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Research Article

The king cobra genome reveals dynamic gene evolution and adaptation in the snake venom system

Freek J. Vonk, Nicholas R. Casewell, Christiaan V. Henkel, Alysha M. Heimberg, Hans J. Jansen, Ryan J. R. McCleary, Harald M. E. Kerkkamp, Rutger A. Vos, Isabel Guerreiro, Juan J. Calvete, Wolfgang Wüster, Anthony E. Woods, Jessica M. Logan, Robert A. Harrison, Todd A. Castoe, A. P. Jason de Koning, David D. Pollock, Mark Yandell, Diego Calderon, Camila Renjifo, Rachel B. Currier, David Salgado, Davinia Pla, Libia Sanz, Asad S. Hyder, José M. C. Ribeiro, Jan W. Arntzen, Guido E. E. J. M. van den Thillart, Marten Boetzer, Walter Pirovano, Ron P. Dirks, Herman P. Spaink, Denis Duboule, Edwina McGlinn, R. Manjunatha Kini, and Michael K. Richardson
  1. aNaturalis Biodiversity Center, 2333 CR, Leiden, The Netherlands;
  2. bInstitute of Biology Leiden, Leiden University, Sylvius Laboratory, Sylviusweg 72, 2300 RA, Leiden, The Netherlands;
  3. cMolecular Ecology and Evolution Group, School of Biological Sciences, Bangor University, Bangor LL57 2UW, United Kingdom;
  4. dAlistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Liverpool L3 5QA, United Kingdom;
  5. eZF-Screens B.V., Bio Partner Center, 2333 CH, Leiden, The Netherlands;
  6. fEuropean Molecular Biology Laboratory Australia, Australian Regenerative Medicine Institute, Monash University, Clayton, 3800, Australia;
  7. gDepartment of Biological Sciences, National University of Singapore, Singapore 117543;
  8. hDepartment of Genetics and Evolution, University of Geneva, CH-1211 Geneva 4, Switzerland;
  9. iInstituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Cientificas (Spain), 11 46010 Valencia, Spain;
  10. jSchool of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA 5000, Australia;
  11. kDepartment of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, CO 80045;
  12. lDepartment of Biology, University of Texas, Arlington, TX 76010;
  13. mDepartment of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary and Alberta Children’s Hospital Research Institute for Child and Maternal Health, Calgary, AB, Canada T2N 4N1;
  14. nDepartment of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT;
  15. oAix-Marseille Université, Inserm, GMGF UMR_S910, 13385 Marseille, France;
  16. pLaboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20892; and
  17. qBaseClear B.V., 2333 CC, Leiden, The Netherlands

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PNAS first published December 2, 2013; https://doi.org/10.1073/pnas.1314702110
Freek J. Vonk
aNaturalis Biodiversity Center, 2333 CR, Leiden, The Netherlands;
bInstitute of Biology Leiden, Leiden University, Sylvius Laboratory, Sylviusweg 72, 2300 RA, Leiden, The Netherlands;
cMolecular Ecology and Evolution Group, School of Biological Sciences, Bangor University, Bangor LL57 2UW, United Kingdom;
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Nicholas R. Casewell
cMolecular Ecology and Evolution Group, School of Biological Sciences, Bangor University, Bangor LL57 2UW, United Kingdom;
dAlistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Liverpool L3 5QA, United Kingdom;
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Christiaan V. Henkel
bInstitute of Biology Leiden, Leiden University, Sylvius Laboratory, Sylviusweg 72, 2300 RA, Leiden, The Netherlands;
eZF-Screens B.V., Bio Partner Center, 2333 CH, Leiden, The Netherlands;
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Alysha M. Heimberg
fEuropean Molecular Biology Laboratory Australia, Australian Regenerative Medicine Institute, Monash University, Clayton, 3800, Australia;
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Hans J. Jansen
eZF-Screens B.V., Bio Partner Center, 2333 CH, Leiden, The Netherlands;
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Ryan J. R. McCleary
gDepartment of Biological Sciences, National University of Singapore, Singapore 117543;
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Harald M. E. Kerkkamp
bInstitute of Biology Leiden, Leiden University, Sylvius Laboratory, Sylviusweg 72, 2300 RA, Leiden, The Netherlands;
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Rutger A. Vos
aNaturalis Biodiversity Center, 2333 CR, Leiden, The Netherlands;
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Isabel Guerreiro
hDepartment of Genetics and Evolution, University of Geneva, CH-1211 Geneva 4, Switzerland;
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Juan J. Calvete
iInstituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Cientificas (Spain), 11 46010 Valencia, Spain;
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Wolfgang Wüster
cMolecular Ecology and Evolution Group, School of Biological Sciences, Bangor University, Bangor LL57 2UW, United Kingdom;
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Anthony E. Woods
jSchool of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA 5000, Australia;
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Jessica M. Logan
jSchool of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA 5000, Australia;
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Robert A. Harrison
dAlistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Liverpool L3 5QA, United Kingdom;
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Todd A. Castoe
kDepartment of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, CO 80045;
lDepartment of Biology, University of Texas, Arlington, TX 76010;
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A. P. Jason de Koning
kDepartment of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, CO 80045;
mDepartment of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary and Alberta Children’s Hospital Research Institute for Child and Maternal Health, Calgary, AB, Canada T2N 4N1;
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David D. Pollock
kDepartment of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, CO 80045;
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Mark Yandell
nDepartment of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT;
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Diego Calderon
nDepartment of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT;
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Camila Renjifo
dAlistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Liverpool L3 5QA, United Kingdom;
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Rachel B. Currier
dAlistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Liverpool L3 5QA, United Kingdom;
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David Salgado
fEuropean Molecular Biology Laboratory Australia, Australian Regenerative Medicine Institute, Monash University, Clayton, 3800, Australia;
oAix-Marseille Université, Inserm, GMGF UMR_S910, 13385 Marseille, France;
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Davinia Pla
iInstituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Cientificas (Spain), 11 46010 Valencia, Spain;
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Libia Sanz
iInstituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Cientificas (Spain), 11 46010 Valencia, Spain;
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Asad S. Hyder
bInstitute of Biology Leiden, Leiden University, Sylvius Laboratory, Sylviusweg 72, 2300 RA, Leiden, The Netherlands;
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José M. C. Ribeiro
pLaboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20892; and
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Jan W. Arntzen
aNaturalis Biodiversity Center, 2333 CR, Leiden, The Netherlands;
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Guido E. E. J. M. van den Thillart
eZF-Screens B.V., Bio Partner Center, 2333 CH, Leiden, The Netherlands;
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Marten Boetzer
qBaseClear B.V., 2333 CC, Leiden, The Netherlands
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Walter Pirovano
qBaseClear B.V., 2333 CC, Leiden, The Netherlands
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Ron P. Dirks
eZF-Screens B.V., Bio Partner Center, 2333 CH, Leiden, The Netherlands;
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Herman P. Spaink
bInstitute of Biology Leiden, Leiden University, Sylvius Laboratory, Sylviusweg 72, 2300 RA, Leiden, The Netherlands;
eZF-Screens B.V., Bio Partner Center, 2333 CH, Leiden, The Netherlands;
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Denis Duboule
hDepartment of Genetics and Evolution, University of Geneva, CH-1211 Geneva 4, Switzerland;
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Edwina McGlinn
fEuropean Molecular Biology Laboratory Australia, Australian Regenerative Medicine Institute, Monash University, Clayton, 3800, Australia;
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R. Manjunatha Kini
gDepartment of Biological Sciences, National University of Singapore, Singapore 117543;
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Michael K. Richardson
bInstitute of Biology Leiden, Leiden University, Sylvius Laboratory, Sylviusweg 72, 2300 RA, Leiden, The Netherlands;
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  • For correspondence: M.K.Richardson@biology.leidenuniv.nl
  1. Edited by David B. Wake, University of California, Berkeley, CA, and approved October 22, 2013 (received for review August 2, 2013)

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Significance

Snake venoms are toxic protein cocktails used for prey capture. To investigate the evolution of these complex biological weapon systems, we sequenced the genome of a venomous snake, the king cobra, and assessed the composition of venom gland expressed genes, small RNAs, and secreted venom proteins. We show that regulatory components of the venom secretory system may have evolved from a pancreatic origin and that venom toxin genes were co-opted by distinct genomic mechanisms. After co-option, toxin genes important for prey capture have massively expanded by gene duplication and evolved under positive selection, resulting in protein neofunctionalization. This diverse and dramatic venom-related genomic response seemingly occurs in response to a coevolutionary arms race between venomous snakes and their prey.

Abstract

Snakes are limbless predators, and many species use venom to help overpower relatively large, agile prey. Snake venoms are complex protein mixtures encoded by several multilocus gene families that function synergistically to cause incapacitation. To examine venom evolution, we sequenced and interrogated the genome of a venomous snake, the king cobra (Ophiophagus hannah), and compared it, together with our unique transcriptome, microRNA, and proteome datasets from this species, with data from other vertebrates. In contrast to the platypus, the only other venomous vertebrate with a sequenced genome, we find that snake toxin genes evolve through several distinct co-option mechanisms and exhibit surprisingly variable levels of gene duplication and directional selection that correlate with their functional importance in prey capture. The enigmatic accessory venom gland shows a very different pattern of toxin gene expression from the main venom gland and seems to have recruited toxin-like lectin genes repeatedly for new nontoxic functions. In addition, tissue-specific microRNA analyses suggested the co-option of core genetic regulatory components of the venom secretory system from a pancreatic origin. Although the king cobra is limbless, we recovered coding sequences for all Hox genes involved in amniote limb development, with the exception of Hoxd12. Our results provide a unique view of the origin and evolution of snake venom and reveal multiple genome-level adaptive responses to natural selection in this complex biological weapon system. More generally, they provide insight into mechanisms of protein evolution under strong selection.

  • genomics
  • phylogenetics
  • serpentes

Footnotes

  • ↵1F.J.V. and N.R.C. contributed equally to this work.

  • ↵2To whom correspondence should be addressed. E-mail: M.K.Richardson{at}biology.leidenuniv.nl.
  • Author contributions: F.J.V., N.R.C., and M.K.R. designed research; F.J.V. acquired samples for sequencing and estimated genome size; H.J.J., and R.P.D. prepared sequencing libraries; M.B., and W.P. developed assembly software; C.V.H. assembled the genome; H.J.J., M.Y., D.C., and H.P.S. annotated the genome; J.M.C.R. assembled and annotated RNA-seq libraries; F.J.V., N.R.C., H.M.E.K., and A.S.H. analyzed RNA-seq libraries; A.M.H., D.S., and E.M. annotated and analyzed small RNA libraries; H.M.E.K., I.G., H.P.S., and D.D. annotated and analyzed Hox genes; F.J.V., N.R.C., C.V.H., R.J.R.M., H.M.E.K., A.S.H., R.P.D., R.M.K., and M.K.R. annotated venom toxin genes and performed synteny analyses; N.R.C., R.A.V., and W.W. analyzed gene family evolution; A.M.H. performed miRNA in situ hybridization; A.E.W., and J.M.L. performed lectin in situ hybridization; N.R.C., R.J.R.M., J.J.C., R.A.H., C.R., R.B.C., D.P., L.S., and R.M.K. analyzed the venom proteome; T.A.C., A.P.J.d.K., and D.D.P. contributed Burmese python genome data and assisted with comparative analyses; H.J.J., J.W.A., G.E.E.J.M.v.d.T., R.P.D., H.P.S., and M.K.R. organized sequencing platforms and facilities; F.J.V., N.R.C., W.W., and M.K.R. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • Data deposition: The king cobra genome assembly and reads reported in this paper have been deposited in the GenBank database (bioproject no. PRJNA201683). The transcriptome sequences reported in this paper have been deposited in the GenBank Short Read Archive database (bioproject no. PRJNA222479). The microRNA sequences reported in this paper have been deposited in miRBase, www.mirbase.org.

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1314702110/-/DCSupplemental.

Freely available online through the PNAS open access option.

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King cobra genome and snake venom evolution
Freek J. Vonk, Nicholas R. Casewell, Christiaan V. Henkel, Alysha M. Heimberg, Hans J. Jansen, Ryan J. R. McCleary, Harald M. E. Kerkkamp, Rutger A. Vos, Isabel Guerreiro, Juan J. Calvete, Wolfgang Wüster, Anthony E. Woods, Jessica M. Logan, Robert A. Harrison, Todd A. Castoe, A. P. Jason de Koning, David D. Pollock, Mark Yandell, Diego Calderon, Camila Renjifo, Rachel B. Currier, David Salgado, Davinia Pla, Libia Sanz, Asad S. Hyder, José M. C. Ribeiro, Jan W. Arntzen, Guido E. E. J. M. van den Thillart, Marten Boetzer, Walter Pirovano, Ron P. Dirks, Herman P. Spaink, Denis Duboule, Edwina McGlinn, R. Manjunatha Kini, Michael K. Richardson
Proceedings of the National Academy of Sciences Dec 2013, 201314702; DOI: 10.1073/pnas.1314702110

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King cobra genome and snake venom evolution
Freek J. Vonk, Nicholas R. Casewell, Christiaan V. Henkel, Alysha M. Heimberg, Hans J. Jansen, Ryan J. R. McCleary, Harald M. E. Kerkkamp, Rutger A. Vos, Isabel Guerreiro, Juan J. Calvete, Wolfgang Wüster, Anthony E. Woods, Jessica M. Logan, Robert A. Harrison, Todd A. Castoe, A. P. Jason de Koning, David D. Pollock, Mark Yandell, Diego Calderon, Camila Renjifo, Rachel B. Currier, David Salgado, Davinia Pla, Libia Sanz, Asad S. Hyder, José M. C. Ribeiro, Jan W. Arntzen, Guido E. E. J. M. van den Thillart, Marten Boetzer, Walter Pirovano, Ron P. Dirks, Herman P. Spaink, Denis Duboule, Edwina McGlinn, R. Manjunatha Kini, Michael K. Richardson
Proceedings of the National Academy of Sciences Dec 2013, 201314702; DOI: 10.1073/pnas.1314702110
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