Uncovering the hidden within shipping containers: Molecular biosurveillance confirms a pathway for introducing multiple regulated and invasive species.

The negative ramifications of invasive alien species (IAS) are considered the second-most cause of biodiversity extinction and endangerment after habitat modification. IAS movements are mainly anthropogenically driven (e.g., transport of shipping containers) and require fast detection to minimize damage and cost. The present study is the first to use molecular biosurveillance of international shipping containers to detect IAS and regulated species identification in Canada. Thirty-eight samples were collected from debris (soil, stems, seeds, individual specimens) found in containers arriving in Canada. A multi-marker approach using COI, ITS, ITS2, and 16S was used to identify four main taxonomic groups: arthropods, fungi, plants, and bacteria, respectively. Eleven IAS species were identified via metabarcoding based on environmental DNA samples, including two arthropods, six fungi, two plants, and one bacteria. The origin of the eDNA detected from each species was linked to their native distribution and country of origin, except for Lymantria dispar . Four physical specimens were also collected from shipping container debris and DNA barcoded, identifying three non-regulated species (two arthropods and one fungus). Altogether, these results demonstrate the importance of integrating molecular identification into current toolkits for the biosurveillance of invasive alien species and provide a set of validated protocols ready to be used in this context. Additionally, it reaffirms international shipping containers as a pathway for multiple invasive aliens and regulated species introduction in Canada. It also highlights the need to establish regular and effective molecular biosurveillance at the Canadian border to avoid new or recurrent invasions.