Recovering mtDNA from long-term formalin-preserved museum arthropods: A pilot study

Background : Natural history collections can be a valuable source of genetic material for evolutionary research. However, numerous samples from zoological collections are fixed in formalin and may remain continuously exposed to formalin over long periods of time. Due to severe DNA degradation caused by formalin fixation, extracting DNA from these collections is highly challenging. Results : Multiple DNA extraction protocols were tested for their ability to recover genetic material from long-term formalin-preserved specimens. We identified a best-performing protocol that provides a straightforward and contamination-resistant method for obtaining high-quality mitochondrial DNA from arthropods, particularly ground beetles (Carabidae) and freshwater crabs (Potamidae), preserved in formalin for up to four decades. Out of the 37 tested samples, 26 produced DNA concentrations of ≥ 0.1 ng/µl at ~ 180 bp (including adapters) after library preparation, which is sufficient for successful Illumina sequencing. Notably, crab samples yielded higher DNA concentrations after library preparation than beetle samples, likely due to differences in tissue structure and preservation history. Results from competitive mapping showed mitochondrial genome coverage ranging from approximately 3X to 17X, despite low endogenous mitochondrial DNA content. Despite the relatively low coverage, the recovered mitochondrial data proved sufficient for downstream phylogenetic analyses. As species-specific genomes were unavailable, coverage was calculated by mapping to the mitochondrial reference genomes of closely-related species of the target taxa. Conclusion : This study demonstrates that high-throughput sequencing is feasible from invertebrate museum specimens preserved in formalin for decades and provides a practical protocol adaptable to a broad taxonomic range.