Description of four new filamentous cyanobacterial taxa from freshwater habitats in the Azores Archipelago.

Simple filamentous cyanobacteria comprise a diverse and polyphyletic group of species, primarily in the orders Leptolyngbyales and Oscillatoriales, that need more sampling to improve their taxonomy. Oceanic islands, such as the Azores archipelago, present unique habitats and biogeographic conditions that harbor an unknown range of diversity of microorganisms. Filamentous cyanobacteria isolated from aquatic habitats in the Azores and maintained in the BACA culture collection were described using morphology, both light and transmission electron microscopy, ecology, and genetic data of the 16S rRNA gene sequences and 16S-23S Internal Transcribed Spacer (ITS) rRNA region secondary structure. Our analyses revealed two new monophyletic genera: Tumidithrix elongata gen. sp. nov. (Pseudanabaenaceae) and Radiculonema aquaticum gen. sp. nov. (Leptolyngbyaceae). In addition, two new species Leptodesmis lacustris sp. nov. (Leptolyngbyaceae) and Pycnacronema lacustrum sp. nov. (Wilmottiaceae) are reported as the first aquatic species for these genera. The description of these new taxa and the genetic study of an isolate of Leptodesmis alaskaensis from the Azores followed the polyphasic approach, identifying diacritical features. Our results reinforce the need for taxonomic studies on cyanobacteria from less-studied habits and geographic regions, which have a potential for new taxa description.

Tenebriella gen. nov. - The dark twin of Oscillatoria.

Oscillatoria has long been known to be polyphyletic. After recent resequencing of the reference strain for this genus, many Oscillatoria-like groups phylogenetically distant from the type species O. princeps remained unresolved. Here we describe one of these groups as a new genus Tenebriella. Most of the studied strains originate from Central Europe, where they are able to form prominent microbial mats. Despite the overall Oscillatoria-like morphology, Tenebriella can be distinguished by darker trichomes and forms a separate monophyletic clade in phylogenies inferred from the 16S rRNA gene and two additional loci (rpoC1, rbcLX). Within Tenebriella we recognize two new species differing from each other by morphological and ecological characteristics. First species does not fit any known taxon description, and thus is described as a new species T. amphibia. The latter one corresponds with the information available for Oscillatoria curviceps Agardh ex Gomont, and thus new combination T. curviceps is proposed. The phylogenetic analyses of the 16S-23S ITS region together with the comparison of the hypothetical secondary structures confirmed recognition of these two species and additionally revealed presence of a morphologically cryptic species Tenebriella sp. The results corroborate frequent recurrence of convergent morphotypes in the evolution of cyanobacteria and justify further exploration even of the intensively studied European freshwaters using molecular phylogenetics to discover new and ecologically relevant taxa.

The effects of macrophytes on the growth of bloom-forming cyanobacteria: Systematic review and experiment.

Macrophytes have often been considered as a prospective tool for the elimination of cyanobacterial bloom, because they may produce chemical compounds that outcompete bloom-forming cyanobacteria. However, a comprehensive, unbiased overview of evidence to support this is missing. Moreover, studies into the effects of individual macrophyte species have often used different methodologies and, thus, cannot be compared. Herein, we firstly carried out a systematic review of studies into the effects of macrophytes on the growth of bloom-forming cyanobacteria. Secondly, we carried out an experiment into the effects of aqueous and ethanol extracts from 19 macrophyte species on the growth of two of the most common cyanobacteria, Aphanizomenon gracile and Microcystis aeruginosa, using a uniform methodological approach. The systematic review revealed that most of the 69 macrophyte species previously studied have shown a combination of inhibitory, stimulatory, and neutral effects. In our own experiment, an inhibitory effect was exhibited only 15 times out of 532 experimental variants, specifically by Chara globularis, Ceratophyllum submersum, Elodea nuttallii, Hydrilla verticillata, Myriophyllum heterophyllum, M. spicatum, and Vallisneria americana. Put together, these results indicate that the practical application of chemical compounds produced by macrophytes to eliminate cyanobacterial bloom may have lower prospects than previously anticipated.

Taxonomic resolution of the genus Cyanothece (Chroococcales, Cyanobacteria), with a treatment on Gloeothece and three new genera, Crocosphaera, Rippkaea, and Zehria.

The systematics of single-celled cyanobacteria represents a major challenge due to morphological convergence and application of various taxonomic concepts. The genus Cyanothece is one of the most problematic cases, as the name has been applied to oval-shaped coccoid cyanobacteria lacking sheaths with little regard to their phylogenetic position and details of morphology and ultrastructure. Hereby we analyze an extensive set of complementary genetic and phenotypic evidence to disentangle the relationships among these cyanobacteria. We provide diagnostic characters to separate the known genera Cyanothece, Gloeothece, and Aphanothece, and provide a valid description for Crocosphaera gen. nov. We describe two new genera, Rippkaea and Zehria, to characterize two distinct phylogenetic lineages outside the previously known genera. We further describe 13 new species in total including Cyanothece svehlovae, Gloeothece aequatorialis, G. aurea, G. bryophila, G. citriformis, G. reniformis, Gloeothece tonkinensis, G. verrucosa, Crocosphaera watsonii, C. subtropica, C. chwakensis, Rippkaea orientalis, and Zehria floridana to recognize the intrageneric diversity as rendered by polyphasic analysis. We discuss the close relationship of free-living cyanobacteria from the Crocosphaera lineage to nitrogen-fixing endosymbionts of marine algae. The current study includes several experimental strains (Crocosphaera and "Cyanothece") important for the study of diazotrophy and the global oceanic nitrogen cycle, and provides evidence suggesting ancestral N2 -fixing capability in the chroococcalean lineage.

Molecular and morphological delimitation and generic classification of the family Oocystaceae (Trebouxiophyceae, Chlorophyta).

The family Oocystaceae (Chlorophyta) is a group of morphologically and ultrastructurally distinct green algae that constitute a well-supported clade in the class Trebouxiophyceae. Despite the family's clear delimitation, which is based on specific cell wall features, only a few members of the Oocystaceae have been examined using data other than morphological. In previous studies of Trebouxiophyceae, after the establishment of molecular phylogeny, the taxonomic status of the family was called into question. The genus Oocystis proved to be paraphyletic and some species were excluded from Oocystaceae, while a few other species were newly redefined as members of this family. We investigated 54 strains assigned to the Oocystaceae using morphological, ultrastructural and molecular data (SSU rRNA and rbcL genes) to clarify the monophyly of and diversity within Oocystaceae. Oonephris obesa and Nephrocytium agardhianum clustered within the Chlorophyceae and thus are no longer members of the Oocystaceae. On the other hand, we transferred the coenobial Willea vilhelmii to the Oocystaceae. Our findings combined with those of previous studies resulted in the most robust definition of the family to date. The division of the family into three subfamilies and five morphological clades was suggested. Taxonomical adjustments in the genera Neglectella, Oocystidium, Oocystis, and Ooplanctella were established based on congruent molecular and morphological data. We expect further taxonomical changes in the genera Crucigeniella, Eremosphaera, Franceia, Lagerheimia, Oocystis, and Willea in the future.

Description of new genera and species of marine cyanobacteria from the Portuguese Atlantic coast.

Aiming at increasing the knowledge on marine cyanobacteria from temperate regions, we previously isolated and characterized 60 strains from the Portuguese foreshore and evaluate their potential to produce secondary metabolites. About 15% of the obtained 16S rRNA gene sequences showed less than 97% similarity to sequences in the databases revealing novel biodiversity. Herein, seven of these strains were extensively characterized and their classification was re-evaluated. The present study led to the proposal of five new taxa, three genera (Geminobacterium, Lusitaniella, and Calenema) and two species (Hyella patelloides and Jaaginema litorale). Geminobacterium atlanticum LEGE 07459 is a chroococcalean that shares morphological characteristics with other unicellular cyanobacterial genera but has a distinct phylogenetic position and particular ultrastructural features. The description of the Pleurocapsales Hyella patelloides LEGE 07179 includes novel molecular data for members of this genus. The filamentous isolates of Lusitaniella coriacea - LEGE 07167, 07157 and 06111 - constitute a very distinct lineage, and seem to be ubiquitous on the Portuguese coast. Jaaginema litorale LEGE 07176 has distinct characteristics compared to their marine counterparts, and our analysis indicates that this genus is polyphyletic. The Synechococcales Calenema singularis possess wider trichomes than Leptolyngbya, and its phylogenetic position reinforces the establishment of this new genus.

Macrochaete gen. nov. (Nostocales, Cyanobacteria), a taxon morphologically and molecularly distinct from Calothrix.

Historically, the genus Calothrix included all noncolonial, tapered, heterocytous filaments within the cyanobacteria. However, recent molecular phylogenies show that "Calothrix" defined in this sense represents five distinct clades. The type species of Calothrix is marine, with solitary basal heterocytes, no akinetes, and distal ends tapering abruptly into short hairs. We examined the morphology and phylogeny of 45 tapering cyanobacteria in the Rivulariaceae, including freshwater and marine representatives of both Calothrix (35 strains) and its sister taxon Rivularia (10 strains). The marine Calothrix fall into two lineages, but we lack the generitype and so cannot identify the clade corresponding to the type species. The freshwater and soil Calothrix fall into the C. parietina clade and are characterized by having a basal heterocyte, no akinetes, and gradual tapering-but not into a long hyaline hair. Macrochaete gen. nov. is a freshwater taxon sister to the Calothrix lineages but clearly separated from Rivularia. The species in this genus differ morphologically from Calothrix by their ability to produce two heteromorphic basal heterocytes and specific secondary structures of the 16S-23S ITS. An additional feature present in most species is the presence of a distal, long hyaline hair, but this character has incomplete penetrance due to its expression only under specific environmental conditions (low phosphate), and in one species appears to be lost. We recognize three species: M. psychrophila (type species) from cold environments (high mountains, Antarctica), M. santannae from wet walls of subtropical South America, and M. lichenoides, a phycobiont of lichens from Europe.

Effects of trophic status on microcystin production and the dominance of cyanobacteria in the phytoplankton assemblage of Mediterranean reservoirs.

The aim of our study was to evaluate the abundance of cyanobacteria and microcystins in four Sardinian reservoirs (Italy) characterised by different trophic status to define a reference picture for future changes. Increasing levels of eutrophication and the abundance of cyanobacteria are expected to occur due to climate change, especially in the southern Mediterranean. Consequently, an in-depth study of the occurrence of harmful cyanobacteria is important to develop appropriate management strategies for water resources at a local scale. Monthly samples were collected at one station in each reservoir over an 18-month period. The Analysis of similarity indicated that cyanobacterial abundance and species composition differed significantly among the reservoirs. The Redundancy analysis highlighted their relationship to trophic, hydrological and seasonal patterns. Spearman's analysis indicated that there were significant correlations among the most important species (Planktothrix agardhii-rubescens group, Aphanizomenon flos-aquae and Dolichospermum planctonicum), nutrients and microcystins. We highlighted that the species composition during periods of maximum microcystin concentrations differed from those typically reported for other Mediterranean sites. We found new potential microcystin producers (Aphanizomenon klebahnii, Dolichospermum macrosporum and Dolichospermum viguieri), which emphasised the high diversity of cyanobacteria in the Mediterranean area and the need for detailed research at the local scale.

Phylogenetic analysis of cultivation-resistant terrestrial cyanobacteria with massive sheaths (Stigonema spp. and Petalonema alatum, Nostocales, Cyanobacteria) using single-cell and filament sequencing of environmental samples.

Molecular assessment of a large portion of traditional cyanobacterial taxa has been hindered by the failure to isolate and grow them in culture. In this study, we developed an optimized protocol for single cell/filament isolation and 16S rRNA gene sequencing of terrestrial cyanobacteria with large mucilaginous sheaths, and applied it to determine the phylogenetic position of typical members of the genera Petalonema and Stigonema. A methodology based on a glass-capillary isolation technique and a semi-nested PCR protocol enabled reliable sequencing of the 16S rRNA gene from all samples analyzed. Ten samples covering seven species of Stigonema from Europe, North and Central America, and Hawaii, and the type species of Petalonema from Slovakia were sequenced. Contrary to some previous studies, which proposed a relationship with heteropolar nostocalean cyanobacteria, Petalonema appeared to belong to the family Scytonemataceae. Analysis of Stigonema specimens recovered a unique coherent phylogenetic cluster, substantially broadening our knowledge of the molecular diversity within this genus. Neither the uni- to biseriate species nor the multiseriate species formed monophyletic subclusters within the genus. Typical multiseriate species of Stigonema clustered in a phylogenetic branch derived from uni- to biseriate S. ocellatum Thuret ex Bornet & Flahault in our analysis, suggesting that species with more complex thalli may have evolved from the more simple ones. We propose the technique tested in this study as a promising tool for a future revision of the molecular taxonomy in cyanobacteria.

True branched nostocalean cyanobacteria from tropical aerophytic habitats and molecular assessment of two species from field samples.

Aerophytic cyanobacteria are commonly found growing on rocks, tree trunks and soil, but the diversity of these organisms is still poorly known. This complex group is very problematic considering the taxonomic arrangement and species circumscription, especially when taking into account tropical populations. In this work, 20 samples of aerophytic cyanobacteria from 15 distinct sampling sites were collected along a tropical rainforest area at the São Paulo State (Brazil). Samples were dried at room temperature after the collection, and once in the laboratory, they were rehydrated and analyzed. The taxonomic study resulted in the record and description of nine species of true-branched cyanobacteria pertaining to the genera Spelaeopogon (one specie), Hapalosiphon (two species) and Stigonema (six species). The similarity of the flora found when compared to surveys conducted in other geographical regions was relatively low. These differences could be addressed to ecological conditions of the habitats, to the extension of the area surveyed or even to taxonomic misinterpretations. The molecular assessment of 16S rDNA on the basis of field material was successful for two morphospecies, Hapalosiphon sp. and Stigonema ocellatum; however, their relationships with other populations and species revealed to be uncertain. The results of the floristic survey and of the molecular approach evidenced the fragile delimitation of some genera and species in the true branched group of cyanobacteria.

Moorea producens gen. nov., sp. nov. and Moorea bouillonii comb. nov., tropical marine cyanobacteria rich in bioactive secondary metabolites.

The filamentous cyanobacterial genus Moorea gen. nov., described here under the provisions of the International Code of Botanical Nomenclature, is a cosmopolitan pan-tropical group abundant in the marine benthos. Members of the genus Moorea are photosynthetic (containing phycocyanin, phycoerythrin, allophycocyanin and chlorophyll a), but non-diazotrophic (lack heterocysts and nitrogenase reductase genes). The cells (discoid and 25-80 µm wide) are arranged in long filaments (<10 cm in length) and often form extensive mats or blooms in shallow water. The cells are surrounded by thick polysaccharide sheaths covered by a rich diversity of heterotrophic micro-organisms. A distinctive character of this genus is its extraordinarily rich production of bioactive secondary metabolites. This is matched by genomes rich in polyketide synthase and non-ribosomal peptide synthetase biosynthetic genes which are dedicated to secondary metabolism. The encoded natural products are sometimes responsible for harmful algae blooms and, due to morphological resemblance to the genus Lyngbya, this group has often been incorrectly cited in the literature. We here describe two species of the genus Moorea: Moorea producens sp. nov. (type species of the genus) with 3L(T) as the nomenclature type, and Moorea bouillonii comb. nov. with PNG5-198(R) as the nomenclature type.

A UNIQUE PSEUDANABAENALEAN (CYANOBACTERIA) GENUS NODOSILINEA GEN. NOV. BASED ON MORPHOLOGICAL AND MOLECULAR DATA(1).

The cyanobacteria are a diverse, ancient lineage of oxygenic, phototrophic bacteria. Ubiquitous in nearly all ecosystems, the alpha-level diversity of these organisms lags behind other algal lineages due to a perceived dearth of phylogenetically useful characters. Recent phylogenetic studies of species within the genus Leptolyngbya have demonstrated that this is a polyphyletic assemblage. One group of strains that fits within the current circumscription of Leptolyngbya is genetically and phylogenetically distinct from Leptolyngbya sensu stricto. Members of this clade possess both a morphological synapomorphy and shared 16S-23S internal transcribed spacer (ITS) secondary structure, allowing the diagnosis of the new cyanobacterial genus Nodosilinea. Members of this genus are united by the unique ability to form nodules along the length of the filament. This trait has been previously observed only in the species Leptolyngbya nodulosa Z. Li et J. Brand, and we have chosen this species as the generitype of Nodosilinea. We currently recognize four species in the genus, N. nodulosa (Z. Li et J. Brand) comb. nov., N. bijugata (Kong.) comb. nov., N. conica sp. nov., and N. epilithica sp. nov.

Comparative analysis of the microbial communities inhabiting halite evaporites of the Atacama Desert.

SUMMARY: Molecular biology and microscopy techniques were used to characterize the microbial communities inside halite evaporites from different parts of the Atacama Desert. Denaturing gradient gel electrophoresis (DGGE) analysis revealed that the evaporite rocks harbor communities predominantly made up of cyanobacteria, along with heterotrophic bacteria and archaea. Different DGGE profiles were obtained for the different sites, with the exception of the cyanobacterial profile, in which only one phylotype was detected across the three sites examined. Chroococcidiopsis-like cells were the only cyanobacterial components of the rock samples, although the phylogenetic study revealed their closer genetic affinity to Halothece genera. Gene sequences of the heterotrophic bacteria and archaea indicated their proximity to microorganisms found in other hypersaline environments. Microorganisms colonizing these halites formed microbial aggregates in the pore spaces between halite crystals, where microbial interactions occur. In this exceptional, salty, porous halite rock habitat, microbial consortia with a community structure probably conditioned by the environmental conditions occupy special microhabitats with physical and chemical properties that promote their survival.

Comparative analysis of the microbial communities inhabiting halite evaporites of the Atacama Desert

Molecular biology and microscopy techniques were used to characterize the microbial communities inside halite evaporites from different parts of the Atacama Desert. Denaturing gradient gel electrophoresis (DGGE) analysis revealed that the evaporite rocks harbor communities predominantly made up of cyanobacteria, along with heterotrophic bacteria and archaea. Different DGGE profiles were obtained for the different sites, with the exception of the cyanobacterial profile, in which only one phylotype was detected across the three sites examined. Chroococcidiopsis-like cells were the only cyanobacterial components of the rock samples, although the phylogenetic study revealed their closer genetic affinity to Halothece genera. Gene sequences of the heterotrophic bacteria and archaea indicated their proximity to microorganisms found in other hypersaline environments. Microorganisms colonizing these halites formed microbial aggregates in the pore spaces between halite crystals, where microbial interactions occur. In this exceptional, salty, porous halite rock habitat, microbial consortia with a community structure probably conditioned by the environmental conditions occupy special microhabitats with physical and chemical properties that promote their survival (AU)

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