Experimental test and refutation of a classic case of molecular adaptation in Drosophila melanogaster
Mohammad A. Siddiq, David W. Loehlin, Kristi L. Montooth & Joseph W. Thornton
Nature Ecology & Evolution 1, Article number: 0025 (2017)
Evolutionary genetics Molecular evolution
Received: 13 September 2016 Accepted: 01 November 2016 Published online:
13 January 2017
Sequence evolution on the phylogeny of D. melanogaster and closely related species.
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 Drosophila melanogaster 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.
A central goal of molecular evolutionary biology is to identify the genes and biological mechanisms that mediated historical adaptation. Rigorously testing hypotheses in this area has been a major challenge. Many studies infer past selection from statistical signatures in genes that are involved in biological processes that might have suited species to their environments 1,2,3,4 . But sequence signatures of selection can be forged by chance or demographic processes and it is difficult to predict from sequence alone how genetic changes affect phenotypes and fitness 5,6,7,8 . Compelling evidence for molecular adaptation therefore requires formulating and testing explicit hypotheses about the causal links between specific evolutionary changes in gene sequence and the resulting changes in molecular function, organismal phenotype and fitness 6,7,8,9,10 . Advances in genetic mapping, experimental studies of molecular function and transgenic engineering have allowed hypotheses of molecular adaptation between recently diverged populations to be tested with increasing rigour 11,12,13,14,15,16 . But hypotheses about adaptive divergence between species or at higher taxonomic levels are explicitly historical, so testing them requires the effect of genetic changes that occurred on phenotype and fitness in specific evolutionary lineages from the distant past to be measured. Here we address this challenge by combining ancestral protein reconstruction 17 with biochemical experiments and physiological analysis of transgenic animals that carry ancestral genes.
We applied this approach to a longstanding hypothesis of molecular adaptation—that changes in the alcohol dehydrogenase (ADH) protein of the fruit fly Drosophila melanogaster increased the catalytic activity of the enzyme and thereby contributed to the adaptation of the species to its ethanol-rich ecological niche 18,19,20 . This hypothesis was articulated decades ago 18,21 and became widely accepted 19,20,22,23 on the basis of several observations that were consistent with it, but did not directly address the putative causal links among historical changes in protein sequence, function and fitness. First, D. melanogaster evolved to colonize ethanol-rich habitats in rotting fruit after it split from its sister species, D. simulans, some two to four million years ago 24,25 . Second, fractionated cell extracts from D. melanogaster catalyse alcohol turnover more rapidly than those from D. simulans 18,26,27 . Third, the first-ever application of the McDonald–Kreitman (MK) test detected an excess of non-synonymous substitutions in an alignment of the ADH coding sequences of D. melanogaster and closely related species 28 , which was interpreted as evidence for adaptive evolution driving the divergence of the ADH protein between D. melanogaster and D. simulans 21,22,29,30 . These observations were integrated into a narrative in which adaptation to ethanol-rich habitats was driven by selection on the ADH protein sequence for increased catalytic activity. Other factors—particularly increases in the expression level 26,31,32,33 of ADH, changes at other genetic loci 34,35,36 and within-species polymorphisms 37,38,39 —also probably contributed to ethanol adaptation in D. melanogaster, but they are independent of and cannot explain the selection signature on the protein-coding sequence of the ADH enzyme found in the MK test.
We focused on the hypothesis of adaptive ADH protein evolution because it is widely accepted on the basis of correlated forms of variation in extant species and because it is particularly amenable to testing using the experimental approaches of ancestral reconstruction, biochemical characterization and engineering of transgenic organisms. The ADH adaptive hypothesis entails specific, testable predictions about how genetic changes that occurred in the ADH protein sequence during the historical divergence of D. melanogaster affect the phenotype at several levels, including molecular function (catalytic turnover of ethanol by pure ADH protein), physiology (ethanol catabolism in the tissues of D. melanogaster) and fitness components (survival in the presence of ethanol) (Fig. 1a). We tested these predictions by reconstructing the ADH protein from the last common ancestor of D. melanogaster and D. simulans (AncMS) and experimentally characterizing how changes in ADH sequence along the D. melanogaster lineage affected ADH function, physiology and fitness.
How to cite this article: Siddiq, M. A., Loehlin, D. W., Montooth, K. L. & Thornton, J. W. Experimental test and refutation of a classic case of molecular adaptation in Drosophila melanogaster. Nat. Ecol. Evol. 1, 0025 (2017).
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.
Department of Ecology and Evolution, University of Chicago, Chicago, Illinois, USA
Mohammad A. Siddiq & Joseph W. Thornton
Laboratory of Cell & Molecular Biology, University of Wisconsin-Madison, Madison, Wisconsin, USA
David W. Loehlin
Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, Wisconsin, USA
David W. Loehlin
School of Biological Sciences, University of Nebraska, Lincoln, Nebraska, USA
Kristi L. Montooth
Department of Human Genetics, University of Chicago, Chicago, Illinois, USA
Joseph W. Thornton
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.
The authors declare no competing financial interests.
Correspondence to Joseph W. Thornton.