Transcriptomic and functional genetic evidence for distinct ecophysiological responses across complex life cycle stages.

Organisms with complex life cycles demonstrate a remarkable ability to change their phenotypes across development, presumably as an evolutionary adaptation to developmentally variable environments. Developmental variation in environmentally sensitive performance, and thermal sensitivity in particular, has been well documented in holometabolous insects. For example, thermal performance in adults and juvenile stages exhibit little genetic correlation (genetic decoupling) and can evolve independently, resulting in divergent thermal responses. Yet, we understand very little about how this genetic decoupling occurs. We tested the hypothesis that genetic decoupling of thermal physiology is driven by fundamental differences in physiology between life stages, despite a potentially conserved cellular stress response. We used RNAseq to compare transcript expression in response to a cold stressor in Drosophila melanogaster larvae and adults and used RNA interference (RNAi) to test whether knocking down nine target genes differentially affected larval and adult cold tolerance. Transcriptomic responses of whole larvae and adults during and following exposure to -5°C were largely unique both in identity of responding transcripts and in temporal dynamics. Further, we analyzed the tissue-specificity of differentially expressed transcripts from FlyAtlas 2 data, and concluded that stage-specific differences in transcription were not simply driven by differences in tissue composition. In addition, RNAi of target genes resulted in largely stage-specific and sometimes sex-specific effects on cold tolerance. The combined evidence suggests that thermal physiology is largely stage-specific at the level of gene expression, and thus natural selection may be acting on different loci during the independent thermal adaptation of different life stages.

Stage-specific genotype-by-environment interactions for cold and heat hardiness in Drosophila melanogaster.

Environments often vary across a life cycle, imposing fluctuating natural selection across development. Such fluctuating selection can favor different phenotypes in different life stages, but stage-specific evolutionary responses will depend on genetic variance, covariance, and their interaction across development and across environments. Thus, quantifying how genetic architecture varies with plastic responses to the environment and across development is vital to predict whether stage-specific adaptation will occur in nature. Additionally, the interaction of genetic variation and environmental plasticity (GxE) may be stage-specific, leading to a three-way interaction between genotype, environment, and development or GxDxE. To test for these patterns, we exposed larvae and adults of Drosophila melanogaster isogenic lines derived from a natural population to extreme heat and cold stress after developmental acclimation to cool (18 °C) and warm (25 °C) conditions and measured genetic variance for thermal hardiness. We detected significant GxE that was specific to larvae and adults for cold and heat hardiness (GxDxE), but no significant genetic correlation across development for either trait at either acclimation temperature. However, cross-development phenotypic correlations for acclimation responses suggest that plasticity itself may be developmentally constrained, though rigorously testing this hypothesis requires more experimentation. These results illustrate the potential for stage-specific adaptation within a complex life cycle and demonstrate the importance of measuring traits at appropriate developmental stages and environmental conditions when predicting evolutionary responses to changing climates.

Constraints, independence, and evolution of thermal plasticity: probing genetic architecture of long- and short-term thermal acclimation.

Seasonal and daily thermal variation can limit species distributions because of physiological tolerances. Low temperatures are particularly challenging for ectotherms, which use both basal thermotolerance and acclimation, an adaptive plastic response, to mitigate thermal stress. Both basal thermotolerance and acclimation are thought to be important for local adaptation and persistence in the face of climate change. However, the evolutionary independence of basal and plastic tolerances remains unclear. Acclimation can occur over longer (seasonal) or shorter (hours to days) time scales, and the degree of mechanistic overlap is unresolved. Using a midlatitude population of Drosophila melanogaster, we show substantial heritable variation in both short- and long-term acclimation. Rapid cold hardening (short-term plasticity) and developmental acclimation (long-term plasticity) are positively correlated, suggesting shared mechanisms. However, there are independent components of these traits, because developmentally acclimated flies respond positively to short-term acclimation. A strong negative correlation between basal cold tolerance and developmental acclimation suggests that basal cold tolerance may constrain developmental acclimation, whereas a weaker negative correlation between basal cold tolerance and short-term acclimation suggests less constraint. Using genome-wide association mapping, we show the genetic architecture of rapid cold hardening and developmental acclimation responses are nonoverlapping at the SNP and corresponding gene level. However, genes associated with each trait share functional similarities, including genes involved in apoptosis and autophagy, cytoskeletal and membrane structural components, and ion binding and transport. These results indicate substantial opportunity for short-term and long-term acclimation responses to evolve separately from each other and for short-term acclimation to evolve separately from basal thermotolerance.

Speed of exposure to rapid cold hardening and genotype drive the level of acclimation response in Drosophila melanogaster.

Comparing methodologies that attempt to mimic natural conditions is important when evaluating thermal tolerances of ectotherms, as exposing animals to different artificial thermal regimes may provide conflicting information of an insect's thermal profile. Rapid cold hardening (RCH) occurs in ectotherms and typically increases survivorship to extreme cold exposure through a short, pre-treatment to a non-lethal cold temperature. Here we assess survivorship in a set of genotypes from the Drosophila melanogaster Reference Panel for direct and ramping RCH pre-treatments at cooling rates occurring under more natural conditions (0.1 °C/min and 0.5 °C/min) in combination with a direct and ramped rewarming treatment post cold exposure. We find that all three pre-treatment exposures alone significantly increase survivorship. We find significant correlations in survivorship among treatments across genotypes, suggesting that regardless of the pre-treatment, individuals of a given genotype have an innate level of acclimation. When rewarming is introduced, survivorship significantly decreased relative to pre-treatment alone and correlations of survival between phenotypes were not significant. Our results suggest that rewarming and slow RCH are costly to survival while a quicker RCH may impart physiological benefits more consistently across genotypes.

Cold adaptation increases rates of nutrient flow and metabolic plasticity during cold exposure in Drosophila melanogaster.

Metabolic flexibility is an important component of adaptation to stressful environments, including thermal stress and latitudinal adaptation. A long history of population genetic studies suggest that selection on core metabolic enzymes may shape life histories by altering metabolic flux. However, the direct relationship between selection on thermal stress hardiness and metabolic flux has not previously been tested. We investigated flexibility of nutrient catabolism during cold stress in Drosophila melanogaster artificially selected for fast or slow recovery from chill coma (i.e. cold-hardy or -susceptible), specifically testing the hypothesis that stress adaptation increases metabolic turnover. Using (13)C-labelled glucose, we first showed that cold-hardy flies more rapidly incorporate ingested carbon into amino acids and newly synthesized glucose, permitting rapid synthesis of proline, a compound shown elsewhere to improve survival of cold stress. Second, using glucose and leucine tracers we showed that cold-hardy flies had higher oxidation rates than cold-susceptible flies before cold exposure, similar oxidation rates during cold exposure, and returned to higher oxidation rates during recovery. Additionally, cold-hardy flies transferred compounds among body pools more rapidly during cold exposure and recovery. Increased metabolic turnover may allow cold-adapted flies to better prepare for, resist and repair/tolerate cold damage. This work illustrates for the first time differences in nutrient fluxes associated with cold adaptation, suggesting that metabolic costs associated with cold hardiness could invoke resource-based trade-offs that shape life histories.

Artificial selection on chill-coma recovery time in Drosophila melanogaster: Direct and correlated responses to selection.

Artificial selection can be used to create populations with extreme phenotypic responses to environmental stressors. When artificial selection is applied to a single component of a stress response, this selection may result in correlated responses in other stress responses, a phenomenon called cross-tolerance, which is ultimately controlled by the genetic correlations among traits. We selected for extreme responses to cold tolerance by selecting for chill-coma recovery time from a single temperate population of Drosophila melanogaster. Chill-coma recovery time is a common metric of low, but non-lethal, cold temperature tolerance. Replicated divergent artificial selection was applied to a genetically variable base population for 31 generations, resulting in two cold resistant, two cold susceptible, and two unselected control lines. To quantify the relationship between selection on chill-coma recovery and other metrics of thermal performance, we also measured survivorship after acute cold exposure, survivorship after chronic cold exposure, survivorship after cold exposure following a pre-treatment period (rapid cold hardening), starvation tolerance, and heat tolerance. We find that chill-coma recovery time is heritable within this population and that there is an asymmetric response to increased and decreased chill-coma recovery time. Surprisingly, we found no cross-tolerances between selection on chill-coma recovery time and the other environmental stress response traits. These results suggest that although artificial selection has dramatically altered chill-coma recovery time, the correlated response to selection on other stress response phenotypes has been negligible. The lack of a correlated response suggests that chill-coma recovery time in these selection lines is likely genetically independent from measures of cold survivorship tested here.

Pleiotropic fitness effects of the Tre1-Gr5a region in Drosophila melanogaster.

The abundance of transposable elements and DNA repeat sequences in mammalian genomes raises the question of whether such insertions represent passive evolutionary baggage or may influence the expression of complex traits. We addressed this question in Drosophila melanogaster, in which the effects of single transposable elements on complex traits can be assessed in genetically identical individuals reared in controlled environments. Here we demonstrate that single P-element insertions in the intergenic region between the gustatory receptor 5a (Gr5a, also known as Tre) and trapped in endoderm 1 (Tre1), which encodes an orphan receptor, exert complex pleiotropic effects on fitness traits, including selective nutrient intake, life span, and resistance to starvation and heat stress. Mutations in this region interact epistatically with downstream components of the insulin signaling pathway. Transposon-induced sex-specific and sex-antagonistic effects further accentuate the complex influences that intergenic transposable elements can contribute to quantitative trait phenotypes.

Ecotypes of an ecologically dominant prairie grass (Andropogon gerardii) exhibit genetic divergence across the U.S. Midwest grasslands' environmental gradient.

Big bluestem (Andropogon gerardii) is an ecologically dominant grass with wide distribution across the environmental gradient of U.S. Midwest grasslands. This system offers an ideal natural laboratory to study population divergence and adaptation in spatially varying climates. Objectives were to: (i) characterize neutral genetic diversity and structure within and among three regional ecotypes derived from 11 prairies across the U.S. Midwest environmental gradient, (ii) distinguish between the relative roles of isolation by distance (IBD) vs. isolation by environment (IBE) on ecotype divergence, (iii) identify outlier loci under selection and (iv) assess the association between outlier loci and climate. Using two primer sets, we genotyped 378 plants at 384 polymorphic AFLP loci across regional ecotypes from central and eastern Kansas and Illinois. Neighbour-joining tree and PCoA revealed strong genetic differentiation between Kansas and Illinois ecotypes, which was better explained by IBE than IBD. We found high genetic variability within prairies (80%) and even fragmented Illinois prairies, surprisingly, contained high within-prairie genetic diversity (92%). Using Bayenv2, 14 top-ranked outlier loci among ecotypes were associated with temperature and precipitation variables. Six of seven BayeScanFST outliers were in common with Bayenv2 outliers. High genetic diversity may enable big bluestem populations to better withstand changing climates; however, population divergence supports the use of local ecotypes in grassland restoration. Knowledge of genetic variation in this ecological dominant and other grassland species will be critical to understanding grassland response and restoration challenges in the face of a changing climate.

Natural selection drives clinal life history patterns in the perennial sunflower species, Helianthus maximiliani.

In plants, ecologically important life history traits often display clinal patterns of population divergence. Such patterns can provide strong evidence for spatially varying selection across environmental gradients but also may result from nonselective processes, such as genetic drift, population bottlenecks and spatially restricted gene flow. Comparison of population differentiation in quantitative traits (measured as Q(ST) ) with neutral molecular markers (measured as F(ST) ) provides a useful tool for understanding the relative importance of adaptive and nonadaptive processes in the formation and maintenance of clinal variation. Here, we demonstrate the existence of geographic variation in key life history traits in the diploid perennial sunflower species Helianthus maximiliani across a broad latitudinal transect in North America. Strong population differentiation was found for days to flowering, growth rate and multiple size-related traits. Differentiation in these traits greatly exceeds neutral predictions, as determined both by partial Mantel tests and by comparisons of global Q(ST) values with theoretical F(ST) distributions. These findings indicate that clinal variation in these life history traits likely results from local adaptation driven by spatially heterogeneous environments.

Genetic variation in heat-stress tolerance among South American Drosophila populations.

Spatial or temporal differences in environmental variables, such as temperature, are ubiquitous in nature and impose stress on organisms. This is especially true for organisms that are isothermal with the environment, such as insects. Understanding the means by which insects respond to temperature and how they will react to novel changes in environmental temperature is important for understanding the adaptive capacity of populations and to predict future trajectories of evolutionary change. The organismal response to heat has been identified as an important environmental variable for insects that can dramatically influence life history characters and geographic range. In the current study we surveyed the amount of variation in heat tolerance among Drosophila melanogaster populations collected at diverse sites along a latitudinal gradient in Argentina (24°-38°S). This is the first study to quantify heat tolerance in South American populations and our work demonstrates that most of the populations surveyed have abundant within-population phenotypic variation, while still exhibiting significant variation among populations. The one exception was the most heat tolerant population that comes from a climate exhibiting the warmest annual mean temperature. All together our results suggest there is abundant genetic variation for heat-tolerance phenotypes within and among natural populations of Drosophila and this variation has likely been shaped by environmental temperature.

Genetic variation in senescence marker protein-30 is associated with natural variation in cold tolerance in Drosophila.

A comprehensive understanding of the genetic basis of phenotypic adaptation in nature requires the identification of the functional allelic variation underlying adaptive phenotypes. The manner in which organisms respond to temperature extremes is an adaptation in many species. In the current study, we investigate the role of molecular variation in senescence marker protein-30 (Smp-30) on natural phenotypic variation in cold tolerance in Drosophila melanogaster. Smp-30 encodes a product that is thought to be involved in the regulation of Ca2+ ion homeostasis and has been shown previously to be differentially expressed in response to cold stress. Thus, we sought to assess whether molecular variation in Smp-30 was associated with natural phenotypic variation in cold tolerance in a panel of naturally derived inbred lines from a population in Raleigh, North Carolina. We identified four non-coding polymorphisms that were strongly associated with natural phenotypic variation in cold tolerance. Interestingly, two polymorphisms that were in close proximity to one another (2 bp apart) exhibited opposite phenotypic effects. Consistent with the maintenance of a pair of antagonistically acting polymorphisms, tests of molecular evolution identified a significant excess of maintained variation in this region, suggesting balancing selection is acting to maintain this variation. These results suggest that multiple mutations in non-coding regions can have significant effects on phenotypic variation in adaptive traits within natural populations, and that balancing selection can maintain polymorphisms with opposite effects on phenotypic variation.

Quantitative genetics of pigmentation development in 2 populations of the common garter snake, Thamnophis sirtalis.

The evolutionary importance of ontogenetic change has been noted since Darwin. However, most analyses of phenotypic evolution focus on single landmark ages. Here, we present an inheritance study that quantifies genetic variation in pigmentation across early-age (i.e., birth to 180 days) development in 2 populations of the common garter snake, Thamnophis sirtalis. The populations are phenotypically distinct and geographically isolated (Manitoba, CA and Northern California, USA). There were highly significant differences between populations for the developmental trajectory of mean pigmentation, with the Manitoba population exhibiting a mean pigmentation level that increased across ontogeny, whereas the California population exhibited mean pigmentation that was invariant across ontogeny. Subsequent quantitative genetic analyses revealed heritable variation at all ages in Manitoba but low levels of phenotypic and genetic variation in California at all ages. A quantitative genetic decomposition of the longitudinal genetic variance-covariance matrix for the age-specific pigmentation phenotypes in the Manitoba population revealed 2 primary orthogonal axes that explained most ( approximately 100%) of the pigmentation variation across ontogeny. The primary axis, explaining 93% of the genetic variation, is an axis of genetic variation whose principal value loadings change from positive to negative across development, suggesting that the most rapid evolutionary response to selection on pigmentation variation will occur in the direction characterized by a tradeoff in early-age versus late-age pigmentation phenotypes. Pigmentation is known to be ecologically important and subject to rapid evolution under selection. Our study shows that significant differences exist between these 2 populations for their capacity to respond to selection on pigmentation which is not only influenced by the population of origin but also by the developmental process. We suggest that developmental timing may be a potential explanatory mechanism for the difference between the populations.

Phenotypic variation and natural selection at catsup, a pleiotropic quantitative trait gene in Drosophila.

Quantitative traits are shaped by networks of pleiotropic genes . To understand the mechanisms that maintain genetic variation for quantitative traits in natural populations and to predict responses to artificial and natural selection, we must evaluate pleiotropic effects of underlying quantitative trait genes and define functional allelic variation at the level of quantitative trait nucleotides (QTNs). Catecholamines up (Catsup), which encodes a negative regulator of tyrosine hydroxylase , the rate-limiting step in the synthesis of the neurotransmitter dopamine, is a pleiotropic quantitative trait gene in Drosophila melanogaster. We used association mapping to determine whether the same or different QTNs at Catsup are associated with naturally occurring variation in multiple quantitative traits. We sequenced 169 Catsup alleles from a single population and detected 33 polymorphisms with little linkage disequilibrium (LD). Different molecular polymorphisms in Catsup are independently associated with variation in longevity, locomotor behavior, and sensory bristle number. Most of these polymorphisms are potentially functional variants in protein coding regions, have large effects, and are not common. Thus, Catsup is a pleiotropic quantitative trait gene, but individual QTNs do not have pleiotropic effects. Molecular population genetic analyses of Catsup sequences are consistent with balancing selection maintaining multiple functional polymorphisms.

Quantitative genomics of aggressive behavior in Drosophila melanogaster.

Aggressive behavior is important for animal survival and reproduction, and excessive aggression is an enormous social and economic burden for human society. Although the role of biogenic amines in modulating aggressive behavior is well characterized, other genetic mechanisms affecting this complex behavior remain elusive. Here, we developed an assay to rapidly quantify aggressive behavior in Drosophila melanogaster, and generated replicate selection lines with divergent levels of aggression. The realized heritability of aggressive behavior was approximately 0.10, and the phenotypic response to selection specifically affected aggression. We used whole-genome expression analysis to identify 1,539 probe sets with different expression levels between the selection lines when pooled across replicates, at a false discovery rate of 0.001. We quantified the aggressive behavior of 19 mutations in candidate genes that were generated in a common co-isogenic background, and identified 15 novel genes affecting aggressive behavior. Expression profiling of genetically divergent lines is an effective strategy for identifying genes affecting complex traits.

Phenology of Drosophila species across a temperate growing season and implications for behavior.

Drosophila community composition is complex in temperate regions with different abundance of flies and species across the growing season. Monitoring Drosophila populations provides insights into the phenology of both native and invasive species. Over a single growing season, we collected Drosophila at regular intervals and determined the number of individuals of the nine species we found in Kansas, USA. Species varied in their presence and abundance through the growing season with peak diversity occurring after the highest seasonal temperatures. We developed models for the abundance of the most common species, Drosophila melanogaster, D. simulans, D. algonquin, and the recent invasive species, D. suzukii. These models revealed that temperature played the largest role in abundance of each species across the season. For the two most commonly studied species, D. melanogaster and D. simulans, the best models indicate shifted thermal optima compared to laboratory studies, implying that fluctuating temperature may play a greater role in the physiology and ecology of these insects than indicated by laboratory studies, and should be considered in global climate change studies.

Antagonistic pleiotropy and mutation accumulation contribute to age-related decline in stress response.

As organisms age, the effectiveness of natural selection weakens, leading to age-related decline in fitness-related traits. The evolution of age-related changes associated with senescence is likely influenced by mutation accumulation (MA) and antagonistic pleiotropy (AP). MA predicts that age-related decline in fitness components is driven by age-specific sets of alleles, nonnegative genetic correlations within trait across age, and an increase in the coefficient of genetic variance. AP predicts that age-related decline in a trait is driven by alleles with positive effects on fitness in young individuals and negative effects in old individuals, and is expected to lead to negative genetic correlations within traits across age. We build on these predictions using an association mapping approach to investigate the change in additive effects of SNPs across age and among traits for multiple stress-response fitness-related traits, including cold stress with and without acclimation and starvation resistance. We found support for both MA and AP theories of aging in the age-related decline in stress tolerance. Our study demonstrates that the evolution of age-related decline in stress tolerance is driven by a combination of alleles that have age-specific additive effects, consistent with MA, as well as nonindependent and antagonistic genetic architectures characteristic of AP.

Cold adaptation does not alter ATP homeostasis during cold exposure in Drosophila melanogaster.

In insects and other ectotherms, cold temperatures cause a coma resulting from loss of neuromuscular function, during which ionic and metabolic homeostasis are progressively lost. Cold adaptation improves homeostasis during cold exposure, but the ultimate targets of selection are still an open question. Cold acclimation and adaptation remodels mitochondrial metabolism in insects, suggesting that aerobic energy production during cold exposure could be a target of selection. Here, we test the hypothesis that cold adaptation improves the ability to maintain rates of aerobic energy production during cold exposure by using 31 P NMR on live flies. Using lines of Drosophila melanogaster artificially selected for fast and slow recovery from a cold coma, we show that cold exposure does not lower ATP levels and that cold adaptation does not alter aerobic ATP production during cold exposure. Cold-hardy and cold-susceptible lines both experienced a brief transition to anaerobic metabolism during cooling, but this was rapidly reversed during cold exposure, suggesting that oxidative phosphorylation was sufficient to meet energy demands below the critical thermal minimum, even in cold-susceptible flies. We thus reject the hypothesis that performance under mild low temperatures is set by aerobic ATP supply limitations in D. melanogaster, excluding oxygen and capacity limitation as a weak link in energy supply. This work suggests that the modulations to mitochondrial metabolism resulting from cold acclimation or adaptation may arise from selection on a biosynthetic product(s) of those pathways rather than selection on ATP supply during cold exposure.

Costs of cold acclimation on survival and reproductive behavior in Drosophila melanogaster.

Fitness is determined by the ability of an organism to both survive and reproduce; however, the mechanisms that lead to increased survival may not have the same effect on reproductive success. We used nineteen natural Drosophila melanogaster genotypes from the Drosophila Genetic Reference Panel to determine if adaptive plasticity following short-term acclimation through rapid cold-hardening (RCH) affects mating behavior and mating success. We confirmed that exposure to the acclimation temperature is beneficial to survival following cold stress; however, we found that this same acclimation temperature exposure led to less efficient male courtship and a significant decrease in the likelihood of mating. Cold tolerance and the capacity to respond plastically to cold stress were not correlated with mating behavior following acclimation, suggesting that the genetic control of the physiological effects of the cold temperature exposure likely differ between survival and behavioral responses. We also tested whether the exposure of males to the acclimation temperature influenced courtship song. This exposure again significantly increased courtship duration; however, courtship song was unchanged. These results illustrate costs of short-term acclimation on survival and reproductive components of fitness and demonstrate the pronounced effect that short-term thermal environment shifts can have on reproductive success.

Quantitative trait loci for locomotor behavior in Drosophila melanogaster.

Locomotion is an integral component of most animal behaviors and many human diseases and disorders are associated with locomotor deficits, but little is known about the genetic basis of natural variation in locomotor behavior. Locomotion is a complex trait, with variation attributable to the joint segregation of multiple interacting quantitative trait loci (QTL), with effects that are sensitive to the environment. We assessed variation in a component of locomotor behavior (locomotor reactivity) in a population of 98 recombinant inbred lines of Drosophila melanogaster and mapped four QTL affecting locomotor reactivity by linkage to polymorphic roo transposable element insertion sites. We used complementation tests of deficiencies to fine map these QTL to 12 chromosomal regions and complementation tests of mutations to identify 13 positional candidate genes affecting locomotor reactivity, including Dopa decarboxylase (Ddc), which catalyzes the final step in the synthesis of serotonin and dopamine. Linkage disequilibrium mapping in a population of 164 second chromosome substitution lines derived from a single natural population showed that polymorphisms at Ddc were associated with naturally occurring genetic variation in locomotor behavior. These data implicate variation in the synthesis of bioamines as a factor contributing to natural variation in locomotor reactivity.