San Francisco Estuary and Watershed Science

Although formalin is commonly used as a preserving reagent for tissue specimens, the fixation process itself damages DNA, which can be detrimental to most downstream genetic analyses. It may still be possible to confirm species identification from archived specimens by targeting short, species-specific genic regions. In this study we genetically verified maternal mitochondrial lineage from 150 hatchery larval Delta Smelt (Hypomesus transpacificus) that were preserved in 10% neutral, buffered formalin and Rose Bengal at room temperature (20–22 °C) for 611 days, 732 days, and 928 days. We targeted a region of the mitochondrial cytochrome b (Cyt-b) gene using a quantitative PCR (qPCR) assay designed to specifically amplify Delta Smelt environmental DNA (eDNA). Because mitochondrial genetic markers used for species identification are generally maternally inherited, detecting hybridization is not possible using species-specific mitochondrial markers if among-species hybrids are present. Because Delta Smelt can hybridize with non-native Wakasagi Smelt (Hypomesus nipponensis), we assumed in this study the method detected maternal lineage. Maternal mitochondrial lineage was confirmed in 100% of our 150 Delta Smelt samples analyzed, although the strength of detection appeared to decline over time. We posit that the short DNA target size (84 base pairs) of the Delta Smelt-specific qPCR assay allowed for successful identification, and we confirmed this by Sanger sequencing with the qPCR assay forward and reverse primers. Our results may be broadly applicable because formalin remains a commonly used fixative in the preservation of fish, reptiles, amphibians, and various invertebrate taxa. Additional testing with both Sanger- and Illumina-based sequencing methods on a subset of the larval extracts did not successfully produce positive identification.