Metabarcoding and post-sampling strategies to discover non-indigenous species: A case study in the estuaries of the central south Bay of Biscay

Estuaries are highly productive habitats that generate more organic material than other areas of comparable size, such as forests, meadows or agricultural lands. They exhibit high biodiversity and host relevant species, which can be at risk because these areas are most affected by human activities. In this study, environmental DNA (eDNA) and metabarcoding analyses were performed in two estuaries within the Bay of Biscay that are important in terms of seafood production (Ría del Eo and Ría de Villaviciosa, Asturias, Northern Spain). The main goal was to assess the potential of these novel tools to detect possible introductions of non-indigenous species. Alien genera that might have been introduced through exotic shellfish cultures in these estuaries were detected despite limited sampling. Of the NIS found, Crepidula fornicata was the only one that was already included in the Spanish invasive species catalogue (BOE, 2013). After the initial invasive species detection through the metabarcoding study, post-NGS samplings and classical DNA barcoding were performed, and they confirmed the presence of the highly invasive species C. fornicata in these estuaries. Although metabarcoding still has some drawbacks, such as a lack of universal PCR primers and reference sequences for all the species in the databases as well as frequent false positives, it represents a powerful tool that can facilitate the monitoring and management of these important ecosystems, especially if it is accompanied by post-NGS samplings to confirm species occurrences.