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data repository of genomic data available for D. melanogaster to date, encompassing 271 previously published and unpublished population samples from over 100 locations in 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.

Genomic Analysis of European Drosophila melanogaster Populations Reveals Longitudinal Structure, Continent-Wide Selection, and Previously Unknown DNA Viruses.

Genetic variation is the fuel of evolution, with standing genetic variation especially important for short-term evolution and local adaptation. To date, studies of spatiotemporal patterns of genetic variation in natural populations have been challenging, as comprehensive sampling is logistically difficult, and sequencing of entire populations costly. Here, we address these issues using a collaborative approach, sequencing 48 pooled population samples from 32 locations, and perform the first continent-wide genomic analysis of genetic variation in European Drosophila melanogaster. Our analyses uncover longitudinal population structure, provide evidence for continent-wide selective sweeps, identify candidate genes for local climate adaptation, and document clines in chromosomal inversion and transposable element frequencies. We also characterize variation among populations in the composition of the fly microbiome, and identify five new DNA viruses in our samples.

Detecting purging of inbreeding depression by a slow rate of inbreeding for various traits: the impact of environmental and experimental conditions.

Inbreeding depression (ID) has since long been recognized as a significant factor in evolutionary biology. It is mainly the consequence of (partially) recessive deleterious mutations maintained by mutation-selection balance in large random mating populations. When population size is reduced, recessive alleles are increasingly found in homozygous condition due to drift and inbreeding and become more prone to selection. Particularly at slow rates of drift and inbreeding, selection will be more effective in purging such alleles, thereby reducing the amount of ID. Here we test assumptions of the efficiency of purging in relation to the inbreeding rate and the experimental conditions for four traits in D. melanogaster. We investigated the magnitude of ID for lines that were inbred to a similar level, F ≈ 0.50, reached either by three generations of full-sib mating (fast inbreeding), or by 12 consecutive generations with a small population size (slow inbreeding). This was done on two different food media. We observed significant ID for egg-to-adult viability and heat shock mortality, but only for egg-to-adult viability a significant part of the expressed inbreeding depression was effectively purged under slow inbreeding. For other traits like developmental time and starvation resistance, however, adaptation to the experimental and environmental conditions during inbreeding might affect the likelihood of purging to occur or being detected. We discuss factors that can affect the efficiency of purging and why empirical evidence for purging may be ambiguous.

Genomic signatures of experimental adaptive radiation in Drosophila.

Abiotic environmental factors play a fundamental role in determining the distribution, abundance and adaptive diversification of species. Empowered by new technologies enabling rapid and increasingly accurate examination of genomic variation in populations, researchers may gain new insights into the genomic background of adaptive radiation and stress resistance. We investigated genomic variation across generations of large-scale experimental selection regimes originating from a single founder population of Drosophila melanogaster, diverging in response to ecologically relevant environmental stressors: heat shock, heat knock down, cold shock, desiccation and starvation. When compared to the founder population, and to parallel unselected controls, there were more than 100,000 single nucleotide polymorphisms (SNPs) displaying consistent allelic changes in response to selective pressures across generations. These SNPs were found in both coding and noncoding sequences, with the highest density in promoter regions, and involved a broad range of functionalities, including molecular chaperoning by heat-shock proteins. The SNP patterns were highly stressor-specific despite considerable variation among line replicates within each selection regime, as reflected by a principal component analysis, and co-occurred with selective sweep regions. Only ~15% of SNPs with putatively adaptive changes were shared by at least two selective regimes, while less than 1% of SNPs diverged in opposite directions. Divergent stressors driving evolution in the experimental system of adaptive radiation left distinct genomic signatures, most pronounced in starvation and heat-shock selection regimes.

Expression of thermal tolerance genes in two Drosophila species with different acclimation capacities.

Heat tolerance increases at higher acclimation temperatures in D. melanogaster, but not in D. subobscura. The two species represent separate lineages of the subgenus Sophophora of Drosophila with contrasting tropical African and temperate Palearctic evolutionary histories. D. melanogaster has five copies of the inducible hsp70 gene distributed in two clusters, named A (with two copies) and B (three copies), while D. subobscura has only two copies arranged similarly to cluster A of D. melanogaster. The hsp70s of the two species also differ in their cis-regulatory regions, with D. melanogaster exhibiting features of a faster and more productive promoter. We predicted that the interspecific variation in acclimation capacity of heat tolerance is explained by evolved variation in expression of the major group of heat shock proteins. To test this prediction, we compared basal levels of gene expression at different developmental temperatures within each of the two species. Furthermore, we explored the heat hardening dynamics by measuring the induction of gene expression during a ramping assay. The prediction of a stronger heat shock protein response in D. melanogaster as compared to D. subobscura was confirmed for both long-term acclimation and short-term hardening. For D. melanogaster the upregulation with temperature ramping ranged from less than two fold (hsp26) to 2500 fold (hsp70A) increase. In all cases induction in D. melanogaster exceeded that of D. subobscura homologs. These differences correlate with structural differences in the regulatory regions of hsp70, and might explain differences in acclimation capacity among species. Finally, in D. melanogaster we found an indication of an inverse relationship between basal and induced levels of hsp70A and hsp83 expression, suggesting a divergent role for thermal adaptation of these genes at benign and stressful temperatures, respectively.

Plasticity for desiccation tolerance across Drosophila species is affected by phylogeny and climate in complex ways.

Comparative analyses of ectotherm susceptibility to climate change often focus on thermal extremes, yet responses to aridity may be equally important. Here we focus on plasticity in desiccation resistance, a key trait shaping distributions of Drosophila species and other small ectotherms. We examined the extent to which 32 Drosophila species, varying in their distribution, could increase their desiccation resistance via phenotypic plasticity involving hardening, linking these responses to environment, phylogeny and basal resistance. We found no evidence to support the seasonality hypothesis; species with higher hardening plasticity did not occupy environments with higher and more seasonal precipitation. As basal resistance increased, the capacity of species to respond via phenotypic plasticity decreased, suggesting plastic responses involving hardening may be constrained by basal resistance. Trade-offs between basal desiccation resistance and plasticity were not universal across the phylogeny and tended to occur within specific clades. Phylogeny, environment and trade-offs all helped to explain variation in plasticity for desiccation resistance but in complex ways. These findings suggest some species have the ability to counter dry periods through plastic responses, whereas others do not; and this ability will depend to some extent on a species' placement within a phylogeny, along with its basal level of resistance.

Linking developmental diet to adult foraging choice in Drosophila melanogaster.

Rather than maximizing intake of available macronutrients, insects increase intake of some nutrients and restrict intake of others. This selective consumption influences, and potentially optimizes, developmental time, reproduction and lifespan of the organism. Studies so far have focused on discriminating between protein and carbohydrate uptake and the consequences on fitness components at different life stages. However, it is largely unknown whether and how the developmental diets, which may entail habitat-specific nutrient restrictions, affect selective consumption in adults. We show that adult female D. melanogaster opt for the same protein to carbohydrate (P:C) ratio regardless of their developmental diet (P:C ratio of 1:1, 1:4 or 1:8). In contrast, males choose a diet that makes up for deficiencies; when protein is low during development, males increase protein consumption despite this being detrimental to starvation resistance. The sexual dimorphism in foraging choice could be due to the different energetic requirements of males and females. To investigate the effect of developmental diet on lifespan once an adult nutritional environment has been established, we also conducted a no-choice experiment. Here, adult lifespan increased as P:C ratio decreased, irrespective of developmental diet, thus demonstrating a 'cancelling out' effect of the nutritional environment experienced during early life stages. Our study provides novel insights into how developmental diet is linked to adult diet by presenting evidence for sexual dimorphism in foraging choice as well as life-stage dependency of diet on lifespan.

Geographic variation in responses of European yellow dung flies to thermal stress.

Climatic conditions can be very heterogeneous even over small geographic scales, and are believed to be major determinants of the abundance and distribution of species and populations. Organisms are expected to evolve in response to the frequency and magnitude of local thermal extremes, resulting in local adaptation. Using replicate yellow dung fly (Scathophaga stercoraria; Diptera: Scathophagidae) populations from cold (northern Europe) and warm climates (southern Europe), we compared 1) responses to short-term heat and cold shocks in both sexes, 2) heat shock protein (Hsp70) expression in adults and eggs, and 3) female reproductive traits when facing short-term heat stress during egg maturation. Contrary to expectations, thermal traits showed minor geographic differentiation, with weak evidence for greater heat resistance of southern flies but no differentiation in cold resistance. Hsp70 protein expression was little affected by heat stress, indicating systemic rather than induced regulation of the heat stress response, possibly related to this fly group's preference for cold climes. In contrast, sex differences were pronounced: males (which are larger) endured hot temperatures longer, while females featured higher Hsp70 expression. Heat stress negatively affected various female reproductive traits, reducing first clutch size, overall reproductive investment, egg lipid content, and subsequent larval hatching. These responses varied little across latitude but somewhat among populations in terms of egg size, protein content, and larval hatching success. Several reproductive parameters, but not Hsp70 expression, exhibited heritable variation among full-sib families. Rather than large-scale clinal geographic variation, our study suggests some local geographic population differentiation in the ability of yellow dung flies to buffer the impact of heat stress on reproductive performance.

Nucleotide diversity inflation as a genome-wide response to experimental lifespan extension in Drosophila melanogaster.

BACKGROUND: Evolutionary theory predicts that antagonistically selected alleles, such as those with divergent pleiotropic effects in early and late life, may often reach intermediate population frequencies due to balancing selection, an elusive process when sought out empirically. Alternatively, genetic diversity may increase as a result of positive frequency-dependent selection and genetic purging in bottlenecked populations. RESULTS: While experimental evolution systems with directional phenotypic selection typically result in at least local heterozygosity loss, we report that selection for increased lifespan in Drosophila melanogaster leads to an extensive genome-wide increase of nucleotide diversity in the selected lines compared to replicate control lines, pronounced in regions with no or low recombination, such as chromosome 4 and centromere neighborhoods. These changes, particularly in coding sequences, are most consistent with the operation of balancing selection and the antagonistic pleiotropy theory of aging and life history traits that tend to be intercorrelated. Genes involved in antioxidant defenses, along with multiple lncRNAs, were among those most affected by balancing selection. Despite the overwhelming genetic diversification and the paucity of selective sweep regions, two genes with functions important for central nervous system and memory, Ptp10D and Ank2, evolved under positive selection in the longevity lines. CONCLUSIONS: Overall, the 'evolve-and-resequence' experimental approach proves successful in providing unique insights into the complex evolutionary dynamics of genomic regions responsible for longevity.

Metabolic and functional characterization of effects of developmental temperature in Drosophila melanogaster.

The ability of ectotherms to respond to changes in their thermal environment through plastic mechanisms is central to their adaptive capability. However, we still lack knowledge on the physiological and functional responses by which ectotherms acclimate to temperatures during development, and in particular, how physiological stress at extreme temperatures may counteract beneficial acclimation responses at benign temperatures. We exposed Drosophila melanogaster to 10 developmental temperatures covering their entire permissible temperature range. We obtained metabolic profiles and reaction norms for several functional traits: egg-to-adult viability, developmental time, and heat and cold tolerance. Females were more heat tolerant than males, whereas no sexual dimorphism was found in cold tolerance. A group of metabolites, mainly free amino acids, had linear reaction norms. Several energy-carrying molecules, as well as some sugars, showed distinct inverted U-shaped norms of reaction across the thermal range, resulting in a positive correlation between metabolite intensities and egg-to-adult viability. At extreme temperatures, low levels of these metabolites were interpreted as a response characteristic of costs of homeostatic perturbations. Our results provide novel insights into a range of metabolites reported to be central for the acclimation response and suggest several new candidate metabolites. Low and high temperatures result in different adaptive physiological responses, but they also have commonalities likely to be a result of the failure to compensate for the physiological stress. We suggest that the regulation of metabolites that are tightly connected to the performance curve is important for the ability of ectotherms to cope with variation in temperature.

Unexpected high genetic diversity in small populations suggests maintenance by associative overdominance.

The effective population size (Ne ) is a central factor in determining maintenance of genetic variation. The neutral theory predicts that loss of variation depends on Ne , with less genetic drift in larger populations. We monitored genetic drift in 42 Drosophila melanogaster populations of different adult census population sizes (10, 50 or 500) using pooled RAD sequencing. In small populations, variation was lost at a substantially lower rate than expected. This observation was consistent across two ecological relevant thermal regimes, one stable and one with a stressful increase in temperature across generations. Estimated ratios between Ne and adult census size were consistently higher in small than in larger populations. The finding provides evidence for a slower than expected loss of genetic diversity and consequently a higher than expected long-term evolutionary potential in small fragmented populations. More genetic diversity was retained in areas of low recombination, suggesting that associative overdominance, driven by disfavoured homozygosity of recessive deleterious alleles, is responsible for the maintenance of genetic diversity in smaller populations. Consistent with this hypothesis, the X-chromosome, which is largely free of recessive deleterious alleles due to hemizygosity in males, fits neutral expectations even in small populations. Our experiments provide experimental answers to a range of unexpected patterns in natural populations, ranging from variable diversity on X-chromosomes and autosomes to surprisingly high levels of nucleotide diversity in small populations.

Reversibility of developmental heat and cold plasticity is asymmetric and has long-lasting consequences for adult thermal tolerance.

The ability of insects to cope with stressful temperatures through adaptive plasticity has allowed them to thrive under a wide range of thermal conditions. Developmental plasticity is generally considered to be a non-reversible phenotypic change, e.g. in morphological traits, while adult acclimation responses are often considered to be reversible physiological responses. However, physiologically mediated thermal acclimation might not follow this general prediction. We investigated the magnitude and rate of reversibility of developmental thermal plasticity responses in heat and cold tolerance of adult flies, using a full factorial design with two developmental and two adult temperatures (15 and 25°C). We show that cold tolerance attained during development is readily adjusted to the prevailing conditions during adult acclimation, with a symmetric rate of decrease or increase. In contrast, heat tolerance is only partly reversible during acclimation and is thus constrained by the temperature during development. The effect of adult acclimation on heat tolerance was asymmetrical, with a general loss of heat tolerance with age. Surprisingly, the decline in adult heat tolerance at 25°C was decelerated in flies developed at low temperatures. This result was supported by correlated responses in two senescence-associated traits and in accordance with a lower rate of ageing after low temperature development, suggesting that physiological age is not reset at eclosion. The results have profound ecological consequences for populations, as optimal developmental temperatures will be dependent on the thermal conditions faced in the adult stage and the age at which they occur.

Mild heat treatments induce long-term changes in metabolites associated with energy metabolism in Drosophila melanogaster.

Heat-induced hormesis, the beneficial effect of mild heat-induced stress, increases the average lifespan of many organisms. Yet little is known about the mechanisms underlying this effect. We used nuclear magnetic resonance spectroscopy to investigate the long-term effects of repeated mild heat treatments on the metabolome of male Drosophila melanogaster. 10 days after the heat treatment, metabolic aging appears to be slowed down, and a treatment response with 40 % higher levels of alanine and lactate and lower levels of aspartate and glutamate were measured. All treatment effects had disappeared 16 days later. Metabolic reprogramming has been associated with the life extending effects of dietary restriction. The metabolite changes induced by the hormetic treatment suggest that the positive effects might not be limited to the repair pathways induced, but that there also is a change in energy metabolism. A possible direct link between changes in energy metabolism and heat induced increase in Hsp70 expression is discussed.

Injuries can prolong lifespan in Drosophila melanogaster males.

Previous studies have shown that a range of different stresses can increase mean lifespan. Here we investigated the effect of injuries and bacterial inoculation on mean lifespan in lines selected for increased longevity and their controls. The three lines from each selection regime were subjected to one of five treatments ranging from control, over perforating the cuticle with a sterile needle, to inoculating with peptidoglycan or one of two strains of live bacteria. The flies were subjected to the infection stress at two ages and the experiment was conducted on both males and females of replicate lines of each selection regime. The individual lines and sexes differed in response to the treatment. However, overall the sterile injury of young males resulted in prolonged mean lifespan from both selection regimes, whereas inoculating had no additional effect to stabbing with a sterile needle. In middle-aged males only treatment with peptidoglycan had a significant hormetic effect and this was only in longevity-selected flies. In females only one of the tested contrasts was significant and here the effect of the treatment was to reduce average lifespan. As could be expected, the results showed a significant interaction between the effects of sex and infection on survival.

Experimental Evolution under Fluctuating Thermal Conditions Does Not Reproduce Patterns of Adaptive Clinal Differentiation in Drosophila melanogaster.

Experimental evolution can be a useful tool for testing the impact of environmental factors on adaptive changes in populations, and this approach is being increasingly used to understand the potential for evolutionary responses in populations under changing climates. However, selective factors will often be more complex in natural populations than in laboratory environments and produce different patterns of adaptive differentiation. Here we test the ability of laboratory experimental evolution under different temperature cycles to reproduce well-known patterns of clinal variation in Drosophila melanogaster. Six fluctuating thermal regimes mimicking the natural temperature conditions along the east coast of Australia were initiated. Contrary to expectations, on the basis of field patterns there was no evidence for adaptation to thermal regimes as reflected by changes in cold and heat resistance after 1-3 years of laboratory natural selection. While laboratory evolution led to changes in starvation resistance, development time, and body size, patterns were not consistent with those seen in natural populations. These findings highlight the complexity of factors affecting trait evolution in natural populations and indicate that caution is required when inferring likely evolutionary responses from the outcome of experimental evolution studies.

Sodium distribution predicts the chill tolerance of Drosophila melanogaster raised in different thermal conditions.

Many insects, including the model holometabolous insect Drosophila melanogaster, display remarkable plasticity in chill tolerance in response to the thermal environment experienced during development or as adults. At low temperatures, many insects lose the ability to regulate Na(+) balance, which is suggested to cause a secondary loss of hemolymph water to the tissues and gut lumen that concentrates the K(+) remaining in the hemolymph. The resultant increase in extracellular [K(+)] inhibits neuromuscular excitability and is proposed to cause cellular apoptosis and injury. The present study investigates whether and how variation in chill tolerance induced through developmental and adult cold acclimation is associated with changes in Na(+), water, and K(+) balance. Developmental and adult cold acclimation improved the chilling tolerance of D. melanogaster in an additive manner. In agreement with the proposed model, these effects were intimately related to differences in Na(+) distribution prior to cold exposure, such that chill-tolerant flies had low hemolymph [Na(+)], while intracellular [Na(+)] was similar among treatment groups. The low hemolymph Na(+) of cold-acclimated flies allowed them to maintain hemolymph volume, prevent hyperkalemia, and avoid injury following chronic cold exposure. These findings extend earlier observations of hemolymph volume disruption during cold exposure to the most ubiquitous model insect (D. melanogaster), highlight shared mechanisms of developmental and adult thermal plasticity and provide strong support for ionoregulatory failure as a central mechanism of insect chill susceptibility.

Inbreeding affects locomotor activity in Drosophila melanogaster at different ages.

The ability to move is essential for many behavioural traits closely related to fitness. Here we studied the effect of inbreeding on locomotor activity (LA) of Drosophila melanogaster at different ages under both dark and light regimes. We expected to find a decreased LA in inbred lines compared to control lines. We also predicted an increased differentiation between lines due to inbreeding. LA was higher in the dark compared to the light regime for both inbred and outbred control lines. As expected, inbreeding increased phenotypic variance in LA, with some inbred lines showing higher and some lower LA than control lines. Moreover, age per se did not affect LA neither in control nor in inbred lines, while we found a strong line by age interaction between inbred lines. Interestingly, inbreeding changed the daily activity pattern of the flies: these patterns were consistent across all control lines but were lost in some inbred lines. The departure in the daily pattern of LA in inbred lines may contribute to the inbreeding depression observed in inbred natural populations.

Upper thermal limits of Drosophila are linked to species distributions and strongly constrained phylogenetically.

Upper thermal limits vary less than lower limits among related species of terrestrial ectotherms. This pattern may reflect weak or uniform selection on upper limits, or alternatively tight evolutionary constraints. We investigated this issue in 94 Drosophila species from diverse climates and reared in a common environment to control for plastic effects that may confound species comparisons. We found substantial variation in upper thermal limits among species, negatively correlated with annual precipitation at the central point of their distribution and also with the interaction between precipitation and maximum temperature, showing that heat resistance is an important determinant of Drosophila species distributions. Species from hot and relatively dry regions had higher resistance, whereas resistance was uncorrelated with temperature in wetter regions. Using a suite of analyses we showed that phylogenetic signal in heat resistance reflects phylogenetic inertia rather than common selection pressures. Current species distributions are therefore more likely to reflect environmental sorting of lineages rather than local adaptation. Similar to previous studies, thermal safety margins were small at low latitudes, with safety margins smallest for species occupying both humid and dry tropical environments. Thus, species from a range of environments are likely to be at risk owing to climate change. Together these findings suggest that this group of insects is unlikely to buffer global change effects through marked evolutionary changes, highlighting the importance of facilitating range shifts for maintaining biodiversity.

Phenotypic plasticity with instantaneous but delayed switches.

Phenotypic plasticity is a widespread phenomenon, allowing organisms to better adapt to changing environments. Most empirical and theoretical studies are restricted to irreversible plasticity where the expression of a specific phenotype is mostly determined during development. However, reversible plasticity is not uncommon; here, organisms are able to switch back and forth between phenotypes. We present two optimization models for the fitness of (i) non-plastic, (ii) irreversibly plastic, and (iii) reversibly plastic genotypes in a fluctuating environment. In one model, the fitness values of an organism during different life phases act together multiplicatively (so as to consider traits that are related to survival). The other model additionally considers additive effects (corresponding to traits related to fecundity). Both models yield qualitatively similar results. If the only costs of reversible plasticity are due to temporal maladaptation while switching between phenotypes, reversibility is virtually always advantageous over irreversibility, especially for slow environmental fluctuations. If reversibility implies an overall decreased fitness, then irreversibility is advantageous if the environment fluctuates quickly or if stress events last relatively short. Our results are supported by observations from different types of organisms and have implications for many basic and applied research questions, e.g., on invasive alien species.