Comparison and evaluation of sampling and eDNA metabarcoding protocols to assess soil biodiversity in Belgian LUCAS Biopoints

Environmental DNA (eDNA) metabarcoding is emerging as a novel tool for monitoring soil biodiversity. Soil biodiversity, critical to soil health and ecosystem services, remains under-monitored due to the lack of standardized and efficient methods. We evaluated whether refinements to sampling protocols (for soil invertebrates and Fungi) and molecular protocols (for soil invertebrates) could improve biodiversity detection. Comparing the 2018 LUCAS soil biodiversity protocol with newly developed national methods, we tested sampling and sequencing surface layers (0-10 cm and forest floor) versus deeper layers, larger soil sample sizes for DNA-extraction, taking more subsamples for composite soil samples, and alternative primer sets across 9 Belgian Biopoints included in the LUCAS 2022 survey. We show that the choice of sampling protocol significantly influences soil biodiversity assessments. The results show that, based on eDNA, we are able to detect significantly more species when sampling and sequencing the upper soil layers separately, while the diversity in the 10–30 cm soil layer is insufficient for annelids and arthropods to serve as indicators of ecological changes. Collembola and Arthropoda richness and diversity generally increased towards less intensely managed soils, when using the national (Cmon), and to a lesser extent the European (LUCAS) sampling protocols. In contrast, sampling and sequencing the 10-30 cm layer failed to capture such a pattern. Overall, the analyses suggest that soil depth has a greater influence on the soil invertebrate diversity captured than sampling intensity, and that the highest diversity is recovered when surface sampling (0–10 cm topsoil and forest floor) is combined with a greater number of subsamples (16 compared to 5 in LUCAS) and a larger sampled area. Additionally, comparison of the universal eukaryotic primers (18S) with primer sets tailored to important soil invertebrate groups, showed that universal 18S primers provide limited resolution for Collembola and Annelida, making them less suitable for accurately assessing the diversity of these groups as a response variable in monitoring ecological changes and biological soil health. With refinement and standardization, eDNA metabarcoding, combined with optimized sampling protocols, could become a powerful and efficient tool for monitoring soil biodiversity in European soils.