TY - BOOK

T1 - Pyrosequencing and genetic diversity of microeukaryotes

AU - Harder, Christoffer Bugge

PY - 2013

Y1 - 2013

N2 - Free-living, heterotrophic protozoa have an important ecological role in mostterrestrial ecosystems by their grazing of bacteria as one of the first links in foodchains and webs. Furthermore, some of them serve as reservoirs for disease-causingbacteria and /or as occasional opportunistic pathogens themselves. Protozoa is amorphological group which occurs in many different eukaryotic phyla, and manyapparently morphologically similar types are very different from each othersgenetically. This complicates the development of good primers for analysis of theirdiversity with modern DNA based methods. Compared to other microorganisms suchas fungi, algae and bacteria, much less is known about protozoa. It has been anessential element of this thesis to to advance our knowledge of protozoa bydeveloping new primers for DNA-based studies of protozoa impact on ecosystems oras indicators of environmental conditions. The ribosomal 18S region is the onlymarker that is sufficiently well known in a broad range of eukaryotic microorganismsto 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 pyrosequencinganalyses of protozoa with newly developed 18S primers. One specifically targetsCercozoa, a particularly abundant phylum of protozoa (Article III), on heath land thathad been subjected to prolonged artificially induced drought in a Danish free-airclimate-manipulation experiment (CLIMAITE). Article III showed that the testatecercozoan forms responded negatively to prolonged drought, and that on just on thisone biotope, an unknown diversity of cercozoans is present, far exceeding the numberof described cercozoan species.The other applied general eukaryotic 18S primers on the aforementioned 11waterworks (Article V). Their eukaryotic community was dominated by protozoa, andwhen we compared it to the bacterial dataset from Article IV, we found that unlike thebacterial community composition, the eukaryotic community composition was notprimarily driven by ecological conditions, but by geographical distances.Pyrosequencing analyses are highly dependent on existing DNA libraries that can beused to identify the thousands of DNA sequences, and Article II contributes to this bya morphological and phylogenetic characterization and naming of two new genera offlagellates found in Danish soil. The fact that it is possible to find two unknown andgenetically divergent lineages in a few grams of soil from a well-studied country is anillustration of the limited knowledge of the microbial diversity.Finally, Article I separates a group of closely related fungi that could not bedetermined by morphology by using a phylogenetic analysis combining three markergenes. Using multiple markers makes it possible to evaluate the explanatory power ofindividual genes and their mutual consistency. The fact that ITS, the most widely usedmarker in fungi, did not show the best taxonomic resolution serves to put the heavyreliance upon a single marker (18S) in protozoology into perspective.

AB - Free-living, heterotrophic protozoa have an important ecological role in mostterrestrial ecosystems by their grazing of bacteria as one of the first links in foodchains and webs. Furthermore, some of them serve as reservoirs for disease-causingbacteria and /or as occasional opportunistic pathogens themselves. Protozoa is amorphological group which occurs in many different eukaryotic phyla, and manyapparently morphologically similar types are very different from each othersgenetically. This complicates the development of good primers for analysis of theirdiversity with modern DNA based methods. Compared to other microorganisms suchas fungi, algae and bacteria, much less is known about protozoa. It has been anessential element of this thesis to to advance our knowledge of protozoa bydeveloping new primers for DNA-based studies of protozoa impact on ecosystems oras indicators of environmental conditions. The ribosomal 18S region is the onlymarker that is sufficiently well known in a broad range of eukaryotic microorganismsto 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 pyrosequencinganalyses of protozoa with newly developed 18S primers. One specifically targetsCercozoa, a particularly abundant phylum of protozoa (Article III), on heath land thathad been subjected to prolonged artificially induced drought in a Danish free-airclimate-manipulation experiment (CLIMAITE). Article III showed that the testatecercozoan forms responded negatively to prolonged drought, and that on just on thisone biotope, an unknown diversity of cercozoans is present, far exceeding the numberof described cercozoan species.The other applied general eukaryotic 18S primers on the aforementioned 11waterworks (Article V). Their eukaryotic community was dominated by protozoa, andwhen we compared it to the bacterial dataset from Article IV, we found that unlike thebacterial community composition, the eukaryotic community composition was notprimarily driven by ecological conditions, but by geographical distances.Pyrosequencing analyses are highly dependent on existing DNA libraries that can beused to identify the thousands of DNA sequences, and Article II contributes to this bya morphological and phylogenetic characterization and naming of two new genera offlagellates found in Danish soil. The fact that it is possible to find two unknown andgenetically divergent lineages in a few grams of soil from a well-studied country is anillustration of the limited knowledge of the microbial diversity.Finally, Article I separates a group of closely related fungi that could not bedetermined by morphology by using a phylogenetic analysis combining three markergenes. Using multiple markers makes it possible to evaluate the explanatory power ofindividual genes and their mutual consistency. The fact that ITS, the most widely usedmarker in fungi, did not show the best taxonomic resolution serves to put the heavyreliance upon a single marker (18S) in protozoology into perspective.

UR - https://soeg.kb.dk/permalink/45KBDK_KGL/fbp0ps/alma99122550976705763

M3 - Ph.D. thesis

BT - Pyrosequencing and genetic diversity of microeukaryotes

PB - Department of Biology, Faculty of Science, University of Copenhagen

ER -