Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Epistasis as the primary factor in molecular evolution


The main forces directing long-term molecular evolution remain obscure. A sizable fraction of amino-acid substitutions seem to be fixed by positive selection1,2,3,4, but it is unclear to what degree long-term protein evolution is constrained by epistasis, that is, instances when substitutions that are accepted in one genotype are deleterious in another. Here we obtain a quantitative estimate of the prevalence of epistasis in long-term protein evolution by relating data on amino-acid usage in 14 organelle proteins and 2 nuclear-encoded proteins to their rates of short-term evolution. We studied multiple alignments of at least 1,000 orthologues for each of these 16 proteins from species from a diverse phylogenetic background and found that an average site contained approximately eight different amino acids. Thus, without epistasis an average site should accept two-fifths of all possible amino acids, and the average rate of amino-acid substitutions should therefore be about three-fifths lower than the rate of neutral evolution. However, we found that the measured rate of amino-acid substitution in recent evolution is 20 times lower than the rate of neutral evolution and an order of magnitude lower than that expected in the absence of epistasis. These data indicate that epistasis is pervasive throughout protein evolution: about 90 per cent of all amino-acid substitutions have a neutral or beneficial impact only in the genetic backgrounds in which they occur, and must therefore be deleterious in a different background of other species. Our findings show that most amino-acid substitutions have different fitness effects in different species and that epistasis provides the primary conceptual framework to describe the tempo and mode of long-term protein evolution.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


Prices may be subject to local taxes which are calculated during checkout


  1. McDonald, J. H. & Kreitman, M. Adaptive protein evolution at the Adh locus in Drosophila. Nature 351, 652–654 (1991)

    Article  ADS  CAS  Google Scholar 

  2. Andolfatto, P. Adaptive evolution of non-coding DNA in Drosophila. Nature 437, 1149–1152 (2005)

    Article  ADS  CAS  Google Scholar 

  3. Charlesworth, J. & Eyre-Walker, A. The rate of adaptive evolution in enteric bacteria. Mol. Biol. Evol. 23, 1348–1356 (2006)

    Article  CAS  Google Scholar 

  4. Boyko, A. R. et al. Assessing the evolutionary impact of amino acid mutations in the human genome. PLoS Genet. 4, e1000083 (2008)

    Article  Google Scholar 

  5. Kimura, M. Evolutionary rate at the molecular level. Nature 217, 624–626 (1968)

    Article  ADS  CAS  Google Scholar 

  6. Li, W. H. Molecular Evolution 419–429 (Sinauer, 1997)

    Google Scholar 

  7. Kimura, M. The role of compensatory neutral mutations in molecular evolution. J. Genet. 64, 7–19 (1985)

    Article  CAS  Google Scholar 

  8. Weinreich, D. M., Watson, R. A. & Chao, L. Sign epistasis and genetic constraint on evolutionary trajectories. Evolution 59, 1165–1174 (2005)

    CAS  PubMed  Google Scholar 

  9. Lehner, B. Molecular mechanisms of epistasis within and between genes. Trends Genet. 27, 323–331 (2011)

    Article  CAS  Google Scholar 

  10. de Visser, J. A., Cooper, T. F. & Elena, S. F. The causes of epistasis. Proc. R. Soc. Lond. B 278, 3617–3624 (2011)

    Article  Google Scholar 

  11. Kondrashov, A. S., Sunyaev, S. & Kondrashov, F. A. Dobzhansky-Muller incompatibilities in protein evolution. Proc. Natl Acad. Sci. USA 99, 14878–14883 (2002)

    Article  ADS  CAS  Google Scholar 

  12. Maynard Smith, J. Natural selection and the concept of a protein space. Nature 225, 563–564 (1970)

    Article  ADS  Google Scholar 

  13. Fitch, W. M. & Markowitz, E. An improved method for determining codon variability in a gene and its application to the rate of fixation of mutations in evolution. Biochem. Genet. 4, 579–593 (1970)

    Article  CAS  Google Scholar 

  14. Gillespie, J. H. Natural selection and the molecular clock. Mol. Biol. Evol. 3, 138–155 (1986)

    CAS  PubMed  Google Scholar 

  15. Povolotskaya, I. S. & Kondrashov, F. A. Sequence space and the ongoing expansion of the protein universe. Nature 465, 922–926 (2010)

    Article  ADS  CAS  Google Scholar 

  16. Poon, A. F. & Chao, L. Functional origins of fitness effect-sizes of compensatory mutations in the DNA bacteriophage phiX174. Evolution 60, 2032–2043 (2006)

    CAS  PubMed  Google Scholar 

  17. Bridgham, J. T., Ortlund, E. A. & Thornton, J. W. An epistatic ratchet constrains the direction of glucocorticoid receptor evolution. Nature 461, 515–519 (2009)

    Article  ADS  CAS  Google Scholar 

  18. Meer, M. V., Kondrashov, A. S., Artzy-Randrup, Y. & Kondrashov, F. A. Compensatory evolution in mitochondrial tRNAs navigates valleys of low fitness. Nature 464, 279–282 (2010)

    Article  ADS  CAS  Google Scholar 

  19. Costanzo, M. S. & Hartl, D. L. The evolutionary landscape of antifolate resistance in Plasmodium falciparum. J. Genet. 90, 187–190 (2011)

    Article  CAS  Google Scholar 

  20. Salverda, M. L. et al. Initial mutations direct alternative pathways of protein evolution. PLoS Genet. 7, e1001321 (2011)

    Article  CAS  Google Scholar 

  21. Woods, R. J. et al. Second-order selection for evolvability in a large Escherichia coli population. Science 331, 1433–1436 (2011)

    Article  ADS  CAS  Google Scholar 

  22. Osada, N. & Akashi, H. Mitochondrial-nuclear interactions and accelerated compensatory evolution: evidence from the primate cytochrome C oxidase complex. Mol. Biol. Evol. 29, 337–346 (2012)

    Article  CAS  Google Scholar 

  23. Kvitek, D. J. & Sherlock, G. Reciprocal sign epistasis between frequently experimentally evolved adaptive mutations causes a rugged fitness landscape. PLoS Genet. 7, e1002056 (2011)

    Article  CAS  Google Scholar 

  24. Silva, R. F. et al. Pervasive sign epistasis between conjugative plasmids and drug-resistance chromosomal mutations. PLoS Genet. 7, e1002181 (2011)

    Article  CAS  Google Scholar 

  25. Notredame, C., Higgins, D. G. & Heringa, J. T-Coffee: a novel method for fast and accurate multiple sequence alignment. J. Mol. Biol. 302, 205–217 (2000)

    Article  CAS  Google Scholar 

  26. Lunzer, M., Golding, G. B. & Dean, A. M. Pervasive cryptic epistasis in molecular evolution. PLoS Genet. 6, e1001162 (2010)

    Article  Google Scholar 

  27. Costanzo, M. et al. The genetic landscape of a cell. Science 327, 425–431 (2010)

    Article  ADS  CAS  Google Scholar 

  28. Tokuriki, N. & Tawfik, D. S. Chaperonin overexpression promotes genetic variation and enzyme evolution. Nature 459, 668–673 (2009)

    Article  ADS  CAS  Google Scholar 

  29. Poelwijk, F. J., de Vos, M. G. & Tans, S. J. Tradeoffs and optimality in the evolution of gene regulation. Cell 146, 462–470 (2011)

    Article  CAS  Google Scholar 

  30. Burga, A. Olivia Casanueva, M. & Lehner, B. Predicting mutation outcome from early stochastic variation in genetic interaction partners. Nature 480, 250–253 (2011)

  31. Benson, D. A., Karsch-Mizrachi, I., Lipman, D. J., Ostell, J. & Wheeler, D. L. GenBank. Nucleic Acids Res. 34, D16–D20 (2006)

    Article  CAS  Google Scholar 

  32. Tatusov, R. L., Koonin, E. V. & Lipman, D. J. A genomic perspective on protein families. Science 278, 631–637 (1997)

    Article  ADS  CAS  Google Scholar 

  33. Katoh, K., Misawa, K., Kuma, K. & Miyata, T. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 30, 3059–3066 (2002)

    Article  CAS  Google Scholar 

  34. Sievers, F. et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011)

    Article  Google Scholar 

  35. Nei, M. & Li, W. H. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl Acad. Sci. USA 76, 5269–5273 (1979)

    Article  ADS  CAS  Google Scholar 

  36. Sayers, E. W. et al. Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 40, D13–D25 (2012)

    Article  CAS  Google Scholar 

  37. Sunyaev, S. et al. Prediction of deleterious human alleles. Hum. Mol. Genet. 10, 591–597 (2001)

    Article  CAS  Google Scholar 

  38. Yang, Z. PAML 4: phylogenetic analysis by maximum likelihood. Mol. Biol. Evol. 24, 1586–1591 (2007)

    Article  CAS  Google Scholar 

Download references


The work was supported by Plan Nacional grants from the Spanish Ministry of Science and Innovation, to F.A.K. and C.N. C.K. was supported by the European Union FP7 project Quantomics (KBBE2A222664). F.A.K. is a European Molecular Biology Organization Young Investigator and Howard Hughes Medical Institute International Early Career Scientist. We thank B. Lehner and T. Warnecke for input and a critical reading of the manuscript.

Author information

Authors and Affiliations



M.S.B., C.K., P.K.V. and F.A.K. participated in obtaining and quality-testing the data; C.K. and C.N. participated in the design of the alignment algorithm; and F.A.K. designed the study and wrote the manuscript with the participation of all authors.

Corresponding author

Correspondence to Fyodor A. Kondrashov.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Tables 1-6 and Supplementary Figures 1-4. (PDF 698 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Breen, M., Kemena, C., Vlasov, P. et al. Epistasis as the primary factor in molecular evolution. Nature 490, 535–538 (2012).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing