Anna Karnkowska

A Eukaryote without a Mitochondrial Organelle

The presence of mitochondria and related organelles in every studied eukaryote supports the view that mitochondria are essential cellular components. Here, we report the genome sequence of a microbial eukaryote, the oxymonad Monocercomonoides sp., which revealed that this organism lacks all hallmark mitochondrial proteins. Crucially, the mitochondrial iron-sulfur cluster assembly pathway, thought to be conserved in virtually all eukaryotic cells, has been replaced by a cytosolic sulfur mobilization system (SUF) acquired by lateral gene transfer from bacteria. In the context of eukaryotic phylogeny, our data suggest that Monocercomonoides is not primitively amitochondrial but has lost the mitochondrion secondarily. This is the first example of a eukaryote lacking any form of a mitochondrion, demonstrating that this organelle is not absolutely essential for the viability of a eukaryotic cell.

Phylogenetic Relationships and Morphological Character Evolution of Photosynthetic Euglenids (Excavata) Inferred from Taxon-rich Analyses of Five Genes

Photosynthetic euglenids acquired chloroplasts by secondary endosymbiosis, which resulted in changes to their mode of nutrition and affected the evolution of their morphological characters. Mapping morphological characters onto a reliable molecular tree could elucidate major trends of those changes. We analyzed nucleotide sequence data from regions of three nuclear‐encoded genes (nSSU, nLSU, hsp90), one chloroplast‐encoded gene (cpSSU) and one nuclear‐encoded chloroplast gene (psbO) to estimate phylogenetic relationships among 59 photosynthetic euglenid species. Our results were consistent with previous works; most genera were monophyletic, except for the polyphyletic genus Euglena, and the paraphyletic genus Phacus. We also analyzed character evolution in photosynthetic euglenids using our phylogenetic tree and eight morphological traits commonly used for generic and species diagnoses, including: characters corresponding to well‐defined clades, apomorphies like presence of lorica and mucilaginous stalks, and homoplastic characters like rigid cells and presence of large paramylon grains. This research indicated that pyrenoids were lost twice during the evolution of phototrophic euglenids, and that mucocysts, which only occur in the genus Euglena, evolved independently at least twice. In contrast, the evolution of cell shape and chloroplast morphology was difficult to elucidate, and could not be unambiguously reconstructed in our analyses.

Distribution of Conventional and Nonconventional Introns in Tubulin (α and β) Genes of Euglenids

The nuclear genomes of euglenids contain three types of introns: conventional spliceosomal introns, nonconventional introns for which a splicing mechanism is unknown (variable noncanonical borders, RNA secondary structure bringing together intron ends), and so-called intermediate introns, which combine features of conventional and nonconventional introns. Analysis of two genes, tubA and tubB, from 20 species of euglenids reveals contrasting distribution patterns of conventional and nonconventional introns—positions of conventional introns are conserved, whereas those of the nonconventional ones are unique to individual species or small groups of closely related taxa. Moreover, in the group of phototrophic euglenids, 11 events of conventional intron loss versus 15 events of nonconventional intron gain were identified. A comparison of all nonconventional intron sequences highlighted the most conserved elements in their sequence and secondary structure. Our results led us to put forward two hypotheses. 1) The first one posits that mutational changes in intron sequence could lead to a change in their excision mechanism—intermediate introns would then be a transitional form between the conventional and nonconventional introns. 2) The second hypothesis concerns the origin of nonconventional introns—because of the presence of inverted repeats near their ends, insertion of MITE-like transposon elements is proposed as a possible source of new introns.

Intermediate introns in nuclear genes of euglenids – are they a distinct type?

BackgroundNuclear genes of euglenids contain two major types of introns: conventional spliceosomal and nonconventional introns. The latter are characterized by variable non-canonical borders, RNA secondary structure that brings intron ends together, and an unknown mechanism of removal. Some researchers also distinguish intermediate introns, which combine features of both types. They form a stable RNA secondary structure and are classified into two subtypes depending on whether they contain one (intermediate/nonconventional subtype) or both (conventional/intermediate subtype) canonical spliceosomal borders. However, it has been also postulated that most introns classified as intermediate could simply be special cases of conventional or nonconventional introns.ResultsSequences of tubB, hsp90 and gapC genes from six strains of Euglena agilis were obtained. They contain four, six, and two or three introns, respectively (the third intron in the gapC gene is unique for just one strain). Conventional introns were present at three positions: two in the tubB gene (at one position conventional/intermediate introns were also found) and one in the gapC gene. Nonconventional introns are present at ten positions: two in the tubB gene (at one position intermediate/nonconventional introns were also found), six in hsp90 (at four positions intermediate/nonconventional introns were also found), and two in the gapC gene.ConclusionsSequence and RNA secondary structure analyses of nonconventional introns confirmed that their most strongly conserved elements are base pairing nucleotides at positions +4, +5 and +6/ -8, −7 and −6 (in most introns CAG/CTG nucleotides were observed). It was also confirmed that the presence of the 5 GT/C end in intermediate/nonconventional introns is not the result of kinship with conventional introns, but is due to evolutionary pressure to preserve the purine at the 5 end. However, an example of a nonconventional intron with GC-AG ends was shown, suggesting the possibility of intron type conversion between nonconventional and conventional. Furthermore, an analysis of conventional introns revealed that the ability to form a stable RNA secondary structure by some introns is probably not a result of their relationship with nonconventional introns. It was also shown that acquisition of new nonconventional introns is an ongoing process and can be observed at the level of a single species. In the recently acquired intron in the gapC gene an extended direct repeats at the intron-exon junctions are present, suggesting that double-strand break repair process could be the source of new nonconventional introns.

Single cell genomics of uncultured marine alveolates shows paraphyly of basal dinoflagellates

Marine alveolates (MALVs) are diverse and widespread early-branching dinoflagellates, but most knowledge of the group comes from a few cultured species that are generally not abundant in natural samples, or from diversity analyses of PCR-based environmental SSU rRNA gene sequences. To more broadly examine MALV genomes, we generated single cell genome sequences from seven individually isolated cells. Genes expected of heterotrophic eukaryotes were found, with interesting exceptions like presence of proteorhodopsin and vacuolar H+-pyrophosphatase. Phylogenetic analysis of concatenated SSU and LSU rRNA gene sequences provided strong support for the paraphyly of MALV lineages. Dinoflagellate viral nucleoproteins were found only in MALV groups that branched as sister to dinokaryotes. Our findings indicate that multiple independent origins of several characteristics early in dinoflagellate evolution, such as a parasitic life style, underlie the environmental diversity of MALVs, and suggest they have more varied trophic modes than previously thought.

Molecular characterization and phylogeny of four new species of the genus Trichonympha (Parabasalia, Trichonymphea) from lower termite hindguts

Members of the genus Trichonympha are among the most well-known, recognizable and widely distributed parabasalian symbionts of lower termites and the wood-eating cockroach species of the genus Cryptocercus. Nevertheless, the species diversity of this genus is largely unknown. Molecular data have shown that the superficial morphological similarities traditionally used to identify species are inadequate, and have challenged the view that the same species of the genus Trichonympha can occur in many different host species. Ambiguities in the literature, uncertainty in identification of both symbiont and host, and incomplete samplings are limiting our understanding of the systematics, ecology and evolution of this taxon. Here we describe four closely related novel species of the genus Trichonympha collected from South American and Australian lower termites: Trichonympha hueyi sp. nov. from Rugitermes laticollis, Trichonympha deweyi sp. nov. from Glyptotermes brevicornis, Trichonympha louiei sp. nov. from Calcaritermes temnocephalus and Trichonympha webbyae sp. nov. from Rugitermes bicolor. We provide molecular barcodes to identify both the symbionts and their hosts, and infer the phylogeny of the genus Trichonympha based on small subunit rRNA gene sequences. The analysis confirms the considerable divergence of symbionts of members of the genus Cryptocercus, and shows that the two clades of the genus Trichonympha harboured by termites reflect only in part the phylogeny of their hosts.

Phylogeny and Evolution

The concept of a phylogeny of parasites is inextricably linked to that of the phylogeny of eukaryotes. Though it can be useful to infer functional principles from similar morphologies and trophic strategies, the evolutionary histories of parasites are most accurately viewed as independent shifts to this lifestyle from a free-living state. This chapter will describe the phylogeny of eukaryotes, the evolutionary positions of various prominent parasites within this framework, and the ways in which genomics has facilitated understanding of the free-living to parasitic transition, both in terms of phylogeny and function. Two major cellular systems of parasitological relevance, mitochondrion-related organelles and endocytic systems, will be explored, highlighting where considering the genomics and molecular cell biology of parasites in the context of their emergence from free-living relatives have helped us to better understand organelle evolution.

The curious case of vanishing mitochondria

Due to their involvement in the energy metabolism, mitochondria are essential for most eukaryotic cells. Microbial eukaryotes living in low oxygen environments possess reduced forms of mitochondria, namely mitochondrion-related organelles (MROs). These do not produce ATP by oxidative phosphorylation on their membranes and some do not produce ATP at all. Still, they are indispensable because of other essential functions such as iron-sulphur (Fe-S) cluster assembly. Recently, the first microbial eukaryote with neither mitochondrion nor MRO was characterized - Monocercomonoides sp. Genome and transcriptome sequencing of Monocercomonoides revealed that it lacks all hallmark mitochondrial proteins. Crucially, the essential mitochondrial pathway for the Fe-S cluster assembly (ISC) was replaced by a bacterial sulphur mobilization (SUF) system. The discovery of such bona fide amitochondriate eukaryote broadens our knowledge about the diversity and plasticity of eukaryotic cells and provides a substantial contribution to our understanding of eukaryotic cell evolution.

Arginine deiminase pathway enzymes: evolutionary history in metamonads and other eukaryotes

BackgroundMultiple prokaryotic lineages use the arginine deiminase (ADI) pathway for anaerobic energy production by arginine degradation. The distribution of this pathway among eukaryotes has been thought to be very limited, with only two specialized groups living in low oxygen environments (Parabasalia and Diplomonadida) known to possess the complete set of all three enzymes. We have performed an extensive survey of available sequence data in order to map the distribution of these enzymes among eukaryotes and to reconstruct their phylogenies.ResultsWe have found genes for the complete pathway in almost all examined representatives of Metamonada, the anaerobic protist group that includes parabasalids and diplomonads. Phylogenetic analyses indicate the presence of the complete pathway in the last common ancestor of metamonads and heterologous transformation experiments suggest its cytosolic localization in the metamonad ancestor. Outside Metamonada, the complete pathway occurs rarely, nevertheless, it was found in representatives of most major eukaryotic clades.ConclusionsPhylogenetic relationships of complete pathways are consistent with the presence of the Archaea-derived ADI pathway in the last common ancestor of all eukaryotes, although other evolutionary scenarios remain possible. The presence of the incomplete set of enzymes is relatively common among eukaryotes and it may be related to the fact that these enzymes are involved in other cellular processes, such as the ornithine-urea cycle. Single protein phylogenies suggest that the evolutionary history of all three enzymes has been shaped by frequent gene losses and horizontal transfers, which may sometimes be connected with their diverse roles in cellular metabolism.

Dynamic evolution of inverted repeats in Euglenophyta plastid genomes

Photosynthetic euglenids (Euglenophyta) are a monophyletic group of unicellular eukaryotes characterized by the presence of plastids, which arose as the result of the secondary endosymbiosis. Many Euglenophyta plastid (pt) genomes have been characterized recently, but they represented mainly one family – Euglenaceae. Here, we report a comparative analysis of plastid genomes from eight representatives of the family Phacaceae. Newly sequenced plastid genomes share a number of features including synteny and gene content, except for genes mat2 and mat5 encoding maturases. The observed diversity of intron number and presence/absence of maturases corroborated previously suggested correlation between the number of maturases in the pt genome and intron proliferation. Surprisingly, pt genomes of taxa belonging to Discoplastis and Lepocinclis encode two inverted repeat (IR) regions containing the rDNA operon, which are absent from the Euglenaceae. By mapping the presence/absence of IR region on the obtained phylogenomic tree, we reconstructed the most probable events in the evolution of IRs in the Euglenophyta. Our study highlights the dynamic nature of the Euglenophyta plastid genome, in particular with regards to the IR regions that underwent losses repeatedly.