Choice of pore size can introduce artefacts when filtering picoeukaryotes for molecular biodiversity studies

Published results of studies based on samples size fractionated by sequential filtration (e.g. 0.2-3 μm) indicate that many ciliate, dinoflagellate and rhizarian phylotypes are found among marine picoeukaryotes. This is somewhat surprising as these protists are typically known as being large organisms (often >10 μm) and no picoplanktonic species have so far been identified. Here, the abundances of ciliate and dinoflagellate phylotypes in published molecular studies of picoeukaryotes are shown to correlate negatively with the pore size chosen for the end filter in the sequential filtrations (i.e. the filter used to collect the microbial biomass). This suggests that extracellular DNA adhering to small particles may be the source of ciliate and dinoflagellate phylotypes in picoplanktonic size fractions. This hypothesis was confirmed using real-time qPCR, which revealed significantly less dinoflagellate 18S rDNA in a 0.8-3-μm size fraction compared to 0.2-3 μm. On average, the abundance of putative extracellular phylotypes decreased by 84-89 % when a 0.8- μm end filter was used rather than a 0.2-μm end filter. A 0.8-μm filter is, however, not sufficient to retain all picoeukaryotic cells. Thus, selection of filter pore size involves a trade-off between avoiding artefacts generated by extracellular DNA and sampling the entire picoeukaryotic community. In contrast to ciliate and dinoflagellate phylotypes, rhizarian phylotypes in the picoplankton size range do not display a pattern consistent with an extracellular origin. This is likely due to the documented existence of picoplanktonic swarmer cells within this group.