Simplifying Macroinvertebrate Biodiversity Assessment: A Standardised Metabarcoding Method for Invertebrate Samples...

DNA metabarcoding of freshwater macroinvertebrate samples can provide rapid and accurate identification of species diversity. Processing samples where macroinvertebrates are not separated from net contents can substantially reduce time and cost. We devised and tested a standardised method for processing combined kick/sweep samples using DNA metabarcoding to provide accurate estimates of macroinvertebrate species diversity. Kick and sweep samples were collected from 10 sites across Greater Melbourne known to vary in catchment land use, macroinvertebrate abundance and diversity as well as the type and volume of sampling debris. A sieving, blending and DNA extraction protocol was developed to reduce large debris, rocks and sand in samples, and to concentrate macroinvertebrates for metabarcoding. Variable percentages of the dry weight of samples from each site were used as a source of DNA for metabarcoding. We found that the macroinvertebrate species diversity recovered was mostly consistent when processing different percentages (20%-40%) of dry weight, but a minimum dry weight would be recommended for samples with low total dry weight. We present a method with a laboratory and bioinformatic workflow that enables the cost-effective and reliable processing of large volume samples, like those encountered in freshwater bioassessment. This metabarcoding approach has potential to be applied to any invertebrate biodiversity analysis where invertebrates are not separated from sampling debris, regardless of volume and composition, and used on samples varying in species abundance, diversity and density.