Protocols for Identifying the Botanical Origin of Pollen and Nectar Collected by Honey Bees (Apis mellifera L.) Using...

DNA metabarcoding has emerged as one of the most powerful tools for large-scale characterization of multiple taxa from complex mixed samples. In recent years, researchers have increasingly adopted this molecular approach to assess both the overall availability and taxonomic composition of floral resources in studies of honey bee foraging ecology. By providing high taxonomic resolution, plant DNA metabarcoding enables accurate species identification and quantitative assessments of mixed samples such as pollen and nectar collected from managed honey bee colonies. Here, we present comprehensive protocols for DNA metabarcoding of pollen and nectar samples. These protocols describe essential laboratory workflows, including sample preparation, DNA extraction, and quantification. In addition, they describe a bioinformatics pipeline using QIIME2 for adapter and primer trimming, filtering low-quality reads, merging forward and reverse reads, and taxonomic assignment of plant taxa. These protocols also address common challenges associated with DNA metabarcoding of pollen and nectar samples, providing guidance to help researchers make informed decisions when designing and implementing DNA metabarcoding studies. Guidance on the statistical analysis and visualization of pollen and nectar community diversity and abundance is also provided. © 2026 Wiley Periodicals LLC. Basic protocol 1: DNA extraction from pollen and nectar samples using the CTAB method for Illumina MiSeq sequencing Basic protocol 2: QIIME2 bioinformatics pipeline for plant DNA metabarcoding analysis Basic protocol 3: Statistical analysis and visualization of pollen and nectar metabarcoding data.