Phylogenetic position of the copepod-infesting parasite Syndinium turbo (Dinoflagellata, Syndinea)

Sequences were determined for the nuclear-encoded small subunit (SSU) rRNA and 5.8S rRNA genes as well as the internal transcribed spacers ITS1 and ITS2 of the parasitic dinoflagellate genus Syndinium from two different marine copepod hosts. Syndinium developed a multicellular plasmodium inside its host and at maturity free-swimming zoospores were released. Syndinium plasmodia in the copepod Paracalanus parvus produced zoospores of three different morphological types. However, full SSU rDNA sequences for the three morphotypes were 100% identical and also their ITS1-ITS2 sequences were identical except for four base pairs. It was concluded that the three morphotypes belong to a single species that was identified as Syndinium turbo, the type species of the dinoflagellate subdivision Syndinea. The SSU rDNA sequence of another Syndinium species infecting Corycaeus sp. was similar to Syndinium turbo except for three base pairs and the ITS1-ITS2 sequences of the two species differed at 34-35 positions. Phylogenetic analyses placed Syndinium as a sister taxon to the blue crab parasite Hematodinium sp. and both parasites were affiliated with the so-called marine alveolate Group II. This corroborates the hypothesis that marine alveolate Group II is Syndinea.     

Ribosomal RNA genes in Scots pine (Pinus sylvestris L.): Chromosomal organization and structure

The nuclear 18S, 5.8S and 25S rRNA genes exist as thousands of rDNA repeats in the Scots pine genome. The number and location of rDNA loci (nucleolus organizers, NORs) were studied by cytological methods, and a restriction map from the coding region of the Scots pine rDNA repeat was constructed using digoxigenin-labeled flax rDNA as a probe. Based on the maximum number of nucleoli and chromosomal secondary constrictions, Scots pine has at least eight NORs in its haploid genome. The size of the Scots pine rDNA repeat unit is approximately 27 kb, two- or threefold larger than the typical angiosperm rDNA unit, but similar in size to other characterized conifer rDNA repeats. The intergenic spacer region (IGS) of the rDNA repeat unit in Scots pine is longer than 20 kb, and the transcribed spacer regions surrounding the 5.8S gene (ITS1 and ITS2) span a region of 2.9 kb. Restriction analysis revealed that although the coding regions of rDNA repeats are homogeneous, heterogeneity exists in the intergenic spacer region between individuals, as well as among the rDNA repeats within individuals.     

The 5S ribosomal RNA gene in Pythium species: two different genomic locations.

The 5S ribosomal RNA (rRNA) genes in eukaryotes may occur either interspersed with the other rRNA genes in the ribosomal DNA (rDNA) repeat, or in separate tandem arrays, or dispersed throughout the genome. In Pythium species and in several related Oomycetes, polymerase chain reaction (PCR) amplification of the nontranscribed spacer (NTS) region with one primer specific for the 5S gene revealed, with several exceptions, that the 5S rRNA gene was present in the rDNA repeat of those species with filamentous sporangia and was absent from the rDNA repeat of those with globose or unknown sporangia. When present, the gene was located approximately 1 kb downstream of the large-subunit rRNA gene and on the strand opposite that on which the other rRNA genes were located. This was confirmed in P. torulosum by sequencing of the gene and its flanking regions. The 5S rRNA genes of P. ultimum were found in tandem arrays outside the rDNA repeat. Evidence of dispersed 5S rRNA sequences was also observed. For many of the species of Pythium having the 5S gene in the NTS, the region between the large-subunit rRNA gene and the 5S gene was found to have length heterogeneity. Oomycetes related to Pythium were also found to have the 5S gene in the NTS, although sometimes in the opposite orientation. This may mean that the presence of the gene in the NTS is ancestral for the Oomycetes and that the absence of the gene in the NTS in those Pythiums with globose sporangia is due to loss of the gene from the rDNA repeat in an ancestor of this group of species. These results favor the view that 5S rRNA gene linkage to the rRNA cistron existed prior to the unlinked arrangement seen in most plants and animals.     

Identity and systematic position of Paradinium poucheti and other Paradinium-like parasites of marine copepods based on morphology and nuclear-encoded SSU rDNA

Paradinium and Paradinium-like parasites were detected in various copepod hosts collected in the NW Mediterranean Sea, the North Atlantic Ocean, and the Godth?bsfjord (Greenland). The identity and systematic position of the parasitic, plasmodial protist Paradinium was investigated on the basis of SSU rDNA and morphology. SSU rDNA sequences were obtained from 3 specimens of Paradinium poucheti isolated from their cyclopoid copepod host, Oithona similis. In addition, a comparable sequence was obtained from a hitherto undescribed species of Paradinium from the harpactacoid copepod Euterpina acutifrons. Finally, SSU rDNA sequences were acquired from 2 specimens of a red plasmodial parasite (RP parasite) isolated from Clausocalanus sp. Both morphological and SSU rDNA sequence data supported that P. poucheti and Paradinium sp. are closely related organisms. In phylogenetic analyses based on SSU rDNA sequences, Paradinium spp. clustered with sequences from an uncultured eukaryote clone from the Pacific Ocean and two sequences from haplosporidian-like parasites of shrimps, Pandalus spp. This Paradinium clade branched as a sister group to a clade comprising the Haplosporidia and the Foraminifera. The RP parasite had a superficial morphological resemblance to Paradinium and has previously been interpreted as a member of this genus. However, several morphological characters contradict this and SSU rDNA sequence data disagree with the RP parasite and Paradinium being related. The phylogenetic analyses suggested that the RP parasite is a fast-evolved alveolate and a member of the so-called marine alveolate Group I (MAGI) and emerging data now suggest that this enigmatic group may, like the syndinian dinoflagellates, consist of heterotrophic parasites.     

Molecular data and the evolutionary history of dinoflagellates

We have sequenced small-subunit (SSU) ribosomal RNA (rRNA) genes from 16 dinoflagellates, produced phylogenetic trees of the group containing 105 taxa, and combined small- and partial large-subunit (LSU) rRNA data to produce new phylogenetic trees. We compare phylogenetic trees based on dinoflagellate rRNA and protein genes with established hypotheses of dinoflagellate evolution based on morphological data. Protein-gene trees have too few species for meaningful in-group phylogenetic analyses, but provide important insights on the phylogenetic position of dinoflagellates as a whole, on the identity of their close relatives, and on specific questions of evolutionary history. Phylogenetic trees obtained from dinoflagellate SSU rRNA genes are generally poorly resolved, but include by far the most species and some well-supported clades. Combined analyses of SSU and LSU somewhat improve support for several nodes, but are still weakly resolved. All analyses agree on the placement of dinoflagellates with ciliates and apicomplexans (=Sporozoa) in a well-supported clade, the alveolates. The closest relatives to dinokaryotic dinoflagellates appear to be apicomplexans, Perkinsus, Parvilucifera, syndinians and Oxyrrhis. The position of Noctiluca scintillans is unstable, while Blastodiniales as currently circumscribed seems polyphyletic. The same is true for Gymnodiniales: all phylogenetic trees examined (SSU and LSU-based) suggest that thecal plates have been lost repeatedly during dinoflagellate evolution. It is unclear whether any gymnodinialean clades originated before the theca. Peridiniales appear to be a paraphyletic group from which other dinoflagellate orders like Prorocentrales, Dinophysiales, most Gymnodiniales, and possibly also Gonyaulacales originated. Dinophysiales and Suessiales are strongly supported holophyletic groups, as is Gonyaulacales, although with more modest support. Prorocentrales is a monophyletic group only in some LSU-based trees. Within Gonyaulacales, molecular data broadly agree with classificatory schemes based on morphology. Implications of this taxonomic scheme for the evolution of selected dinoflagellate features (the nucleus, mitosis, flagella and photosynthesis) are discussed.     

Molecular and morphological characterization of a poorly known marine ciliate, Myoschiston duplicatum precht 1935: implications for phylogenetic relationships between three morphologically similar genera -- Zoothamnium, Myoschiston, and Zoothamnopsis (Ciliophora, Peritrichia, Zoothamniidae)

We studied the morphology and molecular phylogeny of Myoschiston duplicatum, a peritrich ciliate that has been recorded as an epibiont of crustaceans, but which we also identified on marine algae from Korea. The important morphological characteristics revealed by silver staining of Myoschiston species have not been described because they are rarely collected. Using morphological methods, we redescribed the type species of the genus, Myoschiston duplicatum, and provided an improved diagnosis of Myoschiston. In addition, the coding regions for nuclear small subunit (SSU) rRNA and internal transcribed spacer 1‐5.8S‐internal transcribed spacer 2 sequences were sequenced. Phylogenetic analyses that included available SSU rDNA sequences of peritrichs from GenBank strongly supported a position of M. duplicatum within the family Zoothamniidae. In addition, phylogenetic analyses were performed with single datasets (ITS1‐5.8S‐ITS2) and combined datasets (SSU rDNA + ITS1‐5.8S‐ITS2) to explore further the phylogenetic relationship in the family Zoothamniidae between the three morphologically similar genera—Zoothamnium, Myoschiston, and Zoothamnopsis.     

Phylogenetic analysis of the Metastrongyloidea (Nematoda: Strongylida) inferred from ribosomal RNA gene sequences

Phylogenetic relationships among nematodes of the strongylid superfamily Metastrongyloidea were analyzed using partial sequences from the large-subunit ribosomal RNA (LSU rRNA) and small-subunit ribosomal RNA (SSU rRNA) genes. Regions of nuclear ribosomal DNA (rDNA) were amplified by polymerase chain reaction, directly sequenced, aligned, and phylogenies inferred using maximum parsimony. Phylogenetic hypotheses inferred from the SSU rRNA gene supported the monophyly of representative taxa from each of the 7 currently accepted metastrongyloid families. Metastrongyloid taxa formed the sister group to representative trichostrongyloid sequences based on SSU data. Sequences from either the SSU or LSU RNA regions alone provided poor resolution for relationships within the Metastrongyloidea. However, a combined analysis using sequences from all rDNA regions yielded 3 equally parsimonious trees that represented the abursate Filaroididae as polyphyletic, Parafilaroides decorus as the sister species to the monophyletic Pseudaliidae, and a sister group relationship between Oslerus osleri and Metastrongylus salmi. Relationships among 3 members of the Crenosomatidae, and 1 representative of the Skrjabingylidae (Skrjabingylus chitwoodorum) were not resolved by these combined data. However, members of both these groups were consistently resolved as the sister group to the other metastrongyloid families. These relationships are inconsistent with traditional classifications of the Metastrongyloidea and existing hypotheses for their evolution.     

Structural characteristics and possible horizontal transfer of group I introns between closely related plant pathogenic fungi.

We have characterized structural features and the distribution pattern of nuclear group I introns found in ribosomal DNA (rDNA) of closely related plant pathogenic fungi of the family Sclerotiniaceae. Sixteen introns, at two distinct positions in the small-subunit (SSU) and large-subunit (LSU) rDNA, were sequenced and analyzed among the 29 taxa included in the initial screening. Genera found to contain introns were Botrytis, Dumontinia, Encoelia, Grovesinia, Myriosclerotinia, and Sclerotinia. Secondary-structure analyses of the group I introns concluded that all belong to the common IC1 subclass. Interestingly, the SSU rDNA intron from Myriosclerotinia caricisampullacea contains an insertion-like sequence extension which may be a relic of an open reading frame. Incongruent branching patterns of intron-based and rDNA-based (internal transcribed spacer) phylogenetic trees suggest that the fungal host genomes and the group I introns do not share a common evolutionary history. A model to explain how horizontal intron transfers may have occurred among the closely related fungal taxa is proposed.     

rRNA genes from the lower chordate Herdmania momus: structural similarity with higher eukaryotes.

Ascidians, primitive chordates that have retained features of the likely progenitors to all vertebrates, are a useful model to study the evolutionary relationship of chordates to other animals. We have selected the well characterized ribosomal RNA (rRNA) genes to investigate this relationship, and we describe here the cloning and characterization of an entire ribosomal DNA (rDNA) tandem repeat unit from a lower chordate, the ascidian Herdmania momus. rDNA copy number and considerable sequence differences were observed between two H. momus populations. Comparison of rDNA primary sequence and rRNA secondary structures from H. momus with those from other well characterized organisms, demonstrated that the ascidians are more closely related to other chordates than invertebrates. The rDNA tandem repeat makes up a larger percentage (7%) of the genome of this animal than in other higher eukaryotes. The total length of the spacer and transcribed region in H. momus rDNA is small compared to most higher eukaryotes, being less than 8 kb, and the intergenic spacer region consists of smaller internal repeats. Comparative analysis of rDNA sequences has allowed the construction of secondary structures for the 18S, 5.8S and 26S rRNAs.     

Nucleotide sequence of the 17S–25S spacer region from rice rDNA

Summary The nucleotide sequence of a spacer region between rice 17S and 25S rRNA genes (rDNAs) has been determined. The coding regions for the mature 17S, 5.8S and 25S rRNAs were identified by sequencing terminal regions of these rRNAs. The first internal transcribed spacer (ITS1), between 17S and 5.8S rDNAs, is 194–195 bp long. The second internal transcribed spacer (ITS2), between 5.8S and 25S rDNAs, is 233 bp long. Both spacers are very rich in G+C, 72.7% for ITS1 and 77.3% for ITS2. The 5.8S rDNA is 163–164 bp long and similar in primary and secondary structures to other eukaryotic 5.8S rDNAs. The 5.8S rDNA is capable of interacting with the 5′ terminal region of 25S rDNA.     

The crustacean parasites <Emphasis Type="Italic">Ellobiopsis</Emphasis> Caullery, 1910 and <Emphasis Type="Italic">Thalassomyces</Emphasis> Niezabitowski, 1913 form a monophyletic divergent clade within the Alveolata

The Ellobiopsidae are enigmatic parasites of crustaceans that have been grouped together exclusively on the basis of morphological similarities. Ultrastructural studies have revealed their affiliation within the alveolates, which was confirmed by the phylogenetic analysis of the ribosomal RNA gene (SSU rDNA) sequences of two species of Thalassomyces Niezabitowski, 1913. However, their precise systematic position within this group remains unresolved, since they could not be definitively allied with any particular alveolate group. To better determine the systematic position of ellobiopsids by molecular phylogeny, we sequenced the SSU rDNA from the type-species of the Ellobiopsidae, Ellobiopsis chattoni Caullery, 1910. We found E. chattoni infecting various copepod hosts, Acartia clausi Giesbrecht, Centropages typicus Kröyer and Clausocalanus sp., in the Bay of Marseille, NW Mediterranean Sea, which allowed us to study several stages of the parasite development. A single unicellular multinucleate specimen provided two different sequences of the SSU rDNA gene, indicating the existence of polymorphism at this locus within single individuals. Ellobiopsis Caullery, 1910 and Thalassomyces formed a very divergent and well-supported clade in phylogenetic analyses. This clade appears to be more closely related to the dinoflagellates (including the Syndiniales/Marine Alveolate Group II and the Dinokaryota) and Marine Alveolate Group I than to the other alveolates (Ciliophora, Perkinsozoa and Apicomplexa).     

Secondary structure prediction for complete rDNA sequences (18S, 5.8S,and 28S rDNA) of Demodex folliculorum, and comparison of divergent domains structures across Acari

According to base pairing, the rRNA folds into corresponding secondary structures, which contain additional phylogenetic information. On the basis of sequencing for complete rDNA sequences (18S, ITS1, 5.8S, ITS2 and 28S rDNA) of Demodex, we predicted the secondary structure of the complete rDNA sequence (18S, 5.8S, and 28S rDNA) of Demodex folliculorum, which was in concordance with that of the main arthropod lineages in past studies. And together with the sequence data from GenBank, we also predicted the secondary structures of divergent domains in SSU rRNA of 51 species and in LSU rRNA of 43 species from four superfamilies in Acari (Cheyletoidea, Tetranychoidea, Analgoidea and Ixodoidea). The multiple alignment among the four superfamilies in Acari showed that, insertions from Tetranychoidea SSU rRNA formed two newly proposed helixes, and helix c3-2b of LSU rRNA was absent in Demodex (Cheyletoidea) taxa. Generally speaking, LSU rRNA presented more remarkable differences than SSU rRNA did, mainly in D2, D3, D5, D7a, D7b, D8 and D10.     

Complex structure of the ribosomal DNA spacer of Cucumis sativus (cucumber)

Summary The nuclear 18 S, 5.8 S and 25 S ribosomal RNA genes (rDNA) of Cucumis sativus (cucumber) occur in at least four different repeat types of 10.2, 10.5, 11.5, and 12.5 kb in length. The intergenic spacer of these repeats has been cloned and characterized with respect to sequence organization. The spacer structure is very unusual compared to those of other eukaryotes. Duplicated regions of 197 bp and 311 bp containing part of the 3 end of the 25 S rRNA coding region and approximately 470 bp of 25 S rRNA flanking sequences occur in the intergenic spacer. The data from sequence analysis suggest that these duplications originate from recombination events in which DNA sequences of the original rDNA spacer were paired with sequences of the 25 S rRNA coding region. The duplicated 3ends of the 25 S rRNA are separated from each other mostly by a tandemly repeated 30 bp element showing a high GC-content of 87.5%. In addition, another tandemly repeated sequence of 90 bp was found downstream of the 3flanking sequences of the 25 S rRNA coding region. These results suggest that rRNA coding sequences can be involved in the generation of rDNA spacer sequences by unequal crossing over.     

Phylogenetic validation of the genera Angomonas and Strigomonas of trypanosomatids harboring bacterial endosymbionts with the description of new species of trypanosomatids and of proteobacterial symbionts

We comparatively examined the nutritional, molecular and optical and electron microscopical characteristics of reference species and new isolates of trypanosomatids harboring bacterial endosymbionts. Sequencing of the V7V8 region of the small subunit of the ribosomal RNA (SSU rRNA) gene distinguished six major genotypes among the 13 isolates examined. The entire sequences of the SSU rRNA and glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) genes were obtained for phylogenetic analyses. In the resulting phylogenetic trees, the symbiont-harboring species clustered as a major clade comprising two subclades that corresponded to the proposed genera Angomonas and Strigomonas. The genus Angomonas comprised 10 flagellates including former Crithidia deanei and C. desouzai plus a new species. The genus Strigomonas included former Crithidia oncopelti and Blastocrithidia culicis plus a new species. Sequences from the internal transcribed spacer of ribosomal DNA (ITS rDNA) and size polymorphism of kinetoplast DNA (kDNA) minicircles revealed considerable genetic heterogeneity within the genera Angomonas and Strigomonas. Phylogenetic analyses based on 16S rDNA and ITS rDNA sequences demonstrated that all of the endosymbionts belonged to the Betaproteobacteria and revealed three new species. The congruence of the phylogenetic trees of trypanosomatids and their symbionts support a co-divergent host-symbiont evolutionary history.     

Organization of ribosomal genes in Paramecium tetraurelia

The macronuclear ribosomal DNA (rDNA) of the ciliated protozoan Paramecium tetraurelia (stock 51) was analyzed by digestion with restriction endonucleases. The fragments which contained ribosomal RNA (rRNA) coding sequences and spacer sequences were identified. The spacer sequences exhibited some heterogeneity in size. The genes coding for 5.8S RNA, but not for 5S RNA, are linked to the 17S and 25S rRNA genes. Complementary RNA, synthesized from rDNA of stock 51, was hybridized with restriction digests of whole cell DNA from six other allopatric stocks of this species. The restriction patterns of the rDNA from these seven stocks were, in general, very similar, and the sizes of the coding sequences were identical in all seven stocks. Only the restriction pattern of rDNA from stock 127 differed significantly from that of stock 51. The rDNA from stock 127 was isolated and characterized, and with the exception of the restriction pattern of its spacer, it resembled the rDNA from stock 51. It is concluded that the rDNA repeat in Paramecium, including the spacer, has, in general, been conserved during the course of evolution. It is suggested that in some species, even in the absence of genetic exchange among geographically separated populations, selection pressure may act to conserve spacers of tandemly repeated rDNA. The conservation may be related to the number of rDNA copies in the germinal nucleus.     

Phylogenetic and genomic relationships in Setaria italica and its close relatives based on the molecular diversity and chromosomal organization of 5S and 18S-5.8S-25S rDNA genes

We have analyzed the phylogenetic and genomic relationships in the genus Setaria Beauv. including diploid and tetraploid species, by means of the molecular diversity of the 5S rDNA spacer and chromosomal organization of the 5S and 18S-5.8S-25S rDNA genes. PCR amplification of the 5S rDNA sequences gave specific patterns. All the species studied here share a common band of about 340 bp. An additional band of an approximately 300-bp repeat unit was found for Setaria verticillata and the Chinese accessions of Setaria italica and Setaria viridis. An additional band of 450 bp was found in the sole species Setaria faberii. Fluorescent in situ hybridization was used for physical mapping of the 5S and 18S-5.8S-25S rDNA genes and showed that they are localized at two separate loci with no polymorphism of chromosome location among species. Two chromosome pairs carrying the 5S and 18S-5.8S-25S rDNA clusters can now be unambiguously identified using FISH. Phylogenetic trees based on the variation of the amplified 5S rDNA sequences showed a clear separation into four groups. The clustering was dependent on the genomic composition (genome A versus genome B) and confirmed the closest relationship of S. italica and S. viridis accessions from the same geographical region. Our results confirm previous hypotheses on the domestication centers of S. italica. They also show the wide difference between the A and B genomes, and even clarify the taxonomic position of S. verticillata. Received: 28 August 2000 / Accepted: 27 January 2001