Microbial community composition of earthworm-invaded and earthworm-free soils of the Canadian boreal forest

Earthworm invasion in North American forests has the potential to greatly impact soil microbiomes by altering soil physicochemical properties. We characterized and compared microbial communities of earthworm-invaded and non-invaded soils in previously described sites across three major soil types found in the Canadian boreal forest using phospholipid fatty acid (PLFA) analysis and metabarcoding of the 16S rRNA gene (bacteria and archaea) and ITS2 region (fungi)., Samples of mineral soils, forest floors, and earthworm casts were collected in the summer of 2019 in multiple boreal forest sites across Canada.
Phospholipid fatty acids were extracted from freeze-dried samples following a modified Bligh and Dyer extraction method. PLFA identification was performed using the Sherlock Microbial Identification System version 4.5 following standard nomenclature. All unsaturated PLFAs were in cis configuration.
DNA was extracted from 250 mg of ground and freeze-dried soil sample using the DNeasy PowerSoil DNA Isolation Kit. For bacteria and archaea, primers 515F-Y (5’-GTGYCAGCMGCCGCGGTAA-3’) and 926R (5’- CCGYCAATTYMTTTRAGTTT-3’) were used for amplification of the V4-V5 regions of the 16S rRNA gene with 30 PCR cycles and an annealing temperature of 55 ºC. For fungi, primers ITS9F (5’-GAACGCAGCRAAIIGYGA-3’) and ITS4R (5’- TCCTCCGCTTATTGATATGC-3’) were used for amplification of the internal transcriber spacer 2 (ITS2) region with 30 PCR cycles and an annealin...,