False‐negative detections from environmental DNA collected in the presence of large numbers of killer whales (Orcinus orca)

Abstract

While environmental DNA (eDNA) is becoming increasingly established in biodiversity monitoring of freshwater ecosystems, the use of eDNA surveys in the marine environment is still in its infancy. Here, we use two approaches: targeted quantitative PCR (qPCR) and whole-genome enrichment capture followed by shotgun sequencing in an effort to amplify killer whale DNA from seawater samples. Samples were collected in close proximity to killer whales in inshore and offshore waters, in varying sea conditions and from the surface and subsurface but none returned strongly positive detections of killer whale eDNA. We validated our laboratory methodologies by successfully amplifying a dilution series of a positive control of killer whale DNA. Furthermore, DNA of Atlantic mackerel, which was present at all sites during sampling, was successfully amplified from the same seawater samples, with positive detections found in ten of the eighteen eDNA extracts. We discuss the various eDNA collection and amplification methodologies used and the abiotic and biotic factors that influence eDNA detection. We discuss possible explanations for the lack of positive killer whale detections, potential pitfalls, and the apparent limitations of eDNA for genetic research on cetaceans, particularly in offshore regions. K E Y W O R D S eDNA, environmental DNA, metagenomics, Orcinus orca, PCR, Scomber scombrus, wholegenome enrichment | 317 PINFIELD Et aL. S U PP O RTI N G I N FO R M ATI O N Additional supporting information may be found online in the Supporting Information section at the end of the article. How to cite this article: Pinfield R, Dillane E, Runge AKW, et al. False-negative detections from environmental DNA collected in the presence of large numbers of killer whales (Orcinus orca).

Environmental DNA captures the genetic diversity of bowhead whales (Balaena mysticetus) in West Greenland

“…Still, we did also detect target DNA in transect samples when no bowhead whales were visually observed in the close vicinity of the boat, indicating that the false‐negative detections reported by Pinfield et al. (2019) could in part be owing to differences in study design (e.g., primer sensitivity and inhibitors), sampling environment (e.g., temperature and sea state), and/or target species (e.g., size of animal and rate of skin shedding and defecation).…”

“…Our results demonstrated a significant difference in target DNA quantity when comparing footprint and transect sampling, which could in part explain the lack of positive detections in the study of Pinfield et al. (2019). Still, we did also detect target DNA in transect samples when no bowhead whales were visually observed in the close vicinity of the boat, indicating that the false‐negative detections reported by Pinfield et al.…”

“…Intriguingly, another recent eDNA study on killer whales reported false‐negative detection despite animals being visually observed during sampling (Pinfield et al., 2019). The platform used for the study did not allow for footprint sampling; however, the animals were less than 20 m from the boat during sample collection.…”

How does eDNA decay affect metabarcoding experiments?

“…Therefore, understanding eDNA decay is critical to evaluate the link between eDNA detection and contemporaneous species presence. If eDNA decays quickly, then a false negative detection (specifically, not detecting a present organism due to a decay of eDNA signal) is possible (Pinfield et al, 2019;Wood et al, 2018).…”

Genome‐scale target capture of mitochondrial and nuclear environmental DNA from water samples

“…Pinfield et al. (2019) applied whole‐genome enrichment capture with RNA baits followed by subsequent shotgun sequencing of eDNA samples, but not enough killer whale DNA was retrieved to conduct population genetic analyses and infer a potential source population.…”