Genetic Diversity, Morphological Uniformity and Polyketide Production in Dinoflagellates (Amphidinium, Dinoflagellata)

Dinoflagellates are an intriguing group of eukaryotes, showing many unusual morphological and genetic features. Some groups of dinoflagellates are morphologically highly uniform, despite indications of genetic diversity. The species Amphidinium carterae is abundant and cosmopolitan in marine environments, grows easily in culture, and has therefore been used as a ‘model’ dinoflagellate in research into dinoflagellate genetics, polyketide production and photosynthesis. We have investigated the diversity of ‘cryptic’ species of Amphidinium that are morphologically similar to A. carterae, including the very similar species Amphidinium massartii, based on light and electron microscopy, two nuclear gene regions (LSU rDNA and ITS rDNA) and one mitochondrial gene region (cytochrome b). We found that six genetically distinct cryptic species (clades) exist within the species A. massartii and four within A. carterae, and that these clades differ from one another in molecular sequences at levels comparable to other dinoflagellate species, genera or even families. Using primers based on an alignment of alveolate ketosynthase sequences, we isolated partial ketosynthase genes from several Amphidinium species. We compared these genes to known dinoflagellate ketosynthase genes and investigated the evolution and diversity of the strains of Amphidinium that produce them

Distribution, diversity and function of biomarker biosynthesis genes in modern cyanobacteria

The oxygenation of Earth's atmosphere by cyanobacteria was one of the most significant events in the history of the evolution of life on Earth. Although the timing of the Great Oxidation Event (GOE) is fairly well constrained, what is still unclear is how far in advance of this event cyanobacteria and oxygenic photosynthesis evolved. 2-Methylhopanes, geologic remnants of 2-methylhopanoids, have been used extensively as cyanobacterial biomarkers in tracing the history of oxygenic photosynthesis. However, it was recently shown that other bacteria can also produce significant amounts of 2-methylhopanoids, bringing into question the validity of using 2-methylhopanes as cyanobacterial biomarkers. Identification of the gene coding for the enzyme that methylates hopanoids at the C2 position, hpnP, has provided a genetic tool for investigating 2-methylhopanoids. This thesis aimed to provide more information about the distribution, diversity and function of 2-methylhopanoids in modern bacteria in order to more accurately interpret the biomarker evidence for cyanobacteria prior to the GOE. A survey of hopanoid biosynthesis genes in the genomes of available cyanobacteria cultures was undertaken in order to determine the distribution and prevalence of the genetic capacity for hopanoid/2-methylhopanoid production among cyanobacteria, and to develop a database of hpnP sequences from known strains against which to compare environmental isolates. Less than a third of cyanobacteria surveyed were found to possess the hpnP gene, with no clear taxonomic affiliation between them. Next, several environments that can be considered analogous to Archean environments were surveyed for the diversity of hpnP genes. These environments included stromatolite-forming hypersaline microbial mats, desert biological soil crusts, and hotspring microbial mats. A total of fifty-one hpnP operational taxonomic units were recovered from these environments, and all but two of these were shown to originate from cyanobacterial species. Finally, to investigate the function of 2- methylhopanoids in cyanobacteria, hpnP was deleted from the filamentous cyanobacterium Nostoc punctiforme ATCC 29133S. The resulting hpnP deletion mutant was found to be sensitive to salt and pH stress. The results of this work therefore support a cyanobacterial origin for the majority of 2- methylhopanoids produced in the ancient-analog environments sampled, and a role in stress-tolerance for 2-methylhopanoids in cyanobacteria

Polyketide compounds isolated to date from strains of species of <i>Amphidinium</i>.

<p>Polyketide compounds isolated to date from strains of species of <i>Amphidinium</i>.</p

Strains of <i>Amphidinium</i> species used in this study.

<p>Strains of <i>Amphidinium</i> species used in this study.</p

Light micrographs of <i>Amphidinium massartii</i> strain CS-259 and <i>Amphidinium thermaeum</i> strain CS-109, showing general cell shape, plastid, dividing cells, nucleus, pyrenoid.

<p>Scale bars represent 5 µm. (A)–(F), CS-259. (A) <i>A. massartii</i> CS-259 in ventral view, showing shape of the epicone and longitudinal flagellum, arrow points to position of flagellar insertion. (B) Low focus image, arrow points to pyrenoid. (C) Cell in dorsal view showing general cell shape, (D) Motile dividing cells, arrow points to starch-sheathed pyrenoid, (E) Cell in lateral view showing flattening, (F) Cell taken using epifluorescent microscopy, showing the plastid with multiple lobes. (G)–(I), CS-109. (G) Cell in ventral view showing general shape and position of flagellar insertion (arrow), (H), Cell in lateral view, arrow points to flagellar insertion, (I), Motile cells shortly following cell division.</p

Phylogenetic analysis of alignment of <i>Amphidinium</i> partial LSU rDNA sequences (D1–D3 domains), using maximum likelihood.

<p>Values at nodes represent bootstrap support values and Bayesian posterior probability support (BS/PP).</p

Partial alignment of β-ketosynthase protein sequence from bacteria and alveolates, including three conserved active site residues, and showing conserved regions against which degenerate primers were designed.

<p>Partial alignment of β-ketosynthase protein sequence from bacteria and alveolates, including three conserved active site residues, and showing conserved regions against which degenerate primers were designed.</p

Transmission Electron Microscopy images showing body scales in <i>Amphidinium massartii</i>.

<p>(A)A section through <i>Amphidinium massartii</i> CS-259 showing body scales in alveolae (arrow points to alveolae). (B, C) Whole mount preparation of culture suspension showing the body scales (arrows point to scales).</p

Primers used in this study.

<p>Primers used in this study.</p

Results of tBlastx analysis of putative PKS genes from <i>Amphidinium</i> species.

<p>Results of tBlastx analysis of putative PKS genes from <i>Amphidinium</i> species.</p