From the sea to <scp>DNA</scp> barcode: Optimized preservation method for gelatinous zooplankton integrative taxonomy

Abstract Assigning taxonomic identity and thus assessing taxonomic diversity in gelatinous zooplankton is challenging due to their fragility and difficult preservation. Formalin fixation compromises DNA integrity, while flash‐freezing, ethanol, and acetone preservation affect morphological features. In this study, we developed a methodological protocol to optimize morphological and molecular identification of pelagic cnidarians and tunicates from the Gulf of Naples (Mediterranean Sea). Specimens were identified while fresh and preserved using four methods: liquid nitrogen (N), ethanol (EtOH), ethanol replaced with acetone (EA), and formalin followed by ethanol and acetone (FEA). Genomic DNA was extracted and PCR‐amplified targeting the 18S rRNA gene, being subsequently assessed for concentration, integrity, and sequence quality. FEA specimens retained better morphology compared to N, EtOH, or EA samples, while sequencing performance was comparable between methods. DNA concentrations did not significantly differ among treatments (range: 139–1645 ng ind −1 ) and smaller taxa showed higher values per unit size compared to larger ones. DNA integrity was lower in N and FEA samples, and often undetectable in EA and FEA when assessed by gel electrophoresis. Although 18S : gDNA ratio decreased in some FEA samples, this method still produced high‐quality reads (&gt; 500 nucleotides). Our findings show that formalin fixation followed by ethanol and acetone does not compromise molecular analysis, even for species with low DNA content. Unlike other methods, this optimized protocol preserves essential morphological features for accurate taxonomic identification of gelatinous zooplankton while substantially reducing molecular processing time. Overall, the adoption of the proposed protocol could significantly enhance biodiversity assessments of gelatinous taxa.