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. Additionally, four physical specimens were 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. 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.外来入侵物种（Invasive Alien Species，IAS）所引发的负面影响，被认为是仅次于生境改造的生物多样性灭绝与濒危第二大诱因。IAS的传播主要由人为活动驱动（例如集装箱运输），且需开展快速检测以最大限度降低损害与成本。 本研究首次针对国际集装箱开展分子生物监测，用于检测外来入侵物种并完成受管制物种的鉴定，相关工作覆盖加拿大入境场景。研究人员从入境加拿大的集装箱内获取的碎屑（包括土壤、茎秆、种子及单份标本）中采集了38份样本。 本研究采用基于COI、ITS、ITS2及16S的多标记策略，分别鉴定出节肢动物、真菌、植物与细菌四大分类类群。基于环境DNA（environmental DNA，eDNA）样本的宏条形码测序技术共鉴定出11种外来入侵物种，其中包含2种节肢动物、6种真菌、2种植物及1种细菌。 除舞毒蛾（Lymantria dispar）外，所有检测到的eDNA溯源结果均与对应物种的自然分布范围及原产国相符。此外，研究人员从集装箱碎屑中采集了4份实物标本，并通过DNA条形码技术完成测序鉴定，共识别出3种非受管制物种（2种节肢动物与1种真菌）。 综上，本研究结果证实了将分子鉴定技术纳入现有外来入侵物种生物监测工具包的重要意义。同时，本研究再次印证了国际集装箱是加拿大境内多种外来入侵物种与受管制物种传入的关键途径。此外，本研究还强调了在加拿大边境建立常态化、高效的分子生物监测体系的必要性，以防范新型入侵事件或入侵物种的反复定殖。