Small-subunit Ribosomal RNA Gene Sequences Research Articles

Comparing Microsporidia-targeting primers for environmental DNA sequencing.

Metabarcoding is a powerful tool to detect classical, and well-known "long-branch" Microsporidia in environmental samples. Several primer pairs were developed to target these unique microbial parasites, the majority of which remain undetected when using general metabarcoding primers. Most of these Microsporidia-targeting primer pairs amplify fragments of different length of the small subunit ribosomal RNA (SSU-rRNA) gene. However, we lack a broad comparison of the efficacy of those primers. Here, we conducted in silico PCRs with three short-read (which amplify a few-hundred base pairs) and two long-read (which amplify over a thousand base pairs) metabarcoding primer pairs on a variety of publicly available Microsporidia sensu lato SSU-rRNA gene sequences to test which primers capture most of the Microsporidia diversity. Our results indicate that the primer pairs do result in slight differences in inferred richness. Furthermore, some of the reverse primers are also able to bind to microsporidian subtaxa beyond the classical Microsporidia, which include the metchnikovellidan Amphiamblys spp., the chytridiopsid Chytridiopsis typographi and the "short-branch" microsporidian Mitosporidium daphniae.

An insight into misidentification of the small-subunit ribosomal RNA (18S rRNA) gene sequences of Theileria spp. as Theileria annulata

BackgroundThere had been isolated reports of the presence of novel Theileria annulata genotypes based on the 18S rRNA gene sequence data from India, Pakistan and Saudi Arabia; but, these studies were restricted to limited field samples. Additionally, no comparative study has been conducted on all the isolates of this parasite from different countries whose sequences are available in the nucleotide databases. Therefore, we aimed to study the genetic diversity of T. annulata based on all available nearly complete 18S rRNA gene sequences in the GenBank™. Out of a total of 312 gene sequences of T. annulata available in the NCBI database, only 70 nearly complete sequences (> 1527 bp) were used for multiple sequence alignment.ResultsThe maximum likelihood tree obtained using TN93 + G + I model manifested two major clades. All the valid host-cell transforming Theileria species clustered in one clade. The T. annulata designated sequences occupying this clade clustered together, excluding two isolates (DQ287944 and EU083799), and represented the true T. annulata sequences (n = 54). DQ287944 and EU083799 exhibited close association with Theileria lestoquardi. In addition, 14 Indian sequences formed a large monophyletic group with published Theileria orientalis sequences. The broad range of sequence identity (95.8–100%) of T. annulata designated sequences indicated the presence of different Theileria spp. A closer analysis revealed the presence of three Theileria spp., namely, T. annulata, T. orientalis, and two isolates (DQ287944 and EU083799) closely related to T. lestoquardi. The true T. annulata sequences manifested 98.8–100% nucleotide identity within them. EU083799 and 14 misidentified Indian T. annulata sequences exhibited the highest similarity with T. lestoquardi (98.6–98.8%) and T. orientalis (98.0-99.9%) in comparison with the other Theileria spp. of domestic and wild ruminants.ConclusionIn the course of analyzing the genetic diversity of T. annulata, we identified the nearly complete 18S rRNA gene sequences of other Theileria spp. that have not only been misidentified as T. annulata in the GenBank™, but are also published as T. annulata. Moreover, a high level of sequence conservation was noticed in the 18S rRNA gene of true T. annulata and T. orientalis sequences.

Phylogenetic analysis, genetic diversity and geographical distribution of Babesia caballi based on 18S rRNA gene

The present investigation was aimed to study the presence of Babesia caballi clades upon phylogenetic analysis of all available V4 hypervariable 18S rRNA gene sequences in GenBank in addition to the intra- and interclade genetic diversity in B. caballi and the distribution of parasite clades in different countries. Out of altogether 155 small-subunit ribosomal RNA gene sequences of B. caballi available in the database, only 92 sequences with a complete V4 hypervariable region (>293 bp) were used in multiple sequence alignment. The phylogenetic tree placed all the sequences into two distinct clades with high bootstrap values which are designated as B. caballi clades A and B. Clade A was further divided into two subclades A1 and A2 with 98% bootstrap support. On the contrary, clade B contained multiple small subclades which either lacked bootstrap support or did not have enough bootstrap support to further group them into subclades. All the sequences of B. caballi were 91.5-100% identical with each other. Clade B manifested a comparatively higher genetic diversity (95.2-100% identity) amongst sequences as opposed to clade A (97.3-100% identity). Moreover, it indicated 91.5-93.5%, 92.9-94.6% and 91.5-94.6% nucleotide identity with B. caballi subclades A1, A2, and clade A, respectively. Significant nucleotide variations were observed in one region, between nucleotide positions 126-178, in some of the sequences. A total of 21 molecular signature residues were identified in the V4 hypervariable region. The alignment report of the V4 hypervariable region of 18S rRNA gene of clades A and B exhibited nucleotide variation at nine and 24 places, respectively. The distribution map of all the clades of B. caballi is also reported. The number of 18S rRNA gene sequences employed in the study is relatively high compared to previous studies. Therefore, a fair comparison of definite genetic variations between isolates/sequences from different countries was carried out.

New insights into the phylogeny of the dark-spored Myxomycetes (Amoebozoa: Conosa: Myxogastria: Fuscisporidia) and polyphyly of the genus Stemonitis

About a thousand described species of Myxomycetes, colloquially named slime moulds, are present in most terrestrial habitats. Molecular environmental sampling has revealed that a significant proportion of sequences could not be assigned to known species, with some specific groups showing a high hidden diversity. One such group included Stemonitis and allied genera, with only a few closely related reference sequences from known species and many distantly related sequences from environmental sampling. To improve the phylogeny and help define genera in this lineage, we provided nine new small-subunit ribosomal RNA gene sequences of Stemonitis and allied genera, along with microscopy observations. We showed that the genus Stemonitis is polyphyletic, clearly separated into two groups, with the main distinguishing feature being spore ornamentation. The first group included the type species Stemonitis fusca, and was characterized by reticulated spores, while the second contained species with warted spores (e.g. S. flavogenita and S. axifera) and many undetermined environmental sequences. In addition, we show that the related genera Stemonitopsis, Stemonaria and Symphytocarpus were polyphyletic and that their circumscription was based on inconsistent characteristics. Instead, representatives of these genera clustered into the two clades according to their spore ornamentation. Our study provides a first insight into the genetic diversity of Stemonitis and allied genera, and proposes spore ornamentation as an evolutionary informative feature to distinguish genera in this group.

Marinobacter piscensis sp. nov., a moderately halophilic bacterium isolated from salty food in Tunisia.

An aerobic, Gram-negative, moderately halophilic bacterium, oxidase, and catalase positive-designated Abdou3(T), was isolated from salted traditional foods (Anchovies) in Tunisia. Cells were rod-shaped, non-spore-forming and motile. Growth occurred at 15-45°C (optimum, 37°C), pH 5.5-8.75 (optimum, 7.3), and in the presence of 1-15% NaCl (optimum, 10%). Strain Abdou3(T) used glucose, D-arabinose, and sucrose. Strain Abdou3(T) had Q9 as the major respiratory quinone and C18:1 ω9c and C16:0 as predominant fatty acids. The DNA G+C content was 55.2mol%. Phylogenetic analysis of the small-subunit ribosomal RNA (rRNA) gene sequence indicated that strain Abdou3(T) had as its closest relative Marinobacter maritimus (identity of 96%). Based on phenotypic, phylogenetic, and taxonomic characteristics, strain Abdou3(T) is proposed as a novel species of the genus Marinobacter within the order Alteromonadales, for which the name M. piscensis sp. nov. is proposed. The type strain is Abdou3(T) (=DSM 26804(T)).

Molecular analysis of lungworms from European bison (Bison bonasus) on the basis of small subunit ribosomal RNA gene (SSU)

Dictyocaulosis (Nematoda: Trichostrongyloidea) is a widespread parasitosis of the European bison (Bison bonasus) inhabiting Bialowieza Primeval Forest. Bearing in mind the current coexistence of bison with wild cervids, and with domestic ruminants in the 19th and 20th century, the need arose for molecular identification of lungworm species. Molecular analysis was done on adult lungworms that were obtained from the respiratory track of four free-roaming bison euthanized as a part of the population health control program. As the result of the study four identical small subunit-ribosomal RNA gene sequences from the lungworms were obtained and deposited in GenBank as sequence, 1708 bp long (GenBank KC771250). Comparative analysis of the SSU rRNA sequences revealed the European bison to be a host for the bovine lungworm Dictyocaulus viviparus.

Virgibacillus natechei sp. nov., A Moderately Halophilic Bacterium Isolated from Sediment of a Saline Lake in Southwest of Algeria

A novel, Gram-positive, moderately halophilic bacterium, oxidase- and catalase-positive designated FarD(T) was isolated from sediments of a saline lake located in Taghit, 93km from Bechar, southwest of Algeria. Cells were rod-shaped, endospore forming, and motile. Growth occurred at 15-40°C (optimum, 35°C), pH 6.0-12.0 (optimum, 7.0) and in the presence of 1-20% NaCl (optimum, 10%). Strain FarD(T) used glucose, mannitol, melibiose, D-mannose, and 5 ketogluconate. The polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, and three phospholipids; MK-7 is the predominant menaquinone. The predominant cellular fatty acids were anteiso C15:0, anteiso C17:0, C20:0, and anteiso C19:0. The DNA G+C content was 42.1mol%. Phylogenetic analysis of the small-subunit ribosomal RNA gene sequence indicated that strain FarD(T) had as its closest relative Virgibacillus salinus (similarity of 96.3%). Based on phenotypic, phylogenetic, and taxonomic characteristics, strain FarD(T) is proposed as a novel species of the genus Virgibacillus within the order Clostridiales, for which the name V. natechei is proposed. The type strain is FarD(T) (=DSM 25609(T)=CCUG 62224(T)).

Desulfosoma profundi sp. nov., a thermophilic sulfate-reducing bacterium isolated from a deep terrestrial geothermal spring in France

A novel strictly anaerobic bacterium designated SPDX02-08(T) was isolated from a deep terrestrial geothermal spring located in southwest France. Cells (1-2×2-6μm) were non-motile, non sporulating and stained Gram negative. Strain SPDX02-08(T) grew at a temperature between 40 and 60°C (optimum 55°C), pH between 6.3 and 7.3 (optimum 7.2) and a NaCl concentration between 0 and 5g/l (optimum 2g/l). Sulfate, thiosulfate and sulfite were used as terminal electron acceptors, but not elemental sulfur, nitrate, nitrite, Fe (III) or fumarate. In the presence of sulfate, strain SPDX02-08(T) completely oxidized pyruvate, propionate, butyrate, isobutyrate, valerate, isovalerate and hexadecanoate. Stoichiometric measurements revealed a complete oxidation of part of lactate (0.125mol of acetate produced per mole lactate oxidized). Strain SPDX02-08(T) required yeast extract to oxidize formate and H(2) but did not grow autotrophically on H(2). Among the substrates tested, only pyruvate was fermented. The G+C content of the genomic DNA was 57.6mol%. Major cellular fatty acids of strain SPDX02-08(T) were iso-C(15:0), C(15:0), and C(16:0). Phylogenetic analysis of the 16S small-subunit (SSU) ribosomal RNA gene sequence indicated that strain SPDX02-08(T) belongs to the genus Desulfosoma, family Syntrophobacteraceae, having Desulfosoma caldarium as its closest phylogenetic relative (97.6% similarity). The mean DNA/DNA reassociation value between strain SPDX02-08(T) and Desulfosoma caldarium was 16.9±2.7%. Based on the polyphasic differences, strain SPDX02-08(T) is proposed to be assigned as a new species of the genus Desulfosoma, Desulfosoma profundi sp. nov. (DSM 22937(T)=JCM 16410(T)). GenBank accession number for the 16S rRNA gene sequence of strain SPDX02-08(T) is HM056226.

Delimiting operational taxonomic units for assessing ciliate environmental diversity using small‐subunit rRNA gene sequences

Delineating operational taxonomic units (OTUs) is a central element in any culture-independent analysis of environmental microbial eukaryotic diversity. Previous studies either have not justified their choice in sequence distance used to bin small-subunit ribosomal RNA (SSU rRNA) gene sequences amplified from environmental samples into OTUs, or have used a value based on the average across a broad sampling of microbial eukaryotes. Here, we analyse distances (320 922 pairwise comparisons) among sequences just from identified ciliates, and compare these with their taxonomic hierarchy. Our results show that no single sequence similarity value can always and unambiguously delineate species boundaries and higher taxa. Nevertheless, we suggest the use of 98% similarity to delineate ciliate OTUs because this threshold at least accounts for intra-specific polymorphism among multiple rRNA cistron copies. However, we suggest refraining from reconciling SSU rRNA gene-based OTUs and ciliate morphotypes; these OTUs should be used to analyse ciliate phylotype diversity, not ciliate species diversity.

Rapid Shifts in the Structure and Composition of a Protistan Assemblage During Bottle Incubations Affect Estimates of Total Protistan Species Richness

Changes in the structure and composition of a protistan community were characterized through the analysis of small-subunit ribosomal RNA gene (18S) sequences for a 3-day bottle incubation using a single sample collected in the western North Atlantic. Cloning and sequencing was used to investigate changes in perceived species richness and diversity as a consequence of environmental perturbation. The treatments included a control (unamended seawater), inorganic nutrient enrichment, and enrichment with a complex organic mixture. Five clone libraries were constructed and analyzed at the time of collection (t-0 h) and after 24 (t-24 h) and 72 (t-72 h) h for the control, and at t-72 h for the inorganic and organic enrichments, resulting in an analysis of 1,626 partial 18S rDNA sequences that clustered into 238 operational taxonomic units (OTUs). Analysis of the clone libraries revealed that protistan assemblages were highly dynamic and changed substantially at both the OTU level and higher taxonomic classifications during time frames consistent with many oceanographic methods used for measuring biological rates. Changes were most dramatic in enrichments, which yielded community compositions that were strongly dominated by one or a few taxa. Changes in community structure during incubation dramatically influenced estimates of species richness, which were substantially lower with longer incubation and especially with amendment, even though all incubated samples originated from the same aliquot of seawater. Containment and enrichment of the seawater sample led to the detection of otherwise undetected protistan taxa, suggesting that characterization of protistan diversity in a sample only at the time of collection could lead to an underrepresentation of unique taxa. Additionally, the rapid increase in the relative abundance of some members of the "rare biosphere" in our results implies an ecological importance of at least some of the taxa comprising the "rare biosphere."

Caldicoprobacter algeriensis sp. nov. a New Thermophilic Anaerobic, Xylanolytic Bacterium Isolated from an Algerian Hot Spring

A thermophilic anaerobic bacterium (strain TH7C1(T)) was isolated from the hydrothermal hot spring of Guelma in the northeast of Algeria. Strain TH7C1(T) stained Gram-positive, was a non-motile rod appearing singly, in pairs, or as long chains (0.7-1 × 2-6 μm(2)). Spores were never observed. It grew at temperatures between 55 and 75°C (optimum 65°C) and at pH between 6.2 and 8.3 (optimum 6.9). It did not require NaCl for growth, but tolerated it up to 5 g l(-1). Strain TH7C1(T) is an obligatory heterotroph fermenting sugars including glucose, galactose, lactose, raffinose, fructose, ribose, xylose, arabinose, maltose, mannitol, cellobiose, mannose, melibiose, saccharose, but also xylan, and pyruvate. Fermentation of sugars only occurred in the presence of yeast extract (0.1%). The end-products from glucose fermentation were acetate, lactate, ethanol, CO(2), and H(2). Nitrate, nitrite, thiosulfate, elemental sulfur, sulfate, and sulfite were not used as electron acceptors. The G+C content of the genomic DNA was 44.7 mol% (HPLC techniques). Phylogenetic analysis of the small-subunit ribosomal RNA (rRNA) gene sequence indicated that strain TH7C1(T) was affiliated to Firmicutes, order Clostridiales, family Caldicoprobacteraceae, with Caldicoprobacter oshimai (98.5%) being its closest relative. Based on phenotypic, phylogenetic, and genetic characteristics, strain TH7C1(T) is proposed as a novel species of genus Caldicoprobacter, Caldicoprobacter algeriensis, sp. nov. (strain TH7C1(T) = DSM 22661(T) = JCM 16184(T)).

Desulfovibrio legallis sp. nov.: A Moderately Halophilic, Sulfate-Reducing Bacterium Isolated from a Wastewater Digestor in Tunisia

A new moderately halophilic sulfate-reducing bacterium (strain H₁(T) ) was enriched and isolated from a wastewater digestor in Tunisia. Cells were curved, motile rods (2-3 x 0.5 μm). Strain H₁(T) grew at temperatures between 22 and 43°C (optimum 35°C), and at pH between 5.0 and 9.2 (optimum 7.3-7.5). Strain H₁(T) required salt for growth (1-45 g of NaCl/l), with an optimum at 20-30 g/l. Sulfate, sulfite, thiosulfate, and elemental sulfur were used as terminal electron acceptors but not nitrate and nitrite. Strain H₁(T) utilized lactate, pyruvate, succinate, fumarate, ethanol, and hydrogen (in the presence of acetate and CO₂) as electron donors in the presence of sulfate as electron acceptor. The main end-products from lactate oxidation were acetate with H₂ and CO₂. The G + C content of the genomic DNA was 55%. The predominant fatty acids of strain H₁(T) were C(15:0) iso (38.8%), C(16:0) (19%), and C(14:0) iso 3OH (12.2%), and menaquinone MK-6 was the major respiratory quinone. Phylogenetic analysis of the small-subunit (SSU) ribosomal RNA (rRNA) gene sequence indicated that strain H₁(T) was affiliated to the genus Desulfovibrio. On the basis of SSU rRNA gene sequence comparisons and physiological characteristics, strain H₁(T) is proposed to be assigned to a novel species of sulfate reducers of the genus Desulfovibrio, Desulfovibrio legallis sp. nov. (= DSM 19129(T) = CCUG 54389(T)).

Caldinitratiruptor microaerophilus, gen. nov., sp. nov. isolated from a French hot spring (Chaudes-Aigues, Massif Central): a novel cultivated facultative microaerophilic anaerobic thermophile pertaining to the Symbiobacterium branch within the Firmicutes

A novel facultative microaerophilic nitrate-reducing bacterium designated CA62N(T) was isolated from a thermal spring in France. Cells were non-motile rods (2-3 x 0.2 mum) and showed low cytoplasmic density when observed under a phase-contrast microscope. Strain CA62N(T) grew at temperatures between 50 and 75 degrees C (optimum 65 degrees C) and at a pH between 6.3 and 7.9 (optimum 7.0). NaCl was not required for growth but was tolerated up to 10 gl(-1). Sulfate, thiosulfate, elemental sulfur, sulfite, and nitrite were not used as electron acceptors. Nitrate was reduced to nitrite. Strain CA62N(T) used lactate, pyruvate, glucose, mannose, fructose, and casamino acids and some amino acids as electron donors only in the presence of nitrate as electron acceptor. None of these substrates was fermented. The main end-products of glucose oxidation were acetate, CO(2), and traces of H(2). The G + C content of the genomic DNA was 70.3 mol% (HPLC techniques). Phylogenetic analysis of the small-subunit (SSU) ribosomal RNA (rRNA) gene sequence indicated that strain CA62N(T) was affiliated to the Symbiobacterium branch within the Firmicutes and had Symbiobacterium thermophilum and "S. toebii" as its closest phylogenetic relatives. On the basis of phylogenetical and physiological characteristics, strain CA62N(T) is proposed to be the type strain for the novel species in the novel genus, Caldinitratiruptor microaerophilus gen. nov., sp. nov. (DSM 22660, JCM 16183).

High diversity of arbuscular mycorrhizal fungi in a boreal herb‐rich coniferous forest

* Here, the diversity of arbuscular mycorrhizal (AM) fungi was determined in a boreal herb-rich coniferous forest in relation to environmental variables. * Root samples of five plant species (Fragaria vesca, Galeobdolon luteum, Hepatica nobilis, Oxalis acetosella and Trifolium pratense) were analysed from stands differing in age and forest management intensity. * Thirty-four Glomeromycota taxa (small-subunit ribosomal RNA gene (SSU rDNA) sequence groups) were detected from 90 root samples (911 clones), including eight new taxa. Sequence groups related to Glomus intraradices were most common (MO-G3 and MO-G13). Samples of H. nobilis were colonized by more AM fungal taxa (3.68 +/- 0.31) than those of O. acetosella (2.69 +/- 0.34), but did not differ significantly in this respect from those of F. vesca (3.15 +/- 0.38). Effects of forest management, host plant species (except above) or season on the number or composition of fungal taxa in root samples were not detected, and neither were they explained by environmental variables (vegetation, soil and light conditions). * This is the most taxon-rich habitat described to date in terms of root-colonizing Glomeromycota. The data demonstrate the importance of temperate coniferous forests as habitats for AM fungi and plants. Lack of obvious fungal community patterns suggests more complex effects of biotic and abiotic factors, and possibly no adverse effect of common forest management practices on AM fungal diversity.

An Automated Phylogenetic Tree-Based Small Subunit rRNA Taxonomy and Alignment Pipeline (STAP)

Comparative analysis of small-subunit ribosomal RNA (ss-rRNA) gene sequences forms the basis for much of what we know about the phylogenetic diversity of both cultured and uncultured microorganisms. As sequencing costs continue to decline and throughput increases, sequences of ss-rRNA genes are being obtained at an ever-increasing rate. This increasing flow of data has opened many new windows into microbial diversity and evolution, and at the same time has created significant methodological challenges. Those processes which commonly require time-consuming human intervention, such as the preparation of multiple sequence alignments, simply cannot keep up with the flood of incoming data. Fully automated methods of analysis are needed. Notably, existing automated methods avoid one or more steps that, though computationally costly or difficult, we consider to be important. In particular, we regard both the building of multiple sequence alignments and the performance of high quality phylogenetic analysis to be necessary. We describe here our fully-automated ss-rRNA taxonomy and alignment pipeline (STAP). It generates both high-quality multiple sequence alignments and phylogenetic trees, and thus can be used for multiple purposes including phylogenetically-based taxonomic assignments and analysis of species diversity in environmental samples. The pipeline combines publicly-available packages (PHYML, BLASTN and CLUSTALW) with our automatic alignment, masking, and tree-parsing programs. Most importantly, this automated process yields results comparable to those achievable by manual analysis, yet offers speed and capacity that are unattainable by manual efforts.

Severe Scuticociliate (Philasterides dicentrarchi) Infection in a Population of Sea Dragons (Phycodurus eques and Phyllopteryx taeniolatus)

Scuticociliatosis is a disease of fish induced by ciliated parasites of the genus Scuticociliatida. It has been described in sea horses (Hippocampus sp.), flounders (Paralichthys olivaceus), and turbots (Scophthalmus maximus). Here we present a case study of a population of sea dragons chronically infected with scuticociliates identified as Philasterides dicentrarchi by histopathology and PCR. Beginning in 2004, over a period of 19 months, 10 sea dragons (Phycodurus eques and Phyllopteryx taeniolatus) were found dead in an aquarium of the Zoological Garden Basle, Switzerland. Clinically, the animals showed only faint symptoms of disease over a short period of time. At necropsy, macroscopic lesions were confined to the skin with multiple, often hemorrhagic, ulcerations. Histologically, epidermal ulcers were associated with necrosis and inflammation of the underlying dermis and musculature. Numerous ciliates, with a morphology consistent with scuticociliates, were present in these lesions. In several animals these ciliates had invaded blood vessels and were detected in gills and internal organs including kidney, thyroid gland, and central nervous system (CNS). In these organs, mild degenerative lesions and inflammatory reactions were evident. The ciliates were identified as Philasterides dicentrarchi based on small-subunit ribosomal RNA (SSUrRNA) gene sequences obtained by polymerase chain reaction (PCR) on DNA extracted from paraffin-embedded tissue sections. Our report shows that scuticociliate infections of sea dragons can develop into a systemic infection and that both species of sea dragons can be affected.

Morphology and 18S rDNA OF Henneguya gurlei (Myxosporea) from Ameiurus nebulosus (SILURIFORMES) in North Carolina

Henneguya gurlei was isolated from Ameiurus nebulosus captured in North Carolina and redescribed using critical morphological features and 18S small-subunit ribosomal RNA (SSU rDNA) gene sequence. Plasmodia are white, spherical, or subspherical, occur in clusters, measure up to 1.8 mm in length, and are located on the dorsal, pectoral, and anal fins. Histologically, plasmodia are located in the dermis and subdermally, and the larger cysts disrupt the melanocyte pigment layer. The spore body is lanceolate, 18.2 +/- 0.3 microm (range 15.7-20.3) in length, and 5.4 +/- 0.1 microm (range 3.8-6.1) in width in valvular view. The caudal appendages are 41.1 +/- 1.1 microm (range 34.0-49.7) in length. Polar capsules are pyriform and of unequal size. The longer polar capsule measures 6.2 +/- 0.1 microm (range 5.48-7.06), while the shorter is 5.7 +/- 0.1 microm (range 4.8-6.4) in length. Polar capsule width is 1.2 +/- 0.03 microm (range 1.0-1.54). The total length of the spore is 60.9 +/- 1.2 microm (range 48.7-68.5). Morphologically, this species is similar to other species of Henneguya that are known to infect ictalurids. Based on SSU rDNA sequences, this species is most closely related to H. exilis and H. ictaluri, which infect Ictalurus punctatus.

Cylindrotheca closterium is a species complex as was evidenced by the variations of rbcL gene and SSU rDNA

The genus Cylindrotheca consists of a small group of marine diatoms with a few species described. Eleven isolates of diatoms identified as Cylindrotheca closterium morphologically were obtained from Jiaozhou Bay with their nuclear-encoded small-subunit ribosomal RNA (SSU rDNA) and chloroplast-encoded rbcL gene sequences determined in this study. Interestingly, very high sequence divergences of SSU rDNA and rbcL gene were found among these isolates, and numerous nucleotide variation of rbcL gene caused relatively few variation of deduced amino acid sequence. Phylogenetic analyses based on SSU rDNA and rbcL gene, respectively, grouped the isolates into 6 clades. Phylogenetic tree of SSU rDNA placed all the Cylindrotheca isolates together, separating them into two lineages clearly. Lineage I was composed of the eleven C. closterium isolates obtained in this study together with another C. closterium isolate, but some clades were not well supported. Lineage II contained two C. closterium isolates and one C. fusiformis isolate. Phylogenetic analysis of rbcL gene also separated the Cylindrotheca isolates into two well-defined lineages. The eleven C. closterium isolates formed a lineage and all clades were supported strongly. Statistical comparisons of SSU rDNA indicated that the average distance within lineage I was significantly higher than that of other microalgae species (P < 0.01). These results suggested the existence of cryptic species within C. closterium.

Phylogeny of the order Choreotrichida (Ciliophora, Spirotricha, Oligotrichea) as inferred from morphology, ultrastructure, ontogenesis, and SSrRNA gene sequences

The phylogeny within the order Choreotrichida is reconstructed using (i) morphologic, ontogenetic, and ultrastructural evidence for the cladistic approach and (ii) the small subunit ribosomal RNA (SSrRNA) gene sequences, including the new sequence of Rimostrombidium lacustris. The morphologic cladograms and the gene trees converge rather well for the Choreotrichida, demonstrating that hyaline and agglutinated loricae do not characterize distinct lineages, i.e., both lorica types can be associated with the most highly developed ciliary pattern. The position of Rimostrombidium lacustris within the family Strobilidiidae is corroborated by the genealogical analyses. The diagnosis of the genus Tintinnidium is improved, adding cytological features, and the genus is divided into two subgenera based on the structure of the somatic kineties. The diagnosis of the family Lohmanniellidae and the genus Lohmanniella are improved, and Rimostrombidium glacicolum Petz, Song and Wilbert, 1995 is affiliated.

The phylogeny of Myxosporea (Myxozoa) based on small subunit ribosomal RNA gene analysis

The phylogeny of the Myxosporea was studied using the small-subunit ribosomal RNA gene sequences. Maximum parsimony and Bayesian inference were used to determine myxosporean phylogenetic relationships. The analysis included 120 myxosporean sequences retrieved from GenBank and 21 newly obtained sequences of myxosporeans representing nine genera. Members of the genera Palliatus and Auerbachia were sequenced for the first time. The phylogenetic analysis supported a split of myxosporeans into two main lineages separating most of freshwater species from marine ones as described by previous authors. In addition to the two main lineages, a third lineage consisting of three species was found ( Sphaerospora truttae, Sphaerospora elegans and Leptotheca ranae) and additional exceptions to the marine/freshwater myxosporean split were recognised ( Sphaeromyxa hellandi, Sphaeromyxa longa and Myxidium coryphaenoideum). All three myxosporean lineages were characterised by specific lengths of SSU rDNA sequences. The lineage of marine myxosporeans split into five well-defined clades. They consisted of species with a similar site of infection and spore morphology and were referred as the Parvicapsula clade, the Enteromyxum clade, the Ceratomyxa clade, the marine Myxidium clade and the Kudoa clade, respectively. The inner topology of the freshwater clade was more complex but the trend to branch according to site of infection was observed in this clade as well. Due to the number of sequences available, a histozoic ( Myxobolus clade) predominated. Interestingly, five morphologically different species infecting urinary bladder clustered within the histozoic ( Myxobolus) clade. The phylogenetic trees derived from this study differ in a number of respects from the current taxonomy of the myxosporeans, which suggests that several currently utilised characters may be homoplasious or that reliance on a single gene tree may not adequately reflect the phylogeny of the group.