Mating environments mediate the evolution of behavioral isolation during ecological speciation.

The evolution of behavioral isolation is often the first step toward speciation. While past studies show that behavioral isolation will sometimes evolve as a by-product of divergent ecological selection, we lack a more nuanced understanding of factors that may promote or hamper its evolution. The environment in which mating occurs may be important in mediating whether behavioral isolation evolves for two reasons. Ecological speciation could occur as a direct outcome of different sexual interactions being favored in different mating environments. Alternatively, mating environments may vary in the constraint they impose on traits underlying mating interactions, such that populations evolving in a "constraining" mating environment would be less likely to evolve behavioral isolation than populations evolving in a less constraining mating environment. In the latter, mating environment is not the direct cause of behavioral isolation but rather permits its evolution only if other drivers are present. We test these ideas with a set of 28 experimental fly populations, each of which evolved under one of two mating environments and one of two larval environments. Counter to the prediction of ecological speciation by mating environment, behavioral isolation was not maximal between populations evolved in different mating environments. Nonetheless, mating environment was an important factor as behavioral isolation evolved among populations from one mating environment but not among populations from the other. Though one mating environment was conducive to the evolution of behavioral isolation, it was not sufficient: assortative mating only evolved between populations adapting to different-larval environments within that mating environment, indicating a role for ecological speciation. Intriguingly, the mating environment that promoted behavioral isolation is characterized by less sexual conflict compared to the other mating environment. Our results suggest that mating environments play a key role in mediating ecological speciation via other axes of divergent selection.

Among- and Within-Individual Variance in Metabolic Thermal Reaction Norms.

AbstractIn ectotherms, temperature has a strong effect on metabolic rate (MR), yet the extent to which the thermal sensitivity of MR varies among versus within individuals is largely unknown. This is of interest because significant among-individual variation is a prerequisite for the evolution of metabolic thermal sensitivity. Here, we estimated the repeatability (R) of the thermal sensitivity of MR in individual virgin, adult male Drosophila melanogaster (N=316) by taking repeated overnight measures of their MRs at two temperatures (~24°C and ~27°C). At the population level, thermal sensitivity decreased with locomotor activity, and older individuals showed a higher thermal sensitivity of MR than younger individuals. Taking these effects (and body mass) into account, we detected significant repeatability in both the centered intercept (Rint=0.52±0.04) and the slope (Rslp=0.21±0.07) of the metabolic thermal reaction norms, which respectively represent average MR and thermal sensitivity of MR. Furthermore, individuals with a higher overall MR also displayed greater increases in MR as temperature increased from ~24°C to ~27°C (rind=0.32±0.14). Average MR and thermal sensitivity of MR were also positively correlated within individuals (re=0.15±0.07). Our study represents a point of departure for future larger studies, in which more complex protocols (e.g., wider temperature range, breeding design) can be applied to quantify the causal components of variation in thermal sensitivity that are needed to make accurate predictions of adaptive responses to global warming.

The evolution of sexual dimorphism in gene expression in response to a manipulation of mate competition.

Many genes are differentially expressed between males and females and patterns of sex-biased gene expression (SBGE) vary among species. Some of this variation is thought to have evolved in response to differences in mate competition among species that cause varying patterns of sex-specific selection. We used experimental evolution to test this by quantifying SBGE and sex-specific splicing in 15 Drosophila melanogaster populations that evolved for 104 generations in mating treatments that removed mate competition via enforced monogamy, or allowed mate competition in either small, simple, or larger, structurally more complex mating environments. Consistent with sex-specific selection affecting SBGE, initially sex-biased genes diverged in expression more among treatments than unbiased genes, and there was greater expression divergence for male- than female-biased genes. It has been suggested the transcriptome should be "feminized" under monogamy because of the removal of sexual selection on males; we did not observe this, likely because selection differs in additional ways between monogamy vs. polygamy. Significant divergence in average expression dimorphism between treatments was observed and, in some treatment comparisons, the direction of the divergence differed across different sex-bias categories. There was not a generalized reduction in expression dimorphism under enforced monogamy.

An experimental test of the evolutionary consequences of sympatry in Drosophila subquinaria.

Prezygotic isolation is often stronger between sympatric as opposed to allopatric taxa, but the underlying cause can be difficult to infer from comparative studies alone. Experimental evolution, where evolutionary responses to treatments manipulating the presence/absence of heterospecific individuals are tracked, can provide a powerful complementary approach. We used experimental evolution to investigate a naturally occurring pattern of reproductive character displacement in the mushroom-feeding fly, Drosophila subquinaria. In nature, female D. subquinaria from populations sympatric with the closely related Drosophila recens discriminate more strongly against heterospecific males than do females from allopatric populations. Starting with 16 replicate allopatric populations of D. subquinaria, we manipulated the presence/absence of D. recens during mating (experimental sympatry vs. control) and, when present, we allowed hybrids to live or kill them each generation. Across 12 generations, heterospecific offspring production from no-choice mating trials between D. subquinaria females and D. recens males declined in both experimental sympatry treatments relative to the control, suggesting increased sexual isolation. Male cuticular hydrocarbon profiles also evolved, but only in the hybrids killed treatment. Our results strongly imply that the existing reproductive character displacement in wild D. subquinaria populations was an evolutionary response to selection arising from secondary contact with D. recens.

Quantifying male harm and its divergence.

Male harm arises when traits that increase reproductive success in competition with other males also harm females as a side effect. The extent of harm depends on male and female phenotypes, both of which can diverge between populations. Within a population, harm is inferred when increased exposure to males reduces female fitness, but studies of the divergence of male harm rarely manipulate male exposure. Here, we quantify male harm and compare its magnitude between two lab populations of Drosophila serrata that were derived from a common ancestor 7 years earlier and subsequently held under conditions that minimized environmental differences. We manipulated female exposure to males in a factorial design involving all four combinations of males and females from these populations, providing insight into divergence in both sexes. Our results reveal substantial harm to females and provide stronger evidence of divergence in males than in females. Using these and other published data, we discuss conceptual issues surrounding the quantification and comparison of harm that arise because it involves a comparison of multiple quantities (e.g., female fitness under varying male exposure), and we demonstrate the increased insight that is gained by manipulating male exposure to quantify these quantities.

Maternal and Paternal Age Effects on Male Antler Flies: A Field Experiment.

AbstractIn many species, parental age at reproduction can influence offspring performance and life span, but the direction of these effects and the traits affected vary among studies. Data on parental age effects are still scarce in noncaptive populations, especially insects, despite species such as fruit flies being models in laboratory-based aging research. We performed a biologically relevant experimental manipulation of maternal and paternal age at reproduction of antler flies (Protopiophila litigata) in the laboratory and tracked the adult life span and reproductive success of their male offspring released in the wild. Increased paternal, but not maternal, age somewhat increased sons' adult life span, while parental ages did not influence sons' mating rate or reproductive senescence. Our results indicate that while parental age effects do exist in an insect in the field, they may be beneficial in such a short-lived animal, in contrast to results from most wild vertebrates and laboratory invertebrates.

On Male Harm: How It Is Measured and How It Evolves in Different Environments.

AbstractMales can harm the females that they interact with, but populations and species widely vary in the occurrence and extent of harm. We consider the merits and limitations of two common approaches to investigating male harm and apply these to an experimental study of divergence in harm. Different physical environments can affect how the sexes interact, causing plastic and/or evolved changes in harm. If harmful male phenotypes are less likely to evolve in situations where females have more control over sexual interactions, populations evolving in environments in which females have greater control should have less harmful males. We test this idea using experimental populations of Drosophila melanogaster that have evolved in either of two environments that vary in the extent to which females can avoid males or in a third environment without mate competition (i.e., enforced monogamy). We demonstrate an evolved reduction in harm in the absence of mate competition and also in a mate competition environment in which females have greater control. We also show a plastic effect in that otherwise harmful males are no longer so when tested in the environment in which females have greater control. Our results reveal the different perspectives provided by the two methods of studying harm.

Sex-specific genetic (co)variances of standard metabolic rate, body mass and locomotor activity in Drosophila melanogaster.

A longstanding focus in evolutionary physiology concerns the causes and consequences of variation in maintenance metabolism. Insight into this can be gained by estimating the sex-specific genetic architecture of maintenance metabolism alongside other, potentially correlated traits on which selection may also act, such as body mass and locomotor activity. This may reveal potential genetic constraints affecting the evolution of maintenance metabolism. Here, we used a half-sibling breeding design to quantify the sex-specific patterns of genetic (co)variance in standard metabolic rate (SMR), body mass and daily locomotor activity in Drosophila melanogaster. There was detectable additive genetic variance for all traits in both sexes. As expected, SMR and body mass were strongly and positively correlated, with genetic allometry exponents (bA  ± SE) that were close to 2/3 in females (0.66 ± 0.16) and males (0.58 ± 0.32). There was a significant and positive genetic correlation between SMR and locomotor activity in males, suggesting that alleles that increase locomotion have pleiotropic effects on SMR. Sexual differences in the genetic architecture were largely driven by a difference in genetic variance in locomotor activity between the sexes. Overall, genetic variation was mostly shared between males and females, setting the stage for a potential intralocus sexual conflict in the face of sexually antagonistic selection.

Quantifying selection on standard metabolic rate and body mass in Drosophila melanogaster.

Standard metabolic rate (SMR), defined as the minimal energy expenditure required for self-maintenance, is a key physiological trait. Few studies have estimated its relationship with fitness, most notably in insects. This is presumably due to the difficulty of measuring SMR in a large number of very small individuals. Using high-throughput flow-through respirometry and a Drosophila melanogaster laboratory population adapted to a life cycle that facilitates fitness measures, we quantified SMR, body mass, and fitness in 515 female and 522 male adults. We used a novel multivariate approach to estimate linear and nonlinear selection differentials and gradients from the variance-covariance matrix of fitness, SMR, and body mass, allowing traits specific covariates to be accommodated within a single model. In males, linear selection differentials for mass and SMR were positive and individually significant. Selection gradients were also positive but, despite substantial sample sizes, were nonsignificant due to increased uncertainty given strong SMR-mass collinearity. In females, only nonlinear selection was detected and it appeared to act primarily on body size, although the individual gradients were again nonsignificant. Selection did not differ significantly between sexes although differences in the fitness surfaces suggest sex-specific selection as an important topic for further study.

Development time mediates the effect of larval diet on ageing and mating success of male antler flies in the wild.

High-quality developmental environments often improve individual performance into adulthood, but allocating toward early life traits, such as growth, development rate and reproduction, may lead to trade-offs with late-life performance. It is, therefore, uncertain how a rich developmental environment will affect the ageing process (senescence), particularly in wild insects. To investigate the effects of early life environmental quality on insect life-history traits, including senescence, we reared larval antler flies (Protopiophila litigata) on four diets of varying nutrient concentration, then recorded survival and mating success of adult males released in the wild. Declining diet quality was associated with slower development, but had no effect on other life-history traits once development time was accounted for. Fast-developing males were larger and lived longer, but experienced more rapid senescence in survival and lower average mating rate compared to slow developers. Ultimately, larval diet, development time and body size did not predict lifetime mating success. Thus, a rich environment led to a mixture of apparent benefits and costs, mediated by development time. Our results indicate that 'silver spoon' effects can be complex and that development time mediates the response of adult life-history traits to early life environmental quality.

Sex-Specific Among-Individual Covariation in Locomotor Activity and Resting Metabolic Rate in Drosophila melanogaster.

A key endeavor in evolutionary physiology is to identify sources of among- and within-individual variation in resting metabolic rate (RMR). Although males and females often differ in whole-organism RMR due to sexual size dimorphism, sex differences in RMR sometimes persist after conditioning on body mass, suggesting phenotypic differences between males and females in energy-expensive activities contributing to RMR. One potential difference is locomotor activity, yet its relationship with RMR is unclear and different energy budget models predict different associations. We quantified locomotor activity (walking) over 24 h and RMR (overnight) in 232 male and 245 female Drosophila melanogaster that were either mated or maintained as virgins between two sets of measurements. Accounting for body mass, sex, and reproductive status, RMR and activity were significantly and moderately repeatable (RMR: R=0.33±0.06; activity: R=0.58±0.03). RMR and activity were positively correlated among (rind=0.26±0.09) but not within (re=0.05±0.06) individuals. Moreover, activity varied throughout the day and between the sexes. Partitioning our analysis by sex and activity by time of day revealed that all among-individual correlations were positive and significant in males but nonsignificant or even significantly negative in females. Such differences in the RMR-activity covariance suggest fundamental differences in how the sexes manage their energy budget.

Comparing ageing and the effects of diet supplementation in wild vs. captive antler flies, Protopiophila litigata.

Few studies have simultaneously compared ageing within genetically similar populations in both laboratory and natural environments. Such comparisons are important for interpreting laboratory studies, because factors such as diet could affect ageing in environment-dependent ways. Using a natural population of antler flies (Protopiophila litigata), we conducted separate factorial experiments in 2012 and 2013 that compared age-specific male survival and mating success in laboratory cages versus a natural field environment while supplementing their diets with protein or sugar. We found consistent and substantial increases in both survival and mating rates in the laboratory compared to the field, but remarkably, despite these large differences actuarial ageing was only higher in the laboratory than in the field in 2012 and similar in the two environments in 2013. In both years, there was no difference between environments in reproductive ageing. We found that males fed protein had a higher mortality rate than males fed sugar (strong and low support in 2012 and 2013, respectively). In contrast, diet did not strongly impact average mating rates, actuarial ageing or reproductive ageing in either experiment. Our results provide the first evidence that the negative effect of protein on life span reported in many laboratory studies can also occur in wild populations, although perhaps less consistently. They also highlight how laboratory environments can influence life-history traits and suggest caution when extrapolating from the laboratory to the field.

Testing for local adaptation in adult male and female fitness among populations evolved under different mate competition regimes.

Mating/fertilization success and fecundity are influenced by sexual interactions among individuals, the nature and frequency of which can vary among different environments. The extent of local adaptation for such adult fitness components is poorly understood. We allowed 63 populations of Drosophila melanogaster to independently evolve in one of three mating environments that alter sexual interactions: one involved enforced monogamy, while the other two permitted polygamy in either structurally simple standard fly vials or in larger "cages" with added complexity. Adult male and female reproductive fitness were measured after 16 and 28 generations, respectively, via full reciprocal transplants. In males, reciprocal local adaptation was observed between the monogamy and simple polygamy treatments, consistent with the evolution of reproductively competitive males under polygamy that perform poorly under monogamy because they harm their only mate. However, males evolved in the complex polygamy treatment performed similarly or better than all other males in all mating environments, consistent with previous results showing higher genetic quality in this treatment. Differences in female fitness were more muted, suggesting selection on females was less divergent across the mating treatments and echoing a common pattern of greater phenotypic and expression divergence in males than females.

The genetic basis of female pheromone differences between Drosophila melanogaster and D. simulans.

Chemical signals are one means by which many insect species communicate. Differences in the combination of surface chemicals called cuticular hydrocarbons (CHCs) can influence mating behavior and affect reproductive isolation between species. Genes influencing three CHC compounds have been identified in Drosophila melanogaster. However, the genetic basis of other CHC compounds, whether these genes affect species differences in CHCs, and the genes' resulting effect on interspecies mating, remains unknown. We used fine-scale deficiency mapping of the third chromosome to identify 43 genomic regions that influence production of CHCs in both D. melanogaster and Drosophila simulans females. We identified an additional 23 small genomic regions that affect interspecies divergence in CHCs between females of these two species, one of which spans two genes known to influence the production of multiple CHCs within D. melanogaster. By testing these genes individually, we determined that desat1 also affects interspecific divergence in one CHC compound, while desat2 has no effect on interspecific divergence. Thus, some but not all genes affecting intraspecific amounts of CHCs also affect interspecific divergence, but not all genes or all CHCs. Lastly, we find no evidence of a relationship between the CHC profile and female attractiveness or receptivity towards D. melanogaster males.

The contribution of sexual selection to ecological and mutation-order speciation.

Abundant evidence supports a role for sexual selection in the evolution of reproductive isolation, and it is thus unsurprising that much attention has been given, both conceptually and empirically, to understanding its role in speciation. In doing so, debate has arisen on how sexual selection fits within the much used ecological versus mutation-order classification of speciation mechanisms, with sexual selection often presented as a distinct third alternative. We argue that models of speciation by sexual selection include a fundamental role of divergent selection between environments or mutation order in initiating the process. Rather than representing a unique mechanism, sexual selection layers a coevolutionary process between males and females on top of the classic mechanisms such that the evolution of each sex can now be driven by divergent selection, mutation order, and selection arising from interactions with the other sex. In addition to blurring the distinction between ecological and mutation-order speciation, this coevolutionary process is likely to speed divergence. Sexual selection is not unique in this way; coevolutionary processes can similarly arise from ecological interactions between populations or species, with similar results. Ultimately, understanding the contribution of sexual selection to speciation will require identifying the processes that drive the divergence of mating biases.

The effects of male harm vary with female quality and environmental complexity in Drosophila melanogaster.

Mate competition provides the opportunity for sexual selection which often acts strongly on males, but also the opportunity for sexual conflict that can alter natural selection on females. Recent attention has focused on the potential of sexual conflict to weaken selection on females if male sexual attention, and hence harm, is disproportionately directed towards high- over low-quality females, thereby reducing the fitness difference between these females. However, sexual conflict could instead strengthen selection on females if low-quality females are more sensitive to male harm than high-quality females, thereby magnifying fitness differences between them. We quantify the effects of male exposure on low- versus high-quality females in Drosophila melanogaster in each of two environments ('simple' and 'complex') that are known to alter behavioural interactions. We show that the effects of male harm are greater for low- compared to high-quality females in the complex but not the simple environment, consistent with mate competition strengthening selection on females in the former but not in the latter environment.

Competition for mates and the improvement of nonsexual fitness.

Competition for mates can be a major source of selection, not just on secondary sexual traits but across the genome. Mate competition strengthens selection on males via sexual selection, which typically favors healthy, vigorous individuals and, thus, all genetic variants that increase overall quality. However, recent studies suggest another major effect of mate competition that could influence genome-wide selection: Sexual harassment by males can drastically weaken selection on quality in females. Because of these conflicting effects, the net effect of mate competition is uncertain, although perhaps not entirely unpredictable. We propose that the environment in which mate competition occurs mediates the importance of sexual selection relative to sexual conflict and, hence, the net effect of mate competition on nonsexual fitness. To test this, we performed experimental evolution with 63 fruit fly populations adapting to novel larval conditions where each population was maintained with or without mate competition. In half the populations with mate competition, adults interacted in simple, high-density environments. In the remainder, adults interacted in more spatially complex environments in which male-induced harm is reduced. Populations evolving with mate competition in the complex environment adapted faster to novel larval environments than did populations evolving without mate competition or with mate competition in the simple environment. Moreover, mate competition in the complex environment caused a substantial reduction in inbreeding depression for egg-to-adult viability relative to the other two mating treatments. These results demonstrate that the mating environment has a substantial and predictable effect on nonsexual fitness through adaptation and purging.

The purging of deleterious mutations in simple and complex mating environments.

There is a general expectation that sexual selection should align with natural selection to aid the purging of deleterious mutations, yet experiments comparing purging under monogamy versus polygamy have provided mixed results. Recent studies suggest that this may be because the simplified mating environments used in these studies reduce the benefit of sexual selection through males and hamper natural selection through females by increasing costs associated with sexual conflict. To test the effect of the physical mating environment on purging, we use experimental evolution in Drosophila melanogaster to track the frequency of four separate deleterious mutations in replicate populations that experience polygamy under either a simple or structurally complex mating arena while controlling for arena size. Consistent with past results suggesting a greater net benefit of polygamy in a complex environment, two of the mutations were purged significantly faster in this environment. The other two mutations showed no significant difference between environments.

Environmental complexity and the purging of deleterious alleles.

Sexual interactions among adults can generate selection on both males and females with genome-wide consequences. Sexual selection through males is one component of this selection that has been argued to play an important role in purging deleterious alleles. A common technique to assess the influence of sexual selection is by a comparison of experimental evolution under enforced monogamy versus polygamy. Mixed results from past studies may be due to the use of highly simplified laboratory conditions that alter the nature of sexual interactions. Here, we examine the rate of purging of 22 gene disruption mutations in experimental polygamous populations of Drosophila melanogaster in each of two mating environments: a simple, high-density environment (i.e., typical fly vials), and a lower density, more spatially complex environment. Based on past work, we expect sexual interactions in the latter environment to result in stronger selection in both sexes. Consistent with this, we find that mutations tend to be purged more quickly in populations evolving in complex environments. We discuss possible mechanisms by which environmental complexity might modulate the rate at which deleterious alleles are purged and putatively ascribe a role for sexual interactions in explaining the treatment differences in our experiment.

Tissue-specific insulin signaling mediates female sexual attractiveness.

Individuals choose their mates so as to maximize reproductive success, and one important component of this choice is assessment of traits reflecting mate quality. Little is known about why specific traits are used for mate quality assessment nor about how they reflect it. We have previously shown that global manipulation of insulin signaling, a nutrient-sensing pathway governing investment in survival versus reproduction, affects female sexual attractiveness in the fruit fly, Drosophila melanogaster. Here we demonstrate that these effects on attractiveness derive from insulin signaling in the fat body and ovarian follicle cells, whose signals are integrated by pheromone-producing cells called oenocytes. Functional ovaries were required for global insulin signaling effects on attractiveness, and manipulations of insulin signaling specifically in late follicle cells recapitulated effects of global manipulations. Interestingly, modulation of insulin signaling in the fat body produced opposite effects on attractiveness, suggesting a competitive relationship with the ovary. Furthermore, all investigated tissue-specific insulin signaling manipulations that changed attractiveness also changed fecundity in the corresponding direction, pointing to insulin pathway activity as a reliable link between fecundity and attractiveness cues. The cues themselves, cuticular hydrocarbons, responded distinctly to fat body and follicle cell manipulations, indicating independent readouts of the pathway activity from these two tissues. Thus, here we describe a system in which female attractiveness results from an apparent connection between attractiveness cues and an organismal state of high fecundity, both of which are created by lowered insulin signaling in the fat body and increased insulin signaling in late follicle cells.