MBE Advance Access originally published online on December 16, 2005
Molecular Biology and Evolution 2006 23(5):883-886; doi:10.1093/molbev/msj077
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005 |
Molecular Genetics of Natural Populations
Department of Ecology and Evolution, The University of Chicago
E-mail: tonyg{at}uchicago.edu.
 |
Abstract |
|---|
 TOP Abstract IntroductionMolecular Manipulation of...Integrating Molecular Biology,...AcknowledgementsReferences |
|---|
Significant progress in evolutionary genetics has been made by studying, on the one hand, patterns of DNA sequence polymorphism and, on the other, genetic architecture of complex adaptive traits. However, connections between nucleotide variants under selection and adaptively relevant phenotypes are missing. Such connections can be established using precise gene replacement. We review the recent successful introduction of this technique to the analysis of two evolutionarily interesting loci—Odysseus and desaturase2. Both genes have subtle phenotypes that nevertheless could be identified using gene replacement, demonstrating that effects of naturally occurring alleles can be measured in the laboratory. This is an important first step in connecting statistical signatures of selection with adaptation in nature. More candidate genes involved in adaptation, for example, through cloning of genes responsible for reproductive isolation, now need to be identified. Molecular genetic manipulation, DNA polymorphism analysis, and field studies then have to be integrated to provide fresh insights into the mechanisms of evolutionary change.
Key Words: gene replacement • population genetics • adaptation • speciation
|
Introduction |
|---|
|
TOPAbstractIntroductionMolecular Manipulation of...Integrating Molecular Biology,...AcknowledgementsReferences |
|---|
In the decades since the modern synthesis, our understanding of the forces that shape the evolutionary process rapidly expanded. To produce change, these forces have to act on genetic variation. Examination of the patterns of this variation in nature and its phenotypic consequences is thus central to the study of evolution. Progress in evolutionary genetics has traditionally been made through the pursuit of two major lines of inquiry. One focuses on patterns of genetic variation and the other on the genetic architecture of (potentially) adaptive phenotypic traits. The overall goal is to detect signs of the operation of selection against the background of demographic change and accidents of genetic drift.
Characterization of patterns of genetic variation in natural populations has preoccupied several generations of biologists, starting with the classical studies on inversion polymorphism (Dobzhansky and Queal 1938
), allozyme variation (Lewontin and Hubby 1966), and, ultimately, polymorphism in the DNA sequence itself (Kreitman 1983). Availability of the data on DNA sequence variation within and between species allowed the application of increasingly sophisticated statistical techniques (e.g., Tajima 1989; McDonald and Kreitman 1991; Nielsen and Yang 1998; Williamson et al. 2005; Wright et al. 2005; for a review, see Li 1997) in an attempt to assess the relative importance of drift, demography, and selection in shaping the patterns. Although population size change and negative selection clearly play a large role in shaping patterns of DNA sequence variation (Haddrill et al. 2005; Williamson et al. 2005; Wright et al. 2005), signs that positive selection plays an appreciable role can be found in at least some species (Fay, Wyckoff, and Wu 2002; Smith and Eyre-Walker 2002; Williamson et al. 2005). Nevertheless, even when the action of positive selection can be deduced from patterns of variation, it is hard to know what adaptive forces are responsible (Lewontin 1997).
Efforts to connect adaptive phenotypes with DNA sequence variation are within the purview of evolutionary quantitative genetics (Barton and Turelli 1989), although the focus has been mostly on the overall genetic architecture rather than on finding specific genes (Orr and Coyne 1992; Lynch and Walsh 1998, pp. 13–16; Barton and Keightley 2002). In several cases, phenotypic variants have been shown with some confidence to be subject to selection (Endler 1986; Schluter 2000, chapter 5; Hawthorne and Via 2001; Nachman 2005). Nucleotide changes responsible for such phenotypic traits are yet to be identified (Mackay 2001), although success in cloning genes responsible for complex traits involved in artificial selection has been occasionally achieved in plants (Remington, Ungerer, and Purugganan 2001).
We are thus in a curious situation—on the one hand, we have strong evidence that positive selection acts on DNA sequence variants, but we do not know what adaptive forces drive selection. On the other hand, we can identify heritable phenotypic variation that is subject to selection in nature, but we do not know the nucleotide differences responsible. Bridging this gap is a crucial step in understanding the genetic basis of evolutionary change.
|
Molecular Manipulation of Naturally Occurring Alleles |
|---|
|
TOPAbstractIntroductionMolecular Manipulation of...Integrating Molecular Biology,...AcknowledgementsReferences |
|---|
Traditionally, relationships between nucleotide polymorphisms and phenotypes are studied by establishing statistical associations of DNA sequence variants at candidate loci with their presumed phenotypic effects (e.g., Nachman 2005
). However, this approach, while fruitful for generating hypotheses for further testing, does not establish causality. Moreover, linkage disequilibrium between nucleotide polymorphisms often obscures the primary molecular change underlying the association. In principle, it is quite clear what can be done to establish causal connections between nucleotide changes subject to selection and adaptive phenotypes. One allele has to be substituted for another while holding the genetic background constant. The resulting phenotypes then have to be characterized, and their relevance to the environmental conditions experienced by the organisms in nature must be assessed.
The main technical difficulty is that mutations of even small phenotypic effect are likely to be subject to selection in nature. Conventional transgenic technology relies on the introduction of manipulated genes into genomes at random sites (e.g., Rubin and Spradling 1982). Due to effects of chromosome position on the expression of the transgene, the number of independent inserts required to measure subtle phenotypes quickly becomes prohibitively large (Laurie-Ahlberg and Stam 1987). Moreover, transgenes carrying different alleles of a gene cannot be competed against each other in large-scale population experiments because recombination between the inserts will remove competition. An improved method that allows integration of pairs of transgenes at the same chromosomal site can eliminate most of these problems. Such a technique was successfully used to measure fitness effects of a pathogen resistance gene in Arabidopsis thaliana (Tian et al. 2003). However, this approach does not allow manipulation of the endogenous locus. For that, we need precise allele substitution, a method long used by microbial geneticists and now available in Drosophila melanogaster (Rong and Golic 2000).
Two recent studies (Greenberg et al. 2003; Sun, Ting, and Wu 2004) successfully used the gene replacement technique to target evolutionarily important genes. Sun, Ting, and Wu (2004) generated a null allele of Odysseus (Ods), a gene responsible for male sterility in Drosophila simulans/Drosophila mauritiana hybrids (Ting et al. 1998). The deletion has a subtle effect on fertility of young males that is manifested only in the first 3 days of their life. Despite the ephemeral nature of the phenotype, it was readily detected using gene replacement, a testament to the power of this technique.
Sun, Ting, and Wu (2004) aimed to assign a within-species function to a gene causing hybrid incompatibility. They thus created a null allele of Ods in D. melanogaster rather than study the effects of naturally occurring variants at the locus. In contrast, Greenberg et al. (2003), who focused on the desaturase2 (ds2) gene of D. melanogaster, attempted to measure phenotypes caused by alleles segregating in wild populations.
The ds2 locus is associated with a polymorphism in hydrocarbons that are present on cuticles of females (Dallerac et al. 2000; Takahashi et al. 2001). Cuticular hydrocarbons are mating pheromones in Drosophila (Jallon 1984). In particular, dienes are attractive to males (Jallon 1984). Females with high levels of 5,9-heptacosadiene (5,9-HD) express ds2, while females with high levels of 7,11-HD do not (Dallerac et al. 2000). A derived 16-bp deletion upstream of ds2 is responsible both for the lack of expression of the gene and for high levels of 7,11-HD (Takahashi et al. 2001; Greenberg et al. 2003). This deletion allele is at high frequency in populations of D. melanogaster from around the world (cosmopolitan, or M, populations). Conversely, the ancestral insertion allele is prevalent in populations found around Zimbabwe in Africa (Z populations). Z females do not readily mate with M males, while the reciprocal cross is possible (Wu et al. 1995). Some Z populations are polymorphic for the strength of this behavioral isolation (Hollocher et al. 1997). In these populations, both ds2 alleles are present, and the insertion allele is associated with strong Z behavior (Fang, Takahashi, and Wu 2002).
Using precise allele substitution, Greenberg et al. (2003) replaced the deletion allele of ds2 in an M line with the insertion allele. To control for the effects of genetic background, they compared several of these substitution lines with controls that did not receive the insertion (ds2Z) allele but otherwise carried the same set of alleles at loci unrelated to ds2. They found that ds2Z caused a decrease in tolerance to cold, while elevating starvation resistance. Recent results (J. R. Moran, personal communication) suggest that this allele also affects mating behavior, possibly contributing to Z/M isolation.
Cold tolerance was probably important in successful emigration of D. melanogaster out of Africa to Europe. It thus seems reasonable to conclude that ecological forces maintain the polymorphism at ds2. Supporting this idea, patterns of nucleotide polymorphism bear a signature of recent positive selection on the upstream region of ds2 (Takahashi et al. 2001; Greenberg et al. 2006). Selective sweeps can be detected by the departure of the shape of mutation frequency spectra from the neutral expectation. Two tests, Tajima's D (Tajima 1989) and Fay and Wu's H (Fay and Wu 2000), measure such departure in different ways. Significantly negative D implies a deficit of intermediate frequency variants. Conversely, significantly negative H indicates an access of high frequency–derived variants. Both of these statistics for ds2 are compatible with neutral evolution in the Z population (Greenberg et al. 2006). In contrast, both are significantly negative for the ds2 regulatory region in the M population. However, various patterns of population structure and growth may mimic signatures of positive selection. Greenberg et al. (2006) have thus sequenced a nearby gene, sas, as a control. Although Tajima's D was significantly negative for this locus, Fay and Wu's H was compatible with neutral evolution. Demographic history thus does not appear to fully account for the nucleotide polymorphism patterns in the regulatory region of ds2. Moreover, the selection event probably occurred recently. No sign of accelerated evolution could be observed in any part of ds2 since the divergence of the Sophophora and Drosophila clades about 40 MYA (Greenberg et al. 2006).
Bringing together population and molecular genetic data, we conclude that the derived deletion allele of ds2 rose to high frequency in the M population through the action of positive selection driven by ecological adaptation. Pleiotropic effects of the ds2 alleles on mate choice may then contribute to isolation between the Z and M behavioral races.
Although the small number of studies precludes any broad generalizations, it appears that, as expected, mutations in genes involved in evolutionary processes produce rather subtle phenotypes. However, given that precise allele substitution is used, and with proper control of the genetic background (Greenberg et al. 2003), these phenotypic effects can be measured.
|
Integrating Molecular Biology, Ecology, and Evolution |
|---|
|
TOPAbstractIntroductionMolecular Manipulation of...Integrating Molecular Biology,...AcknowledgementsReferences |
|---|
The successful application of gene replacement to the ds2 gene suggests that, in principle, phenotypic effects of naturally occurring alleles can be measured. This is, however, the first step in discerning the adaptive forces that act on nucleotide variants. Adaptation usually involves multiple genes (Orr and Coyne 1992
; Orr 1998). A sufficient number of these loci need to be manipulated in a given system so that most of the genetic effect can be explained. We thus need reliable ways of identifying genes subject to selection, as well as connecting the observed phenotypes to conditions in nature.
An obvious way to isolate genes under selection is by identifying quantitative trait loci (QTLs) that produce adaptive phenotypes, such as host specialization (Hawthorne and Via 2001) or immune response (Lazzaro, Sceurman, and Clark 2004), and cloning the genes that control these QTLs. An alternative approach involves identification of genes responsible for reproductive isolation among recently diverged or incipient species. Although the mechanisms of reproductive isolation per se are of limited evolutionary interest (Lewontin 1997; Wu and Ting 2004), hybrid breakdown and behavioral incompatibilities are the result of the divergence of underlying developmental or behavioral programs. Both theoretical and empirical results (Schluter 2000; Gavrilets 2003; Coyne and Orr 2004; Wu and Ting 2004) suggest that such divergence between species is largely due to adaptation. Indeed, while the QTL approach is being successfully applied in plants (albeit mostly for artificially selected traits, Remington, Ungerer, and Purugganan 2001), it is the cloning of speciation genes that is leading the way in animals (Wu and Ting 2004). All these genes bear signatures of positive selection (Ting et al. 1998; Barbash et al. 2003; Presgraves et al. 2003). The inference of positive selection in these cases is based on the observation of excess of amino acid substitutions over the number expected, given the rate of evolution at silent sites. It thus appears that we can identify genes based on their involvement in reproductive isolation and then study their effects within each species to discern the adaptive forces responsible for differentiation.
Cloning of genes underlying putative adaptations and manipulating them molecularly to introduce various naturally occurring alleles is not straightforward, but the problems are largely technical. In contrast, much creativity will be needed to demonstrate that phenotypic outcomes of gene replacement are relevant to selection events in nature. Success will only come with intimate knowledge of the study organism and the conditions it faces in its habitat. A number of methods for demonstrating selection in the wild are already available (Endler 1986). For example, controlled measurements of fitness under conditions mimicking some aspects of the environment can be very powerful (e.g., Wright and Dobzhansky 1946). However, such experiments have to be verified by field observation and ecological studies (Endler 1986).
Introduction of molecular genetics to the study of evolution should involve more than just the implementation of a suite of techniques. Mayr (1961) divided biology into "functional" (of which molecular genetics is an example) and "evolutionary." Functional biology is concerned with mechanisms of biological processes, while evolutionary biology asks why historically these processes arose (Mayr 1961). However, adoption of molecular genetics, with its emphasis on understanding in extensive mechanistic detail a handful of tractable model systems, can be employed in the service of answering Mayr's "why" questions. Moreover, the experience of developmental genetics suggests that broad generalizations will eventually be possible using the information from model systems as a guide.
|
Acknowledgements |
|---|
|
TOPAbstractIntroductionMolecular Manipulation of...Integrating Molecular Biology,...AcknowledgementsReferences |
|---|
We thank J. L. Huie for critical reading of the manuscript. We are also grateful for financial support from the National Institutes of Health Ruth L. Kirschstein National Research Service Award fellowship to A.J.G. and grants to C.-I.W. This is an Society of Molecular Biology and Evolution Young Investigators Workshop article.
|
Footnotes |
|---|
Marta Wayne, Associate Editor
|
References |
|---|
|
TOPAbstractIntroductionMolecular Manipulation of...Integrating Molecular Biology,...AcknowledgementsReferences |
|---|
Barbash, D. A., D. F. Siino, A. M. Tarone, and J. Roote. 2003. A rapidly evolving MYB-related protein causes species isolation in Drosophila. Proc. Natl. Acad. Sci. USA 100:5302–5307.
Barton, N. H., and P. D. Keightley. 2002. Understanding quantitative genetic variation. Nat. Rev. Genet. 3:11–21.[ISI][Medline]
Barton, N. H., and M. Turelli. 1989. Evolutionary quantitative genetics: how little do we know? Annu. Rev. Genet. 23:337–370.[ISI][Medline]
Coyne, J. A., and H. A. Orr. 2004. Speciation. Sinauer Associates, Sunderland, Mass.
Dallerac, R., C. Labeur, J. M. Jallon, D. C. Knipple, W. L. Roelofs, and C. Wicker-Thomas. 2000. A 
9 desaturase gene with a different substrate specificity is responsible for the cuticular diene hydrocarbon polymorphism in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 97:9449–9454.
Dobzhansky, T., and M. L. Queal. 1938. Genetics of natural populations. I. Chromosome variation in populations of Drosophila pseudoobscura inhabiting isolated mountain ranges. Genetics 23:239–251.
Endler, J. A. 1986. Natural selection in the wild. Princeton University Press, Princeton, N.J.
Fang, S., A. Takahashi, and C.-I Wu. 2002. A mutation in the promoter of desaturase2 is correlated with sexual isolation between Drosophila behavioral races. Genetics 162:781–784.
Fay, J. C., and C.-I Wu. 2000. Hitchhiking under positive Darwinian selection. Genetics 155:1405–1413.
Fay, J. C., G. J. Wyckoff, and C.-I Wu. 2002. Testing the neutral theory of molecular evolution with genomic data from Drosophila. Nature 415:1024–1026.[CrossRef][Medline]
Gavrilets, S. 2003. Models of speciation: what have we learned in 40 years? Evolution 57:2197–2215.[CrossRef][ISI][Medline]
Greenberg, A. J., J. R. Moran, J. A. Coyne, and C.-I Wu. 2003. Ecological adaptation during incipient speciation revealed by precise gene replacement. Science 302:1754–1757.
Greenberg, A. J., J. R. Moran, S. Fang, and C.-I Wu. 2006. Adaptive loss of an old duplicated gene during incipient speciation. Mol. Biol. Evol. (in press).
Haddrill, P. R., K. R. Thornton, B. Charlesworth, and P. Andolfatto. 2005. Multilocus patterns of nucleotide variability and the demographic and selection history of Drosophila melanogaster populations. Genome Res. 15:790–799.
Hawthorne, D. J., and S. Via. 2001. Genetic linkage of ecological specialization and reproductive isolation in pea aphids. Nature 412:904–907.[CrossRef][Medline]
Hollocher, H., C.-T. Ting, F. Pollack, and C.-I Wu. 1997. Incipient speciation by sexual isolation in Drosophila melanogaster: variation in mating preference and correlation between sexes. Evolution 51:1175–1181.[CrossRef]
Jallon, J. M. 1984. A few chemical words exchanged by Drosophila during courtship and mating. Behav. Genet. 14:441–478.[CrossRef][ISI][Medline]
Kreitman, M. 1983. Nucleotide polymorphism at the alcohol dehydrogenase locus of Drosophila melanogaster. Nature 304:412–417.[CrossRef][Medline]
Laurie-Ahlberg, C. C., and L. F. Stam. 1987. Use of P-element-mediated transformation to identify the molecular basis of naturally occurring variants affecting Adh expression in Drosophila melanogaster. Genetics 115:129–140.
Lazzaro, B. P., B. K. Sceurman, and A. G. Clark. 2004. Genetic basis of natural variation in D. melanogaster antibacterial immunity. Science 303:1873–1876.
Lewontin, R. C. 1997. Dobzhansky's genetics and the origin of species: is it still relevant? Genetics 147:351–355.[ISI][Medline]
Lewontin, R. C., and J. L. Hubby. 1966. A molecular approach to the study of genic heterozygosity in natural populations. II. Amount of variation and degree of heterozygosity in natural populations of Drosophila pseudoobscura. Genetics 54:595–609.
Li, W.-H. 1997. Molecular evolution. Sinauer Associates, Sunderland, Mass.
Lynch, M., and B. Walsh. 1998. Genetics and analysis of quantitative traits. Sinauer Associates, Sunderland, Mass.
Mackay, T. F. C. 2001. Quantitative trait loci in Drosophila. Nat. Rev. Genet. 2:11–20.[ISI][Medline]
Mayr, E. 1961. Cause and effect in biology. Science 134:1501–1506.
McDonald, J. H., and M. Kreitman. 1991. Adaptive protein evolution at the Adh locus in Drosophila. Nature 351:652–654.[CrossRef][Medline]
Nachman, M. W. 2005. The genetic basis of adaptation: lessons from concealing coloration in pocket mice. Genetica 123:125–136.[CrossRef][ISI][Medline]
Nielsen, R., and Z. Yang. 1998. Likelihood models for detecting positively selected amino acid sites and applications to the HIV-1 envelope gene. Genetics 148:929–936.
Orr, H. A. 1998. The population genetics of adaptation: the distribution of factors fixed during adaptive evolution. Evolution 52:935–949.[CrossRef][ISI]
Orr, H. A., and J. A. Coyne. 1992. The genetics of adaptation: a reassessment. Am. Nat. 140:725–742.[CrossRef]
Presgraves, D. C., L. Balagopalan, S. M. Abmayr, and H. A. Orr. 2003. Adaptive evolution drives divergence of a hybrid inviability gene between two species of Drosophila. Nature 423:715–719.[CrossRef][Medline]
Remington, D. L., M. C. Ungerer, and M. D. Purugganan. 2001. Map-based cloning of quantitative trait loci: progress and prospects. Genet. Res. 78:213–218.[CrossRef][ISI][Medline]
Rong, Y. S., and K. G. Golic. 2000. Gene targeting by homologous recombination in Drosophila. Science 288:2013–2018.
Rubin, G. M., and A. C. Spradling. 1982. Genetic transformation of Drosophila with transposable element vectors. Science 218:348–353.
Schluter, D. 2000. The ecology of adaptive radiation. Oxford University Press, Oxford.
Smith, N. G. C., and A. Eyre-Walker. 2002. Adaptive protein evolution in Drosophila. Nature 415:1022–1024.[CrossRef][Medline]
Sun, S., C.-T. Ting, and C.-I. Wu. 2004. The normal function of a speciation gene, Odysseus, and its hybrid sterility effect. Science 305:81–83.
Tajima, F. 1989. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123:585–595.
Takahashi, A., S. C. Tsaur, J. A. Coyne, and C.-I Wu. 2001. The nucleotide changes governing cuticular hydrocarbon variation and their evolution in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 98:3920–3925.
Tian, D., M. B. Traw, J. Q. Chen, M. Kreitman, and J. Bergelson. 2003. Fitness costs of R-gene-mediated resistance in Arabidopsis thaliana. Nature 423:74–77.[CrossRef][Medline]
Ting, C.-T., S.-C. Tsaur, M.-L. Wu, and C.-I Wu. 1998. A rapidly evolving homeobox at the site of a hybrid sterility gene. Science 282:1501–1504.
Williamson, S. H., R. Hernandez, A. Fledel-Alon, L. Zhu, R. Nielsen, and C. D. Bustamante. 2005. Simultaneous inference of selection and population growth from patterns of variation in the human genome. Proc. Natl. Acad. Sci. USA 102:7882–7887.
Wright, S., and T. Dobzhansky. 1946. Genetics of natural populations. XII. Experimental reproduction of some of the changes caused by natural selection in certain populations of Drosophila pseudoobscura. Genetics 31:125–156.
Wright, S. I., I. Vroh Bi, S. G. Schroeder, M. Yamasaki, J. F. Doebley, M. D. McMullen, and B. S. Gaut. 2005. The effects of artificial selection on the maize genome. Science 308:1310–1314.
Wu, C.-I, H. Hollocher, D. J. Begun, C. F. Aquadro, Y. Xu, and M.-L. Wu. 1995. Sexual isolation in Drosophila melanogaster—a possible case of incipient speciation. Proc. Natl. Acad. Sci. USA 92:2519–2523.
Wu, C.-I, and C.-T. Ting. 2004. Genes and speciation. Nat. Rev. Genet. 5:114–122.[CrossRef][ISI][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||