Ichthyoplankton monitoring is crucial for stock assessments, offering insights into spawning grounds, stock size, seasons, recruitment, and changes in regional ichthyofauna. This study evaluates the efficiency of multi-marker DNA metabarcoding using mitochondrial cytochrome c oxidase subunit I (COI), 12S rRNA and 16S rRNA gene markers, in comparison to morphology-based methods for fish species identification in ichthyoplankton samples. Two transects with four coastal distance categories were sampled along the southern coast of Portugal, being each sample divided for molecular and morphological analyses. A total of 76 fish species were identified by both approaches, with DNA metabarcoding overperforming morphology—75 versus 11 species-level identifications. Linking species-level DNA identifications with higher taxonomic morphological identifications resolved several uncertainties associated with traditional methods. Multi-marker DNA metabarcoding improved fish species detection by 20–36% compared to using a single marker/amplicon, and identified 38 species in common, reinforcing the validity of our results. PERMANOVA analysis revealed significant differences in species communities based on the primer set employed, transect location, and distance from the coast. Our findings underscore the potential of DNA metabarcoding to assess ichthyoplankton diversity and suggest that its integration into routine surveys could enhance the accuracy and comprehensiveness of fish stock assessments.

To maximize the efficiency of marine fish detection using environmental DNA (eDNA) metabarcoding, methodological factors such as PCR amplification bias and sampled water volume must be carefully considered. In this study, we evaluated the influence of different genetic markers (COI, 12S rRNA, and 16S rRNA) and water sample volumes (0.5, 1.0, 2.5, 5.0, and 10 L) on eDNA-based detection of 60 marine fish species maintained at the Oceanário de Lisboa. Five primer sets were used to generate amplicon libraries from triplicate water samples per tested volume. Overall, the combined use of all markers enabled the detection of 81.7% of the fish species present in the tank ( n = 49), as well as 37 additional exogenous taxa. Detection success varied among primer sets, ranging from 31.7% for COI FishATL_Cocktail1 to 73.3% for Fish16S. When accounting for reference sequence availability, relative detection success reached 82.4% for 12S and 77.2% for 16S. Increasing water volume consistently led to higher species detection across all markers, with detection percentages approaching saturation at the highest volumes tested. When pooling all markers, up to 76.7% of the species present were detected using a water volume of 5 L. These results highlight the limitations of relying on a single genetic marker and demonstrate the strong influence of sampling volume on detection efficiency, a factor likely to be even more critical in open marine environments.

Over the last two decades, the use of DNA barcodes has transformed our ability to identify and assess life on our planet. Both strengths and weaknesses of the method have been exemplified through thousands of peer-reviewed scientific articles. Given the novel sequencing approaches, currently capable of generating millions of reads at low cost, we reflect on the questions: What will the future bring for DNA barcoding? Will identification of species using short, standardized fragments of DNA stand the test of time? We present reflected opinions of early career biodiversity researchers in the form of a SWOT analysis and discuss answers to these questions.