Bycatch in a bottle: what taxa are recoverable from metabarcoding DNA in historical invertebrate collection preservative fluid? Authors: Ajith Seresinghe1, David Herbst2,3,Jen Quick-Cleveland1, Severyn Korneyev4, Benjamin K Maples4, Eva Sofia Horna Lowell5, Malia Mosser1 Robert N Fisher6 , Eric Palkovacs1,7, Daniel Gluesenkamp8, Rachel S Meyer1* Dept of Ecology and Evolutionary Biology, University of California Santa Cruz, Santa Cruz California 95060 Institute of Marine Sciences, University of California Santa Cruz, Santa Cruz California 95060 Sierra Streams Institute, 117 New Mohawk Rd H, Nevada City, CA 95959 California Department of Food and Agriculture, Plant Pest Diagnostics Branch, 3294 Meadowview Road, Sacramento, California 95832-1448, U.S.A. San Diego Natural History Museum, 1788 El Prado Balboa Park, San Diego, CA 92101 U.S. Geological Survey, Western Ecological Research Center, San Diego, CA 92101 Institute for Marine Sciences, University of California Santa Cruz, Santa Cruz, CA 95060 California Institute for Biodiversity, 1400 Shattuck Ave. STE #12 PMB 101, Berkeley, CA 94709 Natural history museum collections are invaluable repositories of biodiversity, offering insights into life on Earth. Genomic approaches provide powerful tools to characterize biodiversity in these collections. However using these collections for genomics without damaging specimens is a challenge. Here, we develop and test non-destructive DNA metabarcoding methods to capture biodiversity from the preservative fluids of archived insect collections (‘Bycatch’). We optimized workflows for extracting and amplifying the partial CO1 locus (CO1) and fungal ITS1 locus (FITS) from ethanol-based preservative fluids, validating ethanol preparation methods, comparing DNA extraction kits, and refining PCR protocols. Our results demonstrate that from museum collections with low DNA yields, CO1 and FITS loci can often be recovered from preservative fluids, and we present detailed methodology and workflows. We test metabarcoding success to recover taxa in several museum collections ranging in age and storage condition. This is to support the State of California’s effort to catalog and sequence all insects and fungi, building baselines of California biodiversity with help from museum collections. And lastly we investigate the complementarity of metabarcoding water versus ethanol and morphological identifications aimed to capture benthic macroinvertebrate biodiversity in streams. Our findings highlight that DNA metabarcoding of the preservative fluid is a non-destructive tool for capturing biodiversity in historical specimens, but there are limitations on the overlaps between DNA results and physical contents, where morphological identification still reigns in taxon counts, but metabarcoding sometimes provides more taxonomic resolution, and can be used to track DNA from other organisms beyond the directly surveyed specimens. This dataset are the Amplicon Sequence Variants and counts for results produced by Tronko (Pipes and Nielsen, 2024) that were subsampled to only ASVs with fewer than 25 mismatches to the reference. The subsampled ones were used for comparing taxonomic profiles of different samples, an analysis published in the Journal of Heredity, esag001, https://doi.org/10.1093/jhered/esag001.