DNA barcode of spirotrich ciliates (Protozoa: Ciliophora) with emphasis on the high resolution among closely related species

Abstract

The mitochondrial cytochrome c oxidase subunit I (COI) gene has been utilized for DNA barcoding to discriminate species. The CO1 barcode was also applied for ciliates and showed high resolution. However, previous primer pairs are not enough to uncover the class Spirotrichea of which one of species-rich groups in Ciliophora. Here we designed new primer pair for ciliate barcoding, especially for the class Spirotrichea. To design new primers, we sequenced mitochondrial genomes of two pseudokeronopsids by using next generation sequencing technique (HiSeq™ 2000). Based on the newly obtained CO1 gene fragments of the pseudokeronopsids, we designed new primer pair for spirotrichean ciliates and successfully sequenced CO1 barcode of 69 ciliate populations composed of 47 species. The CO1 barcode showed great potential to discriminate closely related species. We suggest reconsideration of Euplotes crassus as a distinct species, which was considered as a junior synonym of E. vannus, and found some candidates of cryptic species in Caudiholosticha sylvatica, Diophrys scutum, Euplotes crassus. Additionally, among the successfully sequenced 47 species, common species/genera were included and their barcodes will support to prove or reinforce biogeographical distribution pattern. to uncover the class Spirotrichea of which one of species-rich groups in Ciliophora. Here we designed new primer pair for ciliate barcoding, especially for the class Spirotrichea. To design new primers, we sequenced mitochondrial genomes of two pseudokeronopsids by using next generation sequencing technique (HiSeq™ 2000). Based on the newly obtained CO1 gene fragments of the pseudokeronopsids, we designed new primer pair for spirotrichean ciliates and successfully sequenced CO1 barcode of 69 ciliate populations composed of 47 species. The CO1 barcode showed great potential to discriminate closely related species. We suggest reconsideration of Euplotes crassus as a distinct species, which was considered as a junior synonym of E. vannus, and found some candidates of cryptic species in Caudiholosticha sylvatica, Diophrys scutum, Euplotes crassus. Additionally, among the successfully sequenced 47 species, common species/genera were included and their barcodes will support to prove or reinforce biogeographical distribution pattern.