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The role of natural selection in shaping biological diversity is an area of intense interest in modern biology. To date, studies of positive selection have primarily relied on genomic datasets from contemporary populations, which are susceptible to confounding factors associated with complex and often unknown aspects of population history. In particular, admixture between diverged populations can distort or hide prior selection events in modern genomes, though this process is not explicitly accounted for in most selection studies despite its apparent ubiquity in humans and other species. Through analyses of ancient and modern human genomes, we show that previously reported Holocene-era admixture has masked more than 50 historic hard sweeps in modern European genomes. Our results imply that this canonical mode of selection has probably been underappreciated in the evolutionary history of humans and suggest that our current understanding of the tempo and mode of selection in natural populations may be inaccurate. Ancient human genomes uncover historical hard sweeps Hard sweeps were common in ancient West Eurasian populations A historical hard sweep in the MHC-III region Admixture can obscure historical hard sweeps Evolutionary scenarios underlying sweep signal dilution The mutational basis of the Eurasian hard sweeps Discussion Methods

Pseudoexfoliation syndrome (PEX) is a common systemic disease that results in severe and often irreversible vision loss. Despite considerable research effort, PEX remains incompletely understood. This study sought to perform the first RNAseq study in elucidate the pathophysiology of PEX, and contribute a publicly available transcriptomic data resource for future research.Human ocular lens capsular epithelium samples were collected from 25 patients with PEX and 39 non-PEX controls undergoing cataract surgery. RNA extracted from these specimens was subjected to polyadenylated (mRNA) selection and deep bulk RNA sequencing. Differential expression analysis investigated protein-coding gene transcripts. Exploratory analyses used pathway analysis tools, and curated class- and disease-specific gene sets.Differential expression analysis demonstrated that 2882 genes were differentially expressed according to PEX status. Genes associated with viral gene expression pathways were among the most upregulated, alongside genes encoding ribosomal and mitochondrial respiratory transport chain proteins. Cell adhesion protein transcripts including type 4 collagen subunits were downregulated.This comparative transcriptomic dataset highlights novel and previously recognized pathogenic pathways in PEX and provides the first comprehensive transcriptomic resource, adding an additional layer to build further understanding of PEX pathophysiology.

To investigate the association between the apolipoprotein E (Retrospective analysis of prospective cohort data.This study included all available eyes from participants recruited to the Progression Risk of Glaucoma: Relevant SNPs [single nucleotide polymorphisms] with Significant Association (PROGRESSA) study with genotyping data from whichApolipoprotein E alleles and genotypes were determined in PROGRESSA, and their distributions were compared with an age-matched and ancestrally matched normative cohort, the Blue Mountains Eye Study. Structural parameters of neuroretinal atrophy measured using spectral-domain OCT were compared within the PROGRESSA cohort on the basis ofLongitudinal rates of thinning in the macular ganglion cell-inner plexiform layer (mGCIPL) complex and the peripapillary retinal nerve fiber layer (pRNFL).Rates of mGCIPL complex thinning were faster in participants harboring ≥1 copies of theThe

Central and eastern chimpanzees are infected with Simian Immunodeficiency Virus (SIV) in the wild, typically without developing acute immunodeficiency. Yet the recent zoonotic transmission of chimpanzee SIV to humans, which were naïve to the virus, gave rise to the Human Immunodeficiency Virus (HIV), which causes AIDS and is responsible for one of the deadliest pandemics in human history. Chimpanzees have likely been infected with SIV for tens of thousands of years and have likely evolved to reduce its pathogenicity, becoming semi-natural hosts that largely tolerate the virus. In support of this view, central and eastern chimpanzees show evidence of positive selection in genes involved in SIV/HIV cell entry and immune response to SIV, respectively. We hypothesise that the population first infected by SIV would have experienced the strongest selective pressure to control the lethal potential of zoonotic SIV, and that population genetics will reveal those first critical adaptations. With that aim we used population genetics to investigate signatures of positive selection in the common ancestor of central-eastern chimpanzees. The genes with signatures of positive selection in the ancestral population are significantly enriched in SIV-related genes, especially those involved in the immune response to SIV and those encoding for host genes that physically interact with SIV/HIV (VIPs). This supports a scenario where SIV first infected the central-eastern ancestor and where this population was under strong pressure to adapt to zoonotic SIV. Interestingly, integrating these genes with candidates of positive selection in the two infected subspecies reveals novel patterns of adaptation to SIV. Specifically, we observe evidence of positive selection in numerous steps of the biological pathway responsible for T-helper cell differentiation, including CD4 and multiple genes that SIV/HIV use to infect and control host cells. This pathway is active only in CD4+ cells which SIV/HIV infects, and it plays a crucial role in shaping the immune response so it can efficiently control the virus. Our results confirm the importance of SIV as a selective factor, identify specific genetic changes that may have allowed our closest living relatives to reduce SIV’s pathogenicity, and demonstrate the potential of population genomics to reveal the evolutionary mechanisms used by naïve hosts to reduce the pathogenicity of zoonotic pathogens.

Recent efforts to comprehensively characterize great ape genetic diversity using short-read sequencing and single-nucleotide variants have led to important discoveries related to selection within species, demographic history, and lineage-specific traits. Structural variants (SVs), including deletions and inversions, comprise a larger proportion of genetic differences between and within species, making them an important yet understudied source of trait divergence. Here, we used a combination of long-read and -range sequencing approaches to characterize the structural variant landscape of two additional Pan troglodytes verus individuals, one of whom carries 13% admixture from Pan troglodytes troglodytes. We performed optical mapping of both individuals followed by nanopore sequencing of one individual. Filtering for larger variants (&gt, 10 kbp) and combined with genotyping of SVs using short-read data from the Great Ape Genome Project, we identified 425 deletions and 59 inversions, of which 88 and 36, respectively, were novel. Compared with gene expression in humans, we found a significant enrichment of chimpanzee genes with differential expression in lymphoblastoid cell lines and induced pluripotent stem cells, both within deletions and near inversion breakpoints. We examined chromatin-conformation maps from human and chimpanzee using these same cell types and observed alterations in genomic interactions at SV breakpoints. Finally, we focused on 56 genes impacted by SVs in &gt, 90% of chimpanzees and absent in humans and gorillas, which may contribute to chimpanzee-specific features. Sequencing a greater set of individuals from diverse subspecies will be critical to establish the complete landscape of genetic variation in chimpanzees.

Insecticide resistance is considered a classic model of microevolution, where a strong selective agent is applied to a large natural population, resulting in a change in frequency of alleles that confer resistance. While many insecticide resistance variants have been characterized at the gene level, they are typically single genes of large effect identified in highly resistant pest species. In contrast, multiple variants have been implicated in DDT resistance in Drosophila melanogaster; however, only the Cyp6g1 locus has previously been shown to be relevant to field populations. Here we use genome-wide association studies (GWAS) to identify DDT-associated polygenes and use selective sweep analyses to assess their adaptive significance. We identify and verify two candidate DDT resistance loci. A largely uncharacterized gene, CG10737, has a function in muscles that ameliorates the effects of DDT, while a putative detoxifying P450, Cyp6w1, shows compelling evidence of positive selection.

Chimpanzees, humans’ closest relatives, are in danger of extinction. Aside from direct human impacts such as hunting and habitat destruction, a key threat is transmissible disease. As humans continue to encroach upon their habitats, which shrink in size and grow in density, the risk of inter-population and cross-species viral transmission increases, a point dramatically made in the reverse with the global HIV/AIDS pandemic. Inhabiting central Africa, the four subspecies of chimpanzees differ in demographic history and geographical range, and are likely differentially adapted to their particular local environments. To quantitatively explore genetic adaptation, we investigated the genic enrichment for SNPs highly differentiated between chimpanzee subspecies. Previous analyses of such patterns in human populations exhibited limited evidence of adaptation. In contrast, chimpanzees show evidence of recent positive selection, with differences among subspecies. Specifically, we observe strong evidence of recent selection in eastern chimpanzees, with highly differentiated SNPs being uniquely enriched in genic sites in a way that is expected under recent adaptation but not under neutral evolution or background selection. These sites are enriched for genes involved in immune responses to pathogens, and for genes inferred to differentiate the immune response to infection by simian immunodeficiency virus (SIV) in natural vs. non-natural host species. Conversely, central chimpanzees exhibit an enrichment of signatures of positive selection only at cytokine receptors, due to selective sweeps in CCR3, CCR9 and CXCR6 –paralogs of CCR5 and CXCR4, the two major receptors utilized by HIV to enter human cells. Thus, our results suggest that positive selection has contributed to the genetic and phenotypic differentiation of chimpanzee subspecies, and that viruses likely play a predominate role in this differentiation, with SIV being a likely selective agent. Interestingly, our results suggest that SIV has elicited distinctive adaptive responses in these two chimpanzee subspecies., Author summary Viruses are a major factor driving recent and ongoing natural selection in mammalian genomes. Studying the effects such selection has had on chimpanzee genomes can provide valuable insights into how pathogens are affecting an endangered primate species. As there are many notable examples of cross-species transmission between other primates and humans—including the HIV/AIDS pandemic—these studies can also illuminate mechanisms of adaption to pathogens of medical and economic importance. By investigating patterns of genetic differentiation amongst the four chimpanzee subspecies, we show that genetic differences among some subspecies are due to recent genetic adaptation. The genetic variants selected uniquely in eastern chimpanzees fall disproportionally within genes that differentiate the immune response to infection by simian immunodeficiency virus (SIV) in natural vs. non-natural host species. Thus, we infer that SIV has likely elicited adaptive responses in chimpanzees, building upon a growing body of work suggesting that SIV elicits ongoing selection in African primates.

All four subspecies of chimpanzees are endangered. Differing in their demographic histories and geographical ranges within sub-Saharan Africa, they have likely adapted to different environmental factors. We show that highly differentiated SNPs in eastern chimpanzees are uniquely enriched in genic sites in a way that is expected under recent adaptation. These sites are enriched for genes that differentiate the immune response to infection by simian immunodeficiency virus (SIV) in natural vs. non-natural host species. Conversely, central chimpanzees exhibit selective sweeps at the cytokine receptorsCCR3,CCR9andCXCR6– paralogs ofCCR5andCXCR4,the two major receptors utilized by HIV to enter human cells. Thus, we infer that SIV may be eliciting distinctive adaptive responses in different chimpanzee subspecies. Since central chimpanzee SIV is the source of the global HIV/AIDS pandemic, understanding the mechanisms that limit pathogenicity of SIV in chimpanzees can broaden our understanding of HIV infection in humans.

Manuel, Marc de et al., Our closest living relatives, chimpanzees and bonobos, have a complex demographic history. We analyzed the high-coverage whole genomes of 75 wild-born chimpanzees and bonobos from 10 countries in Africa. We found that chimpanzee population substructure makes genetic information a good predictor of geographic origin at country and regional scales. Multiple lines of evidence suggest that gene flow occurred from bonobos into the ancestors of central and eastern chimpanzees between 200,000 and 550,000 years ago, probably with subsequent spread into Nigeria-Cameroon chimpanzees. Together with another, possibly more recent contact (after 200,000 years ago), bonobos contributed less than 1% to the central chimpanzee genomes. Admixture thus appears to have been widespread during hominid evolution., M.d.M. is supported by a Formació de personal Investigador fellowship from Generalitat de Catalunya (FI_B01111). M.K. is supported by a Deutsche Forschungsgemeinschaft fellowship (KU 3467/1-1). V.C.S., I.D., and L.E. are supported by Swiss National Science Foundation grants 31003A-143393 and 310030B-16660. T.D. is funded by the Gates Cambridge Trust. O.L. is supported by a Ramón y Cajal grant from Ministerio de Economía y Competitividad (MINECO) (RYC-2013-14797) and MINECO grant BFU2015-68759-P [Fondo Europeo de Desarrollo Region (FEDER)]. P.H. is supported by Estonian Research Council grant PUT1036. J.M.S., A.M.A., and S.C. are funded by the Max Planck Society. J.P.-M., C.T.-S., and Y.X. were supported by The Wellcome Trust (098051). J.M.H.-G. is supported by the María de Maeztu Programme (MDM-2014-0370). A.S. is supported by an Isaac Newton Trust/Wellcome Trust Institutional Strategic Support Fund Joint Research Grant. J.N. had support from a U.S. NIH U01CA198933 grant, and B.M.P. is supported by a Swiss National Science Foundation postdoctoral fellowship. A.N. is supported by MINECO grant BFU2015-68649-P. The collection of fecal samples was supported by the Max Planck Society and Krekeler Foundation’s generous funding for the Pan African Programme. T.M.-B. thanks ICREA; the European Molecular Biology Organization Young Investigator Programme 2013; MINECO grants BFU2014-55090-P (FEDER), BFU2015-7116-ERC, and BFU2015-6215-ERCU01; U.S. NIH grant MH106874; Fundacio Zoo Barcelona; and Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya for the support to his laboratory.

Insecticide resistance evolves extremely rapidly, providing an illuminating model for the study of adaptation. With climate change reshaping species distribution, pest and disease vector control needs rethinking to include the effects of environmental variation and insect stress physiology. Here, we assessed how both long-term adaptation of populations to temperature and immediate temperature variation affect the genetic architecture of DDT insecticide response in Drosophila melanogaster. Mortality assays and behavioural assays based on continuous activity monitoring were used to assess the interaction between DDT and temperature on three field-derived populations from climate extremes (Raleigh for warm temperate, Tasmania for cold oceanic and Queensland for hot tropical). The Raleigh population showed the highest mortality to DDT, whereas the Queensland population, epicentre for derived alleles of the resistance gene Cyp6g1, showed the lowest. Interaction between insecticide and temperature strongly affected mortality, particularly for the Tasmanian population. Activity profiles analysed using self-organizing maps show that the insecticide promoted an early response, whereas elevated temperature promoted a later response. These distinctive early or later activity phases revealed similar responses to temperature and DDT dose alone but with more or less genetic variance depending on the population. This change in genetic variance among populations suggests that selection particularly depleted genetic variance for DDT response in the Queensland population. Finally, despite similar (co)variation between traits in benign conditions, the genetic responses across population differed under stressful conditions. This showed how stress-responsive genetic variation only reveals itself in specific conditions and thereby escapes potential trade-offs in benign environments.

Ambient temperature is a critical environmental factor for all living organisms. It was likely an important selective force as modern humans recently colonized temperate and cold Eurasian environments. Nevertheless, as of yet we have limited evidence of local adaptation to ambient temperature in populations from those environments. To shed light on this question, we exploit the fact that humans are a cosmopolitan species that inhabit territories under a wide range of temperatures. Focusing on cold perception–which is central to thermoregulation and survival in cold environments–we show evidence of recent local adaptation on TRPM8. This gene encodes for a cation channel that is, to date, the only temperature receptor known to mediate an endogenous response to moderate cold. The upstream variant rs10166942 shows extreme population differentiation, with frequencies that range from 5% in Nigeria to 88% in Finland (placing this SNP in the 0.02% tail of the FST empirical distribution). When all populations are jointly analyzed, allele frequencies correlate with latitude and temperature beyond what can be explained by shared ancestry and population substructure. Using a Bayesian approach, we infer that the allele originated and evolved neutrally in Africa, while positive selection raised its frequency to different degrees in Eurasian populations, resulting in allele frequencies that follow a latitudinal cline. We infer strong positive selection, in agreement with ancient DNA showing high frequency of the allele in Europe 3,000 to 8,000 years ago. rs10166942 is important phenotypically because its ancestral allele is protective of migraine. This debilitating disorder varies in prevalence across human populations, with highest prevalence in individuals of European descent–precisely the population with the highest frequency of rs10166942 derived allele. We thus hypothesize that local adaptation on previously neutral standing variation may have contributed to the genetic differences that exist in the prevalence of migraine among human populations today., Author summary Some human populations were likely under strong pressure to adapt biologically to cold climates during their colonization of non-African territories in the last 50,000 years. Such putative adaptations required genetic variation in genes that could mediate adaptive responses to cold. TRPM8 is potentially one such gene, being the only known receptor for the sensation of moderate cold temperature. We show that a likely regulatory genetic variant nearby TRPM8 has several signatures of positive selection raising its frequency in Eurasian populations during the last 25,000 years. While the genetic variant was and is rare in Africa, it is now common outside of Africa, with frequencies that strongly correlate with latitude and are highest in northern European populations. Interestingly, this same genetic variant has previously been strongly associated with migraine. This suggests that adaptation to cold has potentially contributed to the variation in migraine prevalence that exists among human groups today.

Patterns of nucleotide polymorphism within populations ofDrosophila melanogastersuggest that insecticides have been the selective agents driving the strongest recent bouts of positive selection. However, there is a need to explicitly link selective sweep loci to the particular insecticide phenotypes that could plausibly account for the drastic selective responses that are observed in these non-target insects. Here, we screen the Drosophila Genetic Reference Panel with two common insecticides; malathion (an organophosphate) and permethrin (a pyrethroid). Genome wide association studies map ‘survival-on-malathion’ to two of the largest sweeps in theD. melanogastergenome;AceandCyp6g1. Malathion survivorship also correlates with lines which have high levels ofCyp12d1andJheh1andJheh2transcript abundance. Permethrin phenotypes map to the largest cluster of P450 genes in the Drosophila genome, however in contrast to a selective sweep driven by insecticide use, the derived state seems to be associated with susceptibility. These results underscore previous findings that highlight the importance of structural variation to insecticide phenotypes:Cyp6g1exhibits copy number variation and transposable element insertions,Cyp12d1is tandemly duplicated, theJhehloci are associated with aBari1transposable element insertion, and aCyp6a17deletion is associated with susceptibility.

Balancing selection maintains advantageous diversity in populations through various mechanisms. Although extensively explored from a theoretical perspective, an empirical understanding of its prevalence and targets lags behind our knowledge of positive selection. Here, we describe the Non-central Deviation (NCD), a simple yet powerful statistic to detect long-term balancing selection (LTBS) that quantifies how close frequencies are to expectations under LTBS, and provides the basis for a neutrality test. NCD can be applied to a single locus or genomic data, and can be implemented considering only polymorphisms (NCD1) or also considering fixed differences with respect to an outgroup (NCD2) species. Incorporating fixed differences improves power, and NCD2 has higher power to detect LTBS in humans under different frequencies of the balanced allele(s) than other available methods. Applied to genome-wide data from African and European human populations, in both cases using chimpanzee as an outgroup, NCD2 shows that, albeit not prevalent, LTBS affects a sizable portion of the genome: ∼0.6% of analyzed genomic windows and 0.8% of analyzed positions. Significant windows (P

Balancing selection maintains advantageous diversity in populations through various mechanisms. While extensively explored from a theoretical perspective, an empirical understanding of its prevalence and targets lags behind our knowledge of positive selection. Here we describe theNon-Central Deviation(NCD), a simple yet powerful statistic to detect long-term balancing selection (LTBS) that quantifies how close frequencies are to expectations under LTBS, and provides the basis for a neutrality test.NCDcan be applied to a single locus or genomic data, and can be implemented considering only polymorphisms (NCD1) or also considering fixed differences with respect to an outgroup (NCD2) species. Incorporating fixed differences improves power, andNCD2has higher power to detect LTBS in humans under different frequencies of the balanced allele(s) than other available methods. Applied to genome-wide data from African and European human populations, in both cases using chimpanzee as an outgroup,NCD2shows that, albeit not prevalent, LTBS affects a sizable portion of the genome: about 0.6% of analyzed genomic windows and 0.8% of analyzed positions. Significant windows (p< 0.0001) contain 1.6% of SNPs in the genome, which disproportionally fall within exons and change protein sequence, but are not enriched in putatively regulatory sites. These windows overlap about 8% of the protein-coding genes, and these have larger number of transcripts than expected by chance even after controlling for gene length. Our catalog includes known targets of LTBS but a majority of them (90%) are novel. As expected, immune-related genes are among those with the strongest signatures, although most candidates are involved in other biological functions, suggesting that LTBS potentially influences diverse human phenotypes.

The Resistance to Dieldrin gene, Rdl , encodes a GABA-gated chloride channel subunit that is targeted by cyclodiene and phenylpyrazole insecticides. The gene was first characterized in Drosophila melanogaster by genetic mapping of resistance to the cyclodiene dieldrin. The 4,000-fold resistance observed was due to a single amino acid replacement, Ala 301 to Ser. The equivalent change was subsequently identified in Rdl orthologs of a large range of resistant insect species. Here, we report identification of a duplication at the Rdl locus in D. melanogaster . The 113-kb duplication contains one WT copy of Rdl and a second copy with two point mutations: an Ala 301 to Ser resistance mutation and Met 360 to Ile replacement. Individuals with this duplication exhibit intermediate dieldrin resistance compared with single copy Ser 301 homozygotes, reduced temperature sensitivity, and altered RNA editing associated with the resistant allele. Ectopic recombination between Roo transposable elements is involved in generating this genomic rearrangement. The duplication phenotypes were confirmed by construction of a transgenic, artificial duplication integrating the 55.7-kb Rdl locus with a Ser 301 change into an Ala 301 background. Gene duplications can contribute significantly to the evolution of insecticide resistance, most commonly by increasing the amount of gene product produced. Here however, duplication of the Rdl target site creates permanent heterozygosity, providing unique potential for adaptive mutations to accrue in one copy, without abolishing the endogenous role of an essential gene.

An intriguing observation from somestudies of adaptive change is allelicseries, where adaptive alleles successive-ly replace each other at a single locus.For instance, at the Cyp6g1 locus ofDrosophila melanogaster, transposable ele-ment insertions and a gene duplicationevent have combined to create at leasttwo adaptive alleles in which the morederived the allele, the greater theinsecticide resistance of its bearer [1].Similarly, insecticide-resistant alleles inCulex mosquitoes have been observedreplacing each other within the periodof a decade [2].Another case of an allelic series ispresented in the paper by Magwire et al.[3], which identifies a new locus affect-ing sigma virus resistance inD. melano-gaster. Multiple alleles exist at this locusand they differ in their extent of genecopy number polymorphism and featurea transposable element thought to gen-erate novel transcripts. Thus, this studycontributes to an emerging picture thatthe mutations associated with recentadaptive events may not involve regula-tory SNPs or coding SNPs, but complexgene rearrangements [1,4,5]. Further-more, the nested nature of these rear-rangements means that the order inwhich they arose can be deduced.The genes featured in the particularrearrangement described by Magwireetal. [3] were originally identified via anovel genome-wide screen to identifytransposable element insertions at highfrequencies in natural populations [6].Unlike the situation in humans andmany other vertebrates, particular trans-posable element insertions are rarely athigh frequencies in Drosophila popula-tions. A survey of insertion site occu-pancy led Aminetzach and colleagues[6] to a gene, which they dubbedCHKov1,thathasaDOC transposableelement inserted into the coding region.This gene is one of a large cluster of 27paralogs that encode proteins withdistant similarity to choline kinases.The pattern of polymorphism aroundthe DOC insertion suggests it was at thecenter of a very recent and strongselective sweep dating to between 25and 240 years ago. What selective agentcould result in such strong selection onan insect species, so recently? The link to‘‘choline’’ motivated Aminetzach et al.[6] to test whether a commonly usedclass of insecticides, the organophos-phates (OPs), which target the insectnervous system by inhibiting the enzymeacetylcholine esterase, could be theselective agent driving this selectivesweep at a locus implied in cholinemetabolism. They found that a linebearing the DOC allele had greaterresistance to an OP than a control linewith a similar genetic background.The new study of Magwire et al. [3]links another adaptive phenotype, viralresistance, to the CHKov genes. Thesigma virus has been found to infect upto 20% of D. melanogaster flies in fieldpopulations. At least six separate genesthat reduce infection rates have beenmapped in D. melanogaster [7]. Sigma-resistant alleles of theref(2)P locus of D.melanogaster have previously been char-acterized and display patterns of poly-morphism consistent with a selectivesweep[8].Magwireetal.[3]usedapositional cloning approach involvingsome of the genetic tools available forD. melanogaster to molecularly character-izethesecondofthesixgenes,ref(3)D.The resistant mutation involves a com-plex rearrangement of the CHKov1 andCHKov2 genes, with gene duplicationsderived from the allele originally char-acterized by Aminetzach et al. [6]. Thusthe naturally occurring allelic seriesinvolves three alleles: the ancestral allelethat is purportedly susceptible to an OPinsecticide and the sigma virus, theDOCinsertion allele characterized as resistantto an OP and moderately resistant tosigma viruses, and a derived, highlyvirus-resistant allele (alleles A, B, andC, respectively, in Figure 1).As in the case of Cyp6g1, it appearsthat the next step in an allelic series hasarisen before the previous step has sweptto fixation. What is the significance ofthis? We might expect that in a specieswith high population substructure, inde-pendent alleles may arise and competeagainst each other depending on thedegree of gene flow. However, D.melanogaster populations are not thoughtof as highly structured and the fact thealleles in an allelic series are notindependent, but are nested, indicatesthat D. melanogaster populations are largeenough to increase the probability ofsubsequent mutation, even while theprevious allele is at a low to moderatefrequency.On the other hand, these results suggestmutation may still be limiting. The mostadaptive allele at a gene may be two,three, or more mutational steps away.This may be because the initial adaptiveallele is negatively correlated with otherimportant traits, while the subsequentalleles ameliorate these trade offs or costs.Alternatively, the allelic series may reflecta ‘‘Red Queen’’ phenomenon, where amolecular arms race between host andpathogen means that new alleles mustarise in the host species, to counter thenew alleles in the pathogen species. In

The increased transcription of the Cyp6g1 gene of Drosophila melanogaster, and consequent resistance to insecticides such as DDT, is a widely cited example of adaptation mediated by cis-regulatory change. A fragment of an Accord transposable element inserted upstream of the Cyp6g1 gene is causally associated with resistance and has spread to high frequencies in populations around the world since the 1940s. Here we report the existence of a natural allelic series at this locus of D. melanogaster, involving copy number variation of Cyp6g1, and two additional transposable element insertions (a P and an HMS-Beagle). We provide evidence that this genetic variation underpins phenotypic variation, as the more derived the allele, the greater the level of DDT resistance. Tracking the spatial and temporal patterns of allele frequency changes indicates that the multiple steps of the allelic series are adaptive. Further, a DDT association study shows that the most resistant allele, Cyp6g1-[BP], is greatly enriched in the top 5% of the phenotypic distribution and accounts for ∼16% of the underlying phenotypic variation in resistance to DDT. In contrast, copy number variation for another candidate resistance gene, Cyp12d1, is not associated with resistance. Thus the Cyp6g1 locus is a major contributor to DDT resistance in field populations, and evolution at this locus features multiple adaptive steps occurring in rapid succession., Author Summary The study of insecticide resistance has greatly enriched our understanding of the genetic basis of adaptation, because it represents some of the most intense selection pressures acting on any natural population of eukaryote. Thus it can inform us about the limits of natural selection, both in terms of the number and type of mutations that can arise and also in terms of the rate at which these spread throughout populations. Fifty years ago, studies in Drosophila melanogaster indicated that many genes contributed to DDT resistance. Subsequent research into the Hikone-R strain indicated much of the resistance in this particular strain could be attributed to a single gene known as Cyp6g1. Here we show that there have been successive DDT resistance mutations occurring at the Cyp6g1 locus. They include an increase in gene copy number and the insertion of transposable elements into the regulatory regions of the Cyp6g1 gene. These mutations have swept to high frequencies in natural populations since World War II, when insecticides were first used. D. melanogaster is not a pest and has not been targeted by insecticides, and yet profound changes are occurring within its genome in response to man-made chemicals in the environment.