CORRESPONDENCE BETWEEN PHYLOGENY AND MORPHOLOGY OF SNOWELLA SPP. AND WORONICHINIA NAEGELIANA,CYANOBACTERIA COMMONLY OCCURRING IN LAKES1

In this study, the first reported isolates of the genera Snowella and Woronichinia were characterized by 16S rRNA gene sequencing and morphological analysis. Phylogenetic studies and sequences for these genera were not available previously. By botanical criteria, the five isolated strains were identified as Snowella litoralis (Häyrén) Komárek et Hindák Snowella rosea (Snow) Elenkin and Woronichinia naegeliana (Unger) Elenkin. This study underlines the identification of freshly isolated cultures, since the Snowella strains lost the colony structure and were not identifiable after extended laboratory cultivation. In the 16S rRNA gene analysis, the Snowella strains formed a monophyletic cluster, which was most closely related to the Woronichinia strain. Thus, our results show that the morphology of the genera Snowella and Woronichinia was in congruence with their phylogeny, and their phylogeny seems to support the traditional botanical classification of these genera. Furthermore, the genera Snowella and Woronichinia occurred commonly and might occasionally be the most abundant cyanobacterial taxa in mainly oligotrophic and mesotrophic Finnish lakes. Woronichinia occurred frequently and also formed blooms in eutrophic Czech reservoirs.     

Characterization of freshwater benthic biofilm‐forming Hydrocoryne (Cyanobacteria) isolates from Antarctica

The aims of this work were to study cyanobacterial isolates resembling the genus Hydrocoryne using a combination of morphology and phylogeny of 16S rRNA and nifH sequences and to investigate genes involved in cyanotoxin and protease inhibitor production. Four new cyanobacterial strains, isolated from biofilm samples collected from King George Island, Antarctica, were studied. In terms of morphology, these new strains share traits similar to true Anabaena morphotypes (benthic ones), whereas phylogenetic analysis of their 16S rRNA gene sequences grouped them with the sequence of the type species Hydrocoryne spongiosa (H. Schwabe ex Bornet and Flahault 1886–1888), but not with sequences of the type species from the genus Anabaena. This cluster is the sister group of Anabaena morphotypes isolated only from the Gulf of Finland. In addition, this cluster is related to two other clusters formed by sequences of Anabaena isolated from different sites. Partial nifH genes were sequenced from two strains and the phylogenetic tree revealed that the Antarctic nifH sequences clustered with sequences from Anabaena. Furthermore, two strains were tested, using PCR with specific primers, for the presence of genes involved in cyanotoxins (microcystin and saxitoxin) and protease inhibitor (aeruginosin, and cyanopeptolin). Only cyanopeptolin was amplified using PCR. These four Hydrocoryne strains are the first to be isolated and sequenced from Antarctica, which improves our knowledge on this poorly defined cyanobacterial genus.     

POLYPHASIC CHARACTERIZATION OF THREE STRAINS OF ANABAENA RENIFORMIS AND APHANIZOMENON APHANIZOMENOIDES (CYANOBACTERIA) AND THEIR RECLASSIFICATION TO SPHAEROSPERMUM GEN. NOV. (INCL. ANABAENA KISSELEVIANA)1

Occurrences of rare cyanobacteria Anabaena reniformis Lemmerm. and Aphanizomenon aphanizomenoides (Forti) Horecká et Komárek were recently detected at several localities in the Czech Republic. Two monoclonal strains of An. reniformis and one strain of Aph. aphanizomenoides were isolated from distant localities and different sampling years. They were characterized by a combination of morphological, genetic, and biochemical approaches. For the first time, partial 16S rRNA gene sequences were obtained for these morphospecies. Based on this gene, all of these strains clustered separately from other planktonic Anabaena and Aphanizomenon strains. They appeared in a cluster with Cylindrospermopsis Seenaya et Subba Raju and Raphidiopsis F. E. Fritsch et M. F. Rich, clustered closely together with two An. kisseleviana Elenkin strains available from GenBank. A new generic entity was defined (Sphaerospermum gen. nov., with the type species S. reniforme, based on the traditional species An. reniformis). These results contribute significantly to the knowledge base about genetic heterogeneity among planktonic Anabaena–like and Aphanizomenon–like morphospecies. Accordingly, the subgenus Dolichospermum, previously proposed for the group of planktonic Anabaena, should be revaluated. Secondary metabolite profiles of the An. reniformis and Aph. aphanizomenoides strains differed considerably from 17 other planktonic Anabaena strains of eight morphospecies isolated from Czech water bodies. Production of puwainaphycin A was found in both of the An. reniformis strains. Despite the relatively short phylogenetic distance from Cylidrospermopsis, the production of cylindrospermopsin was not detected in any of our strains.     

UNRAVELING MOLECULAR DIVERSITY AND PHYLOGENY OF APHANIZOMENON (NOSTOCALES,CYANOBACTERIA) STRAINS ISOLATED FROM CHINA1

Fifty‐three strains of the genus Aphanizomenon isolated from Chinese waters were employed to conduct morphological examination and sequencing of the 16S rRNA gene, rbcLX (RUBISCO), and cpcBA‐IGS gene regions. Based on morphological characteristics, the examined strains were divided into three morphotypes [Aph. flos‐aquae Bréb. ex Bornet et Flahault, Aph. gracile Lemmerm., and Aph. issatchenkoi (Usacer) Proshk.‐Lavr.]. Phylogenetic analysis based on 16S rRNA and rbcLX showed that Aphanizomenon strains could be divided into three main clades (Clade A of Aph. flos‐aquae, Clade B of Aph. gracile, and Clade C of Aph. issatchenkoi), but two additional clades formed by Aph. ovalisporum and Aph. aphanizomenoides were detected in the 16S rDNA‐based topology. All Aph. issatchenkoi strains contained an additional 175 nucleotides from the 779 to 954 nucleotide location in rbcLX region, compared with strains of Aph. flos‐aquae and Aph. gracile. The cpcBA‐IGS‐based phylogenetic tree revealed that Aph. issatchenkoi strains were not discriminated from Aph. flos‐aquae strains; however, a concatenated alignment of 16S rDNA, rbcLX, and cpcBA‐IGS led to the three distinct clades (Aph. flos‐aquae, Aph. gracile, and Aph. issatchenkoi, respectively). It is suggested that the taxonomic revision of Aphanizomenon and Anabaena genera is required to be performed by employing multilocus sequence analysis and polyphasic studies.     

Morphological,biochemical and phylogenetic assessments of water‐bloom‐forming tropical morphospecies of Microcystis (Chroococcales,Cyanobacteria)

Previous polyphasic analyses of five morphospecies of the water‐bloom‐forming cyanobacterial genus Microcystis, Microcystis aeruginosa (Kützing) Lemmermann (=Microcystis aeruginosa (Kützing) Kützing), Microcystis ichthyoblabe Kützing, Microcystis novacekii (Komárek) Compère, Microcystis viridis (A. Braun) Lemmermann, and Microcystis wesenbergii (Komárek) Komárek in Kondratieva, have shown them to be conspecific and they have been proposed to be included under the binomial Microcystis aeruginosa (Kützing) Lemmermann. However, several morphospecies from tropical regions, such as Microcystis bengalensis Banerji, Microcystis panniformis Komárek, Komárková‐Legnerová, Sant'anna, Azevedo & Senna, Microcystis protocystis Crow, Microcystis pseudofilamentosa Crow, Microcystis ramosa Bharadwaya, and Microcystis robusta (Clark) Nygaard, have never been analyzed biochemically or phylogenetically; consequently, their taxonomic status is uncertain. To resolve this issue, we collected 57 strains of Microcystis from Vietnam for taxonomic analysis using a polyphasic approach. Strains were assigned to the six tropical morphospecies listed above or to four morphospecies with cosmopolitan distributions (M. aeruginosa, M. ichthyoblabe, M. novacekii, and M. wesenbergii). Several strains produced colony variants in different culture media; some of these variants had forms that overlapped with those of other morphospecies. Cell diameters varied widely between strains (2.6–9.3 µm) and were unrelated to morphospecies discrimination criteria. Strains of the 10 morphospecies examined had similar fatty acid compositions and closely similar 16S rRNA gene sequences (>99.2% similar). Phylogenetic analyses using 16S rRNA gene and 16S–23S internal transcribed spacer sequences did not identify any clear separations corresponding to morphospecies concepts or microcystin‐producing abilities. Thus, the six tropical morphospecies (M. bengalensis, M. panniformis, M. protocystis, M. pseudofilamentosa, M. ramosa, and M. robusta) are not natural taxonomic units within the genus Microcystis and should be included under M. aeruginosa.     

FUNGAL CELLWALL POLYSACCHARIDES ELICIT AN ANTIFUNGAL SECONDARY METABOLITE (PHYTOALEXIN) IN THE CYANOBACTERIUM SCYTONEMA OCELUTUM2

The addition of fungal cell-wall homogenates of Penicillium notatum and Cylindrocladium spathiphylli markedly stimulated the accumulation of tolytoxin, an antifungal secondary metabolite, in cultures of the cyanobacterium Scytonema ocellatum Lyngbye ex Bornet et Flahault. Evaluation of polysaccharides, proteins, and other polymers established that a limited range of polysaccharides, especially chitin and carboxymthylcellulose, selectively elicited enhanced tolytoxin accumulation in S. ocellatum. The elicitor activity of fungal cell-wall preparations could be correlated with the chitin content of the preparation. Polymeric chitin was half-maximally effective at a concentration (EC₅₀) of 19 mg.L^-1, whereas chitin oligoments were more effective (EC₅₀= 3.3 mg.L^-1) in eliciting enhanced tolytoxin accumulation. The elicitor activity of either purified chitin or an elicitor-active fungal cell-wall preparation could be destroyed by treatment with chitinase. The results suggest an ecological role for tolytoxin as an inducible chemical defense agent (phytoalexin) capable of protecting S. ocellatum against fungal invasion.     

MOLECULAR AND MORPHOLOGICAL CHARACTERIZATION OF TEN POLAR AND NEAR‐POLAR STRAINS WITHIN THE OSCILLATORIALES (CYANOBACTERIA)1

An approximately 1400‐bp region of the 16S rRNA gene was sequenced for 10 polar or near‐polar strains putatively placed in the Oscillatorialean genera Oscillatoria, Phormidium, and Lyngbya obtained from the University of Toronto Culture Collection to assess phylogenetic relationships. The strains were also examined for thylakoid structure and cell division type with TEM as well as traditional morphology with LM. Phylogenetic trees constructed using parsimony, distance, and maximum likelihood methods were similar in topology. If the original epithets applied to the sequenced strains (both polar and those from GenBank) were used, it was clear that taxa were not monophyletic. However, using the revised taxonomic system of Anagnostidis and Komárek, we were able to reassign these strains to their current correct taxa (species, genus, and family). When these assignments were made, it was determined that the molecular sequence data analyses were congruent with morphology and ultrastructure. Nine of the polar strains were found to be new species, and eight were described as such: Arthronema gygaxiana Casamatta et Johansen sp. nov., Pseudanabaena tremula Johansen et Casamatta sp. nov., Leptolyngbya angustata Casamatta et Johansen sp. nov., Phormidium lumbricale Johansen et Casamatta sp. nov., Microcoleus glaciei Johansen et Casamatta sp. nov., Microcoleus rushforthii Johansen et Casamatta sp. nov., Microcoleus antarcticus Casamatta et Johansen sp. nov., Microcoleus acremannii Casamatta et Johansen sp. nov. Some genera (Leptolyngbya and Microcoleus) were clearly not monophyletic and require future revision.     

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.     

TWO SPECIES OF THE CHLOROPHYTE GENUS OSTREOBIUM FROM SKELETONS OF ATLANTIC AND CARIBBEAN REEF CORALS1,2

Two species of the siphonaceous chlorophyte Ostreobium inhabiting the aragonite skeletons of Atlantic and Caribbean reef corals were studied. Ostreobium quekettii Bornet & Flahault has been previously reported from these locations, but the species is here amended to include filament forms previously described under the name O. reineckei Bornet. Ostreobium constrictum sp. n. is described here for the first time. The 2 sympatric species are distinguished on the basis of filament morphology and chloroplast form.     

Hormogonia in two nostocalean cyanophytes (cyanobacteria) from the genera Hapalosiphon and Fischerella

The formation of hormogonia in the nostocalean cyanophytes/cyanobacteria Hapalosiphon fontinalis (C. Agardh) Bornet and Fischerella sp. was studied in natural populations collected from the Klin peatbog, northern Slovakia. Hormogonia were produced terminally in lateral branches of filaments (both species), or also directly on the main branches (Fischerella sp.). In contrast to vegetative filaments, hormogonia were not ramified, lacked heterocytes, were embedded in mucilaginous envelopes, were able to move, and their cells contained aerotopes. They were released by gliding through an opening in the sheath at the end of lateral branches of filaments. Released hormogonia of H. fontinalis were solitary or agglomerated into common fascicles morphologically resembling planktic colonies of Aphanizomenon flos-aquae (L.) Ralfs ex Bornet et Flahault or Dolichospermum affine (Lemmermann) Wacklin, Hoffmann et Komárek (syn. Anabaena affinis Lemmermann). Occasionally, lateral or sessile Nostochopsis-like heterocytes and apical spherical monocytes were formed on the main filaments. Hormogonia of Fischerella sp. were formed not only in apical part of lateral trichomes, but also directly on the main trichomes. Their cells were markedly larger than the vegetative cells and possessed well-developed aerotopes. Released hormogonia remained solitary, and were not agglomerated into fascicles. Apical hormogonia were released by gliding through an opening in the sheath at the end of lateral branches of filaments, and basal hormogonia were released by breaking off the main axis. In contrast to filaments of H. fontinalis which were very common and represented the dominant species of the cyanophyte communities in the locality, filaments of Fischerella sp. were observed only in one sample and for a limited period. This is the first record of a representative of the genus Fischerella in Slovakia.     

Population of <Emphasis Type="Italic">Aphanizomenon</Emphasis> from the Gulf of Gdańsk (Southern Baltic Sea): differences in phenotypic and genotypic characteristics

In this study, one of the major bloom-forming cyanobacteria in the Gulf of Gdańsk (Southern Baltic Sea), Aphanizomenon Morren ex Bornet et Flahaut has been characterized using a polyphasic approach by comparing phenotypic and molecular characteristics. The phenotypic analysis dealt with cell and filament morphology as well as ultrastructure. The molecular phylogenetic analyses were based on sequences of the 16S rRNA gene and the adjacent intergenic transcribed spacer (ITS). We have found the Aphanizomenon population from the Gulf of Gdańsk to be significantly different in ultrastructure, morphology from freshwater A. flos-aquae and according to the traditional approach; it could be assigned to different taxonomic units. However, genetic relationship with regard to sequences of the 16S rRNA gene, showed an high overall sequence identity (97.5–99%) to freshwater isolates. Similarly, ITS sequence identity among populations from the Baltic Sea and different freshwater isolates was as high as 90.3–97.7% suggesting one and the same species. Handling editor: D. Hamilton An erratum to this article can be found at     

SUBPOPULATION DIFFERENTIATION ASSOCIATED WITH NONRIBOSOMAL PEPTIDE SYNTHETASE GENE CLUSTER DYNAMICS IN THE CYANOBACTERIUM PLANKTOTHRIX SPP.1

The Plankthotrix Anagn. et Komárek population in the mesotrophic Lake Steinsfjorden has been intensively studied over several decades. This Planktothrix population produces a number of different classes of oligopetides. However, over the study period, only four main oligopeptide profiles (chemotypes) have been associated with the strains isolated from the lake. The chemotypes show distinct interactions with the environment, demonstrated by shifts in abundance along time series and vertical profiles. Here, we present genetic analysis of nonribosomal peptide synthetase (NRPS) gene regions in strains representing the four Planktothrix chemotypes in Lake Steinsfjorden. On the basis of phylogenetic analyses, we show that the NRPS genes for microcystin (mcy) and cyanopeptolin (oci) display the same clustering as do the chemotypes. Nucleotide diversity in mcy and oci was significantly higher between strains of different chemotypes than between strains of the same chemotype. K_a/K_s (nonsynonymous vs. synonymous mutations) values indicated positive selection in several polymorphic regions of the mcy and oci genes. Notably, incongruence between the phylogenetic trees for different gene segments and split decomposition analyses for segments of oci suggested horizontal gene transfer (HGT) events between strains showing different oligopeptide profiles. The oci HGT region encodes a module responsible for incorporating a variable amino acid in cyanopeptolin and is one of the regions suggested to be under positive selection. Taken together, our data suggest that there are four genetically distinct sympatric subpopulations—displayed as distinct chemotypes—in Lake Steinsfjorden. The diversification process of the chemotypes, and consequently the subpopulations, is driven by HGT and reinforced by positive selection of the corresponding NRPS gene regions.     

Igniting taxonomic curiosity: The amazing story of Amazonocrinis with the description of a new genus Ahomia gen. nov. and novel species of Ahomia,Amazonocrinis, and Dendronalium from the biodiversity-rich northeast region of India

Five cyanobacterial strains exhibiting Nostoc-like morphology were sampled from the biodiversity hotspots of the northeast region of India and characterized using a polyphasic approach. Molecular and phylogenetic analysis using the 16S rRNA gene indicated that the strains belonged to the genera Amazonocrinis and Dendronalium. In the present investigation, the 16S rRNA gene phylogeny clearly demarcated two separate clades of Amazonocrinis. The strain MEG8-PS clustered along with Amazonocrinis nigriterrae CENA67, which is the type strain of the genus. The other three strains ASM11-PS, RAN-4C-PS, and NP-KLS-5A-PS clustered in a different clade that was phylogenetically distinct from the Amazonocrinis sensu stricto clade. Interestingly, while the 16S rRNA gene phylogeny exhibited two separate clusters, the 16S–23S ITS region analysis did not provide strong support for the phylogenetic observation. Subsequent analyses raised questions regarding the resolving power of the 16S–23S ITS region at the genera level and the associated complexities in cyanobacterial taxonomy. Through this study, we describe a novel genus Ahomia to accommodate the members clustering outside the Amazonocrinis sensu stricto clade. In addition, we describe five novel species, Ahomia kamrupensis, Ahomia purpurea, Ahomia soli, Amazonocrinis meghalayensis, and Dendronalium spirale, in accordance with the International Code of Nomenclature for algae, fungi, and plants (ICN). Apart from further enriching the genera Amazonocrinis and Dendronalium, the current study helps to resolve the taxonomic complexities revolving around the genus Amazonocrinis and aims to attract researchers to the continued exploration of the tropical and subtropical cyanobacteria for interesting taxa and lineages.     

Anatoxin‐a occurrence and potential cyanobacterial anatoxin‐a producers in Spanish reservoirs1

The presence of anatoxin‐a (ANA) in Mediterranean region freshwaters has been reported only recently, and this work presents the first survey for ANA on a national scale in such waters. Fourteen reservoirs were sampled over a distance of 550 km from northeast to southwest Spain. Genera of cyanobacteria with ANA‐producing members in other countries were present in all of the Spanish reservoirs, but this toxin was detected in only one reservoir. The maximum ANA concentration detected was 0.31 μg · L^?1, and the most probable producer was Anabaena flos‐aquae (Lyngb.) Bréb. ex Bornet et Flahault. These findings suggest that in spite of the abundant cyanobacterial populations, ANA may be of lower occurrence in the Spanish reservoirs investigated than in other European freshwaters, and different strains of potential ANA producers occur in different ecoregions.     

GEITLERINEMA SPECIES (OSCILLATORIALES,CYANOBACTERIA) REVEALED BY CELLULAR MORPHOLOGY,ULTRASTRUCTURE, AND DNA SEQUENCING1

Geitlerinema amphibium (C. Agardh ex Gomont) Anagn. and G. unigranulatum (Rama N. Singh) Komárek et M. T. P. Azevedo are morphologically close species with characteristics frequently overlapping. Ten strains of Geitlerinema (six of G. amphibium and four of G. unigranulatum) were analyzed by DNA sequencing and transmission electronic and optical microscopy. Among the investigated strains, the two species were not separated with respect to cellular dimensions, and cellular width was the most varying characteristic. The number and localization of granules, as well as other ultrastructural characteristics, did not provide a means to discriminate between the two species. The two species were not separated either by geography or environment. These results were further corroborated by the analysis of the cpcB‐cpcA intergenic spacer (PC‐IGS) sequences. Given the fact that morphology is very uniform, plus the coexistence of these populations in the same habitat, it would be nearly impossible to distinguish between them in nature. On the other hand, two of the analyzed strains were distinct from all others based on the PC‐IGS sequences, in spite of their morphological similarity. PC‐IGS sequences indicate that these two strains could be a different species of Geitlerinema. Using morphology, cell ultrastructure, and PC‐IGS sequences, it is not possible to distinguish G. amphibium and G. unigranulatum. Therefore, they should be treated as one species, G. unigranulatum as a synonym of G. amphibium.     

Drouetiella epilithica sp. nov. and Drouetiella lurida (Oculatellaceae,Synechococcales) isolated in the Republic of Korea based on the polyphasic approach

Five strains of Drouetiella (ACKU666, 667, 668, 669 and 670) were isolated from gravels in water, stone monument and coastal mudflat in Korea, and were studied using morphological and molecular traits. All five strains had thin and simple trichomes and exhibited false branching. From these strains, four strains (ACKU666, 667, 668 and 669) exhibited similar cell lengths with reddish–brown colored cells such as Drouetiella lurida. The 16S rRNA gene phylogeny showed the four strains formed a clade with Drouetiella lurida, and their DNA similarity was calculated to be 99.1–100%. The color of strain ACKU670 appeared to be in bright blue–green color like Drouetiella fasciculata, and their thylakoids showed a parietal arrangement, which is a characteristic feature of the family Oculatellaceae. Strain ACKU670 turned out to be a sister clade to the D. lurida according to the phylogenetic analysis of the 16S rRNA gene. The 16–23S rRNA internal transcribed spacer secondary folding structure (D1–D1′, Box-B and V3 helices) confirmed the uniqueness of strain ACKU670, therefore indicating differences from the related species. Considering all the results, we described our strain ACKU670 as Drouetiella epilithica sp. nov. in accordance with the International Code of Nomenclature for Algae, Fungi and Plants.     

Phylogenetic Analyses and Diterpenoid Production by Marine Bacteria of the Genus <Emphasis Type="Italic">Saprospira</Emphasis>

The relationship between 16S rRNA gene sequence-derived phylogeny and the bacterial production of diterpenoids from 18 isolates of marine bacteria belonging to the genus Saprospira was determined. Restriction fragment length polymorphism (RFLP) analysis of the PCR amplified 16S rRNA genes of these isolates indicated four distinct phylotypes. The terpenoid metabolite profiles of each phylotype, determined by liquid chromatography mass spectrometry (LCMS) and nuclear magnetic resonance (NMR) analyses, indicated that diterpenoid production was restricted to phylotype A, which included the type specimen S. grandis Gross, and the sole member of the closely related phylotype B. The discovery of two new neoverrucosane diterpenoids produced by phylotype B has also been documented.