The genomic basis of adaptive evolution in threespine sticklebacks

Journal name:
Nature
Volume:
484,
Pages:
55–61
Date published:
DOI:
doi:10.1038/nature10944
Received
Accepted
Published online

Abstract

Marine stickleback fish have colonized and adapted to thousands of streams and lakes formed since the last ice age, providing an exceptional opportunity to characterize genomic mechanisms underlying repeated ecological adaptation in nature. Here we develop a high-quality reference genome assembly for threespine sticklebacks. By sequencing the genomes of twenty additional individuals from a global set of marine and freshwater populations, we identify a genome-wide set of loci that are consistently associated with marine–freshwater divergence. Our results indicate that reuse of globally shared standing genetic variation, including chromosomal inversions, has an important role in repeated evolution of distinct marine and freshwater sticklebacks, and in the maintenance of divergent ecotypes during early stages of reproductive isolation. Both coding and regulatory changes occur in the set of loci underlying marine–freshwater evolution, but regulatory changes appear to predominate in this well known example of repeated adaptive evolution in nature.

At a glance

Figures

  1. Genome scans for parallel marine-freshwater divergence.
    Figure 1: Genome scans for parallel marine–freshwater divergence.

    a, Marine (red) and freshwater (blue) stickleback populations were surveyed from diverse locations. b, Morphometric analysis was used to select individuals for re-sequencing. The 20 chosen individuals are from multiple geographically proximate pairs of populations with typical marine and freshwater morphology (solid symbols). Points, population mean morphologies; ellipses, 95% confidence intervals for ecotypes. c, Genomes were analysed using SOM/HMM (top) and CSS (bottom) methods to identify parallel marine–freshwater divergent regions. Across most of the genome, the dominant patterns reflect neutral divergence or geographic structure. In contrast, <0.5% of the genome shows clustering by ecotype, a pattern characteristic of divergent marine and freshwater adaptation via parallel reuse of standing genetic variation11, 12. Mds1 and mds2 represent the first and second major axes of variation extracted from pairwise genetic distance matrices using multidimensional scaling.

  2. Parallel divergence signals at known armour plate locus.
    Figure 2: Parallel divergence signals at known armour plate locus.

    a, Ensembl gene models around EDA. b, Visual genotypes for sequenced fish (homozygous sites for most frequent allele in marine fish (red); homozygous for alternative allele (blue); heterozygous (yellow), or non-variable/missing/repeat-masked data (white)). c, DDC cluster assignments for marine (red) and freshwater populations (blue). Most fish are assigned to cluster k1, except in the boxed region, where freshwater fish are assigned to a distinct cluster (k2). d, SOM/HMM analysis supports patterns of divergence with a marine–freshwater-like tree topology in the centre, but not edges, of the window (trees ad). e, f, Similar support is shown by CSS analysis (e) and its associated P-value (f). The combined analyses define a consensus 16-kb region shared in freshwater fish (vertical shaded box), matching the minimal haplotype known to control repeated low armour evolution in sticklebacks11.

  3. Genome-wide distribution of marine-freshwater divergence regions.
    Figure 3: Genome-wide distribution of marine–freshwater divergence regions.

    Whole-genome profiles of SOM/HMM and CSS analyses reveal many loci distributed on multiple chromosomes (plus unlinked scaffolds, here grouped as ‘ChrUn’). Extended regions of marine–freshwater divergence on chromosomes I, XI and XXI correspond to inversions (red arrows). Marine–freshwater divergent regions detected by CSS are shown as grey peaks with grey points above chromosomes indicating regions of significant marine–freshwater divergence (FDR<0.05). Genomic regions with marine–freshwater-like tree topologies detected by SOM/HMM are shown as green points below chromosomes.

  4. How much of local marine-freshwater adaptation occurs by reuse of global variants?
    Figure 4: How much of local marine–freshwater adaptation occurs by reuse of global variants?

    a, Classic marine and freshwater ecotypes are maintained in downstream and upstream locations of the River Tyne, Scotland, despite extensive hybridization at intermediate sites16. b, Pairwise sequence comparisons identify many genomic regions that show high divergence between upstream and downstream fish (x axis). Many, but not all, of these regions also show high global marine–freshwater divergence (y axis; red points indicate significant CSS FDR<0.05), indicating that both global and local variants contribute to formation and reproductive isolation of a marine–freshwater species pair.

  5. Chromosome inversions and marine-freshwater divergence.
    Figure 5: Chromosome inversions and marine–freshwater divergence.

    a, Multiple marine BAC clones have paired-end reads that place anomalously against the freshwater reference genome (grey arrows below chromosome bars; see Supplementary Methods for BAC names). b, Intrachromosomal inversions on chromosomes I, XI and XXI resolve orientation and size anomalies for all marine clones. c, The chromosome XI inversion breakpoints map inside the exons of KCNH4, a potassium transporter gene. Duplicated 3′ exons lead to different transcript orientations and gene products in marine (red gene model) and freshwater fish (blue gene model). d, The chromosome XXI inversion occurs in a region with separate QTLs controlling armour plate number and body shape11, 30, traits that differ between marine and freshwater fish.

  6. Contributions of coding and regulatory changes to parallel marine-freshwater stickleback adaptation.
    Figure 6: Contributions of coding and regulatory changes to parallel marine–freshwater stickleback adaptation.

    a, A genome-wide set of marine–freshwater divergent loci recovered by both SOM/HMM and CSS analyses includes regions with consistent amino acid substitutions between marine and freshwater ecotypes (yellow sector); regions with no predicted coding sequence (green sector); and regions with both coding and non-coding sequences, but no consistent marine–freshwater amino acid substitutions (grey). b, Genome-wide expression analysis shows that marine–freshwater regions identified by SOM/HMM or CSS analyses are enriched for genes showing significant expression differences in 6 out of 7 tissues between marine LITC and freshwater FTC fish (observed, grey bars; expected, white bars; *P<0.01, **P<0.001, ***P<0.0001, ****Pless double0.00001), consistent with a role for regulatory changes in marine–freshwater evolution.

Accession codes

Primary accessions

Gene Expression Omnibus

References

  1. Stern, D. L. Perspective: Evolutionary developmental biology and the problem of variation. Evolution 54, 10791091 (2000)
  2. Carroll, S. B. Evo-devo and an expanding evolutionary synthesis: a genetic theory of morphological evolution. Cell 134, 2536 (2008)
  3. Wray, G. The evolutionary significance of cis-regulatory mutations. Nature Rev. Genet. 8, 206216 (2007)
  4. Hoekstra, H. E. & Coyne, J. A. The locus of evolution: evo devo and the genetics of adaptation. Evolution 61, 9951016 (2007)
  5. Stern, D. L. & Orgogozo, V. The loci of evolution: how predictable is genetic evolution? Evolution 62, 21552177 (2008)
  6. McKinnon, J. S. & Rundle, H. D. Speciation in nature: the threespine stickleback model systems. Trends Ecol. Evol. 17, 480488 (2002)
  7. Bell, M. A. & Foster, S. A. The Evolutionary Biology of the Threespine Stickleback (Oxford Univ. Press, 1994)
  8. Endler, J. A. Natural selection in the wild. Monogr. Popul. Biol. 21, 1336 (1986)
  9. Kingsley, D. M. & Peichel, C. L. The molecular genetics of evolutionary change in sticklebacks. In Biology of the Threespine Stickleback 41–81 (CRC Press, 2007)
  10. Chan, Y. F. et al. Adaptive evolution of pelvic reduction in sticklebacks by recurrent deletion of a Pitx1 enhancer. Science 327, 302305 (2010)
  11. Colosimo, P. F. et al. Widespread parallel evolution in sticklebacks by repeated fixation of Ectodysplasin alleles. Science 307, 19281933 (2005)
  12. Miller, C. T. et al. cis-Regulatory changes in Kit ligand expression and parallel evolution of pigmentation in sticklebacks and humans. Cell 131, 11791189 (2007)
  13. Hohenlohe, P. A. et al. Population genomics of parallel adaptation in threespine stickleback using sequenced RAD tags. PLoS Genet. 6, e1000862 (2010)
  14. Kitano, J. et al. Adaptive divergence in the thyroid hormone signaling pathway in the stickleback radiation. Curr. Biol. 20, 21242130 (2010)
  15. Hagen, D. Isolating mechanisms in threespine sticklebacks (Gasterosteus). J. Fish. Res. Board Can. 24, 16371692 (1967)
  16. Jones, F., Brown, C., Pemberton, J. & Braithwaite, V. Reproductive isolation in a threespine stickleback hybrid zone. J. Evol. Biol. 19, 15311544 (2006)
  17. Barton, N. H. & Gale, K. S. Genetic analysis of hybrid zones. In Hybrid Zones and the Evolutionary Process 13–45 (Oxford Univ. Press, 1993)
  18. Vinogradov, A. E. Genome size and GC-percent in vertebrates as determined by flow cytometry: the triangular relationship. Cytometry 31, 100109 (1998)
  19. Aparicio, S. et al. Whole-genome shotgun assembly and analysis of the genome of Fugu rubripes. Science 297, 13011310 (2002)
  20. Jaillon, O. et al. Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype. Nature 431, 946957 (2004)
  21. Kasahara, M. et al. The medaka draft genome and insights into vertebrate genome evolution. Nature 447, 714719 (2007)
  22. Star, B. et al. The genome sequence of Atlantic cod reveals a unique immune system. Nature 477, 207210 (2011)
  23. Kawahara, R. & Nishida, M. Extensive lineage-specific gene duplication and evolution of the spiggin multi-gene family in stickleback. BMC Evol. Biol. 7, 209 (2007)
  24. Siepel, A. et al. Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes. Genome Res. 15, 10341050 (2005)
  25. Shapiro, M. D. et al. Genetic and developmental basis of evolutionary pelvic reduction in threespine sticklebacks. Nature 428, 717723 (2004)
  26. Peichel, C. L. et al. The genetic architecture of divergence between threespine stickleback species. Nature 414, 901905 (2001)
  27. Colosimo, P. F. et al. The genetic architecture of parallel armor plate reduction in threespine sticklebacks. PLoS Biol. 2, 635641 (2004)
  28. Cresko, W. A. et al. Parallel genetic basis for repeated evolution of armor loss in Alaskan threespine stickleback populations. Proc. Natl Acad. Sci. USA 101, 60506055 (2004)
  29. Kimmel, C. B. et al. Evolution and development of facial bone morphology in threespine sticklebacks. Proc. Natl Acad. Sci. USA 102, 57915796 (2005)
  30. Albert, A. Y. K. et al. The genetics of adaptive shape shift in stickleback: pleiotropy and effect size. Evolution 62, 7685 (2008)
  31. Barrett, R. D. H., Rogers, S. M. & Schluter, D. Natural selection on a major armor gene in threespine stickleback. Science 322, 255257 (2008)
  32. Allali-Hassani, A. et al. Structural and chemical profiling of the human cytosolic sulfotransferases. PLoS Biol. 5, e97 (2007)
  33. Knecht, A. K., Hosemann, K. E. & Kingsley, D. M. Constraints on utilization of the EDA-signaling pathway in threespine stickleback evolution. Evol. Dev. 9, 141154 (2007)
  34. Yu, J. et al. A Wnt7b-dependent pathway regulates the orientation of epithelial cell division and establishes the cortico-medullary axis of the mammalian kidney. Development 136, 161171 (2009)
  35. Marshall, W. S. & Grosell, M. Ion transport, osmoregulation and acid-base balance. In The Physiology of Fishes 177–230 (CRC Press, 2006)
  36. Kingsley, D. M. et al. New genomic tools for molecular studies of evolutionary change in threespine sticklebacks. Behaviour 141, 13311344 (2004)
  37. Miyake, A., Mochizuki, S., Yokoi, H., Kohda, M. & Furuichi, K. New ether-à-go-go K+ channel family members localized in human telencephalon. J. Biol. Chem. 274, 2501825025 (1999)
  38. Miyake, A. et al. Disruption of the ether-à-go-go K+ channel gene BEC1/KCNH3 enhances cognitive function. J. Neurosci. 29, 1463714645 (2009)
  39. Gutman, G. A. et al. International Union of Pharmacology. LIII. Nomenclature and molecular relationships of voltage-gated potassium channels. Pharmacol. Rev. 57, 473508 (2005)
  40. Kirkpatrick, M. & Barton, N. Chromosome inversions, local adaptation and speciation. Genetics 173, 419434 (2006)
  41. Hoffmann, A. A. & Rieseberg, L. H. Revisiting the impact of inversions in evolution: from population genetic markers to drivers of adaptive shifts and speciation? Annu. Rev. Ecol. Evol. Syst. 39, 2142 (2008)
  42. Lowry, D. B. & Willis, J. H. A widespread chromosomal inversion polymorphism contributes to a major life-history transition, local adaptation, and reproductive isolation. PLoS Biol. 8, e1000500 (2010)
  43. Joron, M. et al. Chromosomal rearrangements maintain a polymorphic supergene controlling butterfly mimicry. Nature 477, 203206 (2011)
  44. Feder, J. L., Roethele, J. B., Filchak, K., Niedbalski, J. & Romero-Severson, J. Evidence for inversion polymorphism related to sympatric host race formation in the apple maggot fly, Rhagoletis pomonella. Genetics 163, 939953 (2003)
  45. Barrick, J. E. et al. Genome evolution and adaptation in a long-term experiment with Escherichia coli. Nature 461, 12431247 (2009)
  46. Kvitek, D. J. & Sherlock, G. Reciprocal sign epistasis between frequently experimentally evolved adaptive mutations causes a rugged fitness landscape. PLoS Genet. 7, e1002056 (2011)
  47. Wittkopp, P. & Haerum, B. K. Regulatory changes underlying expression differences within and between Drosophila species. Nature Genet. 40, 346350 (2008)
  48. Reimchen, T. E., Stinson, E. M. & Nelson, J. S. Multivariate differentiation of parapatric and allopatric populations of threespine stickleback in the Sangan River watershed, Queen Charlotte Islands. Can. J. Zool. 63, 29442951 (1985)
  49. Deagle, B. E. et al. Population genomics of parallel phenotypic evolution in stickleback across stream–lake ecological transitions. Proc. R. Soc.. B 279, 12771286 (2011)
  50. McPhail, J. D. Speciation and the evolution of reproductive isolation in the sticklebacks (Gasterosteus) of south-western British Columbia. In The Evolutionary Biology of the Threespine Stickleback 399–437 (Oxford Univ. Pres, 1994)

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Author information

  1. These authors contributed equally to this work.

    • Felicity C. Jones,
    • Manfred G. Grabherr,
    • Yingguang Frank Chan &
    • Pamela Russell

Affiliations

  1. Department of Developmental Biology, Beckman Center B300, Stanford University School of Medicine, Stanford California 94305, USA

    • Felicity C. Jones,
    • Yingguang Frank Chan,
    • Craig T. Miller,
    • Brian R. Summers,
    • Anne K. Knecht,
    • Shannon D. Brady,
    • Haili Zhang,
    • Alex A. Pollen,
    • Timothy Howes &
    • David M. Kingsley
  2. Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge Massachusetts 02142, USA

    • Manfred G. Grabherr,
    • Pamela Russell,
    • Evan Mauceli,
    • Jeremy Johnson,
    • Ross Swofford,
    • Mono Pirun,
    • Michael C. Zody,
    • Eric S. Lander,
    • Federica Di Palma &
    • Kerstin Lindblad-Toh
  3. Science for Life Laboratory Uppsala, Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala 751 23, Sweden

    • Manfred G. Grabherr &
    • Kerstin Lindblad-Toh
  4. Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK

    • Simon White &
    • Stephen Searle
  5. European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK

    • Ewan Birney
  6. HudsonAlpha Institute for Biotechnology, 601 Genome Way, Huntsville, Alabama 35806, USA

    • Jeremy Schmutz,
    • Jane Grimwood,
    • Mark C. Dickson &
    • Richard M. Myers
  7. Department of Molecular Genetics, Benaroya Research Institute at Virginia Mason, 1201 Ninth Avenue, Seattle Washington 98101, USA

    • Chris Amemiya
  8. Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA

    • David M. Kingsley
  9. Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, Massachusetts 02142, USA.

    • Jen Baldwin,
    • Toby Bloom,
    • David B. Jaffe,
    • Robert Nicol &
    • Jane Wilkinson
  10. Present addresses: Max Planck Institute for Evolutionary Biology, August-Thienemann-Str. 2, Plön 24306, Germany (Y.F.C.); Children's Hospital Boston, Genetic Diagnostic Lab, 300 Longwood Avenue, Boston, Massachusetts 02115, USA (E.M.); Bioinformatics Core, Zuckerman Research Center, New York, New York 10065, USA (M.P.); Department of Molecular & Cell Biology, 142 LSA 3200, University of California, Berkeley, California 94720, USA (C.T.M.).

    • Yingguang Frank Chan,
    • Evan Mauceli,
    • Mono Pirun &
    • Craig T. Miller

Consortia

  1. Broad Institute Genome Sequencing Platform & Whole Genome Assembly Team

    • Jen Baldwin,
    • Toby Bloom,
    • David B. Jaffe,
    • Robert Nicol &
    • Jane Wilkinson

Contributions

K.L.-T., F.D.P., E.S.L. and D.M.K. planned and oversaw the project and K.L.-T. and D.M.K. are co-senior authors. E.M. and M.G.G. assembled, J.S., J.G., M.C.D., A.K.K. and R.M.M. anchored, and S.W., E.B. and S.S. annotated the reference genome. C.A. constructed the BEPA BAC library. F.C.J., Y.F.C., D.M.K., K.L.-T., F.D. and M.G.G. designed the whole-genome re-sequencing experiment. F.C.J. and Y.F.C. performed morphometric analyses. S.D.B. and J.J. prepared and coordinated samples. M.G.G., M.P. and M.C.Z. analysed pilot data and performed simulations to evaluate sequencing strategies. M.G.G., P.R., E.M., F.C.J., Y.F.C., J.J. and R.S. analysed polymorphisms. P.R. and M.G.G. developed and carried out the SOM/HMM analysis. F.C.J. and Y.F.C. developed and carried out the CSS and DDC analysis. P.R. analysed gene and non-coding element density, and performed phylogenetic analysis. T.H. analysed GO term enrichments. F.C.J. and Y.F.C. carried out hybrid zone analysis. C.T.M., B.R.S., J.G., J.S., Y.F.C. and F.C.J. analysed chromosome inversions. F.C.J. and D.M.K. performed analysis of coding and regulatory changes. H.Z., A.A.P. and T.H. performed the whole-genome expression study. D.M.K., F.C.J., Y.F.C., P.R., F.D.P. and K.L.-T. wrote the paper with input from other authors.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to:

UCSC Genome browser tracks showing genome-wide analyses are available at http://sticklebrowser.stanford.edu. Microarray expression data are deposited at the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) under accession number GSE34783. BAC-end sequences are deposited at http://www.ncbi.nlm.nih.gov/dbGSS (accession numbers JS583469 to JS583576).

Author details

Supplementary information

PDF files

  1. Supplementary Information (8.1M)

    This file contains Supplementary Methods and Data, Supplementary Figures 1-10 and Supplementary Tables 1-10. The Supplementary Figures and Tables contain additional details on the genome assembly; gene annotation, sampled populations; morphometric analysis; number and details regarding the SOM/HMM and CSS methods; additional example loci; genome-wide summary of re-sequencing data; specific features of marine–freshwater divergent regions; and chromosome inversions.

Excel files

  1. Supplementary Data (59K)

    This file contains data supporting the conclusions in Figure 6, specifically genes located in the 81 regions jointly identified by both the SOM/HMM and the CSS methods. Annotations on protein coding changes and gene expression differences are also included.

Additional data