The menu varies with metabarcoding practices: A case study with the bat Plecotus auritus.

Metabarcoding of feces has revolutionized the knowledge of animal diets by providing unprecedented resolution of consumed resources. However, it is still unclear how different methodological approaches influence the ecological conclusions that can be drawn from such data. Here, we propose a critical evaluation of several data treatments on the inferred diet of the bat Plecotus auritus using guano regularly collected from various colonies throughout the entire active season. First and unlike previous claims, our data indicates that DNA extracted from large amounts of fecal material issued from guano accumulates yield broader taxonomic diversity of prey than smaller numbers of pellets would do, provided that extraction buffer volumes are adapted to such increased amounts of material. Second, trophic niche analyses based on prey occurrence data uncover strong seasonality in the bat's diet and major differences among neighboring maternity colonies. Third, while the removal of rare prey items is not always warranted as it introduces biases affecting particularly samples with greater prey species richness. Fourth, examination of distinct taxonomic depths in diet analyses highlights different aspects of food consumption providing a better understanding of the consumer's diet. Finally, the biologically meaningful patterns recovered with presence-absence approaches are virtually lost when attempting to quantify prey consumed using relative read abundances. Even in an ideal situation where reference barcodes are available for most potential prey species, inferring realistic patterns of prey consumption remains relatively challenging. Although best practice in metabarcoding analyses will depend on the aims of the study, several previous methodological recommendations seem unwarranted for studying such diverse diets as that of brown long-eared bats.