Harnessing the power of metabarcoding in the ecological interpretation of plant-pollinator DNA data: strategies and consequences of filtering approaches

Although DNA metabarcoding of pollen mixtures has been increasingly used in the field of pollination biology, methodological and interpretation issues arise due to its high sensitivity. Filtering or maintaining false positives, contaminants, and rare taxa or molecular features could lead to different ecological results. Here, we reviewed how this choice has been addressed in 43 studies featuring pollen DNA metabarcoding, which highlighted a very high heterogeneity of filtering methods. We assessed how these strategies shaped pollen assemblage composition, species richness, and interaction networks. To do so, we compared four processing methods: unfiltering, filtering with a proportional 1% of sample reads, a fixed threshold of 100 reads, and the ROC approach (Receiver Operator Characteristic). The results indicated that filtering impacted species composition and reduced species richness, with ROC emerging as a conservative approach. Moreover, in contrast to unfiltered networks, filtering decreased network Connectance and Entropy, and it increased Modularity and Connectivity, indicating that using cut-off thresholds better describes interactions. Overall, unfiltering might compromise reliable ecological interpretations, unless a study targets rare species. We discuss the suitability of each filtering type, plead for justifying filtering strategies on biological or methodological bases and for developing shared approaches to make future studies more comparable.