Utility of mitochondrial CO1 sequences for species discrimination of Spirotrichea ciliates (Protozoa, Ciliophora)

Ciliates are a diverse species group of the Protozoa, and nuclear and mitochondrial genes have been utilized to discover new species and discriminate closely related species. The mitochondrial cytochrome c oxidase subunit 1 (CO1) gene has been used to discriminate metazoan species and has also been applied for some groups in the phylum Ciliophora. However, it is difficult to produce a universal primer as a standard barcode, because unlike metazoans, mitochondrial DNA sequences of ciliates are long and highly variable. Therefore, to design the new primer set, we sequenced the mitochondrial genomes of two pseudokeronopsids in the class Spirotrichea using next-generation sequencing technology (HiSeq™ 2000). Based on putative CO1 gene fragments of the pseudokeronopsids, we designed the new primer set and successfully sequenced the CO1 of 69 populations representing 47 species (five orders, 14 families, and 27 genera). We found that CO1 showed higher resolution for separating congeneric species than did nuclear SSU rRNA gene sequences, and we identified some putative cryptic species.