scholarly journals Ciliate Environmental Diversity Can Be Underestimated by the V4 Region of SSU rDNA: Insights from Species Delimitation and Multilocus Phylogeny of Pseudokeronopsis (Ciliophora, Spirotrichea)

2019 ◽  
Vol 7 (11) ◽  
pp. 493 ◽  
Author(s):  
Zhan ◽  
Li ◽  
Xu
Keyword(s):  
Species Delimitation ◽  
High Throughput Sequencing ◽  
Sequence Similarity ◽  
Large Subunit ◽  
Small Subunit ◽  
Base Change ◽  
Published Data ◽  
Environmental Diversity ◽  
5.8S Rdna ◽  
Eukaryotic Microorganisms

Metabarcoding and high-throughput sequencing methods have greatly improved our understanding of protist diversity. Although the V4 region of small subunit ribosomal DNA (SSU-V4 rDNA) is the most widely used marker in DNA metabarcoding of eukaryotic microorganisms, doubts have recently been raised about its suitability. Here, using the widely distributed ciliate genus Pseudokeronopsis as an example, we assessed the potential of SSU-V4 rDNA and four other nuclear and mitochondrial markers for species delimitation and phylogenetic reconstruction. Our studies revealed that SSU-V4 rDNA is too conservative to distinguish species, and a threshold of 97% and 99% sequence similarity detected only one and three OTUs, respectively, from seven species. On the basis of the comparative analysis of the present and previously published data, we proposed the multilocus marker including the nuclear 5.8S rDNA combining the internal transcribed spacer regions (ITS1-5.8S-ITS2) and the hypervariable D2 region of large subunit rDNA (LSU-D2) as an ideal barcode rather than the mitochondrial cytochrome c oxidase subunit 1 gene, and the ITS1-5.8S-ITS2 as a candidate metabarcoding marker for ciliates. Furthermore, the compensating base change and tree-based criteria of ITS2 and LSU-D2 were useful in complementing the DNA barcoding and metabarcoding methods by giving second structure and phylogenetic evidence.

scholarly journals Species delimitation for the molecular taxonomy and ecology of the widely distributed microbial eukaryote genus Euplotes (Alveolata, Ciliophora)

2018 ◽  
Vol 285 (1871) ◽  
pp. 20172159 ◽  
Author(s):  
Yan Zhao ◽  
Zhenzhen Yi ◽  
Alan Warren ◽  
Weibo B. Song
Keyword(s):  
Species Delimitation ◽  
High Throughput Sequencing ◽  
Large Subunit ◽  
Molecular Taxonomy ◽  
Integrative Taxonomy ◽  
Small Subunit ◽  
Sensu Stricto ◽  
Gene Markers ◽  
Morphological Differences ◽  
Large Subunit Rdna

Recent advances in high-throughput sequencing and metabarcoding technologies are revolutionizing our understanding of the diversity and ecology of microbial eukaryotes (protists). The interpretation of protist diversity and the elucidation of their ecosystem function are, however, impeded by problems with species delimitation, especially as it applies to molecular taxonomy. Here, using the ciliate Euplotes as an example, we describe approaches for species delimitation based on integrative taxonomy by using evolutionary and ecological perspectives and selecting the most appropriate metabarcoding gene markers as proxies for species units. Our analyses show that: Euplotes ( sensu lato ) comprises six distinct clades, mainly as result of ecological speciation; the validity of the genera Euplotes ( sensu stricto ), Euplotoides , Euplotopsis and Moneuplotes are not supported; the vannus -type group, which includes species without distinct morphological differences, seems to be undergoing incipient speciation and contains cryptic species; the hypervariable V4 region of the small subunit rDNA and D1–D2 region of the large subunit rDNA are the promising candidates for general species delimitation in Euplotes .

scholarly journals Molecular Diversity of the Syndinean Genus Euduboscquella Based on Single-Cell PCR Analysis

2011 ◽  
Vol 78 (2) ◽  
pp. 334-345 ◽  
Author(s):  
Tsvetan R. Bachvaroff ◽  
Sunju Kim ◽  
Laure Guillou ◽  
Charles F. Delwiche ◽  
D. Wayne Coats
Keyword(s):  
Large Subunit ◽  
Small Subunit ◽  
Base Change ◽  
Pcr Analysis ◽  
Ssu Rrna ◽  
Compensatory Base Change ◽  
Content Type ◽  
Single Cell Pcr ◽  
Group I ◽  
Lsu Rrna

ABSTRACTThe genusEuduboscquellais 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 withEuduboscquellasampled 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 ofEuduboscquellaand 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 genusEuduboscquellaconsistently 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, inE. cachonithere was variation between ITS to LSU copies at up to 20 sites within an individual, while in a parasite ofTintinnopsisspp., 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.

scholarly journals Fungal Community Analysis by Large-Scale Sequencing of Environmental Samples

2005 ◽  
Vol 71 (9) ◽  
pp. 5544-5550 ◽  
Author(s):  
Heath E. O'Brien ◽  
Jeri Lynn Parrent ◽  
Jason A. Jackson ◽  
Jean-Marc Moncalvo ◽  
Rytas Vilgalys
Keyword(s):  
Large Scale ◽  
High Throughput Sequencing ◽  
Small Subunit ◽  
Its Sequences ◽  
High Rate ◽  
Rrna Genes ◽  
Soil Horizon ◽  
Eukaryotic Microorganisms ◽  
Sequence Types ◽  
Fungal Its

ABSTRACT Fungi are an important and diverse component of soil communities, but these communities have proven difficult to study in conventional biotic surveys. We evaluated soil fungal diversity at two sites in a temperate forest using direct isolation of small-subunit and internal transcribed spacer (ITS) rRNA genes by PCR and high-throughput sequencing of cloned fragments. We identified 412 sequence types from 863 fungal ITS sequences, as well as 112 ITS sequences from other eukaryotic microorganisms. Equal proportions of Basidiomycota and Ascomycota sequences were present in both the ITS and small-subunit libraries, while members of other fungal phyla were recovered at much lower frequencies. Many sequences closely matched sequences from mycorrhizal, plant-pathogenic, and saprophytic fungi. Compositional differences were observed among samples from different soil depths, with mycorrhizal species predominating deeper in the soil profile and saprophytic species predominating in the litter layer. Richness was consistently lowest in the deepest soil horizon samples. Comparable levels of fungal richness have been observed following traditional specimen-based collecting and culturing surveys, but only after much more extensive sampling. The high rate at which new sequence types were recovered even after sampling 863 fungal ITS sequences and the dominance of fungi in our libraries relative to other eukaryotes suggest that the abundance and diversity of fungi in forest soils may be much higher than previously hypothesized.

scholarly journals The “Green” Form I Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from the Nonsulfur Purple BacteriumRhodobacter capsulatus

1999 ◽  
Vol 181 (13) ◽  
pp. 3935-3941 ◽  
Author(s):  
Kempton M. Horken ◽  
F. Robert Tabita
Keyword(s):  
Genetic Manipulation ◽  
Sequence Similarity ◽  
Large Subunit ◽  
Small Subunit ◽  
Amino Acid Sequences ◽  
Kinetic Properties ◽  
Phylogenetic Groups ◽  
Bisphosphate Carboxylase ◽  
Related Organism ◽  
Green Form

ABSTRACT Form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) of the Calvin-Benson-Bassham cycle may be divided into two broad phylogenetic groups, referred to as red-like and green-like, based on deduced large subunit amino acid sequences. Unlike the form I enzyme from the closely related organism Rhodobacter sphaeroides, the form I RubisCO from R. capsulatus is a member of the green-like group and closely resembles the enzyme from certain chemoautotrophic proteobacteria and cyanobacteria. As the enzymatic properties of this type of RubisCO have not been well studied in a system that offers facile genetic manipulation, we purified theR. capsulatus form I enzyme and determined its basic kinetic properties. The enzyme exhibited an extremely low substrate specificity factor, which is congruent with its previously determined sequence similarity to form I enzymes from chemoautotrophs and cyanobacteria. The enzymological results reported here are thus strongly supportive of the previously suggested horizontal gene transfer that most likely occurred between a green-like RubisCO-containing bacterium and a predecessor to R. capsulatus. Expression results from hybrid and chimeric enzyme plasmid constructs, made with large and small subunit genes fromR. capsulatus and R. sphaeroides, also supported the unrelatedness of these two enzymes and were consistent with the recently proposed phylogenetic placement of R. capsulatus form I RubisCO. The R. capsulatus form I enzyme was found to be subject to a time-dependent fallover in activity and possessed a high affinity for CO2, unlike the closely similar cyanobacterial RubisCO, which does not exhibit fallover and possesses an extremely low affinity for CO2. These latter results suggest definite approaches to elucidate the molecular basis for fallover and CO2 affinity.

scholarly journals De novo species delimitation in metabarcoding datasets using ecology and phylogeny

2017 ◽  
Author(s):  
Caitlin Potter ◽  
Cuong Q Tang ◽  
Vera Fonseca ◽  
Delphine Lallias ◽  
John M Gaspar ◽  
...  
Keyword(s):  
Species Delimitation ◽  
Biological Diversity ◽  
De Novo ◽  
Sequence Similarity ◽  
Salinity Gradient ◽  
Small Subunit ◽  
Morphological Identification ◽  
Ecological Groups ◽  
Clustering Methods ◽  
Intraspecific Divergence

Background: Metabarcoding studies allow a wide variety of taxa to be analysed simultaneously in a fraction of the time taken by morphological identification, but currently metabarcoding studies must rely on sequence similarity-based methodologies to delimit operational taxonomic units (OTUs). Similarity-based OTU clustering methodologies can lead to inaccurate estimates of diversity, species’ distributions or responses to change, meaning that there is a critical need for methods to delimit species in metabarcoding datasets. Methods: We introduce SNAPhy (Species delimitation using Niche And PHYlogeny), a novel approach which utilises ecological and phylogenetic information to delimit de novo OTUs in metabarcoding datasets and avoids the problems associated with current OTU clustering methods. Sequencing reads are first divided into ecological groups based on co-occurrence, thereby reducing data complexity and facilitating the use of evolutionary and phylogenetic models (e.g. BEAST and GMYC) to delimit species-level groupings within discrete ecologically informed phylogenies. The utility of SNAPhy is demonstrated using an 18S rDNA nuclear small subunit (nSSU) dataset representing replicated samples taken along the entire length of an estuarine salinity gradient, and SNAPhy is then compared to existing OTU clustering methods. Results: All of the OTU clustering methods compared yielded different numbers of OTUs and a different taxonomic distribution of OTUs, which we suggest is due to the taxon differences that are known to exist in the degree of intraspecific divergence. SNAPhy and UCLUST (with a 98% similarity threshold) gave the most plausible numbers of OTUs, especially within the Nematoda. Additionally, the degree of variation within nematode OTUs delimited by SNAPhy lies within the range of variation in deeply metabarcoded individuals. Discussion: SNAPhy avoids the static clustering threshold problems associated with current OTU clustering methods and instead focuses on genuine biological diversity delimited according to a general lineage species concept. We suggest that the SNAPhy approach should play a crucial role in future sequencing-based biodiversity assessment by providing more accurate estimates of species diversity and distributions than current methods, thereby enabling more accurate impact assessments and better informing managerial decisions.

scholarly journals Archaeal DNA Replication: Identifying the Pieces to Solve a Puzzle

Genetics ◽  
1999 ◽  
Vol 152 (4) ◽  
pp. 1249-1267 ◽  
Author(s):  
Isaac K O Cann ◽  
Yoshizumi Ishino
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Pyrococcus Furiosus ◽  
Sequence Similarity ◽  
Large Subunit ◽  
Dna Polymerases ◽  
Small Subunit ◽  
Accessory Proteins ◽  
Pol Ii

Abstract Archaeal organisms are currently recognized as very exciting and useful experimental materials. A major challenge to molecular biologists studying the biology of Archaea is their DNA replication mechanism. Undoubtedly, a full understanding of DNA replication in Archaea requires the identification of all the proteins involved. In each of four completely sequenced genomes, only one DNA polymerase (Pol BI proposed in this review from family B enzyme) was reported. This observation suggested that either a single DNA polymerase performs the task of replicating the genome and repairing the mutations or these genomes contain other DNA polymerases that cannot be identified by amino acid sequence. Recently, a heterodimeric DNA polymerase (Pol II, or Pol D as proposed in this review) was discovered in the hyperthermophilic archaeon, Pyrococcus furiosus. The genes coding for DP1 and DP2, the subunits of this DNA polymerase, are highly conserved in the Euryarchaeota. Euryarchaeotic DP1, the small subunit of Pol II (Pol D), has sequence similarity with the small subunit of eukaryotic DNA polymerase δ. DP2 protein, the large subunit of Pol II (Pol D), seems to be a catalytic subunit. Despite possessing an excellent primer extension ability in vitro, Pol II (Pol D) may yet require accessory proteins to perform all of its functions in euryarchaeotic cells. This review summarizes our present knowledge about archaeal DNA polymerases and their relationship with those accessory proteins, which were predicted from the genome sequences.

Identification of Giardia species and Giardia duodenalis assemblages by sequence analysis of the 5.8S rDNA gene and internal transcribed spacers

Parasitology ◽  
2010 ◽  
Vol 137 (6) ◽  
pp. 919-925 ◽  
Author(s):  
SIMONE M. CACCIÒ ◽  
RELJA BECK ◽  
ANDRE ALMEIDA ◽  
ANNA BAJER ◽  
EDOARDO POZIO
Keyword(s):  
Sequence Analysis ◽  
Large Subunit ◽  
Practical Importance ◽  
Giardia Duodenalis ◽  
Epidemiological Studies ◽  
Small Subunit ◽  
Ribosomal Gene ◽  
Internal Transcribed Spacers ◽  
Animal Origin ◽  
5.8S Rdna

SUMMARYPCR assays have been developed mainly to assist investigations into the epidemiology of Giardia duodenalis, the only species in the Giardia genus having zoonotic potential. However, a reliable identification of all species is of practical importance, particularly when water samples and samples from wild animals are investigated. The aim of the present work was to genotype Giardia species and G. duodenalis assemblages using as a target the region spanning the 5.8S gene and the 2 flanking internal transcribed spacers (ITS1 and ITS2) of the ribosomal gene. Primers were designed to match strongly conserved regions in the 3′ end of the small subunit and in the 5′ end of the large subunit ribosomal genes. The corresponding region (about 310 bp) was amplified from 49 isolates of both human and animal origin, representing all G. duodenalis assemblages as well as G. muris and G. microti. Sequence comparison and phylogenetic analysis showed that G. ardeae, G. muris, G. microti as well as the 7 G. duodenalis assemblages can be easily distinguished. Since the major subgroups within the zoonotic assemblages A and B can be identified by sequence analysis, this assay is also informative for molecular epidemiological studies.

scholarly journals Integrative Studies on a New Ciliate Campanella sinica n. sp. (Protista, Ciliophora, Peritrichia) Based on the Morphological and Molecular Data, With Notes on the Phylogeny and Systematics of the Family Epistylididae

2021 ◽  
Vol 12 ◽  
Author(s):  
Zhe Wang ◽  
Tong Wu ◽  
Borong Lu ◽  
Yong Chi ◽  
Xue Zhang ◽  
...  
Keyword(s):  
Ribosomal Dna ◽  
Phylogenetic Analyses ◽  
Large Subunit ◽  
Small Subunit ◽  
Internal Transcribed Spacers ◽  
Lsu Rdna ◽  
5.8S Rdna ◽  
Freshwater Pond ◽  
The Family

During an investigation on freshwater peritrichs, a new colonial sessilid ciliate, Campanella sinica n. sp., was isolated from aquatic plants in an artificial freshwater pond in Qingdao, China. Specimen observations of this species were performed both in vivo and using silver staining. C. sinica n. sp. is characterized by the appearance of the mature colony, which is up to 2 cm high and contains more than 1,000 zooids, the asymmetric horn-shaped zooids, strongly everted and multi-layered peristomial lip, the slightly convex peristomial disc, and the well-developed haplokinety and polykinety, which make more than four circuits of the peristome before descending into the infundibulum. The small subunit ribosomal DNA (SSU rDNA), 5.8s rDNA and its flank internal transcribed spacers (ITS1-5.8s rDNA-ITS2), and large subunit ribosomal DNA (LSU rDNA) are sequenced and used for phylogenetic analyses which reveal that the family Epistylididae Kahl, 1933 is non-monophyletic whereas the genus Campanella is monophyletic and nests within the basal clade of the sessilids. The integrative results support the assertion that the genus Campanella represents a separate lineage from other epistylidids, suggesting a further revision of the family Epistylididae is needed. We revise Campanella including the transfer into this genus of a taxon formerly assigned to Epistylis, which we raise to species rank, i.e., Campanella ovata (Nenninger, 1948) n. grad. & n. comb. (original combination Epistylis purneri f. ovataNenninger, 1948). In addition, we provide a key to the identification of the species of Campanella.

cDNA cloning and gene expression of carbonic anhydrase in Chlamydomonas reinhardtii

1991 ◽  
Vol 69 (5) ◽  
pp. 1088-1096 ◽  
Author(s):  
Hideya Fukuzawa ◽  
Sarami Ishida ◽  
Shigetoh Miyachi
Keyword(s):  
Carbonic Anhydrase ◽  
Chlamydomonas Reinhardtii ◽  
Sequence Similarity ◽  
Large Subunit ◽  
Mrna Level ◽  
Nuclear Genome ◽  
Small Subunit ◽  
Nucleotide Sequence Analysis ◽  
Cdna Clones ◽  
Upstream Gene

cDNA and genes encoding periplasmic carbonic anhydrase (CA) polypeptides of Chlamydomonas reinhardtii have been isolated and characterized. Nucleotide sequence analysis of cDNA clones revealed that the large subunit (35 kDa or 36.5 kDa) and the small subunit (4 kDa) are cotranslated as a precursor polypeptide (41 626 Da) with a NH2-terminal hydrophobic signal peptide of 20 amino acids. The amino acid sequence of Chlamydomonas CA showed 20–22% identity with animal CA isozymes (CAI, CAII, CAIII, and CAVII). Three zinc-liganded histidine residues and those forming the hydrogen-bond network to zinc-bound solvent molecules were highly conserved. No significant sequence similarity was observed between Chlamydomonas CA and chloroplast CAs of spinach and pea. Two copies of structurally related CA genes (CAH1 and CAH2) were tandemly clustered in Chlamydomonas nuclear genome and regulated by external CO2 concentration in a reverse manner. The 5′ upstream gene CAH1 encodes the major periplasmic CA whose mRNA level is induced under low CO2 condition in light. Photosynthesis is absolutely required for the accumulation of the CAH1 mRNA. The 3′ downstream gene CAH2 is possibly a gene for another periplasmic CA isozyme, which is induced under high CO2 conditions. Light has an inhibitory effect on the accumulation of the CAH2 mRNA. Key words: photosynthesis, light regulation, zinc, CO2-concentrating mechanism, intracellular processing.

Diploscapter formicidae sp. n. (Rhabditida: Diploscapteridae), from the ant Prolasius advenus (Hymenoptera: Formicidae) in New Zealand

Nematology ◽  
2013 ◽  
Vol 15 (1) ◽  
pp. 109-123 ◽  
Author(s):  
Zeng Qi Zhao ◽  
Kerrie A. Davies ◽  
Evan C. Brenton-Rule ◽  
Julien Grangier ◽  
Monica A.M. Gruber ◽  
...  
Keyword(s):  
New Zealand ◽  
Large Subunit ◽  
Host Record ◽  
Small Subunit ◽  
Published Data ◽  
Rrna Gene ◽  
Scanning Electron Micrographs ◽  
Separate Species ◽  
New Host ◽  
Prolasius Advenus

Diploscapter formicidae sp. n. was collected from the ant Prolasius advenus and its nests in native beech forests of the South Island, New Zealand. This is a new host record for the nematode genus and the first report of an ant associate from the southern hemisphere. Diploscapter formicidae sp. n. appears to be native to New Zealand. No males were found from collections from 16 nests, in agreement with previously published data on the other members of this genus, suggesting that males are absent or very rare. The adult females have bilateral symmetry of the head, characteristic dorsal and ventral projections of the putative cheilostom with paired hook-like structures or hamuli, expansive membranous lateral lip flaps or laciniae, gymnostom and stegostom with parallel walls, a swollen procorpus, large terminal bulb with a strong valve, paired ovaries with medial vulva, and a short conoid tail with slender pointed or spicate tip. Scanning electron micrographs of the structure of the head confirmed that the lateral laciniae with finger-like tines or filopodia are moveable (alternately covering and exposing the mouth). These lateral lip flaps are located posterior to the stoma, but anterior to the pore-like amphidial openings. The anterior margin of the cheilostom possesses apomorphic lateral bell-shaped projections and the hamuli are broader and less pointed than other species that have been examined. Molecular phylogeny of near full-length small subunit, D2/D3 expansion segments of the large subunit rRNA gene and heat shock protein 90 (Hsp90) gene showed that D. formicidae sp. n. is monophyletic with the Diploscapter species and isolates available in GenBank, but is on an independent trajectory supporting separate species status.

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