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.

由于大量特殊物种和独特的环境条件，高山春季生态系统特别容易受到环境变化和人类影响的影响。因此，通过长期栖息地监测来评估生态系统完整性尤为重要，尤其是在保护区。常规生态系统评估和监测的生物指示包括对整个群落进行采样和形态学物种鉴定。然而，这种方法带来了主要缺点，例如高侵入性、低重现性、低特异性和相对耗时。为了克服这些问题，我们开发了一种针对代表性淡水大型无脊椎动物物种的靶向生物指示剂物种 eDNA 检测方法，并将结果与​​通过常规采样进行的检测进行了比较。使用手网对位于瑞士国家公园和联合国教科文组织生物圈保护区 Engiadina Val Müstair 的 15 个泉水的大型无脊椎动物进行采样，并对物种进行了形态学鉴定。我们选择了六种春季品种：Hygrobates norvegicus、Partnunia steinmanni、Dictyogenus fontium、Protonemuralateralis、Lithax niger和Wormaldia occipitalis并设计了新的物种特异性 qPCR 引物和水解探针。Spring eDNA 是通过硝酸纤维素过滤漏斗过滤 1.5 l 水收集的，DNA 提取物通过 qPCR 筛选选定的生物指示剂物种。结果显示传统和基于 eDNA qPCR 的物种检测之间存在一致性。针对L. niger的测定与其他物种相比，eDNA 样品的灵敏度较低，qPCR 性能降低，表明需要仔细选择和评估指标物种。新开发的基于 eDNA 的 qPCR 协​​议允许检测高山泉中的指示物种，并代表了传统生物监测方法的非侵入性、敏感和特异性、成本和时间有效的替代方案。特别是在瑞士国家公园等保护区，在 eDNA 过滤水样本中实施指示物种检测可能是有益的，并促进可持续的淡水生态系统监测和评估。