Metabarcoding and Metagenomics 5: 59–71 DOI 10.3897/mbmg.5.66557 Research Article Beyond fish eDNA metabarcoding: Field replicates disproportionately improve the detection of stream associated vertebrate species Till-Hendrik Macher1, Robin Schütz1, Jens Arle2, Arne J. Beermann1,3, Jan Koschorreck2, Florian Leese1,3 1 University of Duisburg-Essen, Aquatic Ecosystem Research, Universitätsstr. 5, 45141 Essen, Germany 2 German Environment Agency, Wörlitzer Platz 1, 06844 Dessau-Roßlau, Germany 3 University of Duisburg-Essen, Centre for Water and Environmental Research (ZWU), Universitätsstr. 3, 45141 Essen, Germany Corresponding author: Till-Hendrik Macher (
[email protected]) Academic editor: Pieter Boets | Received 26 March 2021 | Accepted 10 June 2021 | Published 13 July 2021 Abstract Fast, reliable, and comprehensive biodiversity monitoring data are needed for environmental decision making and management. Recent work on fish environmental DNA (eDNA) metabarcoding shows that aquatic diversity can be captured fast, reliably, and non-invasively at moderate costs. Because water in a catchment flows to the lowest point in the landscape, often a stream, it can col- lect traces of terrestrial species via surface or subsurface runoff along its way or when specimens come into direct contact with water (e.g., when drinking). Thus, fish eDNA metabarcoding data can provide information on fish but also on other vertebrate species that live in riparian habitats. This additional data may offer a much more comprehensive approach for assessing vertebrate diversity at no additional costs. Studies on how the sampling strategy affects species detection especially of stream-associated communities, however, are scarce. We therefore performed an analysis on the effects of biological replication on both fish as well as (semi-)terrestrial species detection.