Identification of group I introns within the SSU rDNA gene in species of Ceratocystiopsis and related taxa

During a recent phylogenetic study, group I introns were noted that interrupt the nuclear small subunit ribosomal RNA (SSU rDNA) gene in species of Ceratocystiopsis. Group I introns were found to be inserted at the following rDNA positions: S943, S989, and S1199. The introns have been characterized and phylogenetic analysis of the host gene and the corresponding intron data suggest that for S943 vertical transfer and frequent loss appear to be the most parsimonious explanation for the distribution of nuclear SSU rDNA introns among species of Ceratocystiopsis. The SSU rDNA data do suggest that a recent proposal of segregating the genus Ophiostoma sensu lato into Ophiostoma sensu stricto, Grosmannia, and Ceratocystiopsis has some merit but may need further amendments, as the SSU rDNA suggests that Ophiostoma s. str. may now represent a paraphyletic grouping.     

A rapid method for differentiating Saccharomyces sensu stricto strains from other yeast species in an enological environment

During programs for the selection of enological yeasts, several hundred natural isolates are usually screened. The scope of these operations is to isolate strains possessing good fermentative properties without necessarily arriving at a precise species designation: in other words, to detect strains belonging to the Saccharomyces sensu stricto complex. In the present study, a pair of primers, designed within the variable D1/D2 region of the 26S subunit of ribosomal yeast RNA, have been constructed. These generate an amplification fragment of 471 bp that is specific for the seven Saccharomyces sensu stricto species, while no signal was obtained for Saccharomyces sensu lato strains (17 species) or for another 18 selected species commonly found in enological environments. A second pair of primers was also constructed, within the 18S rRNA gene, composed of perfectly conserved sequences common for all 42 yeast species examined, which generate a 900 bp (c.) band for all strains. This was used as a positive experimental control in multiplex PCR analysis using all four primers.     

Metarhiziopsis microspora gen. et sp. nov. associated with the elongate hemlock scale

A sporodochial fungus collected from the elongate hemlock scale, Fiorinia externa (Ferris) in Coventry, Connecticut, is described. This fungus has characteristics of both Metarhizium and Myrothecium but develops setae surrounding white to buff sporodochia and dry conidia in chains, a combination of characters found in neither genus. Phylogenetic analyses of the complete small subunit ribosomal DNA (ssu), partial ef1-alpha, and complete 5.8S ribosomal DNA and internal transcribed spacers (ITS) 1 and 2 shows that the fungus is allied with a subclade within Cordyceps including the species C. agriota, which places this fungus in the Hypocreales, Clavicipitaceae sensu lato or the newly erected Ophioclavicipitaceae. Morphological observation and molecular analysis indicate that this fungus is sufficiently different from Metarhizium and Myrothecium to warrant the erection of a new anamorphic genus. Therefore Metarhiziopsis microspora gen. et sp. nov. is proposed.     

Morphology and phylogeny of a new urostylid ciliate, Monocoronella carnea n. g., n. sp. (Ciliophora, Hypotricha) from Daya Bay, Southern China

The morphology, infraciliature and small subunit ribosomal RNA gene-based phylogeny of an urostylid ciliate, Monocoronella carnea n. g., n. sp., found in coastal areas off Daya Bay, Southern China, were investigated. The new genus Monocoronella n. g. is recognized by the following features: having conspicuous frontal cirri forming a long and single corona; buccal and frontoterminal cirri present; single marginal row on each side; adoral zone, midventral complex and transverse cirri in Pseudokeronopsis mode. The type species M. carnea n. sp. is diagnosed by the combination of marine habitat and brown-reddish color of the cortical granules. Phylogenetic analyses for the new taxon indicate that Monocoronella n. g. is most closely related to Bergeriella, and is located within the core Urostylida clade. A misidentification in previous literature was recognized and a new species, Monocoronella dragescoi n. sp. [Basionym: Holosticha (Keronopsis) monilata (Kahl 1928) sensu Dragesco (1970) et sensu Dragesco and Dragesco-Kernéis (1986), non sensu Kahl (1928)], was suggested.     

Sixteen discrete RNA components in the cytoplasmic ribosome of Euglena gracilis

We have isolated cytoplasmic ribosomes from Euglena gracilis and characterized the RNA components of these particles. We show here that instead of the four rRNAs (17-19 S, 25-28 S, 5.8 S and 5 S) found in typical eukaryotic ribosomes, Euglena cytoplasmic ribosomes contain 16 RNA components. Three of these Euglena rRNAs are the structural equivalents of the 17-19 S, 5.8 S and 5 S rRNAs of other eukaryotes. However, the equivalent of 25-28 S rRNA is found in Euglena as 13 separate RNA species. We demonstrate that together with 5 S and 5.8 S rRNA, these 13 RNAs are all components of the large ribosomal subunit, while a 19 S RNA is the sole RNA component of the small ribosomal subunit. Two of the 13 pieces of 25-28 S rRNA are not tightly bound to the large ribosomal subunit and are released at low (0 to 0.1 mM) magnesium ion concentrations. We present here the complete primary sequences of each of the 14 RNA components (including 5.8 S rRNA) of Euglena large subunit rRNA. Sequence comparisons and secondary structure modeling indicate that these 14 RNAs exist as a non-covalent network that together must perform the functions attributed to the covalently continuous, high molecular weight, large subunit rRNA from other systems.     

Classification of genus Pseudomonas by MALDI-TOF MS based on ribosomal protein coding in S10-spc-alpha operon at strain level

We have proposed a rapid phylogenetic classification at the strain level by MALDI-TOF MS using ribosomal protein matching profiling. In this study, the S10-spc-alpha operon, encoding half of the ribosomal subunit proteins and highly conserved in eubacterial genomes, was selected for construction of the ribosomal protein database as biomarkers for bacterial identification by MALDI-TOF MS analysis to establish a more reliable phylogenetic classification. Our method revealed that the 14 reliable and reproducible ribosomal subunit proteins with less than m/z 15,000, except for L14, coded in the S10-spc-alpha operon were significantly useful biomarkers for bacterial classification at species and strain levels by MALDI-TOF MS analysis of genus Pseudomonas strains. The obtained phylogenetic tree was consisted with that based on genetic sequence (gyrB). Since S10-spc-alpha operons of genus Pseudomonas strains were sequenced using specific primers designed based on nucleotide sequences of genome-sequenced strains, the ribosomal subunit proteins encoded in S10-spc-alpha operon were suitable biomarkers for construction and correction of the database. MALDI-TOF MS analysis using these 14 selected ribosomal proteins is a rapid, efficient, and versatile bacterial identification method with the validation procedure for the obtained results.     

Characterization of Two Perkinsus spp. from the Softshell Clam, Mya arenaria Using the Small Subunit Ribosomal RNA Gene

Sequence analysis and riboprinting of the small subunit ribosomal RNA genes were used to characterize two morphologically different Perkinsus species isolates from the gill (G117) and the hemolymph (H49) of the softshell clam, Mya arenaria. Sequence data of the polymerase chain reaction amplified ribosomal RNA loci of G117 and H49 indicated that these genes are 1803 and 1806 base-pair long, respectively. A sequence similarity of > 98.9% was calculated among ribosomal RNA sequences of the two isolates of this study and the published sequences of Perkinsus marinus from the American eastern oyster, Crassostrea virginica, and Perkinsus sp. from the blood cockle of the Australian mollusc, Anadara trapezia. From a phylogenetic tree obtained from Jukes-Cantor distances of the aligned ribosomal RNA gene sequences of 13 eukaryotic taxa using the Neighbor-Joining method, we showed that G117 and H49 clustered within the genus Perkinsus. Guided by the sequence data of Perkinsus marinus (accession # X75762) and Perkinsus sp. (accession # L07375), restriction endonucleases were selected for restriction fragment analysis of polymerase chain reaction products of the small subunit ribosomal RNA genes (riboprinting). Riboprinting was used to distinguish the four members of the genus Perkinsus from each other.     

Role of deoxyribonucleic acid polymerases and deoxyribonucleic acid ligase in x-ray-induced repair synthesis in toluene-treated Escherichia coli K-12.

The biosynthesis of ribosomal ribonucleic acid (rRNA) In wild-type Neurospora crassa growing at 25 degrees C was investigated by continuous-labeling and pulsechase experiments using [5-3H]uridine. The results of these experiments suggest the following precursor-product relationships: the first RNA molecule to be synthesized in significant quantities is the 2.4 X 10(6)-dalton (2.4-Mdal) ribosomal precursor RNA. This RNA is cleaved to produce two species of RNA with weights of 0.7 and 1.4-Mdal. The former is the mature 17S rRNA of the 37S ribosomal subunit. The 1.4-Mdal RNA is subsequently cleaved to produce the mature 1.27-Mdal (25S) and 61,000-dalton (5.8S) rRNA's of the 60S ribosomal subunit. In the maturation process, approximately 15 to 20% of the 2.4-Mdal ribosomal precursor rRNA molecule is lost. As in other eukaryotes that have been examined, 5S rRNA is not derived from this precursor molecule.     

Molecular taxonomic position of the elephant schistosome, Bivitellobilharzia nairi, newly discovered in Sri Lanka

Bivitellobilharzia nairi (Mudaliar and Ramanujachar, 1945) Dutt and Srivastava, 1955 was first recorded in India. A number of adult worm specimens of this schistosome species were recovered from a domestic elephant, which died in 1999 in Sri Lanka. This is the first report of this schistosome from Sri Lanka. In the present study, in order to clarify the phylogenetic relationship with other species of schistosomes, sequences from the second internal transcribed spacer (ITS2) of the ribosomal gene repeat, part of the 28S ribosomal RNA gene (28S), and part of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene from B. nairi were analyzed. Two intraspecific variations were seen within 13 individuals in the ITS2 region. In the CO1 region of the mitochondrial DNA, there were four haplotypes in the nucleotide sequences and two haplotypes in the amino acid sequences. Phylogenetic analysis using the nuclear DNA showed that B. nairi was basal to all of species of the genus Schistosoma. The 28S tree also showed that the mammalian lineage was monophyletic. However, phylogenetic analysis using the mitochondrial DNA showed that B. nairi was nested within the genus Schistosoma. The taxonomical position for this species as well as the contradiction between the results from the nuclear and mitochondrial genes were discussed.     

Molecular Phylogeny of the Ocelloid-Bearing Dinoflagellates Erythropsidinium and Warnowia (Warnowiaceae, Dinophyceae)

ABSTRACT. Members of the family Warnowiaceae are unarmored phagotrophic dinoflagellates that possess an ocelloid. The genus Erythropsidinium (= Erythropsis ) has also developed a unique dynamic appendage, the piston, which is able to independently retract and extend for at least 2 min after the cell lyses. We provide the first small subunit ribosomal RNA gene sequences of warnowiid dinoflagellates, those of the type Erythropsidinium agile and one species of Warnowia . Phylogenetic analyses show that warnowiid dinoflagellates branch within the Gymnodinium sensu stricto group, forming a cluster separated from the Polykrikos clade and with autotrophic Pheopolykrikos beauchampii as closest relative. This reinforces their classification as unarmored dinoflagellates based on the shape of the apical groove, despite the strong ecological and ultrastructural diversity of the Gymnodinium s.s. group. Other structures, such as the ocelloid and piston, have no systematic value above the genus level.     

Structural organization of the 16S ribosomal RNA from E. coli. Topography and secondary structure.

Extensive studies in our laboratory using different ribonucleases resulted in valuable data on the topography of the E.coli 16S ribosomal RNA within the native 30S subunit, within partially unfolded 30S subunits, in the free state, and in association with individual ribosomal proteins. Such studies have precise details on the accessibility of certain residues and delineated highly accessible RNA regions. Furthermore, they provided evidence that the 16S rRNA is organized in its subunit into four distinct domains. A secondary structure model of the E.coli 16S rRNA has been derived from these topographical data. Additional information from comparative sequence analyses of the small ribosomal subunit RNAs from other species sequenced so far has been used.     

DNA barcoding in the rust genus Chrysomyxa and its implications for the phylogeny of the genus

Chrysomyxa rusts are fungal pathogens widely present in the boreal forest. Taxonomic delimitation and precise species identification are difficult within this genus because several species display similar morphological features. We applied a DNA barcode system based on the ribosomal internal transcribed spacer region (ITS), large subunit (28S) ribosomal RNA gene, mitochondrial cytochrome oxidase 1 (CO1) and mitochondrial NADH dehydrogenase subunit 6 (NAD6) in 86 strains from 16 different Chrysomyxa species, including members of the Chrysomyxa ledi species complex. The nuclear ITS and 28S loci revealed higher resolving power than the mitochondrial genes. Amplification of the full CO1 barcode region failed due to the presence of introns limiting the dataset obtained with this barcode. In most cases the ITS barcodes were in agreement with taxonomic species based on phenotypic characters. Nevertheless we observed genetically distinct (different DNA barcodes) lineages within Chrysomyxa pyrolae and Chrysomyxa rhododendri, providing some evidence for allopatric speciation within these morphologically defined species. This finding, together with the observed pattern of host specificities of the studied rust fungi, suggest that species diversification within the C. ledi species complex might be governed by a set of factors such as specialisation to certain Ericaceae species as telial hosts and to a lesser extent specialization to different spruce species as aecial hosts. Moreover allopatric speciation by geographic disruption of species also seems to take place. When our data were integrated into a broader phylogenetic framework the Chrysomyxa genus unexpectedly was not resolved as a monophyletic group. Indeed the spruce cone rusts C. pyrolae and C. monesis coalesced with the pine needle rusts belonging to the genus Coleosporium, whereas the microcyclic species Chrysomyxa weirii was embedded within a clade comprising the genus Melampsora.     

Resolving evolutionary relationships in the lichen-forming genus Porpidia and related allies (Porpidiaceae, Ascomycota)

The lichen-forming genus Porpidia (Porpidiaceae, Ascomycota) provides excellent opportunities for evolutionary, reproductive, and ecological studies of crustose epilithic lichen symbioses. However, despite the fact that the genus itself seemed to be clearly delimited, the group was thought to be a hopeless case with regard to intrageneric relationships and species delimitations due to apparently rampant homoplasy throughout most character systems. Contrary to the situation for non-molecular data, a robust and generally well-resolved phylogeny was recovered based on DNA-sequence data. Separate and combined analyses of nuclear ribosomal RNA large subunit and nuclear beta-tubulin gene fragments were performed using maximum parsimony, maximum likelihood, and Bayesian approaches. Branch support was estimated using non-parametic bootstrapping and posterior probabilities, while monophyly of a priori defined groups was tested using posterior probabilities. The results reveal a highly supported "Porpidia sensu lato," however, Porpidia itself is not monophyletic. Several smaller genera of the Porpidiaceae and probably the large genus Lecidea (Lecideaceae) are nested within the group. Most taxa analyzed fall into one of four major subgroups within Porpidia s.l., though the basal relationships among these subgroups could not be supported. This phylogeny will make it possible to re-evaluate morphological and chemical characters in the group, and to conduct detailed studies of species delimitations within the monophyletic subgroups.     

Molecular diagnostics of economically important Ceratitis fruit fly species (Diptera: Tephritidae) in Africa using PCR and RFLP analyses

The predominantly Afrotropical fruit fly genus Ceratitis contains many species of agricultural importance. Consequently, quarantine of Ceratitis species is a major concern for governmental regulatory agencies. Although diagnostic keys exist for identification of all described Ceratitis species, these tools are based on adult characters. Flies intercepted at ports of entry are usually immatures, and Ceratitis species cannot be diagnosed based on larval morphology. To facilitate identification of Ceratitis pests at ports of entry, this study explores the utility of DNA-based diagnostic tools for a select group of Ceratitis species and related tephritids, some of which infest agriculturally important crops in Africa. The application of the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method to analyse three mitochondrial genes (12S ribosomal RNA, 16S ribosomal RNA, and NADH-dehydrogenase subunit 6) is sufficient to diagnose 25 species and two species clusters. PCR analysis of the internal transcribed spacer region 1 (ITS-1) is able to distinguish three of the five species left unresolved by mitochondrial DNA analysis.     

Amoeba at attention: phylogenetic affinity of Sappinia pedata

The genus Sappinia, a taxon of free-living amoebae with trophozoites that typically have two closely appressed nuclei, contains two named species, Sappinia pedata, the type species, and S. diploidea. The amoebae of both species are essentially identical according to the literature. The two species are distinguished by S. pedata having a standing amoeba stage, incorrectly interpreted as a cyst, and S. diploidea having sessile, bicellular cysts. Using four isolates of S. pedata collected from around the world, we present detailed light micrographic illustrations of all stages of its life cycle. We confirm that the standing amoeba lacks a cell wall. In two isolates of S. pedata, there are bicellular cysts indistinguishable from those of S. diploidea. Using sequence data from the nuclear small subunit ribosomal RNA gene, we conclude that S. pedata and the published neotype of S. diploidea are congeneric but not conspecific. The genus branches within Thecamoebidae. Sequencing of the actin gene confirms the inclusion of Sappinia in Thecamoebidae. Resolving the taxonomy of Sappinia is gaining importance because it has recently been attributed as an opportunistic human pathogen.     

Molecular phylogeny of labyrinthulids and thraustochytrids based on the sequencing of 18S ribosomal RNA gene

Labyrinthulids and thraustochytrids are unicellular heterotrophs, formerly considered as fungi, but presently are recognized as members in the stramenopiles of the kingdom Protista sensu lato. We determined the 18S ribosomal RNA gene sequences of 14 strains from different species of the six genera and analyzed the molecular phylogenetic relationships. The results conflict with the current classification based on morphology, at the genus and species levels. These organisms are separated, based on signature sequences and unique inserted sequences, into two major groups, which were named the labyrinthulid phylogenetic group and the thraustochytrid phylogenetic group. Although these groupings are in disagreement with many conventional taxonomic characters, they correlated better with the sugar composition of the cell wall. Thus, the currently used taxonomic criteria need serious reconsideration.     

Identifying monophyletic groups within Bugula sensu lato (Bryozoa,Buguloidea)

Species in the genus Bugula are globally distributed. They are most abundant in tropical and temperate shallow waters, but representatives are found in polar regions. Seven species occur in the Arctic and one in the Antarctic and species are represented in continental shelf or greater depths as well. The main characters used to define the genus include bird's head pedunculate avicularia, erect colonies, embryos brooded in globular ooecia and branches comprising two or more series of zooids. Skeletal morphology has been the primary source of taxonomic information for many calcified bryozoan groups, including the Buguloidea. Several morphological characters, however, have been suggested to be homoplastic at distinct taxonomic levels, in the light of molecular phylogenies. Our purpose was to investigate the phylogenetic interrelationships of the genus Bugula, based on molecular phylogenetics and morphology. A Bayesian molecular phylogeny was constructed using original and previously published sequences of the mitochondrial genes cytochrome c oxidase subunit 1 (COI) and the large ribosomal RNA subunit (16S). Morphological characteristics from scanning electron and light microscopy were used to confirm the clades detected by the molecular phylogeny. Our results suggest that the genus is composed of four clades, for which we provide diagnoses: Bugula sensu stricto (30 species), Bugulina (24 species), Crisularia (23 species) and the monotypic Virididentula gen. n. Ten species could not be assigned to any of those genera, so they remain as genus incertae sedis. Our findings highlight the importance of using molecular phylogenies in association with morphological characters in systematic revisions of bryozoan taxa.     

Phylogeny and taxonomy of the genus Cylindrocladiella

The genus Cylindrocladiella was established to accommodate Cylindrocladium-like fungi that have small, cylindrical conidia and aseptate stipe extensions. Contemporary taxonomic studies of these fungi have relied on morphology and to a lesser extent on DNA sequence comparisons of the internal transcribed spacer regions (ITS 1, 2 and 5.8S gene) of the ribosomal RNA and the ??-tubulin gene regions. In the present study, the identity of several Cylindrocladiella isolates collected over two decades was determined using morphology and phylogenetic inference. A phylogeny constructed for these isolates employing the ??-tubulin, histone H3, ITS, 28S large subunit and translation elongation factor 1-alpha gene regions resulted in the identification of several cryptic species in the genus. In spite of the 18 new Cylindrocladiella species described in this study based on morphological and sequence data, several species complexes remain unresolved.     

Phylogenetic relationships of the Gomphales based on nuc-25S-rDNA, mit-12S-rDNA, and mit-atp6-DNA combined sequences

Phylogenetic relationships among Geastrales, Gomphales, Hysterangiales, and Phallales were estimated via combined sequences: nuclear large subunit ribosomal DNA (nuc-25S-rDNA), mitochondrial small subunit ribosomal DNA (mit-12S-rDNA), and mitochondrial atp6 DNA (mit-atp6-DNA). Eighty-one taxa comprising 19 genera and 58 species were investigated, including members of the Clathraceae, Gautieriaceae, Geastraceae, Gomphaceae, Hysterangiaceae, Phallaceae, Protophallaceae, and Sphaerobolaceae. Although some nodes deep in the tree could not be fully resolved, some well-supported lineages were recovered, and the interrelationships among Gloeocantharellus, Gomphus, Phaeoclavulina, and Turbinellus, and the placement of Ramaria are better understood. Both Gomphus sensu lato and Ramaria sensu lato comprise paraphyletic lineages within the Gomphaceae. Relationships of the subgenera of Ramaria sensu lato to each other and to other members of the Gomphales were clarified. Within Gomphus sensu lato, Gomphus sensu stricto, Turbinellus, Gloeocantharellus and Phaeoclavulina are separated by the presence/absence of clamp connections, spore ornamentation (echinulate, verrucose, subreticulate or reticulate), and basidiomal morphology (fan-shaped, funnel-shaped or ramarioid). Gautieria, a sequestrate genus in the Gautieriaceae, was recovered as monophyletic and nested with members of Ramaria subgenus Ramaria. This agrees with previous observations of traits shared by these two ectomycorrhizal taxa, such as the presence of fungal mats in the soil. Clavariadelphus was recovered as a sister group to Beenakia, Kavinia, and Lentaria. The results reaffirm relationships between the Geastrales, Gomphales, Hysterangiales, and the Phallales, suggesting extensive convergence in basidiomal morphology among members of these groups. A more extensive sampling that focuses on other loci (protein-coding genes have been shown to be phylogenetically informative) may be useful to answer questions about evolutionary relationships among these fungal groups.