Barcoding ciliates: a comprehensive study of 75 isolates of the genus Tetrahymena

Abstract

The mitochondrial cytochrome-c oxidase subunit 1 (cox1) gene has been proposed as a DNA barcode to identify animal species. To test the applicability of the cox1 gene in identifying ciliates, 75 isolates of the genus Tetrahymena and three non-Tetrahymena ciliates that are close relatives of Tetrahymena, Colpidium campylum, Colpidium colpoda and Glaucoma chattoni, were selected. All tetrahymenines of unproblematic species could be identified to the species level using 689 bp of the cox1 sequence, with about 11 % interspecific sequence divergence. Intraspecific isolates of Tetrahymena borealis, Tetrahymena lwoffi, Tetrahymena patula and Tetrahymena thermophila could be identified by their cox1 sequences, showing ,0.65 % intraspecific sequence divergence. In addition, isolates of these species were clustered together on a cox1 neighbour-joining (NJ) tree. However, strains identified as Tetrahymena pyriformis and Tetrahymena tropicalis showed high intraspecific sequence divergence values of 5.01 and 9.07 %, respectively, and did not cluster together on a cox1 NJ tree. This may indicate the presence of cryptic species. The mean interspecific sequence divergence of Tetrahymena was about 11 times greater than the mean intraspecific sequence divergence, and this increased to 58 times when all isolates of species with high intraspecific sequence divergence were excluded. This result is similar to DNA barcoding studies on animals, indicating that congeneric sequence divergences are an order of magnitude greater than conspecific sequence divergences. Our analysis also demonstrated low sequence divergences of ,1.0 % between some isolates of T. pyriformis and Tetrahymena setosa on the one hand and some isolates of Tetrahymena furgasoni and T. lwoffi on the other, suggesting that the latter species in each pair is a junior synonym of the former. Overall, our study demonstrates the feasibility of using the mitochondrial cox1 gene as a taxonomic marker for 'barcoding' and identifying Tetrahymena species and some other ciliated protists.Abbreviations: CVP, contractile vacuole pore; K2P, Kimura two-parameter; LSU, large subunit; NJ, neighbour-joining; PBG, polar basal granule; SSU, small subunit. The GenBank/EMBL/DDBJ accession numbers for the cox1 and SSU rDNA sequences determined in this study are EF070242-EF070328, as detailed in Supplementary Table S1.Details of the strains examined in this study, including sequence accession numbers, details of the nucleotide compositions of the cox1 and SSU rDNA sequences, values of overall, within-genus and between-genera divergence of datasets of the cox1 sequences and alignments of cox1 and SSU rDNA sequences are available as supplementary material with the online version of this paper.

Comparing Potential COI and SSU rDNA Barcodes for Assessing the Diversity and Phylogenetic Relationships of Cyphoderiid Testate Amoebae (Rhizaria: Euglyphida)

“…Mean inter-population COI sequence dissimilarities were indeed always higher than mean inter-population SSU rDNA sequence dissimilarities while COI mean intrapopulation sequence dissimilarities were lower than the SSU rDNA sequence dissimilarities. The apparently fast evolutionary rate of COI gene in Cyphoderiidae testate amoeba and lack of COI intra-population heterogeneity concur with previously reported data in most animals (Hebert et al 2003b;Remigio and Hebert 2003), and the other studied groups of protists (Chantangsi et al 2007;Evans et al 2007;Nassonova et al 2010). Our data suggest that the COI intra-individual polymorphism is non-existent while SSU rDNA intra-individual polymorphism remains likely.…”

“…The high COI sequence dissimilarities among Cyphoderia populations (>3%), in agreement with SSU DNA and to some extent with morphological data demonstrate that the five C. ampulla populations examined in this study represent distinct species. Such COI sequence dissimilarities values are indeed similar to most threshold values usually used for discriminating protist species (Chantangsi et al 2007;Evans et al 2007;Nassonova et al 2010). In agreement with the recent morphological and SSU rDNA molecular phylogenetic studies reported by Todorov et al (2009) andHeger et al (2010), our COI sequence data confirm the presence of a hidden diversity (i.e.…”

“…Although mainly based on animals, the potential use of COI marker as a barcoding tool was also demonstrated for several groups of protist (Barth et al 2006;Chantangsi et al 2007;Lin et al 2009;Nassonova et al 2010) and red algae (Saunders 2005). However, COI sequences are available for relatively few groups of eukaryotes.…”

“…This stresses the need of testing any potential barcode for species delimitation before utilizing the genetic marker in contingent research (e.g., a study on biogeography or paleoecology). Some recent studies have demonstrated the usefulness of the COI for species identification in different groups of microbial eukaryotes, such as ciliates (Barth et al 2006;Chantangsi et al 2007;Chantangsi and Lynn 2008), diatoms (Evans et al 2007;Evans et al 2008; Evans and Mann 2009), dinoflagellates (Lin et al 2009) and amoebozoans (Nassonova et al 2010). The utility of the COI marker for species level discrimination and phylogenetic inferences has yet to be tested within a member of the Rhizaria, the eukaryotic supergroup to which the euglyphid testate amoebae belong.…”

Environmental DNA COI barcoding for quantitative analysis of protists communities: A test using the Nebela collaris complex (Amoebozoa; Arcellinida; Hyalospheniidae)

“…The advantage of the COI-based metabarcoding approach is to provide a much finer level of taxonomic resolution than that the SSU rRNA gene usually used in environmental DNA studies. COI has also been shown to give high taxonomic resolution in many microbial eukary-otic groups (Barth et al 2006;Chantangsi et al 2007;Heger et al 2010;Lin et al 2009;Nassonova et al 2010). For exam-ple, in the Hyalospheniidae, SSU rRNA does not allow to discriminating among closely related taxa (Lara et al 2008) while COI does (Kosakyan et al 2012(Kosakyan et al , 2013.…”