MOLECULAR SYSTEMATICS OF CYANOBACTERIA Submitted by Elvina Lee Mei Hui, BSc (Hons) This thesis is presented for the degree of Doctor of Philosophy of Murdoch University i DECLARATION I declare that this thesis is my own account of my research and contains as its main content work, which has not been previously submitted for a degree at any tertiary education institution. ______________________________ Elvina Lee January 2016 ii ABSTRACT Cyanobacteria constitute a phylum of ubiquitous cosmopolitan bacteria with the ability to perform oxygenic photosynthesis. Their ancient origins, ecological and economic potential, biotechnological applications and impact on water systems have attracted much interest from the academia, industry, health authorities and regulators. Despite this, cyanobacteria classification and nomenclature still remains difficult. One of the aims of this project was to further our understanding of cyanobacteria systematics by (1) testing polyphasic characterization methods and (2) examining the effect of various phylogenetic reconstruction strategies. Additionally, (3) Next Generation Sequencing (NGS) assays using novel cyanobacteria 16S rDNA targeted primers were implemented to provide better taxa resolution than that offered by “universal” 16S rDNA primers. Cyanobacteria strains from various water sources in Australia were isolated, characterised at four loci commonly used for cyanobacteria molecular classification, and tested for the presence of genes implicated in toxin and terpene (odour) production. A total of 55 novel cyanobacterial strains were isolated and maintained in culture, forming the first known collection of cyanobacteria isolates from Western Australia. Comparison of molecular– and morphology– based identifications not only showed the limitations of the current methods (only 45% of the isolates showed agreement) but also provided the opportunity to suggest guidelines and conceive a way forward towards more effective identification approaches. Examination of alternative phylogenetic markers, workflows and stringencies showed that between alignment algorithms, alignment curations and tree building methods, the latter had the greatest effect on tree topology. This result was consistent regardless of locus, alignment and curation strategy employed. Finally, two sets of novel cyanobacteria-targeted primers were designed for use with NGS technologies. As compared to the universal 16S rRNA primers, these primers showed higher specificity and preferential amplification of cyanobacteria and proteobacteria DNA. Of the sequences obtained using these two new primer pairs, cyanobacteria sequences comprised 50.5% and 54.4%, while proteobacteria sequences comprised 44.5% and 40.3% respectively. In comparison, with the universal 16S rRNA primers, cyanobacteria and proteobacteria comprised 15.3% and 33.4% respectively of the sequences analysed. iii Using morphological and molecular methods, this project provides a snapshot of the as yet unstudied freshwater cyanobacterial diversity found in Western Australia using polyphasic methods. The limitations of the current identification approaches, uncovered during the first phase of the project, were harnessed to develop a method to assess the variability of phylogenetic reconstructions. Finally, novel cyanobacteria specific NGS primers demonstrated how adopting the latest NGS technology represents a promising advance in the molecular investigation of cyanobacteria. iv TABLE OF CONTENTS Declaration ........................................................................................................................... ii Abstract .............................................................................................................................. iii Table of Contents ................................................................................................................. v Acknowledgements ............................................................................................................ vii Table of Figures ............................................................................................................... viii List of Tables ....................................................................................................................... xi List of Abbreviations ......................................................................................................... xii CHAPTER 1. General Introduction ............................................................................ 1 1.1. Introduction ............................................................................................................... 1 1.2. Characteristics and habitat ...................................................................................... 2 1.3. Public and environmental implications of cyanobacteria ...................................... 5 1.3.1. Effect of blooms ............................................................................................... 5 1.3.2. Toxins and their effects .................................................................................... 8 1.3.3. Taste and odour .............................................................................................. 13 1.4. Management of cyanobacteria ............................................................................... 14 1.4.1. Chemical methods .......................................................................................... 14 1.4.2. Physical methods ........................................................................................... 15 1.4.3. Biological and management methods ............................................................ 16 1.5. Taxonomy: difficulties in nomenclature and identification................................. 16 1.6. Methods used to identify and detect cyanobacteria ............................................. 20 1.6.1. Morphological identification ......................................................................... 20 1.6.2. Molecular methods ........................................................................................ 21 1.6.3. Polyphasic approaches ................................................................................... 27 1.7. Aims .......................................................................................................................... 29 CHAPTER 2. General Materials & Methods ........................................................... 30 2.1. Water Sources .......................................................................................................... 30 2.2. Isolation and maintenance of cyanobacteria ......................................................... 30 2.3. Extraction of cyanobacterial DNA ......................................................................... 31 2.4. PCR amplification and sequencing of target genes .............................................. 31 2.4.1. Conventional Standard PCR .......................................................................... 31 2.4.2. Quantitative PCR (qPCR) .............................................................................. 32 2.5. Sequencing and analysis of amplified products .................................................... 32 2.5.1. Sanger sequencing ......................................................................................... 32 2.5.2. Analysis of sequenced chromatograms .......................................................... 32 CHAPTER 3. Polyphasic identification of cyanobacterial isolates from Australia34 3.1. Introduction ............................................................................................................. 34 3.2. Materials and Methods ........................................................................................... 35 3.2.1. Isolation and cultivation of cyanobacterial strains ......................................... 35 3.2.2. Morphological identification ......................................................................... 36 3.2.3. Molecular and phylogenetic analyses ............................................................ 36 3.3. Results ...................................................................................................................... 37 3.3.1. Comparison of morphological data ................................................................ 39 3.3.2. Comparison of molecular identification and phylogenetic analysis at different loci 45 3.3.3. Comparison between morphological and molecular identifications .............. 51 3.3.4. Identification of novel isolates ....................................................................... 52 3.4. Discussion ................................................................................................................. 52 CHAPTER 4. Effect of alignment, curation and reconstruction methods on the molecular systematics of Cyanobacteria ......................................................................... 57 4.1. Introduction ............................................................................................................. 57 4.2. Materials and Methods ........................................................................................... 59 4.2.1. Isolation, cultivation and sequencing of cyanobacterial isolates ................... 59 4.2.2. Multiple sequence alignment and curation .................................................... 59 v 4.2.3. Tree-building and multivariate analyses .......................................................