Inter- and intrasporal nuclear ribosomal gene sequence variation within one isolate of arbuscular mycorrhizal fungus, Diversispora sp.

Most molecular ecological studies of arbuscular mycorrhizal fungi (AMF) have been based on the rRNA gene sequences. However, information about intraspecific nucleotide variation is still limited in these fungi. In this study, we calculated the inter- and intrasporal nucleotide variation of Diversispora sp. EE1 using 78 cloned sequences from four spores within a ca 4960 bp fragment of the nuclear ribosomal operon spanning the near full length small ribosomal subunit (SSU) rRNA gene, the full internal transcribed spacer (ITS: ITS1-5.8S-ITS2) and ca 2740 bp of the large ribosomal subunit (LSU) rRNA gene. Data for each marker region (SSU, ITS and LSU) originated from the very same spores. Sequence variation resulting from point mutations and small indels was recorded in all regions. Highest sequence variation was observed in the ITS region at both the inter- and intrasporal levels. The ITS1 component was more variable than ITS2, whilst the 5.8S gene was the least variable component of the ITS region. Evolutionary divergence of gene copies between spores was intermediate for the LSU and lowest for the SSU. The SSU and the LSU genes had relatively similar evolutionary divergence per spore. Sequence variant richness was not exhaustive for any of the marker regions, indicating that multiple sequences per spore from multiple spores are needed when characterizing a species. This study provides reference sequences for ecological studies, permitting identification of AMF using any of the ribosomal regions or primer systems.     

Phylogeography of the freshwater raphidophyte Gonyostomum semen confirms a recent expansion in northern Europe by a single haplotype

Gonyostmum semen is a freshwater raphidophyte that has increased in occurrence and abundance in several countries in northern Europe since the 1980s. More recently, the species has expanded rapidly also in north‐eastern Europe, and it is frequently referred to as invasive. To better understand the species history, we have explored the phylogeography of G. semen using strains from northern Europe, United States, and Japan. Three regions of the ribosomal RNA gene (small subunit [SSU], internal transcribed spacer [ITS] and large subunit [LSU]) and one mitochondrial DNA marker (cox1) were analyzed. The SSU and partial LSU sequences were identical in all strains, confirming that they belong to the same species. The ITS region differentiated the American from the other strains, but showed high intra‐strain variability. In contrast, the mitochondrial marker cox1 showed distinct differences between the European, American, and Japanese strains. Interestingly, only one cox1 haplotype was detected in European strains. The overall low diversity and weak geographic structure within northern European strains supported the hypothesis of a recent invasion of new lakes by G. semen. Our data also show that the invasive northern European lineage is genetically distinct from the lineages from the other continents. Finally, we concluded that the mitochondrial cox1 was the most useful marker in determining large‐scale biogeographic patterns in this species.     

Phylogenetic analysis of ribosomal RNA operons from uncultivated coastal marine bacterioplankton

Analyses of small subunit ribosomal RNA genes (SSU rDNAs) have significantly influenced our understanding of the composition of aquatic microbial assemblages. Unfortunately, SSU rDNA sequences often do not have sufficient resolving power to differentiate closely related species. To address this general problem for uncultivated bacterioplankton taxa, we analysed and compared sequences of polymerase chain reaction (PCR)-generated and bacterial artificial chromosome (BAC)-derived clones that contained most of the SSU rDNAs, the internal transcribed spacer (ITS) and the large subunit ribosomal RNA gene (LSU rDNA). The phylogenetic representation in the rRNA operon PCR library was similar to that reported previously in coastal bacterioplankton SSU rDNA libraries. We observed good concordance between the phylogenetic relationships among coastal bacterioplankton inferred from SSU or LSU rDNA sequences. ITS sequences confirmed the close intragroup relationships among members of the SAR11, SAR116 and SAR86 clades that were predicted by SSU and LSU rDNA sequence analyses. We also found strong support for homologous recombination between the ITS regions of operons from the SAR11 clade.     

Phylogenetic analysis of Glomeromycota by partial LSU rDNA sequences

We analyzed the large subunit ribosomal RNA (rRNA) gene [LSU ribosomal DNA (rDNA)] as a phylogenetic marker for arbuscular mycorrhizal (AM) fungal taxonomy. Partial LSU rDNA sequences were obtained from ten AM fungal isolates, comprising seven species, with two new primers designed for Glomeromycota LSU rDNA. The sequences, together with 58 sequences available from the databases, represented 31 AM fungal species. Neighbor joining and parsimony analyses were performed with the aim of evaluating the potential of the LSU rDNA for phylogenetic resolution. The resulting trees indicated that Archaeosporaceae are a basal group in Glomeromycota, Acaulosporaceae and Gigasporaceae belong to the same clade, while Glomeraceae are polyphyletic. The results support data obtained with the small subunit (SSU) rRNA gene, demonstrating that the LSU rRNA gene is a useful molecular marker for clarifying taxonomic and phylogenetic relationships in Glomeromycota.     

Evolution according to large ribosomal subunit RNA

Evolutionary trees were constructed, by distance methods, from an alignment of 225 complete large subunit (LSU) rRNA sequences, representing Eucarya, Archaea, Bacteria, plastids, and mitochondria. A comparison was made with trees based on sets of small subunit (SSU) rRNA sequences. Trees constructed on the set of 172 species and organelles for which the sequences of both molecules are known had a very similar topology, at least with respect to the divergence order of large taxa such as the eukaryotic kingdoms and the bacterial divisions. However, since there are more than ten times as many SSU as LSU rRNA sequences, it is possible to select many SSU rRNA sequence sets of equivalent size but different species composition. The topologies of these trees showed considerable differences according to the particular species set selected.The effect of the dataset and of different distance correction methods on tree topology was tested for both LSU and SSU rRNA by repetitive random sampling of a single species from each large taxon. The impact of the species set on the topology of the resulting consensus trees is much lower using LSU than using SSU rRNA. This might imply that LSU rRNA is a better molecule for studying wide-range relationships. The mitochondria behave clearly as a monophyletic group, clustering with the Proteobacteria. Gram-positive bacteria appear as two distinct groups, which are found clustered together in very few cases. Archaea behave as if monophyletic in most cases, but with a low confidence.Abbreviations LSU rRNA large subunit ribosomal RNA - SSU rRNA small subunit ribosomal RNA - JC Jukes and Cantor - JN Jin and Nei Correspondence to: R. De Wachter     

Complete sequence and structure of ribosomal RNA gene of Heterosporis anguillarum

The ribosomal RNA (rRNA) gene region of the microsporidium Heterosporis anguillarum has been examined. Complete DNA sequence data (4060 bp, GenBank Accession No. AF402839) of the rRNA gene of H. anguillarum are presented for the small subunit gene (SSU rRNA: 1359 bp), the internal transcribed spacer (ITS: 37 bp), and the large subunit gene (LSU rRNA: 2664 bp). The secondary structures of the H. anguillarum SSU and LSU rRNA genes are constructed and described. This is the first complete sequence of an rRNA gene published for a fish-infecting microsporidian species. In the phylogenetic analysis, the sequences, including partial SSU rRNA, ITS, and partial LSU rRNA sequences of the fish-infecting microsporidia, were aligned and analysed. The taxonomic position of H. anguillarum as suggested by Lom et al. (2000; Dis Aquat Org 43:225-231) is confirmed in this paper.     

Structure of ribosomal RNA gene and phytogeny ofNosema isolates in Korea

The ribosomal RNA (rRNA) gene region of the fourNosema sp. isolates (C01, C02, C03 and C04) fromPieris rapae in Korea has been examined. Complete DNA sequence data (3779 bp) of The rRNA gene ofNosema sp. C01 are presented for the small subunit gene (SSU rRNA: 1236 bp), the internal transcribed spacer (ITS: 37 bp), and the large subunit gene (LSU rRNA 2506 bp). The secondary structures ofNosema sp. COI SSU and LSU rRNA genes are constructed and described. The SSU rRNA showed a hypervariable V4 region identified four additional stems including a pseudoknot. Phylogenetic analysis based on the SSU rRNA suggests that the four isolates belong to the ‘true’Nosema group. In contrast to theNosema/Vairimorpha clade, the members of the group are highly divergent.     

New taxa refresh the phylogeny and classification of pleurostomatid ciliates (Ciliophora,Litostomatea)

A high diversity of pleurostomatid ciliates has been discovered in the last decade, and their systematics needs to be improved in the light of new findings concerning their morphology and molecular phylogeny. In this work, a new genus, Protolitonotus gen. n., and two new species, Protolitonotus magnus sp. n. and Protolitonotus longus sp. n., were studied. Furthermore, 19 novel nucleotide sequences of SSU rDNA, LSU rDNA and ITS1‐5.8S‐ITS2 were collected to determine the phylogenetic relationships and systematic positions of the pleurostomatid ciliates in this study. Based on both molecular and morphological data, the results demonstrated that: (i) as disclosed by the sequence analysis of SSU rDNA, LSU rDNA and ITS1‐5.8S‐ITS2, Protolitonotus gen. n. is sister to all other pleurostomatids and thus represents an independent lineage and a separate family, Protolitonotidae fam. n., which is defined by the presence of a semi‐suture formed by the right somatic kineties near the dorsal margin of the body; (ii) the families Litonotidae and Kentrophyllidae are both monophyletic based on both SSU rDNA and LSU rDNA sequences, whereas Amphileptidae are non‐monophyletic in trees inferred from SSU rDNA sequences; and (iii) the genera Loxophyllum and Kentrophyllum are both monophyletic, whereas Litonotus is non‐monophyletic based on SSU rDNA analyses. ITS1‐5.8S‐ITS2 sequence data were used for the phylogenetic analyses of pleurostomatids for the first time; however, species relationships were less well resolved than in the SSU rDNA and LSU rDNA trees. In addition, a major revision to the classification of the order Pleurostomatida is suggested and a key to its families and genera is provided.     

Molecular genetic variation within and among isolates of QPX (Thraustochytridae), a parasite of the hard clam Mercenaria mercenaria

The thraustochytrid known as QPX (Quahog Parasite Unknown) has sporadically caused disease in the hard clam Mercenaria mercenaria along the east coast of North America since the 1960s. We hypothesized that genetically distinct QPX strains might be responsible for outbreaks of QPX disease in different areas and tested this hypothesis by comparing several QPX isolates recovered from the recent outbreak in Raritan Bay, New York with QPX strains isolated from 2 outbreaks in Massachusetts, USA. There was no variation in small subunit rDNA (SSU rDNA), 5.8S rDNA, or 4 mitochondrial gene sequences. In contrast, both of the ribosomal ribonucleic acid (rRNA) operon intergenic spacers, internal transcribed spacers 1 and 2 (ITS1 and ITS2), revealed substantial sequence variation. However, strain-specific sequences were not detected because the ITS sequence variation within QPX isolates was comparable to the variation between isolates. ITS1 sequences recovered from an infected clam by amplification with a QPX ITS2-specific primer were identical to those recovered from the QPX isolates.     

Using the SSU,ITS, and Ribosomal DNA Operon Arrangement to Characterize Two Microsporidia Infecting Bruce Spanworm,Operophtera bruceata (Lepidoptera: Geometridae)

Research pertaining to the two closely‐related microsporidian genera Nosema and Vairimorpha is hindered by inconsistencies in species differentiation within and between the two clades. One proposal to better delimit these genera is to restructure the Nosema around a “True Nosema” clade, consisting of species that share a characteristic reversed ribosomal DNA operon arrangement and small subunit (SSU) ribosomal DNA sequences similar to that of the Nosema type species, N. bombycis. Using this framework, we assess two distinct microsporidia recovered from the forest insect Bruce spanworm (Operophtera bruceata) by sequencing their SSU and internal transcribed spacer regions. Phylogenetic analyses place one of our isolates within the proposed True Nosema clade close to N. furnacalis and place the other in the broader Nosema/Vairimorpha clade close to N. thomsoni. We found that 25% of Bruce spanworm cadavers collected over the four‐year study period were infected with microsporidia, but no infections were detected in cadavers of the Bruce spanworm's invasive congener, the winter moth (O. brumata), collected over the same period. We comment on these findings as they relate to the population dynamics of the Bruce spanworm‐winter moth system in this region, and more broadly, on the value of ribosomal DNA operon arrangement in Nosema systematics.     

Internal Transcribed Spacer Sequence Analysis of Puccinia helianthi Schw. and Its Application in Detection of Sunflower Rust

Two basidiomycete‐specific primers ITS1‐F and ITS4‐B were used in identification of the genus Puccinia. The primers showed good specificity for the genus with an 816‐bp product that was amplified exclusively. Twenty sequences of internal transcribed spacer (ITS) regions of Puccinia helianthi isolates from China remain unchanged. The whole ITS length (including ITS1 sequence 194 bp, 5.8S rRNA gene 156 bp, ITS2 sequence 206 bp) was 556 bp. By comparing the aligned ITS sequences of several Puccinia isolates from China, Spain and the United States, ITS homogeneity among these sunflower rust isolates was >99%. Genetic homology and phylogeny of P. helianthi with other Puccinia spp. was investigated. Nineteen sequences of rDNA ITS1 and ITS2 were determined and used as phylogenetic markers. Phylogenetic analysis of ITS regions showed that Puccinia spp. of sunflower was clustered in one clade with P. komarovii and P. violae, divergent from Puccinia spp. of Chrysanthemum, P. tenaceti of tansy (Tanacetum vulgare) and Puccina spp. of big sagebrush (Artemisia tridentate) indicating sunflower rust had distant phylogenetic relationships with other Compositae rusts. With the specified primers SR‐1 and SR‐2, either from purified urediniospores or symptomless (but infected) sunflower leaves could be examined specifically. Therefore, results of this study help in detection and polygenetic study of rust fungi occurring on sunflower.     

Description of a New Freshwater Ciliate Epistylis wuhanensis n. sp. (Ciliophora,Peritrichia) from China,with a Focus on Phylogenetic Relationships within Family Epistylididae

Two populations of Epistylis wuhanensis n. sp., a new freshwater peritrich ciliate, were isolated from different freshwater ponds located in Hubei, China. Their morphological characteristics were investigated using live observation, protargol impregnation, and scanning electron microscopy (SEM). Specimens from the two populations showed identical arrangement of the infraciliature and identical small subunit ribosomal RNA (SSU rRNA) gene and ITS1‐5.8S‐ITS2 sequences. The zooids present bell‐shaped and 90–175 × 27–54 μm in vivo. Macronucleus is variable in shape and located in the middle of cell. Pellicle is usually smooth with 139–154 and 97–105 striations above and below the trochal band, respectively. SSU rRNA gene and ITS1‐5.8S‐ITS2 sequences of E. wuhanensis n. sp. did not match any available sequences in GenBank. Phylogenetically, E. wuhanensis n. sp. clusters with the other Epistylis within the family Epistylididae, but is distinct from the major clades of Epistylis. Above all, the morphological characteristics and molecular analyses support that the present Epistylis is a new species. Expanded phylogenetic analyses of sessilids based on both SSU rRNA gene sequences and ITS1‐5.8S‐ITS2 sequences reveal that the genus Epistylis consists of Epistylis morphospecies and taxonomic revision of the genus is needed.     

Complete rRNA gene sequences reveal that the microsporidium Nosema bombi infects diverse bumblebee (Bombus spp.) hosts and contains multiple polymorphic sites

Characterisation of microsporidian species and differentiation among genetic variants of the same species has typically relied on ribosomal RNA (rRNA) gene sequences. We characterised the entire rRNA gene of a microsporidium from 11 isolates representing eight different European bumblebee (Bombus) species. We demonstrate that the microsporidium Nosema bombi infected all hosts that originated from a wide geographic area. A total of 16 variable sites (all single nucleotid polymorphisms (SNPs)) was detected in the small subunit (SSU) rRNA gene and 42 (39 SNPs and 3 indels) in the large subunit (LSU) rRNA sequence. Direct sequencing of PCR-amplified DNA products of the internal transcribed spacer (ITS) region revealed identical sequences in all isolates. In contrast, ITS fragment length determined by PAGE and sequencing of cloned amplicons gave better resolution of sequences and revealed multiple SNPs across isolates and two fragment sizes in each isolate (six short and seven long amplicon variants). Genetic variants were not unique to individual host species. Moreover, two or more sequence variants were obtained from individual bumblebee hosts, suggesting the existence of multiple, variable copies of rRNA in the same microsporidium, and contrary to that expected for a class of multi-gene family under concerted evolution theory. Our data on within-genome rRNA variability call into question the usefulness of rRNA sequences to characterise intraspecific genetic variants in the Microsporidia and other groups of unicellular organisms.     

Prototheca tumulicola sp. nov., a novel achlorophyllous,yeast-like microalga isolated from the stone chamber interior of the Takamatsuzuka Tumulus

Two achlorophyllous microalgal strains were isolated from the soil and white moldy colony collected inside the stone chamber of the Takamatsuzuka Tumulus in Japan. Phylogenetic analyses of the small subunit ribosomal RNA (SSU rRNA) and Dl/D2 large subunit ribosomal RNA (LSU rRNA) gene sequences, and concatenated gene sequences of the SSU and D1/D2 LSU rRNA genes indicated that our two isolates were the members of the non-photosynthetic, yeast-like microalgal Chlorellaceous genus Prototheca (Chlorellales, Trebouxiophyceae, Chlorophyta) but well distinguished from known species. Based on phenotypic and genotypic characteristics, isolates T6713-13-10^T and T61213-7-11 are proposed to represent a novel species in Prototheca, P. tumulicola, with the type strain JCM 31123^T (isolate T6713-13-10^T).     

Phylogeny and species delineation in the marine diatom Pseudo‐nitzschia (Bacillariophyta) using cox1, LSU,and ITS2 rRNA genes: A perspective in character evolution

Analyses of the mitochondrial cox1, the nuclear‐encoded large subunit (LSU), and the internal transcribed spacer 2 (ITS2) RNA coding region of Pseudo‐nitzschia revealed that the P. pseudodelicatissima complex can be phylogenetically grouped into three distinct clades (Groups I–III), while the P. delicatissima complex forms another distinct clade (Group IV) in both the LSU and ITS2 phylogenetic trees. It was elucidated that comprehensive taxon sampling (sampling of sequences), selection of appropriate target genes and outgroup, and alignment strategies influenced the phylogenetic accuracy. Based on the genetic divergence, ITS2 resulted in the most resolved trees, followed by cox1 and LSU. The morphological characters available for Pseudo‐nitzschia, although limited in number, were overall in agreement with the phylogenies when mapped onto the ITS2 tree. Information on the presence/absence of a central nodule, number of rows of poroids in each stria, and of sectors dividing the poroids mapped onto the ITS2 tree revealed the evolution of the recently diverged species. The morphologically based species complexes showed evolutionary relevance in agreement with molecular phylogeny inferred from ITS2 sequence–structure data. The data set of the hypervariable region of ITS2 improved the phylogenetic inference compared to the cox1 and LSU data sets. The taxonomic status of P. cuspidata and P. pseudodelicatissima requires further elucidation.     

Characterization of Two Fungal Isolates from Cotton and Evaluation of their Potential for Biocontrol of Verticillium Wilt of Cotton

Two isolates (CVd‐WHw and CVn‐WHg) recovered from Verticillium‐wilt‐symptomatic cotton grown in Hubei Province of China were identified based on their morphology, growth characteristics in culture, specific amplification and identification of internal transcribed spacer (ITS) rDNA sequence. According to the morphological characteristics, specific PCR amplification and ITS sequences, CVd‐WHw was identified as V. dahliae and CVn‐WHg as Gibellulopsis nigrescens. In bioassays, the two isolates had significantly lower pathogenicity to cotton plant than V. dahliae isolate CVd‐AYb. Cotton pre‐inoculated with isolate CVn‐WHg or CVd‐WHw exhibited reduced disease indices of Verticillium wilt compared with those inoculated with CVd‐AYb alone. Cotton co‐inoculated with CVn‐WHg or CVd‐WHw and CVd‐AYb provided increased protection from subsequent CVd‐AYb inoculation. These results suggest that the two isolates have the potential to be developed as biocontrol agents for the control of Verticillium wilt in cotton. To our knowledge, this is the first report of a cross‐protection phenomenon using Gibellulopsis nigrescens against Verticillium wilt caused by V. dahliae on cotton.     

Gymnodinium smaydae n. sp., a New Planktonic Phototrophic Dinoflagellate from the Coastal Waters of Western Korea: Morphology and Molecular Characterization

The marine phototrophic dinoflagellate Gymnodinium smaydae n. sp. is described from cells prepared for light, scanning, and transmission electron microscopy. Also, sequences of the small (SSU) and large subunits (LSU) and the internal transcribed spacer region (ITS1–5.8S–ITS2) of ribosomal DNA were analyzed. This newly isolated dinoflagellate possessed nuclear chambers, nuclear fibrous connective, an apical groove running in a counterclockwise direction around the apex, and a major accessory pigment peridinin, which are four key features for the genus Gymnodinium. The epicone was conical with a round apex, while the hypocone was ellipsoid. Cells growing photosynthetically were 6.3–10.9 μm long and 5.1–10.0 μm wide, and therefore smaller than any other Gymnodinium species so far reported except Gymnodinium nanum. Cells were covered with polygonal amphiesmal vesicles arranged in 11 horizontal rows, and the vesicles were smaller than those of the other Gymnodinium species. This dinoflagellate had a sharp and elongated ventral ridge reaching half way down the hypocone, unlike other Gymnodinium species. Moreover, displacement of the cingulum was 0.4–0.6 × cell length while in other known Gymnodinium species it is less than 0.3 × cell length. In addition, the new species possessed a peduncle, permanent chloroplasts, pyrenoids, trichocysts, pusule systems, and small knobs along the apical furrow, but it lacked an eyespot, nematocysts, and body scales. The sequence of the SSU, ITS1–5.8S–ITS2, and LSU rDNA region differed by 1.5–3.8%, 6.0–17.4%, and 9.1–17.5%, respectively, from those of the most closely related species. The phylogenetic trees demonstrated that the new species belonged to the Gymnodinium clade at the base of a clade consisting of Gymnodinium acidotum, Gymnodinium dorsalisulcum, Gymnodinium eucyaneum, etc. Based on morphological and molecular data, we suggest that the taxon represents a new species, Gymnodinium smaydae n. sp.     

Molecular diversity of the syndinean genus Euduboscquella based on single-cell PCR analysis

The genus Euduboscquella is one of a few described genera within the syndinean dinoflagellates, an enigmatic lineage with abundant diversity in marine environmental clone libraries based on small subunit (SSU) rRNA. The region composed of the SSU through to the partial large subunit (LSU) rRNA was determined from 40 individual tintinnid ciliate loricae infected with Euduboscquella sampled from eight surface water sites in the Northern Hemisphere, producing seven distinct SSU sequences. The corresponding host SSU rRNA region was also amplified from eight host species. The SSU tree of Euduboscquella and syndinean group I sequences from environmental clones had seven well-supported clades and one poorly supported clade across data sets from 57 to 692 total sequences. The genus Euduboscquella consistently formed a supported monophyletic clade within a single subclade of group I sequences. For most parasites with identical SSU sequences, the more variable internal transcribed spacer (ITS) to LSU rRNA regions were polymorphic at 3 to 10 sites. However, in E. cachoni there was variation between ITS to LSU copies at up to 20 sites within an individual, while in a parasite of Tintinnopsis spp., variation between different individuals ranged up to 19 polymorphic sites. However, applying the compensatory base change model to the ITS2 sequences suggested no compensatory changes within or between individuals with the same SSU sequence, while one to four compensatory changes between individuals with similar but not identical SSU sequences were found. Comparisons between host and parasite phylogenies do not suggest a simple pattern of host or parasite specificity.