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 revolutionisedmicrobiology, 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.