Investigating the use of DNA metabarcoding to quantify bee foraging and effects of threshold selection

DNA metabarcoding of pollen is a useful tool for studying bee foraging ecology.However, several questions about this method remain unresolved, including the extentto which sequence read data is quantitative, which type of sequence count removalthreshold to use and how that choice affects our ability to detect rare flower visits, andhow sequence artefacts may confound conclusions about bee foraging behavior. Toaddress these questions, we isolated pollen from five plant species and createdtreatments comprised of pollen from each species alone and combinations of pollenfrom multiple species that varied in richness and evenness. We used ITS2 and rbcLmetabarcoding to identify plant species in the samples, compared the proportion ofpollen by mass to the proportion of sequencing reads for each plant species in eachtreatment, and analyzed the sequencing data using both liberal and conservativethresholds. We collected pollen from foraging bees, analyzed metabarcoding data fromthose samples using each threshold, and compared the differences in the pollinatornetworks constructed from the data. Regardless of the threshold used, the relationshipbetween the proportion of pollen by mass and sequencing reads was inconsistent,suggesting that the number of sequence reads is a poor proxy for pollen abundance inmixed-species samples. Using a liberal threshold resulted in greater detection oforiginal plant species in mixtures but also detected additional species in mixtures andsingle-species samples. The conservative threshold reduced the number of additionalplant species detected, but several species in mixtures were not detected above thethreshold, resulting in false negatives. Pollinator networks produced using the twothresholds differed and illustrated tradeoffs between detection of rare species andestimation of network complexity. Threshold selection can have a major effect onconclusions drawn from studies using metabarcoding of bee pollen to study plant-pollinator interactions.