The Magdalena River basin harbors a large biodiversity of vertebrates, with numerous endemic species, many of which are threatened with extinction. Following the same trend as most freshwater ecosystems globally, the Magdalena faces threats from human activities, causing habitat degradation and the decline of its biodiversity. Monitoring the freshwater biodiversity is crucial to detect early impacts and guide conservation actions in this river system, which is of vital ecological importance for Colombia. Here, we used environmental DNA (eDNA) metabarcoding, with two primer sets designed to target mitochondrial DNA regions, specifically the 12S ribosomal RNA gene, targeting fish as well as amphibians, reptiles, birds, and mammals in the Magdalena River. We detected a total of 158 vertebrate taxa, not only aquatic but also terrestrial, arboreal, and aerial. The diversity of these vertebrates increases as the river mouth is approached, accompanied by a change in the composition of the aquatic vertebrate assemblages, with two distinct groups linked to the geomorphology of the lower Magdalena basin. The assemblage was characterized by a high turnover of taxa between the sites closest to the mouth of the river and a marked nesting of taxa when comparing the sites closest to the river mouth with those farther away. We conclude that eDNA metabarcoding allows characterizing vertebrate assemblages in large rivers, assessing conservation status, and elucidating biodiversity patterns with minimal ecosystem disturbance. The information obtained through this approach can contribute to the sustainable management and preservation of vital freshwater ecosystems and their associated biodiversity.

Human activities can degrade the quality of coral reefs, cause a decline in fish species richness and functional diversity and an erosion of the ecosystem services provided. Environmental DNA metabarcoding (eDNA) has been proposed as an alternative to Underwater Visual Census (UVC) to offer more rapid assessment of marine biodiversity to meet management demands for ecosystem health indices. Taxonomic information derived from sequenced eDNA can be combined with functional traits and phylogenetic positions to generate a variety of ecological indices describing ecosystem functioning. Here, we inventoried reef fish assemblages of two contrasted coastal areas of Curaçao, (i) in close proximity to the island’s capital city and (ii) in a more remote area under more limited anthropogenic pressure. We sampled eDNA by filtering large volumes of sea water (2 x 30L) along 2km boat transects, which we coupled with species ecological properties related to habitat use, trophic level and body size to investigate the difference in fish taxonomic composition, functional and phylogenetic indices recovered from eDNA metabarcoding between these two distinct coastal areas. Despite no marked difference in species richness, we found a higher phylogenetic diversity in proximity to the city, but a higher functional diversity on the more isolated reef. Composition differences between coastal areas were associated with different frequencies of reef fish families. Because of a partial reference database, eDNA only partly matched those detected with UVC, but eDNA surveys nevertheless provided rapid and robust species occupancy responses to contrasted environments. eDNA metabarcoding coupled with functional and phylogenetic diversity assessment can serve the management of coastal habitats under increasing threat from global changes.