Christoffer Bugge Harder:
Pyrosequencing and genetic diversity of microeukaryotes

Date: 17-06-2013    Supervisor: Flemming Ekelund

Free-living, heterotrophic protozoa have an important ecological role in most terrestrial ecosystems by their grazing of bacteria as one of the first links in food chains and webs. Furthermore, some of them serve as reservoirs for disease-causing bacteria and /or as occasional opportunistic pathogens themselves. Protozoa is a morphological group which occurs in many different eukaryotic phyla, and many apparently morphologically similar types are very different from each others genetically. This complicates the development of good primers for analysis of their diversity with modern DNA based methods. Compared to other microorganisms such as fungi, algae and bacteria, much less is known about protozoa. It has been an essential element of this thesis to to advance our knowledge of protozoa by developing new primers for DNA-based studies of protozoa impact on ecosystems or as indicators of environmental conditions. The ribosomal 18S region is the only marker that is sufficiently well known in a broad range of eukaryotic microorganisms to be widely applicable in protozoa, and this defined the frame for the work. Pyrosequencing of environmental DNA from environmental DNA has revolutionised microbiology, as it enables the exploration and description of diversity (also the one that is not cultivable) on a hitherto unprecedented scale. In the bacterial microbiogy the technique is standardized and well established, and this was used in a study of bacterial diversity in sand filters at 11 Danish carefully selected waterworks (Article IV), where the bacterial metabolic diversity and its important for water purification was described.

Building on this, the most important part of the thesis consists of two pyrosequencing analyses of protozoa with newly developed 18S primers. One specifically targets Cercozoa, a particularly abundant phylum of protozoa (Article III), on heath land that had been subjected to prolonged artificially induced drought in a Danish free-air climate-manipulation experiment (CLIMAITE). Article III showed that the testate cercozoan forms responded negatively to prolonged drought, and that on just on this one biotope, an unknown diversity of cercozoans is present, far exceeding the number of described cercozoan species.

The other applied general eukaryotic 18S primers on the aforementioned 11 waterworks (Article V). Their eukaryotic community was dominated by protozoa, and when we compared it to the bacterial dataset from Article IV, we found that unlike the bacterial community composition, the eukaryotic community composition was not primarily driven by ecological conditions, but by geographical distances.

Pyrosequencing analyses are highly dependent on existing DNA libraries that can be used to identify the thousands of DNA sequences, and Article II contributes to this by a morphological and phylogenetic characterization and naming of two new genera of flagellates found in Danish soil. The fact that it is possible to find two unknown and genetically divergent lineages in a few grams of soil from a well-studied country is an illustration of the limited knowledge of the microbial diversity.

Finally, Article I separates a group of closely related fungi that could not be determined by morphology by using a phylogenetic analysis combining three marker genes. Using multiple markers makes it possible to evaluate the explanatory power of individual genes and their mutual consistency. The fact that ITS, the most widely used marker in fungi, did not show the best taxonomic resolution serves to put the heavy reliance upon a single marker (18S) in protozoology into perspective.