Experimental test and refutation of a classic case of molecular adaptation in Drosophila melanogaster


Identifying the genetic basis for adaptive differences between species requires explicit tests of historical hypotheses concerning the effects of past changes in gene sequence on molecular function, organismal phenotype and fitness. We address this challenge by combining ancestral protein reconstruction with biochemical experiments and physiological analysis of transgenic animals that carry ancestral genes. We tested a widely held hypothesis of molecular adaptation—that changes in the alcohol dehydrogenase protein (ADH) along the lineage leading to Drosophilamelanogaster increased the catalytic activity of the enzyme and thereby contributed to the ethanol tolerance and adaptation of the species to its ethanol-rich ecological niche. Our experiments strongly refute the predictions of the adaptive ADH hypothesis and caution against accepting intuitively appealing accounts of historical molecular adaptation that are based on correlative evidence. The experimental strategy we employed can be used to decisively test other adaptive hypotheses and the claims they entail about past biological causality.

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Figure 1: Predictions of the classic hypothesis of ADH adaptive evolution.
Figure 2: Effects of ADH sequence divergence on the activity of purified enzymes.
Figure 3: Effects of ADH sequence divergence on ethanol catabolism and fitness in transgenic flies.
Figure 4: Sequence evolution on the phylogeny of D. melanogaster and closely related species.


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We thank L. Picton, K. O’Brien, K. Gordon and members of the C. Meiklejohn and K. Montooth laboratories for technical assistance. We thank D. Matute for providing polymorphism data for D.yakuba. We thank M. Kreitman, members of the J. Thornton laboratory and D. Anderson for comments and suggestions that enriched the project. The project was supported by a National Science Foundation (NSF) grant (DEB-1501877; J.W.T./M.A.S.), an NSF graduate research fellowship (M.A.S.), National Institutes of Health (NIH) grant (R01-GM104397; J.W.T.), NSF CAREER Award (1505247; K.L.M.) and an NIH training grant (T32-GM007197; M.A.S.). D.W.L. was supported by a Howard Hughes Medical Institute postdoctoral fellowship from the Life Sciences Research Foundation and an investigatorship to S. B. Carroll from the Howard Hughes Medical Institute.

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M.A.S. and J.W.T. conceived the project. All authors participated in the experimental design. M.A.S. performed the phylogenetic and population genetic analyses. D.W.L. constructed the transgenic animals. M.A.S., D.W.L. and K.L.M. performed the functional experiments. All authors participated in data analysis and interpretation. M.A.S. and J.W.T. wrote the paper with contributions from D.W.L. and K.L.M.

Corresponding author

Correspondence to Joseph W. Thornton.

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Siddiq, M., Loehlin, D., Montooth, K. et al. Experimental test and refutation of a classic case of molecular adaptation in Drosophila melanogaster. Nat Ecol Evol 1, 0025 (2017). https://doi.org/10.1038/s41559-016-0025

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