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 optimization of existing methodologies. In particular, most studies utilize only 0.5-1 gram 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, firstly, modified existing protocols for the pretreatment of large soil volumes for DNA extraction. The main modification involves a combined procedures of treating soil samples with a phosphate-based buffer and concentrating the extracted DNA on a nitrocellulose membrane, enabling final purification using standard commercial kits. Secondly, 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. In the soil of a temperate spruce forest, the developed pretreatment method allowed for the detection of 2-2.5 times more invertebrate genera compared to commercial DNA extraction kits. The diversity of unicellular eukaryotes did not differ between the extraction methods employed. For the majority of invertebrate taxa, eDNA metabarcoding revealed a higher diversity compared to classical morphological method. However, the diversity of Sarcoptiformes and Mesostigmata was underestimated. For most of the studied groups, occurrence frequency correlated with animal abundance estimated using traditional morphological identification. However, this correlation was only detected in samples where DNA was extracted from 10 g of soil. The suggested approach allows for a more representative assessments of soil invertebrate diversity and, for a range of taxa, provides quantitative data on the abundance. The ability to scale up the soil sample processed offers the potential to identify a greater diversity of large or rare organisms.