Evidence for polyphyletic origin of the members of the orders of Oscillatoriales and Pleurocapsales as determined by 16S rDNA analysis.

Phylogenetic analyses were carried out on six pleurocapsalean strains and 12 oscillatorialean strains by sequence determination of the 16S rDNA (16S rRNA gene). Although heterocyst-forming strains of the orders Nostocales and Stigonematales were shown to be monophyletic, unicellular strains of the orders Chroococcales and filamentous Oscillatoriales were shown to be polyphyletic, as reported earlier. Moreover, unicellular and baeocyte-forming strains of the order Pleurocapsales, which were thought to be monophyletic, were newly found to be polyphyletic. The results strongly indicate that even morphology was not necessarily reflected in the phylogenetic relationships at the order level. A need exists to reconstruct the taxonomy of cyanobacteria at the order level.

Phylogenetic analyses of Synechococcus strains (cyanobacteria) using sequences of 16S rDNA and part of the phycocyanin operon reveal multiple evolutionary lines and reflect phycobilin content.

The genus Synechococcus (cyanobacteria), while containing morphologically similar isolates, is polyphyletic and organisms presently classified as such require reclassification into several independent genera. Studies based on analysis of 16S rRNA gene sequences have shown that members of the genus Synechococcus are affiliated to three of seven deeply branching cyanobacterial lineages. In addition, some strains do not appear to be associated with any of these lineages and may represent novel clades. In this report, a cyanobacterial phylogeny based on 16S rDNA sequences, including 14 newly sequenced Synechococcus isolates, is presented. One newly sequenced Synechococcus strain (PCC 7902) did not have any close relatives amongst cyanobacterial isolates currently contained in 16S rDNA sequence databases and was only loosely affiliated to a cyanobacterial lineage in which no other Synechococcus strains were found. Three hot-spring Synechococcus isolates, including two that were newly sequenced in this study (PCC 6716 and PCC 6717), formed an additional cyanobacterial lineage. These results indicated that Synechococcus species are affiliated to five of eight deeply branching cyanobacterial lineages. Part of the phycocyanin (PC) gene sequence (cpc), including the intergenic spacer (IGS) between cpcB and cpcA and the corresponding flanking regions (cpcBA-IGS), was used to investigate relationships between closely related Synechococcus isolates. Previously described PCR primers did not amplify this region from the majority of strains under investigation, so a new set of primers was designed that allowed amplification and sequencing of the cpcBA-IGS and flanking regions from 38 Synechococcus species. Phylogenetic analysis of this region was largely consistent with that obtained from 16S rDNA sequence analysis and revealed a relationship between the primary PC DNA sequence and the phycobilin content of cells.

A proposal for the unification of five species of the cyanobacterial genus Microcystis Kützing ex Lemmermann 1907 under the rules of the Bacteriological Code.

Genomic DNA homologies were examined from six Microcystis (cyanobacteria) strains, including five different species, Microcystis aeruginosa, Microcystis ichthyoblabe, Microcystis novacekii, Microcystis viridis and Microcystis wesenbergii. All DNA-DNA reassociation values between two strains of M. aeruginosa and the other four species exceeded 70%, which is considered high enough for them to be classified within the same bacterial species. It is proposed to unify these five species into M. aeruginosa under the Rules of the Bacteriological Code and NIES843T (= IAM M-247T) is proposed as the type strain. Two other species, Microcystis flos-aquae and Microcystis pseudofilamentosa, should be regarded as morphological variations of this unified M. aeruginosa. The current taxonomy of cyanobacteria depends too much upon morphological characteristics and must be reviewed by means of bacteriological methods as well as traditional botanical methods.

Fatty acid profiles and their chemotaxonomy in planktonic species of Anabaena (Cyanobacteria) with straight trichomes.

Twenty-four axenic strains of planktonic Anabaena with straight trichomes, assigned to 7 species, were investigated by analyzing the pattern and content of their fatty acid composition and comparing their fatty acid composition with their morphological properties. In general, the fatty acids in planktonic Anabaena contained 14:0, 16:0, 16:1(cis-), 18:0, 18:1, 18:2, and 18:3(alpha) as their major components, and were classified as Type 2 according to the Kenyon-Murata system. These strains were further divided into 2 subtypes: 18 strains belonging to Type 2A, which contains 16:2 and 16:3, and 6 strains to Type 2B, which lacks 16:2 and 16:3. Fatty acid compositions of strains of A. solitaria, A. smithii, and A. kisseleviana closely corresponded to morphological properties; however, 10 strains of A. planctonica were divided into 4 clusters, and 3 strains of A. affinis into 2 clusters. These clusters should be taxonomically evaluated based on other aspects such as genetic characteristics.

Isolation and identification of the cyanotoxin cylindrospermopsin and deoxy-cylindrospermopsin from a Thailand strain of Cylindrospermopsis raciborskii (Cyanobacteria).

A strain of Cylindrospermopsis (Cyanobacteria) isolated from a fishpond in Thailand was examined for its taxonomy based upon morphology and 16S rRNA gene sequence. It was also examined for production of the hepatotoxic cyanotoxin called cylindrospermopsin (CYN) and deoxycylindrospermopsin (deoxy-CYN). The strain (CY-Thai) was identified as C. raciborskii (Woloszynska) Seenaya and Subba Raju based upon morphological examination which was confirmed by 16S rRNA gene sequences and phylogenetic comparisons based upon its 16S rRNA gene. The alkaloid heptatotoxin CYN was confirmed using mouse bioassay, HPLC and HPLC-MS/MS while deoxy-CYN was confirmed using HPLC-MS/MS. The mouse bioassay gave a minimum lethal dose at 250mg dry weight cells/kg body weight within 24h and 125mg/kg at 72h, with signs of poisoning the same as in literature reports for CYN. HPLC chromatographic comparison of the CY-Thai toxin with standard CYN gave the same retention time and an absorbance maximum at 262nm. HPLC-MS/MS confirmed the presence of CYN (M+H 416) and deoxy-CYN (M+H 400). The CYN content in strain CY-Thai was estimated at 1.02mg/g and approximately 1/10 of this amount for deoxy-CYN. This is the first report from Asia of a CYN, deoxy-CYN producing Cylindrospermopsis raciborskii.

Origin and evolution of the colonial volvocales (Chlorophyceae) as inferred from multiple, chloroplast gene sequences.

A combined data set of DNA sequences (6021 bp) from five protein-coding genes of the chloroplast genome (rbcL, atpB, psaA, psaB, and psbC genes) were analyzed for 42 strains representing 30 species of the colonial Volvocales (Volvox and its relatives) and 5 related species of green algae to deduce robust phylogenetic relationships within the colonial green flagellates. The 4-celled family Tetrabaenaceae was robustly resolved as the most basal group within the colonial Volvocales. The sequence data also suggested that all five volvocacean genera with 32 or more cells in a vegetative colony (all four of the anisogamous/oogamous genera, Eudorina, Platydorina, Pleodorina, and Volvox, plus the isogamous genus Yamagishiella) constituted a large monophyletic group, in which 2 Pleodorina species were positioned distally to 3 species of Volvox. Therefore, most of the evolution of the colonial Volvocales appears to constitute a gradual progression in colonial complexity and in types of sexual reproduction, as in the traditional volvocine lineage hypothesis, although reverse evolution must be considered for the origin of certain species of Pleodorina. Data presented here also provide robust support for a monophyletic family Goniaceae consisting of two genera: Gonium and Astrephomene.

Phylogenetic relationships between toxic and non-toxic strains of the genus Microcystis based on 16S to 23S internal transcribed spacer sequence.

16S to 23S ribosomal DNA internal transcribed spacer sequences of 47 strains of the genus Microcystis were determined. Derived maximum likelihood and DNA distance trees indicated that Microcystis can be divided into three clusters. The first cluster included toxic and non-toxic strains, the second only toxic ones, and the third only non-toxic ones. The tree topologies were not necessarily correlated with morphospecies distinction or phycobilin pigment composition, and one genotype may have more than one morphotype. Phylogenetic analysis based on intergenic spacer sequences was thought to be effective for understanding relationships among closely related species and strains.

Thioic O-acid ester in sulfolipid isolated from freshwater picoplankton cyanobacterium, Synechococcus sp.

A thioic O-acid ester-containing sulfolipid (thionsulfolipid) was isolated from cells of picoplankton cyanobacterium, Synechococcus sp. The lipid accounted for about 0.2% of the lyophilized cells. The lipid was subjected to mild alkaline hydrolysis, and the structures of the hydrolysis products were identified by infrared spectra, mass and nuclear magnetic resonance spectrometries, as fatty acids and hexadecane-, hexadecene- and tetradecanethioic S-acids. Thioic S-acid was further confirmed by the synthesis of hexadecanethioic S-acid from palmitoylchloride and hydrogen sulfide. The positional distribution of the thioic acid ester in the lipid was determined by beta-galactosidase, sulphur-oxygen exchange reaction using silver nitrate, and lipase hydrolysis of the diacylglycerol derived from the lipid. The structure of the thionsulfolipid was identified as 6-sulfo-alpha-D-quinovopyranosyl(1-->1')-2'-O-acyl-3'-O-thioacy l -2-glycerol. When cells of HL 60, as a human lymphoma, were cultured with thionsulfolipid, 61% of the cell growth was inhibited at the concentration of 200 micrograms/ml. The lipid was toxic against minnows (Tanichtys albonubes). The LD50 was 20 ppm. Thioic O-acid ester-containing lipid (thionsulfolipid) has not been found in any other photosynthetic organisms.