Large soil samples for large soil organisms – modification of eDNA extraction method for investigation of soil invertebrate communities

Although soil eDNA metabarcoding is widely used in both applied and fundamental research, this approach requires the optimization of existing methodologies. In particular, most studies utilize only 0.2-1.0 g of soil for DNA extraction, which is insufficient for a representative assessment of soil invertebrate communities. Furthermore, eDNA metabarcoding does not yield reliable data on soil fauna abundance. In this study, we first modified existing protocols for the pretreatment of large soil volumes for total DNA extraction. The main modification involves a combined procedure of treating soil samples with a phosphate-based lysis buffer and concentrating the extracted DNA on a nitrocellulose membrane, enabling final purification using standard commercial kits. Second, we tested the feasibility of assessing soil fauna abundance via metabarcoding based on the frequency of taxon occurrence and relative read abundance in a series of analyzed soil samples. We found that in the soil of a temperate spruce forest, the developed pretreatment method allowed for the detection of 2-2.5 times more invertebrate genera than commercial DNA extraction kits, while the diversity of unicellular eukaryotes did not differ between the extraction methods employed. For the majority of invertebrate taxa, eDNA metabarcoding has revealed a higher diversity than traditional morphological identification. However, the diversity of Sarcoptiformes and Mesostigmata mites has been underestimated. We also showed that for most of the studied groups, the frequency of occurrence correlated with animal abundance estimated using morphological identification. However, this correlation was only detected in samples where DNA was extracted from 10 g of soil. The suggested approach allows for more representative assessments of soil invertebrate diversity and provides quantitative data on abundance for a range of taxa. The ability to increase the volume of the processed soil sample offers the potential to identify a greater diversity of large or rare organisms. This is especially important in the large-scale biodiversity monitoring programs.