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The hyporheic zone, i.e. the ecotone between surface water and the groundwater, is a rarely studied freshwater ecosystem. Hyporheic taxa are often meiofaunal (&lt;1 mm) in size and difficult to identify based on morphology. Metabarcoding approaches are promising for the study of these environments and taxa, but it is yet unclear if commonly applied metabarcoding primers and replication strategies can be used. In this study, we took sediment cores from two near natural upstream ( NNU ) and two ecologically improved downstream ( EID ) sites in the Boye catchment (Emscher River, Germany), metabarcoding their meiofaunal communities. We evaluated the usability of a commonly used, highly degenerate  COI  primer pair (BF2/BR2) and tested how sequencing three PCR replicates per sample and removing  MOTUs  present in only one out of three replicates impacts the inferred community composition. A total of 22,514  MOTUs  were detected, of which only 263 were identified as   Metazoa  . Our results highlight the gaps in reference databases for meiofaunal taxa and the potential problems of using highly degenerate primers for studying samples containing a high number of non-metazoan taxa. Alpha diversity was higher in  EID  sites and showed higher community similarity when compared to  NNU  sites. Beta diversity analyses showed that removing  MOTUs  detected in only one out of three replicates per site greatly increased community similarity in samples. Sequencing three sample replicates and removing rare  MOTUs  is seen as a good compromise between retaining too many false-positives and introducing too many false-negatives. We conclude that metabarcoding hyporheic communities using highly degenerate  COI  primers can provide valuable first insights into the diversity of these ecosystems and highlight some potential application scenarios.

A DNA metabarcoding protocol for hyporheic freshwater meiofauna: Evaluating highly degenerate COI primers and replication strategy