Genome-skimming provides accurate quantification for pollen mixtures

Studies on foraging partitioning in pollinators can provide critical information to the understanding of food-web niche and pollination functions, thus aiding conservation. Metabarcoding based on PCR amplification and high-throughput sequencing has seen increasing applications in characterizing pollen loads carried by pollinators. However, amplification bias across taxa could lead to unpredictable artefacts in estimation of pollen compositions. We examined the efficacy of a genome-skimming method based on direct shotgun sequencing in quantifying mixed pollen, using mock samples (five and 14 mocks of flower and bee pollen, respectively). The results demonstrated a high level of repeatability and accuracy in identifying pollen from mixtures of varied species ratios. All pollen species were detected in all mocks, and pollen frequencies estimated from the number of sequence reads of each species were significantly correlated with pollen count proportions (linear model, R²  = 86.7%, p = 2.2e-16). For >97% of the mixed taxa, pollen proportion could be quantified by sequencing to the correct order of magnitude, even for species which constituted only 0.2% of the total pollen. In addition, DNA extracted from pollen grains equivalent to those collected from a single honeybee corbicula was sufficient for genome-skimming. We conclude that genome-skimming is a feasible approach to identifying and quantifying mixed pollen samples. By providing reliable and sensitive taxon identification and relative abundance, this method is expected to improve our understanding in studies that involve plant-pollinator interactions, such as pollen preference in corbiculate bees, pollen diet analyses and identification of landscape pollen resource use from beehives.