Dynamics and stage-specificity of between-population gene expression divergence in the Drosophila melanogaster larval fat body.

Gene expression variation is pervasive across all levels of organismal organization, including development. Few studies, however, have examined variation in developmental transcriptional dynamics among populations, or how it contributes to phenotypic divergence. Indeed, the evolution of gene expression dynamics when both the evolutionary and temporal timescale are comparatively short remains relatively uncharacterized. Here, we examined coding and non-coding gene expression in the fat body of an ancestral African and a derived European Drosophila melanogaster population across three developmental stages spanning ten hours of larval development. Between populations, expression divergence was largely stage-specific. We detected higher expression variation during the late wandering stage, which may be a general feature of this stage. During this stage, we also detected higher and more extensive lncRNA expression in Europe, suggesting that lncRNA expression may be more important in derived populations. Interestingly, the temporal breadth of protein-coding and lncRNA expression became more restricted in the derived population. Taken together with the signatures of potential local adaptation that we detected at the sequence level in 9-25% of candidate genes (those showing evidence of expression divergence between populations), this finding suggests that gene expression becomes more developmental stage-specific during adaptation to new environments. We further used RNAi to identify several candidate genes that likely contribute to known phenotypic divergence between these populations. Our results shed light on the evolution and dynamics of expression variation over short developmental and evolutionary timescales, and how this variation contributes to population and phenotypic divergence.

From whole bodies to single cells: A guide to transcriptomic approaches for ecology and evolutionary biology.

RNA sequencing (RNAseq) methodology has experienced a burst of technological developments in the last decade, which has opened up opportunities for studying the mechanisms of adaptation to environmental factors at both the organismal and cellular level. Selecting the most suitable experimental approach for specific research questions and model systems can, however, be a challenge and researchers in ecology and evolution are commonly faced with the choice of whether to study gene expression variation in whole bodies, specific tissues, and/or single cells. A wide range of sometimes polarised opinions exists over which approach is best. Here, we highlight the advantages and disadvantages of each of these approaches to provide a guide to help researchers make informed decisions and maximise the power of their study. Using illustrative examples of various ecological and evolutionary research questions, we guide the readers through the different RNAseq approaches and help them identify the most suitable design for their own projects.

Rapid evolutionary change, constraints and the maintenance of polymorphism in natural populations of Drosophila melanogaster.

Allele frequencies can shift rapidly within natural populations. Under certain conditions, repeated rapid allele frequency shifts can lead to the long-term maintenance of polymorphism. In recent years, studies of the model insect Drosophila melanogaster have suggested that this phenomenon is more common than previously believed and is often driven by some form of balancing selection, such as temporally fluctuating or sexually antagonistic selection. Here we discuss some of the general insights into rapid evolutionary change revealed by large-scale population genomic studies, as well as the functional and mechanistic causes of rapid adaptation uncovered by single-gene studies. As an example of the latter, we consider a regulatory polymorphism of the D. melanogaster fezzik gene. Polymorphism at this site has been maintained at intermediate frequency over an extended period of time. Regular observations from a single population over a period of 7 years revealed significant differences in the frequency of the derived allele and its variance across collections between the sexes. These patterns are highly unlikely to arise from genetic drift alone or from the action of sexually antagonistic or temporally fluctuating selection individually. Instead, the joint action of sexually antagonistic and temporally fluctuating selection can best explain the observed rapid and repeated allele frequency shifts. Temporal studies such as those reviewed here further our understanding of how rapid changes in selection can lead to the long-term maintenance of polymorphism as well as improve our knowledge of the forces driving and limiting adaptation in nature.

Sexual Antagonism, Temporally Fluctuating Selection, and Variable Dominance Affect a Regulatory Polymorphism in Drosophila melanogaster.

Understanding how genetic variation is maintained within species is a major goal of evolutionary genetics that can shed light on the preservation of biodiversity. Here, we examined the maintenance of a regulatory single-nucleotide polymorphism (SNP) of the X-linked Drosophila melanogaster gene fezzik. The derived variant at this site is at intermediate frequency in many worldwide populations but absent in populations from the ancestral species range in sub-Saharan Africa. We collected and genotyped wild-caught individuals from a single European population biannually over a period of 5 years, which revealed an overall difference in allele frequency between the sexes and a consistent change in allele frequency across seasons in females but not in males. Modeling based on the observed allele and genotype frequencies suggested that both sexually antagonistic and temporally fluctuating selection may help maintain variation at this site. The derived variant is predicted to be female-beneficial and mostly recessive; however, there was uncertainty surrounding our dominance estimates and long-term modeling projections suggest that it is more likely to be dominant. By examining gene expression phenotypes, we found that phenotypic dominance was variable and dependent upon developmental stage and genetic background, suggesting that dominance may be variable at this locus. We further determined that fezzik expression and genotype are associated with starvation resistance in a sex-dependent manner, suggesting a potential phenotypic target of selection. By characterizing the mechanisms of selection acting on this SNP, our results improve our understanding of how selection maintains genetic and phenotypic variation in natural populations.

Drosophila Evolution over Space and Time (DEST): A New Population Genomics Resource.

Drosophila melanogaster is a leading model in population genetics and genomics, and a growing number of whole-genome data sets from natural populations of this species have been published over the last years. A major challenge is the integration of disparate data sets, often generated using different sequencing technologies and bioinformatic pipelines, which hampers our ability to address questions about the evolution of this species. Here we address these issues by developing a bioinformatics pipeline that maps pooled sequencing (Pool-Seq) reads from D. melanogaster to a hologenome consisting of fly and symbiont genomes and estimates allele frequencies using either a heuristic (PoolSNP) or a probabilistic variant caller (SNAPE-pooled). We use this pipeline to generate the largest data repository of genomic data available for D. melanogaster to date, encompassing 271 previously published and unpublished population samples from over 100 locations in >20 countries on four continents. Several of these locations have been sampled at different seasons across multiple years. This data set, which we call Drosophila Evolution over Space and Time (DEST), is coupled with sampling and environmental metadata. A web-based genome browser and web portal provide easy access to the SNP data set. We further provide guidelines on how to use Pool-Seq data for model-based demographic inference. Our aim is to provide this scalable platform as a community resource which can be easily extended via future efforts for an even more extensive cosmopolitan data set. Our resource will enable population geneticists to analyze spatiotemporal genetic patterns and evolutionary dynamics of D. melanogaster populations in unprecedented detail.

Functional characterization of adaptive variation within a cis-regulatory element influencing Drosophila melanogaster growth.

Gene expression variation is a major contributor to phenotypic diversity within species and is thought to play an important role in adaptation. However, examples of adaptive regulatory polymorphism are rare, especially those that have been characterized at both the molecular genetic level and the organismal level. In this study, we perform a functional analysis of the Drosophila melanogaster CG9509 enhancer, a cis-regulatory element that shows evidence of adaptive evolution in populations outside the species' ancestral range in sub-Saharan Africa. Using site-directed mutagenesis and transgenic reporter gene assays, we determined that 3 single nucleotide polymorphisms are responsible for the difference in CG9509 expression that is observed between sub-Saharan African and cosmopolitan populations. Interestingly, while 2 of these variants appear to have been the targets of a selective sweep outside of sub-Saharan Africa, the variant with the largest effect on expression remains polymorphic in cosmopolitan populations, suggesting it may be subject to a different mode of selection. To elucidate the function of CG9509, we performed a series of functional and tolerance assays on flies in which CG9509 expression was disrupted. We found that CG9509 plays a role in larval growth and influences adult body and wing size, as well as wing loading. Furthermore, variation in several of these traits was associated with variation within the CG9509 enhancer. The effect on growth appears to result from a modulation of active ecdysone levels and expression of growth factors. Taken together, our findings suggest that selection acted on 3 sites within the CG9509 enhancer to increase CG9509 expression and, as a result, reduce wing loading as D. melanogaster expanded out of sub-Saharan Africa.

An Indel Polymorphism in the MtnA 3' Untranslated Region Is Associated with Gene Expression Variation and Local Adaptation in Drosophila melanogaster.

Insertions and deletions (indels) are a major source of genetic variation within species and may result in functional changes to coding or regulatory sequences. In this study we report that an indel polymorphism in the 3' untranslated region (UTR) of the metallothionein gene MtnA is associated with gene expression variation in natural populations of Drosophila melanogaster. A derived allele of MtnA with a 49-bp deletion in the 3' UTR segregates at high frequency in populations outside of sub-Saharan Africa. The frequency of the deletion increases with latitude across multiple continents and approaches 100% in northern Europe. Flies with the deletion have more than 4-fold higher MtnA expression than flies with the ancestral sequence. Using reporter gene constructs in transgenic flies, we show that the 3' UTR deletion significantly contributes to the observed expression difference. Population genetic analyses uncovered signatures of a selective sweep in the MtnA region within populations from northern Europe. We also find that the 3' UTR deletion is associated with increased oxidative stress tolerance. These results suggest that the 3' UTR deletion has been a target of selection for its ability to confer increased levels of MtnA expression in northern European populations, likely due to a local adaptive advantage of increased oxidative stress tolerance.

The discovery, distribution, and diversity of DNA viruses associated with Drosophila melanogaster in Europe.

Drosophila melanogaster is an important model for antiviral immunity in arthropods, but very few DNA viruses have been described from the family Drosophilidae. This deficiency limits our opportunity to use natural host-pathogen combinations in experimental studies, and may bias our understanding of the Drosophila virome. Here, we report fourteen DNA viruses detected in a metagenomic analysis of 6668 pool-sequenced Drosophila, sampled from forty-seven European locations between 2014 and 2016. These include three new nudiviruses, a new and divergent entomopoxvirus, a virus related to Leptopilina boulardi filamentous virus, and a virus related to Musca domestica salivary gland hypertrophy virus. We also find an endogenous genomic copy of galbut virus, a double-stranded RNA partitivirus, segregating at very low frequency. Remarkably, we find that Drosophila Vesanto virus, a small DNA virus previously described as a bidnavirus, may be composed of up to twelve segments and thus represent a new lineage of segmented DNA viruses. Two of the DNA viruses, Drosophila Kallithea nudivirus and Drosophila Vesanto virus are relatively common, found in 2 per cent or more of wild flies. The others are rare, with many likely to be represented by a single infected fly. We find that virus prevalence in Europe reflects the prevalence seen in publicly available datasets, with Drosophila Kallithea nudivirus and Drosophila Vesanto virus the only ones commonly detectable in public data from wild-caught flies and large population cages, and the other viruses being rare or absent. These analyses suggest that DNA viruses are at lower prevalence than RNA viruses in D.melanogaster, and may be less likely to persist in laboratory cultures. Our findings go some way to redressing an earlier bias toward RNA virus studies in Drosophila, and lay the foundation needed to harness the power of Drosophila as a model system for the study of DNA viruses.

Population Genetic and Functional Analysis of a cis-Regulatory Polymorphism in the DrosophilamelanogasterMetallothionein A gene.

Although gene expression can vary extensively within and among populations, the genetic basis of this variation and the evolutionary forces that maintain it are largely unknown. In Drosophilamelanogaster, a 49-bp insertion/deletion (indel) polymorphism in the Metallothionein A (MtnA) gene is associated with variation in MtnA expression and oxidative stress tolerance. To better understand the functional and evolutionary significance of this polymorphism, we investigated it in several worldwide populations. In a German population, the deletion was present at a high and stable frequency over multiple seasons and years, and was associated with increased MtnA expression. There was, however, no evidence that the polymorphism was maintained by overdominant, seasonally fluctuating, or sexually antagonistic selection. The deletion was rare in a population from the species' ancestral range in sub-Saharan Africa and is likely the result of non-African admixture, suggesting that it spread to high frequency following the species' out-of-Africa expansion. Using data from a North American population, we found that the deletion was associated with MtnA expression and tolerance to oxidative stress induced by menadione sodium bisulfite. Our results are consistent with the deletion being selectively favored in temperate populations due to the increased MtnA expression and oxidative stress tolerance that it confers.

Gene Regulatory Variation in Drosophila melanogaster Renal Tissue.

Genetic variation influencing levels of gene expression is abundant in natural populations, and may exert its effects through complex mechanisms that depend on an organism's genetic background and the tissue in which expression is measured. We investigated natural variation in gene expression in the Malpighian tubules of three inbred Drosophila melanogaster strains and their F1 hybrids. One of the strains was from a population in the species' ancestral range (Zambia), while the other two were from a more recently derived population (Sweden). Although closely related, the two Swedish strains differed greatly in terms of their expression inheritance when hybridized with the Zambian strain, with one Swedish strain showing a large excess of genes with recessive expression inheritance, as well as a large number of genes with overdominant inheritance. Although most expression variation could be attributed to trans-regulation, there were ∼200 genes that showed allele-specific expression differences in each of the between-population hybrids, indicating that cis-regulation contributes as well. The cis-regulated genes were enriched with cytochrome P450 genes, and the upstream regions of six of these genes were incorporated into transgenic reporter gene constructs to test their effects on expression. Differential expression was observed for five of the six reporter genes in the Malpighian tubule, suggesting that a large proportion of cis-regulatory variation lies directly upstream of the affected gene. In most cases, the differential expression was specific to the Malpighian tubule or greater in this tissue than in the rest of the body, highlighting the importance of single-tissue studies of gene expression variation.

Adaptive divergence of a transcriptional enhancer between populations of Drosophila melanogaster.

As species colonize new habitats they must adapt to the local environment. Much of this adaptation is thought to occur at the regulatory level; however, the relationships among genetic polymorphism, expression variation and adaptation are poorly understood. Drosophila melanogaster, which expanded from an ancestral range in sub-Saharan Africa around 15 000 years ago, represents an excellent model system for studying regulatory evolution. Here, we focus on the gene CG9509, which differs in expression between an African and a European population of D. melanogaster. The expression difference is caused by variation within a transcriptional enhancer adjacent to the CG9509 coding sequence. Patterns of sequence variation indicate that this enhancer was the target of recent positive selection, suggesting that the expression difference is adaptive. Analysis of the CG9509 enhancer in new population samples from Europe, Asia, northern Africa and sub-Saharan Africa revealed that sequence polymorphism is greatly reduced outside the ancestral range. A derived haplotype absent in sub-Saharan Africa is at high frequency in all other populations. These observations are consistent with a selective sweep accompanying the range expansion of the species. The new data help identify the sequence changes responsible for the difference in enhancer activity.