Joshua M. Schmidt

Copy number variation and transposable elements feature in recent, ongoing adaptation at the Cyp6g1 locus.

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.

Modification of Phytohormone Response by a Peptide Encoded by ENOD40 of Legumes and a Nonlegume

The gene ENOD40 is expressed during early stages of legume nodule development. A homolog was isolated from tobacco, which, as does ENOD40 from legumes, encodes an oligopeptide of about 10 amino acids. In tobacco protoplasts, these peptides change the response to auxin at concentrations as low as 10−12 to 10−16 M. The peptides encoded by ENOD40 appear to act as plant growth regulators. Sequence alignment of full ENDO40 gene sequences from soybean, pea, alfalfa, and tocacco plants.

Chimpanzee genomic diversity reveals ancient admixture with bonobos

Of chimpanzees and bonobos Modern non-African human genomes contain genomic remnants that suggest that there was interbreeding between ancient humans and archaic hominoid lineages. Now, de Manuel et al. show similar ancestral interbreeding between the ancestors of todays chimpanzees and bonobos (see the Perspective by Hoelzel). The study also provides population-specific genetic markers that may be valuable for conservation efforts. Science, this issue p. 477; see also p. 414 Genome sequences reveal ancient interbreeding between chimpanzees and bonobos. 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.

Gene duplication in the major insecticide target site, Rdl, in Drosophila melanogaster.

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, Ala301 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 Ala301 to Ser resistance mutation and Met360 to Ile replacement. Individuals with this duplication exhibit intermediate dieldrin resistance compared with single copy Ser301 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 Ser301 change into an Ala301 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.

Perception and Signal Transduction of Rhizobial NOD Factors

Referee: Dr. Gary Stacey, Director, Center for Legume Research, Department of Microbiology, M409 Walters Life Science Bldg., University of Tennessee, Knoxville, TN 37966-0845 Soil bacteria belonging to genera Rhizobium, Bradyrhizobium, Allorhizobium, Azorhizobium, Mesorhizobium, and Sinorhizobium are able to induce nodule formation on the roots of leguminous plants. In the differentiated root nodules bacteria fix as bacteroids atmospheric nitrogen and deliver it to the host plant. The interaction between bacteria and host plant starts with a complex signal exchange. After induction by plant flavonoids, rhizobia synthesize and secrete lipo-chitooligosaccharides (LCOs), known as Nod factors, which induce morphological changes and expression of early nodulin genes in the roots of host plants. Specific recognition of Nod factors by host plants and early stages of signal transduction are discussed.

Genomic and Transcriptomic Associations Identify a New Insecticide Resistance Phenotype for the Selective Sweep at the Cyp6g1 Locus of Drosophila melanogaster.

Scans of the Drosophila melanogaster genome have identified organophosphate resistance loci among those with the most pronounced signature of positive selection. In this study, the molecular basis of resistance to the organophosphate insecticide azinphos-methyl was investigated using the Drosophila Genetic Reference Panel, and genome-wide association. Recently released full transcriptome data were used to extend the utility of the Drosophila Genetic Reference Panel resource beyond traditional genome-wide association studies to allow systems genetics analyses of phenotypes. We found that both genomic and transcriptomic associations independently identified Cyp6g1, a gene involved in resistance to DDT and neonicotinoid insecticides, as the top candidate for azinphos-methyl resistance. This was verified by transgenically overexpressing Cyp6g1 using natural regulatory elements from a resistant allele, resulting in a 6.5-fold increase in resistance. We also identified four novel candidate genes associated with azinphos-methyl resistance, all of which are involved in either regulation of fat storage, or nervous system development. In Cyp6g1, we find a demonstrable resistance locus, a verification that transcriptome data can be used to identify variants associated with insecticide resistance, and an overlap between peaks of a genome-wide association study, and a genome-wide selective sweep analysis.

An adaptive allelic series featuring complex gene rearrangements.

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

Insights into DDT Resistance from the Drosophila melanogaster Genetic Reference Panel

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.

Behavioural response to combined insecticide and temperature stress in natural populations of Drosophila melanogaster

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.

Human local adaptation of the TRPM8 cold receptor along a latitudinal cline

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.