Pepinelli Lab

Background

Plant and pollinator communities depend on each other: flowering plants provide essential food, while pollinators help plants reproduce. However, pollinator populations are declining everywhere, which may threaten plant communities. This study uses environmental DNA (eDNA) from wildflowers native to Northern Ontario to identify pollinators and map plant-pollinator networks. Working with Northern Wildflowers, we are aiming to better understand species interactions and how to support pollinator communities in the region.

Field work

Flowers from the Northern Wildflower farm were collected weekly, over a five week period during the 2025 season. Five individual flowers of eleven different species were collected and transported to the laboratory. In the laboratory samples were kept in the freezer, a cold and dark environment to mitigate degradation of DNA until processing. A total of 270 wildflower samples had been collected and analyzed.

Lab work – DNA extraction

Total DNA for each sample was extracted in a three-day process. The first step was to get all eDNA from the flowers into solution by adding PBS buffer and shaking it in a 45C incubator for 24 hours. The second step was to concentrate and lysis the DNA. The third step was to extract DNA using a Blood & Tissue Kit from Qiagen. After 3 days, total DNA was extracted from each flower sample.

Lab work – PCR optimization

We tested four combinations of COI primers to evaluate
their effectiveness at detecting pollinator eDNA on
flowers. Only one primer combination was consistently
successful at amplifying the target DNA across samples

Lab work – Sequencing

Once the CO1 gene has been successfully amplified the
samples undergo sequencing for the following 72 hours using
Oxford Nanopore MinION. We estimate that the sequencer will
provide us with approximately 10-20 million reads of
eDNA native pollinators.

Bioinformatics – Analysing results

With a tailored local pollinator genomic reference
database each read will be cross-referenced for
identification. Given that Sudbury is located in the
transitional area between the southern mixed deciduous
and northern boreal forest, we expected to identify
several pollinator species interacting with the flowers.

Expected Findings

The data collected will provide the identification of pollinators per
flower, providing deeper insights as to which species of flowers attract greater diversity of insects. This information will help increase
effective pollinator conservation.