Preservative DNA metabarcoding of benthic macroinvertebrates from the Vjosa catchment

Reliable biodiversity data are crucial for environmental research and management. Unfortunately, data paucity prevails for many regions and organismal groups such as aquatic invertebrates. High-throughput DNA-based identification, in particular DNA metabarcoding, has accelerated biodiversity data generation. However, in the process of metabarcoding, specimens are usually destroyed, excluding later specimen-based analyses. Metabarcoding of DNA released into the preservative ethanol has been proposed as a non-destructive alternative, but proof-of-principle studies have yielded ambiguous results, reporting variance in detection probability for various taxa and methodological biases. In this study, we tested the performance of ethanol-based metabarcoding of aquatic invertebrates in comparison to a classical morpho-taxonomic assessment. Samples from one of Europe's last free flowing rivers, the Vjosa, were analyzed. Multihabitat samples were collected at 43 sites in two seasons and stored in ethanol. Both methods recovered similar proportions of major macrozoobenthos groups (Diptera, Ephemeroptera, Plecoptera, Trichoptera). In comparison, ethanol-based metabarcoding yielded greater taxonomic diversity but also missed some taxa due to incomplete reference data and primer bias. The metabarcoding data revealed distinct local partitioning of macroinvertebrate communities in the Vjosa catchment and similar beta biodiversity patterns could be observed with both approaches. Overall, our results indicate that preservative-based metabarcoding provides valuable biodiversity data. The method is robust and reliable, even though samples were taken under harsh field-conditions and stored for a prolonged period without cooling. Further, our results emphasise the need for reliable DNA barcoding reference libraries. In fact, preservative-based metabarcoding shows its biggest advantage by maintaining intact vouchers for subsequent specimen-based analyses.