From Nets to Molecules: A Comparative Study of Stream Fish Diversity Recovery Using Different Sampling Methods in Eastern Amazonia

The Neotropical freshwaters of South America host an exceptional level of ichthyofaunal diversity with over 5,160 species, making it the richest continental fauna worldwide. Despite their richness, these freshwater ecosystems face severe threats from human activities, leading to significant declines in fish populations. Traditional fish sampling techniques, such as netting, have been fundamental to ichthyology, offering insights into species richness and abundance. However, the complexity of stream environments limits the effectiveness of conventional sampling tools. As a result, more elusive or niche species are often missed. In recent years, water environmental DNA (eDNA) has emerged as a complementary method to traditional sampling. It allows for detection of aquatic organisms from water samples, expanding the scope of biodiversity assessments. Nevertheless, eDNA filtration faces challenges, especially in turbid waters, including the likelihood of co-extracting inhibitors that can affect amplification and detection processes, as well as the downstream flow of eDNA signals, which means that samples predominantly detect upstream fauna. To address these limitations, the use of bulk samples, such as stomach contents, provides a robust alternative by directly analyzing biological tissues and leveraging the bidirectional mobility of organisms within the stream, enabling the detection of taxa from both upstream and downstream regions. Given these issues, this study combines traditional netting, water eDNA analysis, and dietary metabarcoding to assess the fish biodiversity in three Neotropical streams in the Capim River basin, Pará, Brazil. The integration of multiple sampling techniques offers a more accurate picture of biodiversity, helping to overcome the limitations of each individual method and providing essential insights for conservation efforts.