Ancient DNA from speleothems: opportunity or challenge?

Ancient environmental DNA retrieved from sedimentary records (sedaDNA) can complement fossil-morphological approaches for char- acterizing Quaternary biodiversity changes. PCR-based DNA metabarcoding is so far the most widely used method in environmental DNA studies, including sedaDNA. However, degradation of ancient DNA and potential contamination, together with the PCR amplification drawbacks, have to be carefully considered. Here we tested this approach on speleothems from an Alpine cave that, according to a previous palynomorphological study, have shown to contain abundant pollen grains. This offers a unique opportunity for comparing the two meth- ods and, indirectly, trying to validate DNA-based results. The plant taxa identified by sedaDNA are fewer than those by pollen analysis, and success rate of PCR replicates is low. Despite extensive work performed following best practice for sedaDNA, our results are suboptimal and accompanied by a non-negligible uncertainty. Our preliminary data seem to indicate that paleoenvironmental DNA may be isolated from speleothems, but the intrinsic weakness of PCR-based metabarcoding poses a challenge to its exploitation. We suggest that newly developed methods such as hybridization capture, being free from PCR drawbacks and offering the opportunity to directly assess aDNA authenticity, may overcome these limitations, allowing a proper exploitation of speleothems as biological archives.