In the search for natural reservoirs of hepatitis C virus (HCV), a broad diversity of non-human viruses within the Hepacivirus genus has been uncovered. However, the evolutionary dynamics that shaped the diversity and timescale of hepaciviruses evolution remain elusive. To gain further insights into the origins and evolution of this genus, we screened a large dataset of wild mammal samples (n = 1,672) from Africa and Asia, and generated 34 full-length hepacivirus genomes. Phylogenetic analysis of these data together with publicly available genomes emphasizes the importance of rodents as hepacivirus hosts and we identify 13 rodent species and 3 rodent genera (in Cricetidae and Muridae families) as novel hosts of hepaciviruses. Through co-phylogenetic analyses, we demonstrate that hepacivirus diversity has been affected by cross-species transmission events against the backdrop of detectable signal of virus-host co-divergence in the deep evolutionary history. Using a Bayesian phylogenetic multidimensional scaling approach, we explore the extent to which host relatedness and geographic distances have structured present-day hepacivirus diversity. Our results provide evidence for a substantial structuring of mammalian hepacivirus diversity by host as well as geography, with a somewhat more irregular diffusion process in geographic space. Finally, using a mechanistic model that accounts for substitution saturation, we provide the first formal estimates of the timescale of hepacivirus evolution and estimate the origin of the genus to be about 22 million years ago. Our results offer a comprehensive overview of the micro- and macroevolutionary processes that have shaped hepacivirus diversity and enhance our understanding of the long-term evolution of the Hepacivirus genus.

Animal Resource Science and Management Group Faculty of Resource Science and Technology Universiti Malaysia Sarawak 94300 Malaysia

College of Life Sciences Liaocheng University Liaocheng 252000 China

College of Life Sciences Linyi University Linyi 276000 China

Department of Biology University of Oxford Oxford OX1 UK

Department of Biostatistics University of California Los Angeles CA 90095 USA

Department of Hygiene Epidemiology and Medical Statistics Medical School National and Kapodistrian University of Athens Athens 11527 Greece

Department of Microbiology Immunology and Transplantation KU Leuven Rega Institute KU Leuven Leuven 3000 Belgium

Department of Pathobiology and Population Sciences the Royal Veterinary College University of London Herts AL9 7TA UK

Department of Science and Environmental Studies The Education University of Hong Kong Hong Kong 999077 China

EcoHealth Alliance New York NY 10018 USA

Evolutionary Ecology group Department of Biology University of Antwerp Antwerp 2020 Belgium

Faculty of Sciences University of Kisangani Kisangani Democratic Republic of the Congo

Institute of Vertebrate Biology The Czech Academy of Sciences Květná 8 603 65 Brno Czech Republic

Laboratory of Biodiversity Parasitology and Ecology of Aquatic Ecosystems Department of Biology Faculty of Sciences of Tunis University of Tunis El Manar Tunis 2092 Tunisia

Marine College Shandong University Weihai 264209 China

School of Biodiversity One Health and Veterinary Medicine College of Medical Veterinary and Life Sciences University of Glasgow Glasgow G61 1QH UK

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