SAG culture collection left about half of the cyanobacteria OTUs still unidentified. Most of the diatom OTUs could be identified at least at the generic level. To improve identification also cultures of cyanobacteria and diatoms were established that allowed even species identification of some diatoms, but also revealed additional cyanobacteria hard to identify which were not recovered in the clone libraries. A significant correlation of the relative OTU abundances in clone libraries with values of SI calcite was found and, therefore, redundancy analysis distinguished highly calcified sites far from the spring from those less calcified closer to the spring. The noncalcified spring sites were clearly distinct from all other sites by the presence of four cyanobacteria OTUs exclusively retrieved and that no diatoms could be recovered from there. Four cyanobacteria and three diatom OTUs were recovered whose increasing relative abundance per clone library was correlated with increasing calcification. This may indicate that not only cyanobacteria, but also diatoms are more directly involved in the biogenic impact on tufa formation than assumed previously.

Akinetes are the dormant cells of Nostocales (cyanobacteria) that enable the organisms to survive harsh environmental conditions while resting in bottom sediments. The germination of akinetes assists the dispersal and persistence of the species. The assessment of the akinete pool in lake sediments is essential to predict the bloom formation of the Nostocales population. We present here the implementation of an improved catalysed reporter deposition (CARD)-fluorescence in situ hybridization (FISH) protocol to assist the identification and quantification of akinetes in sediment samples. Several 16S rRNA gene oligonucleotide probes were evaluated for labelling akinetes of various species of Anabaena, Aphanizomenon and Cylindrospermopsis. Akinetes of all the taxa studied were successfully labelled and could be easily detected by their bright fluorescence signal. The probes' specificity was tested with 32 strains of different taxa. All six Cylindrospermopsis raciborskii strains were labelled with a specific probe for its 16S rRNA gene. A more general probe labelled 73% of the Anabaena and Aphanizomenon strains. The counting data of field samples obtained with CARD-FISH and the regular light microscopy approach did not differ significantly, confirming the suitability of both methods. The CARD-FISH approach was found to be less time-consuming because of better visibility of akinetes.

For the first time the diversity of microscopic green algae (Chlorophyta) from calcified biofilms of karstic streams was analyzed using a combined approach based on pure cultures, i.e., 18S rRNA gene sequencing and microscopic analyses. Our study focused on two creeks in Germany. A considerable diversity of 34 species of green microalgae comprising three classes, the Trebouxiophyceae, Chlorophyceae and Ulvophyceae, was recovered. The biofilms of both streams were rather different in their species compositions which may reflect that they are exposed to differed hydrochemical conditions. The shallow Westerh€ ofer creek harbored predominantly Trebouxiophyceae and exhibited higher Mg 2C and SO 4 2¡ concentrations. In contrast, the deeper, longer and spatially more heterogeneous Deinschwanger creek harbored numerous species of Chlorophyceae. A lower number of species from the Ulvophyceae were spread on both studied streams. The closest relatives of the identified species were from other freshwater habitats, but mostly from phytoplankton. However, also several species we recovered from freshwater for the first time; so far they have been known from terrestrial habitats only. Less than half of the recovered species could be identified with names at the species level based on high sequence identities with available sequences from named reference strains. Most recovered species could not be identified with names to species level, i.e., they may represent species for which no 18S rRNA gene sequence have become available yet or which have been retrieved for the first time. The genus Gongrosira K€ utzing, often reported from freshwater tufa-stromatolites, was found to represent most likely a collective morphotype formed by several genera nested within the Ulvophyceae.