Inversions Can Accumulate Balanced Sexual Antagonism: Evidence from Simulations and Drosophila Experiments.

Chromosomal inversion polymorphisms can be common, but the causes of their persistence are often unclear. We propose a model for the maintenance of inversion polymorphism, which requires that some variants contribute antagonistically to two phenotypes, one of which has negative frequency-dependent fitness. These conditions yield a form of frequency-dependent disruptive selection, favoring two predominant haplotypes segregating alleles that favor opposing antagonistic phenotypes. An inversion associated with one haplotype can reduce the fitness load incurred by generating recombinant offspring, reinforcing its linkage to the haplotype and enabling both haplotypes to accumulate more antagonistic variants than expected otherwise. We develop and apply a forward simulator to examine these dynamics under a tradeoff between survival and male display. These simulations indeed generate inversion-associated haplotypes with opposing sex-specific fitness effects. Antagonism strengthens with time, and can ultimately yield karyotypes at surprisingly predictable frequencies, with striking genotype frequency differences between sexes and between developmental stages. To test whether this model may contribute to well-studied yet enigmatic inversion polymorphisms in Drosophila melanogaster, we track inversion frequencies in laboratory crosses to test whether they influence male reproductive success or survival. We find that two of the four tested inversions show significant evidence for the tradeoff examined, with In(3R)K favoring survival and In(3L)Ok favoring male reproduction. Additionally, all inversions show survival differences between sexes, and paternal success depends on maternal genotype. Based on this work, we expect that balancing selection on antagonistically pleiotropic traits may provide a significant and underappreciated contribution to the maintenance of natural inversion polymorphism.

Genomes from historical Drosophila melanogaster specimens illuminate adaptive and demographic changes across more than 200 years of evolution.

The ability to perform genomic sequencing on long-dead organisms is opening new frontiers in evolutionary research. These opportunities are especially notable in the case of museum collections, from which countless documented specimens may now be suitable for genomic analysis-if data of sufficient quality can be obtained. Here, we report 25 newly sequenced genomes from museum specimens of the model organism Drosophila melanogaster, including the oldest extant specimens of this species. By comparing historical samples ranging from the early 1800s to 1933 against modern-day genomes, we document evolution across thousands of generations, including time periods that encompass the species' initial occupation of northern Europe and an era of rapidly increasing human activity. We also find that the Lund, Sweden population underwent local genetic differentiation during the early 1800s to 1933 interval (potentially due to drift in a small population) but then became more similar to other European populations thereafter (potentially due to increased migration). Within each century-scale time period, our temporal sampling allows us to document compelling candidates for recent natural selection. In some cases, we gain insights regarding previously implicated selection candidates, such as ChKov1, for which our inferred timing of selection favors the hypothesis of antiviral resistance over insecticide resistance. Other candidates are novel, such as the circadian-related gene Ahcy, which yields a selection signal that rivals that of the DDT resistance gene Cyp6g1. These insights deepen our understanding of recent evolution in a model system, and highlight the potential of future museomic studies.

Genomic Diversity Illuminates the Environmental Adaptation of Drosophila suzukii.

Biological invasions carry substantial practical and scientific importance, and represent natural evolutionary experiments on contemporary timescales. Here, we investigated genomic diversity and environmental adaptation of the crop pest Drosophila suzukii using whole-genome sequencing data and environmental metadata for 29 population samples from its native and invasive range. Through a multifaceted analysis of this population genomic data, we increase our understanding of the D. suzukii genome, its diversity and its evolution, and we identify an appropriate genotype-environment association pipeline for our data set. Using this approach, we detect genetic signals of local adaptation associated with nine distinct environmental factors related to altitude, wind speed, precipitation, temperature, and human land use. We uncover unique functional signatures for each environmental variable, such as a prevalence of cuticular genes associated with annual precipitation. We also infer biological commonalities in the adaptation to diverse selective pressures, particularly in terms of the apparent contribution of nervous system evolution to enriched processes (ranging from neuron development to circadian behavior) and to top genes associated with all nine environmental variables. Our findings therefore depict a finer-scale adaptive landscape underlying the rapid invasion success of this agronomically important species.

Hybrid breakdown in male reproduction between recently diverged Drosophila melanogaster populations has a complex and variable genetic architecture.

Secondary contact between formerly isolated populations may result in hybrid breakdown, in which untested allelic combinations in hybrids are maladaptive and limit genetic exchange. Studying early-stage reproductive isolation may yield key insights into the genetic architectures and evolutionary forces underlying the first steps toward speciation. Here, we leverage the recent worldwide expansion of Drosophila melanogaster to test for hybrid breakdown between populations that diverged within the last 13,000 years. We found clear evidence for hybrid breakdown in male reproduction, but not female reproduction or viability, supporting the prediction that hybrid breakdown affects the heterogametic sex first. The frequency of non-reproducing F2 males varied among different crosses involving the same southern African and European populations, as did the qualitative effect of cross direction, implying a genetically variable basis of hybrid breakdown and a role for uniparentally inherited factors. The levels of breakdown observed in F2 males were not recapitulated in backcrossed individuals, consistent with the existence of incompatibilities with at least three partners. Thus, some of the very first steps toward reproductive isolation could involve incompatibilities with complex and variable genetic architectures. Collectively, our findings emphasize this system's potential for future studies on the genetic and organismal basis of early-stage reproductive isolation.

Maximum SNP FST Outperforms Full-Window Statistics for Detecting Soft Sweeps in Local Adaptation.

Local adaptation can lead to elevated genetic differentiation at the targeted genetic variant and nearby sites. Selective sweeps come in different forms, and depending on the initial and final frequencies of a favored variant, very different patterns of genetic variation may be produced. If local selection favors an existing variant that had already recombined onto multiple genetic backgrounds, then the width of elevated genetic differentiation (high FST) may be too narrow to detect using a typical windowed genome scan, even if the targeted variant becomes highly differentiated. We, therefore, used a simulation approach to investigate the power of SNP-level FST (specifically, the maximum SNP FST value within a window, or FST_MaxSNP) to detect diverse scenarios of local adaptation, and compared it against whole-window FST and the Comparative Haplotype Identity statistic. We found that FST_MaxSNP had superior power to detect complete or mostly complete soft sweeps, but lesser power than full-window statistics to detect partial hard sweeps. Nonetheless, the power of FST_MaxSNP depended highly on sample size, and confident outliers depend on robust precautions and quality control. To investigate the relative enrichment of FST_MaxSNP outliers from real data, we applied the two FST statistics to a panel of Drosophila melanogaster populations. We found that FST_MaxSNP had a genome-wide enrichment of outliers compared with demographic expectations, and though it yielded a lesser enrichment than window FST, it detected mostly unique outlier genes and functional categories. Our results suggest that FST_MaxSNP is highly complementary to typical window-based approaches for detecting local adaptation, and merits inclusion in future genome scans and methodologies.

Interactions Between Natural Selection and Recombination Shape the Genomic Landscape of Introgression.

Hybridization between lineages that have not reached complete reproductive isolation appears more and more like a common phenomenon. Indeed, speciation genomic studies have now extensively shown that many species' genomes have hybrid ancestry. However, genomic patterns of introgression are often heterogeneous across the genome. In many organisms, a positive correlation between introgression levels and recombination rate has been observed. It is usually explained by the purging of deleterious introgressed material due to incompatibilities. However, the opposite relationship was observed in a North American population of Drosophila melanogaster with admixed European and African ancestry. In order to explore how directional and epistatic selection can impact the relationship between introgression and recombination, we performed forward simulations of whole D. melanogaster genomes reflecting the North American population's history. Our results revealed that the simplest models of positive selection often yield negative correlations between introgression and recombination such as the one observed in D. melanogaster. We also confirmed that incompatibilities tend to produce positive introgression-recombination correlations. And yet, we identify parameter space under each model where the predicted correlation is reversed. These findings deepen our understanding of the evolutionary forces that may shape patterns of ancestry across genomes, and they strengthen the foundation for future studies aimed at estimating genome-wide parameters of selection in admixed populations.

Gene Regulatory Evolution in Cold-Adapted Fly Populations Neutralizes Plasticity and May Undermine Genetic Canalization.

The relationships between adaptive evolution, phenotypic plasticity, and canalization remain incompletely understood. Theoretical and empirical studies have made conflicting arguments on whether adaptive evolution may enhance or oppose the plastic response. Gene regulatory traits offer excellent potential to study the relationship between plasticity and adaptation, and they can now be studied at the transcriptomic level. Here, we take advantage of three closely related pairs of natural populations of Drosophila melanogaster from contrasting thermal environments that reflect three separate instances of cold tolerance evolution. We measure the transcriptome-wide plasticity in gene expression levels and alternative splicing (intron usage) between warm and cold laboratory environments. We find that suspected adaptive changes in both gene expression and alternative splicing tend to neutralize the ancestral plastic response. Further, we investigate the hypothesis that adaptive evolution can lead to decanalization of selected gene regulatory traits. We find strong evidence that suspected adaptive gene expression (but not splicing) changes in cold-adapted populations are more vulnerable to the genetic perturbation of inbreeding than putatively neutral changes. We find some evidence that these patterns may reflect a loss of genetic canalization accompanying adaptation, although other processes including hitchhiking recessive deleterious variants may contribute as well. Our findings augment our understanding of genetic and environmental effects on gene regulation in the context of adaptive evolution.

The evolution of larger size in high-altitude Drosophila melanogaster has a variable genetic architecture.

Important uncertainties persist regarding the genetic architecture of adaptive trait evolution in natural populations, including the number of genetic variants involved, whether they are drawn from standing genetic variation, and whether directional selection drives them to complete fixation. Here, we take advantage of a unique natural population of Drosophila melanogaster from the Ethiopian highlands, which has evolved larger body size than any other known population of this species. We apply a bulk segregant quantitative trait locus mapping approach to 4 unique crosses between highland Ethiopian and lowland Zambian populations for both thorax length and wing length. Results indicated a persistently variable genetic basis for these evolved traits (with largely distinct sets of quantitative trait loci for each cross), and at least a moderately polygenic architecture with relatively strong effects present. We complemented these mapping experiments with population genetic analyses of quantitative trait locus regions and gene ontology enrichment analysis, generating strong hypotheses for specific genes and functional processes that may have contributed to these adaptive trait changes. Finally, we find that the genetic architectures indicated by our quantitative trait locus mapping results for size traits mirror those from similar experiments on other recently evolved traits in this species. Collectively, these studies suggest a recurring pattern of polygenic adaptation in this species, in which causative variants do not approach fixation and moderately strong effect loci are present.

A Population Genomic Assessment of Three Decades of Evolution in a Natural Drosophila Population.

Population genetics seeks to illuminate the forces shaping genetic variation, often based on a single snapshot of genomic variation. However, utilizing multiple sampling times to study changes in allele frequencies can help clarify the relative roles of neutral and non-neutral forces on short time scales. This study compares whole-genome sequence variation of recently collected natural population samples of Drosophila melanogaster against a collection made approximately 35 years prior from the same locality-encompassing roughly 500 generations of evolution. The allele frequency changes between these time points would suggest a relatively small local effective population size on the order of 10,000, significantly smaller than the global effective population size of the species. Some loci display stronger allele frequency changes than would be expected anywhere in the genome under neutrality-most notably the tandem paralogs Cyp6a17 and Cyp6a23, which are impacted by structural variation associated with resistance to pyrethroid insecticides. We find a genome-wide excess of outliers for high genetic differentiation between old and new samples, but a larger number of adaptation targets may have affected SNP-level differentiation versus window differentiation. We also find evidence for strengthening latitudinal allele frequency clines: northern-associated alleles have increased in frequency by an average of nearly 2.5% at SNPs previously identified as clinal outliers, but no such pattern is observed at random SNPs. This project underscores the scientific potential of using multiple sampling time points to investigate how evolution operates in natural populations, by quantifying how genetic variation has changed over ecologically relevant timescales.

Ethanol resistance in Drosophila melanogaster has increased in parallel cold-adapted populations and shows a variable genetic architecture within and between populations.

Understanding the genetic properties of adaptive trait evolution is a fundamental crux of biological inquiry that links molecular processes to biological diversity. Important uncertainties persist regarding the genetic predictability of adaptive trait change, the role of standing variation, and whether adaptation tends to result in the fixation of favored variants. Here, we use the recurrent evolution of enhanced ethanol resistance in Drosophila melanogaster during this species' worldwide expansion as a promising system to add to our understanding of the genetics of adaptation. We find that elevated ethanol resistance has evolved at least three times in different cooler regions of the species' modern range-not only at high latitude but also in two African high-altitude regions. Applying a bulk segregant mapping framework, we find that the genetic architecture of ethanol resistance evolution differs substantially not only between our three resistant populations, but also between two crosses involving the same European population. We then apply population genetic scans for local adaptation within our quantitative trait locus regions, and we find potential contributions of genes with annotated roles in spindle localization, membrane composition, sterol and alcohol metabolism, and other processes. We also apply simulation-based analyses that confirm the variable genetic basis of ethanol resistance and hint at a moderately polygenic architecture. However, these simulations indicate that larger-scale studies will be needed to more clearly quantify the genetic architecture of adaptive evolution and to firmly connect trait evolution to specific causative loci.

Broad geographic sampling reveals the shared basis and environmental correlates of seasonal adaptation in Drosophila.

To advance our understanding of adaptation to temporally varying selection pressures, we identified signatures of seasonal adaptation occurring in parallel among Drosophila melanogaster populations. Specifically, we estimated allele frequencies genome-wide from flies sampled early and late in the growing season from 20 widely dispersed populations. We identified parallel seasonal allele frequency shifts across North America and Europe, demonstrating that seasonal adaptation is a general phenomenon of temperate fly populations. Seasonally fluctuating polymorphisms are enriched in large chromosomal inversions, and we find a broad concordance between seasonal and spatial allele frequency change. The direction of allele frequency change at seasonally variable polymorphisms can be predicted by weather conditions in the weeks prior to sampling, linking the environment and the genomic response to selection. Our results suggest that fluctuating selection is an important evolutionary force affecting patterns of genetic variation in Drosophila.

Parallel and population-specific gene regulatory evolution in cold-adapted fly populations.

Changes in gene regulation at multiple levels may comprise an important share of the molecular changes underlying adaptive evolution in nature. However, few studies have assayed within- and between-population variation in gene regulatory traits at a transcriptomic scale, and therefore inferences about the characteristics of adaptive regulatory changes have been elusive. Here, we assess quantitative trait differentiation in gene expression levels and alternative splicing (intron usage) between three closely related pairs of natural populations of Drosophila melanogaster from contrasting thermal environments that reflect three separate instances of cold tolerance evolution. The cold-adapted populations were known to show population genetic evidence for parallel evolution at the SNP level, and here we find evidence for parallel expression evolution between them, with stronger parallelism at larval and adult stages than for pupae. We also implement a flexible method to estimate cis- vs trans-encoded contributions to expression or splicing differences at the adult stage. The apparent contributions of cis- vs trans-regulation to adaptive evolution vary substantially among population pairs. While two of three population pairs show a greater enrichment of cis-regulatory differences among adaptation candidates, trans-regulatory differences are more likely to be implicated in parallel expression changes between population pairs. Genes with significant cis-effects are enriched for signals of elevated genetic differentiation between cold- and warm-adapted populations, suggesting that they are potential targets of local adaptation. These findings expand our knowledge of adaptive gene regulatory evolution and our ability to make inferences about this important and widespread process.

Recurrent Collection of Drosophila melanogaster from Wild African Environments and Genomic Insights into Species History.

A long-standing enigma concerns the geographic and ecological origins of the intensively studied vinegar fly, Drosophila melanogaster. This globally distributed human commensal is thought to originate from sub-Saharan Africa, yet until recently, it had never been reported from undisturbed wilderness environments that could reflect its precommensal niche. Here, we document the collection of 288 D. melanogaster individuals from multiple African wilderness areas in Zambia, Zimbabwe, and Namibia. The presence of D. melanogaster in these remote woodland environments is consistent with an ancestral range in southern-central Africa, as opposed to equatorial regions. After sequencing the genomes of 17 wilderness-collected flies collected from Kafue National Park in Zambia, we found reduced genetic diversity relative to town populations, elevated chromosomal inversion frequencies, and strong differences at specific genes including known insecticide targets. Combining these genomes with existing data, we probed the history of this species' geographic expansion. Demographic estimates indicated that expansion from southern-central Africa began ∼10,000 years ago, with a Saharan crossing soon after, but expansion from the Middle East into Europe did not begin until roughly 1,400 years ago. This improved model of demographic history will provide an important resource for future evolutionary and genomic studies of this key model organism. Our findings add context to the history of D. melanogaster, while opening the door for future studies on the biological basis of adaptation to human environments.

Wild African Drosophila melanogaster Are Seasonal Specialists on Marula Fruit.

Although the vinegar fly Drosophila melanogaster is arguably the most studied organism on the planet, fundamental aspects of this species' natural ecology have remained enigmatic [1]. We have here investigated a wild population of D. melanogaster from a mopane forest in Zimbabwe. We find that these flies are closely associated with marula fruit (Sclerocarya birrea) and propose that this seasonally abundant and predominantly Southern African fruit is a key ancestral host of D. melanogaster. Moreover, when fruiting, marula is nearly exclusively used by D. melanogaster, suggesting that these forest-dwelling D. melanogaster are seasonal specialists, in a similar manner to, e.g., Drosophila erecta on screw pine cones [2]. We further demonstrate that the main chemicals released by marula activate odorant receptors that mediate species-specific host choice (Or22a) [3, 4] and oviposition site selection (Or19a) [5]. The Or22a-expressing neurons-ab3A-respond strongly to the marula ester ethyl isovalerate, a volatile rarely encountered in high amounts in other fruit. We also show that Or22a differs among African populations sampled from a wide range of habitats, in line with a function associated with host fruit usage. Flies from Southern Africa, most of which carry a distinct allele at the Or22a/Or22b locus, have ab3A neurons that are more sensitive to ethyl isovalerate than, e.g., European flies. Finally, we discuss the possibility that marula, which is also a culturally and nutritionally important resource to humans, may have helped the transition to commensalism in D. melanogaster.

Impacts of Recurrent Hitchhiking on Divergence and Demographic Inference in Drosophila.

In species with large population sizes such as Drosophila, natural selection may have substantial effects on genetic diversity and divergence. However, the implications of this widespread nonneutrality for standard population genetic assumptions and practices remain poorly resolved. Here, we assess the consequences of recurrent hitchhiking (RHH), in which selective sweeps occur at a given rate randomly across the genome. We use forward simulations to examine two published RHH models for D. melanogaster, reflecting relatively common/weak and rare/strong selection. We find that unlike the rare/strong RHH model, the common/weak model entails a slight degree of Hill-Robertson interference in high recombination regions. We also find that the common/weak RHH model is more consistent with our genome-wide estimate of the proportion of substitutions fixed by natural selection between D. melanogaster and D. simulans (19%). Finally, we examine how these models of RHH might bias demographic inference. We find that these RHH scenarios can bias demographic parameter estimation, but such biases are weaker for parameters relating recently diverged populations, and for the common/weak RHH model in general. Thus, even for species with important genome-wide impacts of selective sweeps, neutralist demographic inference can have some utility in understanding the histories of recently diverged populations.

Directional selection reduces developmental canalization against genetic and environmental perturbations in Drosophila wings.

Natural selection may enhance or weaken the robustness of phenotypes against genetic or environmental perturbations. However, important aspects of the relationship between adaptive evolution and canalization remain unclear. Recent work showed that the evolution of larger wing size in a high altitude natural population of Drosophila melanogaster was accompanied by decanalized wing development--specifically a loss of robustness to genetic perturbation. But this study did not address environmental robustness, and it compared populations that may have numerous biological differences. Here, we perform artificial selection on this same trait in D. melanogaster (larger wing length) and directly test whether this directional selection resulted in decanalization. We find that in general, size-selected replicates show greater frequencies of wing defects than control replicates both after mutagenesis (genetic perturbation) and when subjected to high temperature stress (environmental perturbation), although the increase in defect frequency varies importantly among replicates. These results support the hypothesis that directional selection may result in the loss of both genetic and environmental robustness-offering a rare window into the relationship between adaptation and canalization.

PopFly: the Drosophila population genomics browser.

SUMMARY: The recent compilation of over 1100 worldwide wild-derived Drosophila melanogaster genome sequences reassembled using a standardized pipeline provides a unique resource for population genomic studies (Drosophila Genome Nexus, DGN). A visual display of the estimated metrics describing genome-wide variation and selection patterns would allow gaining a global view and understanding of the evolutionary forces shaping genome variation. AVAILABILITY AND IMPLEMENTATION: Here, we present PopFly, a population genomics-oriented genome browser, based on JBrowse software, that contains a complete inventory of population genomic parameters estimated from DGN data. This browser is designed for the automatic analysis and display of genetic variation data within and between populations along the D. melanogaster genome. PopFly allows the visualization and retrieval of functional annotations, estimates of nucleotide diversity metrics, linkage disequilibrium statistics, recombination rates, a battery of neutrality tests, and population differentiation parameters at different window sizes through the euchromatic chromosomes. PopFly is open and freely available at site http://popfly.uab.cat . CONTACT: sergi.hervas@uab.cat or antonio.barbadilla@uab.cat.

Parallel Evolution of Cold Tolerance within Drosophila melanogaster.

Drosophila melanogaster originated in tropical Africa before expanding into strikingly different temperate climates in Eurasia and beyond. Here, we find elevated cold tolerance in three distinct geographic regions: beyond the well-studied non-African case, we show that populations from the highlands of Ethiopia and South Africa have significantly increased cold tolerance as well. We observe greater cold tolerance in outbred versus inbred flies, but only in populations with higher inversion frequencies. Each cold-adapted population shows lower inversion frequencies than a closely-related warm-adapted population, suggesting that inversion frequencies may decrease with altitude in addition to latitude. Using the FST-based "Population Branch Excess" statistic (PBE), we found only limited evidence for parallel genetic differentiation at the scale of ∼4 kb windows, specifically between Ethiopian and South African cold-adapted populations. And yet, when we looked for single nucleotide polymorphisms (SNPs) with codirectional frequency change in two or three cold-adapted populations, strong genomic enrichments were observed from all comparisons. These findings could reflect an important role for selection on standing genetic variation leading to "soft sweeps". One SNP showed sufficient codirectional frequency change in all cold-adapted populations to achieve experiment-wide significance: an intronic variant in the synaptic gene Prosap. Another codirectional outlier SNP, at senseless-2, had a strong association with our cold trait measurements, but in the opposite direction as predicted. More generally, proteins involved in neurotransmission were enriched as potential targets of parallel adaptation. The ability to study cold tolerance evolution in a parallel framework will enhance this classic study system for climate adaptation.

A Thousand Fly Genomes: An Expanded Drosophila Genome Nexus.

The Drosophila Genome Nexus is a population genomic resource that provides D. melanogaster genomes from multiple sources. To facilitate comparisons across data sets, genomes are aligned using a common reference alignment pipeline which involves two rounds of mapping. Regions of residual heterozygosity, identity-by-descent, and recent population admixture are annotated to enable data filtering based on the user's needs. Here, we present a significant expansion of the Drosophila Genome Nexus, which brings the current data object to a total of 1,121 wild-derived genomes. New additions include 305 previously unpublished genomes from inbred lines representing six population samples in Egypt, Ethiopia, France, and South Africa, along with another 193 genomes added from recently-published data sets. We also provide an aligned D. simulans genome to facilitate divergence comparisons. This improved resource will broaden the range of population genomic questions that can addressed from multi-population allele frequencies and haplotypes in this model species. The larger set of genomes will also enhance the discovery of functionally relevant natural variation that exists within and between populations.