Sampling strategy matters: eDNA-based assessment and extrapolation of myxozoan diversity in a model stream system

Determining true species diversity is particularly challenging for parasitic organisms. Environmental DNA (eDNA) metabarcoding has revolutionized diversity studies by increasing both efficiency and precision. This approach is especially relevant for parasitic species, like myxozoans that spend part of their life cycle outside their hosts, often during hostswitching stages. However, the adequacy of sampling depth and methodology in capturing total diversity often remains uncertain. To address this, we used a small stream as a model system and collected 60 small-volume aquatic sediment samples. Using an eDNA metabarcoding approach with myxozoan-specific primers, including newly designed primers for Malacosporea and the Paramyxidium group, we identified 30 myxozoan OTUs. These were predominantly from the Myxobolus clade but represented a broad spectrum of freshwater myxozoans, including Malacosporea. Rarefaction and extrapolation analyses estimated the potential maximum myxozoan diversity at 40 OTUs, 25% higher than the observed diversity. Although approximately a quarter of OTUs were probably missed, our analysis suggested that additional sampling would not be efficient, as most newly detected OTUs would overlap with those already identified or belong to very rare species. We demonstrated that eDNA-based methods reliably detect myxozoan diversity in small sediment volumes and that statistical approaches can effectively estimate true diversity and assess the need for further sampling at a given locality.