Effects of intragenomic polymorphism in the SSU rRNA gene on estimating marine microeukaryotic diversity: A test for ciliates using single-cell high-throughput DNA sequencing

Diversity assessments using the small subunit (SSU) rRNA gene allow for rapid assessments of microeukaryotic communities. Yet, for microeukaryotes, only limited information is available on the extent to which sequence variations within individual genomes exist at this locus. We investigated 14 marine species belonging to four major ciliate groups by single-cell high-throughput DNA sequencing to estimate the extent of intragenomic polymorphism in the V4 region of the SSU rRNA gene. Based on these results, we evaluated the potential effect of polymorphism on metabarcoding studies. The intragenomic variability was generally below 3% among about 1000 unique sequences obtained from each studied cell, except of Protocruzia sp., whose intragenomic variability was as high as 15.6% among about 2000 unique sequences. Intragenomic variations significantly correlated with the number of macronuclei, but not with the biovolume of cell or macronuclei, indicating the occurrence of nonuniform evolution within the macronuclei. The removal of rare unique sequences greatly decreased the number of variations. For diversity studies, we therefore propose to conservatively discard rare amplicons. Considering the intragenomic variation within the V4 region, a sequence similarity cutoff of 3% to delimit marine ciliate operational taxonomic units could be substantiated, as at least this threshold could exclude most effect of intragenomic polymorphisms, although it may be too strict for some particular groups.