Summary The accelerating decline of biodiversity, particularly in freshwater ecosystems, highlights an urgent need for frequent and reliable biomonitoring to detect species loss and ecosystem degradation at an early stage. Early and accurate assessments are essential to assess the impacts of anthropogenic stressors on biodiversity and ecosystem functioning, as they provide the basis to inform appropriate conservation and restoration measures. However, biomonitoring within current regulatory frameworks, such as the EU Water Framework Directive (WFD, 2000/60/EC), remains limited, as morpho-taxonomic assessments are time-consuming and costly. As a result, conducting comprehensive assessments across all streams remains challenging, resulting in many water bodies being insufficiently surveyed. Genetic-based approaches offer a promising alternative to overcome these limitations by enabling high-frequency biomonitoring and the simultaneous processing of hundreds to thousands of samples. These approaches offer the potential to generate biodiversity data at unprecedented spatial and temporal resolution. For metabarcoding of organismal bulk samples intercalibration with current regulatory requirements has already been demonstrated, indicating that this approach can be integrated without major modifications. However, the benefits of large-scale data generation for regulatory biomonitoring must ultimately serve the goals of biodiversity and ecosystem conservation. In this context, environmental DNA (eDNA) metabarcoding provides a promising non-invasive alternative that allows for rapid sample collection and processing while reducing the harm to organisms. Despite these advantages, current eDNA metabarcoding approaches cannot yet be fully integrated into regulatory frameworks, as they are not directly compatible with established site-specific assessment methodologies. This results in fundamental differences between site-specific regulatory morpho-taxonomic assessments and the more spatially comprehensive eDNA-based assessments. Therefore, the aim of this thesis was to develop and validate novel strategies that combine the strength of both invasive and non-invasive monitoring approaches to improve the spatial specificity of biodiversity assessments while utilizing minimally invasive methods. The following strategies were employed: I) metabarcoding of eRNA obtained from a water sample taken directly from the stream; II) metabarcoding of eDNA obtained from organisms collected locally using multi-habitat (MH) kicknet sampling after a short incubation in water; III) metabarcoding of eDNA obtained from locally colonised natural substrate exposures (NSEs) after a short incubation in water. These three strategies were selected as the most promising approaches for minimally invasive assessments of local stream macroinvertebrate communities. It is hypothesised that environmental RNA (eRNA) metabarcoding represents metabolically active organisms and, by extension, the organisms that are present at a site. The incubation-based strategies assume that the DNA released by the organisms into their environment will be enriched in the incubation-medium and will overlay the regional DNA signal from the stream water. The incubation-based strategies were developed in this thesis and therefore had not been tested before and thus required careful evaluation to determine the optimal incubation time. The incubation time needed to be long enough to ensure sufficient DNA release for species detection, but short enough to prevent harm to the incubated organisms and to remain suitable for practical application into routine biomonitoring. In Chapter 1 the incubation-based strategy proved highly effective in detecting local macroinvertebrate communities regardless of the length of the incubation period. These results provided the foundation for a more extensive application and evaluation of the incubation-based strategy, as presented in Chapter 2. In Chapter 2, the three strategies were directly compared to each other for their potential for local bioassessment in streams. While both incubation-based strategies improved the detection of local macroinvertebrate communities compared to eDNA metabarcoding of direct stream samples, eRNA metabarcoding underperformed in comparison. Surprisingly, the incubation-based strategy using NSEs detected more species than the strategy using MH sampling, despite sampling only selected microhabitats and a smaller sampling area. Thus, using NSEs for the collection of macroinvertebrates seems to be the preferable option. In addition, the method is more passive in the sampling process, which reduces the impact on the surrounding microhabitats and organisms compared to the more disruptive MH sampling. However, for integration into current regulatory frameworks, methods must also align with existing assessment procedures. Chapter 3 showed that both incubation-based strategies were broadly congruent with regulatory morpho-taxonomic assessments and metabarcoding of bulk samples. Moreover, the incubation-based strategies demonstrated the potential of eDNA-based metabarcoding to detect fine-scale spatial differences in trait composition, highlighting their potential to support future trait-based bioassessment. A more comprehensive integration of trait-based metrics into regulatory biomonitoring may enable an earlier and more precise detection of changes in ecosystem functioning than is achievable with current taxonomy-based metrics. In conclusion, the incubation-based strategies developed in this thesis show strong potential for minimally invasive, locally resolved, and WFD-compliant biomonitoring of stream macroinvertebrates. In particular, the NSE-based incubation strategy allows for efficient and non-disruptive assessments of local macroinvertebrate communities, making it well suited for evaluating stressor impacts in restored, highly impacted, or sensitive ecosystems. For a rapid and straightforward integration into regulatory frameworks, the incubation-based strategy in combination with standardized МН sampling offers an effective complementary or alternative approach to traditional morpho-taxonomic assessments. Although broader application of genetic approaches beyond current regulatory limits will require further refinement, validation, and standardization, the findings of this thesis demonstrate the potential of genetic approaches for enabling more flexible, comprehensive, and future-oriented freshwater biomonitoring.
Mandy Sander (Wed,) studied this question.