Jeunen, G.-J.; Mills, S.; Bailie, M.; Mauvisseau, Q.; Lamare, M.; Mariani, S.; Pearman, W.; Zavodna, M.; Treece, J.; Ferreira, S.; Gemmell, N.

Recent technical advances have significantly enhanced the value of museum specimens for molecular research, with metagenomic and metabarcoding approaches expanding further the utility of museum collections. However, given the finite number of specimens, there is a critical need to move past destructive DNA extraction approaches and to explore non-destructive techniques. In this study, we evaluated the feasibility of extracting historical fish eDNA from the ethanol preservative used to store Antarctic museum specimens. We compared a variety of extraction methods, including centrifugation, evaporation, filtration, and precipitation, using ten replicate samples per treatment for statistical analyses. To assess potential differences in preservative-derived eDNA recovery across different filter-feeding taxonomic groups, we included a Bryozoa and two Porifera, i.e., Demospongiae and Hexactinellida. Comparative analyses with tissue biopsies revealed that the total genomic DNA was significantly reduced for non-destructive approaches. However, 10 ml ethanol filtration performed equal to, or in some instances, outperformed tissue biopsies when investigating the historical eDNA of Antarctic fish using a 16S rRNA metabarcoding approach, both for the number of species detected (-diversity) and community characterisation ({beta}-diversity). Our findings demonstrate the potential of ethanol preservative as a valuable, non-destructive source of historical fish eDNA from museum-stored filter-feeding specimens. These results highlight the viability of non-destructive sampling for molecular research on museum collections, preserving specimen integrity while enabling biodiversity assessments. Further refinement of non-destructive eDNA extraction could expand its applicability across taxa, collection types, and preservation methods, ensuring the long-term sustainability of museum-based genomic research.