Specimen sampling and DNA extraction
Ethanol-stored and pinned-dry invertebrate specimens were selected from a variety of taxa, primarily earthworms (132 specimens) and insects (315 specimens, mainly beetles and wasps), as well as individual millipede, spider and mite specimens; 450 specimens in total (Table 1). We randomly selected 1-4 individuals per species, depending on availability. Earthworm specimens were variously collected between 2004 and 2014, and arthropod samples from 2009 to 2019 using a range of techniques, including malaise traps, pitfall traps, sweeping and hand collection. Most specimens were stored in 95 % ethanol at -20 °C, but some were in 70 % ethanol, and some recent samples were stored at room temperature.
Total genomic DNA of specimens was extracted in a sterile environment with the following variations, depending on the size and availability of specimens for destructive sampling. Briefly, specimens were either soaked whole (112 specimens: 93 wasps, ten beetles, and nine moths) or crushed whole (48 small specimens: 25 beetles, 12 flies, nine other insects, one spider, and one mite) in lysis buffer, or a piece of tissue was sampled (290 specimens; all 132 earthworms, 125 beetles, 32 other insects, and one millipede) and added to the lysis buffer. DNA extraction was carried out using one of the following kits following the manufacturer’s instructions: DX reagents kit on the X-tractor Gene (Qiagen, Germantown, Maryland, USA) (211 specimens; 115 earthworms, 96 insects, and one mite, spider, and millipede); QIAamp 96 DNA QIAcube HT Kit on the QIAcube HT (Qiagen, Germantown, Maryland, USA) (222 insects, including the 112 soaked specimens); or the AquaPure Genomic DNA Isolation kit (BioRad, Hercules, California, USA) (17 earthworms). Extracted DNA was stored at -20°C until amplicon library construction.