Wanted not, wasted not: Searching for non-target taxa in environmental DNA metabarcoding by-catch

Metabarcoding of environmental DNA is based on primers that are specific to the target taxa (e.g. bacteria, zooplankton, fishes). However, due to the nature of the commonly used protocols, regardless of the chosen primers, several sequences of non-target species will inevitably be generated, but are usually discarded in commonly used bioinformatics pipelines. These non-target sequences might contain important biological information about the presence of other species in the studied habitats and its potential for ecological studies is still poorly understood. Here, we analyzed the presence of mammal and bird species in aquatic environmental samples that were originally amplified targeting teleost fish species. After all cleaning and checking steps, we kept 21 amplicon sequence variants (ASVs) belonging to mammals and ten to birds. Most ASVs were taxonomic assigned to farm/domestic animals, such as cats, cows, and ducks. Yet, we were able to identify a native semi-aquatic mammal, the capybara, in the samples. Four native bird species and a non-native potentially invasive bird (Corvus sp.) were also detected. Although the data derived from these samples for mammals and birds are of limited use for diversity analyses, our results demonstrate the potential of aquatic samples to characterize non-aquatic birds and highlight the presence of a potentially invasive species that had not been recorded before in the region.