DNA Extraction and mtDNA genome sequencing protocol from museum voucher specimens v1

Museum voucher specimens represent an invaluable resource for biodiversity research, serving as permanent, verifiable records of species identities, morphology, and provenance (Suarez & Tsutsui, 2004). In recent decades, these archived specimens have also become critical molecular reservoirs, particularly for DNA-based taxonomic and phylogenetic studies (Wandeler et al., 2007). Among genetic markers, mitochondrial DNA (mtDNA) is especially suited for taxonomic assessment due to its maternal inheritance, relatively rapid mutation rate, and high copy number per cell, which enhances its retrievability even from degraded or historical samples (Avise, 2000; Paabo et al., 2004). In mammals, sequencing mitochondrial genomes or key regions (e.g., cytochrome b, COI) from voucher specimens enables retrospective species identification, clarifies cryptic diversity, and facilitates phylogeographic and conservation assessments (Tobe et al., 2010; Lorenzen et al., 2011). Integrating molecular data from authenticated voucher specimens strengthens taxonomic resolution and provides reproducibility across studies, underscoring their dual importance in both classical and modern systematics. Our protocol offers multiple extraction methods for variously stored samples (formallin or ethanol fixed). It makes it widely usable but the method was mainly tested on mammal samples. References Avise, J. C. (2000). Phylogeography: The History and Formation of Species. Harvard University Press. Lorenzen, E. D., et al. (2011). Species-specific responses of Late Quaternary megafauna to climate and humans. Nature, 479(7373), 359–364. Paabo, S., et al. (2004). Genetic analyses from ancient DNA. Annual Review of Genetics, 38, 645–679. Suarez, A. V., & Tsutsui, N. D. (2004). The value of museum collections for research and society. BioScience, 54(1), 66–74. Tobe, S. S., Kitchener, A. C., & Linacre, A. M. T. (2010). Reconstructing mammalian phylogenies: mitochondrial DNA versus morphology. Molecular Phylogenetics and Evolution, 55(2), 727–733. Wandeler, P., Hoeck, P. E., & Keller, L. F. (2007). Back to the future: museum specimens in population genetics. Trends in Ecology & Evolution, 22(12), 634–642.