Multi-locus metabarcoding and intensive sampling reveal extraordinary diversity carried in the ballast water of a single vessel

Understanding risks of biological invasions associated with ballast water (BW) requires full understanding of the biodiversity transported in ballast tanks. Here we characterize the remarkable level of diversity that can be carried in the BW of a single vessel. To maximize our ability to capture BW diversity we: 1) utilized DNA-based methods to describe biodiversity, including both native and non-native taxa; 2) exploited multiple primer sets targeting multiple genomic loci with different expectations for taxonomic coverage; 3) assessed multiple tanks on a single vessel to capture different communities present in different tanks; and 4) sampled those tanks with far higher-than-usual replication both to improve representation of the diversity present and to enable statistical estimation of total richness. Using this approach, we found extraordinarily high diversity associated with a single vessel. Across all loci, we estimate a total of 272 taxa that can be assigned species names; looking more broadly at unnamed molecular operational taxonomic units, our estimates are between 425 and 742 individual taxa, depending on the locus. We confirm that only a fraction of this diversity would be captured with typical sampling efforts. We found that different loci capture different snapshots of biodiversity and that our ability to detect taxa of interest (e.g., non-native species) depends on sampling effort and genomic locus. Our results expand upon previous studies describing highly diverse BW communities and add to a growing literature that demonstrates the value of molecular methods for characterizing those communities and assessing the associated risk of non-native species introduction.