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Brian S Leander - One of the best experts on this subject based on the ideXlab platform.

  • The evolution of paralogous enzymes MAT and MATX within the Euglenida and beyond
    BMC Evolutionary Biology, 2014
    Co-Authors: Jana Szabova, Naoji Yubuki, Brian S Leander, Richard E Triemer, Vladimir Hampl
    Abstract:

    Background Methionine adenosyltransferase (MAT) is a ubiquitous essential enzyme that, in eukaryotes, occurs in two relatively divergent paralogues: MAT and MATX. MATX has a punctate distribution across the tree of eukaryotes and, except for a few cases, is mutually exclusive with MAT. This phylogenetic pattern could have arisen by either differential loss of old paralogues or the spread of one of these paralogues by horizontal gene transfer. Our aim was to map the distribution of MAT/MATX genes within the Euglenida in order to more comprehensively characterize the evolutionary history of MATX. Results We generated 26 new sequences from 23 different lineages of euglenids and one prasinophyte alga Pyramimonas parkeae . MATX was present only in photoautotrophic euglenids. The mixotroph Rapaza viridis and the prasinophyte alga Pyramimonas parkeae , which harbors chloroplasts that are most closely related to the chloroplasts in photoautotrophic euglenids, both possessed only the MAT paralogue. We found both the MAT and MATX paralogues in two photoautotrophic species ( Phacus orbicularis and Monomorphina pyrum ). The significant conflict between eukaryotic phylogenies inferred from MATX and SSU rDNA data represents strong evidence that MATX paralogues have undergone horizontal gene transfer across the tree of eukaryotes. Conclusions Our results suggest that MATX entered the euglenid lineage in a single horizontal gene transfer event that took place after the secondary endosymbiotic origin of the euglenid chloroplast. The origin of the MATX paralogue is unclear, and it cannot be excluded that it arose by a gene duplication event before the most recent common ancestor of eukaryotes.

  • Ultrastructure and molecular phylogenetic position of Heteronema scaphurum: a eukaryovorous euglenid with a cytoproct.
    Journal of Eukaryotic Microbiology, 2013
    Co-Authors: Susana A. Breglia, Naoji Yubuki, Brian S Leander
    Abstract:

    Euglenids comprise a distinct clade of flagellates with diverse modes of nutrition, including phagotrophy, osmotrophy and phototrophy. Much of the previous research on euglenids has focused on phototrophic species because of their ecological abundance and significance as indicators for the health of aquatic ecosystems. Although largely understudied, phagotrophic species probably represent the majority of euglenid diversity. Phagotrophic euglenids tend to be either bacterivorous or eukaryovorous and use an elaborate feeding apparatus for capturing prey cells. We characterized the ultrastructure and molecular phylogenetic position of Heteronema scaphurum, a eukaryovorous euglenid collected in freshwater. This species was equipped with a distinct cytoproct through which waste products were eliminated in the form of faecal pellets; a cytoproct has not been reported in any other member of the Euglenida. Heteronema scaphurum also had a novel predatory mode of feeding. The euglenid ensnared and corralled several green algal prey cells (i.e. Chlamydomonas) with hook-like flagella covered in mucous before engulfing the bundle of prey cells whole. Molecular phylogenetic analyses inferred from small subunit rDNA sequences placed this species with other eukaryovorous euglenids, which was consistent with ultrastructural features associated with the feeding apparatus, flagellar apparatus, extrusomes, and pellicle.

  • Reconciling the bizarre inheritance of microtubules in complex (euglenid) microeukaryotes
    Protoplasma, 2012
    Co-Authors: Naoji Yubuki, Brian S Leander
    Abstract:

    We introduce a hypothetical model that explains how surface microtubules in euglenids are generated, integrated and inherited with the flagellar apparatus from generation to generation. The Euglenida is a very diverse group of single-celled eukaryotes unified by a complex cell surface called the “pellicle”, consisting of proteinaceous strips that run along the longitudinal axis of the cell and articulate with one another along their lateral margins. The strips are positioned beneath the plasma membrane and are reinforced with subtending microtubules. Euglenids reproduce asexually, and the two daughter cells inherit pellicle strips and associate microtubules from the parent cell in a semi-conservative pattern. In preparation for cell division, nascent pellicle strips develop from the anterior end of the cell and elongate toward the posterior end between two parent (mature) strips, so that the total number of pellicle strips and underlying microtubules is doubled in the predivisional cell. Each daughter cell inherits an alternating pattern of strips consisting of half of the nascent strips and half of the parent (mature) strips. This observation combined with the fact that the microtubules underlying the strips are linked to the flagellar apparatus created a cytoskeletal riddle: how do microtubules associated with an alternating pattern of nascent strips and mature strips maintain their physical relationship to the flagellar apparatus when the parent cell divides? The model of microtubular inheritance articulated here incorporates known patterns of cytoskeletal semi-conservatism and two new inferences: (1) a multigenerational “pellicle microtubule organizing center” (pMTOC) extends from the dorsal root of the flagellar apparatus, encircles the flagellar pocket, and underpins the microtubules of the pellicle; and (2) prior to cytokinesis, nascent pellicle microtubules fall within one of two “left/right” constellations that are linked to one of the two new dorsal basal bodies.

  • Morphostasis in a novel eukaryote illuminates the evolutionary transition from phagotrophy to phototrophy: description of Rapaza viridis n. gen. et sp. (Euglenozoa, Euglenida)
    BMC Evolutionary Biology, 2012
    Co-Authors: Aika Yamaguchi, Naoji Yubuki, Brian S Leander
    Abstract:

    Background Morphostasis of traits in different species is necessary for reconstructing the evolutionary history of complex characters. Studies that place these species into a molecular phylogenetic context test hypotheses about the transitional stages that link divergent character states. For instance, the transition from a phagotrophic mode of nutrition to a phototrophic lifestyle has occurred several times independently across the tree of eukaryotes; one of these events took place within the Euglenida, a large group of flagellates with diverse modes of nutrition. Phototrophic euglenids form a clade that is nested within lineages of phagotrophic euglenids and that originated through a secondary endosymbiosis with green algae. Although it is clear that phototrophic euglenids evolved from phagotrophic ancestors, the morphological disparity between species representing these different nutritional modes remains substantial. Results We cultivated a novel marine euglenid, Rapaza viridis n. gen. et sp. ("green grasper"), and a green alga, Tetraselmis sp., from the same environment. Cells of R. viridis were comprehensively characterized with light microscopy, SEM, TEM, and molecular phylogenetic analysis of small subunit rDNA sequences. Ultrastructural and behavioral observations demonstrated that this isolate habitually consumes a specific strain of Tetraselmis prey cells and possesses a functional chloroplast that is homologous with other phototrophic euglenids. A novel feeding apparatus consisting of a reduced rod of microtubules facilitated this first and only example of mixotrophy among euglenids. R. viridis also possessed a robust photoreception apparatus, two flagella of unequal length, euglenoid movement, and a pellicle consisting of 16 strips and one (square-shaped) whorl of posterior strip reduction. The molecular phylogenetic data demonstrated that R. viridis branches as the nearest sister lineage to phototrophic euglenids. Conclusions The unusual combination of features in R. viridis combined with its molecular phylogenetic position completely conforms to the expected transitional stage that occurred during the early evolution of phototrophic euglenids from phagotrophic ancestors. The marine mixotrophic mode of nutrition, the preference for green algal prey cells, the structure of the feeding apparatus, and the organization of the pellicle are outstanding examples of morphostasis that clarify pivotal stages in the evolutionary history of this diverse group of microbial eukaryotes.

  • morphostasis in a novel eukaryote illuminates the evolutionary transition from phagotrophy to phototrophy description of rapaza viridis n gen et sp euglenozoa Euglenida
    BMC Evolutionary Biology, 2012
    Co-Authors: Aika Yamaguchi, Naoji Yubuki, Brian S Leander
    Abstract:

    Background Morphostasis of traits in different species is necessary for reconstructing the evolutionary history of complex characters. Studies that place these species into a molecular phylogenetic context test hypotheses about the transitional stages that link divergent character states. For instance, the transition from a phagotrophic mode of nutrition to a phototrophic lifestyle has occurred several times independently across the tree of eukaryotes; one of these events took place within the Euglenida, a large group of flagellates with diverse modes of nutrition. Phototrophic euglenids form a clade that is nested within lineages of phagotrophic euglenids and that originated through a secondary endosymbiosis with green algae. Although it is clear that phototrophic euglenids evolved from phagotrophic ancestors, the morphological disparity between species representing these different nutritional modes remains substantial.

Angelika Preisfeld - One of the best experts on this subject based on the ideXlab platform.

  • Phylogenetic position and inter-relationships of the osmotrophic euglenids based on SSU rDNA data, with emphasis on the Rhabdomonadales (Euglenozoa).
    International journal of systematic and evolutionary microbiology, 2020
    Co-Authors: Angelika Preisfeld, Ingo Busse, M Klingberg, S Talke, H G Ruppel
    Abstract:

    In order to reconstruct the evolution of euglenid flagellates, euglenozoan SSU rDNA data have been used to investigate phylogenetic relationships with a focus on osmotrophic taxa and especially on the Rhabdomonadales. The dataset consisting of the SSU rDNAs of osmotrophic, phagotrophic and phototrophic taxa was used in parsimony, maximum-likelihood and distance analyses. Five genera make up the Rhabdomonadales, all of them osmotrophic: Gyropaigne, Menoidium, Parmidium, Rhabdomonas and Rhabdospira. According to our analyses they form a strongly supported monophyletic assemblage which is characterized by a low sequence divergence compared to the euglenids in general. Closest relatives are the members of the osmotrophic genus Distigma. All primary osmotrophic species constitute a larger monophyletic group with the phototrophic euglenids and the phagotroph Peranema trichophorum. The combination of three rhabdomonadalian species Rhabdomonas gibba, Rhabdomonas spiralis and Rhabdospira spiralis with nearly identical SSU rDNA sequences is strongly recommended. The phagotroph Petalomonas cantuscygni branches at the bottom of the euglenid subtree with significantly weaker support. The inter-relationship of the three distinct euglenozoan taxa (euglenids, kinetoplastids and diplonemids) could not be convincingly resolved by this study.

  • Application of spectral analysis to examine phylogenetic signal among euglenid SSU rDNA data sets (Euglenozoa)
    Organisms Diversity & Evolution, 2020
    Co-Authors: Ingo Busse, Angelika Preisfeld
    Abstract:

    Abstract Euglenid flagellates as a common and widespread group of protists display a broad morphological variety. Against the background of pronounced genetic diversity and varying sequence characteristics of SSU rDNA sequences among different euglenid subgroups we analyzed the content and distribution of phylogenetic signal and noise within different euglenozoan data sets. Two statistical approaches, PTP-test and RASA, were employed to achieve a measure of overall signal content. Spectral analyses were used to evaluate support and conflict for given bipartitions of the data sets. These investigations revealed a large amount of phylogenetic information present in the molecular data. Convincing support could be found for primary osmotrophic euglenids and corresponding subgroups, a taxon mainly based on molecular data. On the other hand, in agreement with weak corroboration from morphological data, euglenid monophyly and interrelationships of phagotrophs, phototrophs and osmotrophs were not supported. Focusing on the primary osmotrophic subclade Rhabdomonadina spectral analysis revealed only few well supported splits. Generally, the application of sequence evolution models in maximum likelihood and spectral analyses of euglenid SSU rDNA data sets did not lead to significant amplification of split supporting signal. Phylogenetic hypotheses are discussed in regard to the evolution of morphological and ultrastructural characters.

  • Ultrastructural and immunocytochemical investigation of paramylon combined with new 18S rDNA-based secondary structure analysis clarifies phylogenetic affiliation of Entosiphon sulcatum (Euglenida: Euglenozoa)
    Organisms Diversity & Evolution, 2017
    Co-Authors: Stefan Paerschke, Almut H. Vollmer, Angelika Preisfeld
    Abstract:

    The phylogeny of phagotrophic euglenids is widely based on nuclear small subunit ribosomal DNA (18S rDNA) sequence data, but most analyses suffer from weakness in statistical support regarding the “connecting backbone” between monophyletic clades. Moreover, the position of Entosiphon has remained unclear. Testing the 18S rDNA capability for a phylogeny of phagotrophic euglenids, we isolated sequences of Peranema sp. and Ploeotia edaphica and utilized secondary structure data as a prerequisite for recognition of homologous positions. We found a unique, clade-specific nucleotide substitution in the deduced 18S rDNA helix 44. Since our 18S rDNA phylogenies could only in part resolve positions of phagotrophic lineages, but did not verify that of Entosiphon , we investigated the phagotrophic key taxa Peranema trichophorum , Petalomonas cantuscygni , Ploeotia costata , and Entosiphon sulcatum ultrastructurally. Additionally, we explored the presence or absence of the euglenid reserve carbohydrate paramylon by specific staining with monoclonal anti-β-1,3-glucan antibodies. Paramylon was found to be clearly present in P. trichophorum and E. sulcatum , but was absent in Pt. cantuscygni and Pl. costata . Combined results of our molecular, ultrastructural, and immunocytochemical investigations suggest that Entosiphon sulcatum is the sister taxon of a monophyletic euglenid crown clade, characterized by a helical pellicle, which we propose to rename. This phylogenetic affiliation is confirmed by a clade-specific primary absence of the unique nucleotide substitution in helix 44 and by the common presence of paramylon.

  • the chloroplast genome of euglena mutabilis cluster arrangement intron analysis and intrageneric trends
    Journal of Eukaryotic Microbiology, 2017
    Co-Authors: Nadja Dabbagh, Angelika Preisfeld
    Abstract:

    A comparative analysis of the chloroplast genome of Euglena mutabilis underlined a high diversity in the evolution of plastids in euglenids. Gene clusters in more derived Euglenales increased in complexity with only a few, but remarkable changes in the genus Euglena. Euglena mutabilis differed from other Euglena species in a mirror-inverted arrangement of 12 from 15 identified clusters, making it very likely that the emergence at the base of the genus Euglena, which has been considered a long branch artifact, is truly a probable position. This was corroborated by many similarities in gene arrangement and orientation with Strombomonas and Monomorphina, rendering the genome organization of E. mutabilis in certain clusters as plesiomorphic feature. By RNA analysis exact exon-intron boundaries and the type of the 77 introns identified were mostly determined unambiguously. A detailed intron study of psbC pointed at two important issues: First, the number of introns varied even between species, and no trend from few to many introns could be observed. Second, mat1 was localized in Eutreptiales exclusively in intron 1, and mat2 was not identified. With the emergence of Euglenaceae in most species, a new intron containing mat2 inserted in front of the previous intron 1 and thereby became intron 2 with mat1.

  • PHYLOGENY OF PHAGOTROPHIC EUGLENIDS (EUGLENOZOA): A MOLECULAR APPROACH BASED ON CULTURE MATERIAL AND ENVIRONMENTAL SAMPLES1
    Journal of Phycology, 2003
    Co-Authors: Ingo Busse, David J. Patterson, Angelika Preisfeld
    Abstract:

    Molecular studies based on small subunit (SSU) rDNA sequences addressing euglenid phylogeny hitherto suffered from the lack of available data about phagotrophic species. To extend the taxon sampling, SSU rRNA genes from species of seven genera of phagotrophic euglenids were investigated. Sequence analyses revealed an increasing genetic diversity among euglenid SSU rDNA sequences compared with other well-known eukaryotic groups, reflecting an equally broad diversity of morphological characters among euglenid phagotrophs. Phylogenetic inference using standard parsimony and likelihood approaches as well as Bayesian inference and spectral analyses revealed no clear support for euglenid monophyly. Among phagotrophs, monophyly of Petalomonas cantuscygni and Notosolenus ostium, both comprising simple ingestion apparatuses, is strongly supported. A moderately supported clade comprises phototrophic euglenids and primary osmotrophic euglenids together with phagotrophs, exhibiting a primarily flexible pellicle composed of numerous helically arranged strips and a complex ingestion apparatus with two supporting rods and four curved vanes. Comparison of molecular and morphological data is used to demonstrate the difficulties to formulate a hypothesis about how the ingestion apparatus evolved in this group.

Naoji Yubuki - One of the best experts on this subject based on the ideXlab platform.

  • The evolution of paralogous enzymes MAT and MATX within the Euglenida and beyond
    BMC Evolutionary Biology, 2014
    Co-Authors: Jana Szabova, Naoji Yubuki, Brian S Leander, Richard E Triemer, Vladimir Hampl
    Abstract:

    Background Methionine adenosyltransferase (MAT) is a ubiquitous essential enzyme that, in eukaryotes, occurs in two relatively divergent paralogues: MAT and MATX. MATX has a punctate distribution across the tree of eukaryotes and, except for a few cases, is mutually exclusive with MAT. This phylogenetic pattern could have arisen by either differential loss of old paralogues or the spread of one of these paralogues by horizontal gene transfer. Our aim was to map the distribution of MAT/MATX genes within the Euglenida in order to more comprehensively characterize the evolutionary history of MATX. Results We generated 26 new sequences from 23 different lineages of euglenids and one prasinophyte alga Pyramimonas parkeae . MATX was present only in photoautotrophic euglenids. The mixotroph Rapaza viridis and the prasinophyte alga Pyramimonas parkeae , which harbors chloroplasts that are most closely related to the chloroplasts in photoautotrophic euglenids, both possessed only the MAT paralogue. We found both the MAT and MATX paralogues in two photoautotrophic species ( Phacus orbicularis and Monomorphina pyrum ). The significant conflict between eukaryotic phylogenies inferred from MATX and SSU rDNA data represents strong evidence that MATX paralogues have undergone horizontal gene transfer across the tree of eukaryotes. Conclusions Our results suggest that MATX entered the euglenid lineage in a single horizontal gene transfer event that took place after the secondary endosymbiotic origin of the euglenid chloroplast. The origin of the MATX paralogue is unclear, and it cannot be excluded that it arose by a gene duplication event before the most recent common ancestor of eukaryotes.

  • Ultrastructure and molecular phylogenetic position of Heteronema scaphurum: a eukaryovorous euglenid with a cytoproct.
    Journal of Eukaryotic Microbiology, 2013
    Co-Authors: Susana A. Breglia, Naoji Yubuki, Brian S Leander
    Abstract:

    Euglenids comprise a distinct clade of flagellates with diverse modes of nutrition, including phagotrophy, osmotrophy and phototrophy. Much of the previous research on euglenids has focused on phototrophic species because of their ecological abundance and significance as indicators for the health of aquatic ecosystems. Although largely understudied, phagotrophic species probably represent the majority of euglenid diversity. Phagotrophic euglenids tend to be either bacterivorous or eukaryovorous and use an elaborate feeding apparatus for capturing prey cells. We characterized the ultrastructure and molecular phylogenetic position of Heteronema scaphurum, a eukaryovorous euglenid collected in freshwater. This species was equipped with a distinct cytoproct through which waste products were eliminated in the form of faecal pellets; a cytoproct has not been reported in any other member of the Euglenida. Heteronema scaphurum also had a novel predatory mode of feeding. The euglenid ensnared and corralled several green algal prey cells (i.e. Chlamydomonas) with hook-like flagella covered in mucous before engulfing the bundle of prey cells whole. Molecular phylogenetic analyses inferred from small subunit rDNA sequences placed this species with other eukaryovorous euglenids, which was consistent with ultrastructural features associated with the feeding apparatus, flagellar apparatus, extrusomes, and pellicle.

  • Reconciling the bizarre inheritance of microtubules in complex (euglenid) microeukaryotes
    Protoplasma, 2012
    Co-Authors: Naoji Yubuki, Brian S Leander
    Abstract:

    We introduce a hypothetical model that explains how surface microtubules in euglenids are generated, integrated and inherited with the flagellar apparatus from generation to generation. The Euglenida is a very diverse group of single-celled eukaryotes unified by a complex cell surface called the “pellicle”, consisting of proteinaceous strips that run along the longitudinal axis of the cell and articulate with one another along their lateral margins. The strips are positioned beneath the plasma membrane and are reinforced with subtending microtubules. Euglenids reproduce asexually, and the two daughter cells inherit pellicle strips and associate microtubules from the parent cell in a semi-conservative pattern. In preparation for cell division, nascent pellicle strips develop from the anterior end of the cell and elongate toward the posterior end between two parent (mature) strips, so that the total number of pellicle strips and underlying microtubules is doubled in the predivisional cell. Each daughter cell inherits an alternating pattern of strips consisting of half of the nascent strips and half of the parent (mature) strips. This observation combined with the fact that the microtubules underlying the strips are linked to the flagellar apparatus created a cytoskeletal riddle: how do microtubules associated with an alternating pattern of nascent strips and mature strips maintain their physical relationship to the flagellar apparatus when the parent cell divides? The model of microtubular inheritance articulated here incorporates known patterns of cytoskeletal semi-conservatism and two new inferences: (1) a multigenerational “pellicle microtubule organizing center” (pMTOC) extends from the dorsal root of the flagellar apparatus, encircles the flagellar pocket, and underpins the microtubules of the pellicle; and (2) prior to cytokinesis, nascent pellicle microtubules fall within one of two “left/right” constellations that are linked to one of the two new dorsal basal bodies.

  • Morphostasis in a novel eukaryote illuminates the evolutionary transition from phagotrophy to phototrophy: description of Rapaza viridis n. gen. et sp. (Euglenozoa, Euglenida)
    BMC Evolutionary Biology, 2012
    Co-Authors: Aika Yamaguchi, Naoji Yubuki, Brian S Leander
    Abstract:

    Background Morphostasis of traits in different species is necessary for reconstructing the evolutionary history of complex characters. Studies that place these species into a molecular phylogenetic context test hypotheses about the transitional stages that link divergent character states. For instance, the transition from a phagotrophic mode of nutrition to a phototrophic lifestyle has occurred several times independently across the tree of eukaryotes; one of these events took place within the Euglenida, a large group of flagellates with diverse modes of nutrition. Phototrophic euglenids form a clade that is nested within lineages of phagotrophic euglenids and that originated through a secondary endosymbiosis with green algae. Although it is clear that phototrophic euglenids evolved from phagotrophic ancestors, the morphological disparity between species representing these different nutritional modes remains substantial. Results We cultivated a novel marine euglenid, Rapaza viridis n. gen. et sp. ("green grasper"), and a green alga, Tetraselmis sp., from the same environment. Cells of R. viridis were comprehensively characterized with light microscopy, SEM, TEM, and molecular phylogenetic analysis of small subunit rDNA sequences. Ultrastructural and behavioral observations demonstrated that this isolate habitually consumes a specific strain of Tetraselmis prey cells and possesses a functional chloroplast that is homologous with other phototrophic euglenids. A novel feeding apparatus consisting of a reduced rod of microtubules facilitated this first and only example of mixotrophy among euglenids. R. viridis also possessed a robust photoreception apparatus, two flagella of unequal length, euglenoid movement, and a pellicle consisting of 16 strips and one (square-shaped) whorl of posterior strip reduction. The molecular phylogenetic data demonstrated that R. viridis branches as the nearest sister lineage to phototrophic euglenids. Conclusions The unusual combination of features in R. viridis combined with its molecular phylogenetic position completely conforms to the expected transitional stage that occurred during the early evolution of phototrophic euglenids from phagotrophic ancestors. The marine mixotrophic mode of nutrition, the preference for green algal prey cells, the structure of the feeding apparatus, and the organization of the pellicle are outstanding examples of morphostasis that clarify pivotal stages in the evolutionary history of this diverse group of microbial eukaryotes.

  • morphostasis in a novel eukaryote illuminates the evolutionary transition from phagotrophy to phototrophy description of rapaza viridis n gen et sp euglenozoa Euglenida
    BMC Evolutionary Biology, 2012
    Co-Authors: Aika Yamaguchi, Naoji Yubuki, Brian S Leander
    Abstract:

    Background Morphostasis of traits in different species is necessary for reconstructing the evolutionary history of complex characters. Studies that place these species into a molecular phylogenetic context test hypotheses about the transitional stages that link divergent character states. For instance, the transition from a phagotrophic mode of nutrition to a phototrophic lifestyle has occurred several times independently across the tree of eukaryotes; one of these events took place within the Euglenida, a large group of flagellates with diverse modes of nutrition. Phototrophic euglenids form a clade that is nested within lineages of phagotrophic euglenids and that originated through a secondary endosymbiosis with green algae. Although it is clear that phototrophic euglenids evolved from phagotrophic ancestors, the morphological disparity between species representing these different nutritional modes remains substantial.

Julius Lukes - One of the best experts on this subject based on the ideXlab platform.

  • A uniquely complex mitochondrial proteome from Euglena gracilis.
    Molecular Biology and Evolution, 2020
    Co-Authors: Michael J Hammond, Eva Dobáková, Martin Zoltner, Anna Nenarokova, Anzhelika Butenko, Mark C. Field, Julius Lukes
    Abstract:

    : Euglena gracilis is a metabolically flexible, photosynthetic and adaptable free-living protist of considerable environmental importance and biotechnological value. By label-free LC-MSMS, a total of 1,786 proteins were identified from the E. gracilis purified mitochondria, representing one of the largest mitochondrial proteomes so far described. Despite this apparent complexity, protein machinery responsible for the extensive RNA editing, splicing and processing in the sister clades diplonemids and kinetoplastids, is absent. This strongly suggests that the complex mechanisms of mitochondrial gene expression in diplonemids and kinetoplastids occurred late in euglenozoan evolution, arising independently. By contrast, the alternative oxidase pathway and numerous ribosomal subunits presumed to be specific for parasitic trypanosomes are present in E. gracilis. We investigated the evolution of unexplored protein families, including import complexes, cristae formation proteins and translation termination factors, as well as canonical and unique metabolic pathways. We additionally compare this mitoproteome with the transcriptome of Eutreptiella gymnastica, illuminating conserved features of Euglenida mitochondria as well as those exclusive to E. gracilis. This is the first mitochondrial proteome of a free-living protist from the Excavata, and one of few available for protists as a whole. This study alters our views of the evolution of the mitochondrion, and indicates early emergence of complexity within euglenozoan mitochondria, independent of parasitism.

  • Evolution of metabolic capabilities and molecular features of diplonemids, kinetoplastids, and euglenids
    BMC Biology, 2020
    Co-Authors: Anzhelika Butenko, Fred R. Opperdoes, Vladimir Hampl, Pavel Flegontov, Olga Flegontova, Aleš Horák, Patrick Keeling, Ryan M. R. Gawryluk, Denis Tikhonenkov, Julius Lukes
    Abstract:

    Background The Euglenozoa are a protist group with an especially rich history of evolutionary diversity. They include diplonemids, representing arguably the most species-rich clade of marine planktonic eukaryotes; trypanosomatids, which are notorious parasites of medical and veterinary importance; and free-living euglenids. These different lifestyles, and particularly the transition from free-living to parasitic, likely require different metabolic capabilities. We carried out a comparative genomic analysis across euglenozoan diversity to see how changing repertoires of enzymes and structural features correspond to major changes in lifestyles. Results We find a gradual loss of genes encoding enzymes in the evolution of kinetoplastids, rather than a sudden decrease in metabolic capabilities corresponding to the origin of parasitism, while diplonemids and euglenids maintain more metabolic versatility. Distinctive characteristics of molecular machines such as kinetochores and the pre-replication complex that were previously considered specific to parasitic kinetoplastids were also identified in their free-living relatives. Therefore, we argue that they represent an ancestral rather than a derived state, as thought until the present. We also found evidence of ancient redundancy in systems such as NADPH-dependent thiol-redox. Only the genus Euglena possesses the combination of trypanothione-, glutathione-, and thioredoxin-based systems supposedly present in the euglenozoan common ancestor, while other representatives of the phylum have lost one or two of these systems. Lastly, we identified convergent losses of specific metabolic capabilities between free-living kinetoplastids and ciliates. Although this observation requires further examination, it suggests that certain eukaryotic lineages are predisposed to such convergent losses of key enzymes or whole pathways. Conclusions The loss of metabolic capabilities might not be associated with the switch to parasitic lifestyle in kinetoplastids, and the presence of a highly divergent (or unconventional) kinetochore machinery might not be restricted to this protist group. The data derived from the transcriptomes of free-living early branching prokinetoplastids suggests that the pre-replication complex of Trypanosomatidae is a highly divergent version of the conventional machinery. Our findings shed light on trends in the evolution of metabolism in protists in general and open multiple avenues for future research.

  • Evolution of metabolic capabilities and molecular features of diplonemids, kinetoplastids, and euglenids.
    BMC Biology, 2020
    Co-Authors: Anzhelika Butenko, Fred R. Opperdoes, Patrick J. Keeling, Vladimir Hampl, Pavel Flegontov, Olga Flegontova, Aleš Horák, Ryan M. R. Gawryluk, Denis Tikhonenkov, Julius Lukes
    Abstract:

    BACKGROUND: The Euglenozoa are a protist group with an especially rich history of evolutionary diversity. They include diplonemids, representing arguably the most species-rich clade of marine planktonic eukaryotes; trypanosomatids, which are notorious parasites of medical and veterinary importance; and free-living euglenids. These different lifestyles, and particularly the transition from free-living to parasitic, likely require different metabolic capabilities. We carried out a comparative genomic analysis across euglenozoan diversity to see how changing repertoires of enzymes and structural features correspond to major changes in lifestyles. RESULTS: We find a gradual loss of genes encoding enzymes in the evolution of kinetoplastids, rather than a sudden decrease in metabolic capabilities corresponding to the origin of parasitism, while diplonemids and euglenids maintain more metabolic versatility. Distinctive characteristics of molecular machines such as kinetochores and the pre-replication complex that were previously considered specific to parasitic kinetoplastids were also identified in their free-living relatives. Therefore, we argue that they represent an ancestral rather than a derived state, as thought until the present. We also found evidence of ancient redundancy in systems such as NADPH-dependent thiol-redox. Only the genus Euglena possesses the combination of trypanothione-, glutathione-, and thioredoxin-based systems supposedly present in the euglenozoan common ancestor, while other representatives of the phylum have lost one or two of these systems. Lastly, we identified convergent losses of specific metabolic capabilities between free-living kinetoplastids and ciliates. Although this observation requires further examination, it suggests that certain eukaryotic lineages are predisposed to such convergent losses of key enzymes or whole pathways. CONCLUSIONS: The loss of metabolic capabilities might not be associated with the switch to parasitic lifestyle in kinetoplastids, and the presence of a highly divergent (or unconventional) kinetochore machinery might not be restricted to this protist group. The data derived from the transcriptomes of free-living early branching prokinetoplastids suggests that the pre-replication complex of Trypanosomatidae is a highly divergent version of the conventional machinery. Our findings shed light on trends in the evolution of metabolism in protists in general and open multiple avenues for future research.

  • From simple to supercomplex: mitochondrial genomes of euglenozoan protists.
    F1000Research, 2016
    Co-Authors: Drahomíra Faktorová, Eva Dobáková, Priscila Peña-diaz, Julius Lukes
    Abstract:

    Mitochondria are double membrane organelles of endosymbiotic origin, best known for constituting the centre of energetics of a eukaryotic cell. They contain their own mitochondrial genome, which as a consequence of gradual reduction during evolution typically contains less than two dozens of genes. In this review, we highlight the extremely diverse architecture of mitochondrial genomes and mechanisms of gene expression between the three sister groups constituting the phylum Euglenozoa - Euglenida, Diplonemea and Kinetoplastea. The earliest diverging euglenids possess a simplified mitochondrial genome and a conventional gene expression, whereas both are highly complex in the two other groups. The expression of their mitochondrial-encoded proteins requires extensive post-transcriptional modifications guided by complex protein machineries and multiple small RNA molecules. Moreover, the least studied diplonemids, which have been recently discovered as a highly abundant component of the world ocean plankton, possess one of the most complicated mitochondrial genome organisations known to date.

  • Mitochondrial Genomes of Photosynthetic Euglenids and Alveolates
    Advances in Botanical Research, 2012
    Co-Authors: Pavel Flegontov, Julius Lukes
    Abstract:

    Euglenida belong to the eukaryotic supergroup Excavata, the members of which possess the most varied mitochondrial genomes in terms of their structure and gene content. Heterotrophic protists represent the majority of Excavata, as only the Euglenida contain a green plastid, apparently acquired by secondary endosymbiosis. The sister group of Euglenida, the mostly parasitic Kinetoplastida, have an extremely complex mitochondrial DNA (kinetoplast DNA), which is usually composed of thousands of mutually interlocked DNA circles. Most mRNAs encoded by this genome are rendered translatable only after they undergo intricate editing via insertions and/or deletions of uridines. The mitochondrial DNA of the other sister group, Diplonemida, is unique as its transcripts must be massively trans-spliced before translation. None of these complex mechanisms has so far been found in the mitochondrial genome and transcriptome of Euglena gracilis, the best studied member of Euglenida. Its mitochondrial DNA exists in the form of numerous differently sized linear fragments. Their significant fraction is non-coding and full of various repeats, which intersperse fragments of a handful of protein-coding genes. Mostly photosynthetic dinoflagellates and parasitic apicomplexans with a relic plastid constitute a large and diverse group within alveolates. All species of this group share the most reduced mitochondrial genome found, containing just three, and in some cases probably two, protein-coding genes along with highly fragmented rRNA genes, and no tRNA genes. Mitochondrial genomes of dinoflagellates and those of smaller groups within the apicomplexa–dinoflagellata assemblage, perkinsids and chromerids, in all cases have a recombining, highly scrambled sequence, and frequently demonstrate other non-canonical features in structure and expression: fused genes, extensive RNA editing, trans-splicing, 5′ oligoU caps, loss of start and stop codons, extensive translational frameshifting. Some of these oddities apparently appeared in several groups independently, probably due to relaxed selective constraints in tiny organellar genomes.

Bożena Zakryś - One of the best experts on this subject based on the ideXlab platform.

  • Taxon-rich phylogeny and taxonomy of the genus Phacus (Euglenida) based on morphological and molecular data.
    Journal of Phycology, 2020
    Co-Authors: Maja Łukomska-kowalczyk, Rafał Milanowski, Katarzyna Chaber, Alicja Fells, Bożena Zakryś
    Abstract:

    : Morphological and molecular features were analyzed for species of Phacus to better understand the phylogenetic relationships among them and establish the taxonomy; the phylogenetic analyses were based on nSSU rDNA; the research resulted in 55 new sequences. The study included species available in algae collections and those isolated directly from the environment in Poland and Czech Republic. As a result, the obtained phylogenetic tree of Phacus includes 50 species, out of which 7 are represented on the tree for the first time (Phacus anacoelus, P. anomalus, P. curvicauda, P. elegans, P. lismorensis, P. minutus and P. stokesii) and many have been taxonomically verified. For all verified species, diagnostic descriptions were amended, the naming was reordered and epitypes were designated.

  • Culture purification and DNA extraction procedures suitable for next-generation sequencing of euglenids
    Journal of Applied Phycology, 2018
    Co-Authors: Natalia Gumińska, Bożena Zakryś, Magdalena Płecha, Halszka Walkiewicz, Paweł Hałakuc, Rafał Milanowski
    Abstract:

    In the present study, five different DNA extraction procedures were examined to determine their effectiveness for extracting DNA suitable for NGS applications. This included two silica-membrane spin column kits, phenol:chloroform, and two CTAB-based methods. Spectrophotometric and fluorimetric measurements as well as standard gel electrophoresis were used as criteria for evaluating the quantity and quality of the isolated DNA prior to the sequencing. Herein, the method of establishing and maintaining axenic Euglena cultures is also presented. The modified CTAB-based method proved to be highly efficient. In terms of DNA quantity and purity (according to the absorbance ratios), the chosen method resulted in DNA of high molecular weight and quality, which fulfills the library construction requirements. Genomic DNA of Euglena hiemalis (CCAP 1224/35) and E. longa (CCAP 1204-17a) isolated using the suggested protocol had been successfully sequenced on the Illumina HiSeq platform. A modified, rapid CTAB-based method of total DNA isolation from Euglena has been described. In terms of the DNA quantity and quality, the protocol devised involving the washing step with DMSO:acetonitrile proved superior to the commonly used, commercially manufactured kits and isolation with phenol:chloroform. The method is also less labor-intensive and time-consuming than the traditional CTAB-based protocol.

  • PCR identification of toxic euglenid species Euglena sanguinea
    Journal of Applied Phycology, 2018
    Co-Authors: Agata Kulczycka, Bożena Zakryś, Maja Łukomska-kowalczyk, Rafał Milanowski
    Abstract:

    Euglena sanguinea Ehrenberg is the only known species of euglenids which forms toxic blooms causing tangible losses to fish farms. Euglena sanguinea produces euglenophycin, a toxin similar in structure to solenopsin, an alkaloid found in fire ant venom. It was proved that euglenophycin exhibits not only ichthyotoxic but also herbicidal and anticancer activity. Recently, a specific mass spectrometric method of identification and quantitation of euglenophycin was developed to facilitate monitoring of that toxin in freshwater ponds. Despite the recent taxonomic verifications, proper identification of E. sanguinea is still difficult, especially for less experienced researchers. Herein, we describe a simple method based on nested PCR amplification of the nSSU rDNA fragments to identify a single E. sanguinea cell and its detection in a sample of water. The method will further facilitate monitoring of water reservoirs, especially estimating the risk of toxic blooms.

  • Evolutionary Origin of Euglena
    Advances in Experimental Medicine and Biology, 2017
    Co-Authors: Bożena Zakryś, Rafał Milanowski, Anna Karnkowska
    Abstract:

    Euglenids (Excavata, Discoba, Euglenozoa, Euglenida) is a group of free-living, single-celled flagellates living in the aquatic environments. The uniting and unique morphological feature of euglenids is the presence of a cell covering called the pellicle. The morphology and organization of the pellicle correlate well with the mode of nutrition and cell movement. Euglenids exhibit diverse modes of nutrition, including phagotrophy and photosynthesis. Photosynthetic species (Euglenophyceae) constitute a single subclade within euglenids. Their plastids embedded by three membranes arose as the result of a secondary endosymbiosis between phagotrophic eukaryovorous euglenid and the Pyramimonas-related green alga. Within photosynthetic euglenids three evolutionary lineages can be distinguished. The most basal lineage is formed by one mixotrophic species, Rapaza viridis. Other photosynthetic euglenids are split into two groups: predominantly marine Eutreptiales and freshwater Euglenales. Euglenales are divided into two families: Phacaceae, comprising three monophyletic genera (Discoplastis, Lepocinclis, Phacus) and Euglenaceae with seven monophyletic genera (Euglenaformis, Euglenaria, Colacium, Cryptoglena, Strombomonas, Trachelomonas, Monomorphina) and polyphyletic genus Euglena. For 150 years researchers have been studying Euglena based solely on morphological features what resulted in hundreds of descriptions of new taxa and many artificial intra-generic classification systems. In spite of the progress towards defining Euglena, it still remains polyphyletic and morphologically almost undistinguishable from members of the recently described genus Euglenaria; members of both genera have cells undergoing metaboly (dynamic changes in cell shape), large chloroplasts with pyrenoids and monomorphic paramylon grains. Model organisms Euglena gracilis Klebs, the species of choice for addressing fundamental questions in eukaryotic biochemistry, cell and molecular biology, is a representative of the genus Euglena.

  • Intermediate introns in nuclear genes of euglenids – are they a distinct type?
    BMC Evolutionary Biology, 2016
    Co-Authors: Rafał Milanowski, Anna Karnkowska, Natalia Gumińska, Takao Ishikawa, Bożena Zakryś
    Abstract:

    Background Nuclear 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. Results Sequences 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. Conclusions Sequence 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.