Seven years in formalin: the burden of metabarcoding mesozooplankton preserved in buffered formalin from the northern...

Historical mesozooplankton collections preserved in formalin are valuable archives for reconstructing past biodiversity, but formalin-induced DNA damage has long limited their molecular use. We evaluated the feasibility of metabarcoding on mesozooplankton samples collected in 2017 from the northern Adriatic Sea and stored in 4% buffered formalin for approximately seven years at room temperature and dark conditions. We applied a hot alkaline lysis protocol to improve DNA recovery from formalin-fixed material and tested three metabarcoding markers differing in amplicon length and taxonomic resolution (COI, 18SV4, and 18SV9), using a bioinformatic pipeline adapted for highly degraded templates. The three markers performed differently. COI retained the strongest metazoan signal, while 18SV9 recovered the broadest metazoan diversity and showed the highest concordance with morphology. By contrast, 18SV4 was strongly biased toward non-metazoan amplification, with fungal reads dominating several samples. Of the 86 taxa identified by stereomicroscopy, COI recovered 16 taxa and 18SV9 recovered 59, with only nine shared between markers. These results show that samples preserved in buffered formalin for 7 years can still yield informative metabarcoding data, although taxonomic recovery is strongly shaped by marker choice.