The relationship between neutral genetic diversity and performance in wild arthropod populations.

Larger effective populations (Ne) are characterized by higher genetic diversity, which is expected to predict population performance (average individual performance that influences fitness). Empirical studies of the relationship between neutral diversity and performance mostly represent species with small Ne, while there is limited data from the species-rich and ecologically important arthropods that are assumed to have large Ne but are threatened by massive declines. We performed a systematic literature search and used meta-analytical models to test the prediction of a positive association between neutral genetic diversity and performance in wild arthropods. From 14 relevant studies of 286 populations, we detected a weak (r = 0.15) but nonsignificant positive association both in the full data set (121 effect sizes) and a reduced data set accounting for dependency (14 effect sizes). Theory predicts that traits closely associated with fitness show a relatively stronger correlation with neutral diversity; this relationship was upheld for longevity and marginally for reproduction. Our analyses point to major knowledge gaps in our understanding of relationships between neutral diversity and performance. Future studies using genome-wide data sets across populations could guide more powerful designs to evaluate relationships between adaptive, deleterious and neutral diversity and performance.

Adaptation to fluctuating temperatures across life stages in endotherms.

Accelerating rates of climate change have intensified research on thermal adaptation. Increasing temperature fluctuations, a prominent feature of climate change, means that the persistence of many species depends on both heat and cold tolerance across the entire life cycle. In endotherms, research has focused on specific life stages, with changes in thermoregulation across life rarely being examined. Consequently, there is a need to (i) analyse how heat and cold tolerance mechanisms coevolve, and (ii) test whether antagonistic effects between heat and cold tolerance across different life stages limit thermal adaptation. Information on genes influencing heat and cold tolerance and how they are expressed through life will enable more accurate modelling of species vulnerabilities to future climatic volatility.

Heritable variation in thermal profiles is associated with reproductive success in the world's largest bird.

Organisms inhabiting extreme thermal environments, such as desert birds, have evolved spectacular adaptations to thermoregulate during hot and cold conditions. However, our knowledge of selection for thermoregulation and the potential for evolutionary responses is limited, particularly for large organisms experiencing extreme temperature fluctuations. Here we use thermal imaging to quantify selection and genetic variation in thermoregulation in ostriches (Struthio camelus), the world's largest bird species that is experiencing increasingly volatile temperatures. We found that females who are better at regulating their head temperatures ("thermoregulatory capacity") had higher egg-laying rates under hotter conditions. Thermoregulatory capacity was both heritable and showed signatures of local adaptation: females originating from more unpredictable climates were better at regulating their head temperatures in response to temperature fluctuations. Together these results reveal that past and present evolutionary processes have shaped genetic variation in thermoregulatory capacity, which appears to protect critical organs, such as the brain, from extreme temperatures during reproduction.

Systematic approaches to assessing high-temperature limits to fertility in animals.

Critical thermal limits (CTLs) gauge the physiological impact of temperature on survival or critical biological function, aiding predictions of species range shifts and climatic resilience. Two recent Drosophila species studies, using similar approaches to determine temperatures that induce sterility (thermal fertility limits [TFLs]), reveal that TFLs are often lower than CTLs and that TFLs better predict both current species distributions and extinction probability. Moreover, many studies show fertility is more sensitive at less extreme temperatures than survival (thermal sensitivity of fertility [TSF]). These results present a more pessimistic outlook on the consequences of climate change. However, unlike CTLs, TFL data are limited to Drosophila, and variability in TSF methods poses challenges in predicting species responses to increasing temperature. To address these data and methodological gaps, we propose 3 standardized approaches for assessing thermal impacts on fertility. We focus on adult obligate sexual terrestrial invertebrates but also provide modifications for other animal groups and life-history stages. We first outline a "gold-standard" protocol for determining TFLs, focussing on the effects of short-term heat shocks and simulating more frequent extreme heat events predicted by climate models. As this approach may be difficult to apply to some organisms, we then provide a standardized TSF protocol. Finally, we provide a framework to quantify fertility loss in response to extreme heat events in nature, given the limitations in laboratory approaches. Applying these standardized approaches across many taxa, similar to CTLs, will allow robust tests of the impact of fertility loss on species responses to increasing temperatures.

The evolutionary maintenance of ancient recombining sex chromosomes in the ostrich.

Sex chromosomes have evolved repeatedly across the tree of life and often exhibit extreme size dimorphism due to genetic degeneration of the sex-limited chromosome (e.g. the W chromosome of some birds and Y chromosome of mammals). However, in some lineages, ancient sex-limited chromosomes have escaped degeneration. Here, we study the evolutionary maintenance of sex chromosomes in the ostrich (Struthio camelus), where the W remains 65% the size of the Z chromosome, despite being more than 100 million years old. Using genome-wide resequencing data, we show that the population scaled recombination rate of the pseudoautosomal region (PAR) is higher than similar sized autosomes and is correlated with pedigree-based recombination rate in the heterogametic females, but not homogametic males. Genetic variation within the sex-linked region (SLR) (π = 0.001) was significantly lower than in the PAR, consistent with recombination cessation. Conversely, genetic variation across the PAR (π = 0.0016) was similar to that of autosomes and dependent on local recombination rates, GC content and to a lesser extent, gene density. In particular, the region close to the SLR was as genetically diverse as autosomes, likely due to high recombination rates around the PAR boundary restricting genetic linkage with the SLR to only ~50Kb. The potential for alleles with antagonistic fitness effects in males and females to drive chromosome degeneration is therefore limited. While some regions of the PAR had divergent male-female allele frequencies, suggestive of sexually antagonistic alleles, coalescent simulations showed this was broadly consistent with neutral genetic processes. Our results indicate that the degeneration of the large and ancient sex chromosomes of the ostrich may have been slowed by high recombination in the female PAR, reducing the scope for the accumulation of sexually antagonistic variation to generate selection for recombination cessation.

Impaired immune function accompanies social evolution in spiders.

An efficient immune system is essential to the survival of many animals. Sociality increases risk of pathogen transmission, which should select for enhanced immune function. However, two hypotheses instead predict a weakened immune function: relaxed selection caused by social immunity/protection, and reduced efficacy of selection due to inbreeding, reproductive skew and female bias in social species that reduce effective population size and accelerate genetic drift. We assessed the effect of social evolution on immune function in a comparative study of two social spider species and their closely related subsocial sister species (genus Stegodyphus). The haemolymph of social species was less efficient in inhibiting bacterial growth of the potentially pathogenic bacteria Bacillus subtilis than that of subsocial species. Reduced efficacy of selection in social species was supported by comparative genomic analysis showing substantially elevated non-synonymous substitutions in immune genes in one of the social species. We propose that impaired immune function results from reduced efficacy of selection because the evolution of sociality in spiders is accompanied by demographic processes that elevate genetic drift. Positive feedback between pathogen-induced local extinctions and the resulting elevation of genetic drift may further weaken responses to selection by pathogens, and threaten species persistence.

Experimental evidence that group size generates divergent benefits of cooperative breeding for male and female ostriches.

Cooperative breeding allows the costs of parental care to be shared, but as groups become larger, such benefits often decline as competition increases and group cohesion breaks down. The counteracting forces of cooperation and competition are predicted to select for an optimal group size, but variation in groups is ubiquitous across cooperative breeding animals. Here, we experimentally test if group sizes vary because of sex differences in the costs and benefits of cooperative breeding in captive ostriches, Struthio camelus, and compare this to the distribution of group sizes in the wild. We established 96 groups with different numbers of males (1 or 3) and females (1, 3, 4, or 6) and manipulated opportunities for cooperation over incubation. There was a clear optimal group size for males (one male with four or more females) that was explained by high costs of competition and negligible benefits of cooperation. Conversely, female reproductive success was maximised across a range of group sizes due to the benefits of cooperation with male and female group members. Reproductive success in intermediate sized groups was low for both males and females due to sexual conflict over the timing of mating and incubation. Our experiments show that sex differences in cooperation and competition can explain group size variation in cooperative breeders.

Being a parent is hard work. The unrelenting demand for food and protection is exhausting. Now imagine being a parent on the hot African savannah. Food and water are scarce, and whenever you leave your offspring, they overheat, or something eats them. This is the reality for ostriches. They, like humans, cope with the challenges of parenthood by sharing childcare responsibilities. Ostriches live in groups, breed in a communal nest, and take it in turns to incubate their eggs. This helps to maximize the survival of their offspring, but it has its downsides. The bigger a group gets, the more its members have to compete over mates and space for their eggs in the nest. The balance between cooperation and competition should, in theory, result in one 'optimal' group size. But this pattern does not seem to hold true: in the wild, ostrich families vary wildly in size and composition. To find out why, Melgar et al. set up dozens of groups of breeding ostriches and gave them different opportunities to cooperate. For males, there was one group size that maximized the number of offspring they produced (reproductive success): a single male with four or more females. Males did not benefit much from cooperation, and suffered greatly from competing with other males for mates. For females, however, the story was different. They benefited much more than males from cooperation and did best in bigger groups where they could share egg care with other individuals. Middle-sized groups were not good for either sex because reproduction was hard to coordinate: males continued to pursue copulations after females had initiated incubation, resulting in eggs being exposed and broken. The different priorities of males and females explain why there is no single optimal group size for ostriches. How groups balance competition and cooperation is a fundamental question in biology. Why do some organisms prefer to live alone, while others thrive in large groups? Understanding more about the balance of priorities within a group could hold the answers. It could also help to inform conservation work and animal breeding by showing how different social pressures influence breeding success.

The role of inter-individual intolerance in group cohesion and the transition to sociality in spiders.

Conspecific tolerance is key for maintaining group cohesion in animals. Understanding shifts from conspecific tolerance to intolerance is therefore important for understanding transitions to sociality. Subsocial species disperse to a solitary lifestyle after a gregarious juvenile phase and display conspecific intolerance as adults as a mechanism to maintain a solitary living. The development of intolerance towards group members is hypothesized to play a role in dispersal decisions in subsocial species. One hypothesis posits that dispersal is triggered by factors such as food competition with the subsequent development of conspecific intolerance, rather than conspecific intolerance developing prior to and potentially driving dispersal. Consistent with this hypothesis, we show that intolerance (inferred by inter-individual distance) developed post-dispersal in the subsocial spider Stegodyphus lineatus. The development of conspecific intolerance was delayed when maintaining spiders in groups showing plasticity in this trait, which is advantageous when trade-offs are not fixed over time. However, major evolutionary transitions, such as the transition to sociality, can permanently modify trade-offs and cause derived adaptations by the evolution of new or modified traits or evolutionary loss of traits that become redundant. Sociality in spiders has evolved repeatedly from subsocial ancestors, and social life in family groups combined with a lack of interaction with competing groups suggests relaxed selection for the development of conspecific intolerance. In the social Stegodyphus sarasinorum we found no evidence for the development of conspecific intolerance, consistent with the loss of this trait. Instead, we found evidence for conspecific attraction, which is likely to govern group cohesion.

Evolutionary trade-offs between heat and cold tolerance limit responses to fluctuating climates.

The evolutionary potential of species to cope with short-term temperature fluctuations during reproduction is critical to predicting responses to future climate change. Despite this, vertebrate research has focused on reproduction under high or low temperatures in relatively stable temperate climates. Here, we characterize the genetic basis of reproductive thermal tolerance to temperature fluctuations in the ostrich, which lives in variable environments in tropical and subtropical Africa. Both heat and cold tolerance were under selection and heritable, indicating the potential for evolutionary responses to mean temperature change. However, we found evidence for a negative, genetic correlation between heat and cold tolerance that should limit the potential for adaptation to fluctuating temperatures. Genetic constraints between heat and cold tolerance appear a crucial, yet underappreciated, factor influencing responses to climate change.

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.

The importance of environmental microbes for Drosophila melanogaster during seasonal macronutrient variability.

Experiments manipulating the nutritional environment and the associated microbiome of animals have demonstrated their importance for key fitness components. However, there is little information on how macronutrient composition and bacterial communities in natural food sources vary across seasons in nature and on how these factors affect the fitness components of insects. In this study, diet samples from an orchard compost heap, which is a natural habitat for many Drosophila species and other arthropods, were collected over 9 months covering all seasons in a temperate climate. We developed D. melanogaster on diet samples and investigated stress resistance and life-history traits as well as the microbial community of flies and compost. Nutrient and microbial community analysis of the diet samples showed marked differences in macronutrient composition and microbial community across seasons. However, except for the duration of development on these diet samples and Critical Thermal maximum, fly stress resistance and life-history traits were unaffected. The resulting differences in the fly microbial community were also more stable and less diverse than the microbial community of the diet samples. Our study suggests that when D. melanogaster are exposed to a vastly varying nutritional environment with a rich, diverse microbial community, the detrimental consequences of an unfavourable macronutrient composition are offset by the complex interactions between microbes and nutrients.

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.

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.

Extreme temperatures compromise male and female fertility in a large desert bird.

Temperature has a crucial influence on the places where species can survive and reproduce. Past research has primarily focused on survival, making it unclear if temperature fluctuations constrain reproductive success, and if so whether populations harbour the potential to respond to climatic shifts. Here, using two decades of data from a large experimental breeding programme of the iconic ostrich (Struthio camelus) in South Africa, we show that the number of eggs females laid and the number of sperm males produced were highly sensitive to natural temperature extremes (ranging from -5 °C to 45 °C). This resulted in reductions in reproductive success of up to 44% with 5 °C deviations from their thermal optimum. In contrast, gamete quality was largely unaffected by temperature. Extreme temperatures also did not expose trade-offs between gametic traits. Instead, some females appeared to invest more in reproducing at high temperatures, which may facilitate responses to climate change. These results show that the robustness of fertility to temperature fluctuations, and not just temperature increases, is a critical aspect of species persistence in regions predicted to undergo the greatest change in climate volatility.

Pronounced Plastic and Evolutionary Responses to Unpredictable Thermal Fluctuations in Drosophila simulans.

Organisms are exposed to temperatures that vary, for example on diurnal and seasonal time scales. Thus, the ability to behaviorally and/or physiologically respond to variation in temperatures is a fundamental requirement for long-term persistence. Studies on thermal biology in ectotherms are typically performed under constant laboratory conditions, which differ markedly from the variation in temperature across time and space in nature. Here, we investigate evolutionary adaptation and environmentally induced plastic responses of Drosophila simulans to no fluctuations (constant), predictable fluctuations or unpredictable fluctuations in temperature. We whole-genome sequenced populations exposed to 20 generations of experimental evolution under the three thermal regimes and examined the proteome after short-term exposure to the same three regimes. We find that unpredictable fluctuations cause the strongest response at both genome and proteome levels. The loci showing evolutionary responses were generally unique to each thermal regime, but a minor overlap suggests either common laboratory adaptation or that some loci were involved in the adaptation to multiple thermal regimes. The evolutionary response, i.e., loci under selection, did not coincide with induced responses of the proteome. Thus, genes under selection in fluctuating thermal environments are distinct from genes important for the adaptive plastic response observed within a generation. This information is key to obtain a better understanding and prediction of the effects of future increases in both mean and variability of temperatures.

Patterns of environmental variance across environments and traits in domestic cattle.

The variance in phenotypic trait values is a product of environmental and genetic variation. The sensitivity of traits to environmental variation has a genetic component and is likely to be under selection. However, there are few studies investigating the evolution of this sensitivity, in part due to the challenges of estimating the environmental variance. The livestock literature provides a wealth of studies that accurately partition components of phenotypic variance, including the environmental variance, in well-defined environments. These studies involve breeds that have been under strong selection on mean phenotype in optimal environments for many generations, and therefore represent an opportunity to study the potential evolution of trait sensitivity to environmental conditions. Here, we use literature on domestic cattle to examine the evolution of micro-environmental variance (CVR-the coefficient of residual variance) by testing for differences in expression of CVR in animals from the same breed reared in different environments. Traits that have been under strong selection did not follow a null expectation of an increase in CVR in heterogenous environments (e.g., grazing), a pattern that may reflect evolution of increased uniformity in heterogeneous environments. When comparing CVR across environments of different levels of optimality, here measured by trait mean, we found a reduction in CVR in the more optimal environments for both life history and growth traits. Selection aimed at increasing trait means in livestock breeds typically occurs in the more optimal environments, and we therefore suspect that the decreased CVR is a consequence of evolution of the expression of micro-environmental variance in this environment. Our results highlight the heterogeneity in micro-environmental variance across environments and point to possible connections to the intensity of selection on trait means.

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.

Prey to predator body size ratio in the evolution of cooperative hunting-a social spider test case.

One of the benefits of cooperative hunting may be that predators can subdue larger prey. In spiders, cooperative, social species can capture prey many times larger than an individual predator. However, we propose that cooperative prey capture does not have to be associated with larger caught prey per se, but with an increase in the ratio of prey to predator body size. This can be achieved either by catching larger prey while keeping predator body size constant, or by evolving a smaller predator body size while maintaining capture of large prey. We show that within a genus of relatively large spiders, Stegodyphus, subsocial spiders representing the ancestral state of social species are capable of catching the largest prey available in the environment. Hence, within this genus, the evolution of cooperation would not provide access to otherwise inaccessible, large prey. Instead, we show that social Stegodyphus spiders are smaller than their subsocial counterparts, while catching similar sized prey, leading to the predicted increase in prey-predator size ratio with sociality. We further show that in a genus of small spiders, Anelosimus, the level of sociality is associated with an increased size of prey caught while predator size is unaffected by sociality, leading to a similar, predicted increase in prey-predator size ratio. In summary, we find support for our proposed 'prey to predator size ratio hypothesis' and discuss how relaxed selection on large body size in the evolution of social, cooperative living may provide adaptive benefits for ancestrally relatively large predators.

Fluctuations in nutrient composition affect male reproductive output in Drosophila melanogaster.

Insects are known to selectively balance their intake of protein and carbohydrate to optimize reproduction and survival. For insects who feed on decomposing fruit, fluctuations in macronutrient composition occur as fruits ripe and decomposition progresses which may challenge optimal resource allocation. Using Drosophila melanogaster, we tested the effect of macronutrient fluctuations and the variability of these fluctuations on starvation resistance and components of reproductive output; traits known to be sensitive to different protein to carbohydrate (P:C) ratios in the diet. For 8 days, flies were fed the same protein to carbohydrate (P:C) ratio (constant feeding), or fed diets with fluctuations in P:C ratio on each day; these fluctuations being regular (predictably fluctuating) or irregular (unpredictably fluctuating). The three feeding regimes yielded the same average P:C ratio across the duration of the experiment. We found no difference in starvation resistance across the feeding regimes. Interestingly, there was a sexual dimorphism in the effect on reproductive output with males performing worst in the unpredictable feeding regime, and with no effect of feeding regime on female performance. Our study provides evidence for means of adapting to fluctuating macronutrient composition and suggests females are more tactful than males in storing and allocating resources for reproduction.

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.