Internal Transcribed Spacers as Phylogenetic Markers Enable Species-level Metataxonomic Analysis of Ciliated Protozoa

BackgroundThe conventional morphology-based classification of ciliates is often inaccurate and time-consuming. To address this issue, sequencing, and analysis of the 18S rRNA gene of ciliates have been used as an alternative. However, this method has limitations because the highly conserved nature of this gene makes it challenging to achieve species-level resolution. This study assesses the capability of two internal transcribed spacers, ITS1 and ITS2, along with the 28S rRNA gene, to enhance the taxonomic resolution beyond that offered by the 18S rRNA gene in free-living and host-associated ciliates. ResultsWe compared sequences of ITSI, ITS2, and the 18S and the 28S rRNA genes downloaded from public databases and found that ITS1 and ITS2 are more divergent at both inter- and intra-specific levels than the 18S rRNA gene. We designed universal primers specific to the two ITS regions and the 28S rRNA gene for free-living and rumen ciliates. We then systematically evaluated these primers using in-silico analysis, PCR assays, and metataxonomic or metabarcoding analysis and compared them to universal 18S rRNA gene primers. We found that the new primers are specific and inclusive, with an inclusiveness rate of over 80% based on in-silico analysis and confirmed their specificity using PCR evaluation. We validated the new primers with metagenomic DNA from freshwater samples and from rumen samples. Our metataxonomic analysis demonstrated that the ITS regions and the 28S rRNA gene could reveal greater ciliate diversity than the 18S rRNA gene in both environments. In particular, ITS1 detected the highest number of ciliate species, including species and genera that were not detected by the 18S rRNA gene. ConclusionsThe ITS regions, particularly ITS1, offer superior taxonomic resolution, and the NCBI ITS RefSeq database allows more species to be classified. Therefore, ITS1, and to a lesser extent ITS2, is recommended for enhancing metataxonomic analysis of ciliate communities in both freshwater and rumen environments.