Optimizing a metabarcoding marker portfolio for species detection from complex mixtures of globally diverse fishes

DNA metabarcoding is used to enumerate and identify taxa in both environmental samples and tissue mixtures, but the effectiveness of particular markers depends on their sensitivity to the taxa involved. Using multiple primer sets that amplify different genes can mitigate biases in amplification efficiency, sequence resolution, and reference data availability, but few empirical studies have evaluated markers for complementary performance. Here, we assess the individual and joint performance of 22 markers for detecting species in a DNA pool of 98 species of marine and freshwater bony fishes from geographically and phylogenetically diverse origins. We find that a portfolio of four markers targeting 12S, 16S, and two regions of COI identifies 100% of reference taxa to family and nearly 60% to species. We then use these four markers to evaluate metabarcoding of heterogeneous tissue mixtures, using experimental fishmeal to test: (1) the tissue input threshold to ensure detection; (2) how read depth scales with tissue abundance; and (3) the effect of non‐target material in the mixture on recovery of target taxa. We consistently detect taxa that makeup >1% of fishmeal mixtures and can detect taxa at the lowest input level of 0.01%, but rare taxa (1% of fishmeal mixtures, but rare taxa (