Biological invasions are a global threat to biodiversity especially for aquatic resources. The distribution of alien species is associated with human activities; therefore, exotic species tend to accumulate near big urban areas through different invasion vectors such as ballast water, hull fouling, aquarium and pet releases. The Rhine River region is one of the most economically important in Europe. Around 60 million people live in the river basin that is connected with other large European rivers via the Rhine–Main–Danube shipping canal. The Alpine Rhine flows to Lake Constance, which is the second largest subalpine lake in Europe.

Here, eDNA metabarcoding was employed to inventory aquatic species from water samples in six riverine and four lake localities within Lake Constance region. A 313 bp fragment within cytochrome c oxidase subunit I gene was PCR amplified using generalist primers for metazoan and sequenced with MiSeq High-Throughput Sequencing platform. Seven invertebrate invasive species and the invasive fish Oncorhynchus mykiss were detected from eDNA. Species-specific primers were employed to confirm metabarcoded species. Most of the invasive species detected in this study correspond to samples from areas around lake ports, followed by other lake and degraded downstream river areas. Samples taken upstream of Lake Constance were free of invertebrate aliens. To establish common regulation and management actions regarding aquatic invasions in the three countries that share Lake Constance is recommended.

生物入侵是对生物多样性的全球威胁，特别是对水生资源而言。外来物种的分布与人类活动有关；因此，外来物种倾向于通过不同的入侵媒介（例如压舱水，船体结垢，水族馆和宠物释放）在大城市附近积累。莱茵河地区是欧洲最重要的经济地区之一。流域中有大约6000万人，该河流域通过莱茵-美因多瑙河的运输运河与其他欧洲大型河流相连。高山莱茵河流向康斯坦茨湖，这是欧洲第二大亚高山湖泊。

在这里，eDNA元条形码技术被用于清点康斯坦茨湖地区六个河流和四个湖泊地区的水样中的水生物种。使用后生动物的通用引物PCR扩增了细胞色素C氧化酶亚基I基因中的313 bp片段，并使用MiSeq高通量测序平台进行了测序。七个无脊椎动物入侵物种和入侵鱼类Oncorhynchus mykiss从eDNA中检测到。使用物种特异性引物来确认超条形码物种。在这项研究中检测到的大多数入侵物种与湖港周围地区的样本相对应，其次是其他湖泊和退化的下游河区。在康斯坦茨湖上游采集的样本中没有无脊椎动物。建议在共享康斯坦茨湖的三个国家中建立关于水生生物入侵的共同法规和管理措施。