Resolving the phylogenetic tree of eukaryotes is an ongoing challenge for evolutionary biologists. One of the most intriguing questions is the phylogenetic status of Excavata, a group that is well supported by morphological evidence, yet usually not recovered as a clade in molecular phylogenies. The most problematic group of excavates are diplomonads (e.g., Giardia), which tend to have very highly divergent gene sequences, making any phylogenetic analyses that include these protists very susceptible to long branch attraction artifact. This thesis first explores which organisms are most closely related to diplomonads. Phylogenies of three marker genes demonstrate that enteromonads, formerly considered a possible sister group to diplomonads, are a polyphyletic group within diplomonads, suggesting complex evolution of cell morphology in this lineage. However, a large diversity of Carpediemonas-like organisms (CLOs) was discovered from marine/saline samples. Most of the major clades of CLOs had not been detected by previous environmental PCR studies. SSU rRNA gene phylogenies show that CLOs form a series of relatively short branches at the base of diplomonads. Phylogenomic analysis of eukaryotes (161 genes), incorporating EST data from 5 excavates, including 3 CLOs, shows that the non-monophyly of Excavata in phylogenomic studies is likely caused by long branch attraction artifact, since most of the methods used to suppress long branch attraction significantly weaken support for this topology. Furthermore, the shorter-branching CLOs represent valuable replacements for the long branching diplomonads; we recovered a robustly supported monophyletic Excavata, when long branches, including diplomonads (and parabasalids), were removed from the analysis. Subsequently, comparative analysis of the putative proteomes of three CLO isolates, the retortamonad Chilomastix, diplomonads and parabasalids was performed. Several putative evolutionary steps leading to the extremely reduced mitochondrial organelle of diplomonads were derived through the comparative analysis of predicted organellar proteomes. This thesis shows the importance of taxon sampling for inferring deep eukaryotic evolution. The more robust understanding of the phylogeny of Excavata, especially diplomonads and parabasalids, and the new availability of a number of deep branching relatives of diplomonads, provide a framework for comparative analyses exploring the evolution of anaerobic organelles or parasitism.

Eukaryotic organisms are divided into about 30 groups today, yet there still reamain many groups whose classification in the eukaryotic tree we are not sure about. This study focused on jacobids, a evolutionary important protist group from the Excavata taxa. Together with Heterolobosea, Euglenozoa and Tsukubamonadida, jacobids constitue a Discoba subgroup of Excavata taxa. Jacobids are significant mainly for their mitochondrial genome, which structurally resembles the original α-proteobacterial genome of the free-living ancestor of mitochondria. Some jacobids have their mitochondrial genome simplified, making them potential candidates for a model group used to study the mitochondrial evolution. However, too few members of jacobid group have been identified so far and the exact phylogeny of the group must be thoroughly understood for the detailed study of mitochondria evolution. This study focuses on broadening the taxonomic coverage of the jacobid excavate protists Andalucia. Twenty new strains of Andalucia have been established into stable culture, their morphology was assessed and SSU rDNA sequenced. Based on the phylogenetic analysis, Andalucia incarcarata species divides into three morphologically almost indistinguishable lines representing three species. Further, this study characterized a...

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Trichomonads (Parabasalia) are anaerobic microeukaryotes classified in the supergroup Excavata. Inclusion of parabasalids within Excavata is exclusively based on the molecular- phylogenetic evidence. Over 400 species of parabasalids have been described so far, and the vast majority of them are endobiotic. In contrast, only few species of free-living parabasalids forming four independent lineages have been described (Pseudotrichomonas keilini, Ditrichomonas honigbergii, Monotrichomonas carabina, Honigbergiella sp., Tetratrichomonas undula, and Lacusteria cypriaca). Lacusteria cypriaca is a new species and genus described in our recent paper. In this paper we published the first two sequences of SSU rDNA from Pseudotrichomonas keilini as well. All of these lineages are likely secondarily free-living, and they developed from endobiotic ancestors. In addition to the already published Lacusteria cypriaca and Pseudotrichomonas keilini strains, we have recently obtained seven another isolates of free-living trichomonads (LAGOS2D, E2NT, CK, LAGOS2M, GR8, GOU23 LIVADIAN, and VAV1A1); from all of these isolates we sequenced SSU rDNA and performed phylogenetic analyses. These isolates split into four independent evolutionary lineages, which indicate that free-living parabasalids are more diversed and...