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Ultrastructure and 18S rDNA sequence analysis of Wobblia lunata gen. et sp. nov., a new heterotrophic flagellate (Stramenopiles, Incertae sedis).

A new heterotrophic flagellate Wobblia lunata gen. et sp. nov. is described. This organism usually attaches to the substratum showing a wobbling motion, and sometimes glides on the substratum or swims freely in the medium. W. lunata has various features characteristic of the stramenopiles. These include a hairy flagellum with tripartite tubular hairs, a mitochondrion with tubular cristae, arrangement of flagellar apparatus components and a double helix in the flagellar transition zone. W. lunata shares a double helix with heterotrophic stramenopiles, including Developayella elegans, oomycetes, hyphochytrids, opalinids and proteromonads, and could be placed in the phylum Bigyra Cavalier-Smith. However, from 18S rDNA tree analysis, these organisms form two distantly-related clades in the stramenopiles, and Wobblia appears at the base of the stramenopiles. Evaluation of morphological features and comparison of 18S rDNA sequences indicate that W. lunata is a member of the stramenopiles, but it is distinct from any other stramenopiles so far described. Its phylogenetic position within the stramenopiles is uncertain and therefore W. lunata is described as a stramenopile incertae sedis.

Animals↗

Phylogenetic and ecological analysis of novel marine stramenopiles.

Culture-independent molecular analyses of open-sea microorganisms have revealed the existence and apparent abundance of novel eukaryotic lineages, opening new avenues for phylogenetic, evolutionary, and ecological research. Novel marine stramenopiles, identified by 18S ribosomal DNA sequences within the basal part of the stramenopile radiation but unrelated to any previously known group, constituted one of the most important novel lineages in these open-sea samples. Here we carry out a comparative analysis of novel stramenopiles, including new sequences from coastal genetic libraries presented here and sequences from recent reports from the open ocean and marine anoxic sites. Novel stramenopiles were found in all major habitats, generally accounting for a significant proportion of clones in genetic libraries. Phylogenetic analyses indicated the existence of 12 independent clusters. Some of these were restricted to anoxic or deep-sea environments, but the majority were typical components of coastal and open-sea waters. We specifically identified four clusters that were well represented in most marine surface waters (together they accounted for 74% of the novel stramenopile clones) and are the obvious targets for future research. Many sequences were retrieved from geographically distant regions, indicating that some organisms were cosmopolitan. Our study expands our knowledge on the phylogenetic diversity and distribution of novel marine stramenopiles and confirms that they are fundamental members of the marine eukaryotic picoplankton.

Animals↗

Homologs of the sexually induced gene 1 (sig1) product constitute the stramenopile mastigonemes.

The tripartite tubular mastigoneme on the anterior flagellum is a morphological feature that characterizes the stramenopiles. Mastigonemes are significant and potentially informative structures not only from the viewpoint of systematics, but also of cell biology. Nevertheless, few biochemical studies have been reported on stramenopile mastigonemes. The flagella of Scytosiphon lomentaria (Phaeophyceae) were successfully isolated and analyzed using SDS-PAGE followed by protein sequencing. The partial amino acid sequence of one flagellar protein (115kDa) showed high similarity with the sexually induced gene 1 (sig1) product of centric diatoms. A polyclonal antibody against the 115-kDa protein reacted not only to the shaft of mastigonemes in Scytosiphon lomentaria, but also another distinctly different stramenopile flagellate, Sulcochrysis biplastida (Dictyochophyceae). Therefore, we propose that the 115-kDa protein (i.e. Sig1 homologs) is a constituent of the tubular shaft of the mastigoneme.

Amino Acid Sequence↗

A new class of the stramenopiles, Placididea Classis nova: description of Placidia cafeteriopsis gen. et sp. nov.

A marine flagellate resembling Cafeteria roenbergensis (bicosoecids, stramenopiles) in cell shape and behavior of the cell while attached to substratum was collected from the coast of Japan. The flagellate was examined by light and electron microscopy, and the 18S rDNA was sequenced to elucidate its taxonomic and phylogenetic position. Ultrastructural features suggested that the flagellate is not a bicosoecid, but a relative of the recently described stramenopile, Wobblia lunata. 18S rDNA phylogenetic trees also revealed that the flagellate forms a monophyletic clade with W. lunata and that it is distantly related to Cafeteria and other bicosoecids. The flagellate differs from W. lunata due to its lack of wobbling motion as well as intracellular features such as the number of mitochondria, flagellar apparatus architecture, the presence of a paranuclear body and cytoplasmic microtubules. The similarity of 18S rDNA sequences was 81% between the flagellate and W. lunata. This new flagellate was described as Placidia cafeteriopsis gen. et sp. nov. Because the phylogenetic lineage comprised of W. lunata and P. cafeteriopsis was one of the major, deep-branching clades of the stramenopiles, the class Placididea (= Placidiophyceae) classis nova was proposed.

Animals↗

The mitochondrial genome of the stramenopile alga Chrysodidymus synuroideus. Complete sequence, gene content and genome organization.

This is the first report of a complete mitochondrial genome sequence from a photosynthetic member of the stramenopiles, the chrysophyte alga Chrysodidymus synuroideus. The circular-mapping mitochondrial DNA (mtDNA) of 34 119 bp contains 58 densely packed genes (all without introns) and five unique open reading frames (ORFs). Protein genes code for components of respiratory chain complexes, ATP synthase and the mitoribosome, as well as one product of unknown function, encoded in many other protist mtDNAs (YMF16). In addition to small and large subunit ribosomal RNAs, 23 tRNAs are mtDNA-encoded, permitting translation of all codons present in protein-coding genes except ACN (Thr) and CGN (Arg). The missing tRNAs are assumed to be imported from the cytosol. Comparison of the C.SYNUROIDEUS: mtDNA with that of other stramenopiles allowed us to draw conclusions about mitochondrial genome organization, expression and evolution. First, we provide evidence that mitochondrial ORFs code for highly derived, unrecognizable versions of ribosomal or respiratory genes otherwise 'missing' in a particular mtDNA. Secondly, the observed constraints in mitochondrial genome rearrangements suggest operon-based, co-ordinated expression of genes functioning in common biological processes. Finally, stramenopile mtDNAs reveal an unexpectedly low variability in genome size and gene complement, testifying to substantial differences in the tempo of mtDNA evolution between major eukaryotic lineages.

Algal Proteins↗

Vestigial chloroplasts in heterotrophic stramenopiles Pteridomonas danica and Ciliophrys infusionum (Dictyochophyceae).

Two heterotrophic members of the Dictyochophyceae (stramenopiles), Pteridomonas danica and Ciliophrys infusionum, were investigated. An undescribed organelle bounded by four membranes and closely associated with the nucleus was detected in P. danica. The outermost membrane was continuous with the outer nuclear membrane. These features strongly suggested that this organelle was a vestigial chloroplast. A photosynthetic gene, rbcL, was successfully amplified by polymerase chain reaction (PCR) from P. danica and C. infusionum. These sequences were readily and well aligned with those of photosynthetic stramenopiles. Phylogenetic trees of 18S rDNA and rbcL were constructed. In all the trees obtained, P. danica and C. infusionum appeared in two different clades, the Pedinellales clade and the Ciliophryales/Rhizochromulinales clade, each of which contained photosynthetic members as well as heterotrophic members. The results indicated that the loss of photosynthetic ability occurred independently in P. danica and C. infusionum. This is the first report of the presence of a vestigial chloroplast (leucoplast) in colorless dictyochophytes.

Animals↗

Structure of the large subunit rDNA from a diatom, and comparison between small and large subunit ribosomal RNA for studying stramenopile evolution.

The aim of this study was to compare the usefulness of complete small and large subunit rRNA, and a combination of both molecules, for reconstructing stramenopile evolution. To this end, phylogenies from species of which both sequences are known were constructed with the neighbor-joining, maximum parsimony, and maximum likelihood methods. Also the use of structural features of the rRNAs was evaluated. The large subunit rRNA from the diatom Skeletonema pseudocostatum was sequenced in order to have a more complete taxon sampling, and a group I intron was identified. Our results indicated that heterokont algae are monophyletic, with diatoms diverging first. However, as the analysis was restricted to a particular data set containing merely six taxa, the outcome has limited value for elucidating stramenopile relationships. On the other hand, this approach permits comparison of the performance of both rRNA molecules without interference from other factors, such as a different species selection for each molecule. For the taxa used, the large subunit rRNA clearly contained more phylogenetic information than the small subunit rRNA. Although this result can definitely not be generalized and depends on the phylogeny to be studied, in some cases determining complete large subunit rRNA sequences certainly seems worthwhile.

Base Sequence↗

Phylogenetic position of Blastocystis hominis and of stramenopiles inferred from multiple molecular sequence data.

Blastocystis hominis, a parasite of the human intestine, has recently been positioned within stramenopiles by the small subunit rRNA phylogeny. To further confirm its phylogenetic position using multiple molecular sequence data, we determined the nucleotide sequences putatively encoding small subunit ribosomal RNA, cytosolic-type 70-kDa heat shock protein, translation elongation factor 2, and the non-catalytic 'B' subunit of vacuolar ATPase of B. hominis (HE87-1 strain). Moreover, we determined the translation elongation factor 2 sequence of an apicomplexan parasite, Plasmodium falciparum, that belongs to alveolates. The maximum likelihood analyses of small subunit rRNA and cytosolic-type 70-kDa heat shock protein clearly demonstrated that B. hominis (HE87-1 strain) is positioned within stramenopiles, being congruent with the previous small subunit rRNA analysis, including the sequences of B. hominis (Nand strain) and a Blastocystis isolate from guinea pig. Although no clear resolution among major eukaryotic groups was obtained by the individual phylogenies based on the four molecules analyzed here, a combined analysis of various molecules, including these, clearly indicated that Blastocystis/stramenopiles are the closest relatives of alveolates.

Animals↗

The hAT -like DNA transposon DodoPi resides in a cluster of retro- and DNA transposons in the stramenopile Phytophthora infestans.

A family of transposable elements belonging to the hAT group of DNA transposons is described from an oomycete, the plant pathogen Phytophthora infestans. The family, named DodoPi, was identified by studying a hotspot for retro- and DNA transposon insertions adjacent to the mating type locus. The DodoPi family comprises a small number of full-length copies, each of which is 2.7 kb long and predicted to encode a transposase-like protein consisting of 617 amino acids, and several truncated copies. Both types contain 12-bp terminal inverted repeats and are flanked by 8-bp target site duplications. Despite the detection of a DodoPi transcript and of many polymorphisms between isolates, conclusive evidence of recent transposition was not obtained. A phylogenetic analysis indicated that DodoPi was novel, with only modest similarity to some elements from plants and fungi. Relatives were detected in only some members of the genus. This is the first DNA transposon identified in the stramenopile group of eukaryotes.

Base Sequence↗

Evolutionary relationships among heterokont algae (the autotrophic stramenopiles) based on combined analyses of small and large subunit ribosomal RNA.

In order to study the phylogenetic relationships within the stramenopiles, and particularly among the heterokont algae, we have determined complete or nearly complete large-subunit ribosomal RNA sequences for different species of raphidophytes, phaeophytes, xanthophytes, chrysophytes, synurophytes and pinguiophytes. With the small- and large-subunit ribosomal RNA sequences of representatives for nearly all known groups of heterokont algae, phylogenetic trees were constructed from a concatenated alignment of both ribosomal RNAs, including more than 5,000 positions. By using different tree construction methods, inferred phylogenies showed phaeophytes and xanthophytes as sister taxa, as well as the pelagophytes and dictyochophytes, and the chrysophytes/synurophytes and eustigmatophytes. All these relationships are highly supported by bootstrap analysis. However, apart from these sister group relationships, very few other internodes are well resolved and most groups of heterokont algae seem to have diverged within a relatively short time frame.

Eukaryota↗

The evolution of stramenopiles and alveolates as derived by "substitution rate calibration" of small ribosomal subunit RNA.

The substitution rate of the individual positions in an alignment of 750 eukaryotic small ribosomal subunit RNA sequences was estimated. From the resulting rate distribution, an equation was derived that gives a more precise relationship between sequence dissimilarity and evolutionary distance than hitherto available. Trees constructed on the basis of evolutionary distances computed by this new equation for small ribosomal subunit RNA sequences from ciliates, apicomplexans, dinoflagellates, oomycetes, hyphochytriomycetes, bicosoecids, labyrinthuloids, and heterokont algae show a more consistent tree topology than trees constructed in the absence of "substitution rate calibration." In particular, they do not suffer from anomalies caused by the presence of extremely long branches.

Animals↗

Analysis of the ITS region and partial ssu and lsu rRNA genes of Blastocystis and Proteromonas lacertae.

Blastocystis is a common single-celled enteric parasite found in a large variety of hosts. Recent molecular analysis supports the concept that this eukaryotic organism is a stramenopile most closely related to Proteromonas lacertae, a parasite of reptiles. In this study, the internal transcribed spacer region, partial small subunit rRNA and large subunit rRNA genes from 7 Blastocystis isolates (5 human, 1 pig and 1 sheep), and a Proteromonas lacertae isolate were amplified by PCR, cloned and sequenced. Blastocystis was found to be a typical eukaryote with both ITS1 and ITS2 regions present. Phylogenetic analysis based on the entire PCR amplicon revealed that the Blastocystis isolates did not segregate according to host or geographic origin. The highest sequence identities with the conserved Blastocystis 5.8S rDNA sequence were with the stramenopiles Fibrocapsa japonica, Chattonella marina, Cylindrotheca closterium and Hyphochytrium catenoides. The most parsimonious tree based on the 5.8S rDNA sequence from P. lacertae, 11 other stramenopiles, 2 fungi, 3 algae and 3 alveolates showed Blastocystis positioned within the stramenopiles, with P. lacertae as its closest relative. This work therefore supports the hypothesis that Blastocystis is most closely related to P. lacertae, and that it should be regarded as an unusual stramenopile.

Animals↗

Unveiling the organisms behind novel eukaryotic ribosomal DNA sequences from the ocean.

Despite the fact that the smallest eukaryotes (cells less than 5 micro m in diameter) play key roles in marine food webs, particularly in open oligotrophic areas, the study of their in situ diversity started just one year ago. Perhaps the most remarkable finding of the most recent studies has been the discovery of completely new phylogenetic lineages, such as novel clades belonging to the stramenopile and alveolate phyla. The two new groups account for a significant fraction of clones in genetic libraries from North Atlantic, equatorial Pacific, Antarctic, and Mediterranean Sea waters. However, the identities and ecological relevance of these organisms remain unknown. Here we investigate the phylogenetic relationships, morphology, in situ abundance, and ecological role of novel stramenopiles. They form at least eight independent clades within the stramenopile basal branches, indicating a large phylogenetic diversity within the group. Two lineages were visualized and enumerated in field samples and enrichments by fluorescent in situ hybridization using specific rRNA-targeted oligonucleotide probes. The targeted organisms were 2- to 3- micro m-diameter, round-shaped, nonpigmented flagellates. Further, they were found to be bacterivorous. One lineage accounted for up to 46% (average during an annual cycle, 19%) of heterotrophic flagellates in a coastal environment, providing evidence that novel stramenopiles are important and unrecognized components of the total stock of bacterial grazers.

Animals↗

Large-subunit rRNA sequence of the chytridiomycete Blastocladiella emersonii, and implications for the evolution of zoosporic fungi.

The 5.8S and 28S ribosomal RNA sequences of the chytridiomycete Blastocladiella emersonii were determined. These data were combined with 18S rRNA sequences in order to carry out a phylogenetic analysis based on distance matrix, parsimony, and maximum likelihood methods. The new data confirmed that chytridiomycetes are true fungi and not protists, as was already suggested on the basis of biochemical, ultrastructural, and 18S rRNA data. Within the fungal clade, B. emersonii formed the first line of divergence. The position of the fungi within the eukaryotic "crown" taxa was also reassessed, and the alveolate-stramenopile cluster appeared as their sister group. The stramenopiles also comprise a number of zoosporic fungi, which resemble chytridiomycetes in so many respects, e.g., production of motile spores, thallus morphology, and absorptive nutrition, that they have been classified together with them in the past. This suggests that the possible common ancestor of the fungi, stramenopiles, and alveolates may have been a zoosporic fungus, which would mean that zoosporic fungi are paraphyletic instead of polyphyletic as previously suggested.

Biological Evolution↗

Evolutionary relationships among the eukaryotic crown taxa taking into account site-to-site rate variation in 18S rRNA.

In this study we constructed a bootstrapped distance tree of 500 small subunit ribosomal RNA sequences from organisms belonging to the so-called crown of eukaryote evolution. Taking into account the substitution rate of the individual nucleotides of the rRNA sequence alignment, our results suggest that (1) animals, true fungi, and choanoflagellates share a common origin: The branch joining these taxa is highly supported by bootstrap analysis (bootstrap support [BS] > 90%), (2) stramenopiles and alveolates are sister groups (BS = 75%), (3) within the alveolates, dinoflagellates and apicomplexans share a common ancestor BS > 95%), while in turn they both share a common origin with the ciliates (BS > 80%), and (4) within the stramenopiles, heterokont algae, hyphochytriomycetes, and oomycetes form a monophyletic grouping well supported by bootstrap analysis (BS > 85%), preceded by the well-supported successive divergence of labyrinthulomycetes and bicosoecids. On the other hand, many evolutionary relationships between crown taxa are still obscure on the basis of 18S rRNA. The branching order between the animal-fungal-choanoflagellates clade and the chlorobionts, the alveolates and stramenopiles, red algae, and several smaller groups of organisms remains largely unresolved.When among-site rate variation is not considered, the inferred tree topologies are inferior to those where the substitution rate spectrum for the 18S rRNA is taken into account. This is primarily indicated by the erroneous branching of fast-evolving sequences. Moreover, when different substitution rates among sites are not considered, the animals no longer appear as a monophyletic grouping in most distance trees.

Animals↗

Two types of FtsZ proteins in mitochondria and red-lineage chloroplasts: the duplication of FtsZ is implicated in endosymbiosis.

The ancestors of plastids and mitochondria were once free-living bacteria that became organelles as a result of endosymbiosis. According to this theory, a key bacterial division protein, FtsZ, plays a role in plastid division in algae and plants as well as in mitochondrial division in lower eukaryotes. Recent studies have shown that organelle division is a process that combines features derived from the bacterial division system with features contributed by host eukaryotic cells. Two nonredundant versions of FtsZ, FtsZ1 and FtsZ2, have been identified in green-lineage plastids, whereas most bacteria have a single ftsZ gene. To examine whether there is also more than one type of FtsZ in red-lineage chloroplasts (red algal chloroplasts and chloroplasts that originated from the secondary endosymbiosis of red algae) and in mitochondria, we obtained FtsZ sequences from the complete sequence of the primitive red alga Cyanidioschyzon merolae and the draft sequence of the stramenopile (heterokont) Thalassiosira pseudonana. Phylogenetic analyses that included known FtsZ proteins identified two types of chloroplast FtsZ in red algae (FtsZA and FtsZB) and stramenopiles (FtsZA and FtsZC). These analyses also showed that FtsZB emerged after the red and green lineages diverged, while FtsZC arose by the duplication of an ftsZA gene that in turn descended from a red alga engulfed by the ancestor of stramenopiles. A comparison of the predicted proteins showed that like bacterial FtsZ and green-lineage FtsZ2, FtsZA has a short conserved C-termmal sequence (the C-terminal core domain), whereas FtsZB and FtsZC, like the green-lineage FtsZ1, lack this sequence. In addition, the Cyanidioschyzon and Dictyostelium genomes encode two types of mitochondrial FtsZ proteins, one of which lacks the C-terminal variable domain. These results suggest that the acquisition of an additional FtsZ protein with a modified C terminus was common to the primary and secondary endosymbioses that produced plastids and that this also occurred during the establishment of mitochondria, presumably to regulate the multiplication of these organelles.

Algal Proteins↗

Complete large subunit ribosomal RNA sequences from the heterokont algae Ochromonas danica, Nannochloropsis salina, and Tribonema aequale, and phylogenetic analysis.

The large subunit ribosomal RNA sequences from the heterokont algae Ochromonas danica, Nannochloropsis salina, and Tribonema aequale were determined. These sequences were combined with small subunit ribosomal RNA sequences in order to carry out a phylogenetic analysis based on neighbor-joining, maximum parsimony, and maximum likelihood methods. Our results indicate that heterokont fungi and heterokont algae each are monophyletic, and confirm that they together form a monophyletic group called "stramenopiles." Within the heterokont algae, the eustigmatophyte Nannochloropsis salina either clusters with the chrysophyte Ochromonas danica or forms a sister group to a cluster comprising the phaeophyte Scytosiphon lomentaria and the xanthophyte Tribonema aequale. The alveolates were identified as the closest relatives of the stramenopiles, but the exact order of divergence between the eukaryotic crown taxa could not be established with confidence.

Animals↗

Genetic diversity of microbial eukaryotes in anoxic sediment around fumaroles on a submarine caldera floor based on the small-subunit rDNA phylogeny.

Recent culture-independent molecular analyses have shown the diversity and ecological importance of microbial eukaryotes (protists) in various marine environments. In the present study we directly extracted DNA from anoxic sediment near active fumaroles on a submarine caldera floor at a depth of 200 m and constructed genetic libraries of PCR-amplified eukaryotic small-subunit (SSU) rDNA. By sequencing cloned SSU rDNA of the libraries and their phylogenetic analyses, it was shown that most sequences have affiliations with known major lineages of eukaryotes (Cercozoa, Alveolata, stramenopiles and Opisthokonta). In particular, some sequences were closely related to those of representatives of eukaryotic parasites, such as Phagomyxa and Cryothecomonas of Cercozoa, Pirsonia of stramenopiles and Ichthyosporea of Opisthokonta, although it is not clear whether the organisms occur in free-living or parasitic forms. In addition, other sequences did not seem to be related to any described eukaryotic lineages suggesting the existence of novel eukaryotes at a high-taxonomic level in the sediment. The community composition of microbial eukaryotes in the sediment we surveyed was different overall from those of other anoxic marine environments previously investigated.

Animals↗