Investigating the use of pollen 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 extent to which sequence read data is quantitative, which type of sequence count removal threshold to use and how that choice affects our ability to detect rare flower visits, and how sequence artefacts may confound conclusions about bee foraging behavior. To address these questions, we isolated pollen from five plant species and created treatments comprised of pollen from each species alone and combinations of pollen from multiple species that varied in richness and evenness. We used ITS2 and rbcL metabarcoding to identify plant species in the samples, compared the proportion of pollen by mass to the proportion of sequencing reads for each plant species in each treatment, and analyzed the sequencing data using both liberal and conservative thresholds. We collected pollen from foraging bees, analyzed metabarcoding data from those samples using each threshold, and compared the differences in the pollinator networks constructed from the data. Regardless of the threshold used, the relationship between 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 in mixed-species samples. Using a liberal threshold resulted in greater detection of original plant species in mixtures but also detected additional species in mixtures and single-species samples. The conservative threshold reduced the number of additional plant species detected, but several species in mixtures were not detected above the threshold, resulting in false negatives. Pollinator networks produced using the two thresholds differed and illustrated tradeoffs between detection of rare species and estimation of network complexity. Threshold selection can have a major effect on conclusions drawn from studies using metabarcoding of bee pollen to study plant-pollinator interactions.

Capabilities and limitations of using DNA metabarcoding to study plant-pollinator interactions.

Many pollinator populations are experiencing declines, emphasizing the need for a better understanding of the complex relationship between bees and flowering plants. Using DNA metabarcoding to describe plant-pollinator interactions eliminates many challenges associated with traditional methods and has the potential to reveal a more comprehensive understanding of foraging behaviour and pollinator life history. Here we use DNA metabarcoding of ITS2 and rbcL gene regions to identify plant species present in pollen loads of 404 bees from three habitats in eastern Oregon. Our specific objectives were to (i) determine whether plant species identified using DNA metabarcoding are consistent with plant species identified using observations, (ii) compare characterizations of diet breadth derived from foraging observations to those based on plant species assignments obtained using DNA metabarcoding, and (iii) compare plant species assignments produced by DNA metabarcoding using a "regional" reference database to those produced using a "local" database. At the three locations, 31%-86% of foraging observations were consistent with DNA metabarcoding data, 8%-50% of diet breadth characterizations based on observations differed from those based on DNA metabarcoding data, and 22%-25% of plant species detected using the regional database were not known to occur in the study area in question. Plant-pollinator networks produced from DNA metabarcoding data had higher sampling completeness and significantly lower specialization than networks based on observations. Here, we examine some strengths and limitations of using DNA metabarcoding to identify plant species present in bee pollen loads, make ecological inferences about foraging behaviour and provide guidance for future research.