Alena Lukešová

Soil biota and upper soil layer development in two contrasting post-mining chronosequences

Abstract Density and community composition of a wide spectrum of soil organisms (fungi, algae, testate amoebae, nematodes, enchytraeids, lumbricids, oribatid mites, diplopods, terrestrial isopods, collembolans and dipteran larvae), direct counts of bacteria, rate of cellulose decomposition and microstructure of upper soil layers were studied in two chronosequences of plots reclaimed from open-cast coal mining near Cottbus (Germany) and near Sokolov (Czech Republic). German plots were characterized by acidic sandy soils and afforested with pine, while the Czech plots possessed alkaline clay soils and were afforested with alder. In both chronosequences, density and species richness in most of investigated groups of soil biota gradually increased with increasing succession age. Typical pioneer species were found in initial stands of both chronosequences. Nevertheless, the initial stands supported poorer assemblages of soil biota in the Cottbus area than did those in the Sokolov area. The Cottbus area was characterized by a moor type of humus and by gradual uniform increase of abundances and species numbers of most studied groups of soil biota. On the contrary, moder type of humus and a more variable pattern of soil biota development during the course of succession were found in the Sokolov area. Slower development of soil biota in initial stages of succession in Cottbus is caused by unfavourable physical and chemical conditions of heap substrates, and by lower input and poor quality of litter in pine plantations.

Diversity and abundance of soil algae in the polar desert, Sverdrup Pass, central Ellesmere Island

Cyanobacteria and eukaryotic algae were investigated during three seasons in 18 plots established across Sverdrup Pass valley of central Ellesmere Island, 79°N, Canada. The sites differed in altitude, substratum, and other characteristics. A high species diversity totalled 136 taxa. Cyanobacteria accounted for 52 and eukaryotic algae 84 species. In both groups, numerous species did not correspond to any taxa described. However, high diversity did not always coincide with high algal abundance or biomass. On older and stable landscapes, visible crusts developed, containing mostly cyanobacteria, fungi, and other microbial components. Considerable variation in algal diversity and abundance was found among the sites. Also the southern, granitic portion of the pass was richer in green algae compared to its northern, dolomitic portion where motile cyanobacteria were more prominent. These micro-autotrophs occupied the soil profile to a depth of 7 cm. Their highest density was not at the surface but at 3–4 cm depth. One plot was contaminated by windblown copper-rich dust from a nearby outcrop and soil here was poorest in content of photosynthetic pigments, suggesting a local heavy-metal toxicity.

Differential patterns of evolution and distribution of the symbiotic behaviour in nostocacean cyanobacteria

Many cyanobacteria commonly identified as belonging to the genus Nostoc are well-known cyanobionts (symbionts) of a wide variety of plants and fungi. They form symbioses with bryophytes, pteridophytes, gymnosperms and angiosperms that are considerably different in the type of reciprocal interaction between the host and the cyanobiont. The phylogenetic and taxonomic relationships among cyanobionts isolated from different hosts and Nostoc strains isolated from free-living conditions are still not well understood. We compared phylogeny and morphology of symbiotic cyanobacteria originating from different host plants (genera Gunnera, Azolla, Cycas, Dioon, Encephalartos, Macrozamia and Anthoceros) with free-living Nostoc isolates originating from different habitats. After preliminary clustering with ARDRA (amplified rDNA restriction analysis), phylogeny was reconstructed on the basis of 16S rRNA gene sequences and compared with morphological characterization, obtaining several supported clusters. Two main Nostoc clusters harboured almost all cyanobionts of Gunnera, Anthoceros and of several cycads, together with free-living strains of the species Nostoc muscorum, Nostoc calcicola, Nostoc edaphicum, Nostoc ellipsosporum and strains related to Nostoc commune. We suggest that the frequent occurrence of symbiotic strains within these clusters is explained by the intensive hormogonia production that was observed in many of the strains studied. However, no evidence for discrimination between symbiotic and free-living strains, either by molecular or morphological approaches, could be found. Sequences of Azolla cyanobiont filaments, taken directly from leaf cavities, clustered tightly with sequences from the planktic cyanobacterium Cylindrospermopsis raciborskii, from the benthic Anabaena cylindrica 133 and from Anabaena oscillarioides HINDAK 1984/43, with high bootstrap values. The phylogenetic analysis showed that two distinct patterns of evolution of symbiotic behaviour might exist for the nostocacean cyanobacteria, one leading to symbioses of Nostoc species with a wide variety of plants, the other leading to the association of a unique cyanobacterial type with the water fern Azolla.

Molecular and morphological criteria for revision of the genus Microcoleus (Oscillatoriales, Cyanobacteria).

Ninety‐two strains of Microcoleus vaginatus (=nomenclatural‐type species of the genus Microcoleus Desmazières ex Gomont) and Phormidium autumnale Trevisan ex Gomont from a wide diversity of regions and biotopes were examined using a combination of morphological and molecular methods. Phylogenies based on the 16S rDNA and 16S‐23S ITS (partial) demonstrated that the 92 strains, together with a number of strains in GenBank, were members of a highly supported monophyletic clade of strains (Bayesian posterior probability = 1.0) distant from the species‐cluster containing the generitype of Phormidium. Similarity of the 16S rRNA gene exceeded 95.5% among all members of the Microcoleus clade, but was less than 95% between any Microcoleus strains and species outside of the clade (e.g., Phormidium sensu stricto). These findings, which are in agreement with earlier studies on these taxa, necessitate the revision of Microcoleus to include P. autumnale. Furthermore, the cluster of Phormidium species in the P. autumnale group (known as Group VII) must be moved into Microcoleus as well, and these nomenclatural transfers are included in this study. The main diacritical characters defining Microcoleus are related to the cytomorphology of trichomes, including: narrowed trichome ends, calyptra, cells shorter than wide up to more or less isodiametric, and facultative presence of sheaths. The majority of species are 4–10 μm in diameter. The possession of multiple trichomes in a common sheath is present facultatively in many but not all species.

Aulosira Bohemensis sp. nov.: Further Phylogenetic Uncertainty at The Base of The Nostocales (Cyanobacteria)

A. Lukešová, J.R. Johansen, M.P. Martin and D.A. Casamatta. 2009. Aulosira bohemensis sp. nov.: further phylogenetic uncertainty at the base of the Nostocales (Cyanobacteria). Phycologia 48: 118–129. DOI: 10.2216/08-56.1 Aulosira bohemensis sp. nov. was isolated from a wet meadow soil in South Bohemia, Czech Republic. It shares all the features of the majority of other Aulosira species, including isopolar development with intercalary heterocytes and apoheterocytic akinete development. It differs from all other species through the production of hormogonia perpendicular to the trichome axis following akinete germination. Morphologically the genus Aulosira appears closest to Nodularia, however, phylogenetic placement of A. bohemensis based on 16S rRNA was distant from that taxon. Aulosira falls within the Nostocaceae, with possible sister taxa in Trichormus, Mojavia and Nostoc. Despite the variety of phylogenetic analyses performed, we were unable to obtain bootstrap support for its position in any tree, and its correct phylogenetic position within the Nostocaceae remains unresolved.

Cytotoxicity and secondary metabolites production in terrestrial Nostoc strains, originating from different climatic/geographic regions and habitats: Is their cytotoxicity environmentally dependent?

Extensive selection of cyanobacterial strains (82 isolates) belonging to the genus Nostoc, isolated from different climatic regions and habitats, were screened for both their secondary metabolite content and their cytotoxic effects to mammalian cell lines. The overall occurrence of cytotoxicity was found to be 33%, which corresponds with previously published data. However, the frequency differs significantly among strains, which originate from different climatic regions and microsites (particular localities). A large fraction of intensely cytotoxic strains were found among symbiotic strains (60%) and temperate and continental climatic isolates (45%); compared with the less significant incidences in strains originating from cold regions (36%), deserts (14%), and tropical habitats (9%). The cytotoxic strains were not randomly distributed; microsites that clearly had a higher occurrence of cytotoxicity were observed. Apparently, certain natural conditions lead to the selection of cytotoxic strains, resulting in a high cytotoxicity occurrence, and vice versa. Moreover, in strains isolated from a particular microsite, the cytotoxic effects were caused by different compounds. This result supports our hypothesis for the environmental dependence of cytotoxicity. It also contradicts the hypothesis that clonality and lateral gene transfer could be the reason for this phenomenon. Enormous variability in the secondary metabolites was detected within the studied Nostoc extracts. According to their molecular masses, only 26% of these corresponded to any known structures; thus, pointing to the high potential for the use of many terrestrial cyanobacteria in both pharmacology and biotechnology.

Morphological and molecular characterization within 26 strains of the genus Cylindrospermum (Nostocaceae, Cyanobacteria), with descriptions of three new species

Twenty‐six strains morphologically identified as Cylindrospermum as well as the closely related taxon Cronbergia siamensis were examined microscopically as well as phylogenetically using sequence data for the 16S rRNA gene and the 16S‐23S internal transcribed spacer (ITS) region. Phylogenetic analysis of the 16S rRNA revealed three distinct clades. The clade we designate as Cylindrospermum sensu stricto contained all five of the foundational species, C. maius, C. stagnale, C. licheniforme, C. muscicola, and C. catenatum. In addition to these taxa, three species new to science in this clade were described: C. badium, C. moravicum, and C. pellucidum. Our evidence indicated that Cronbergia is a later synonym of Cylindrospermum. The phylogenetic position of Cylindrospermum within the Nostocaceae was not clearly resolved in our analyses. Cylindrospermum is unusual among cyanobacterial genera in that the morphological diversity appears to be more evident than sequence divergence. Taxa were clearly separable using morphology, but had very high percent similarity among ribosomal sequences. Given the high diversity we noted in this study, we conclude that there is likely much more diversity remaining to be described in this genus.

A Broad Phylogenetic Survey Unveils the Diversity and Evolution of Telomeres in Eukaryotes

Telomeres, ubiquitous and essential structures of eukaryotic chromosomes, are known to come in a variety of forms, but knowledge about their actual diversity and evolution across the whole phylogenetic breadth of the eukaryotic life remains fragmentary. To fill this gap, we employed a complex experimental approach to probe telomeric minisatellites in various phylogenetically diverse groups of algae. Our most remarkable results include the following findings: 1) algae of the streptophyte class Klebsormidiophyceae possess the Chlamydomonas-type telomeric repeat (TTTTAGGG) or, in at least one species, a novel TTTTAGG repeat, indicating an evolutionary transition from the Arabidopsis-type repeat (TTTAGGG) ancestral for Chloroplastida; 2) the Arabidopsis-type repeat is also present in telomeres of Xanthophyceae, in contrast to the presence of the human-type repeat (TTAGGG) in other ochrophytes studied, and of the photosynthetic alveolate Chromera velia, consistent with its phylogenetic position close to apicomplexans and dinoflagellates; 3) glaucophytes and haptophytes exhibit the human-type repeat in their telomeres; and 4) ulvophytes and rhodophytes have unusual telomere structures recalcitrant to standard analysis. To obtain additional details on the distribution of different telomere types in eukaryotes, we performed in silico analyses of genomic data from major eukaryotic lineages, utilizing also genome assemblies from our on-going genome projects for representatives of three hitherto unsampled lineages (jakobids, malawimonads, and goniomonads). These analyses confirm the human-type repeat as the most common and possibly ancestral in eukaryotes, but alternative motifs replaced it along the phylogeny of diverse eukaryotic lineages, some of them several times independently.

Dynamic Evolution of Telomeric Sequences in the Green Algal Order Chlamydomonadales

Telomeres, which form the protective ends of eukaryotic chromosomes, are a ubiquitous and conserved structure of eukaryotic genomes but the basic structural unit of most telomeres, a repeated minisatellite motif with the general consensus sequence TnAmGo, may vary between eukaryotic groups. Previous studies on several species of green algae revealed that this group exhibits at least two types of telomeric sequences, a presumably ancestral type shared with land plants (Arabidopsis type, TTTAGGG) and conserved in, for example, Ostreococcus and Chlorella species, and a novel type (Chlamydomonas type, TTTTAGGG) identified in Chlamydomonas reinhardtii. We have employed several methodical approaches to survey the diversity of telomeric sequences in a phylogenetically wide array of green algal species, focusing on the order Chlamydomonadales. Our results support the view that the Arabidopsis-type telomeric sequence is ancestral for green algae and has been conserved in most lineages, including Mamiellophyceae, Chlorodendrophyceae, Trebouxiophyceae, Sphaeropleales, and most Chlamydomonadales. However, within the Chlamydomonadales, at least two independent evolutionary changes to the Chlamydomonas type occurred, specifically in a subgroup of the Reinhardtinia clade (including C. reinhardtii and Volvox carteri) and in the Chloromonadinia clade. Furthermore, a complex structure of telomeric repeats, including a mix of the ancestral Arabidopsis-type motifs and derived motifs identical to the human-type telomeric repeats (TTAGGG), was found in the chlamydomonadalean clades Dunaliellinia and Stephanosphaeria. Our results indicate that telomere evolution in green algae, particularly in the order Chlamydomonadales, is far more dynamic and complex than thought before. General implications of our findings for the mode of telomere evolution are discussed.

Cyanobacterial cytotoxicity versus toxicity to brine shrimp Artemia salina.

Heterocytous cyanobacteria from various habitats were screened for toxicity to brine shrimp Artemia salina and the murine lymphoblastic cell line Sp/2 in order to compare these two testing models for evaluation of risk posed by cyanobacteria to human health. Methanol extracts of biomass and cultivation media were tested for toxicity and selected extracts were fractionated to determine the active fraction. We found a significant toxic effect to A. salina and to Sp/2 cells in 5.2% and 31% of studied extracts, respectively. Only 8.6% of the tested strains were highly toxic to both A. salina and the Sp/2 cell line, and only two of the tested strains were toxic to A. salina and not to the murine cell line. Therefore, it is likely that the toxic effect of cyanobacterial secondary metabolites mostly targets basal metabolic pathways present in mammal cells and so is not manifested in A. salina. We conclude that it is insufficient to monitor cytotoxicity of cyanobacteria using only the brine shrimp bioassay as was usual in the past, since cytotoxicity is a more frequent feature in cyanobacteria in comparison with toxicity to A. salina. A. salina toxicity test should not be used when estimating the possible health risk for humans. We suggest that in vitro mammal cells be used for these purposes.