Ancient DNA analysis identifies marine mollusc shells as new metagenomic archives of the past.

This is the author accepted manuscript. The final version is available from Wiley via the DOI in this record.Marine mollusc shells enclose a wealth of information on coastal organisms and their environment. Their life history traits as well as (palaeo-) environmental conditions, including temperature, food availability, salinity and pollution, can be traced through the analysis of their shell (micro-) structure and biogeochemical composition. Adding to this list, the DNA entrapped in shell carbonate biominerals potentially offers a novel and complementary proxy both for reconstructing palaeoenvironments and tracking mollusc evolutionary trajectories. Here, we assess this potential by applying DNA extraction, high-throughput shotgun DNA sequencing and metagenomic analyses to marine mollusc shells spanning the last ~7,000 years. We report successful DNA extraction from shells, including a variety of ancient specimens, and find that DNA recovery is highly dependent on their biomineral structure, carbonate layer preservation and disease state. We demonstrate positive taxonomic identification of mollusc species using a combination of mitochondrial DNA genomes, barcodes, genome-scale data and metagenomic approaches. We also find shell biominerals to contain a diversity of microbial DNA from the marine environment. Finally, we reconstruct genomic sequences of organisms closely related to the Vibrio tapetis bacteria from Manila clam shells previously diagnosed with Brown Ring Disease. Our results reveal marine mollusc shells as novel genetic archives of the past, which opens new perspectives in ancient DNA research, with the potential to reconstruct the evolutionary history of molluscs, microbial communities and pathogens in the face of environmental changes. Other future applications include conservation of endangered mollusc species and aquaculture management.We thank Tom Schiøtte and Martin Vinther Sørensen at the Zoological Museum of Copenhagen for providing historical shell samples from the Invertebrate Collection. We thank Adeline Bidault for Vibrio DNA extraction, Kristian Hanghøj for technical assistance, Mikkel Schubert and Gabriel Renaud for fruitful discussions, the PALEOMIX group and the staff of the Danish National High-Throughput DNA Sequencing Centre for support. This work was supported by the Danish Council for Independent Research, Natural Sciences (FNU, 4002-00152B); the Danish National Research Foundation (DNRF94); the EPT PROXACHEOBIO from Université Européenne de Bretagne (2010–2012); the APEGE initiative PaleoCOO of the Centre National de la Recherche Scientifique; the cluster of excellence LabexMER (ANR-10-LABX-19; METHOMOL) under the program “Investissements d'Avenir”; the UK Natural Environment Research Council (NE/H023356/1); the EU Marie Curie ARAMACC Initial Training Network (FP7-PEOPLE-2013-ITN 604802); the “Chaires d'Attractivité 2014” IDEX, University of Toulouse, France (OURASI)