Small Subunit Ribosomal RNA Sequences Research Articles

The twilight of Heliozoa and rise of Rhizaria, an emerging supergroup of amoeboid eukaryotes.

Recent molecular phylogenetic studies revealed the extraordinary diversity of single-celled eukaryotes. However, the proper assessment of this diversity and accurate reconstruction of the eukaryote phylogeny are still impeded by the lack of molecular data for some major groups of easily identifiable and cultivable protists. Among them, amoeboid eukaryotes have been notably absent from molecular phylogenies, despite their diversity, complexity, and abundance. To partly fill this phylogenetic gap, we present here combined small-subunit ribosomal RNA and actin sequence data for the three main groups of "Heliozoa" (Actinophryida, Centrohelida, and Desmothoracida), the heliozoan-like Sticholonche, and the radiolarian group Polycystinea. Phylogenetic analyses of our sequences demonstrate the polyphyly of heliozoans, which branch either as an independent eukaryotic lineage (Centrohelida), within stramenopiles (Actinophryida), or among cercozoans (Desmothoracida), in broad agreement with previous ultrastructure-based studies. Our data also provide solid evidence for the existence of the Rhizaria, an emerging supergroup of mainly amoeboid eukaryotes that includes desmothoracid heliozoans, all radiolarians, Sticholonche, and foraminiferans, as well as various filose and reticulose amoebae and some flagellates.

Lateral gene transfer and the origins of prokaryotic groups.

Lateral gene transfer (LGT) is now known to be a major force in the evolution of prokaryotic genomes. To date, most analyses have focused on either (a) verifying phylogenies of individual genes thought to have been transferred, or (b) estimating the fraction of individual genomes likely to have been introduced by transfer. Neither approach does justice to the ability of LGT to effect massive and complex transformations in basic biology. In some cases, such transformation will be manifested as the patchy distribution of a seemingly fundamental property (such as aerobiosis or nitrogen fixation) among the members of a group classically defined by the sharing of other properties (metabolic, morphological, or molecular, such as small subunit ribosomal RNA sequence). In other cases, the lineage of recipients so transformed may be seen to comprise a new group of high taxonomic rank ("class" or even "phylum"). Here we review evidence for an important role of LGT in the evolution of photosynthesis, aerobic respiration, nitrogen fixation, sulfate reduction, methylotrophy, isoprenoid biosynthesis, quorum sensing, flotation (gas vesicles), thermophily, and halophily. Sometimes transfer of complex gene clusters may have been involved, whereas other times separate exchanges of many genes must be invoked.

An ultrastructural and molecular study of Hyperamoeba dachnaya, n. sp., and its relationship to the mycetozoan slime moulds

Hyperamoeba dachnaya, a new taxon affiliated with Hyperamoeba flagellata, is described from freshwater anaerobic sediments. Organisms exist as amoeboid flagellates, amoebae and cysts. The flagellates of H. dachnaya are distinguished from those of H. flagellata by larger size and a longer anteriorly-directed finger-shaped pseudopodium. H. dachnaya cysts have bilaminar walls with connectives between the outer and inner layers appearing to be T-shaped spines, while cyst walls of H. flagellata are smooth. H. dachnaya has two basal bodies and a single anteriorly-directed flagellum emerging from the anterior one (BB1). A fibrillar rootlet with a microtubule organising centre (MTOC; ‘R1’) arises from a diaphragm-shaped basal plate at the base of BB1; a short striated fibre (SSF) covers the dorsal side of BB1 and initiates microtubular rootlets ‘R2’ and ‘R3’ and the fibrillar bridge leading to microtubular rootlet ‘R4’. ‘R5’ is composed of 2 microtubules splitting from R4 and 2 others initiating by BB1. The posterior basal body is associated with a fibrillar posterior parakinetosomal structure. A long striated fibre arises in association with the right end of the SSF. A Golgi apparatus is associated with the flagellar apparatus. The flagellar apparatus is present internally in amoebae. All three stages have mitochondria with tubular cristae and an electron-dense central body. Analysis of small subunit ribosomal RNA sequences shows H. dachnaya branching with the myxogastrids within the mycetozoa, but polyphyletic with a nominal Hyperamoeba sp. isolate that is morphologically similar to H. flagellata. We reject the suggestion that H. flagellata and this Hyperamoeba sp. isolate of Zaman et al. (1999) are conspecific. The study reveals extensive similarities between Hyperamoeba and the flagellated stages of myxogastrids.

Phylogenetic position of Licnophora, Lechriopyla, and Schizocaryum, three unusual ciliates (phylum Ciliophora) endosymbiotic in echinoderms (phylum Echinodermata).

Various echinoderms are colonized by species from several classes of the Phylum Ciliophora, indicating that the echinoderm "habitat" has been invaded independently on numerous occasions throughout evolutionary history. Two "echinoderm" ciliates whose phylogenetic positions have been problematic are Licnophora macfarlandi Stevens, 1901 and Schizocaryum dogieli Poljansky and Golikova, 1957. Licnophora macfarlandi is an endosymbiont of the respiratory trees of holothuroids, and S. dogieli is found in the esophagus of echinoids. A third species, Lechriopyla mystax Lynch, 1930, is a plagiopylid ciliate found in the intestine of echinoids. Host echinoderms were collected near the Friday Harbor Laboratories, San Juan Island, WA. Specimens of S. dogieli and L. mystax were obtained from the esophagus and intestine, respectively, of the sea urchin Strongylocentrotus pallidus. Specimens of L. macfarlandi were collected from the fluid obtained from the respiratory trees of Parastichopus californicus. Using small subunit ribosomal RNA (SSrRNA) sequences of these three ciliates and a global alignment of SSrRNA sequences of other ciliates, we established the following. 1) Licnophora is a spirotrich ciliate, clearly related to the hypotrichs and stichotrichs; this is corroborated by its possession of macronuclear replication bands. 2) Lechriopyla is the sister genus to Plagiopyla and is a member of the Class Plagiopylea, which was predicted based on its cytology. 3) Schizocaryum clusters in the Class Oligohymenophorea and is most closely related to the scuticociliates; there are currently no morphological features known to relate Schizocaryum to the scuticociliates.

Molecular phylogenetic analysis of the avian malarial parasite Plasmodium (Novyella) juxtanucleare.

Plasmodium (Novyella) juxtanucleare is a widely distributed parasite that primarily infects chickens (Gallus gallus domesticus). All species of Novyella are characterized by very small schizonts, which in the case of P. juxtanucleare are always found juxtaposed to the erythrocyte nucleus, hence its name. Nearly complete small-subunit ribosomal RNA sequences have been obtained from 2 isolates of this species, and comparisons with other Plasmodium species have been made. Phylogenetic analysis reveals that this parasite is closely related to other avian-infecting Plasmodium species and that molecular relationships among the avian-infecting plasmodia do not correspond to their morphology-based subgeneric classifications.

Molecular characterisation of Babesia canis canis and Babesia canis vogeli from naturally infected European dogs

The morphologically small Babesia species isolated from naturally infected dogs in Europe, Japan, and US are described as Babesia gibsoni despite the fact that molecular techniques show that they should be assigned to two or three separate taxons. The morphologically large Babesia isolated from dogs in Europe, Africa, and US were generally classified as B. canis until it was proposed to distinguish three related, albeit genetically distinct subspecies of this genus, namely B. canis canis, B. canis rossi, and B. canis vogeli. The insight into the molecular taxonomy of canine piroplasms is, however, limited because only partial small subunit ribosomal RNA (ssrRNA) sequence data exist for two species from the B. canis group. In this work, we molecularly characterised natural Babesia infections in 11 dogs from Croatia, France, Italy, and Poland. These infections were diagnosed as caused by B. canis canis and B. canis vogeli based on the analysis of the complete sequence of the ssrRNA genes. Phylogenetic analysis confirmed that the large Babesia species of dogs belong the to the Babesia sensu stricto clade, which includes species characterised by transovarial transmission in the tick vectors and by exclusive development inside the mammalian host erythrocytes. The new data facilitate the reliable molecular diagnosis of the subspecies of B. canis.

A new phylum of Archaea represented by a nanosized hyperthermophilic symbiont.

According to small subunit ribosomal RNA (ss rRNA) sequence comparisons all known Archaea belong to the phyla Crenarchaeota, Euryarchaeota, and--indicated only by environmental DNA sequences--to the 'Korarchaeota'. Here we report the cultivation of a new nanosized hyperthermophilic archaeon from a submarine hot vent. This archaeon cannot be attached to one of these groups and therefore must represent an unknown phylum which we name 'Nanoarchaeota' and species, which we name 'Nanoarchaeum equitans'. Cells of 'N. equitans' are spherical, and only about 400 nm in diameter. They grow attached to the surface of a specific archaeal host, a new member of the genus Ignicoccus. The distribution of the 'Nanoarchaeota' is so far unknown. Owing to their unusual ss rRNA sequence, members remained undetectable by commonly used ecological studies based on the polymerase chain reaction. 'N. equitans' harbours the smallest archaeal genome; it is only 0.5 megabases in size. This organism will provide insight into the evolution of thermophily, of tiny genomes and of interspecies communication.

Morphological, small subunit rRNA, and physiological characterization of Trimyema minutum (Kahl, 1931), an anaerobic ciliate from submarine hydrothermal vents growing from 28 degrees C to 52 degrees C.

A thermophilic strain of Trimyema minutum was isolated from the hydrothermally heated sea floor at Vulcano Island (Italy) and cultivated monoxenically on Marinobacter sp. and Methanococcus thermolithotrophicus. It can be propagated strictly anaerobically and is sensitive to oxygen: if exposed to air at 48 degrees C all cells die within 60 min. It grows from 0.45-7.2% (w/v) salt and at pH 6.0-8.0. The isolate is the most extreme thermophilic ciliate which ever has been cultivated, exhibiting an optimal growth temperature of 48 degrees C (doubling time 6 h). Growth occurs between 28 degrees C and 52 degrees C. Trimyema minutum is redescribed using live observation and silver impregnation. Its morphology and the small subunit ribosomal RNA sequence is distinctly different from that of T. compressum, but morphology is highly similar to that of T. shoalsia Nerad et al. 1995, which is thus probably a junior synonym of T. minutum. To stabilize the bewildering species taxonomy in Trimyema, we suggest to recognize our population as a neotype of T. minutum.

Detection of Haplosporidium nelsoni in Pacific oyster Crassostrea gigas in Japan.

One hundred spat of Crassostrea gigas obtained from a coastal area in north-eastern Japan were examined for the protistan parasite Haplosporidium nelsoni. Haplosporidium-like plasmodia were histologically observed in two spat and positively reacted with a H. nelsoni-specific probe in in situ hybridization. Four spat including the two spat in which the plasmodia were found showed positive reaction in PCR analysis for the detection of H. nelsoni.The small subunit ribosomal RNA sequence amplified from the spat was virtually identical (99.7%) to the sequence of H. nelsoni previously reported. These results demonstrate that H. nelsoni is distributed in Japan.

The European database on small subunit ribosomal RNA.

The European database on SSU rRNA can be consulted via the World WideWeb at http://rrna.uia.ac.be/ssu/ and compiles all complete or nearly complete small subunit ribosomal RNA sequences. Sequences are provided in aligned format. The alignment takes into account the secondary structure information derived by comparative sequence analysis of thousands of sequences. Additional information such as literature references, taxonomy, secondary structure models and nucleotide variability maps, is also available.

Post-transcriptional modification in archaeal tRNAs: identities and phylogenetic relations of nucleotides from mesophilic and hyperthermophilic Methanococcales.

Post-transcriptional modifications in archaeal RNA are known to be phylogenetically distinct but relatively little is known of tRNA from the Methanococci, a lineage of methanogenic marine euryarchaea that grow over an unusually broad temperature range. Transfer RNAs from Methanococcus vannielii, Methanococcus maripaludis, the thermophile Methanococcus thermolithotrophicus, and hyperthermophiles Methanococcus jannaschii and Methanococcus igneus were studied to determine whether modification patterns reflect the close phylogenetic relationships inferred from small ribosomal subunit RNA sequences, and to examine modification differences associated with temperature of growth. Twenty-four modified nucleosides were characterized, including the complex tricyclic nucleoside wyosine characteristic of position 37 in tRNA(Phe) and known previously only in eukarya, plus two new wye family members of presently unknown structure. The hypermodified nucleoside 5-methylaminomethyl-2-thiouridine, reported previously only in bacterial tRNA at the first position of the anticodon, was identified by liquid chromatography-electrospray ionization mass spectrometry in four of the five organisms. The ribose-methylated nucleosides, 2'-O-methyladenosine, N(2),2'-O-dimethylguanosine and N(2),N(2),2'-O-trimethylguanosine, were found only in hyperthermophile tRNA, consistent with their proposed roles in thermal stabilization of tRNA.

Molecular characterisation of the Theileria buffeli/orientalis group

Benign bovine Theileria parasites known as either Theileria buffeli, Theileria orientalis or Theileria sergenti are classified on basis of their morphology, vector specificity, pathogenicity and 18S small subunit ribosomal RNA or major piroplasm protein (MPSP) sequences. Since most isolates have been characterized on only some of these criteria and the existing confusion in nomenclature, an analysis was performed on eight different isolates to combine 18S rRNA data with MPSP data and the results were compared with available biological parameters. A polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) approach for both genes was used in combination with reverse line blot hybridisation for the 18S rRNA gene. Both MPSP and 18S rRNA genes were cloned and sequenced from parasites displaying aberrant MPSP RFLP profiles. Phylogeny based on published and determined 18S rRNA and MPSP sequences did correlate within the same isolate but there was no obvious correlation between molecular and biological data. Based on these findings, we suggest that the appropriate name for all these parasites is Theileria buffeli. A more specific nomenclature should be assigned when new molecular markers may become available.

Phylogenetic analysis of the small subunit ribosomal RNA gene of the hyphochytrid Rhizidiomyces apophysatus

The potential relationship of the uniflagellate hyphochytrids with biflagellate stramenopiles is still not clearly resolved. Thus, we have expanded the number of isolates and taxa of hyphochytrids compared. The phylogenetic analysis of the small subunit ribosomal RNA sequence of Rhizidiomyces apophysatus Zopf based on neighbour-joining and parsimony methods showed that Hyphochytrium catenoides Karling and R. apophysatus are monophyletic and probably the closest relatives of the oomycetes. Our data also confirmed the monophyly of the stramenopiles, which includes heterokont algae along with nonphotosynthetic fungallike organisms, namely the Oomycota, Hyphochytriomycota, and Labyrinthulomycota.Key words: Hyphochytriomycota, heterokont algae, phylogeny, small subunit ribosomal gene.

Ribosomal small subunit sequence variation within spores of an arbuscular mycorrhizal fungus, Scutellospora sp.

Roots of bluebell (Hyacinthoides nonscripta) were sampled from a woodland in Yorkshire, UK and spores of an arbuscular mycorrhizal fungus Scutellospora sp., were obtained from the surrounding soil. Partial small subunit (SSU) ribosomal RNA sequences were amplified from both roots and spores using either the universal forward primer SS38 or the Glomales-specific primer VANS1, with the reverse Gigasporaceae-specific primer VAGIGA. Amplified products were cloned and sequenced. Both spores and roots yielded sequences related to those known from fungi within the Glomales, with up to four distinct SSU sequences obtained from individual spores. The VANS1 primer-binding site varied considerably in sequence and only a subset of Scutellospora sequences were amplified when the VANS1 primer was used. In addition to glomalean sequences, a number of different sequences, apparently from ascomycetes, were obtained from both root and spore samples.

The Rice Inoculant Strain Alcaligenes faecalis A15 is a Nitrogen-fixing Pseudomonas stutzeri

The taxonomic position of the nitrogen-fixing rice isolate A15, previously classified as Alcaligenes faecalis, was reinvestigated. On the basis of its small subunit ribosomal RNA (16S rRNA) sequence this strain identifies as Pseudomonas stutzeri. Phenotyping and fatty acid profiling confirm this result. DNA:DNA hybridisations, using the optical renaturation rate method, between strain A15 and Pseudomonas stutzeri LMG 11199T revealed a mean DNA-binding of 77%. The identification was further corroborated by comparative sequence analysis of the oprF gene, which encodes the major outer membrane protein of rRNA homology group I pseudomonads. Furthermore we determined the nifH sequence of this strain and of two putative diazotrophic Pseudomonas spp. and made a comparative analysis with sequences of other diazotrophs. These Pseudomonas NifH sequences cluster with NifH sequences isolated from the rice rhizosphere by PCR and of proteobacteria from the beta and gamma subclasses.

Phylogenetic analysis of Theileria and Babesia equi in relation to the establishment of parasite populations within novel host species and the development of diagnostic tests

The divergence of parasites is important for maintenance within an established host and spread to novel host species. In this paper we have carried out phylogenetic analyses of Theileria parasites isolated from different host species. This was performed with small subunit ribosomal RNA sequences available in the data bases and a novel sequence amplified from Theileria lestoquardi DNA. Similar phylogenetic studies were carried out with sequences representing the major merozoite/piroplasm surface antigen (mMPSA) from the data base, and novel sequences representing 2 mMPSA alleles from T. lestoquardi, a full length sequence of a Theileria taurotragi mMPSA gene and partial sequences of two new allelic variants of the Babesia equi mMPSA gene homologue. The analysis indicated that the pathogenic sheep parasite T. lestoquardi has most probably evolved from a common ancestor of T. annulata. Interestingly, the level of mMPSA sequence diversity found for T. lestoquardi was surprisingly low, while diversity between the B. equi sequences was higher than that found within any of the classical Theileria species. The possible implications of these results for the establishment of Theileria parasites within novel species are discussed. Extensive cross-reactivity of a range of antisera was found when tested against recombinant mMPSA polypeptides from different Theileria (including B. equi) species. The cross-reactivity between mMPSA polypeptides and sequence diversity are relevant for the development of species specific diagnostic tests.

Phylogenetic identification of the symbiotic hypermastigote Trichonympha agilis in the hindgut of the termite Reticulitermes speratus based on small-subunit rRNA sequence.

The phylogeny of a symbiotic hypermastigote Trichonympha agilis (class Parabasalia; order Hypermastigida) in the hindgut of the lower termite Reticulitermes speratus was examined by a strategy that does not rely on cultivation. From mixed-population DNA obtained from the termite gut, small subunit (16S-like) ribosomal RNA sequences were directly amplified by the polymerase chain reaction method using primers specific for eukaryotes. Comparative sequence analysis of the clones revealed two kinds of sequences, one from the termite itself and the other from a symbiotic protist. A fluorescent-labeled oligonucleotide probe for the latter sequence was designed and used in whole-cell hybridization experiments to provide direct visual evidence that the sequence originated from a larger hypermastigote in the termite hindgut, Trichonympha agilis. According to the phylogenetic trees constructed, the hypermastigote represented one of the deepest branches of eukaryotes. The hypermastigote along with members of the order Trichomonadida formed a monophyletic lineage, indicating that this hypermastigote and trichomonads shared a recent common ancestry.

Patterns and governing forces in aquatic microbial communities

In this review we survey recent publications employing molecular techniques to investigate the distribution of microbial species in aquatic environments. We analyzed the occurrence of microbial phyla in freshwater and marine habitats and observed patterns of distribution that could be explained by the adaptation of microorganisms to physical and biological parameters that vary in aquatic habitats. The gram-positive bacteria, the Verrucomicrobiales and the -a nd-subdivisions of the Proteobacteria are distributed throughout a range of aquatic habitats, while other phylogenetic groups appear to be adapted to more narrowly defined environmental niches such as anoxic water and sediments (-Proteobacteria) or floating aggregates ( Cytophaga-Flexibacter-Bacteroidesphylum). proteobacterial sequence types have been detected throughout freshwater habitats, but these organisms are largely absent from open ocean environments. Within several of these divisions, clusters of closely related small subunit ribosomal RNA sequence types have been detected in geographically disparate environments, suggesting that some microbial species are globally distributed. In addition to physical variables such as salinity and pH, biological variables also influence microbial community composition. This was illustrated by changes that occurred in the eukaryotic and bacterial species composition in laboratory mesocosms after a viral outburst. We conclude that physical and biological forces govern the composition of aquatic microbial communities and result in divergent evolutionary histories of the indigenous microbial species.

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.

Phylogenesis of pit-plug-associated features in the Rhodophyta: inferences from molecular systematic data

Small-subunit ribosomal RNA sequence data are presented for Rhodogorgon carriebowensis Norris et Bucher (Rhodogorgonales, Rhodophyta) and Galaxaura marginata (Ellis et Solander) Lamouroux (Nemaliales, Rhodophyta). Data for these species were included in a matrix consisting of small-subunit ribosomal RNA sequences for 70 taxonomically diverse red algal species. Distance, parsimony, and maximum-likelihood analyses of these data all strongly support (100% of bootstrap replicates for distance and parsimony) a close alliance between the Rhodogorgonales and Corallinales. Our alignment includes 19 species representing six florideophyte orders (Acrochaetiales, Batrachospermales, Corallinales, Nemaliales, Palmariales, Rhodogorgonales) in which pit plugs are characterized by two cap layers. Distance and parsimony analyses resolve a monophyletic lineage including all "two-cap-layer" species. Our parsimony-derived molecular phylogeny was used to test a published hypothesis of pit-plug evolution. Four aspects of Pueschel's hypothesis are consistent with the results of our molecular systematic study: (i) "naked" plugs represent the ancestral type, (ii) outer cap layers are homologous structures, (iii) domed outer caps are ancestral to plate-like outer caps, and (iv) cap membranes are a derived feature within the two-cap-layer lineage. Directions for future research are discussed. Key words: Acrochaetiales, Batrachospermales, Corallinales, molecular systematics, Nemaliales, Palmariales, phylogeny, pit plug, Rhodogorgonales, Rhodophyta.