Suitability of PCR primers for characterizing invertebrate communities from soil and leaf litter targeting metazoan 18S ribosomal or cytochrome oxidase I (COI) genes

- Universal 18S primers are accurate in identification of animal voucher DNA and eDNA.
- Degenerate COI primers consistently amplify bacterial eDNA rather than animal eDNA.
- A wide breadth of invertebrate taxa can be amplified by universal 18S PCR primers.

Sequencing of environmental DNA (eDNA) for community analysis (i.e., eDNA metabarcoding) is already well established and applied in molecular microbial ecology. However, molecular methods for use in soil animal studies require further development before high-throughput sequencing can be considered a reliable technique in community ecology. To aid in this effort, we compare primers that target two frequently used genetic markers, mitochondrial cytochrome oxidase (COI) and ribosomal 18S genes, to determine their utility in broad soil animal eDNA studies. DNA was analyzed from individually identified invertebrates to test the efficiency of the primer sets in successfully targeting animal DNA and identification through sequencing. Primers were also tested for amplification of faunal genes from forest soil and leaf litter eDNA. Targeting the 18S gene resulted in the most successful amplification and correct identification of a wide range of individual invertebrate taxa, and was the most reliable primer set for use in eDNA analysis of invertebrate communities spanning several phyla. In contrast, the selected COI primers were inefficient in identifying a wide range of invertebrates, and amplified mostly bacterial sequences from eDNA.