Targeted non-invasive bioindicator species detection in eDNA water samples to assess and monitor the integrity of vulnerable alpine freshwater environments

Due to a high number of specialized species and unique environmental conditions, alpine spring ecosystems are particularly vulnerable to environmental change and human impact. Therefore, the assessment of ecosystem integrity through habitat monitoring over long periods of time is of particular importance, especially in protected areas. Bioindication by conventional ecosystem assessment and monitoring includes sampling whole communities and morphological species identification. This approach, however, brings along major drawbacks such as high invasiveness, low reproducibility, low specificity and is relatively time-consuming. To overcome these issues, we developed a targeted bioindicator species eDNA detection approach for representative freshwater macroinvertebrate species and compared the results with detection through conventional sampling. Macroinvertebrates of 15 springs, located in the Swiss National Park and the UNESCO biosphere reserve Engiadina Val Müstair, were sampled using a hand-net and species were morphologically identified. We selected six spring-bound species: Hygrobates norvegicus , Partnunia steinmanni , Dictyogenus fontium , Protonemura lateralis , Lithax niger, and Wormaldia occipitalis and designed novel, species-specific qPCR primers and hydrolysis probes. Spring eDNA was collected by filtering 1.5 l water through cellulose nitrate filter funnels and DNA extracts were screened by qPCR for the selected bioindicator species. Results showed congruence between conventional and eDNA qPCR-based species detection. The assay targeting L. niger was less sensitive and qPCR performance in eDNA samples was decreased compared to the other species, indicating the necessity for careful indicator species choice and evaluation. The newly developed eDNA-based qPCR protocols allow detecting indicator species in alpine springs and represent a non-invasive, sensitive and specific, cost- and time-effective alternative to conventional biomonitoring approaches. Particularly in protected areas such as the Swiss National Park, the implementation of indicator species detection in eDNA filtered water samples can be beneficial and fosters sustainable freshwater ecosystem monitoring and assessment.