Is DNA metabarcoding an option for formaldehyde‐preserved zooplankton time series?

Abstract The recovery of amplifiable DNA from formaldehyde‐fixed (FF) zooplankton samples has long been considered problematic, but advances in degraded‐DNA retrieval have renewed interest in FF samples. To access the information stored in long‐term zooplankton time series, we evaluated methods for extracting amplifiable DNA from community samples preserved for up to 28 yr in formaldehyde at room temperature. A method previously reported as successful in FF zooplankton stored at 4°C proved ineffective here, likely due to the differing storage conditions. In contrast, by adapting two protocols developed for FF museum specimens—a harsher (Hahn–hot alkaline [HHA]) and a gentler (Hahn–proteinase K column [HPC]) extraction approach—we were able to amplify and sequence a subset of samples. As expected, DNA integrity and sample pH decreased with preservation time, and only short DNA fragments were recoverable, ruling out standard ≥ 300 bp metabarcoding markers. While DNA integrity appeared to be a better predictor than DNA yield for amplification success, the presence of a gel band of the expected size did not always guarantee congruence with microscopy assessments. Although amplifiable DNA was recovered from most samples, including some of the oldest, community compositions concordant with microscopy were consistently recovered only from samples preserved for up to 2 yr. Beyond this point, the HHA and HPC methods produced divergent results, reflecting a trade‐off between removing formaldehyde‐induced cross‐linkages and avoiding additional DNA damage. Among the three small universal markers tested (~ 120–170 bp), only the 18S rRNA V9 region consistently amplified. We conclude by providing recommendations aimed at improving the methods assessed here.