Chrysosporum ovalisporum is synonymous with the true-branching cyanobacterium Umezakia natans (Nostocales/Aphanizomenonaceae).

True branching is a facultative characteristic only known from two cyanobacteria in the Aphanizomenonaceae, Umezakia natans and Dolichospermum brachiatum. In both cases, its expression has been associated with environmental stress, and its practical use as a diacritical feature has been previously evaluated. In this study, we undertook further evaluation of the phylogeny of Umezakia natans and its relationship to Chrysosporum ovalisporum as a previous study suggested the two were potentially congeneric. We used combined morphological, phylogenetic, and phylogenomic approaches to determine their relatedness using new strains available from a broad geographic range. Phylogenetic analysis based on 16S rRNA gene sequences showed that Australian C. ovalisporum and Japanese U. natans strains clustered together with accessions of C. ovalisporum originating from Australia, Israel, and Spain, with high p-distance similarity values (99.5%-99.9%). Additionally, differences between the two species in the 16S-23S ITS region was low (0%-2.5%). The average nucleotide identity of the U. natans and C. ovalisporum strains was also high (ANI of > 99.5 and AF > 0.9) and supported a genus-level separation from Chrysosporum bergii (83 ANI between clusters). Furthermore, in culture, strains of both species grown in vitamin-free media showed facultative true branching, a feature not previously known in C. ovalisporum. Collectively, the results support unification of C. ovalisporum and U. natans according to the principle of priority as Umezakia ovalisporum.

Cryptic diversity within the Scytonema complex: Characterization of the paralytic shellfish toxin producer Heterosyctonema crispum, and the establishment of the family Heteroscytonemataceae (Cyanobacteria/Nostocales).

Strains of the freshwater filamentous, benthic cyanobacterium Scytonema crispum Agardh isolated from six sites in subtropical south-east Queensland were characterised using a combination of phenotypic and genetic traits. Morphologically, the strains were consistent with the description of Scytonemataceae sensu stricto, and the description of Scytonema crispum. However, phylogenetic analysis of the 16S rRNA gene, the 16S-23S rRNA operon, and the nifH gene revealed that these strains and three others from outside Australia formed a monophyletic clade distinct from Scytonema and other species in the Scytonemataceae. Collectively, this data suggests this group is sufficiently evolutionarily distinct to be placed in a new family, Heteroscytonemataceae fam. nov. Accordingly, the taxon previously known as S. crispum has been transferred to a new genus Heteroscytonema gen nov., as H. crispum. Some strains of H. crispum exhibited facultative production of paralytic shellfish toxins (PSTs). The concentration of PSTs produced by individual strains varied widely, from 2.7 µg g-1 to 171.3 µg g-1, and included C toxins, decarbamoyl saxitoxin (dcSTX), gonyautoxins (GTX2, GTX3 and GTX5), saxitoxin (STX) and uncharacterised PSTs. The majority of the Australian strains produced dcSTX as the dominant saxitoxin analogue, a significant finding given that dcSTX has approximately half the relative toxicity of STX. The PST profile varied within and between Australian strains of H. crispum and in strains collected from New Zealand and the United States. The sxtA gene, one of the determinants for the production of PSTs, was present in all strains in which PSTs were detected. The discovery of PST-producing H. crispum in the headwaters of a major drinking water reservoir presents a serious risk for potential human and animal exposure to these neurotoxic compounds and further highlights the importance of monitoring benthic cyanobacteria populations for potentially toxigenic species.

Cyanotoxins: Which detection technique for an optimum risk assessment?

The presence of toxigenic cyanobacteria (blue-green algae) in drinking water reservoirs poses a risk to human and animal health worldwide. Guidelines and health alert levels have been issued in the Australian Drinking Water Guidelines for three major toxins, which are therefore the subject of routine monitoring: microcystin, cylindrospermopsin and saxitoxin. While it is agreed that these toxic compounds should be monitored closely, the routine surveillance of these bioactive chemicals can be done in various ways and deciding which technique to use can therefore be challenging. This study compared several assays available for the detection of these toxins and their producers in environmental samples: microscopy (for identification and enumeration of cyanobacteria), ELISA (Enzyme-Linked ImmunoSorbant Assay), PPIA (Protein phosphatase inhibition assay), PSI (Protein synthesis inhibition), chemical analysis and PCR (Polymerase Chain Reaction). Results showed that there was generally a good correlation between the presence of potentially toxigenic cyanobacteria and the detection of the toxin by ELISA. Nevertheless data suggest that cell numbers and toxin concentrations measured in bioassays do not necessarily correlate and that enumeration of potentially toxic cyanobacteria by microscopy, while commonly used for monitoring and risk assessment, is not the best indicator of real toxin exposure. The concentrations of saxitoxins quantified by ELISA were significantly different than those measured by LC-MS, while results were comparable in both assays for microcystin and cylindrospermopsin. The evaluation of these analytical methods led to the conclusion that there is no "gold standard" technique for the detection of the aforementioned cyanotoxins but that the choice of detection assay depends on cost, practicality, reliability and comparability of results and essentially on the question to be answered, notably on toxin exposure potential.

Iningainema pulvinus gen nov., sp nov. (Cyanobacteria, Scytonemataceae) a new nodularin producer from Edgbaston Reserve, north-eastern Australia.

A new nodularin producing benthic cyanobacterium Iningainema pulvinus gen nov., sp nov. was isolated from a freshwater ambient spring wetland in tropical, north-eastern Australia and characterised using combined morphological and phylogenetic attributes. It formed conspicuous irregularly spherical to discoid, blue-green to olive-green cyanobacterial colonies across the substratum of shallow pools. Morphologically Iningainema is most similar to Scytonematopsis Kiseleva and Scytonema Agardh ex Bornet & Flahault. All three genera have isopolar filaments enveloped by a firm, often layered and coloured sheath; false branching is typically geminate, less commonly singly. Phylogenetic analyses using partial 16S rRNA sequences of three clones of Iningainema pulvinus strain ES0614 showed that it formed a well-supported monophyletic clade. All three clones were 99.7-99.9% similar, however they shared less than 93.9% nucleotide similarity with other cyanobacterial sequences including putatively related taxa within the Scytonemataceae. Amplification of a fragment of the ndaF gene involved in nodularin biosynthesis from Iningainema pulvinus confirmed that it has this genetic determinant. Consistent with these results, analysis of two extracts from strain ES0614 by HPLC-MS/MS confirmed the presence of nodularin at concentrations of 796 and 1096µgg-1 dry weight. This is the third genus of cyanobacteria shown to produce the cyanotoxin nodularin and the first report of nodularin synthesis from the cyanobacterial family Scytonemataceae. These new findings may have implications for the aquatic biota at Edgbaston Reserve, a spring complex which has been identified as a priority conservation area in the central Australian arid and semiarid zones, based on patterns of endemicity.

Phylogeny and toxicology of Lyngbya wollei (Cyanobacteria, Oscillatoriales) from north-eastern Australia, with a description of Microseira gen. nov.

Three populations of the freshwater filamentous cyanobacterium Lyngbya wollei (Farlow ex Gomont) Speziale and Dyck have been putatively identified from north-eastern Australia and found to produce the potent cyanotoxin cylindrospermopsin (CYN) and its analog deoxy-cylindrospermopsin (deoxy-CYN). We investigated the phylogeny and toxicology of strains and mats isolated from two of these populations using a combination of molecular and morphological techniques. Morphologically the strains corresponded to the type description, however, the frequency of false-branching was low, and variable over time. Strains and mat samples from both sites were positive for the cyrF and cyrJ genes associated with CYN biosynthesis. Phylogenetic analysis of these genes from Australian L. wollei sequences and comparable cyanobacterial sequences revealed that the genes in L. wollei were more closely related to homologous genes in Oscillatoria sp. PCC 6506 than to homologs in Nostocalean CYN-producers. These data suggest a common evolutionary origin of CYN biosynthesis in L. wollei and Oscillatoria. In both the 16S rRNA and nifH phylogenies, the Australian L. wollei strains formed well-supported clades with United States L. wollei (= Plectonema wollei) strains. Pair-wise sequence similarities within the 16S rRNA clade containing all eleven L. wollei strains were high, ranging from 97% to 100%. This group was distantly related (<92% nucleotide similarity) to other taxa within the group previously considered under the genus Lyngbya sensu lato (C. Agardh ex Gomont). Collectively, these results suggest that this toxigenic group is evolutionarily distinct and sufficiently distant as to be considered a separate genus, which we have described as Microseira gen. nov. and hence transfer to it the type M. wollei comb. nov.

Rapid, multiplex-tandem PCR assay for automated detection and differentiation of toxigenic cyanobacterial blooms.

Cyanobacterial blooms are a major water quality issue and potential public health risk in freshwater, marine and estuarine ecosystems globally, because of their potential to produce cyanotoxins. To date, a significant challenge in the effective management of cyanobacterial has been an inability of classical microscopy-based approaches to consistently and reliably detect and differentiate toxic from non-toxic blooms. The potential of cyanobacteria to produce toxins has been linked to the presence of specific biosynthetic gene clusters. Here, we describe the application of a robotic PCR-based assay for the semi-automated and simultaneous detection of toxin biosynthesis genes of each of the toxin classes characterized to date for cyanobacteria [i.e., microcystins (MCYs), nodularins (NODs), cylindrospermopsins (CYNs) and paralytic shellfish toxins (PSTs)/saxitoxins (SXTs)]. We demonstrated high sensitivity and specificity for each assay using well-characterized, cultured isolates, and establish its utility as a quantitative PCR using DNA, clone and cell-based dilution series. In addition, we used 206 field-collected samples and 100 known negative controls to compare the performance of each assay with conventional PCR and direct toxin detection. We report a diagnostic specificity of 100% and a sensitivity of ≥97.7% for each assay.

First report of a toxic Nodularia spumigena (Nostocales/ Cyanobacteria) bloom in sub-tropical Australia. I. Phycological and public health investigations.

Cyanobacterial blooms represent one of the most conspicuous and widespread waterborne microbial hazards to human and ecosystem health. Investigation of a cyanobacterial bloom in a shallow brackish water recreational cable ski lake in south-eastern Queensland, Australia revealed the dominance of the toxigenic species Nodularia spumigena. The bloom spanned three months, during which time cell concentrations exceeded human guideline thresholds for recreational risk, and concentrations of the hepatotoxic cyanotoxin nodularin exceeded 200 µg L(-1). Cyanotoxin origin and identification was confirmed by amplification of the ndaF-specific PCR product and sequencing of the 16S rRNA gene. From the limited data available leading up to, and throughout the bloom, it was not possible to establish the set of causative factors responsible for its occurrence. However a combination of factors including salinity, hydraulic retention time and nutrient status associated with an extended period of drought are likely to have contributed. This was the first known occurrence of this species in bloom proportions from sub-tropical Australia and as such represents a hitherto uncharacterized risk to human and ecosystem health. It highlights the need for adaptive monitoring regimes to ensure a comprehensive understanding of the potentially toxic cyanobacteria likely to inhabit any given region. Such monitoring needs to recognize that cyanobacteria have a significant capacity for range expansion that has been facilitated by recent changes in global climate.