Klughammer, Johanna (CeMM Research Center for Molecular Medicine / Ludwig-Maximilians-Universität München) Bock, Christoph (CeMM Research Center for Molecular Medicine / Medical University of Vienna) Skjærven, Kaja (Norwegian Institute of Marine Research) Farlik, Matthias (CeMM Research Center for Molecular Medicine) Schmidl, Christian (CeMM Research Center for Molecular Medicine) Fortelny, Nikolaus (CeMM Research Center for Molecular Medicine) Datlinger, Paul (CeMM Research Center for Molecular Medicine) Ergüner, Bekir (CeMM Research Center for Molecular Medicine) Kirchberger, Stefanie (St. Anna Children's Cancer Research Institute) Printz, Dieter (St. Anna Children's Cancer Research Institute) Evankow, Ann (Northeastern University) Kosack, Lindsay (CeMM Research Center for Molecular Medicine) Kübber-Heiss, Anna (University of Veterinary Medicine Vienna) Lugo Ramos, Juan Sebastian (Max Planck Institute for Evolutionary Biology) Keinath, Melissa C. (University of Kentucky) Schuster, Linda C. (CeMM Research Center for Molecular Medicine) Seid, Charlotte A. (Northeastern University) Posautz, Annika (University of Veterinary Medicine Vienna) Romanovskaia, Daria (CeMM Research Center for Molecular Medicine) Nemc, Amelie (CeMM Research Center for Molecular Medicine) Distel, Daniel L. (Northeastern University) Distel, Martin (St. Anna Children's Cancer Research Institute) Hermann, Marcela (Medical University of Vienna) Burger, Pamela A. (University of Veterinary Medicine Vienna) Thaller, Denise (University of Veterinary Medicine Vienna) Liedvogel, Miriam (Max Planck Institute for Evolutionary Biology / Institute of Avian Research) Bergthaler, Andreas (CeMM Research Center for Molecular Medicine / Medical University of Vienna) Nature Communications 14(1) (2023) CC BY 4.0 International http://creativecommons.org/licenses/by/4.0/ 2023 application/pdf Animals; DNA Methylation Genetics; Genome Genetics; Invertebrates Genetics; Vertebrates Genetics Metabolism; Epigenesis, Genetic; DNA Metabolism doi:10.1038/s41467-022-34828-y https://phaidra.vetmeduni.ac.at/o:3301 Nature Portfolio Methylation of cytosines is a prototypic epigenetic modification of the DNA. It has been implicated in various regulatory mechanisms across the animal kingdom and particularly in vertebrates. We mapped DNA methylation in 580 animal species (535 vertebrates, 45 invertebrates), resulting in 2443 genome-scale DNA methylation profiles of multiple organs. Bioinformatic analysis of this large dataset quantified the association of DNA methylation with the underlying genomic DNA sequence throughout vertebrate evolution. We observed a broadly conserved link with two major transitions-once in the first vertebrates and again with the emergence of reptiles. Cross-species comparisons focusing on individual organs supported a deeply conserved association of DNA methylation with tissue type, and cross-mapping analysis of DNA methylation at gene promoters revealed evolutionary changes for orthologous genes. In summary, this study establishes a large resource of vertebrate and invertebrate DNA methylomes, it showcases the power of reference-free epigenome analysis in species for which no reference genomes are available, and it contributes an epigenetic perspective to the study of vertebrate evolution. Comparative analysis of genome-scale, base-resolution DNA methylation profiles across 580 animal species eng article