New cyanobacterial genus Argonema is hidding in soil crusts around the world.

Cyanobacteria are crucial primary producers in soil and soil crusts. However, their biodiversity in these habitats remains poorly understood, especially in the tropical and polar regions. We employed whole genome sequencing, morphology, and ecology to describe a novel cyanobacterial genus Argonema isolated from Antarctica. Extreme environments are renowned for their relatively high number of endemic species, but whether cyanobacteria are endemic or not is open to much current debate. To determine if a cyanobacterial lineage is endemic is a time consuming, elaborate, and expensive global sampling effort. Thus, we propose an approach that will help to overcome the limits of the sampling effort and better understand the global distribution of cyanobacterial clades. We employed a Sequencing Read Archive, which provides a rich source of data from thousands of environmental samples. We developed a framework for a characterization of the global distribution of any microbial species using Sequencing Read Archive. Using this approach, we found that Argonema is actually cosmopolitan in arid regions. It provides further evidence that endemic microbial taxa are likely to be much rarer than expected.

On the relativity of species, or the probabilistic solution to the species problem.

For centuries, both scientists and philosophers have discussed the nature of species resulting in c. 35 species concepts proposed to date. However, in our opinion, none of them incorporated neither recent advances in evolutionary genomics nor dimensionality of species in befitting depth. Our attempt to do so resulted in the following conclusions. Due to the continuous nature of evolution (regardless of its rate and constancy), species are inevitably undefinable as natural discontinuous units (except those originating in saltatory speciation) whenever the time dimension is taken into consideration. Therefore, the very existence of species as a natural discontinuous entity is relative to its dimensionality. A direct consequence of the relativity of species is the duality of speciators (e.g., incipient species) meaning that, in a given time, they may be perceived as both being and not being a species. Finally, the most accurate way to reflect both the relativity of species and the duality of speciators in species delimitation is probabilistic. While the novelty of these ideas may be questionable, they still deserve more extensive attention from the biological community. Here, we hope to draw such attention by outlining one of the possible pathways towards a new kind of probabilistic species delimitation methods based on the probability of irreversible divergence of evolutionary lineages. We anticipate that our probabilistic view of speciation has the potential to facilitate some of the most serious and universal issues of current taxonomy and to ensure unity of the species-level taxonomy across the tree of life.

A polyphasic approach to the delimitation of diatom species: a case study for the genus Pinnularia (Bacillariophyta).

Diatoms are one of the most abundant and arguably the most species-rich group of protists. Diatom species delimitation has often been based exclusively on the recognition of morphological discontinuities without investigation of other lines of evidence. Even though DNA sequences and reproductive experiments have revealed several examples of (pseudo)cryptic diversity, our understanding of diatom species boundaries and diversity remains limited. The cosmopolitan pennate raphid diatom genus Pinnularia represents one of the most taxon-rich diatom genera. In this study, we focused on the delimitation of species in one of the major clades of the genus, the Pinnularia subgibba group, based on 105 strains from a worldwide origin. We compared genetic distances between the sequences of seven molecular markers and selected the most variable pair, the mitochondrial cox1 and nuclear encoded LSU rDNA, to formulate a primary hypothesis on the species limits using three single-locus automated species delimitation methods. We compared the DNA-based primary hypotheses with morphology and with other available lines of evidence. The results indicate that our data set comprised 15 species of the P. subgibba group. The vast majority of these taxa have an uncertain taxonomic identity, suggesting that several may be unknown to science and/or members of (pseudo)cryptic species complexes within the P. subgibba group.

Brasilonema lichenoides sp. nov. and Chroococcidiopsis lichenoides sp. nov. (Cyanobacteria): two novel cyanobacterial constituents isolated from a tripartite lichen of headstones.

Cyanolichens are an assemblage of fungi and cyanobacteria from diverse, cosmopolitan habitats. Typically composed of a single species of cyanobacterium, with or without another eukaryotic alga, here we present two novel cyanobionts isolated from an undescribed tripartite lichen. This endolithic lichen was isolated from a granite cemetery tombstone from Jacksonville, FL, and contains two potentially nitrogen-fixing cyanobionts. Employing a total evidence approach, we characterized the cyanobionts using molecular (the 16S rDNA and ITS gene region), morphological, and ecological data. Phylogenetic analyses revealed two novel taxa: Brasilonema lichenoides and Chroococcidiopsis lichenoides, both of which fell within well-supported clades. To our knowledge, this represents the first instance of a tripartite lichen with two cyanobacterial and no eukaryotic members. These types of lichens may well represent an unexplored reservoir of cyanobacterial diversity. The specific epithets are proposed under the provisions of the International Code of Nomenclature for algae, fungi, and plants.

Ardissonea crystallina has a type of sexual reproduction that is unusual for centric diatoms.

Molecular phylogenetic analyses place Ardissonea crystallina (C. Agardh) Grunow and all Toxariids among the bi- and multipolar centric diatoms, almost always recovered as a derived lineage sister to Lampriscus. In all centrics where sexual reproduction has been documented, oogamy, with larger immobile eggs and smaller flagellated sperm has been observed. We were able to initiate both homothallic and heterothallic reproduction in A. crystallina. The heterothallic reproduction turned out to be non-oogamous; gametes were more or less equal in size but no flagellated cells were detected. At the same time, two mating types ("male" and "female") were recognized by the distinct morphology and behaviour of the gametes. While no flagella were observed, periodically thin cytoplasmic projections arose on the surface of the "male" gametes. These projections similar to those found in some pennate diatoms facilitated contact with the "female" cells. In each gametangial cell, regardless of the mating type, only one gamete was formed. Thus, the Toxariids may represent a unique evolutionary group, at least in respect to their reproductive biology. The hypothesis discussed is that non-oogamous mode of reproduction could have evolved in Ardissonea (and possibly in other Toxariids) independently of the pennate lineage of diatoms.

Species complexes within epiphytic diatoms and their relevance for the bioindication of trophic status.

The popularity of aquatic bioassessments has increased in Europe and worldwide, with a considerable number of methods being based on benthic diatoms. Recent evidence from molecular data and mating experiments has shown that some traditional diatom morphospecies represent species complexes, containing several to many cryptic species. This case study is based on epiphytic diatom and environmental data from shallow fishponds, investigating whether the recognition and use of fine taxonomic resolution (cryptic species) can improve assessment of community response to environmental drivers and increase sharpness of classification, compared to coarse taxonomic resolution (genus level and species level with unresolved species complexes). Secondly, trophy bioindication based on a species matrix divided into two compartments (species complexes and remaining species) was evaluated against the expectation that species complexes would be poor trophy indicators, due to their expected wide ecological amplitude. Finally, the response of species complexes and their members (cryptic species) to a trophic gradient (phosphorus) were compared. Multivariate analyses showed similar efficiency of all three taxonomic resolutions in depicting community patterns and their environmental correlates, suggesting that even genus level resolution is sufficient for routine bioassessment of shallow fishponds with a wide trophic range. However, after controlling for coarse taxonomic matrices, fine taxonomic resolution (with resolved cryptic species) still showed sufficient variance related to the environmental variable (habitat groups), and increased the sharpness of classification, number of indicator species for habitat categories, and gave better separation of habitat categories in the ordination space. Regression analysis of trophic bioindication and phosphorus concentration showed a weak relationship for species complexes but a close relationship for the remaining taxa. GLM models also showed that no species complex responded to phosphorus concentration. It follows that the studied species complexes have wide tolerances to, and no apparent optima for, phosphorus concentrations. In contrast, various responses (linear, unimodal, or no response) of cryptic species within species complexes were found to total phosphorus concentration. In some cases, fine taxonomic resolution to species level including cryptic species has the potential to improve data interpretation and extrapolation, supporting recent views of species surrogacy.

Synechococcus: 3 billion years of global dominance.

Cyanobacteria are among the most important primary producers on the Earth. However, the evolutionary forces driving cyanobacterial species diversity remain largely enigmatic due to both their distinction from macro-organisms and an undersampling of sequenced genomes. Thus, we present a new genome of a Synechococcus-like cyanobacterium from a novel evolutionary lineage. Further, we analyse all existing 16S rRNA sequences and genomes of Synechococcus-like cyanobacteria. Chronograms showed extremely polyphyletic relationships in Synechococcus, which has not been observed in any other cyanobacteria. Moreover, most Synechococcus lineages bifurcated after the Great Oxidation Event, including the most abundant marine picoplankton lineage. Quantification of horizontal gene transfer among 70 cyanobacterial genomes revealed significant differences among studied genomes. Horizontal gene transfer levels were not correlated with ecology, genome size or phenotype, but were correlated with the age of divergence. All findings were synthetized into a novel model of cyanobacterial evolution, characterized by serial convergence of the features, that is multicellularity and ecology.

DNA content variation and its significance in the evolution of the genus Micrasterias (Desmidiales, Streptophyta).

It is now clear that whole genome duplications have occurred in all eukaryotic evolutionary lineages, and that the vast majority of flowering plants have experienced polyploidisation in their evolutionary history. However, study of genome size variation in microalgae lags behind that of higher plants and seaweeds. In this study, we have addressed the question whether microalgal phylogeny is associated with DNA content variation in order to evaluate the evolutionary significance of polyploidy in the model genus Micrasterias. We applied flow-cytometric techniques of DNA quantification to microalgae and mapped the estimated DNA content along the phylogenetic tree. Correlations between DNA content and cell morphometric parameters were also tested using geometric morphometrics. In total, DNA content was successfully determined for 34 strains of the genus Micrasterias. The estimated absolute 2C nuclear DNA amount ranged from 2.1 to 64.7 pg; intraspecific variation being 17.4-30.7 pg in M. truncata and 32.0-64.7 pg in M. rotata. There were significant differences between DNA contents of related species. We found strong correlation between the absolute nuclear DNA content and chromosome numbers and significant positive correlation between the DNA content and both cell size and number of terminal lobes. Moreover, the results showed the importance of cell/life cycle studies for interpretation of DNA content measurements in microalgae.

Tracing decadal environmental change in ombrotrophic bogs using diatoms from herbarium collections and transfer functions.

Central European mountain bogs, among the most valuable and threatened of habitats, were exposed to intensive human impact during the 20th century. We reconstructed the subrecent water chemistry and water-table depths using diatom based transfer functions calibrated from modern sampling. Herbarium Sphagnum specimens collected during the period 1918-1998 were used as a source of historic diatom samples. We classified samples into hummocks and hollows according to the identity of dominant Sphagnum species, to reduce bias caused by uneven sampling of particular microhabitats. Our results provide clear evidence for bog pollution by grazing during the period 1918-1947 and by undocumented aerial liming in the early 90-ies. We advocate use of herbarized epibryon as a source of information on subrecent conditions in recently polluted mires.

Phylogeography of the Microcoleus vaginatus (Cyanobacteria) from three continents--a spatial and temporal characterization.

It has long been assumed that cyanobacteria have, as with other free-living microorganisms, a ubiquitous occurrence. Neither the geographical dispersal barriers nor allopatric speciation has been taken into account. We endeavoured to examine the spatial and temporal patterns of global distribution within populations of the cyanobacterium Microcoleus vaginatus, originated from three continents, and to evaluate the role of dispersal barriers in the evolution of free-living cyanobacteria. Complex phylogeographical approach was applied to assess the dispersal and evolutionary patterns in the cyanobacterium Microcoleus vaginatus (Oscillatoriales). We compared the 16S rRNA and 16S-23S ITS sequences of strains which had originated from three continents (North America, Europe, and Asia). The spatial distribution was investigated using a phylogenetic tree, network, as well as principal coordinate analysis (PCoA). A temporal characterization was inferred using molecular clocks, calibrated from fossil DNA. Data analysis revealed broad genetic diversity within M. vaginatus. Based on the phylogenetic tree, network, and PCoA analysis, the strains isolated in Europe were spatially separated from those which originated from Asia and North America. A chronogram showed a temporal limitation of dispersal barriers on the continental scale. Dispersal barriers and allopatric speciation had an important role in the evolution of M. vaginatus. However, these dispersal barriers did not have a permanent character; therefore, the genetic flow among populations on a continental scale was only temporarily present. Furthermore, M. vaginatus is a recently evolved species, which has been going through substantial evolutionary changes.

Barcoding diatoms: exploring alternatives to COI-5P.

Diatoms are a diverse lineage with species that can be difficult to identify or cryptic, but DNA barcoding, a molecular technique, can assist identification and facilitate studies of speciation and biogeography. The most common region used for DNA barcoding, COI-5P, can distinguish diatom species, but has not displayed universality (i.e., successful PCR amplification from diverse taxa). Therefore, we have assessed the following alternative markers: ∼1400bp of rbcL; 748bp at the 3' end of rbcL (rbcL-3P); LSU D2/D3 and UPA. Sellaphora isolates were used to determine each marker's ability to discriminate among closely related species and culture collection material was utilized to explore further marker universality. All of the alternative markers investigated have greater universality than COI-5P. Both full and partial (3P) rbcL regions had the power to discriminate between all species, but rbcL-3P can be sequenced more easily. LSU D2/D3 could distinguish between all but the most closely related species (96%), whereas UPA only distinguished 20% of species. Our observations suggest that rbcL-3P should be used as the primary marker for diatom barcoding, while LSU D2/D3 should be sequenced as a secondary marker to facilitate environmental surveys.

SCALY INCUNABULA, AUXOSPORE DEVELOPMENT, AND GIRDLE POLYMORPHISM IN SELLAPHORA MARVANII SP. NOV. (BACILLARIOPHYCEAE)(1).

Uniparental auxosporulation was observed in a monoclonal culture of a Sellaphora clone isolated from the epipelon of a fishpond in the Czech Republic. The cox1 sequence for the clone confirmed that it belonged to the Sellaphora pupula-bacillum species complex but showed significant differences from all previously characterized Sellaphora species, and it is therefore described as S. marvanii sp. nov. Protoplast, valve, and girdle structure resembled those of other Sellaphora species, but a novel finding for all diatoms was a change in girdle structure during the life cycle: the most advalvar girdle band (valvocopula) bore a single line of pores in enlarged postauxospore cells but was entirely plain in small cells and gametangia. The young auxospores were covered by incunabula containing large, delicate, ± circular scales, resembling those of centric diatom auxospores; similar scales have been reported in a few other raphid diatoms (Pseudo-nitzschia multiseries, Diploneis sp.) but contrast with the strip incunabula of some Nitzschia and Pinnularia and the helmet-like caps of Neidium. The scales persisted during auxospore expansion, mostly as two caps over the auxospore poles. The transverse perizonium comprised a very wide, closed primary band, flanked by numerous secondary bands whose open ends were strongly incurved toward the center. Initial valves were differentiated from their immediate descendants by the very strong external demarcation of the raphe sternum, irregular shape, and curved transapical profile.

Pseudocryptic diversity versus cosmopolitanism in diatoms: a case study on Navicula cryptocephala Kütz. (Bacillariophyceae) and morphologically similar taxa.

Despite the significance of diatoms in biomonitoring, many aspects of their biodiversity and geographical distribution are poorly understood. Recent evidence from molecular data has shown that traditional cosmopolitan and euryvalent morphospecies are often heterogeneous, containing cryptic or pseudo-cryptic species. It is important to establish whether these more finely differentiated species are also cosmopolitan or show restricted distributions. According to the standard freshwater diatom floras, Navicula cryptocephala and morphologically similar species (N. veneta, N. trivialis, N. gregaria and N. cryptotenella) are common, cosmopolitan freshwater pennate diatoms. Although allopatric and even sympatric populations of N. cryptocephala are extremely similar morphologically, they have previously been found to be highly polymorphic with respect to reproductive and nuclear characteristics; however, molecular data supporting the existence of cryptic diversity were lacking. Phylogenetic analyses (LSU rDNA, ITS of the rRNA operon) of 52 strains of N. cryptocephala-like diatoms confirmed the existence of genetically distinct lineages within N. cryptocephala, and revealed a close relationship between N. trivialis and N. cryptocephala. Cytological, reproductive and morphological variation, investigated by means of landmark-based geometric morphometrics, were in congruence with molecular data. Two pseudo-cryptic species within N. cryptocephala coexist sympatrically and are widely distributed, occurring in both European and Australian lakes.

HIGH-THROUGHPUT SCREENING TECHNOLOGY FOR MONITORING PHYTOHORMONE PRODUCTION IN MICROALGAE(1).

New miniaturized techniques for multiplying microalgae and estimating their phytohormone production were developed; in these methods, the strains to be tested are cultivated in microtitre plates, and the phytohormones in suspensions of the cultures are measured by direct ELISAs. Specific and sensitive ELISAs for determining abscisic acid (ABA), indole-3-acetic acid (IAA), cis- and trans-zeatin riboside, isopentenyladenosine (iPR), and other less common cytokinins were developed for this purpose. Polyclonal antibodies used in the ABA and IAA assays were raised against C1- and C1'- conjugates of the compounds with BSA, respectively, and thus were specific for the free acids and their respective C1-derivatives. The use of cytokinin ribosides coupled via their sugar residues to BSA as haptens generally led to antibodies that bound free bases, 9-glycosides and nucleotides, but with high specificity for the corresponding N(6) -side chains. Using internal standards, dilution assays, and authentic [(2) H] and [(3) H] recovery markers, it was shown that the ELISAs could be used to estimate contents of the selected phytohormones in the cultures. The ELISAs provided reliable and very fast estimates of the selected phytohormones, at concentrations ranging from 0.01 to 10 pmol · mL(-1) in various microalgal strains. In addition, a recently developed HPLC selected ion monitoring mass spectrometry (HPLC-SIM-MS) method was used to calibrate and validate the ELISA results and confirm the presence of the detected phytohormones in immunoaffinity-purified extracts. Where independent validation of results is deemed necessary, the use of quantitative HPLC-MS is recommended for each new microalgal strain to be tested.

AUTOGAMOUS AUXOSPORULATION IN PINNULARIA NODOSA (BACILLARIOPHYCEAE)(1).

Auxosporulation of the freshwater epipelic diatom Pinnularia nodosa (Ehrenb.) W. Sm. was studied in a clonal culture. Interphase cells possessed two chloroplasts with invaginated pyrenoids. The nucleus contained a single small body of heterochromatin at one end, also visible during most of meiotic prophase. During auxosporulation, induced by transfer of stationary-phase cells to fresh medium and suppressed by high nitrogen (N), an unpaired mother cell produced a single auxospore. Although meiosis II and nuclear fusion were not observed, indirect evidence indicated that auxosporulation was autogamous (rarely reported in pennate diatoms), rather than apomictic; paedogamy was excluded. The protoplast produced after meiosis either (1) matured into a "pseudozygote," via an asymmetrical contraction after meiosis I to form a single spherical cell at one end of the mother cell (pathway 1); or (2) constricted into two spherical cells (pathway 2). In pathway 2, the "pseudogametes" never fused and only one or none developed into a pseudozygote and then into an auxospore. Pathway 2 could be suppressed by continuous light. During metamorphosis of the spherical pseudozygote into an elongate young auxospore, a complete covering of thin siliceous incunabular strips was formed, separate from the organic wall formed around the pseudozygote when first formed and from the perizonium. Mature auxospores produced via pathway 2 had 60% of the volume as those produced via pathway 1 and had smaller chloroplasts (through loss of fragments during protoplast cleavage), but they achieved exactly the same lengths, suggesting that absolute length is monitored during expansion.