Size does matter: tiny microbes slipping out 0.2 μm pores may alter standard biodiversity assessment

In the last decades, advances in sequencing technologies have enabled a better understanding of prokaryotic dynamics in the Ocean by providing unprecedented details about their biodiversity. In amplicon sequencing studies of planktonic prokaryotes, while we are still far from a global consensus protocol regarding target regions (e.g. choice of primers) and approach (long vs. short reads), biomass collection using 0.2 μm pore membranes for DNA extraction is an undisputed standard procedure. In this study, we challenged this dogma by analyzing the 0.2 μm filtrate of samples collected monthly over two years at a coastal station in the northern Adriatic Sea. Surprisingly, about 2.5% of the total free-living community escaped the standard 0.2 μm pores. The biodiversity of small microbes was limited, with the most abundant121 eight Amplicon Sequence Variants accounting for 75% of the total, on average. These were made up of well-known small bacteria such as SAR11 clade I and II and C. Actinomarina as well as by members of the mostly uncultured NS5 group of the Flavobacteriaceae and Marine Group II archaea. Overall, our results suggest caution in the use of 0.2 μm pore membranes for the study of these taxa and for rare biosphere-oriented investigations.