Filtering Microbial Diversity: Evaluation of Different Pore Sizes for <scp>eDNA</scp> Community Profiling

ABSTRACT Environmental DNA (eDNA) metabarcoding is widely used in biodiversity monitoring, and its popularity is also growing because of its potential to simultaneously detect multiple taxonomic groups, allowing holistic community assessments. When working with water samples, the choice of the filter type is one of the key methodological factors influencing community characterization, with 0.22 μm pore size filters typically used for microbial communities and larger filters for eDNA of larger organisms. However, as holistic community assessments are increasingly adopted, the use of a single filter pore size would help to optimize sampling and experimental efforts. Yet, it remains unclear whether filters with large pore sizes can effectively capture both microbial and macro‐organism eDNA. This study evaluates the use of 0.45 μm filters, commonly used for metazoan eDNA metabarcoding, in assessing microbial diversity across different coastal environments. Replicates of water samples from the Venice Lagoon and nearby waters were filtered independently using both 0.45 μm and the standard 0.22 μm pore size filters and analyzed through 16S rRNA gene metabarcoding. Both filters capture a shared core microbial community, but they also retain distinct taxa. Alpha diversity was significantly higher in samples collected with the 0.45 μm filters, which also showed a more effective recovery of particle‐associated microbes. Our work contributes to optimizing eDNA‐based methodologies for large‐scale multi‐taxa biodiversity monitoring, demonstrating that 0.45 μm filters can effectively capture microbial diversity and supporting their use in holistic studies in aquatic environments.