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Insecticide resistance in Anopheles mosquitoes is a major obstacle in maintaining the momentum in reducing the malaria burden; mitigating strategies require improved understanding of the underlying mechanisms. Mutations in the target site of insecticides (the voltage gated sodium channel for the most widely used pyrethroid class) and over-expression of detoxification enzymes are commonly reported, but their relative contribution to phenotypic resistance remain poorly understood. Here we present a genome editing pipeline to introduce single nucleotide polymorphisms in An. gambiae which we have used to study the effect of the classical kdr mutation L1014F (L995F based on An. gambiae numbering), one of the most widely distributed resistance alleles. Introduction of 1014F in an otherwise fully susceptible genetic background increased levels of resistance to all tested pyrethroids and DDT ranging from 9.9-fold for permethrin to >24-fold for DDT. The introduction of the 1014F allele was sufficient to reduce mortality of mosquitoes after exposure to deltamethrin treated bednets, even as the only resistance mechanism present. When 1014F was combined with over-expression of glutathione transferase Gste2, resistance to permethrin increased further demonstrating the critical combined effect between target site resistance and detoxification enzymes in vivo. We also show that mosquitoes carrying the 1014F allele in homozygosity showed fitness disadvantages including increased mortality at the larval stage and a reduction in fecundity and adult longevity, which can have consequences for the strength of selection that will apply to this allele in the field., Author summary Escalation of pyrethroid resistance in Anopheles mosquitoes threatens to reduce the effectiveness of our most important tools in malaria control. Studying the mechanisms underlying insecticide resistance is critical to design mitigation strategies. Here, using genome modified mosquitoes, we functionally characterize the most prevalent mutation in resistant mosquitoes, showing that it confers substantial levels of resistance to all tested pyrethroids and undermines the performance of pyrethroid-treated nets. Furthermore, we show that combining this mutation with elevated levels of a detoxification enzyme further increases resistance. The pipeline we have developed provides a robust approach to quantifying the contribution of different combinations of resistance mechanisms to the overall phenotype, providing the missing link between resistance monitoring and predictions of resistance impact.

Balancer chromosomes are multiply inverted and rearranged chromosomes that are widely used in Drosophila genetics. First described nearly 100 years ago, balancers are used extensively in stock maintenance and complex crosses. Recently, the complete molecular structures of several commonly used balancers were determined by whole-genome sequencing. This revealed a surprising amount of variation among balancers derived from a common progenitor, identified genes directly affected by inversion breakpoints, and cataloged mutations shared by balancers. These studies emphasized that it is important to choose the optimal balancer, because different inversions suppress meiotic recombination in different chromosomal regions. In this review, we provide a brief history of balancers in Drosophila, discuss how they are used today, and provide examples of unexpected recombination events involving balancers that can lead to stock breakdown.

Social interactions have large effects on individual physiology and fitness. In the immediate sense, social stimuli are often highly salient and engaging. Over longer time scales, competitive interactions often lead to distinct social ranks and differences in physiology and behavior. Understanding how initial responses lead to longer-term effects of social interactions requires examining the changes in responses over time. Here we examined the effects of social interactions on transcriptomic signatures at two times, at the end of a 45-minute interaction and 4 hours later, in female Polistes fuscatus paper wasp foundresses. Female P. fuscatus have variable facial patterns that are used for visual individual recognition, so we separately examined the transcriptional dynamics in the optic lobe and the non-visual brain. Results demonstrate much stronger transcriptional responses to social interactions in the non-visual brain compared to the optic lobe. Differentially regulated genes in response to social interactions are enriched for memory-related transcripts. Comparisons between winners and losers of the encounters revealed similar overall transcriptional profiles at the end of an interaction, which significantly diverged over the course of 4 hours, with losers showing changes in expression levels of genes associated with aggression and reproduction in paper wasps. On nests, subordinate foundresses are less aggressive, do more foraging and lay fewer eggs compared to dominant foundresses and we find losers shift expression of many genes in the non-visual brain, including vitellogenin, related to aggression, worker behavior, and reproduction within hours of losing an encounter. These results highlight the early neurogenomic changes that likely contribute to behavioral and physiological effects of social status changes in a social insect., Author summary Aggressive interactions often create inequalities–some individuals win while others lose. Winning versus losing can lead to large physiological differences between individuals, including different neurogenomic profiles between winners and losers. How this information about contest outcome leads to distinct neurogenomic profiles is poorly understood. Here we examine gene expression in response to aggressive social encounters in paper wasps, which naturally form dominance hierarchies on their nests in the wild. Shortly following encounters winners and losers have similar expression profiles, likely because similar mechanisms are engaged by social experiences. Four hours later, we find divergent neurogenomic profiles between winners and losers, with losers showing larger shifts in expression compared to winners. Many of the most dynamically expressed genes have been previously associated with dominance and caste differences in paper wasps showing how a single interaction can engage many of the same genomic networks that are involved in mediating more dramatic differences in queen-worker behavioral differences are also involved in responses shortly following social interactions.

Patterns of genomic architecture across insects remain largely undocumented or decoupled from a broader phylogenetic context. For instance, it is unknown whether translocation rates differ between insect orders. We address broad scale patterns of genome architecture across Insecta by examining synteny in a phylogenetic framework from open-source insect genomes. To accomplish this, we add a chromosome level genome to a crucial lineage, Coleoptera. Our assembly of the Pachyrhynchus sulphureomaculatus genome is the first chromosome scale genome for the hyperdiverse Phytophaga lineage and currently the largest insect genome assembled to this scale. The genome is significantly larger than those of other weevils, and this increase in size is caused by repetitive elements. Our results also indicate that, among beetles, there are instances of long-lasting (>200 Ma) localization of genes to a particular chromosome with few translocation events. While some chromosomes have a paucity of translocations, intra-chromosomal synteny was almost absent, with gene order thoroughly shuffled along a chromosome. This large amount of reshuffling within chromosomes with few inter-chromosomal events contrasts with patterns seen in mammals in which the chromosomes tend to exchange larger blocks of material more readily. To place our findings in an evolutionary context, we compared syntenic patterns across Insecta in a phylogenetic framework. For the first time, we find that synteny decays at an exponential rate relative to phylogenetic distance. Additionally, there are significant differences in decay rates between insect orders, this pattern was not driven by Lepidoptera alone which has a substantially different rate., Author summary Patterns of genomic architecture across insects remain largely undocumented or decoupled from a broader evolutionary context. For instance, it is unknown whether rates of gene order decay differ between insect orders. We address broad scale patterns of genome architecture across Insecta by examining synteny (shared gene order) in a phylogenetic framework from open-source insect genomes (143 complete chromosome assemblies in total). To accomplish this, we add a chromosome level genome to a crucial lineage, Coleoptera (beetles). Our assembly of the Easter Egg Weevil Pachyrhynchus sulphureomaculatus genome is the first chromosome scale genome for the hyperdiverse Phytophaga lineage and currently the largest insect genome assembled to this scale. We are the first to identify in beetles that genes stay localized on chromosomes for hundreds of millions of years, while their order along chromosomes gets completely shuffled over time. We are also the first to empirically demonstrate that synteny decay rates different significantly between insect orders and that this pattern in not driven solely by Lepidoptera (moths and butterflies), which has a substantially different rate.

Illicit use of psychostimulants, such as cocaine and methamphetamine, constitutes a significant public health problem. Whereas neural mechanisms that mediate the effects of these drugs are well-characterized, genetic factors that account for individual variation in susceptibility to substance abuse and addiction remain largely unknown. Drosophila melanogaster can serve as a translational model for studies on substance abuse, since flies have a dopamine transporter that can bind cocaine and methamphetamine, and exposure to these compounds elicits effects similar to those observed in people, suggesting conserved evolutionary mechanisms underlying drug responses. Here, we used the D. melanogaster Genetic Reference Panel to investigate the genetic basis for variation in psychostimulant drug consumption, to determine whether similar or distinct genetic networks underlie variation in consumption of cocaine and methamphetamine, and to assess the extent of sexual dimorphism and effect of genetic context on variation in voluntary drug consumption. Quantification of natural genetic variation in voluntary consumption, preference, and change in consumption and preference over time for cocaine and methamphetamine uncovered significant genetic variation for all traits, including sex-, exposure- and drug-specific genetic variation. Genome wide association analyses identified both shared and drug-specific candidate genes, which could be integrated in genetic interaction networks. We assessed the effects of ubiquitous RNA interference (RNAi) on consumption behaviors for 34 candidate genes: all affected at least one behavior. Finally, we utilized RNAi knockdown in the nervous system to implicate dopaminergic neurons and the mushroom bodies as part of the neural circuitry underlying experience-dependent development of drug preference., Author summary Illicit use of cocaine and methamphetamine is a major public health problem. Whereas the neurological effects of these drugs are well characterized, it remains challenging to determine genetic risk factors for substance abuse in human populations. The fruit fly, Drosophila melanogaster, presents an excellent model for identifying evolutionarily conserved genes that affect drug consumption, since genetic background and exposure can be controlled precisely. We took advantage of natural variation in a panel of inbred wild derived fly lines with complete genome sequences to assess the extent of genetic variation among these lines for voluntary consumption of cocaine and methamphetamine and to explore whether some genetic backgrounds might show experience-dependent development of drug preference. The drug consumption traits were highly variable among the lines with strong sex-, drug- and exposure time-specific components. We identified candidate genes and gene networks associated with variation in consumption of cocaine and methamphetamine and development of drug preference. Using tissue-specific suppression of gene expression, we were able to functionally implicate candidate genes that affected at least one consumption trait in at least one drug and sex. In humans, the mesolimbic dopaminergic projection plays a role in drug addiction. We asked whether in Drosophila the mushroom bodies could play an analogous role, as they are integrative brain centers associated with experience-dependent learning. Indeed, our results suggest that variation in consumption and development of preference for both cocaine and methamphetamine is mediated, at least in part, through a neural network that comprises dopaminergic projections to the mushroom bodies.

Like other domesticates, the efficient utilization of nitrogen resources is also important for the only fully domesticated insect, the silkworm. Deciphering the way in which artificial selection acts on the silkworm genome to improve the utilization of nitrogen resources and to advance human-favored domestication traits, will provide clues from a unique insect model for understanding the general rules of Darwin's evolutionary theory on domestication. Storage proteins (SPs), which belong to a hemocyanin superfamily, basically serve as a source of amino acids and nitrogen during metamorphosis and reproduction in insects. In this study, through blast searching on the silkworm genome and further screening of the artificial selection signature on silkworm SPs, we discovered a candidate domestication gene, i.e., the methionine-rich storage protein 1 (SP1), which is clearly divergent from other storage proteins and exhibits increased expression in the ova of domestic silkworms. Knockout of SP1 via the CRISPR/Cas9 technique resulted in a dramatic decrease in egg hatchability, without obvious impact on egg production, which was similar to the effect in the wild silkworm compared with the domestic type. Larval development and metamorphosis were not affected by SP1 knockout. Comprehensive ova comparative transcriptomes indicated significant higher expression of genes encoding vitellogenin, chorions, and structural components in the extracellular matrix (ECM)-interaction pathway, enzymes in folate biosynthesis, and notably hormone synthesis in the domestic silkworm, compared to both the SP1 mutant and the wild silkworm. Moreover, compared with the wild silkworms, the domestic one also showed generally up-regulated expression of genes enriched in the structural constituent of ribosome and amide, as well as peptide biosynthesis. This study exemplified a novel case in which artificial selection could act directly on nitrogen resource proteins, further affecting egg nutrients and eggshell formation possibly through a hormone signaling mediated regulatory network and the activation of ribosomes, resulting in improved biosynthesis and increased hatchability during domestication. These findings shed new light on both the understanding of artificial selection and silkworm breeding from the perspective of nitrogen and amino acid resources., Author summary Like other domesticates, nitrogen resources are also important for the only fully domesticated insect, the silkworm. Deciphering the way in which artificial selection acts on the silkworm genome to improve the utilization of nitrogen resources, thereby advancing human-favored domestication traits, will provide clues from a unique insect model for understanding the general rules of Darwin's theory on artificial selection. However, the mechanisms of domestication in the silkworm remain largely unknown. In this study, we focused on one important nitrogen resource, the storage protein (SP). We discovered that the methionine-rich storage protein 1 (SP1), which is divergent from other SPs, is the only target of artificial selection. Based on functional evidence, together with key findings from the comprehensive comparative transcriptome, we propose that artificial selection favored higher expression of SP1 in the domestic silkworm, which would influence the genes or pathways vital for egg development and eggshell formation. Artificial selection also consistently favored activated ribosome activities and improved amide and peptide biosynthesis in the ova, like what they may act in the silk gland to increase silk-cocoon yield. We highlighted a novel case in which artificial selection could directly act on a nitrogen resource protein associated with a human-desired domestication trait.

When environments change, populations may adapt surprisingly fast, repeatedly and even at microgeographic scales. There is increasing evidence that such cases of rapid parallel evolution are fueled by standing genetic variation, but the source of this genetic variation remains poorly understood. In the saltmarsh beetle Pogonus chalceus, short-winged ‘tidal’ and long-winged ‘seasonal’ ecotypes have diverged in response to contrasting hydrological regimes and can be repeatedly found along the Atlantic European coast. By analyzing genomic variation across the beetles’ distribution, we reveal that alleles selected in the tidal ecotype are spread across the genome and evolved during a singular and, likely, geographically isolated divergence event, within the last 190 Kya. Due to subsequent admixture, the ancient and differentially selected alleles are currently polymorphic in most populations across its range, which could potentially allow for the fast evolution of one ecotype from a small number of random individuals, as low as 5 to 15, from a population of the other ecotype. Our results suggest that cases of fast parallel ecological divergence can be the result of evolution at two different time frames: divergence in the past, followed by repeated selection on the same divergently evolved alleles after admixture. These findings highlight the importance of an ancient and, likely, allopatric divergence event for driving the rate and direction of contemporary fast evolution under gene flow. This mechanism is potentially driven by periods of geographic isolation imposed by large-scale environmental changes such as glacial cycles., Author summary Evidence has accumulated that populations may adapt surprisingly fast to changing environments. This rapid, and often parallel, ecological adaptation is presumed to be facilitated when selection acts on preexisting genetic variation. However, the origin of this variation remains to be identified. In our work on genome-wide parallel divergence in a mosaic of two beetle ecotypes, we identify the genomic regions involved in adaptation. We show that the origin of the divergent alleles at different loci can be traced back to a singular, ancient divergence event. This event likely occurred in geographic isolation after which admixture of the diverged populations resulted in a polymorphic population that survived during the most recent glacial maxima. The alleles involved in adaptation to the alternative environments are currently present in much higher frequencies in the populations than generally assumed. Therefore, when habitats become available, the presence of these alleles may result in rapid and parallel ecological differentiation by the reassembly of these ancient alleles. We suggest that this mechanism may be common to examples of parallel evolution and might reconcile different views on the role of geographical isolation in ecological divergence.

Topologically associating domains, or TADs, are functional units that organize chromosomes into 3D structures of interacting chromatin. TADs play an important role in regulating gene expression by constraining enhancer-promoter contacts and there is evidence that deletion of TAD boundaries leads to aberrant expression of neighboring genes. While the mechanisms of TAD formation have been well-studied, current knowledge on the patterns of TAD evolution across species is limited. Due to the integral role TADs play in gene regulation, their structure and organization is expected to be conserved during evolution. However, more recent research suggests that TAD structures diverge relatively rapidly. We use Hi-C chromosome conformation capture to measure evolutionary conservation of whole TADs and TAD boundary elements between D. melanogaster and D. triauraria, two early-branching species from the melanogaster species group which diverged ∼15 million years ago. We find that the majority of TADs have been reorganized since the common ancestor of D. melanogaster and D. triauraria, via a combination of chromosomal rearrangements and gain/loss of TAD boundaries. TAD reorganization between these two species is associated with a localized effect on gene expression, near the site of disruption. By separating TADs into subtypes based on their chromatin state, we find that different subtypes are evolving under different evolutionary forces. TADs enriched for broadly expressed, transcriptionally active genes are evolving rapidly, potentially due to positive selection, whereas TADs enriched for developmentally-regulated genes remain conserved, presumably due to their importance in restricting gene-regulatory element interactions. These results provide novel insight into the evolutionary dynamics of TADs and help to reconcile contradictory reports related to the evolutionary conservation of TADs and whether changes in TAD structure affect gene expression., Author summary Chromosomes are organized inside the three-dimensional nucleus in a hierarchical fashion, from the wrapping of DNA around nucleosomes to the presence of compartments of active and inactive chromatin. Here, we focus on domains of interacting chromatin known as topologically associating domains, or TADs. TADs are dozens to hundreds of kilobases in size and tend to contain genes with similar epigenetic states. While much research has been done on the mechanisms of TAD formation, less is known about the role of TADs in genome evolution. The research that does exist is contradictory, with some studies claiming that TADs are highly conserved and others reporting that TADs diverge rapidly. We compared the TAD locations between two Drosophila species and found that the majority have been reorganized. We also identified TAD subtypes that differ in terms of their evolutionary conservation: TADs that contain developmental genes are less likely to be reorganized while TADs that contain broadly expressed genes evolve rapidly. We found that TAD reorganization has an effect on gene expression, but only for genes located near the site of disruption. These results provide new insight into the evolution of 3D genome organization and help to reconcile previous contradictions in the literature.

Organisms living in seasonally variable environments utilize cues such as light and temperature to induce plastic responses, enabling them to exploit favorable seasons and avoid unfavorable ones. Local adapation can result in variation in seasonal responses, but the genetic basis and evolutionary history of this variation remains elusive. Many insects, including Drosophila melanogaster, are able to undergo an arrest of reproductive development (diapause) in response to unfavorable conditions. In D. melanogaster, the ability to diapause is more common in high latitude populations, where flies endure harsher winters, and in the spring, reflecting differential survivorship of overwintering populations. Using a novel hybrid swarm-based genome wide association study, we examined the genetic basis and evolutionary history of ovarian diapause. We exposed outbred females to different temperatures and day lengths, characterized ovarian development for over 2800 flies, and reconstructed their complete, phased genomes. We found that diapause, scored at two different developmental cutoffs, has modest heritability, and we identified hundreds of SNPs associated with each of the two phenotypes. Alleles associated with one of the diapause phenotypes tend to be more common at higher latitudes, but these alleles do not show predictable seasonal variation. The collective signal of many small-effect, clinally varying SNPs can plausibly explain latitudinal variation in diapause seen in North America. Alleles associated with diapause are segregating in Zambia, suggesting that variation in diapause relies on ancestral polymorphisms, and both pro- and anti-diapause alleles have experienced selection in North America. Finally, we utilized outdoor mesocosms to track diapause under natural conditions. We found that hybrid swarms reared outdoors evolved increased propensity for diapause in late fall, whereas indoor control populations experienced no such change. Our results indicate that diapause is a complex, quantitative trait with different evolutionary patterns across time and space., Author summary Animals exhibit diverse strategies to cope with unfavorable conditions in temperate, seasonally varying environments. The model fly, Drosophila melanogaster, can enter a physiological state known as diapause under winter-like conditions. Diapause is characterized by an absence of egg maturation in females and is thought to conserve energy for survival during stressful times. The ability to diapause is more common in flies from higher latitudes and in offspring from flies that have recently overwintered. Therefore, diapause has been thought to be a recent adaptation to temperate climates. We identified hundreds of genetic variants that affect diapause and found that some vary predictably across latitudes in North America. We found little signal of repeated seasonality in diapause-associated genetic variants, but our populations evolved an increased ability to diapause in the winter when they were exposed to natural conditions. Combined, our results suggest that diapause-associated variants evolve differently across space and time. We find little evidence that diapause evolved recently in temperate environments; rather, alleles associated with diapause are as common as expected in Zambia, suggesting that diapause may promote survival under stresses other than cold. Our results provide future targets for research into the genetic underpinnings of this complex, ecologically relevant trait.

Hybridization and gene flow between species appears to be common. Even though it is clear that hybridization is widespread across all surveyed taxonomic groups, the magnitude and consequences of introgression are still largely unknown. Thus it is crucial to develop the statistical machinery required to uncover which genomic regions have recently acquired haplotypes via introgression from a sister population. We developed a novel machine learning framework, called FILET (Finding Introgressed Loci via Extra-Trees) capable of revealing genomic introgression with far greater power than competing methods. FILET works by combining information from a number of population genetic summary statistics, including several new statistics that we introduce, that capture patterns of variation across two populations. We show that FILET is able to identify loci that have experienced gene flow between related species with high accuracy, and in most situations can correctly infer which population was the donor and which was the recipient. Here we describe a data set of outbred diploid Drosophila sechellia genomes, and combine them with data from D. simulans to examine recent introgression between these species using FILET. Although we find that these populations may have split more recently than previously appreciated, FILET confirms that there has indeed been appreciable recent introgression (some of which might have been adaptive) between these species, and reveals that this gene flow is primarily in the direction of D. simulans to D. sechellia., Author summary Understanding the extent to which species or diverged populations hybridize in nature is crucially important if we are to understand the speciation process. Accordingly numerous research groups have developed methodology for finding the genetic evidence of such introgression. In this report we develop a supervised machine learning approach for uncovering loci which have introgressed across species boundaries. We show that our method, FILET, has greater accuracy and power than competing methods in discovering introgression, and in addition can detect the directionality associated with the gene flow between species. Using whole genome sequences from Drosophila simulans and Drosophila sechellia we show that FILET discovers quite extensive introgression between these species that has occurred mostly from D. simulans to D. sechellia. Our work highlights the complex process of speciation even within a well-studied system and points to the growing importance of supervised machine learning in population genetics.

Adenosine-to-inosine (A-to-I) editing is hypothesized to facilitate adaptive evolution by expanding proteomic diversity through an epigenetic approach. However, it is challenging to provide evidences to support this hypothesis at the whole editome level. In this study, we systematically characterized 2,114 A-to-I RNA editing sites in female and male brains of D. melanogaster, and nearly half of these sites had events evolutionarily conserved across Drosophila species. We detected strong signatures of positive selection on the nonsynonymous editing sites in Drosophila brains, and the beneficial editing sites were significantly enriched in genes related to chemical and electrical neurotransmission. The signal of adaptation was even more pronounced for the editing sites located in X chromosome or for those commonly observed across Drosophila species. We identified a set of gene candidates (termed “PSEB” genes) that had nonsynonymous editing events favored by natural selection. We presented evidence that editing preferentially increased mutation sequence space of evolutionarily conserved genes, which supported the adaptive evolution hypothesis of editing. We found prevalent nonsynonymous editing sites that were favored by natural selection in female and male adults from five strains of D. melanogaster. We showed that temperature played a more important role than gender effect in shaping the editing levels, although the effect of temperature is relatively weaker compared to that of species effect. We also explored the relevant factors that shape the selective patterns of the global editomes. Altogether we demonstrated that abundant nonsynonymous editing sites in Drosophila brains were adaptive and maintained by natural selection during evolution. Our results shed new light on the evolutionary principles and functional consequences of RNA editing., Author summary Adenosine-to-inosine (A-to-I) RNA editing is an evolutionarily conserved mechanism that alters RNA sequences at the co-transcriptional or post-transcriptional level. RNA editing is hypothesized to facilitate adaptation in that it expands the transcriptomic and proteomic diversity. However, evidence for adaptation of RNA editing at the whole editome level is still lacking. In this study we systematically identified A-to-I RNA editing sites in female and male brains of three Drosophila species at different temperatures. With evolutionary analysis from different perspectives, we provide lines of evidence to demonstrate that the nonsynonymous editing sites in Drosophila brains are generally adaptive. The signals of adaptation for the editing sites are significantly enriched in genes related to chemical and electrical neurotransmission. We show that the RNA editing events might interplay with gene expression plasticity in temperature stress responses. Furthermore, we demonstrated that the expression level of Adar, together with the expression profiles of a set of genes that have editing sites favored by natural selection, were important in shaping the overall selective patterns of the global editomes at different developmental stages (or tissues) of D. melanogaster. Altogether our results support the hypothesis that A-to-I editing provides a driving force for adaptive evolution in Drosophila from different aspects.

Admixture—the mixing of genomes from divergent populations—is increasingly appreciated as a central process in evolution. To characterize and quantify patterns of admixture across the genome, a number of methods have been developed for local ancestry inference. However, existing approaches have a number of shortcomings. First, all local ancestry inference methods require some prior assumption about the expected ancestry tract lengths. Second, existing methods generally require genotypes, which is not feasible to obtain for many next-generation sequencing projects. Third, many methods assume samples are diploid, however a wide variety of sequencing applications will fail to meet this assumption. To address these issues, we introduce a novel hidden Markov model for estimating local ancestry that models the read pileup data, rather than genotypes, is generalized to arbitrary ploidy, and can estimate the time since admixture during local ancestry inference. We demonstrate that our method can simultaneously estimate the time since admixture and local ancestry with good accuracy, and that it performs well on samples of high ploidy—i.e. 100 or more chromosomes. As this method is very general, we expect it will be useful for local ancestry inference in a wider variety of populations than what previously has been possible. We then applied our method to pooled sequencing data derived from populations of Drosophila melanogaster on an ancestry cline on the east coast of North America. We find that regions of local recombination rates are negatively correlated with the proportion of African ancestry, suggesting that selection against foreign ancestry is the least efficient in low recombination regions. Finally we show that clinal outlier loci are enriched for genes associated with gene regulatory functions, consistent with a role of regulatory evolution in ecological adaptation of admixed D. melanogaster populations. Our results illustrate the potential of local ancestry inference for elucidating fundamental evolutionary processes., Author Summary When divergent populations hybridize, their offspring obtain portions of their genomes from each parent population. Although the average ancestry proportion in each descendant is equal to the proportion of ancestors from each of the ancestral populations, the contribution of each ancestry type is variable across the genome. Estimating local ancestry within admixed individuals is a fundamental goal for evolutionary genetics, and here we develop a method for doing this that circumvents many of the problems associated with existing methods. Briefly, our method can use short read data, rather than genotypes and can be applied to samples with any number of chromosomes. Furthermore, our method simultaneously estimates local ancestry and the number of generations since admixture—the time that the two ancestral populations first encountered each other. Finally, in applying our method to data from an admixture zone between ancestral populations of Drosophila melanogaster, we find many lines of evidence consistent with natural selection operating to against the introduction of foreign ancestry into populations of one predominant ancestry type. Because of the generality of this method, we expect that it will be useful for a wide variety of existing and ongoing research projects.

The term heterochromatin has been long considered synonymous with gene silencing, but it is now clear that the presence of transcribed genes embedded in pericentromeric heterochromatin is a conserved feature in the evolution of eukaryotic genomes. Several studies have addressed the epigenetic changes that enable the expression of genes in pericentric heterochromatin, yet little is known about the evolutionary processes through which this has occurred. By combining genome annotation analysis and high-resolution cytology, we have identified and mapped 53 orthologs of D. melanogaster heterochromatic genes in the genomes of two evolutionarily distant species, D. pseudoobscura and D. virilis. Our results show that the orthologs of the D. melanogaster heterochromatic genes are clustered at three main genomic regions in D. virilis and D. pseudoobscura. In D. virilis, the clusters lie in the middle of euchromatin, while those in D. pseudoobscura are located in the proximal portion of the chromosome arms. Some orthologs map to the corresponding Muller C element in D. pseudoobscura and D. virilis, while others localize on the Muller B element, suggesting that chromosomal rearrangements that have been instrumental in the fusion of two separate elements involved the progenitors of genes currently located in D. melanogaster heterochromatin. These results demonstrate an evolutionary repositioning of gene clusters from ancestral locations in euchromatin to the pericentromeric heterochromatin of descendent D. melanogaster chromosomes. Remarkably, in both D. virilis and D. pseudoobscura the gene clusters show a conserved association with the HP1a protein, one of the most highly evolutionarily conserved epigenetic marks. In light of these results, we suggest a new scenario whereby ancestral HP1-like proteins (and possibly other epigenetic marks) may have contributed to the evolutionary repositioning of gene clusters into heterochromatin., Author Summary This study concerns the evolutionary dynamics underlying the emergence of heterochromatic single-copy genes in D. melanogaster heterochromatin. By combining genome annotation analysis and high-resolution cytology, we have performed a comparative mapping of the orthologs of 53 single-copy genes of D. melanogaster heterochromatin, in the genomes of D. pseudoobscura and D. virilis evolutionarily distant species. The results of our work are consistent with a scenario where the D. melanogaster heterochromatin genes arose through an evolutionary repositioning from a euchromatic location (D. virilis) to heterochromatin, passing through an intermediate location in regions associated with distal heterochromatin (D. pseudoobscura). The previously unanticipated observation, that in both D. virilis and D. pseudoobscura the gene clusters show a striking association with the HP1a protein, is remarkable in that it implies that the HP1 epigenetic mark may have contributed to the the success of repositioning of genes to pericentromeric heterochromatin by protecting them against silencing effects.

Dosage compensation is an essential process that equalizes transcript levels of X-linked genes between sexes by forming a domain of coordinated gene expression. Throughout the evolution of Diptera, many different X-chromosomes acquired the ability to be dosage compensated. Once each newly evolved X-chromosome is targeted for dosage compensation in XY males, its active genes are upregulated two-fold to equalize gene expression with XX females. In Drosophila melanogaster, the CLAMP zinc finger protein links the dosage compensation complex to the X-chromosome. However, the mechanism for X-chromosome identification has remained unknown. Here, we combine biochemical, genomic and evolutionary approaches to reveal that expansion of GA-dinucleotide repeats likely accumulated on the X-chromosome over evolutionary time to increase the density of CLAMP binding sites, thereby driving the evolution of dosage compensation. Overall, we present new insight into how subtle changes in genomic architecture, such as expansions of a simple sequence repeat, promote the evolution of coordinated gene expression., Author Summary From stem cells to neurons, regulation of gene dosage is essential in all tissues and species in which it has been studied. Gene dosage must be balanced largely because it is critical to maintain the stoichiometry of components of multi-protein complexes that are encoded at diverse locations throughout the genome. The X-chromosome in many heterogametic species is a natural case where there is a seeming imbalance in gene dosage between sexes. How do cells correct imbalances in gene dosage? The key first step in dosage compensation across species is distinguishing the X-chromosome from the autosomes. Here we report a new mechanism by which newly evolved X-chromosomes become dosage compensated in Diptera: the expansion of simple GA dinucleotide repeats increases the density of the conserved CLAMP zinc finger protein that recruits the dosage compensation complex to the X-chromosome. Expansion of dinucleotide repeats can easily occur via slippage of DNA polymerase providing a simple mechanism for nucleating a domain of coordinated gene expression.

The regulatory architecture of gene expression remains an area of active research. Here, we studied how the interplay of genetic and environmental variation affects gene expression by exposing Drosophila melanogaster strains to four different developmental temperatures. At 18°C we observed almost complete canalization with only very few allelic effects on gene expression. In contrast, at the two temperature extremes, 13°C and 29°C a large number of allelic differences in gene expression were detected due to both cis- and trans-regulatory effects. Allelic differences in gene expression were mainly dominant, but for up to 62% of the genes the dominance swapped between 13 and 29°C. Our results are consistent with stabilizing selection causing buffering of allelic expression variation in non-stressful environments. We propose that decanalization of gene expression in stressful environments is not only central to adaptation, but may also contribute to genetic disorders in human populations.

Germline stem cell (GSC) self-renewal and differentiation are required for the sustained production of gametes. GSC differentiation in Drosophila oogenesis requires expression of the histone methyltransferase dSETDB1 by the somatic niche, however its function in this process is unknown. Here, we show that dSETDB1 is required for the expression of a Wnt ligand, Drosophila Wingless type mouse mammary virus integration site number 4 (dWnt4) in the somatic niche. dWnt4 signaling acts on the somatic niche cells to facilitate their encapsulation of the GSC daughter, which serves as a differentiation cue. dSETDB1 is known to repress transposable elements (TEs) to maintain genome integrity. Unexpectedly, we found that independent upregulation of TEs also downregulated dWnt4, leading to GSC differentiation defects. This suggests that dWnt4 expression is sensitive to the presence of TEs. Together our results reveal a chromatin-transposon-Wnt signaling axis that regulates stem cell fate., Author Summary Every multicellular organism is made up of tissues that are maintained by stem cells, due to their capacity to both self-renew and differentiate into terminal cell types. Loss of either of these processes can lead to aging, progression towards degenerative diseases and cancers. Insight into how self-renewal and differentiation are regulated will have tremendous therapeutic impact. Drosophila is an excellent model system for stem cell study due to the availability of various mutants, markers and RNAi technology. We study the ovaries of the female Drosophila, whose stem cell population gives rise to gametes. The tissue from which these gametes arise is surrounded by somatic cells, which provide signaling cues required for proper self-renewal and differentiation. While the mechanisms by which these somatic signals regulate stem cell self-renewal is known, how somatic cues regulate differentiation remains unclear. Here, we have identified that transposons, selfish genetic elements, modulate dWnt4 expression in the somatic cells. We demonstrate that dWnt4 promotes the somatic encapsulation of the stem cell daughter by regulating adherens junction proteins, thereby promoting differentiation. Transposons have been linked to cancers, and therefore establishing how transposons regulate genes essential for differentiation can provide new perspectives on their role in cancer.

Understanding the role of chromosomal inversions in speciation is a fundamental problem in evolutionary genetics. Here, we perform a comprehensive reconstruction of the evolutionary histories of the chromosomal inversions in Drosophila persimilis and D. pseudoobscura. We provide a solution to the puzzling origins of the selfish Sex-Ratio arrangement in D. persimilis and uncover surprising patterns of phylogenetic discordance on this chromosome. These patterns show that, contrary to widely held views, all fixed chromosomal inversions between D. persimilis and D. pseudoobscura were already present in their ancestral population long before the species split. Our results suggest that patterns of higher genomic divergence and an association of reproductive isolation genes with chromosomal inversions may be a direct consequence of incomplete lineage sorting of ancestral polymorphisms. These findings force a reconsideration of the role of chromosomal inversions in speciation, not as protectors of existing hybrid incompatibilities, but as fertile grounds for their formation., Author summary Studies on chromosomal inversions and reproductive isolation between Drosophila persimilis and D. pseudoobscura have played a profound role in shaping our understanding of inversions, speciation and selfish chromosomes. In this study, we reconstruct the evolutionary histories of chromosomal inversions in D. persimilis and D. pseudoobscura to show that, contrary to widely accepted ideas, these inversions existed as polymorphisms in the ancestor of both species before their initial split. These findings force a reconsideration of the role of chromosomal inversions in speciation and raise the possibility that the higher genetic divergence of sequences spanning these chromosomal inversions and an association with hybrid incompatibility genes may be an emergent property of the long-term segregation of these inversions.

While most miRNA knockouts exhibit only subtle defects, a handful of miRNAs are profoundly required for development or physiology. A particularly compelling locus is Drosophila mir-279, which was reported as essential to restrict the emergence of CO2-sensing neurons, to maintain circadian rhythm, and to regulate ovarian border cells. The mir-996 locus is located near mir-279 and bears a similar seed, but they otherwise have distinct, conserved, non-seed sequences, suggesting their evolutionary maintenance for separate functions. We generated single and double deletion mutants of the mir-279 and mir-996 hairpins, and cursory analysis suggested that miR-996 was dispensable. However, discrepancies in the strength of individual mir-279 deletion alleles led us to uncover that all extant mir-279 mutants are deficient for mature miR-996, even though they retain its genomic locus. We therefore engineered a panel of genomic rescue transgenes into the double deletion background, allowing a pure assessment of miR-279 and miR-996 requirements. Surprisingly, detailed analyses of viability, olfactory neuron specification, and circadian rhythm indicate that miR-279 is completely dispensable. Instead, an endogenous supply of either mir-279 or mir-996 suffices for normal development and behavior. Sensor tests of nine key miR-279/996 targets showed their similar regulatory capacities, although transgenic gain-of-function experiments indicate partially distinct activities of these miRNAs that may underlie that co-maintenance in genomes. Altogether, we elucidate the unexpected genetics of this critical miRNA operon, and provide a foundation for their further study. More importantly, these studies demonstrate that multiple, vital, loss-of-function phenotypes can be rescued by endogenous expression of divergent seed family members, highlighting the importance of this miRNA region for in vivo function., Author Summary Amongst the small number of miRNA knockouts that exhibit substantially overt phenotypes, mutants of Drosophila mir-279 are notable. Previous studies have uncovered its essential requirements in a range of developmental and behavioral assays. Surprisingly, we find that the phenotypes attributed to mir-279 deletions depend on the unanticipated loss of expression of the downstream locus mir-996, whose genomic locus is retained in extant mir-279 mutants. These miRNAs share their seed regions but are divergent elsewhere in the mature sequences. We use precise genetic engineering to show that a single endogenous copy of either mir-279 or mir-996 can fully rescue viability, olfactory neuron, and circadian rhythm defects of double deletion animals. These data and genetic reagents set a new foundation for developmental and behavioral studies of this critical miRNA locus. More generally, these data demonstrate that multiple loss-of-function phenotypes can be rescued by endogenous expression of divergent seed family members, highlighting the importance and potentially sufficiency of this region for in vivo function.

In the Drosophila germline, transposable elements (TEs) are silenced by PIWI-interacting RNA (piRNA) that originate from distinct genomic regions termed piRNA clusters and are processed by PIWI-subfamily Argonaute proteins. Here, we explore the variation in the ability to restrain an alien TE in different Drosophila strains. The I-element is a retrotransposon involved in the phenomenon of I-R hybrid dysgenesis in Drosophila melanogaster. Genomes of R strains do not contain active I-elements, but harbour remnants of ancestral I-related elements. The permissivity to I-element activity of R females, called reactivity, varies considerably in natural R populations, indicating the existence of a strong natural polymorphism in defense systems targeting transposons. To reveal the nature of such polymorphisms, we compared ovarian small RNAs between R strains with low and high reactivity and show that reactivity negatively correlates with the ancestral I-element-specific piRNA content. Analysis of piRNA clusters containing remnants of I-elements shows increased expression of the piRNA precursors and enrichment by the Heterochromatin Protein 1 homolog, Rhino, in weak R strains, which is in accordance with stronger piRNA expression by these regions. To explore the nature of the differences in piRNA production, we focused on two R strains, weak and strong, and showed that the efficiency of maternal inheritance of piRNAs as well as the I-element copy number are very similar in both strains. At the same time, germline and somatic uni-strand piRNA clusters generate more piRNAs in strains with low reactivity, suggesting the relationship between the efficiency of primary piRNA production and variable response to TE invasions. The strength of adaptive genome defense is likely driven by naturally occurring polymorphisms in the rapidly evolving piRNA pathway proteins. We hypothesize that hyper-efficient piRNA production is contributing to elimination of a telomeric retrotransposon HeT-A, which we have observed in one particular transposon-resistant R strain., Author summary Transposon activity in the germline is suppressed by the PIWI-interacting RNA (piRNA) pathway. The resistance of natural Drosophila strains to transposon invasion varies considerably, but the nature of this variability is unknown. We discovered that natural variation in the efficiency of primary piRNA production in the germline causes dramatic differences in the susceptibility to expansion of a newly invaded transposon. A high level of piRNA production in the germline is achieved by increased expression of piRNA precursors. In one of the most transposon-resistant strains, increased content of primary piRNA is observed in both the germline and ovarian somatic cells. We suggest that polymorphisms in piRNA pathway factors are responsible for increased piRNA production. piRNA pathway proteins have been shown to be evolving rapidly under selective pressure. Our data are the first to describe a phenotype that might be caused by this kind of polymorphism. We also demonstrate a likely explanation as to why an overly active piRNA pathway can cause more harm than good in Drosophila: Highly efficient piRNA processing leads to elimination of domesticated telomeric retrotransposons essential for telomere elongation, an effect which has been observed in a natural strain that is extremely resistant to transposon invasion.

Environmental heterogeneity has been hypothesized to influence levels of genetic variation but the effect of heterogeneity depends on (i) the form of heterogeneity, (ii) whether ecologically relevant or neutral loci are being considered, and (iii) the genetic basis of ecological adaptation. We surveyed genome-wide SNP diversity in replicate experimental Drosophila melanogaster populations with equal census sizes that evolved for 42 generations under one of four selection regimes: (i) salt-enriched environment (Salt), (ii) cadmium-enriched environment (Cad), (iii) temporally (Temp) or (iv) spatially (Spatial) variable environments. There was significant differentiation between all pairs of treatments but the greatest differentiation occurred between the two homogenous treatments (Cad and Salt). For sites likely under differential ecological selection (and those closely linked to them), the pattern of within-population diversity π followed the expectation from classic antagonistic selection theory: Spatial>Temp>Salt≈Cad. However, neutral diversity unlinked to selected sites followed a different pattern: Spatial>Salt≈Cad>Temp. As implicated by the latter result, measures of FST among replicate populations within treatments are consistent with differences in effective population sizes among selective regimes despite equal census sizes. Though there are clear changes in the rank order of treatments when contrasting selected and neutral sites with respect to π, the rank ordering of treatments with respect to FST appears reasonably consistent between site categories. These results demonstrate that alternative selective regimes affect within- and among-population diversity differently for different site types., Author Summary Evolutionary biologists seek to understand the factors affecting genetic variation. While it is intuitive that environmental heterogeneity should increase levels of variation, theoretical models showed that spatial and temporal heterogeneity differ in how likely they are to maintain polymorphisms affecting fitness. We evolved experimental populations of fruit flies in constant environments or in temporally or spatially varying environments, then examined levels of sequence variation across the genome. For sites associated with ecological selection, polymorphism patterns matched the theoretical expectations with variation greatest in populations evolving in spatially heterogeneous environments, less variation in populations evolving in temporally heterogeneous environments, and least variation in populations evolving in constant environments. However, a different pattern was observed at sites not associated with differential ecological selection (i.e., most of the genome). For these sites, levels of variation were highest at spatially heterogeneous populations but lowest for temporally heterogeneous populations. Populations evolving under temporal heterogeneity also showed the greatest differentiation from one another, suggesting that this selection regime caused more genetic drift than other selection regimes. These results illustrate that environmental heterogeneity affects levels of variation not only at sites subject to differential ecological selection but also genome-wide, though spatial and temporal heterogeneity affect diversity differently.

Modern functional genomics uncovered numerous functional elements in metazoan genomes. Nevertheless, only a small fraction of the typical non-exonic genome contains elements that code for function directly. On the other hand, a much larger fraction of the genome is associated with significant evolutionary constraints, suggesting that much of the non-exonic genome is weakly functional. Here we show that in flies, local (30–70 bp) conserved sequence elements that are associated with multiple regulatory functions serve as focal points to a pattern of punctuated regional increase in G/C nucleotide frequencies. We show that this pattern, which covers a region tenfold larger than the conserved elements themselves, is an evolutionary consequence of a shift in the balance between gain and loss of G/C nucleotides and that it is correlated with nucleosome occupancy across multiple classes of epigenetic state. Evidence for compensatory evolution and analysis of SNP allele frequencies show that the evolutionary regime underlying this balance shift is likely to be non-neutral. These data suggest that current gaps in our understanding of genome function and evolutionary dynamics are explicable by a model of sparse sequence elements directly encoding for function, embedded into structural sequences that help to define the local and global epigenomic context of such functional elements., Author Summary A key challenge in functional genomics is to predict evolutionary dynamics from functional annotation of the genome and vice versa. Modern epigenomic studies helped assign function to numerous new sequence elements, but left most of the genome essentially uncharacterized. Evolutionary genomics, on the other hand, consistently suggests that a much larger fraction of the un-annotated genome evolves under selective pressure. We hypothesize that this function-selection gap can be attributed to sequences that facilitate the physical organization of functional elements, such as transcription factor binding sites, within chromosomes. We exemplify this by studying in detail the sequences embedding small conserved elements (CEs) in Drosophila. We show that, while CEs have typically high AT content, high GC content levels around them are maintained by a non-neutral evolutionary balance between gain and loss of GC nucleotides. This non-uniform pattern is highly correlated with nucleosome organization around CEs, potentially imposing an evolutionary constraint on as much as one quarter of the genome. We suggest this can at least partly explain the above function-selection gap. Weak evolutionary constraints on “structural” sequences (at scales ranging from one nucleosome to recently described multi-megabase topological domains) may affect genome evolution just like structural motifs shape protein evolution.

The reproductive ground plan hypothesis (RGPH) proposes that the physiological pathways regulating reproduction were co-opted to regulate worker division of labor. Support for this hypothesis in honeybees is provided by studies demonstrating that the reproductive potential of workers, assessed by the levels of vitellogenin (Vg), is linked to task performance. Interestingly, contrary to honeybees that have a single Vg ortholog and potentially fertile nurses, the genome of the harvester ant Pogonomyrmex barbatus harbors two Vg genes (Pb_Vg1 and Pb_Vg2) and nurses produce infertile trophic eggs. P. barbatus, thus, provides a unique model to investigate whether Vg duplication in ants was followed by subfunctionalization to acquire reproductive and non-reproductive functions and whether Vg reproductive function was co-opted to regulate behavior in sterile workers. To investigate these questions, we compared the expression patterns of P. barbatus Vg genes and analyzed the phylogenetic relationships and molecular evolution of Vg genes in ants. qRT-PCRs revealed that Pb_Vg1 is more highly expressed in queens compared to workers and in nurses compared to foragers. By contrast, the level of expression of Pb_Vg2 was higher in foragers than in nurses and queens. Phylogenetic analyses show that a first duplication of the ancestral Vg gene occurred after the divergence between the poneroid and formicoid clades and subsequent duplications occurred in the lineages leading to Solenopsis invicta, Linepithema humile and Acromyrmex echinatior. The initial duplication resulted in two Vg gene subfamilies preferentially expressed in queens and nurses (subfamily A) or in foraging workers (subfamily B). Finally, molecular evolution analyses show that the subfamily A experienced positive selection, while the subfamily B showed overall relaxation of purifying selection. Our results suggest that in P. barbatus the Vg gene underwent subfunctionalization after duplication to acquire caste- and behavior- specific expression associated with reproductive and non-reproductive functions, supporting the validity of the RGPH in ants., Author Summary One of the main features of social insects is the division of labor, whereby queens monopolize reproduction while sterile workers perform all of the tasks related to colony maintenance. The workers usually do so in an age-dependent sequence: young workers tend to nurse the brood inside the nest and older workers are more likely to forage for food. Previous studies revealed that vitellogenin, a yolk protein typically involved in the regulation of reproduction in solitary insects, has been co-opted to regulate division of labor in the honeybee. In this study, we investigate such a role of vitellogenin in another group of social insects: the ants. We first use phylogenetic analyses to reveal the existence of multiple vitellogenin genes in most of the sequenced ant genomes. Then we compare the expression of the two vitellogenin genes (Pb_Vg1 and Pb_Vg2) among queens, nurses and foragers in the seed-harvester ant Pogonomyrmex barbatus. Our results suggest that, after the initial duplication in ants, the vitellogenin genes acquired caste and behavioral specific expression associated with reproductive and non-reproductive nutritionally related functions. This study also shows that ants and bees, despite having evolved sociality independently, have conserved similar mechanisms to regulate division of labor.

Polycomb group response elements (PREs) in Drosophila are DNA-elements that recruit Polycomb proteins (PcG) to chromatin and regulate gene expression. PREs are easily recognizable in the Drosophila genome as strong peaks of PcG-protein binding over discrete DNA fragments; many small but statistically significant PcG peaks are also observed in PcG domains. Surprisingly, in vivo deletion of the four characterized strong PREs from the PcG regulated invected-engrailed (inv-en) gene complex did not disrupt the formation of the H3K27me3 domain and did not affect inv-en expression in embryos or larvae suggesting the presence of redundant PcG recruitment mechanism. Further, the 3D-structure of the inv-en domain was only minimally altered by the deletion of the strong PREs. A reporter construct containing a 7.5kb en fragment that contains three weak peaks but no large PcG peaks forms an H3K27me3 domain and is PcG-regulated. Our data suggests a model for the recruitment of PcG-complexes to Drosophila genes via interactions with multiple, weak PREs spread throughout an H3K27me3 domain., Author Summary Polycomb group proteins (PcGs) regulate growth and development of multi-cellular organisms by modifying histones and inhibiting chromatin remodeling resulting in gene silencing. PcGs act via binding to cis-regulatory DNA sequences known as Polycomb response elements (PREs). In genome wide studies, PREs are recognized as strong binding sites for PcG proteins, often near the promoter but many, weaker PcG binding sites are often spread throughout a PcG-domain. It was not known if these weak peaks were functional. We deleted the strong PcG peaks from the PcG-domain containing the invected and engrailed genes and show that a PcG-domain still forms. Further analysis showed these weak sites can act as PREs by recruiting PcGs and modifying histone H3. These weak peaks interact with each other and the target genes to maintain the three-dimensional structure of the PcG domain in the absence of strong peaks. We propose a model, where presence of several weak PREs along with strong ones ensures uniform distribution of histones modification over a PcG domain.

It is widely documented that hybridisation occurs between many closely related species, but the importance of introgression in adaptive evolution remains unclear, especially in animals. Here, we have examined the role of introgressive hybridisation in transferring adaptations between mimetic Heliconius butterflies, taking advantage of the recent identification of a gene regulating red wing patterns in this genus. By sequencing regions both linked and unlinked to the red colour locus, we found a region that displays an almost perfect genotype by phenotype association across four species, H. melpomene, H. cydno, H. timareta, and H. heurippa. This particular segment is located 70 kb downstream of the red colour specification gene optix, and coalescent analysis indicates repeated introgression of adaptive alleles from H. melpomene into the H. cydno species clade. Our analytical methods complement recent genome scale data for the same region and suggest adaptive introgression has a crucial role in generating adaptive wing colour diversity in this group of butterflies., Author Summary Hybridisation occurs between many animal species, however its evolutionary relevance is still a matter of great debate. While some argue that hybridisation leads to maladaptive gene combinations, and therefore to an evolutionary dead end, others consider interspecific hybridisation as a process with great potential to fuel evolution. We examine this question by exploring the origins of red wing colouration, a trait under natural selection, in the adaptive radiation of closely related species of Heliconius butterflies. By sequencing genetic regions both linked and unlinked to the red wing pattern locus, we found experimental evidence supporting multiple hybridisation events that have mediated the acquisition of colour adaptations from H. melpomene to H. timareta. This introgression has allowed H. timareta to colonise new fitness peaks in the Müllerian mimicry landscape. In this way, our results support the idea that interspecific hybridisation in animals constitutes a source of genetic variation that promotes diversification.

Glioblastoma, the most common primary malignant brain tumor, is incurable with current therapies. Genetic and molecular analyses demonstrate that glioblastomas frequently display mutations that activate receptor tyrosine kinase (RTK) and Pi-3 kinase (PI3K) signaling pathways. In Drosophila melanogaster, activation of RTK and PI3K pathways in glial progenitor cells creates malignant neoplastic glial tumors that display many features of human glioblastoma. In both human and Drosophila, activation of the RTK and PI3K pathways stimulates Akt signaling along with other as-yet-unknown changes that drive oncogenesis. We used this Drosophila glioblastoma model to perform a kinome-wide genetic screen for new genes required for RTK- and PI3K-dependent neoplastic transformation. Human orthologs of novel kinases uncovered by these screens were functionally assessed in mammalian glioblastoma models and human tumors. Our results revealed that the atypical kinases RIOK1 and RIOK2 are overexpressed in glioblastoma cells in an Akt-dependent manner. Moreover, we found that overexpressed RIOK2 formed a complex with RIOK1, mTor, and mTor-complex-2 components, and that overexpressed RIOK2 upregulated Akt signaling and promoted tumorigenesis in murine astrocytes. Conversely, reduced expression of RIOK1 or RIOK2 disrupted Akt signaling and caused cell cycle exit, apoptosis, and chemosensitivity in glioblastoma cells by inducing p53 activity through the RpL11-dependent ribosomal stress checkpoint. These results imply that, in glioblastoma cells, constitutive Akt signaling drives RIO kinase overexpression, which creates a feedforward loop that promotes and maintains oncogenic Akt activity through stimulation of mTor signaling. Further study of the RIO kinases as well as other kinases identified in our Drosophila screen may reveal new insights into defects underlying glioblastoma and related cancers and may reveal new therapeutic opportunities for these cancers., Author Summary Glioblastomas, the most common primary brain tumor, harbor mutations in receptor tyrosine kinases (RTKs), such as EGFR, and components of the Pi-3 kinase (PI3K) signaling pathway. However, the genes that act downstream of RTK and PI3K signaling to drive glioblastoma remain unclear. To investigate the genetic and molecular basis of this disease, we created a glioblastoma model in the fruit fly Drosophila melanogaster. To identify new genes involved in glioblastoma development, we performed a screen for the genes required for tumor cell proliferation using our Drosophila glioblastoma model and then functionally assessed the activity of human versions of novel genes identified in this screen. Our results revealed that the RIO kinases become overexpressed in human glioblastomas but not in normal human glial or neuronal cells. We found that overexpression of the RIO kinases promotes and maintains signals that drive tumor cell proliferation and survival in RTK- and PI3K-dependent human glioblastoma, and reduction of RIO kinase expression decreased proliferation and prompted cell death and chemosensitivity in glioblastoma cells. Therefore, disruption of the RIO kinases may provide new therapeutic opportunities to target glioblastoma and other RTK- or PI3K-dependent cancers.

The extent and nature of genetic incompatibilities between incipient races and sibling species is of fundamental importance to our view of speciation. However, with the exception of hybrid inviability and sterility factors, little is known about the extent of other, more subtle genetic incompatibilities between incipient species. Here we experimentally demonstrate the prevalence of such genetic incompatibilities between two young allopatric sibling species, Drosophila simulans and D. sechellia. Our experiments took advantage of 12 introgression lines that carried random introgressed D. sechellia segments in different parts of the D. simulans genome. First, we found that these introgression lines did not show any measurable sterility or inviability effects. To study if these sechellia introgressions in a simulans background contained other fitness consequences, we competed and genetically tracked the marked alleles within each introgression against the wild-type alleles for 20 generations. Strikingly, all marked D. sechellia introgression alleles rapidly decreased in frequency in only 6 to 7 generations. We then developed computer simulations to model our competition results. These simulations indicated that selection against D. sechellia introgression alleles was high (average s = 0.43) and that the marker alleles and the incompatible alleles did not separate in 78% of the introgressions. The latter result likely implies that most introgressions contain multiple genetic incompatibilities. Thus, this study reveals that, even at early stages of speciation, many parts of the genome diverge to a point where introducing foreign elements has detrimental fitness consequences, but which cannot be seen using standard sterility and inviability assays., Author Summary Determining the extent of genomic incompatibilities is a pivotal issue in understanding the process of speciation. A controversial topic that has recently sparked debate is whether there are few isolated genetic regions (so-called “genomic islands of speciation”) or extensive genetic regions (“genomic continents of speciation”) responsible for species divergence. To answer this question, most work has focused on species divergence at the DNA sequence level. Here, we present a new perspective by shifting the focus to the fitness and functional aspects of foreign genomic introgression. To illustrate our point, we performed an introgression experiment on two sibling species, D. sechellia and D. simulans. After introgressing random genomic segments of D. sechellia into D. simulans genetic background, a 20-generation competition experiment revealed that, even at the early stages of speciation, there are virtually always detrimental fitness consequences to introducing random foreign elements from one genome to another. This implies that incipient speciation may be characterized by widespread accumulation of genomic incompatibilities rather than a few isolated genes. This study shows that we should move beyond the sterility and inviability assays in order to understand the full extent of genetic incompatibilities during speciation.

Drosophila melanogaster has played a pivotal role in the development of modern population genetics. However, many basic questions regarding the demographic and adaptive history of this species remain unresolved. We report the genome sequencing of 139 wild-derived strains of D. melanogaster, representing 22 population samples from the sub-Saharan ancestral range of this species, along with one European population. Most genomes were sequenced above 25X depth from haploid embryos. Results indicated a pervasive influence of non-African admixture in many African populations, motivating the development and application of a novel admixture detection method. Admixture proportions varied among populations, with greater admixture in urban locations. Admixture levels also varied across the genome, with localized peaks and valleys suggestive of a non-neutral introgression process. Genomes from the same location differed starkly in ancestry, suggesting that isolation mechanisms may exist within African populations. After removing putatively admixed genomic segments, the greatest genetic diversity was observed in southern Africa (e.g. Zambia), while diversity in other populations was largely consistent with a geographic expansion from this potentially ancestral region. The European population showed different levels of diversity reduction on each chromosome arm, and some African populations displayed chromosome arm-specific diversity reductions. Inversions in the European sample were associated with strong elevations in diversity across chromosome arms. Genomic scans were conducted to identify loci that may represent targets of positive selection within an African population, between African populations, and between European and African populations. A disproportionate number of candidate selective sweep regions were located near genes with varied roles in gene regulation. Outliers for Europe-Africa FST were found to be enriched in genomic regions of locally elevated cosmopolitan admixture, possibly reflecting a role for some of these loci in driving the introgression of non-African alleles into African populations., Author Summary Improvements in DNA sequencing technology have allowed genetic variation to be studied at the level of fully sequenced genomes. We have sequenced more than 100 D. melanogaster genomes originating from sub-Saharan Africa, which is thought to contain the ancestral range of this model organism. We found evidence for recent and substantial non-African gene flow into African populations, which may be driven by natural selection. The data also helped to refine our understanding of the species' history, which may have involved a geographic expansion from southern central Africa (e.g. Zambia). Lastly, we identified a large number of genes and functions that may have experienced recent adaptive evolution in one or more populations. An understanding of genomic variation in ancestral range populations of D. melanogaster will improve our ability to make population genetic inferences for worldwide populations. The results presented here should motivate statistical, mathematical, and computational studies to identify evolutionary models that are most compatible with observed data. Finally, the potential signals of natural selection identified here should facilitate detailed follow-up studies on the genetic basis of adaptive evolutionary change.

Estimating fine-scale recombination maps of Drosophila from population genomic data is a challenging problem, in particular because of the high background recombination rate. In this paper, a new computational method is developed to address this challenge. Through an extensive simulation study, it is demonstrated that the method allows more accurate inference, and exhibits greater robustness to the effects of natural selection and noise, compared to a well-used previous method developed for studying fine-scale recombination rate variation in the human genome. As an application, a genome-wide analysis of genetic variation data is performed for two Drosophila melanogaster populations, one from North America (Raleigh, USA) and the other from Africa (Gikongoro, Rwanda). It is shown that fine-scale recombination rate variation is widespread throughout the D. melanogaster genome, across all chromosomes and in both populations. At the fine-scale, a conservative, systematic search for evidence of recombination hotspots suggests the existence of a handful of putative hotspots each with at least a tenfold increase in intensity over the background rate. A wavelet analysis is carried out to compare the estimated recombination maps in the two populations and to quantify the extent to which recombination rates are conserved. In general, similarity is observed at very broad scales, but substantial differences are seen at fine scales. The average recombination rate of the X chromosome appears to be higher than that of the autosomes in both populations, and this pattern is much more pronounced in the African population than the North American population. The correlation between various genomic features—including recombination rates, diversity, divergence, GC content, gene content, and sequence quality—is examined using the wavelet analysis, and it is shown that the most notable difference between D. melanogaster and humans is in the correlation between recombination and diversity., Author Summary Recombination is a process by which chromosomes exchange genetic material during meiosis. It is important in evolution because it provides offspring with new combinations of genes, and so estimating the rate of recombination is of fundamental importance in various population genomic inference problems. In this paper, we develop a new statistical method to enable robust estimation of fine-scale recombination maps of Drosophila, a genus of common fruit flies, in which the background recombination rate is high and natural selection has been prevalent. We apply our method to produce fine-scale recombination maps for a North American population and an African population of D. melanogaster. For both populations, we find extensive fine-scale variation in recombination rate throughout the genome. We provide a quantitative characterization of the similarities and differences between the recombination maps of the two populations; our study reveals high correlation at broad scales and low correlation at fine scales, as has been documented among human populations. We also examine the correlation between various genomic features. Furthermore, using a conservative approach, we find a handful of putative recombination “hotspot” regions with solid statistical support for a local elevation of at least 10 times the background recombination rate.

Predicting organismal phenotypes from genotype data is important for plant and animal breeding, medicine, and evolutionary biology. Genomic-based phenotype prediction has been applied for single-nucleotide polymorphism (SNP) genotyping platforms, but not using complete genome sequences. Here, we report genomic prediction for starvation stress resistance and startle response in Drosophila melanogaster, using ∼2.5 million SNPs determined by sequencing the Drosophila Genetic Reference Panel population of inbred lines. We constructed a genomic relationship matrix from the SNP data and used it in a genomic best linear unbiased prediction (GBLUP) model. We assessed predictive ability as the correlation between predicted genetic values and observed phenotypes by cross-validation, and found a predictive ability of 0.239±0.008 (0.230±0.012) for starvation resistance (startle response). The predictive ability of BayesB, a Bayesian method with internal SNP selection, was not greater than GBLUP. Selection of the 5% SNPs with either the highest absolute effect or variance explained did not improve predictive ability. Predictive ability decreased only when fewer than 150,000 SNPs were used to construct the genomic relationship matrix. We hypothesize that predictive power in this population stems from the SNP–based modeling of the subtle relationship structure caused by long-range linkage disequilibrium and not from population structure or SNPs in linkage disequilibrium with causal variants. We discuss the implications of these results for genomic prediction in other organisms., Author Summary The ability to accurately predict values of complex phenotypes from genotype data will revolutionize plant and animal breeding, personalized medicine, and evolutionary biology. To date, genomic prediction has utilized high-density single-nucleotide polymorphism (SNP) genotyping arrays, but the availability of sequence data opens new frontiers for genomic prediction methods. This article is the first application of genomic phenotype prediction using whole-genome sequence data in a substantial sample of a higher eukaryote. We use ∼2.5 million SNPs with minor allele frequency greater than 2.5% derived from genomic sequences of the “Drosophila Genetic Reference Panel” to predict phenotypes for two traits, starvation resistance and startle-induced locomotor behavior. We systematically address prediction within versus across sexes, genomic best linear unbiased prediction (GBLUP) versus a Bayesian approach, and the effect of SNP density. We find that (i) genomic prediction can be efficiently implemented using sequence data via GBLUP, (ii) there is little gain in predictive ability if the number of SNPs is increased above 150,000, and (iii) neither implicit nor explicit marker selection substantially improves the predictive ability. Although the findings must be seen against the background of small sample sizes, the results illustrate both the potential of the approach and the challenges ahead.

The extent to which hybridization and the resulting interspecific gene flow (introgression) contribute to adaptation is a matter of great debate. On the one hand, fertile hybrids have the potential to transfer beneficial alleles between species [1] or even to spawn new species [2]. On the other hand, hybrids tend to have reduced fitness relative to parental species [3], which will often make them an evolutionary dead end. Only a handful of examples of adaptive introgression are known, such as recent evidence for the transfer of warfarin resistance between mouse species [4]. However, one place where introgression of beneficial alleles could be common is in adaptive radiations [5]. These explosions of phenotypic and species diversity may be just the place to look for adaptive introgression because they often contain closely related, hybridizing species, and introgression could provide the raw genetic material for their exceptional rates of diversification. Two new papers, one in this issue of PLoS Genetics [6] and another in Nature [7], provide long-awaited evidence for a direct role of introgression in fueling a particularly striking adaptive radiation, the mimetic wing pattern radiation of Heliconius butterflies. The Neotropical genus Heliconius is a diverse clade of brightly colored and chemically defended butterflies. This group is well-known for mimicry, in which different species evolve nearly identical wing patterns as a means of protection from predators [8]. Rapid evolution of wing pattern diversity in Heliconius, combined with convergence due to mimicry, has resulted in a group of closely related and hybridizing species, some of which look very different and others that look nearly identical. These two new papers show that alleles for wing patterning have moved across species boundaries multiple times, effectively transferring mimicry from one species to another. This discovery of adaptive introgression in Heliconius builds upon five important prior advances. First, close to a decade ago, Larry Gilbert used results from a multitude of interspecific crosses to propose a model whereby Heliconius mimicry evolved by repeated interspecific transfer of color patterning alleles [9]. Second, surveys of wild-caught specimens have revealed many instances of natural hybridization in Heliconius [10], and molecular analyses based on neutral markers detected signatures of relatively widespread introgression among closely related species [11], [12]. Third, recent discoveries of cryptic species have provided multiple examples of sympatric, co-mimetic, and potentially hybridizing species [13]; species among which mimicry transfer might be particularly likely. Fourth, there has been a concerted effort by those studying Heliconius to map and characterize their mimicry loci [14]. This recently culminated in the identification of optix as the red patterning gene [15], with high-resolution SNP data revealing strong associations just upstream of the gene [16]. Finally, new population genetic data from optix itself revealed that, within polymorphic species like H. erato and H. melpomene, similar wing patterns in distinct subspecies share a common origin [17]. This result, while focused on within-species variation, showed that apparent convergence can result from shared ancestry of mimicry alleles. The two new studies build on this foundation to explore adaptive introgression of mimicry using different but highly complementary approaches. Pardo-Diaz et al. [6] use amplicon sequencing from targeted portions around optix, combined with phylogenetic- and coalescent-based tests for gene flow. The Heliconius Genome Consortium [7] present a reference genome sequence for H. melpomene, and then use RAD markers, targeted resequencing, and an ABBA-BABA statistical approach to examine introgression genome-wide, with a special emphasis on the optix interval, as well as a second region that controls yellow color patterning. The results of the two studies are highly congruent—genetic variation from a narrow genomic interval just upstream of optix groups populations and species by red wing patterning rather than known phylogenetic relationships (Figure 1). Amazingly, this even applies to H. elevatus, a “rayed” pattern species from the silvaniform clade, a group that generally displays “tiger-stripe” patterns. Importantly, The Heliconius Genome Consortium [7] show that these signatures of introgression ultimately encompass hundreds of SNPs, ruling out the possibility that these groupings are the result of convergent molecular evolution. Figure 1 Two new papers show that wing patterning has been swapped among Heliconius butterfly species via introgressive hybridization. This evidence for adaptive introgression is striking, yet there is much that remains unknown. For instance, H. besckei is the only silvaniform species with a “red band” phenotype, but it remains unknown if it swapped mimicry genes with other red banded species (Figure 1). Furthermore, there are potential instances of more subtle introgression that have yet to be explored; cases in which a single pattern element, as opposed to an entire phenotype, appears to be shared between species [9]. Other important questions include: In which species did mimicry alleles originally arise and when did they spread? How frequently does mimicry introgression precipitate speciation? How do these closely related, sympatric, co-mimetic taxa remain distinct given color pattern's important role in generating reproductive isolation [18]? What about adaptive introgression of other traits and genes not related to mimicry? Has introgression contributed to diversity and mimicry in other Heliconius clades? Beyond Heliconius, it is important that we better understand the frequency and taxonomic distribution of adaptive introgression, as well as its potential link to adaptive radiation. These new results from Heliconius reveal that introgression has played an essential role in driving adaptive evolution across an entire clade. Whether we find a more general role for introgression in facilitating adaptive radiation remains to be seen. However, the fact that the first comprehensive examination revealed widespread adaptive introgression certainly makes this a very real possibility.

Base-excision repair and control of nucleotide pools safe-guard against permanent uracil accumulation in DNA relying on two key enzymes: uracil–DNA glycosylase and dUTPase. Lack of the major uracil–DNA glycosylase UNG gene from the fruit fly genome and dUTPase from fruit fly larvae prompted the hypotheses that i) uracil may accumulate in Drosophila genomic DNA where it may be well tolerated, and ii) this accumulation may affect development. Here we show that i) Drosophila melanogaster tolerates high levels of uracil in DNA; ii) such DNA is correctly interpreted in cell culture and embryo; and iii) under physiological spatio-temporal control, DNA from fruit fly larvae, pupae, and imago contain greatly elevated levels of uracil (200–2,000 uracil/million bases, quantified using a novel real-time PCR–based assay). Uracil is accumulated in genomic DNA of larval tissues during larval development, whereas DNA from imaginal tissues contains much less uracil. Upon pupation and metamorphosis, uracil content in DNA is significantly decreased. We propose that the observed developmental pattern of uracil–DNA is due to the lack of the key repair enzyme UNG from the Drosophila genome together with down-regulation of dUTPase in larval tissues. In agreement, we show that dUTPase silencing increases the uracil content in DNA of imaginal tissues and induces strong lethality at the early pupal stages, indicating that tolerance of highly uracil-substituted DNA is also stage-specific. Silencing of dUTPase perturbs the physiological pattern of uracil–DNA accumulation in Drosophila and leads to a strongly lethal phenotype in early pupal stages. These findings suggest a novel role of uracil-containing DNA in Drosophila development and metamorphosis and present a novel example for developmental effects of dUTPase silencing in multicellular eukaryotes. Importantly, we also show lack of the UNG gene in all available genomes of other Holometabola insects, indicating a potentially general tolerance and developmental role of uracil–DNA in this evolutionary clade., Author Summary The usual paradigm confines “normal” DNA of living cells to a well-defined restricted chemical space populated with only four bases (adenine, thymine, guanine, and cytosine) and some of their methylated derivatives (e.g. 5′-methyl-cytosine). Uracil is not considered to be a “normal” DNA base, except in several bacteriophages. On the contrary, uracil is generally considered to be an error in DNA. We show that Drosophila cells interpret uracil-substituted DNA as normal DNA, due to lack of two repair enzymes. Importantly, this unusual trait is under developmental control and applies only for animals before pupation. Metamorphosis is drastically perturbed by silencing of dUTPase, responsible for keeping uracil out of DNA. Our results argue that in Drosophila, and perhaps in other Holometabola insects as well, uracil–DNA plays a dedicated physiological role.

Transposable elements (TEs) are mobile genetic elements that parasitize genomes by semi-autonomously increasing their own copy number within the host genome. While TEs are important for genome evolution, appropriate methods for performing unbiased genome-wide surveys of TE variation in natural populations have been lacking. Here, we describe a novel and cost-effective approach for estimating population frequencies of TE insertions using paired-end Illumina reads from a pooled population sample. Importantly, the method treats insertions present in and absent from the reference genome identically, allowing unbiased TE population frequency estimates. We apply this method to data from a natural Drosophila melanogaster population from Portugal. Consistent with previous reports, we show that low recombining genomic regions harbor more TE insertions and maintain insertions at higher frequencies than do high recombining regions. We conservatively estimate that there are almost twice as many “novel” TE insertion sites as sites known from the reference sequence in our population sample (6,824 novel versus 3,639 reference sites, with on average a 31-fold coverage per insertion site). Different families of transposable elements show large differences in their insertion densities and population frequencies. Our analyses suggest that the history of TE activity significantly contributes to this pattern, with recently active families segregating at lower frequencies than those active in the more distant past. Finally, using our high-resolution TE abundance measurements, we identified 13 candidate positively selected TE insertions based on their high population frequencies and on low Tajima's D values in their neighborhoods., Author Summary Transposable elements (TE's) are parasitic genetic elements that spread by replicating themselves within a host genome. Most organisms are burdened with transposable elements; in fact, up to 80% of some genomes can consist of TE–derived DNA. Here, we use new sequencing technology to examine variation in genomic TE composition within a population at a finer scale and in a more unbiased fashion than has been possible before. We study a Portuguese population of D. melanogaster and find a large number of TE insertions, most of which occur in few individuals. Our analysis confirms that TE insertions are subject to purifying selection that counteracts their spread, and it suggests that the genome records waves of past TE invasions, with recently active elements occurring at low population frequency. We also find indications that TE insertions may sometimes have beneficial effects.

Why gene order is conserved over long evolutionary timespans remains elusive. A common interpretation is that gene order conservation might reflect the existence of functional constraints that are important for organismal performance. Alteration of the integrity of genomic regions, and therefore of those constraints, would result in detrimental effects. This notion seems especially plausible in those genomes that can easily accommodate gene reshuffling via chromosomal inversions since genomic regions free of constraints are likely to have been disrupted in one or more lineages. Nevertheless, no empirical test has been performed to this notion. Here, we disrupt one of the largest conserved genomic regions of the Drosophila genome by chromosome engineering and examine the phenotypic consequences derived from such disruption. The targeted region exhibits multiple patterns of functional enrichment suggestive of the presence of constraints. The carriers of the disrupted collinear block show no defects in their viability, fertility, and parameters of general homeostasis, although their odorant perception is altered. This change in odorant perception does not correlate with modifications of the level of expression and sex bias of the genes within the genomic region disrupted. Our results indicate that even in highly rearranged genomes, like those of Diptera, unusually high levels of gene order conservation cannot be systematically attributed to functional constraints, which raises the possibility that other mechanisms can be in place and therefore the underpinnings of the maintenance of gene organization might be more diverse than previously thought., Author Summary Eukaryotic genomes have been reshaped by chromosomal rearrangements during evolution. However, the comparison of distantly related species has uncovered unusually large genomic regions with conserved gene organization. A widely accepted explanation is that, in those regions, there exist genes with joint and/or intricate regulation, which, if altered, might affect the performance of the carriers. We used a system that allowed us to precisely disrupt one of the largest genomic regions that has been conserved in the fruit flies since ∼63 million years ago and performed a variety of assays devised to detect differences in organismal performance and anomalous gene expression. Despite the overrepresentation of genes with expression profiles related to male fertility and detection of chemical stimuli in this genomic region, as well as the presence of genes expressed during development and putatively regulated by long distantly located sequences, we do not find evidence of diminished viability and fertility or of anomalous levels of expression. These results lead us to propose that regulatory constraints might not suffice to explain the maintenance of the integrity of some of the best candidate regions in one of the most dynamic eukaryotic genomes.

Transcription factors that drive complex patterns of gene expression during animal development bind to thousands of genomic regions, with quantitative differences in binding across bound regions mediating their activity. While we now have tools to characterize the DNA affinities of these proteins and to precisely measure their genome-wide distribution in vivo, our understanding of the forces that determine where, when, and to what extent they bind remains primitive. Here we use a thermodynamic model of transcription factor binding to evaluate the contribution of different biophysical forces to the binding of five regulators of early embryonic anterior-posterior patterning in Drosophila melanogaster. Predictions based on DNA sequence and in vitro protein-DNA affinities alone achieve a correlation of ∼0.4 with experimental measurements of in vivo binding. Incorporating cooperativity and competition among the five factors, and accounting for spatial patterning by modeling binding in every nucleus independently, had little effect on prediction accuracy. A major source of error was the prediction of binding events that do not occur in vivo, which we hypothesized reflected reduced accessibility of chromatin. To test this, we incorporated experimental measurements of genome-wide DNA accessibility into our model, effectively restricting predicted binding to regions of open chromatin. This dramatically improved our predictions to a correlation of 0.6–0.9 for various factors across known target genes. Finally, we used our model to quantify the roles of DNA sequence, accessibility, and binding competition and cooperativity. Our results show that, in regions of open chromatin, binding can be predicted almost exclusively by the sequence specificity of individual factors, with a minimal role for protein interactions. We suggest that a combination of experimentally determined chromatin accessibility data and simple computational models of transcription factor binding may be used to predict the binding landscape of any animal transcription factor with significant precision., Author Summary During early stages of development, regulatory proteins bind DNA and control the expression of nearby genes, thereby driving spatial and temporal patterns of gene expression during development. But the biochemical forces that determine where these regulatory proteins bind are poorly understood. We gathered experimental data on the activities of several key regulators of early development of the fruit fly (Drosophila melanogaster) and developed a computational method to predict where and how strongly they will bind. We find that competition, cooperativity, and other interactions among individual regulatory proteins have a limited effect on their binding, while the global accessibility of DNA to protein binding has a significant impact on the binding of all factors. Our results suggest a practical method for predicting regulatory binding by combining experimental DNA accessibility assays with computational algorithms to determine where will binding occur among the accessible regions of the genome.

Transposons and other selfish DNA elements can be found in all phyla, and mobilization of these elements can compromise genome integrity. The piRNA (PIWI-interacting RNA) pathway silences transposons in the germline, but it is unclear if this pathway has additional functions during development. Here we show that mutations in the Drosophila piRNA pathway genes, armi, aub, ago3, and rhi, lead to extensive fragmentation of the zygotic genome during the cleavage stage of embryonic divisions. Additionally, aub and armi show defects in telomere resolution during meiosis and the cleavage divisions; and mutations in lig-IV, which disrupt non-homologous end joining, suppress these fusions. By contrast, lig-IV mutations enhance chromosome fragmentation. Chromatin immunoprecipitation studies show that aub and armi mutations disrupt telomere binding of HOAP, which is a component of the telomere protection complex, and reduce expression of a subpopulation of 19- to 22-nt telomere-specific piRNAs. Mutations in rhi and ago3, by contrast, do not block HOAP binding or production of these piRNAs. These findings uncover genetically separable functions for the Drosophila piRNA pathway. The aub, armi, rhi, and ago3 genes silence transposons and maintain chromosome integrity during cleavage-stage embryonic divisions. However, the aub and armi genes have an additional function in assembly of the telomere protection complex., Author Summary Transposons and other selfish genetic elements make up a significant fraction of all eukaryotic genomes, and the piRNA pathway appears to have a conserved function in transposon silencing and genome maintenance. However, other functions for this pathway have not been fully explored. Telomeres must be protected from recognition as DNA breaks by the repair machinery, which can covalently ligate unprotected chromosome ends and thus disrupt meiotic and mitotic chromosome segregation. We show that mutations in a subset of piRNA pathway genes disrupt meiotic and mitotic chromosome separation and that these segregation defects are suppressed by a mutation that blocks ligation of non-homologous DNA ends. These mutations also disrupt assembly of the telomere protection complex and reduce expression of a subpopulation of 19- to 22-nt telomere-specific RNA. We therefore propose that a subpopulation of short piRNAs direct assembly of the telomere protection complex.

Spinal Muscular Atrophy (SMA) is caused by diminished function of the Survival of Motor Neuron (SMN) protein, but the molecular pathways critical for SMA pathology remain elusive. We have used genetic approaches in invertebrate models to identify conserved SMN loss of function modifier genes. Drosophila melanogaster and Caenorhabditis elegans each have a single gene encoding a protein orthologous to human SMN; diminished function of these invertebrate genes causes lethality and neuromuscular defects. To find genes that modulate SMN function defects across species, two approaches were used. First, a genome-wide RNAi screen for C. elegans SMN modifier genes was undertaken, yielding four genes. Second, we tested the conservation of modifier gene function across species; genes identified in one invertebrate model were tested for function in the other invertebrate model. Drosophila orthologs of two genes, which were identified originally in C. elegans, modified Drosophila SMN loss of function defects. C. elegans orthologs of twelve genes, which were originally identified in a previous Drosophila screen, modified C. elegans SMN loss of function defects. Bioinformatic analysis of the conserved, cross-species, modifier genes suggests that conserved cellular pathways, specifically endocytosis and mRNA regulation, act as critical genetic modifiers of SMN loss of function defects across species., Author Summary Spinal Muscular Atrophy (SMA) is a common, untreatable, and often fatal neuromuscular disease predominately caused by reduced Survival Motor Neuron (SMN) protein function. Here, we use invertebrate models to identify and validate conserved genes that play a critical role in SMN loss of function neuromuscular defects. Decreased SMN function causes growth defects in the nematode Caenorhabditis elegans and in the fruit fly Drosophila melanogaster—as well as behavioral or synaptic connectivity defects between neurons and muscles, respectively. We found that a genetic modifier of SMA in patients, plastin, also affects SMN function in these invertebrate models. We undertook a genome-wide RNAi screen to identify genes whose perturbation alters the growth defects of C. elegans lacking SMN. These genes were validated in neuromuscular assays in nematode and fly models of SMA. Additionally, we used the C. elegans model to test SMN modifier genes previously identified in the Drosophila SMA model. Combined, these cross-species approaches identified fifteen genes that are important in both species when SMN function is decreased. Related mammalian proteins and the pathways in which they act (including endocytosis and RNA transport/translational control) are likely important players in SMA.

Transcriptional regulation is one of the most important processes for modulating gene expression. Though much of this control is attributed to transcription factors, histones, and associated enzymes, it is increasingly apparent that the spatial organization of chromosomes within the nucleus has a profound effect on transcriptional activity. Studies in yeast indicate that the nuclear pore complex might promote transcription by recruiting chromatin to the nuclear periphery. In higher eukaryotes, however, it is not known whether such regulation has global significance. Here we establish nucleoporins as a major class of global regulators for gene expression in Drosophila melanogaster. Using chromatin-immunoprecipitation combined with microarray hybridisation, we show that Nup153 and Megator (Mtor) bind to 25% of the genome in continuous domains extending 10 kb to 500 kb. These Nucleoporin-Associated Regions (NARs) are dominated by markers for active transcription, including high RNA polymerase II occupancy and histone H4K16 acetylation. RNAi–mediated knock-down of Nup153 alters the expression of ∼5,700 genes, with a pronounced down-regulatory effect within NARs. We find that nucleoporins play a central role in coordinating dosage compensation—an organism-wide process involving the doubling of expression of the male X chromosome. NARs are enriched on the male X chromosome and occupy 75% of this chromosome. Furthermore, Nup153-depletion abolishes the normal function of the male-specific dosage compensation complex. Finally, by extensive 3D imaging, we demonstrate that NARs contribute to gene expression control irrespective of their sub-nuclear localization. Therefore, we suggest that NAR–binding is used for chromosomal organization that enables gene expression control., Author Summary The eukaryotic genome is spatially distributed in a highly organized manner, with chromosomal regions localizing to well-defined sub-nuclear positions. This organization could have a profound effect on chromatin accessibility and transcriptional activity on a genome-wide level. Using high-resolution, genome-wide, chromatin-binding profiles we show that the nuclear pore components Nup153 and Megator bind to quarter of the Drosophila genome in form of chromosomal domains. These domains represent active regions of the genome. Interestingly, comparison of male and female cells revealed enrichment of these domains on the male X chromosome, which represents an exceptionally active chromosome that is under dosage compensation control to equalize gene expression due to differences in X chromosome number between males and females. Based on extensive 3D image analysis, we show that these chromosomal domains are contributed by both peripheral as well as intranuclear pool of these proteins. We suggest that chromosomal organization by nucleoporins could contribute to global gene expression control.

In Drosophila, genes expressed in males tend to accumulate on autosomes and are underrepresented on the X chromosome. In particular, genes expressed in testis have been observed to frequently relocate from the X chromosome to the autosomes. The inactivation of X-linked genes during male meiosis (i.e., meiotic sex chromosome inactivation—MSCI) was first proposed to explain male sterility caused by X-autosomal translocation in Drosophila, and more recently it was suggested that MSCI might provide the conditions under which selection would favor the accumulation of testis-expressed genes on autosomes. In order to investigate the impact of MSCI on Drosophila testis-expressed genes, we performed a global gene expression analysis of the three major phases of D. melanogaster spermatogenesis: mitosis, meiosis, and post-meiosis. First, we found evidence supporting the existence of MSCI by comparing the expression levels of X- and autosome-linked genes, finding the former to be significantly reduced in meiosis. Second, we observed that the paucity of X-linked testis-expressed genes was restricted to those genes highly expressed in meiosis. Third, we found that autosomal genes relocated through retroposition from the X chromosome were more often highly expressed in meiosis in contrast to their X-linked parents. These results suggest MSCI as a general mechanism affecting the evolution of some testis-expressed genes., Author Summary During the course of Drosophila evolution, genes expressed in males have accumulated on the autosomes. Meiotic sex chromosome X inactivation in males was proposed, among other hypotheses, as a selective force favoring the accumulation of testis-expressed genes on the autosomes. Under such a model, the inactivation of X-linked genes would favor the accumulation of testis-expressed genes in autosomes, wherein these genes would still be expressed. In this study, we observed meiotic expression reduction for X-linked genes in D. melanogaster through a global gene expression analysis in different phases of spermatogenesis, in agreement with MSCI. In order to test the effects of MSCI on the chromosomal distribution of testis-expressed genes, we analyzed their expression pattern throughout spermatogenesis. First, X chromosome underrepresentation was restricted to testis-biased genes over-expressed in meiosis. Second, we observed that the autosomal genes retroposed from the X chromosome more often showed complementary expression in meiosis to their X-linked parents. These results support MSCI in Drosophila, suggesting its mechanistic role in the evolution of testis-expressed genes.

In nature, closely related species may hybridize while still retaining their distinctive identities. Chromosomal regions that experience reduced recombination in hybrids, such as within inversions, have been hypothesized to contribute to the maintenance of species integrity. Here, we examine genomic sequences from closely related fruit fly taxa of the Drosophila pseudoobscura subgroup to reconstruct their evolutionary histories and past patterns of genic exchange. Partial genomic assemblies were generated from two subspecies of Drosophila pseudoobscura (D. ps.) and an outgroup species, D. miranda. These new assemblies were compared to available assemblies of D. ps. pseudoobscura and D. persimilis, two species with overlapping ranges in western North America. Within inverted regions, nucleotide divergence among each pair of the three species is comparable, whereas divergence between D. ps. pseudoobscura and D. persimilis in non-inverted regions is much lower and closer to levels of intraspecific variation. Using molecular markers flanking each of the major chromosomal inversions, we identify strong crossover suppression in F1 hybrids extending over 2 megabase pairs (Mbp) beyond the inversion breakpoints. These regions of crossover suppression also exhibit the high nucleotide divergence associated with inverted regions. Finally, by comparison to a geographically isolated subspecies, D. ps. bogotana, our results suggest that autosomal gene exchange between the North American species, D. ps. pseudoobscura and D. persimilis, occurred since the split of the subspecies, likely within the last 200,000 years. We conclude that chromosomal rearrangements have been vital to the ongoing persistence of these species despite recent hybridization. Our study serves as a proof-of-principle on how whole genome sequencing can be applied to formulate and test hypotheses about species formation in lesser-known non-model systems., Author Summary The transformation of populations into distinct species depends on whether hybridization, recombination, and subsequent gene introgression can be suppressed between diverging species. We use partial genome sequences to reconstruct this evolutionary process in the Drosophila pseudoobscura species subgroup, which includes the hybridizing species pair D. pseudoobscura pseudoobscura and D. persimilis. Recent models suggest that chromosomal inversions can facilitate the persistence of hybridizing species because of their effects on recombination, whereby inverted regions would exhibit higher nucleotide divergence than non-inverted regions. Indeed, D. pseudoobscura-D. persimilis nucleotide divergence outside these inverted regions is lower than within or near inversions, resembling D. ps. pseudoobscura levels of within-species nucleotide diversity. We also observe that recombination suppression in F1 hybrids extends greater than 2 Mbp outside the inversion breakpoints. Furthermore, when genomic sequence of D. persimilis is compared to two sister subspecies—the hybridizing subspecies, D. ps. pseudoobscura, and a non-hybridizing control subspecies, D. ps. bogotana—autosomal divergence is lower in the former, demonstrating recent gene exchange. These lines of evidence support a speciation model in which the two hybridizing species persist despite the presence of recent genic introgression in collinear regions of the genome because of the reduced recombinational effects of the inversions that distinguish them.

Over the past four decades, the predominant view of molecular evolution saw little connection between natural selection and genome evolution, assuming that the functionally constrained fraction of the genome is relatively small and that adaptation is sufficiently infrequent to play little role in shaping patterns of variation within and even between species. Recent evidence from Drosophila, reviewed here, suggests that this view may be invalid. Analyses of genetic variation within and between species reveal that much of the Drosophila genome is under purifying selection, and thus of functional importance, and that a large fraction of coding and noncoding differences between species are adaptive. The findings further indicate that, in Drosophila, adaptations may be both common and strong enough that the fate of neutral mutations depends on their chance linkage to adaptive mutations as much as on the vagaries of genetic drift. The emerging evidence has implications for a wide variety of fields, from conservation genetics to bioinformatics, and presents challenges to modelers and experimentalists alike.

Damage initiates a pleiotropic cellular response aimed at cellular survival when appropriate. To identify genes required for damage survival, we used a cell-based RNAi screen against the Drosophila genome and the alkylating agent methyl methanesulphonate (MMS). Similar studies performed in other model organisms report that damage response may involve pleiotropic cellular processes other than the central DNA repair components, yet an intuitive systems level view of the cellular components required for damage survival, their interrelationship, and contextual importance has been lacking. Further, by comparing data from different model organisms, identification of conserved and presumably core survival components should be forthcoming. We identified 307 genes, representing 13 signaling, metabolic, or enzymatic pathways, affecting cellular survival of MMS–induced damage. As expected, the majority of these pathways are involved in DNA repair; however, several pathways with more diverse biological functions were also identified, including the TOR pathway, transcription, translation, proteasome, glutathione synthesis, ATP synthesis, and Notch signaling, and these were equally important in damage survival. Comparison with genomic screen data from Saccharomyces cerevisiae revealed no overlap enrichment of individual genes between the species, but a conservation of the pathways. To demonstrate the functional conservation of pathways, five were tested in Drosophila and mouse cells, with each pathway responding to alkylation damage in both species. Using the protein interactome, a significant level of connectivity was observed between Drosophila MMS survival proteins, suggesting a higher order relationship. This connectivity was dramatically improved by incorporating the components of the 13 identified pathways within the network. Grouping proteins into “pathway nodes” qualitatively improved the interactome organization, revealing a highly organized “MMS survival network.” We conclude that identification of pathways can facilitate comparative biology analysis when direct gene/orthologue comparisons fail. A biologically intuitive, highly interconnected MMS survival network was revealed after we incorporated pathway data in our interactome analysis., Author Summary Cellular damage is known to elicit a pleiotropic response, but the relative importance of the constituent components in cell survival is poorly understood. To provide an unbiased identification of the proteins utilized in damage survival, we performed an RNAi survival screen in fly cells with methyl methanesulfonate (MMS). The genes identified are involved in 13 biologically diverse pathways. Comparison with analogous yeast data demonstrated a lack of conservation of the individual MMS survival genes but a conservation of pathways. We went on to demonstrate the MMS responsiveness for five representative pathways in both fly and mouse cells. We conclude that identification of pathways can facilitate comparative biology analysis when direct gene/orthologue comparisons fail. Incorporation of pathway data in interactome analysis also improved connectivity and, more importantly, revealed a biologically intuitive, highly inter-connected “MMS survival network.” This pathway conservation and inter-connectivity implies extensive interaction between pathways; for diseases such as cancer, such crosstalk may dictate disparate cellular responses not necessarily expected and confound treatments that are not tailored to the individual molecular context.