Single-fly assemblies fill major phylogenomic gaps across the Drosophilidae Tree of Life.

Long-read sequencing is driving rapid progress in genome assembly across all major groups of life, including species of the family Drosophilidae, a longtime model system for genetics, genomics, and evolution. We previously developed a cost-effective hybrid Oxford Nanopore (ONT) long-read and Illumina short-read sequencing approach and used it to assemble 101 drosophilid genomes from laboratory cultures, greatly increasing the number of genome assemblies for this taxonomic group. The next major challenge is to address the laboratory culture bias in taxon sampling by sequencing genomes of species that cannot easily be reared in the lab. Here, we build upon our previous methods to perform amplification-free ONT sequencing of single wild flies obtained either directly from the field or from ethanol-preserved specimens in museum collections, greatly improving the representation of lesser studied drosophilid taxa in whole-genome data. Using Illumina Novaseq X Plus and ONT P2 sequencers with R10.4.1 chemistry, we set a new benchmark for inexpensive hybrid genome assembly at US $150 per genome while assembling genomes from as little as 35 ng of genomic DNA from a single fly. We present 183 new genome assemblies for 179 species as a resource for drosophilid systematics, phylogenetics, and comparative genomics. Of these genomes, 62 are from pooled lab strains and 121 from single adult flies. Despite the sample limitations of working with small insects, most single-fly diploid assemblies are comparable in contiguity (>1Mb contig N50), completeness (>98% complete dipteran BUSCOs), and accuracy (>QV40 genome-wide with ONT R10.4.1) to assemblies from inbred lines. We present a well-resolved multi-locus phylogeny for 360 drosophilid and 4 outgroup species encompassing all publicly available (as of August 2023) genomes for this group. Finally, we present a Progressive Cactus whole-genome, reference-free alignment built from a subset of 298 suitably high-quality drosophilid genomes. The new assemblies and alignment, along with updated laboratory protocols and computational pipelines, are released as an open resource and as a tool for studying evolution at the scale of an entire insect family.

Assessing the effects of a drought experiment on the reproductive phenology and ecophysiology of a wet tropical rainforest community.

Climate change is expected to increase the intensity and occurrence of drought in tropical regions, potentially affecting the phenology and physiology of tree species. Phenological activity may respond to a drying and warming environment by advancing reproductive timing and/or diminishing the production of flowers and fruits. These changes have the potential to disrupt important ecological processes, with potentially wide-ranging effects on tropical forest function. Here, we analysed the monthly flowering and fruiting phenology of a tree community (337 individuals from 30 species) over 7 years in a lowland tropical rainforest in northeastern Australia and its response to a throughfall exclusion drought experiment (TFE) that was carried out from 2016 to 2018 (3 years), excluding approximately 30% of rainfall. We further examined the ecophysiological effects of the TFE on the elemental (C:N) and stable isotope (δ13C and δ15N) composition of leaves, and on the stable isotope composition (δ13C and δ18O) of stem wood of four tree species. At the community level, there was no detectable effect of the TFE on flowering activity overall, but there was a significant effect recorded on fruiting and varying responses from the selected species. The reproductive phenology and physiology of the four species examined in detail were largely resistant to impacts of the TFE treatment. One canopy species in the TFE significantly increased in fruiting and flowering activity, whereas one understory species decreased significantly in both. There was a significant interaction between the TFE treatment and season on leaf C:N for two species. Stable isotope responses were also variable among species, indicating species-specific responses to the TFE. Thus, we did not observe consistent patterns in physiological and phenological changes in the tree community within the 3 years of TFE treatment examined in this study.

Chromosome Comparisons of Australian Scaptodrosophila Species.

The Scaptodrosophila represent a diverse group of Diptera closely related to Drosophila. Although they have radiated extensively in Australia, they have been the focus of few studies. Here, we characterized the karyotypes of 12 Scaptodrosophila species from several species groups and showed that they have undergone similar types of karyotypic change to those seen in Drosophila. This includes heterochromatin amplification involved in length changes of the sex and 'dot' chromosomes as well as the autosomes, particularly in the coracina group of species. Numerous weak points along the arms of the polytene chromosomes suggest the presence of internal repetitive sequence DNA, but these regions did not C-band in mitotic chromosomes, and their analysis will depend on DNA sequencing. The nucleolar organizing regions (NORs) are at the same chromosome positions in Scaptodrosophila as in Drosophila, and the various mechanisms responsible for changing arm configurations also appear to be the same. These chromosomal studies provide a complementary resource to other investigations of this group, with several species currently being sequenced.

Into the wild-a field study on the evolutionary and ecological importance of thermal plasticity in ectotherms across temperate and tropical regions.

Understanding how environmental factors affect the thermal tolerance of species is crucial for predicting the impact of thermal stress on species abundance and distribution. To date, species' responses to thermal stress are typically assessed on laboratory-reared individuals and using coarse, low-resolution, climate data that may not reflect microhabitat dynamics at a relevant scale. Here, we examine the daily temporal variation in heat tolerance in a range of species in their natural environments across temperate and tropical Australia. Individuals were collected in their habitats throughout the day and tested for heat tolerance immediately thereafter, while local microclimates were recorded at the collection sites. We found high levels of plasticity in heat tolerance across all the tested species. Both short- and long-term variability of temperature and humidity affected plastic adjustments of heat tolerance within and across days, but with species differences. Our results reveal that plastic changes in heat tolerance occur rapidly at a daily scale and that environmental factors on a relatively short timescale are important drivers of the observed variation in thermal tolerance. Ignoring such fine-scale physiological processes in distribution models might obscure conclusions about species' range shifts with global climate change. This article is part of the theme issue 'Species' ranges in the face of changing environments (part 1)'.

Characterization of the first Wolbachia from the genus Scaptodrosophila, a male-killer from the rainforest species S. claytoni.

The Scaptodrosophila genus represents a large group of drosophilids with a worldwide distribution and a predominance of species in Australia, but there is little information on the presence and impacts of Wolbachia endosymbionts in this group. Here we describe the first Wolbachia infection from this group, wClay isolated from Scaptodrosophila claytoni (van Klinken), a species from the east coast of Australia. The infection is polymorphic in natural populations, occurring at a frequency of around 6%-10%. wClay causes male killing, producing female-biased lines; most lines showed 100% male killing, though in 1 line it was <80%. The lines need to be maintained through the introduction of males unless the infection is removed by tetracycline treatment. wClay is transmitted at a high fidelity (98.6%) through the maternal lineage and has been stable in 2 laboratory lines across 24 generations, suggesting it is likely to persist in populations. The infection has not been previously described but is closely related to the male-killing Wolbachia recently described from Drosophila pandora based on multilocus sequence typing and the wsp gene. Male-killing Wolbachia are likely to be common in drosophilids but remain difficult to detect because the infections can often be at a low frequency.

Phylogenomic analyses of the genus Drosophila reveals genomic signals of climate adaptation.

Many Drosophila species differ widely in their distributions and climate niches, making them excellent subjects for evolutionary genomic studies. Here, we have developed a database of high-quality assemblies for 46 Drosophila species and one closely related Zaprionus. Fifteen of the genomes were newly sequenced, and 20 were improved with additional sequencing. New or improved annotations were generated for all 47 species, assisted by new transcriptomes for 19. Phylogenomic analyses of these data resolved several previously ambiguous relationships, especially in the melanogaster species group. However, it also revealed significant phylogenetic incongruence among genes, mainly in the form of incomplete lineage sorting in the subgenus Sophophora but also including asymmetric introgression in the subgenus Drosophila. Using the phylogeny as a framework and taking into account these incongruences, we then screened the data for genome-wide signals of adaptation to different climatic niches. First, phylostratigraphy revealed relatively high rates of recent novel gene gain in three temperate pseudoobscura and five desert-adapted cactophilic mulleri subgroup species. Second, we found differing ratios of nonsynonymous to synonymous substitutions in several hundred orthologues between climate generalists and specialists, with trends for significantly higher ratios for those in tropical and lower ratios for those in temperate-continental specialists respectively than those in the climate generalists. Finally, resequencing natural populations of 13 species revealed tropics-restricted species generally had smaller population sizes, lower genome diversity and more deleterious mutations than the more widespread species. We conclude that adaptation to different climates in the genus Drosophila has been associated with large-scale and multifaceted genomic changes.

Climate stress resistance in male Queensland fruit fly varies among populations of diverse geographic origins and changes during domestication.

BACKGROUND: The highly polyphagous Queensland fruit fly (Bactrocera tryoni Froggatt) expanded its range substantially during the twentieth century and is now the most economically important insect pest of Australian horticulture, prompting intensive efforts to develop a Sterile Insect Technique (SIT) control program. Using a "common garden" approach, we have screened for natural genetic variation in key environmental fitness traits among populations from across the geographic range of this species and monitored changes in those traits induced during domestication. RESULTS: Significant variation was detected between the populations for heat, desiccation and starvation resistance and wing length (as a measure of body size). Desiccation resistance was correlated with both starvation resistance and wing length. Bioassay data for three resampled populations indicate that much of the variation in desiccation resistance reflects persistent, inherited differences among the populations. No latitudinal cline was detected for any of the traits and only weak correlations were found with climatic variables for heat resistance and wing length. All three stress resistance phenotypes and wing length changed significantly in certain populations with ongoing domestication but there was also a strong population by domestication interaction effect for each trait. CONCLUSIONS: Ecotypic variation in heat, starvation and desiccation resistance was detected in Australian Qfly populations, and these stress resistances diminished rapidly during domestication. Our results indicate a need to select source populations for SIT strains which have relatively high climatic stress resistance and to minimise loss of that resistance during domestication.

Genomic changes associated with adaptation to arid environments in cactophilic Drosophila species.

BACKGROUND: Insights into the genetic capacities of species to adapt to future climate change can be gained by using comparative genomic and transcriptomic data to reconstruct the genetic changes associated with such adaptations in the past. Here we investigate the genetic changes associated with adaptation to arid environments, specifically climatic extremes and new cactus hosts, through such an analysis of five repleta group Drosophila species. RESULTS: We find disproportionately high rates of gene gains in internal branches in the species' phylogeny where cactus use and subsequently cactus specialisation and high heat and desiccation tolerance evolved. The terminal branch leading to the most heat and desiccation resistant species, Drosophila aldrichi, also shows disproportionately high rates of both gene gains and positive selection. Several Gene Ontology terms related to metabolism were enriched in gene gain events in lineages where cactus use was evolving, while some regulatory and developmental genes were strongly selected in the Drosophila aldrichi branch. Transcriptomic analysis of flies subjected to sublethal heat shocks showed many more downregulation responses to the stress in a heat sensitive versus heat resistant species, confirming the existence of widespread regulatory as well as structural changes in the species' differing adaptations. Gene Ontology terms related to metabolism were enriched in the differentially expressed genes in the resistant species while terms related to stress response were over-represented in the sensitive one. CONCLUSION: Adaptations to new cactus hosts and hot desiccating environments were associated with periods of accelerated evolutionary change in diverse biochemistries. The hundreds of genes involved suggest adaptations of this sort would be difficult to achieve in the timeframes projected for anthropogenic climate change.

A Wolbachia infection from Drosophila that causes cytoplasmic incompatibility despite low prevalence and densities in males.

Wolbachia bacteria are common insect endosymbionts transmitted maternally and capable of spreading through insect populations by cytoplasmic incompatibility (CI) when infected males cause embryo death after mating with uninfected females. Selection in the Wolbachia endosymbiont occurs on female hosts and is expected to favour strong maternal transmission to female offspring, even at the cost of reduced CI. With maternal leakage, nuclear genes are expected to be selected to suppress cytoplasmic incompatibility caused by males while also reducing any deleterious effects associated with the infection. Here we describe a new type of Wolbachia strain from Drosophila pseudotakahashii likely to have arisen from evolutionary processes on host and/or Wolbachia genomes. This strain is often absent from adult male offspring, but always transmitted to females. It leads to males with low or non-detectable Wolbachia that nevertheless show CI. When detected in adult males, the infection has a low density relative to that in females, a phenomenon not previously seen in Wolbachia infections of Drosophila. This Wolbachia strain is common in natural populations, and shows reduced CI when older (infected) males are crossed. These patterns highlight that endosymbionts can have strong sex-specific effects and that high frequency Wolbachia strains persist through effects on female reproduction. Female-limited Wolbachia infections may be of applied interest if the low level of Wolbachia in males reduces deleterious fitness effects on the host.

Separating multiple sources of variation on heat resistance in Drosophila hydei.

While numerous insect studies have demonstrated the effects environmental conditions, genetic variation and other factors have on thermal resistance, often showing patterns consistent with adaptive plasticity and local adaptation, few experiments have considered the effects of multiple factors simultaneously. Here however, we have investigated the impact of sex, rearing conditions, hardening, population, and laboratory rearing period on adult heat resistance in stocks of Drosophila hydei, a cosmopolitan species that occurs across a range of climatic zones. We show that population and putative laboratory adaptation effects are larger than those associated with rearing temperature and hardening, although there was also a notable interaction between hardening and sex, in that females showed a cost of hardening that was not present in males. In separate experiments, we found that environmental effects across a generation were small and similar in magnitude to those within a generation. These findings suggest multiple sources of variation on heat resistance and place potential genetic versus environmental sources in context.

Tropical Drosophila pandora carry Wolbachia infections causing cytoplasmic incompatibility or male killing.

Wolbachia infections have been described in several Drosophila species, but relatively few have been assessed for phenotypic effects. Cytoplasmic incompatibility (CI) is the most common phenotypic effect that has been detected, while some infections cause male killing or feminization, and many Wolbachia infections have few host effects. Here, we describe two new infections in a recently described species, Drosophila pandora, one of which causes near-complete CI and near-perfect maternal transmission (the "CI" strain). The other infection is a male killer (the "MK" strain), which we confirm by observing reinitiation of male production following tetracycline treatment. No incompatibility was detected in crosses between CI strain males and MK strain females, and rare MK males do not cause CI. Molecular analyses indicate that the CI and MK infections are distantly related and the CI infection is closely related to the wRi infection of Drosophila simulans. Two population surveys indicate that all individuals are infected with Wolbachia, but the MK infection is uncommon. Given patterns of incompatibility among the strains, the infection dynamics is expected to be governed by the relative fitness of the females, suggesting that the CI infection should have a higher fitness. This was evidenced by changes in infection frequencies and sex ratios in population cages initiated at different starting frequencies of the infections.

Assessing the relative importance of environmental effects, carry-over effects and species differences in thermal stress resistance: a comparison of Drosophilids across field and laboratory generations.

There is increasing interest in comparing species of related organisms for their susceptibility to thermal extremes in order to evaluate potential vulnerability to climate change. Comparisons are typically undertaken on individuals collected from the field with or without a period of acclimation. However, this approach does not allow the potential contributions of environmental and carry-over effects across generations to be separated from inherent species differences in susceptibility. To assess the importance of these different sources of variation, we here considered heat and cold resistance in Drosophilid species from tropical and temperate sites in the field and across two laboratory generations. Resistance in field-collected individuals tended to be lower when compared with F1 and F2 laboratory generations, and species differences in field flies were only weakly correlated to differences established under controlled rearing conditions, unlike in F1-F2 comparisons. This reflected large environmental effects on resistance associated with different sites and conditions experienced within sites. For the 8 h cold recovery assay there was no strong evidence of carry-over effects, whereas for the heat knockdown and 2 h cold recovery assays there was some evidence for such effects. However, for heat these were species specific in direction. Variance components for inherent species differences were substantial for resistance to heat and 8 h cold stress, but small for 2 h cold stress, though this may be a reflection of the species being considered in the comparisons. These findings highlight that inherent differences among species are difficult to characterise accurately without controlling for environmental sources of variation and carry-over effects. Moreover, they also emphasise the complex nature of carry-over effects that vary depending on the nature of stress traits and the species being evaluated.

Coevolution of male and female reproductive structures in Drosophila.

The morphology of male genitalia whilst stable within species, exhibits huge interspecific variation. This variation is likely to be as a result of sexual selection due to the direct involvement of these reproductive structures in mating and sperm transfer. In contrast, internal soft tissue components of the genitalia are generally poorly investigated as they are not directly involved in physical and mechanical adequacy during sperm transfer. However, these soft tissue structures may also drive differential male-female interactions, particularly in internally fertilising organisms where females have the ability to store sperm and bias male reproductive success. In this paper we use the drosophila model to investigate the role of male and female reproductive elements in sexual selection. Our meta-analysis supplemented with additional new data clearly shows that within species, sperm length versus testis length, and sperm length versus seminal receptacle length, are highly correlated. Thus, independent of the phylogenetic relationship among species, gamete evolution is likely to result in sexual selection interactions that drive the evolution of internal reproductive components in both sexes. Our results and discussion of the literature highlight the importance of considering internal soft structures that may influence fertilisation, when investigating selective forces acting on the evolution of reproductive traits.

Testing evolutionary hypotheses about species borders: patterns of genetic variation towards the southern borders of two rainforest Drosophila and a related habitat generalist.

Several evolutionary hypotheses help explain why only some species adapt readily to new conditions and expand distributions beyond borders, but there is limited evidence testing these hypotheses. In this study, we consider patterns of neutral (microsatellite) and quantitative genetic variation in traits in three species of Drosophila from the montium species group in eastern Australia. We found little support for restricted or asymmetrical gene flow in any species. In rainforest-restricted Drosophila birchii, there was evidence of selection for increased desiccation and starvation resistance towards the southern border, and a reduction in genetic diversity in desiccation resistance at this border. No such patterns existed for Drosophila bunnanda, which has an even more restricted distribution. In the habitat generalist Drosophila serrata, there was evidence for geographic selection for wing size and development time, although clinal patterns for increased cold and starvation resistance towards the southern border could not be differentiated from neutral expectations. These findings suggest that borders in these species are not limited by low overall genetic variation but instead in two of the species reflect patterns of selection and genetic variability in key traits limiting borders.

Developing and testing a diagnostic probe for grape phylloxera applicable to soil samples.

Grape phylloxera, Daktulosphaira vitifoliae (Fitch) (Hemiptera Phylloxeridae) is a damaging pest of grapevines (Vitis spp.) around the world, and the management of this pest requires early detection of infestations. Here, we describe the development and validation of a sensitive DNA test for grape phylloxera that can be applied to soil. Species-specific primers were developed for grape phylloxera in the internal transcribed space region 2, and their specificity was confirmed after thorough screening by using a wide range of vineyard organisms and aphid genera. Preliminary testing of the detection limits of the grape phylloxera-specific primers was conducted using field-sourced soil types spiked with a known number of grape phylloxera. The assay was converted to a real-time polymerase chain reaction format (TaqMan MGB). This assay, in combination with DNA extraction from soil, can detect phylloxera crawlers added to soil. The assay was evaluated in the field at a recently detected grape phylloxera infestation site from the Yarra Valley in Victoria, Australia. The DNA assay proved to be substantially more sensitive than a standard ground survey for detecting grape phylloxera presence on vine roots in the infested vineyard. Moreover, unlike the ground survey, the assay provided quantitative information on grape phylloxera infestations, because grape phylloxera DNA concentrations in samples from vines closely matched the numbers of grape phylloxera crawlers collected with emergence traps placed at the base of vines. Unlike other detection techniques, the method can be applied at any time of the year, and it can be potentially modified to provide specific information on the virulence levels of the particular grape phylloxera genotypes responsible for any new infestations.

Lack of genetic structure among ecologically adapted populations of an Australian rainforest Drosophila species as indicated by microsatellite markers and mitochondrial DNA sequences.

Although fragmented rainforest environments represent hotspots for invertebrate biodiversity, few genetic studies have been conducted on rainforest invertebrates. Thus, it is not known if invertebrate species in rainforests are highly genetically fragmented, with the potential for populations to show divergent selection responses, or if there are low levels of gene flow sufficient to maintain genetic homogeneity among fragmented populations. Here we use microsatellite markers and DNA sequences from the mitochondrial ND5 locus to investigate genetic differences among Drosophila birchii populations from tropical rainforests in Queensland, Australia. As found in a previous study, mitochondrial DNA diversity was low with no evidence for population differentiation among rainforest fragments. The pattern of mitochondrial haplotype variation was consistent with D. birchii having undergone substantial past population growth. Levels of nuclear genetic variation were high in all populations while F(ST) values were very low, even for flies from geographically isolated areas of rainforest. No significant differentiation was observed between populations on either side of the Burdekin Gap (a long-term dry corridor), although there was evidence for higher gene diversity in low-latitude populations. Spatial autocorrelation coefficients were low and did not differ significantly from random, except for one locus which revealed a clinal-like pattern. Comparisons of microsatellite differentiation contrasted with previously established clinal patterns in quantitative traits in D. birchii, and indicate that the patterns in quantitative traits are likely to be due to selection. These results suggest moderate gene flow in D. birchii over large distances. Limited population structure in this species appears to be due to recent range expansions or cycles of local extinctions followed by recolonizations/expansions. Nevertheless, patterns of local adaptation have developed in D. birchii that may result in populations showing different selection responses when faced with environmental change.

The contrasting genetic architecture of wing size, viability, and development time in a rainforest species and its more widely distributed relative.

Divergence among populations can occur via additive genetic effects and/or because of epistatic interactions among genes. Here we use line-cross analysis to compare the importance of epistasis in divergence among two sympatric Drosophila species from eastern Australia, one (D. serrata) distributed continuously and the other (D. birchii) confined to rainforest habitats that are often disjunct. For D. serrata, crosses indicated that development time and wing size differences were due to additive genetic effects, while for viability there were digenic epistatic effects. Crosses comparing geographically close populations as well as those involving the most geographically distant populations (including the southern species border) revealed epistatic interactions, whereas crosses at an intermediate distance showed no epistasis. In D. birchii, there was no evidence of epistasis for viability, although for development time and wing size there was epistasis in the cross between the most geographically diverged populations. Strong epistasis has not developed among the D. birchii populations, and this habitat specialist does not show stronger epistasis than D. serrata. Given that epistasis has been detected in crosses with other species from eastern Australia, including the recently introduced D. melanogaster, the results point to epistasis not being directly linked to divergence times among populations.

Immunocontraception for population control: will resistance evolve?

The prospect for successful biocontrol using immunocontraception is threatened if there is adaptation to the vaccine through natural selection of individuals that are genetically resistant to the contraceptive agent. To assess this possibility we examined the literature and found that little relevant data are available for any species on the appropriate trait, fertility variation among immunized individuals, or about appropriate population and genetic parameters influencing the likelihood of a selection response. Some data are available on variation in antibody response to immunocontraceptives, but the relationship between antibody response and fertility levels is poorly documented. The antibody response data indicate low heritability for this trait suggesting that fertility levels of contraceptive-resistant individuals will also have a low heritability. Slow evolution of contraception resistance might therefore be anticipated. The absence of information about relevant parameters makes the construction of quantitative models premature. We discuss factors in particular need of investigation if predictions about resistance evolution are to be made. These include: 1. the genetic basis of fertility retention, 2. the proportion of the population resistant to the contraceptive agent and how this is affected by gene flow from refuge populations, 3. the genetically-based fitness tradeoffs of resistant individuals that often accompany selection, 4. cross-generation effects that can thwart the effects of selection, and 5. the efficiency of delivery of the contraceptive agent. An understanding of the above for particular species, and the development of appropriate divergently acting multiple vaccines that can be used in temporal rotation or in mixtures, should facilitate the development of management options to minimize resistance evolution.

Clinal variation in Drosophila serrata for stress resistance and body size.

Clines for size and stress resistance traits have been described for several Drosophila species and replicable clines across different species may indicate climatic selection. Here we consider clines in stress resistance traits in an Australian endemic species, D. serrata, by comparing levels of variation within and among isofemale lines initiated with flies collected from the eastern coast of Australia. We also consider clinical variation in chill coma recovery, a trait that has recently been shown to exhibit high levels of variation among Drosophila species. Patterns were compared with those in the cosmopolitan species D. melanogaster from the same area. Both desiccation and starvation resistance showed no clinical pattern despite heritable variation among isofemale lines. In contrast chill coma resistance exhibited a linear cline in the anticipated direction, resistance increasing with latitude. Body size was measured as wing length and body weight. Both traits showed geographic variation and strong non-linear clines with a sharp reduction in size in the tropics. These results are discussed in the context of climatic selection and evolutionary processes limiting species borders.

CHANGES IN THE HERITABILITY OF FIVE MORPHOLOGICAL TRAITS UNDER COMBINED ENVIRONMENTAL STRESSES IN DROSOPHILA MELANOGASTER.

Heritabilities and evolvabilities for morphological traits were compared between two environments in Drosophila melanogaster using parent-offspring comparisons. One of the environments was favorable. The other stressful environment involved a combination of repeated cold shocks, poor nutrition, and ethanol added to the medium, which markedly decreased viability. For wing traits, heritabilities were relatively lower in the stressful environment, while heritabilities for bristle traits were not influenced by conditions. Heritability changes were largely due to an increase in the environmental variance under stress, whereas levels of additive genetic variance were relatively constant. Evolvabilities were similar between environments except for crossvein length.