Considerations for metabarcoding-based port biological baseline surveys aimed at marine nonindigenous species monitoring and risk assessments.

Monitoring introduction and spread of nonindigenous species via maritime transport and performing risk assessments require port biological baseline surveys. Yet, the comprehensiveness of these surveys is often compromised by the large number of habitats present in a port, the seasonal variability, and the time-consuming morphological approach used for taxonomic identification. Metabarcoding represents a promising alternative for rapid comprehensive port biological baseline surveys, but its application in this context requires further assessments.We applied metabarcoding (based on barcodes of the cytochrome c oxidase subunit I and of the 18S ribosomal RNA gene) to 192 port samples collected (a) from diverse habitats (water column-including environmental DNA and zooplankton, sediment, and fouling structures), (b) at different sites (from inner to outer estuary), and iii) during the four seasons of the year.By comparing the biodiversity metrics derived from each sample group, we show that each sampling method resulted in a distinct community profile and that environmental DNA alone cannot substitute for organismal sampling, and that, although sampling at different seasons and locations resulted in higher observed biodiversity, operational results can be obtained by sampling selected locations and seasons.By assessing the taxonomic composition of the samples, we show that metabarcoding data allowed the detection of previously recorded nonindigenous species as well as to reveal presence of new ones, even if in low abundance. Our comprehensive assessment of metabarcoding for port biological baseline surveys sets the basics for cost-effective, standardized, and comprehensive monitoring of nonindigenous species and for performing risk assessments in ports. This development will contribute to the implementation of the recently entered into force International Convention for the Control and Management of Ships' Ballast Water and Sediments.