Establishing insect community composition using metabarcoding of soil samples, and preservative ethanol and homogenate from Malaise trap catches: surprising inconsistencies between methods.

DNA metabarcoding allows the analysis of insect communities faster and more efficiently than ever before. However, metabarcoding can be conducted through several alternative approaches, and the consistency of results across methods has rarely been studied. We compare the results obtained by DNA metabarcoding of the same communities using two different markers - COI and 16S - and three different sampling methods - homogenized Malaise trap samples (homogenate), preservative ethanol from the same samples, and soil samples. Our results indicate that COI and 16S offer partly complementary information on Malaise trap samples, with each marker detecting a significant number of species not detected by the other. Different sampling methods offer highly divergent estimates of community composition. The community recovered from preservative ethanol of Malaise trap samples is quite distinct from that recovered from homogenate. Small and weakly sclerotized insects tend to be overrepresented in ethanol, with some exceptions that could be related to taxon-specific traits. For soil samples, highly degenerate COI primers pick up large amounts of non-target DNA and only 16S provides adequate analyses of insect diversity. However, even with 16S, very little overlap in MOTU content was found between the trap and the soil samples. Our results demonstrate that no metabarcoding approach is all-comprehensive in itself. For instance, DNA extraction from preservative ethanol is not a valid replacement for destructive bulk extraction but a complement. In future metabarcoding studies, both should ideally be used together to achieve comprehensive representation of the target community.