Tracking marine invaders in Portuguese marinas by DNA metabarcoding from coast to archipelagos

Marine non-indigenous species (NIS), introduced primarily through shipping in marinas and ports, pose significant threats to coastal biodiversity and ecosystem functioning, requiring effective management strategies, especially for monitoring aimed at early detection. This study aimed to capture a comprehensive snapshot of the geographic variation of marine invertebrate assemblages and NIS occurrence, across 10 recreational marinas in Portugal (6 on the mainland - Viana do Castelo, Porto, Aveiro (2 marinas), and Lisbon (2 marinas) - and 2 in each archipelago, Madeira and the Azores). In each marina, hard substrates, zooplankton, and water samples (for eDNA) were collected in triplicate during summer for metabarcoding profiling, yielding 111 samples. Using Illumina MiSeq sequencing of the mitochondrial cytochrome c oxidase subunit I (COI) gene (313 bp) and the V4 region of the small subunit 18S rRNA gene (18S, approximately 400 bp), we detected 641 species from 21 phyla, 39 of which were NIS (7 phyla). Less than 1% of the species and 5% of NIS were detected in all marinas. The Azores had the highest numbers of both exclusive native species (97) and NIS (6). In contrast, Aveiro had the lowest number of exclusive native species (38), while the lowest numbers of exclusive NIS were observed in both the North and Aveiro (1 each). Detection success also varied among sample types: zooplankton samples recovered the highest proportion of total species (64%), whereas hard substrates in the islands and water samples in mainland Portugal recovered the highest proportions of NIS. A Principal Coordinate Analysis indicated a distinct separation among communities forming three main groups: (1) Viana do Castelo, Porto, and Aveiro; (2) Lisbon; and (3) Madeira and the Azores. Similarity between these groups is negatively correlated with geographic distance and overlaps with ecoregions previously identified within Lusitania. In the Azores, 9 NIS were recorded for the first time, and one in Madeira. We also recorded a considerable number of potential range expansions of NIS, predominantly along the mainland coast, in both North (N), South (S) or S-N directions, which suggests a high potential for DNA metabarcoding to be included in future national marine biomonitoring campaigns. • Environmental DNA metabarcoding detected 641 invertebrate species, across 10 marinas. • 39 non-indigenous species (NIS) were identified, including first molecular records for the Azores and Madeira. • Community structure reflects three main regional groupings based on biogeographic differences. • A multi-component (e)DNA approach enhances NIS early detection improving marine biosecurity.