Differences in planktonic microbial communities associated with three types of macrophyte stands in a shallow lake.

Little is known about how various substances from living and decomposing aquatic macrophytes affect the horizontal patterns of planktonic bacterial communities. Study sites were located within Lake Kolon, which is a freshwater marsh and can be characterised by open-water sites and small ponds with different macrovegetation (Phragmites australis, Nymphea alba and Utricularia vulgaris). Our aim was to reveal the impact of these macrophytes on the composition of the planktonic microbial communities using comparative analysis of environmental parameters, microscopy and pyrosequencing data. Bacterial 16S rRNA gene sequences were dominated by members of phyla Proteobacteria (36%-72%), Bacteroidetes (12%-33%) and Actinobacteria (5%-26%), but in the anoxic sample the ratio of Chlorobi (54%) was also remarkable. In the phytoplankton community, Cryptomonas sp., Dinobryon divergens, Euglena acus and chrysoflagellates had the highest proportion. Despite the similarities in most of the measured environmental parameters, the inner ponds had different bacterial and algal communities, suggesting that the presence and quality of macrophytes directly and indirectly controlled the composition of microbial plankton.

Evolutionary Origin of Euglena.

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.

Euglena Transcript Processing.

RNA transcript processing is an important stage in the gene expression pathway of all organisms and is subject to various mechanisms of control that influence the final levels of gene products. RNA processing involves events such as nuclease-mediated cleavage, removal of intervening sequences referred to as introns and modifications to RNA structure (nucleoside modification and editing). In Euglena, RNA transcript processing was initially examined in chloroplasts because of historical interest in the secondary endosymbiotic origin of this organelle in this organism. More recent efforts to examine mitochondrial genome structure and RNA maturation have been stimulated by the discovery of unusual processing pathways in other Euglenozoans such as kinetoplastids and diplonemids. Eukaryotes containing large genomes are now known to typically contain large collections of introns and regulatory RNAs involved in RNA processing events, and Euglena gracilis in particular has a relatively large genome for a protist. Studies examining the structure of nuclear genes and the mechanisms involved in nuclear RNA processing have revealed that indeed Euglena contains large numbers of introns in the limited set of genes so far examined and also possesses large numbers of specific classes of regulatory and processing RNAs, such as small nucleolar RNAs (snoRNAs). Most interestingly, these studies have also revealed that Euglena possesses novel processing pathways generating highly fragmented cytosolic ribosomal RNAs and subunits and non-conventional intron classes removed by unknown splicing mechanisms. This unexpected diversity in RNA processing pathways emphasizes the importance of identifying the components involved in these processing mechanisms and their evolutionary emergence in Euglena species.

A rare case of plastid protein-coding gene duplication in the chloroplast genome of Euglena archaeoplastidiata (Euglenophyta).

Gene duplication is an important evolutionary process that allows duplicate functions to diverge, or, in some cases, allows for new functional gains. However, in contrast to the nuclear genome, gene duplications within the chloroplast are extremely rare. Here, we present the chloroplast genome of the photosynthetic protist Euglena archaeoplastidiata. Upon annotation, it was found that the chloroplast genome contained a novel tandem direct duplication that encoded a portion of RuBisCO large subunit (rbcL) followed by a complete copy of ribosomal protein L32 (rpl32), as well as the associated intergenic sequences. Analyses of the duplicated rpl32 were inconclusive regarding selective pressures, although it was found that substitutions in the duplicated region, all non-synonymous, likely had a neutral functional effect. The duplicated region did not exhibit patterns consistent with previously described mechanisms for tandem direct duplications, and demonstrated an unknown mechanism of duplication. In addition, a comparison of this chloroplast genome to other previously characterized chloroplast genomes from the same family revealed characteristics that indicated E. archaeoplastidiata was probably more closely related to taxa in the genera Monomorphina, Cryptoglena, and Euglenaria than it was to other Euglena taxa. Taken together, the chloroplast genome of E. archaeoplastidiata demonstrated multiple characteristics unique to the euglenoid world, and has justified the longstanding curiosity regarding this enigmatic taxon.

The Chloroplast Genome of Euglena mutabilis-Cluster Arrangement, Intron Analysis, and Intrageneric Trends.

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.

Fatty acid and metabolomic profiling approaches differentiate heterotrophic and mixotrophic culture conditions in a microalgal food supplement 'Euglena'.

BACKGROUND: Microalgae have been recognized as a good food source of natural biologically active ingredients. Among them, the green microalga Euglena is a very promising food and nutritional supplements, providing high value-added poly-unsaturated fatty acids, paramylon and proteins. Different culture conditions could affect the chemical composition and food quality of microalgal cells. However, little information is available for distinguishing the different cellular changes especially the active ingredients including poly-saturated fatty acids and other metabolites under different culture conditions, such as light and dark. RESULTS: In this study, together with fatty acid profiling, we applied a gas chromatography-mass spectrometry (GC-MS)-based metabolomics to differentiate hetrotrophic and mixotrophic culture conditions. CONCLUSIONS: This study suggests metabolomics can shed light on understanding metabolomic changes under different culture conditions and provides a theoretical basis for industrial applications of microalgae, as food with better high-quality active ingredients.

Selection and characterization of Euglena anabaena var. minor as a new candidate Euglena species for industrial application.

Euglena gracilis is a microalgae used as a model organism. Recently, mass cultivation of this species has been achieved for industrial applications. The genus Euglena includes more than 200 species that share common useful features, but the potential industrial applications of other Euglena species have not been evaluated. Thus, we conducted a pilot screening study to identify other species that proliferate at a sufficiently rapid rate to be used for mass cultivation; we found that Euglena anabaena var. minor had a rapid growth rate. In addition, its cells accumulated more than 40% weight of carbohydrate, most of which is considered to be a euglenoid specific type of beta-1-3-glucan, paramylon. Carbohydrate is stored in E. anabaena var. minor cells during normal culture, whereas E. gracilis requires nitrogen limitation to facilitate paramylon accumulation. These results suggest the potential industrial application of E. anabaena var. minor.

Ultrastructure of five Euglena species positioned in the subdivision Serpentes.

Within the genus Euglena, the subgroup "Serpentes" is characterised by species with long, slim cell bodies, which move without flagellum by snake-like locomotion in the detritus or in the mud, or swim freely in the water with a flagellum. Two major groups can be distinguished. The first is centred around the species Euglena satelles, with Euglena carterae, Euglena adhaerens and others, and is characterised by a straight-ended anterior part of the cell without a protruding flagellum. The second group is centred around the species Euglena deses, with its varieties, and Euglena ehrenbergii, and is characterised by a lateral canal opening at the anterior end with one flagellum protruding sideways. The representatives of the whole Serpentes group have various (15-30) large chloroplasts containing characteristic naked pyrenoids. The exception is Euglena ehrenbergii, which possesses innumerable small chloroplasts without pyrenoids. To better characterise this whole subgroup, to better taxonomically distinguish between the diverse species and to provide a basis for further molecular-genetic analysis of the phylogeny of and relationship between the Euglena species, we used transmission and scanning electron microscopy to investigate the five selected species. One important distinguishing feature among the species is the form of the pellicle. It can differ in thickness or cross-sectional shape (e.g. A-, M-or plateau-like shape) and can have various arrangements of microtubules and endoplasmic reticulum mucus vesicles. We show that the group is more heterogeneous than expected and that some species have very individual features that poorly fit into a common Serpentes group, particularly the above-mentioned Euglena ehrenbergii. Euglena carterae, formerly named Euglena deses var. carterae, with its typical straight-ended canal opening, does not fit into the Euglena deses varieties, as has already been confirmed by molecular genetic methods.

Macroevolution of complex cytoskeletal systems in euglenids.

Euglenids comprise a group of single-celled eukaryotes with diverse modes of nutrition, including phagotrophy and photosynthesis. The level of morphological diversity present in this group provides an excellent system for demonstrating evolutionary transformations in morphological characters. This diversity also provides compelling evidence for major events in eukaryote evolution, such as the punctuated effects of secondary endosymbiosis and mutations in underlying developmental mechanisms. In this essay, we synthesize evidence for the origin, adaptive significance and diversification of the euglenid cytoskeleton, especially pellicle ultrastructure, pellicle surface patterns, pellicle strip number and the feeding apparatus. We also highlight holes in our knowledge that must be filled before we are able to confidently describe euglenid cell biology and infer the earliest stages in euglenid evolution. Nonetheless, by possessing combinations of characters resulting from adaptive change and morphostasis, euglenids have retained key pieces of evidence necessary for reconstructing the early evolution and diversification of eukaryotic life.

[A new species of Euglena (Euglenozoa: Euglenales) isolated from extreme environments in "boiling mudflats" of Rincón de la Vieja volcano, Costa Rica]. / Una nueva especie de Euglena (Euglenozoa: Euglenales) aislada de ambientes extremófilos en las Pailas de Barro del Volcán Rincón de la Vieja, Costa Rica.

A new species of euglena isolated from a hot and acid mud pool located in Las Pailas de Barro, Volcán Rincón de la Vieja, Costa Rica is described. This species inhabits hot and acid environments. Euglena pailasensis sp. nov. main features are: the absence of flagella, the presence filaments like "pilis", the presence of chloroplasts with pyrenoids crossed by several tylakoids, and acid and heat tolerance. Molecular phylogeny studies using 18S rDNA and Gap C genes indicated that the new species is related to E. mutabilis. Its taxonomic characters based on morphology, biology and sequence of the 18S rDNA and Gap C genes are discussed and compared with other closely related species of the genus.

[Ultrastructural description of Euglena pailasensis (Euglenozoa) from Rincón de la Vieja volcano, Guanacaste, Costa Rica]. / Descripción ultraestructural de Euglena pailasensis (Euglenozoa) del Volcán Rincón de la Vieja, Guanacaste, Costa Rica.

The euglenoids are unicellular eukaryotic flagellates living in a diversity of soils and aquatic environments and ecosystems. This study describes the ultrastructure of an euglenoid isolated from the surface of a boiling mud pool with temperatures ranging from 38 to 98 degrees C and pH 2 - 4. The hot mud pool is located in Area de Pailas de Barro, Las Pailas, Rincón de la Vieja Volcano, Guanacaste, Costa Rica. The morphological characterization of the Euglena pailasensis was performed by SEM and TEM. It was determined that, although the euglenoid was obtained from an extreme volcanic environment, the general morphology corresponds to that of a typical member of Euglena of 30-45 microm long and 8-10 microm wide, with membrane, pellicle, chloroplasts, mitochondria, nucleus, pigments and other cytoplasmic organelles. E. pailasensis is delimited by a membrane and by 40 to 90 pellicle strips. It was observed up to 5 elongated chloroplasts per cell. The chloroplast contains several osmiophilic globules and a pyrenoid penetrated by few thylakoid pairs. The nutritious material is reserved in numerous small paramylon grains located at the center of the cell, mitocondria are characterized by the presence of crests in radial disposition toward the interior of the lumen. It was also observed around the external surface "pili" like filaments originating from the pellicle strips. There is no evidence for the presence of flagella in the ampulla (reservoir/canal area), a fact confirmed by negative staining, and a difference regarding other species of Euglena. The observed ultrastructural characteristics are not sufficient to explain the adaptation of this species to acid and hot environments.

Phylogeny of the photosynthetic euglenophytes inferred from the nuclear SSU and partial LSU rDNA.

Previous studies using the nuclear SSU rDNA have indicated that the photosynthetic euglenoids are a monophyletic group; however, some of the genera within the photosynthetic lineage are not monophyletic. To test these results further, evolutionary relationships among the photosynthetic genera were investigated by obtaining partial LSU nuclear rDNA sequences. Taxa from each of the external clades of the SSU rDNA-based phylogeny were chosen to create a combined dataset and to compare the individual LSU and SSU rDNA datasets. Conserved areas of the aligned sequences for both the LSU and SSU rDNA were used to generate parsimony, log-det, maximum-likelihood and Bayesian trees. The SSU and LSU rDNA consistently generated the same seven terminal clades; however, the relationship among those clades varied depending on the type of analysis and the dataset used. The combined dataset generated a more robust phylogeny, but the relationships among clades still varied. The addition of the LSU rDNA dataset to the euglenophyte phylogeny supports the view that the genera Euglena, Lepocinclis and Phacus are not monophyletic and substantiates the existence of several well-supported clades. A secondary structural model for the D2 region of the LSU rDNA was proposed on the basis of compensatory base changes found in the alignment.

Phylogeny and taxonomic revision of plastid-containing euglenophytes based on SSU rDNA sequence comparisons and synapomorphic signatures in the SSU rRNA secondary structure.

Sequence comparisons and a revised classification of the Euglenophyceae were based on 92 new SSU rDNA sequences obtained from strains of Euglena, Astasia, Phacus, Trachelomonas, Colacium, Cryptoglena, Lepocinclis, Eutreptia, Eutreptiella and Tetreutreptia. Sequence data also provided molecular signatures for taxa from genus to class level in the SSU rRNA secondary structure, revealed by a novel approach (search for non-homoplasious synapomorphies) and used for taxonomic diagnoses. Photosynthetic euglenoids and secondary heterotrophs formed a clade, designated as Euglenophyceae (emend.) with two orders: Euglenales and Eutreptiales. The mostly marine Eutreptiales (Eutreptia, Eutreptiella; not Distigma) comprised taxa with two or four emergent flagella (the quadriflagellate Tetreutreptia was integrated within Eutreptiella). The Euglenales (freshwater genera with < or = one emergent flagellum) formed nine clades and two individual branches (single strains); however, only two clades were congruent with traditional genera: Trachelomonas (incl. Strombomonas) and Colacium. Euglena was polyphyletic and diverged into four independent clades (intermixed with Astasia, Khawkinea and Lepocinclis) and two individual branches (e.g. E. polymorpha). Phacus was also subdivided into Phacus s. str. and two combined lineages (mixed with Lepocinclis spp. or Cryptoglena). In consequence, Euglena (s. str.), Phacus and other genera were emended and one lineage (mixed Phacus/Lepocinclis-clade) was recognized as the previously neglected genus Monomorphina Mereschkowsky (1877). The sister clade of Phacus s. str. (mixed Euglena/Lepocinclis-clade) was identified as Lepocinclis Perty (emended).

Phylogenetic position and inter-relationships of the osmotrophic euglenids based on SSU rDNA data, with emphasis on the Rhabdomonadales (Euglenozoa).

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.

Phylogenetic analysis of chloroplast small-subunit rRNA genes of the genus Euglena Ehrenberg.

Almost complete sequences of plastid SSU rDNA (16S rDNA) from 17 species belonging to the order Euglenales (sensu Nemeth, 1997; Shi et al., 1999) were determined and used to infer phylogenetic relationships between 10 species of Euglena, three of Phacus, and one of each of Colacium, Lepocinclis, Strombomonas, Trachelomonas and Eutreptia. The maximum-parsimony (MP), maximum-likelihood (ML) and distance analyses of the unambiguously aligned sequence fragments imply that the genus Euglena is not monophyletic. Parsimony and distance methods divide Euglenaceae into two sister groups. One comprises of representatives from the subgenera Phacus, Lepocinclis and Discoglena (sensu Zakrys, 1986), whereas the other includes members of Euglena and Calliglena subgenera (sensu Zakrys, 1986), intermixed with representatives of Colacium, Strombomonas and Trachelomonas. In all analyses subgenera Euglena--together with Euglena polymorpha (representative of the subgenus Calliglena)--and Discoglena--together with Phacus and Lepocinclis--form two well-defined clades. The data clearly indicate that a substantial revision of euglenoid systematics is very much required, nevertheless it must await while more information can be gathered, allowing resolution of outstanding relationships.

Phylogenetic analysis of phagotrophic, photomorphic and osmotrophic euglenoids by using the nuclear 18S rDNA sequence.

Phylogenetic analyses of 35 strains including 25 previously published sequences and 10 which have been newly sequenced, representing two species of Euglena, five species of Phacus and three species of Astasia, were carried out using the SSU rDNA. Parsimony, distance and maximum-likelihood inferred phylogenies support (1) monophyly of the euglenoids, (2) kinetoplastids as the sister group, (3) the phagotrophic Petalomonas cantuscygni Cann et Pennick anchoring the base of the euglenoid lineage, (4) evolution of phototrophy within the euglenoids from a single event, (5) multiple origins of osmotrophic euglenoids and (6) polyphyly of the genera Euglena Ehrenberg and Phacus Dujardin. Analyses also indicate that Lepocinclis Perty, Trachelomonas Ehrenberg and Astasia Dujardin are polyphyletic. In addition, the results suggest that neither the Euglenales nor the Eutreptiales form a monophyletic lineage, thus questioning currently available classifications. Concerning the phagotrophic mode of nutrition, the data suggest that the feeding apparatus arose multiple times.

Trends in the evolution of the euglenid pellicle.

Trends in the evolution of the euglenid pellicle were described using phylogenetic methods on 18S rDNA, morphological, and combined data from 25 mostly phototrophic taxa. The tree topology from a total-evidence analysis formed a template for a synthetic tree that took into account conflicting results derived from the partitioned datasets. Pellicle character states that can only be observed with the assistance of transmission and scanning electron microscopy were phylogenetically mapped onto the synthetic tree to test a set of previously established homology statements (inferences made independently from a cladogram). The results permitted us to more confidently infer the ancestral-derived polarities of character state transformations and provided a framework for understanding the key cytoskeletal innovations associated with the evolution of phototrophic euglenids. We specifically addressed the character evolution of (1) the maximum number of pellicle strips around the cell periphery; (2) the patterns of terminating strips near the cell posterior end; (3) the substructural morphology of pellicle strips; (4) the morphology of the cell posterior tip; and (5) patterns of pellicle pores on the cell surface.

Flagellar systems in the euglenoid flagellates.

The flagellar apparatus of euglenoids consists of two functional basal bodies, three unequal microtubular roots subtending the reservoir, and a fourth band of microtubules nucleated from one of the flagellar roots and subtending the reservoir membrane. The flagellar apparatus of some euglenoids may contain additional basal bodies, striated roots ("rhizoplasts"), fibrous roots, striated connecting fibers between basal bodies, layered structures, or various electron-dense connective substances. With the possible exception of Petalomonas cantuscygni, nearly all euglenoids are biflagellate although the length of one flagellum may be highly reduced. The flagellar transition zone and number of basal bodies are highly variable among species. In recent years a cytoplasmic pocket that branches off from the reservoir has been discovered. The microtubules of the ventral flagellar root are continuous with the microtubules which line this pocket. Based on positional and structural similarities, this structure is believed to be homologous with the MTR/cytostome of bodonids. Coupled with other ultrastructural and biochemical data, the fine structure of the flagellar apparatus supports the belief that the euglenoid flagellates are descendant from bodonid ancestors.

A cytostome/cytopharynx in green euglenoid flagellates (Euglenales) and its phylogenetic implications.

The green, phototrophic euglenoid, Colacium libellae, has a vestigial cytostome and cytopharynx. The membrane forming the simple pocket is decorated with a dense, microfilamentous mesh. The mesh binds the pocket to a band of reinforcing microtubules which is homologous with the bodonid MTR. The opening of the cytostome at the reservoir-canal transition zone implies that the phototrophic euglenoid canal is formed by the invagination of the vestibulum of the phagotrophic euglenoids. Our observations support the hypothesis that the phagotrophic euglenoids arose from a bondonid ancestor and gave rise to the phototrophs by chloroplast acquisition.