22Reliable identification of cyanobacterial isolates has significant 23 socio-economic implications as many bloom-forming species 24 affect the aesthetics and safety of drinking water, through the 25 production of taste and odour compounds or toxic metabolites. 26The limitations of morphological identification have promoted 27 the application of molecular tools, and encouraged the adoption 28 of combined (polyphasic) approaches that include both 29 microscopy-and DNA-based analyses. In this context, the 30 rapid expansion of available sequence data is expected to allow 31 increasingly reliable identification of cyanobacteria, and 32 ultimately resolve current discrepancies between the two 33 approaches. 34In the present study morphological and molecular 35 characterisations of cyanobacterial isolates (n=39), collected 36 from various freshwater sites in Australia, were compared. 37Sequences were obtained for the small ribosomal subunit RNA 38 gene (16S rDNA) (n=36), the DNA-dependent RNA 39 polymerase gene (rpoC1) (n=22), and the phycocyanin operon, 40 with its intergenic spacer region (cpcBA-IGS) (n= 19).

Next generation sequencing (NGS) has rapidly become an invaluable tool for the detection, identification and relative quantification of environmental microorganisms. Here, we demonstrate two new 16S rDNA primer sets, which are compatible with NGS approaches and are primarily for use in water quality studies. Compared to 16S rRNA gene based universal primers, in silico and experimental analyses demonstrated that the new primers showed increased specificity for the Cyanobacteria and Proteobacteria phyla, allowing increased sensitivity for the detection, identification and relative quantification of toxic bloom-forming microalgae, microbial water quality bioindicators and common pathogens. Significantly, Cyanobacterial and Proteobacterial sequences accounted for ca. 95% of all sequences obtained within NGS runs (when compared to ca. 50% with standard universal NGS primers), providing higher sensitivity and greater phylogenetic resolution of key water quality microbial groups. The increased selectivity of the new primers allow the parallel sequencing of more samples through reduced sequence retrieval levels required to detect target groups, potentially reducing NGS costs by 50% but still guaranteeing optimal coverage and species discrimination.

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• Morphological characterisation:Unique from all other reported Eimeria species.• Molecular characterisation at 18S, 28S rRNA and COI loci: Related to Eimeria arnyi. ). Molecular analysis was conducted at three loci; the 18S and 28S ribosomal RNA (rRNA) and the mitochrondial cytochrome oxidase gene (COI). At the 18S rRNA locus, E. collieie n. sp. shared 96.4% and 98.3% genetic similarity to E. ranae (GenBank accession number: EU717219) and E. arnyi (AY613853). At 28S rRNA locus, E. collieie n. sp. shared 91.6% genetic similarity to E. papillata (GenBank accession number: GU593706) and phylogenetic analysis at this locus placed E. collieie n. sp. in a separate clade. At the COI locus, E. collieie n. sp. shared 92.7% genetic similarity to Eimeria setonicis (GenBank accession number: KF225638) from a quokka (Setonix brachyurus) in Western Australia. However reptile-derived sequences were not available for the 28S rRNA and the COI loci. Based on morphological and molecular data, this isolate is a new species of coccidian parasite that, to date, has only been found in western long-necked turtles.

Molecular systematics uses currently available data to produce the best approximation to the true (un-observable) phylogeny of a taxon. Molecular phylogeny complements morphological identification and classification of organisms, in order to infer their evolutionary relationships. In the current era dominated by cultivation-independent surveys, testing the potential technical and analytical pitfalls and limitations of environmental DNA surveys appears crucial. Sequence-based phylogenetic reconstructions rely on three main steps: alignment, alignment curation and tree building. Several independent options and settings can be adopted at each step, but it is well known that their choice (or combination) can significantly affect the topology of the phylogenetic tree obtained and skew the reliability of the resultant systematics. For the present study, five alignment algorithms, two curation options and three tree-building methods were used to infer the phylogeny of three orders of cyanobacteria, based on four validated markers widely used for this phylum: 16S rRNA, 16S-23S ITS, cpcBA-IGS and rpoC1. Compared to the alignment algorithm or the curation stringency used, the tree-building method was found to have the greatest effect on the resultant tree topology. This result was consistent for all loci, including the genetically-constrained (protein-coding) locus rpoC1. The reproducibility of the tree topology was clearly visualized and measured for each locus. This paper presents pitfalls in cyanobacteria systematics and implements a simple and rapid method, applicable to any locus and organism, to identify aberrant results and assess the reproducibility of phylogenetic reconstructions.