DNA metabarcoding improves the detection of multiple stressor responses of stream invertebrates to increased salinity, fine sediment deposition and reduced flow velocity.

Worldwide, multiple stressors affect stream ecosystems and frequently lead to complex and non-linear biological responses. These combined stressor effects on ecologically diverse and functionally important macroinvertebrate communities are often difficult to assess, in particular species-specific responses across many species and effects of different stressors and stressor levels in concert. A central limitation in many studies is the taxonomic resolution applied for specimen identification. DNA metabarcoding can resolve taxonomy and provide greater insights into multiple stressor effects. This was detailed by results of a recent multiple stressor mesocosm experiment, where only for the dipteran family Chironomidae 183 Operational Taxonomic Units (OTUs) could be distinguished. Numerous OTUs showed very different response patterns to multiple stressors. In this study, we applied DNA metabarcoding to assess multiple stressor effects on all non-chironomid invertebrates from the same experiment. In the experiment, we applied three stressors (increased salinity, deposited fine sediment, reduced flow velocity) in a full-factorial design. We compared stressor responses inferred through DNA metabarcoding of the mitochondrial COI gene to responses based on morphotaxonomic taxa lists. We identified 435 OTUs, of which 122 OTUs were assigned to EPT (Ephemeroptera, Plecoptera, Trichoptera) taxa. The most common 35 OTUs alone showed 15 different response patterns to the experimental manipulation, ranging from insensitivity to any applied stressor to sensitivity to single and multiple stressors. These response patterns even comprised differences within one family. The species-specific taxonomic resolution and the inferred response patterns to stressors highlights the potential of DNA metabarcoding in the context of multiple stressor research, even for well-known taxa such as EPT species.