Spatio-temporal metabarcoding surveys in ports reveal homogenised communities of non-indigenous species with high genetic diversity and connectivity.

Large commercial ports facilitate the introduction of non-indigenous species (NIS), while smaller harbours and marinas promote their regional spread, making harbour networks key drivers of biological invasions. We analysed spatio-temporal patterns of metazoan communities over one year in four medium-size harbours, as well as at an outside reference point, along the NW Mediterranean coast using standardised biological collectors and COI metabarcoding. We identified 1,774 metazoan molecular operational taxonomic units (MOTUs), of which 75 were classified as NIS. Although less diverse than native species, NIS accounted for 34-70% of reads in harbours, with the southernmost harbour showing the highest abundance, likely due to proximity to aquaculture facilities. Spatial structure varied among harbours, but NIS showed consistently low genetic differentiation and shared more MOTUs among sites than native species. Seasonal patterns affected both NIS and native communities. NIS also exhibited higher haplotype diversity and lower genetic differentiation across harbours, suggesting spread via local boating and recurrent introductions. These results highlight contrasting dynamics between NIS and native species in artificial environments and underscore the need for continued monitoring of harbour networks.