Does filter pore size introduce bias in DNA sequence-based plankton community studies?

The cell size of microbial eukaryotic plankton normally ranges from 0.2 to 200 μm. During the past decade, high-throughput sequencing of DNA has been revolutionizing their study on an unprecedented scale. Nonetheless, it is currently unclear whether we can accurately, effectively, and quantitatively depict the microbial eukaryotic plankton community using size-fractionated filtration combined with environmental DNA (eDNA) molecular methods. Here we assessed the microbial eukaryotic plankton communities with two filtering strategies from two subtropical reservoirs, that is one-step filtration (0.2-200 μm) and size-fractionated filtration (0.2-3 and 3-200 μm). The difference of 18S rRNA gene copy abundance between the two filtering treatments was less than 50% of the 0.2-200 μm microbial eukaryotic community for 95% of the total samples. Although the microbial eukaryotic plankton communities within the 0.2-200 μm and the 0.2-3 and 3-200 μm size fractions had approximately identical 18S rRNA gene copies, there were significant differences in their community composition. Furthermore, our results demonstrate that the systemic bias introduced by size-fractionation filtration has more influence on unique OTUs than shared OTUs, and the significant differences in abundance between the two eukaryotic plankton communities largely occurred in low-abundance OTUs in specific seasons. This work provides new insights into the use of size-fractionation in molecular studies of microbial eukaryotes populating the plankton.