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ABSTRACT Microbiomes are vast communities of microorganisms and viruses that populate all natural ecosystems. Viruses have been considered to be the most variable component of microbiomes,

as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared with that of other

environments. Here, we investigate the origin, evolution and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboration, we obtained DNA sequences of crAssphage

from more than one-third of the world’s countries and showed that the phylogeography of crAssphage is locally clustered within countries, cities and individuals. We also found fully

colinear crAssphage-like genomes in both Old-World and New-World primates, suggesting that the association of crAssphage with primates may be millions of years old. Finally, by exploiting a

large cohort of more than 1,000 individuals, we tested whether crAssphage is associated with bacterial taxonomic groups of the gut microbiome, diverse human health parameters and a wide

range of dietary factors. We identified strong correlations with different clades of bacteria that are related to Bacteroidetes and weak associations with several diet categories, but no

significant association with health or disease. We conclude that crAssphage is a benign cosmopolitan virus that may have coevolved with the human lineage and is an integral part of the

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support SIMILAR CONTENT BEING VIEWED BY OTHERS PHYLOGENY AND DISEASE ASSOCIATIONS OF A WIDESPREAD AND ANCIENT INTESTINAL BACTERIOPHAGE LINEAGE Article Open access 27 July 2024 METAGENOMIC

COMPENDIUM OF 189,680 DNA VIRUSES FROM THE HUMAN GUT MICROBIOME Article Open access 24 June 2021 METAGENOMIC ANALYSIS REVEALS UNEXPLORED DIVERSITY OF ARCHAEAL VIROME IN THE HUMAN GUT Article

Open access 29 December 2022 DATA AVAILABILITY Sequence data that support the findings of this study have been deposited in GenBank under BioProject accession PRJNA510571 and at

https://github.com/linsalrob/crAssphage. Each of the samples has a unique BioSample accession number (SAMN10656826–SAMN10658627, SAMN10658653 and SAMN10659294). The SRA runs used in this

analysis are included in Supplementary File 5. The data that support the findings of this study are also available from the corresponding authors on reasonable request. CODE AVAILABILITY The

code used to generate the data can be accessed at https://github.com/linsalrob/crAssphage. The current release81 is v.2.0. REFERENCES * Sender, R., Fuchs, S. & Milo, R. Are we really

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Dutilh, Bas E., and Edwards, Robert A. _crAssphage Data Repository on GitHu_b (Github, 2018); https://doi.org/10.5281/zenodo.1230436 Download references ACKNOWLEDGEMENTS We thank R.

Matthews, M. Wright, J. Alexander, S. Arredondo, N. Branch, D. Campbell, R. Chea, D. McDougle, J. Parks and V. Vipatapat for providing access to wastewater treatment samples; the members of

the Mountain Gorilla Veterinary Project and the staff of Maryland Zoo for collecting the gorilla faecal samples in Rwanda; G. Britton for collecting the baboon faecal samples in Ethiopia;

staff of the CSWCT, the UWA and the UNCST for collecting the chimpanzee faecal samples in Uganda; J. Manor at Central Virology Laboratory, Chaim Sheba Medical Center, Tel-Hashomer Hospital

and G. Steward, Department of Oceanography, University of Hawai’i at Manoa for help with sample collection; the COMPARE and LifeLines-DEEP projects for sharing data; O.D.N. thanks G.

Steward, University of Hawai’i, Manoa for support. P.C.F. thanks C. Taylor for support with the PCR. Primate samples were provided by the PMC at the University of Illinois Urbana-Champaign;

D.T.M. thanks the Australian Research Council’s Linkage Project LP160100408, Melbourne Water and EPA Victoria for funding the collection of samples in Melbourne. Gorilla samples were

originally obtained by M.K. and the Mountain Gorilla Veterinary Project in Rwanda. G.R. and N.J.D. provided the wild baboon samples from Ethiopia. Howler samples were provided by M.K. and

lemur samples were provided by R.E.J. and M.T.I., R.M.S. and L.M. provided the chimpanzee samples with permission from the CSWCT, the UWA and the UNCST. The primate microbiome project was

supported by NSF BCS 0935347 to S.L., R. Stumpf, B.W. and K. Nelson. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the

manuscript. This work used the XSEDE Jetstream resources at Indiana University and Texas Advanced Computing Center through allocation MCB170036 to R.A.E., which is supported by National

Science Foundation grant number ACI-1548562. Some of this work was supported by San Diego State University Grants Programs to R.A.E., including the Summer Undergraduate Research Program.

This work was supported by National Science Foundation grant numbers MCB-1441985 to R.A.E. and DUE-1323809 to E.A.D; the Department of Energy Lawrence Livermore National Laboratory grant

B618146 to R.A.E., P.A.d.J. and B.E.D. were supported by the NWO Vidi grant 864.14.004; F.L.N. by the NWO Veni grant 016.Veni.181.092; S.J.J.B. by the European Research Council Stg grant

(638707) and the Vidi grant 864.11.005; O.C. and K. Mazankova by the Ministry of Health of the Czech Republic grant numbers 15-31426A and 15-29078A; P.C.F. by a Rutherford Discovery

Fellowship from the Royal Society of New Zealand. J.J.B. by the ARC Discovery Early Career Researcher Award (DE170100525); S.L.D.M. by an NIH Pathway to Independence Fellowship

(1K99AI119401-01A1); K.B. by award number 1510925 from the United States National Science Foundation; M.T.I. by National Geographic Society (CRE) and NSERC; and C.D. by the Agence Nationale

de la Recherche JCJC grant ANR-13-JSV6-0004 and Investissements d’Avenir Méditerranée Infection 10-IAHU-03. The LifeLines-DEEP sample collection and analysis was funded by the Netherlands

Heart Foundation (IN-CONTROL CVON grant 2012-03) to A.Z. and J.F., by the Top Institute Food and Nutrition, Wageningen, the Netherlands (TiFN GH001) to C.W., by NWO Vidi grants 864.13.013 to

J.F. and 016.178.056 to A.Z., NWO Spinoza Prize SPI 92-266 to C.W., and by the ERC FP7/2007-2013/ERC Advanced Grant agreement 2012-322698 to C.W., ERC Starting Grant 715772 to A.Z. A.Z.

also holds a Rosalind Franklin Fellowship from the University of Groningen. The COMPARE data collection was funded by The Novo Nordisk Foundation (NNF16OC0021856). AUTHOR INFORMATION AUTHORS

AND AFFILIATIONS * Department of Biology, San Diego State University, San Diego, CA, USA Robert A. Edwards, Alejandro A. Vega, Holly M. Norman, Maria Ohaeri, Elizabeth A. Dinsdale, Emma K.

Billings, Tess Condeff, Michael P. Doane, John M. Haggerty, Scott T. Kelley, David Lipson, Megan Morris, Kim Reasor & Pedro J. Torres * The Viral Information Institute, San Diego State

University, San Diego, CA, USA Robert A. Edwards * Department of Computer Science, San Diego State University, San Diego, CA, USA Kyle Levi * Department of Pediatrics, 2nd Faculty of

Medicine, Charles University in Prague, Prague, Czech Republic Ondrej Cinek & Karla Mazankova * Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Cairo,

Egypt Ramy K. Aziz * Computational Sciences Research Center, San Diego State University, San Diego, CA, USA Katelyn McNair, Vito Adrian Cantu & Daniel A. Cuevas * School of Biological

Sciences, Monash University, Clayton, Victoria, Australia Jeremy J. Barr * Civil and Environmental Engineering and Earth Sciences, University of Notre Dame, Notre Dame, IN, USA Kyle Bibby *

Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands Stan J. J. Brouns, Patrick A. de Jonge & Franklin L. Nobrega *

Institute of Infection and Global Health, University of Liverpool, Liverpool, UK Adrian Cazares * Theoretical Biology and Bioinformatics, Science4Life, Utrecht University, Utrecht, The

Netherlands Patrick A. de Jonge, Alessandro Rossi & Bas E. Dutilh * MEPHI, Aix-Marseille Université, IRD, AP-HM, CNRS, IHU Méditerranée Infection, Marseille, France Christelle Desnues *

Mediterranean Institute of Oceanography, Aix-Marseille Université, Université de Toulon, CNRS, IRD, UM 110, Marseille, France Christelle Desnues * Center for Genomics and Systems Biology

& Department of Biology, New York University, New York, NY, USA Samuel L. Díaz Muñoz, Jane M. Carlton, Elodie Ghedin, Kristen M. Gulino, Julia M. Maritz & Alan Twaddle * Department

of Microbiology and Molecular Genetics, University of California, Davis, Davis, CA, USA Samuel L. Díaz Muñoz * Department of Microbiology and Immunology, University of Otago, Dunedin, New

Zealand Peter C. Fineran * Department of Genetics, University Medical Center Groningen, Groningen, The Netherlands Alexander Kurilshikov, Alexandra Zhernakova & Cisca Wijmenga *

Department of Biosystems, KU Leuven, Leuven, Belgium Rob Lavigne & Jeroen Wagemans * EPHM Lab, Civil Engineering Department, Monash University, Clayton, Victoria, Australia David T.

McCarthy * Max Planck Tandem Group in Computational Biology, Departamento de Ciencias Biológicas, Universidad de los Andes, Bogotá, Colombia Alejandro Reyes Muñoz * Department of Child

Health, Norwegian Institute of Public Health, Oslo, Norway German Tapia & Lars C. Stene * GEMA Center for Genomics, Ecology & Environment, Universidad Mayor, Huechuraba, Chile Nicole

Trefault * Laboratory of Bioinformatics, Federal Research and Clinical Center of Physical-Chemical Medicine, Moscow, Russia Alexander V. Tyakht * Department of Informational Technologies,

ITMO University, Saint Petersburg, Russia Alexander V. Tyakht * Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, Mexico Pablo Vinuesa & Daniel Cazares *

National Food Institute, Research Group for Genomic Epidemiology, Technical University of Denmark, Kongens Lyngby, Denmark Frank M. Aarestrup & Rene S. Hendriksen * Endocrine Centre

Baku, Baku, Azerbaijan Gunduz Ahmadov * Department of Pediatrics, School of Medicine, University of Jordan, Amman, Jordan Abeer Alassaf & Rasha Odeh * Department of Physiology, Genetics

and Microbiology, University of Alicante, Alicante, Spain Josefa Anton * Carl R. Woese Institute of Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA Abigail

Asangba, Rebecca Stumpf & Bryan White * Department of Microbiology and Biotechnology, Max Rubner-Institut, Federal Research Institute of Nutrition and Food, Kiel, Germany Gyu-Sung Cho, 

Charles Franz & Horst Neve * Departament de Genètica i de Microbiologia, Universitat Autònoma De Barcelona, Barcelona, Spain Pilar Cortés & Montserrat Llagostera * Wildlife Health

Center, University of California, Davis, Davis, CA, USA Mike Cranfield * Departamento de Genética Molecular y Microbiología, Pontificia Universidad Católica de Chile, Santiago, Chile Rodrigo

De la Iglesia * Department of Bacterial Genetics, Institute of Microbiology, Faculty of Biology, University of Warsaw, Warsaw, Poland Przemyslaw Decewicz & Lukasz Dziewit * Department

of Anthropology, Dartmouth College, Hanover, NH, USA Nathaniel J. Dominy & Gillian A. O. Rice * Department of Pediatrics and Child Health, Faculty of Medicine, University of Khartoum,

Khartoum, Sudan Bashir Mukhtar Elwasila * Department of Medicine, University of Chicago, Chicago, IL, USA A. Murat Eren * Department of Pediatrics, University Medical Center Groningen,

Groningen, The Netherlands Jingyuan Fu * Department of Genetics, Microbiology and Statistics, Universitat de Barcelona, Barcelona, Spain Cristina Garcia-Aljaro, Juan Jofre & Maite

Muniesa * Next Generation Sequencing and Microarray Core Facility, The Scripps Research Institute, La Jolla, CA, USA Steven R. Head * School of Microbiology, University College Cork, Cork,

Ireland Colin Hill * Department of Virology, School of Medicine, University of Tampere, Tampere, Finland Heikki Hyöty * Department of Molecular Biology and Genetics, Federal Research and

Clinical Center of Physical-Chemical Medicine, Moscow, Russia Elena N. Ilina * Department of Anthropology, Northern Illinois University, DeKalb, IL, USA Mitchell T. Irwin * School of Science

and Health, Western Sydney University, Penrith, New South Wales, Australia Thomas C. Jeffries * Department of Animal Health, Columbus Zoo and Aquarium, Powell, OH, USA Randall E. Junge *

Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada Mohammadali Khan Mirzaei * Department Estacion Biologica Corrientes, Institution Museo Arg. Cs.

Naturales-CONICET, Corrientes, Argentina Martin Kowalewski * UWA School of Agriculture and Environment, University of Western Australia, Perth, Western Australia, Australia Deepak Kumaresan,

 Benjamin Moreira-Grez & Andy Whiteley * Department of Anthropology, University of Colorado, Boulder, CO, USA Steven R. Leigh * Department of Research and Development, Lytech Ltd.,

Moscow, Russia Eugenia S. Lisitsyna * Department of Civil and Environmental Engineering, Virginia Tech, Blacksburg, VA, USA Linsey C. Marr, Aaron J. Prussin II & John Shimashita * APC

Microbiome Institute, University College Cork, Cork, Ireland Angela McCann & Ronan Strain * Clinical Microbiology & Immunology, Sackler school of Medicine, Tel Aviv University, Tel

Aviv, Israel Shahar Molshanski-Mor * Laboratorio de Analises, Instituto Superior Tecnico, Universidade Lisboa, Lisboa, Portugal Silvia Monteiro & Ricardo Santos * CEHA, Kampala, Uganda

Lawrence Mugisha * COVAB, Makerere University, Kampala, Uganda Lawrence Mugisha * Computer Science and Engineering, University of California, San Diego, La Jolla, CA, USA Nam-phuong Nguyen *

College of Natural and Computational Sciences, Hawai’i Pacific University, Kaneohe, HI, USA Olivia D. Nigro * Department of Molecular Biosciences, Stockholm University, Stockholm, Sweden

Anders S. Nilsson * Biological and Medical Informatics Program, San Diego State University, San Diego, CA, USA Taylor O’Connell * Department of Molecular Biology & Biochemistry,

University of California, Irvine, Irvine, CA, USA Andrew Oliver, Stephen Wandro & Katrine L. Whiteson * Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales,

Universidad de Buenos Aires, Buenos Aires, Argentina Mariana Piuri * Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel Udi

Qimron * Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, Fudan University, Shanghai, China Zhe-Xue Quan * Centre of Epidemiology and Microbiology,

National Institute of Public Health, Prague, Czech Republic Petra Rainetova * Molecular Genetics, Corporación Corpogen, Bogotá, Colombia Adán Ramírez-Rojas & Maria M. Zambrano * CERELA,

Tucumán, Argentina Raul Raya * Department of Biology, University of Padova, Padova, Italy Alessandro Rossi * Swanson School of Engineering, University of Pittsburgh, Pittsburgh, PA, USA

Elyse N. Stachler * Department of Pediatrics, Federal Teaching Hospital Abakaliki, Ebonyi State University, Abakaliki, Nigeria MaryAnn Ugochi Ibekwe * Escuela de Tecnología Médica,

Universidad Andres Bello, Santiago, Chile Nicolás Villagra * The Bioinformatics Centre, Department of Biology, University of Copenhagen, Copenhagen, Denmark Henrike Zschach * Centre for

Molecular and Biomolecular Informatics, Radboud Institute for Molecular Life Sciences, Radboud University Medical Centre, Nijmegen, The Netherlands Bas E. Dutilh Authors * Robert A. Edwards

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Dutilh View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS B.E.D. and R.A.E. conceived the study, performed the experiments and bioinformatics,

and wrote the paper with input from all authors. A.A.V. performed the volunteer experiments and sampled San Diego wastewater treatment plants. F.L.N., H.M.N., M.O. and P.A.d.J. performed

human volunteer experiments. A.M.E., A.R., A.T., D.A.C., J.M.H., K.L., K.McNair, T.C. and V.A.C. performed bioinformatics analysis. A.A.R.R., A.Alassaf, A.C., A.M., A.O., A.R.M., A.S.N.,

A.W., B.M.-G., B.M.E., C.D., C.F., C.H., D.C., D.K., D.T.M., E.A.D., E.B., E.N.I., E.N.S., E.S.L., G.A., G.C.-A., G.-S.C., G.T., H.H., H.N., J.A., J.J.B., J.J.T., J.M.C., J.M.M., J.W., K.B.,

K.L.W., K.Mazankova, L.C.S., L.D., M.A.U.I., M.K.M., M.L., M.M.Z., M.Morris, M.Muniesa, M.P., M.P.D., N.T., N.V., O.C., O.D.N., P.C., P.C.F., P.D., P.R., P.V., R.d.l.I., R.K.A., R.L., R.O.,

R.R., R.Santos, R.Strain, S.J.J.B., S.L.D.M., S.M., S.M.-M., S.W., T.C., T.J., U.Q. and Z.-X.Q. performed sampling, PCR and sequencing. A.K., A.Z., C.W. and J.F. performed the Lifelines

analysis. F.M.A., H.Z. and R.S.H. provided and analysed COMPARE project data. A.Asangba, B.W., G.A.O.R., N.J.D., N.-p.N., R.Stumpf and S.L. analysed and provided the non-human primate

sequences. M.C. collected gorilla samples. A.T., E.G. and K.M.G. performed the NYC sewage sampling and data analysis. A.J.P., J.S., L.C.M., P.J.T., S.R.H. and S.T.K. examined crAssphage

transfer among infants. M.T.I. and R.E.J. collected lemur samples. M.K. collected howler monkey samples. D.L., K.R. created the map of the world figure. L.M. collected chimpanzee samples.

CORRESPONDING AUTHORS Correspondence to Robert A. Edwards or Bas E. Dutilh. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION

PUBLISHER’S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION

Supplementary Figs. 1–9, Supplementary Tables 1–6 and Supplementary References. REPORTING SUMMARY SUPPLEMENTARY FILE 1 Global sampling of crAssphage: the metadata and sequence data for each

of the amplicon regions. SUPPLEMENTARY FILE 2 Gretel strains: number of strains identified from all of the different samples in the SRA. SUPPLEMENTARY FILE 3 Lifelines phenotype

correlations: correlation, _P_ value and adjusted _P_ value for 207 exogenous and intrinsic human variables, and the presence of crAssphage in stools. SUPPLEMENTARY FILE 4 Lifelines

microbial correlations: correlation, _P_ value and adjusted _P_ value for the presence of 491 bacterial isolates and the presence of crAssphage in stools. SUPPLEMENTARY FILE 5 SRA Runs: the

identities of all runs in the SRA with matches to crAssphage, including the number of sequences that match, the total bases aligned and the average coverage. RIGHTS AND PERMISSIONS Reprints

and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Edwards, R.A., Vega, A.A., Norman, H.M. _et al._ Global phylogeography and ancient evolution of the widespread human gut virus

crAssphage. _Nat Microbiol_ 4, 1727–1736 (2019). https://doi.org/10.1038/s41564-019-0494-6 Download citation * Received: 14 October 2018 * Accepted: 22 May 2019 * Published: 08 July 2019 *

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