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

Chitchai Chantangsi^1,, Denis H. Lynn¹, Maria T. Brandl², Jeffrey C. Cole³, Neil Hetrick³ and Pranvera Ikonomi⁴

¹ Department of Integrative Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada
² United States Department of Agriculture, Agriculture Research Service, Western Regional Research Center, Food Safety and Health Unit, 800 Buchanan St, Albany, CA 94710, USA
³ Protistology Department, American Type Culture Collection, 10801 University Blvd, Manassas, VA 20110-2209, USA
⁴ Molecular Authentication Resource Center, American Type Culture Collection, 10801 University Blvd, Manassas, VA 20110-2209, USA

Correspondence
Denis H. Lynn
ddr{at}uoguelph.ca

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.

Present address: Department of Zoology, University of British Columbia, Biological Sciences Bldg, 6270 University Blvd, Vancouver, British Columbia V6T 1Z4, Canada.

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.

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