Vertebrates have greatly elaborated the basic chordate body plan and evolved highly distinctive genomes that have been sculpted by two whole-genome duplications. Here we sequence the genome of the Mediterranean amphioxus (Branchiostoma lanceolatum) and characterize DNA methylation, chromatin accessibility, histone modifications and transcriptomes across multiple developmental stages and adult tissues to investigate the evolution of the regulation of the chordate genome. Comparisons with vertebrates identify an intermediate stage in the evolution of differentially methylated enhancers, and a high conservation of gene expression and its cis-regulatory logic between amphioxus and vertebrates that occurs maximally at an earlier mid-embryonic phylotypic period. We analyse regulatory evolution after whole-genome duplications, and find that-in vertebrates-over 80% of broadly expressed gene families with multiple paralogues derived from whole-genome duplications have members that restricted their ancestral expression, and underwent specialization rather than subfunctionalization. Counter-intuitively, paralogues that restricted their expression increased the complexity of their regulatory landscapes. These data pave the way for a better understanding of the regulatory principles that underlie key vertebrate innovations.

Australian Research Council Centre of Excellence in Plant Energy Biology School of Molecular Sciences The University of Western Australia Crawley Western Australia Australia

Biologie Intégrative des Organismes Marins BIOM Observatoire Océanologique CNRS and Sorbonne Université Banyuls sur Mer France

Biology and Evolution of Marine Organisms Stazione Zoologica Anton Dohrn Napoli Naples Italy

Biomedical Sciences Research Complex School of Biology University of St Andrews St Andrews UK

Center for Autoimmune Genomics and Etiology Divisions of Biomedical Informatics and Developmental Biology Cincinnati Children's Hospital Medical Center Cincinnati OH USA

Centre for Genomic Regulation Barcelona Spain

Centro Andaluz de Biología del Desarrollo CSIC Universidad Pablo de Olavide Junta de Andalucía Seville Spain

CNRS UMR 8197 Paris France

Computational Regulatory Genomics MRC London Institute of Medical Sciences London UK

Department of Genetics Microbiology and Statistics Faculty of Biology and Institut de Biomedicina University of Barcelona Barcelona Spain

Department of Genetics Microbiology and Statistics Faculty of Biology and Institut de Recerca de la Biodiversitat University of Barcelona Barcelona Spain

Department of Molecular Developmental Biology Faculty of Science Radboud Institute for Molecular Life Sciences Radboud University Nijmegen The Netherlands

Department of Pediatrics University of Cincinnati College of Medicine Cincinnati OH USA

Department of Zoology University of Cambridge Cambridge UK

Department of Zoology University of Oxford Oxford UK

Genomics and Epigenetics Division Garvan Institute of Medical Research Sydney New South Wales Australia

Génomique Métabolique Genoscope Institut de biologie François Jacob Commissariat à l'Energie Atomique CNRS Université Evry Université Paris Saclay Evry France

Genoscope Institut de biologie François Jacob Commissariat à l'Energie Atomique Université Paris Saclay Evry France

Harry Perkins Institute of Medical Research Nedlands Western Australia Australia

Inserm U1024 Paris France

INSERM U830 Équipe Labellisée LNCC SIREDO Oncology Centre Institut Curie PSL Research University Paris France

Institut de Biologie de l'ENS IBENS Ecole Normale Supérieure Paris France

Institute of Cellular and Organismic Biology Academia Sinica Taipei Taiwan

Institute of Clinical Sciences Faculty of Medicine Imperial College London London UK

Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czech Republic

Interdisciplinary Centre of Marine and Environmental Research Department of Biology University of Porto Porto Portugal

IRD APHM Microbe Evolution PHylogénie Infection IHU Méditerranée Infection and CNRS Aix Marseille University Marseille France

Laboratoire de Biométrie et Biologie Evolutive CNRS and Université Lyon 1 Villeurbanne France

Laboratory for Transcriptome Technology RIKEN Center for Integrative Medical Sciences Yokohama Japan

Molecular Genetics Unit Okinawa Institute of Science and Technology Graduate University Onna son Japan

RIKEN Center for Life Science Technologies Yokohama Japan

Sars International Centre for Marine Molecular Biology University of Bergen Bergen Norway

School of Life Sciences Beijing University of Chinese Medicine Beijing China

School of Medical Sciences Faculty of Biology Medicine and Health University of Manchester Manchester UK

Sorbonne Université CNRS Laboratoire de Biologie du Développement de Villefranche sur Mer Institut de la Mer de Villefranche sur Mer Villefranche sur Mer France

St Vincent's Clinical School Faculty of Medicine University of New South Wales Sydney New South Wales Australia

State Key Laboratory of Biocontrol School of Life Sciences Sun Yat sen University Guangzhou China

The Scottish Oceans Institute Gatty Marine Laboratory University of St Andrews St Andrews UK

UMR 9002 CNRS Institut de Génétique Humaine Université de Montpellier Montpellier France

Universitat Pompeu Fabra Barcelona Spain

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