Nuclear Small Subunit rRNA Gene Research Articles

The chloroplast and mitochondrial genomes of two Gomphonema parvulum (Bacillariophyta) environmental isolates from South Carolina (United States) and Virginia (United States).

Gomphonema parvulum is a cosmopolitan freshwater diatom that is used as an indicator in water quality biomonitoring. In this study, we report the culturing of two geographically separated isolates from southeastern North America, their morphology, and the sequencing and assembly of their mitochondrial and chloroplast genomes. Morphologically, both strains fit G. parvulum sensu lato, but the frustules from a protected habitat in South Carolina were smaller than those cited in the historic data of this species from the same location as well as a second culture from Virginia. Phylogenetic analyses using the rbcL gene placed both within a clade with G. parvulum. Genetic markers, including full chloroplast and mitochondrial genomes and the nuclear small subunit rRNA gene region were assembled from each isolate. The organellar genomes of the two strains varied slightly in size due to small differences in intergenic regions with chloroplast genomes of 121,035 bp and 121,482 bp and mitochondrial genomes of 34,639 bp and 34,654 bp. The intraspecific pairwise identities of the chloroplast and mitochondrial genomes of these two isolates were 97.9% and 95.4%, respectively. Multigene phylogenetic analysis demonstrated a close relationship between G. parvulum, Gomphoneis minuta, and Didymosphenia geminata.

Aonchotheca (Nematoda: Capillariidae) is validated as a separated genus from Capillaria by both mitochondrial and nuclear ribosomal DNA

BackgroundThe family Capillariidae is a group of thread-like nematodes of 27 genera and over 300 species that infect a great variety of hosts including humans. Among these, some taxa such as the genus Aonchotheca have remained controversial regarding their systematic status for decades. The aim of the current study was to verify Aonchotheca’s systemic status and to further determine whether it is a distinct genus from Capillaria using molecular and phylogenetic analyses.ResultsWe sequenced the mitochondrial (mt) genome and nuclear small subunit (18S) rRNA gene of Aonchotheca putorii, a representative species of the genus, and investigated its systematic status in Trichinellida using maximum likelihood and Bayesian inference. The differences in amino acid sequences of 13 protein-coding genes were 12.69–67.35% among Aonchotheca, Capillaria, Eucoleus, and Pseudocapillaria with cox1 (12.69%) and atp8 (67.35%) as the most and the least conserved gene, respectively, and the difference of two mt rRNAs was 18.61–34.15%. Phylogenetic analyses of the complete mt genome and 18S rRNAs unequivocally showed that Aonchotheca was a distinct genus from Capillaria.ConclusionsLarge difference exists among Aonchotheca, Capillaria, Eucoleus, and Pseudocapillarias. Aonchotheca putorii is the first species in the genus Aonchotheca for which a complete mitogenome has been sequenced. These data are useful for phylogenetics, systematics and the evolution of Capillariidae.Graphical

PCR and culture for diagnosis of Acanthamoeba keratitis

Background/AimsAcanthamoeba keratitis (AK) is a rare but sight-threatening infection. Molecular diagnosis of corneal scraping has improved the diagnosis of AK. Different molecular targets and conditions have been used in diagnosis...

Two New Benthic Diatoms of the Genus Achnanthidium (Bacillariophyceae) from the Hangang River, Korea

Two new benthic freshwater species belonging to the genus Achnanthidium were found in Korea. Achnanthidium ovale sp. nov. and A. cavitatum sp. nov. are described as new species based on light and scanning electron microscopy observations and molecular analyses. Both species are compared with the type material of morphologically similar taxa. Achnanthidium ovale differs from other species belonging to the A. pyrenaicum complex in outline, striation pattern, raphe central endings, and freestanding areolae at the apices. Achnanthidium cavitatum differs from other species in the A. minutissimum complex in outline, broad axial central area in the raphel ess valve, and slit-like areolae near the axial central area. We assessed their molecular characteristics by analyzing nuclear small subunit (SSU) rRNA and chloroplast-encoded rbcL gene sequences. Both the morphological comparison and the SSU and rbcL sequence analyses provide strong evidence to support the recognition of A. ovale and A. cavitatum as new species.

A new species and two new Chinese records of Ochroconis from sugarcane and banana rhizosphere in Guangxi, China

Abstract Ochroconis guangxiensis isolated from sugarcane and banana rhizosphere, was described as a new species based on morphological characteristics and phylogenetic analysis using sequence data of the nuclear small subunit rRNA gene (SSU), internal transcribed spacer (ITS) region and large subunit (LSU) rRNA gene. Taxonomic and phylogenetic remarks are also provided for O. minima and O. ramosa. The latter two species are newly recorded for China. These three Ochroconis species, as dark septate endophytes, inhabit rhizosphere and can form a symbiosis with sugarcane and banana.

Evidence of Intra-individual SSU Polymorphisms in Dark-spored Myxomycetes (Amoebozoa)

The nuclear small subunit rRNA gene (SSU or 18S) is a marker frequently used in phylogenetic and barcoding studies in Amoebozoa, including Myxomycetes. Despite its common usage and the confirmed existence of divergent copies of ribosomal genes in other protists, the potential presence of intra-individual SSU variability in Myxomycetes has never been studied before. Here we investigated the pattern of nucleotide polymorphism in the 5' end fragment of SSU by cloning and sequencing a total of 238 variants from eight specimens, each representing a species of the dark-spored orders Stemonitidales and Physarales. After excluding singletons, a relatively low SSU intra-individual variability was found but our data indicate that this might be a widely distributed phenomenon in Myxomycetes as all samples analyzed possessed various ribotypes. To determine if the occurrence of multiple SSU variants within a single specimen has a negative effect on the circumscription of species boundaries, we conducted phylogenetic analyses that revealed that clone variation may be detrimental for inferring phylogenetic relationships among some of the specimens analyzed. Despite that intra-individual variability should be assessed in additional taxa, our results indicate that special care should be taken for species identification when working with closely related species.

Natural Products among Brown Algae: The Case of Cystoseira schiffneri Hamel (Sargassaceae, Phaeophyceae).

A chemotaxonomic study on the marine brown alga Cystoseira schiffneri collected from the Tunisian marine coast allowed us to identify kjellmanianone (1) and a new isololiolide derivative named schiffnerilolide (2). The structure elucidation and the assignment of relative configurations of the isolated natural products were based on advanced mass spectrometric and nuclear magnetic resonance techniques. This outcome suggested a close phylogenetic relationship of C. schiffneri with brown algae belonging to genus Sargassum C. Agardh. Molecular characterization using the nuclear small subunit rRNA (SSU rRNA) gene (18S) sequence as genetic marker was made. Pigment analysis showed a significant seasonal change of carotenoids, in particular of fucoxanthin and fucoxanthinol. Also galactolipids, the main constituents of the thylakoid membranes, showed remarkable seasonal changes.

The phylogenetic position of eriophyoid mites (superfamily Eriophyoidea) in Acariformes inferred from the sequences of mitochondrial genomes and nuclear small subunit (18S) rRNA gene

Eriophyoid mites (superfamily Eriophyoidea) comprise >4400 species worldwide. Despite over a century of study, the phylogenetic position of these mites within Acariformes is still poorly resolved. Currently, Eriophyoidea is placed in the order Trombidiformes. We inferred the high-level phylogeny of Acari with the mitochondrial (mt) genome sequences of 110 species including four eriophyoid species, and the nuclear small subunit (18S) rRNA gene sequences of 226 species including 25 eriophyoid species. Maximum likelihood (ML), Bayesian inference (BI) and Maximum parsimony (MP) methods were used to analyze the sequence data. Divergence times were estimated for major lineages of Acari using Bayesian approaches. Our analyses consistently recovered the monophyly of Eriophyoidea but rejected the monophyly of Trombidiformes. The eriophyoid mites were grouped with the sarcoptiform mites, or were the sister group of sarcoptiform mites+non-eriophyoid trombidiform mites, depending on data partition strategies. Eriophyoid mites diverged from other mites in the Devonian (384Mya, 95% HPD, 352–410Mya). The origin of eriophyoid mites was dated to the Permian (262Mya, 95% HPD 230–307Mya), mostly prior to the radiation of gymnosperms (Triassic-Jurassic) and angiosperms (early Cretaceous). We propose that the placement of Eriophyoidea in the order Trombidiformes under the current classification system should be reviewed.

Sequence variation in nuclear ribosomal small subunit, internal transcribed spacer and large subunit regions of Rhizophagus irregularis and Gigaspora margarita is high and isolate-dependent.

Arbuscular mycorrhizal (AM) fungi are known to exhibit high intra-organism genetic variation. However, information about intra- vs. interspecific variation among the genes commonly used in diversity surveys is limited. Here, the nuclear small subunit (SSU) rRNA gene, internal transcribed spacer (ITS) region and large subunit (LSU) rRNA gene portions were sequenced from 3 to 5 individual spores from each of two isolates of Rhizophagus irregularis and Gigaspora margarita. A total of 1482 Sanger sequences (0.5Mb) from 239 clones were obtained, spanning ~4370bp of the ribosomal operon when concatenated. Intrasporal and intra-isolate sequence variation was high for all three regions even though variant numbers were not exhausted by sequencing 12-40 clones per isolate. Intra-isolate nucleotide variation levels followed the expected order of ITS>LSU>SSU, but the values were strongly dependent on isolate identity. Single nucleotide polymorphism (SNP) densities over 4SNP/kb in the ribosomal operon were detected in all four isolates. Automated operational taxonomic unit picking within the sequence set of known identity overestimated species richness with almost all cut-off levels, markers and isolates. Average intraspecific sequence similarity values were 99%, 96% and 94% for amplicons in SSU, LSU and ITS, respectively. The suitability of the central part of the SSU as a marker for AM fungal community surveys was further supported by its level of nucleotide variation, which is similar to that of the ITS region; its alignability across the entire phylum; its appropriate length for next-generation sequencing; and its ease of amplification in single-step PCR.

Ecology and population structure of the hyperbenthic copepod Mesaiokeras hurei Kršinić, 2003 (Calanoida: Mesaiokeratidae) from an isolated marine lake (Mljet island, Southern Adriatic Sea, Croatia)

The monogeneric family Mesaiokeratidae (Copepoda: Calanoida) is exclusively bentho-pelagic and comprises nine small hyperbenthic species. Previous knowledge of these species has been limited to the description of their morphology and habitat. We provide new information about the ecology of Mesaiokeras hurei Krsinic, 2003, one of the small-size species of the family and found only in an isolated marine lake on the southern Adriatic island of Mljet (Croatia). Genetic markers were used to investigate its phylogenetic relationships with other copepods. The abundance of M. hurei abundance was highly variable over the years and seasons sampled. Densities were higher in spring and autumn. It was particularly abundant in 2011, averaging 629 ± 800 individuals/m3, and with a maximum of 1971 individuals m−3 in November, but averaged 74 ± 71 individuals/m3 and 66 ± 71 individuals/m3 in 2001 and 2008, respectively. Specimens were found in a range of temperatures, from 10.5°C to 18.3°C, but a majority of individuals (67%) was found in the 12-14°C layer. In addition to temperature, oxygen was a significant predictor ( p < 0 . 001 ) of the vertical micro-distribution of M. hurei. The DNA barcode sequence for the species is reported here for the first time. Analysis of the nuclear small subunit rRNA gene fragment (18S) showed a genetic similarity with representatives of the superfamily Clausocalanoidea.

Molecular Identification of Fungal Isolates from Textile Wastewater by ITS-RFLP Method

Eight strains isolated from textile post-finishing wastewaters were subjected to molecular identification by ITS-RFLP (Internal Transcriber Spacer-Restriction Fragment Length Polymorphism) method, using ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) DNA amplification primers for ITS region of the nuclear small-subunit rRNA gene. Restriction fragments analysis was conducted post PCR amplification, and cleaved with HaeIII, HinfI, HhaI, MseI and SduI restriction enzymes. Differences in the lengths of the obtained amplicons were detected after gel electrophoresis, and restriction analyses of the PCR fragments led to identification of six out of eight isolates as belonging to fungi T. parceramosum/ T. reesei/ T. longi, Polyporus squamosus, Fusarium oxysporum and Trichoderma atroviride groups. The PCR ITS-RFLP molecular identification technique can be used successfully for routine screening of fungal isolates from various environments.

Novel Discovery of Two Heterotrichid Ciliates, Climacostomum virens and Fabrea salina (Ciliophora: Heterotrichea: Heterotrichida) in Korea

Two heterotrichid ciliates, Climacostomum virens (Ehrenberg, 1838) Stein, 1859 from brackish water and freshwater, and Fabrea salina Henneguy, 1890 from a solar saltern, were collected in Korea. They are novelly investigated in Korea by means of live observation, protargol staining and nuclear small subunit (SSU) rRNA gene sequencing. Climacostomum virens is characterized by pouch-like body shape, body length of <TEX>$200-370{\mu}m$</TEX> in vivo, conspicuous cytopharyngeal tube, macronuclei ribbon-like shape, and one to four in number, with or without symbiont algae in cytoplasm, 34-66 somatic kineties, 67-113 adoral zone of membranelles, 8-42 peristomial kineties, 24-37 apical membranelles. SSU rDNA sequence size is 1,591 bp and GC contents 48.52%. Fabrea salina is also characterized by scoop-like body shape with proboscis, body length of <TEX>$190-240{\mu}m$</TEX> in vivo, one to two rod-shaped macronuclei, oval micronuclei, grayish green cortical granules, 104-186 somatic kineties, 4-8 preoral kineties, 7-19 peristomial kineties and fragmented paroral membrane. SSU rDNA sequence size is 1,598 bp and GC contents 47.50%.

Comparison of potential diatom 'barcode' genes (the 18S rRNA gene and ITS, COI, rbcL) and their effectiveness in discriminating and determining species taxonomy in the Bacillariophyta.

Diatoms form an enormous group of photoautotrophic micro-eukaryotes and play a crucial role in marine ecology. In this study, we evaluated typical genes to determine whether they were effective at different levels of diatom clustering analysis to assess the potential of these regions for barcoding taxa. Our test genes included nuclear rRNA genes (the nuclear small-subunit rRNA gene and the 5.8S rRNA gene+ITS-2), a mitochondrial gene (cytochrome c-oxidase subunit 1, COI), a chloroplast gene [ribulose-1,5-biphosphate carboxylase/oxygenase large subunit (rbcL)] and the universal plastid amplicon (UPA). Calculated genetic divergence was highest for the internal transcribed spacer (ITS; 5.8S+ITS-2) (p-distance of 1.569, 85.84% parsimony-informative sites) and COI (6.084, 82.14%), followed by the 18S rRNA gene (0.139, 57.69%), rbcL (0.120, 42.01%) and UPA (0.050, 14.97%), which indicated that ITS and COI were highly divergent compared with the other tested genes, and that their nucleotide compositions were variable within the whole group of diatoms. Bayesian inference (BI) analysis showed that the phylogenetic trees generated from each gene clustered diatoms at different phylogenetic levels. The 18S rRNA gene was better than the other genes in clustering higher diatom taxa, and both the 18S rRNA gene and rbcL performed well in clustering some lower taxa. The COI region was able to barcode species of some genera within the Bacillariophyceae. ITS was a potential marker for DNA based-taxonomy and DNA barcoding of Thalassiosirales, while species of Cyclotella, Skeletonema and Stephanodiscus gathered in separate clades, and were paraphyletic with those of Thalassiosira. Finally, UPA was too conserved to serve as a diatom barcode.

The complete mitochondrial genomes of three parasitic nematodes of birds: a unique gene order and insights into nematode phylogeny

BackgroundAnalyses of mitochondrial (mt) genome sequences in recent years challenge the current working hypothesis of Nematoda phylogeny proposed from morphology, ecology and nuclear small subunit rRNA gene sequences, and raise the need to sequence additional mt genomes for a broad range of nematode lineages.ResultsWe sequenced the complete mt genomes of three Ascaridia species (family Ascaridiidae) that infest chickens, pigeons and parrots, respectively. These three Ascaridia species have an identical arrangement of mt genes to each other but differ substantially from other nematodes. Phylogenetic analyses of the mt genome sequences of the Ascaridia species, together with 62 other nematode species, support the monophylies of seven high-level taxa of the phylum Nematoda: 1) the subclass Dorylaimia; 2) the orders Rhabditida, Trichinellida and Mermithida; 3) the suborder Rhabditina; and 4) the infraorders Spiruromorpha and Oxyuridomorpha. Analyses of mt genome sequences, however, reject the monophylies of the suborders Spirurina and Tylenchina, and the infraorders Rhabditomorpha, Panagrolaimomorpha and Tylenchomorpha. Monophyly of the infraorder Ascaridomorpha varies depending on the methods of phylogenetic analysis. The Ascaridomorpha was more closely related to the infraorders Rhabditomorpha and Diplogasteromorpha (suborder Rhabditina) than they were to the other two infraorders of the Spirurina: Oxyuridorpha and Spiruromorpha. The closer relationship among Ascaridomorpha, Rhabditomorpha and Diplogasteromorpha was also supported by a shared common pattern of mitochondrial gene arrangement.ConclusionsAnalyses of mitochondrial genome sequences and gene arrangement has provided novel insights into the phylogenetic relationships among several major lineages of nematodes. Many lineages of nematodes, however, are underrepresented or not represented in these analyses. Expanding taxon sampling is necessary for future phylogenetic studies of nematodes with mt genome sequences.

Examining new phylogenetic markers to uncover the evolutionary history of early-diverging fungi: comparing MCM7, TSR1 and rRNA genes for single- and multi-gene analyses of the &lt;I&gt;Kickxellomycotina&lt;/I&gt;

The recently recognised protein-coding genes MCM7 and TSR1 have shown significant promise for phylogenetic resolution within the Ascomycota and Basidiomycota, but have remained unexamined within other fungal groups (except for Mucorales). We designed and tested primers to amplify these genes across early-diverging fungal clades, with emphasis on the Kickxellomycotina, zygomycetous fungi with characteristic flared septal walls forming pores with lenticular plugs. Phylogenetic tree resolution and congruence with MCM7 and TSR1 were compared against those inferred with nuclear small (SSU) and large subunit (LSU) rRNA genes. We also combined MCM7 and TSR1 data with the rDNA data to create 3- and 4-gene trees of the Kickxellomycotina that help to resolve evolutionary relationships among and within the core clades of this subphylum. Phylogenetic inference suggests that Barbatospora, Orphella, Ramicandelaber and Spiromyces may represent unique lineages. It is suggested that these markers may be more broadly useful for phylogenetic studies among other groups of early-diverging fungi.

Molecular phylogenetic analyses based on the nuclear rRNA genes and the intron–exon structures of the nuSSU rRNA gene in Dictyocatenulata alba (anamorphic Ascomycota)

Molecular phylogenies inferred from the nuclear small subunit rRNA gene (nuSSU), nuclear large subunit rRNA gene D1/D2 region (nuLSU), and ITS-5.8S rRNA gene (ITS) indicated that five cultures of the lichenized hyphomycete Dictyocatenulata alba isolated from Japan form a monophyletic clade with high bootstrap support, and a close relationship to the Ostropomycetidae (Lecanoromycetes, Pezizomycotina, Ascomycota). Insertion sequences were found in the nuSSU of all isolates [e.g., nine insertions in the strain JCM 5358 (Japan Collection of Microorganisms)], some of which were group I introns. Five new insertion positions were found among the D. alba isolates. Using BLAST, none of the insertion sequences of D. alba were closely related to those of fungi or other organisms deposited in public DNA databases. Insertion positions were similar in some isolates, and two positions were common to all isolates. Although all phylogenetic analyses based on nuSSU, nuLSU, and ITS revealed the monophyly of D. alba, the isolates were divided into two (in the nuSSU tree) or three (in the nuLSU and ITS trees) groups. Based on the phylogenetic analyses and the intron–exon structures, the five isolates identified as D. alba belong to three cryptic species and therefore D. alba should be considered a species complex. The very slow-growing, tough agar colonies of the isolates, the occurrence of the species on both slightly lichenized and nonlichenized surfaces of trees, or pebbles (rarely on soil), suggest that the members of the D. alba complex may be lichenized. The photobiont was not clearly identified in this study.

Babesia microtiin Rodents and Raccoons from Northeast Florida

Human babesiosis in the United States is caused most commonly by the intraerythrocytic protozoan parasite, Babesia microti . Although a few reports have described evidence of Babesia species in animals in Florida, to date Babesia microti specifically has not been reported from Florida or most other southern states. To determine if the organism is present in vertebrates in the region, small mammals were trapped and sampled at 2 sites in northeastern Florida, and DNA extracts from blood samples were screened for B. microti DNA via PCR assays targeting portions of the nuclear small subunit rRNA (18S rDNA) and beta-tubulin genes. Amplified fragments from representative samples of PCR-positive hosts were sequenced and compared phylogenetically to reference strains of Babesia species. The B. microti strains found in cotton rats ( Sigmodon hispidus ) most closely resembles B. microti sensu stricto strains that are pathogenic to humans, and strains found in raccoons ( Procyon lotor ) most closely resembles previously described raccoon-related strains of B. microti sensu lato. The results of this study suggest that B. microti is prevalent among cotton rats and raccoons at some sites in northeast Florida and may pose a risk to humans in the region.

Identification of a Novel T17 Genotype of Acanthamoeba from Environmental Isolates and T10 Genotype Causing Keratitis in Thailand

We analyzed the nuclear small-subunit rRNA genes of Acanthamoeba isolates from freshwater sources (n=16) and from patients (n=6) in Bangkok and surrounding areas. The T10 genotype from a keratitis patient and a novel T17 genotype from water samples were diagnosed for the first time in this study.

Genome size and phylogenetic analysis of the A and L races of Botryococcus braunii

Botryococcus braunii (Chlorophyta, Botryococcaceae) is a colony-forming green microalga that produces large amounts of liquid hydrocarbons, which can be converted into transportation fuels. There are three different races of B. braunii, A, B, and L, that are distinguished based on the type of hydrocarbon each produces. Each race also has many strains that are distinguished by the location from which they were collected. While B. braunii has been well studied for the chemistry of the hydrocarbon production, very little is known about the molecular biology of B. braunii. To begin to address this problem, we determined the genome size of the A race, Yamanaka strain, and the L race, Songkla Nakarin strain, of B. braunii. Flow cytometry analysis indicates that the A race of B. braunii has a genome size of 166.0 ± 0.4 Mb, while the L race has a substantially larger genome size at 211.3 ± 1.7 Mb. We also used phylogenetic analysis with the nuclear small subunit (18S) rRNA gene to classify strains of the A and B races that have not yet been compared evolutionarily to previously published B. braunii phylogenetics. The analysis suggests that the evolutionary relationship between B. braunii races is correlated with the type of liquid hydrocarbon they produce.

Isolation and genotyping of Acanthamoeba strains from corneal infections in Italy

Acanthamoeba keratitis (AK) is a corneal disease caused by members of a genus of free-living amoebae and is associated predominantly with contact lens (CL) use. This study reports 16 cases of culture-proven AK diagnosed in northern Italy. Genotype identification was carried out with a PCR assay based on sequence analysis of the 18S rRNA gene, and sensitivity and specificity were evaluated in comparison with traditional parasitological techniques. A 405&emsp14;bp region of the 18S rRNA gene (ASA.S1) including diagnostic fragment 3 (DF3) was amplified using the genus-specific primers JDP1 and JDP2. Genotype assignment was based on phenetic analysis of the ASA.S1 subset of the nuclear small-subunit rRNA gene sequence excluding the highly variable DF3 region. Phylogenetic analysis was also performed on the sequences obtained. All patients complained of monolateral infection; 11 (68.75%) admitted improper CL disinfection. In 14/16 (87.5 %) subjects, corneal scrapings were stained with calcofluor white and haematoxylin and eosin and, in ten cases (62.5 %), microscopy was positive for Acanthamoeba cysts. In vitro culture on 3 % non-nutrient agar plates was obtained in all cases (100 %), whereas cloning and axenic growth were positive for 14 amoebic stocks (87.5 %). PCR analysis had 100 % sensitivity and specificity compared with in vitro axenic culture, showing positive amplification from 15 isolates. All Acanthamoeba strains belonged to the T4 genotype, the main AK-related genotype worldwide. These results confirmed the importance of a complete diagnostic protocol, including a PCR assay, for the clinical diagnosis of AK on biological samples. Genotyping allowed inclusion of all isolates in the T4 group, thus demonstrating the prevalence of this genotype in northern Italy.