D3283: Collection and preservation methods of wildflowers for DNA metabarcoding of insects

Reported declines in insect biodiversity and abundance are mounting across the globe, yet obtaining reliable data on these changes using traditional biodiversity surveys is time consuming, expensive, and limited by taxonomic expertise. Molecular methods using DNA from the environment, or eDNA, may provide an alternative way to survey insect biodiversity. Recent studies have shown promise in using wildflowers as a source of DNA for DNA metabarcoding of pollinators, although more validation of this method is needed. Our goal is to validate detections of butterfly species from floral associated DNA with tandem traditional surveys. A pilot study was conducted to test field and laboratory methods of eDNA metabarcoding of wildflowers using the cytochrome oxidase subunit I (COI) barcode to identify insect species. Three montane meadows were surveyed for butterfly species abundance and wildflowers of six different species were collected to test for butterfly eDNA. Methods of flower and insect eDNA collection and preservation were tested including whole flower heads either frozen or dried in silica and three different flower swab collection techniques. Whole flower heads preserved in silica and then frozen returned higher average sequence reads for all insect eDNA than flower heads that were only frozen, and showed higher diversity in amplicon sequence variants (ASVs) than all other collection methods tested. Alpha and beta diversity of ASVs varied across flower species, suggesting surveying diverse flower communities is warranted to detect the breadth of insect biodiversity. Further analysis will assess species detections of butterflies with eDNA compared to traditional surveys.