Sexually discordant selection is associated with trait specific morphological changes and a complex genomic response.

Sexes often have differing fitness optima, potentially generating intra-locus sexual conflict, as each sex bears a genetic 'load' of alleles beneficial to the other sex. One strategy to evaluate conflict in the genome is to artificially select populations discordantly, against established sexual dimorphism, reintroducing attenuated conflict. We investigate a long-term artificial selection experiment reversing sexual size dimorphism in Drosophila melanogaster during ~350 generations of sexually discordant selection. We explore morphological and genomic changes to identify loci under selection between the sexes in discordantly and concordantly size selected treatments. Despite substantial changes to overall size, concordant selection maintained ancestral sexual dimorphism. However, discordant selection altered size dimorphism in a trait-specific manner. We observe multiple, possible soft selective sweeps in the genome, with size related genes showing signs of selection. Patterns of genomic differentiation between the sexes within lineages identified potential sites maintained by sexual conflict. One discordant selected lineage shows a pattern of elevated genomic differentiation between males and females, on chromosome 3L, consistent with the maintenance of sexual conflict. Our results suggest visible signs of conflict and differentially segregating alleles between the sexes due to discordant selection.

Genetic correlation between aggressive signals and fighting.

Theoretical analyses indicate that aggressive signals should positively correlate with the signallers' willingness and abilities to fight. Few experimental studies, however, have tested this prediction. In two experiments employing distinct, ecologically realistic protocols, we quantified the association between aggressive signals and fighting in fruit fly genotypes and found high positive genetic correlations between threat and fighting (rG = 0.80 and 0.74). Our results add to the growing body of experimental work indicating that aggressive signals have relatively high informational value.

Complexities of recapitulating polygenic effects in natural populations: replication of genetic effects on wing shape in artificially selected and wild-caught populations of Drosophila melanogaster.

Identifying the genetic architecture of complex traits is important to many geneticists, including those interested in human disease, plant and animal breeding, and evolutionary genetics. Advances in sequencing technology and statistical methods for genome-wide association studies have allowed for the identification of more variants with smaller effect sizes, however, many of these identified polymorphisms fail to be replicated in subsequent studies. In addition to sampling variation, this failure to replicate reflects the complexities introduced by factors including environmental variation, genetic background, and differences in allele frequencies among populations. Using Drosophila melanogaster wing shape, we ask if we can replicate allelic effects of polymorphisms first identified in a genome-wide association studies in three genes: dachsous, extra-macrochaete, and neuralized, using artificial selection in the lab, and bulk segregant mapping in natural populations. We demonstrate that multivariate wing shape changes associated with these genes are aligned with major axes of phenotypic and genetic variation in natural populations. Following seven generations of artificial selection along the dachsous shape change vector, we observe genetic differentiation of variants in dachsous and genomic regions containing other genes in the hippo signaling pathway. This suggests a shared direction of effects within a developmental network. We also performed artificial selection with the extra-macrochaete shape change vector, which is not a part of the hippo signaling network, but showed a largely shared direction of effects. The response to selection along the emc vector was similar to that of dachsous, suggesting that the available genetic diversity of a population, summarized by the genetic (co)variance matrix (G), influenced alleles captured by selection. Despite the success with artificial selection, bulk segregant analysis using natural populations did not detect these same variants, likely due to the contribution of environmental variation and low minor allele frequencies, coupled with small effect sizes of the contributing variants.

Regulation at Drosophila's Malic Enzyme highlights the complexity of transvection and its sensitivity to genetic background.

Transvection, a type of trans-regulation of gene expression in which regulatory elements on one chromosome influence elements on a paired homologous chromosome, is itself a complex biological phenotype subject to modification by genetic background effects. However, relatively few studies have explored how transvection is affected by distal genetic variation, perhaps because it is strongly influenced by local regulatory elements and chromosomal architecture. With the emergence of the "hub" model of transvection and a series of studies showing variation in transvection effects, it is becoming clear that genetic background plays an important role in how transvection influences gene transcription. We explored the effects of genetic background on transvection by performing two independent genome wide association studies (GWASs) using the Drosophila genetic reference panel (DGRP) and a suite of Malic enzyme (Men) excision alleles. We found substantial variation in the amount of transvection in the 149 DGRP lines used, with broad-sense heritability of 0.89 and 0.84, depending on the excision allele used. The specific genetic variation identified was dependent on the excision allele used, highlighting the complex genetic interactions influencing transvection. We focussed primarily on genes identified as significant using a relaxed P-value cutoff in both GWASs. The most strongly associated genetic variant mapped to an intergenic single nucleotide polymorphism (SNP), located upstream of Tiggrin (Tig), a gene that codes for an extracellular matrix protein. Variants in other genes, such transcription factors (CG7368 and Sima), RNA binding proteins (CG10418, Rbp6, and Rig), enzymes (AdamTS-A, CG9743, and Pgant8), proteins influencing cell cycle progression (Dally and Eip63E) and signaling proteins (Atg-1, Axo, Egfr, and Path) also associated with transvection in Men. Although not intuitively obvious how many of these genes may influence transvection, some have been previously identified as promoting or antagonizing somatic homolog pairing. These results identify several candidate genes to further explore in the understanding of transvection in Men and in other genes regulated by transvection. Overall, these findings highlight the complexity of the interactions involved in gene regulation, even in phenotypes, such as transvection, that were traditionally considered to be primarily influenced by local genetic variation.

The genetic basis of variation in sexual aggression: Evolution versus social plasticity.

Male sexual aggression towards females is a form of sexual conflict that can result in increased fitness for males through forced copulations (FCs) or coercive matings at the cost of female lifetime fitness. We used male fruit flies (Drosophila melanogaster) as a model system to uncover the genomic contributions to variation in FC, both due to standing variation in a wild population, and due to plastic changes associated with variation in social experience. We used RNAseq to analyse whole-transcriptome differential expression (DE) in male head tissue associated with evolved changes in FC from lineages previously selected for high and low FC rate and in male flies with varying FC rates due to social experience. We identified hundreds of genes associated with evolved and plastic variation in FC, however only a small proportion (27 genes) showed consistent DE due to both modes of variation. We confirmed this trend of low concordance in gene expression effects across broader sets of genes significant in either the evolved or plastic analyses using multivariate approaches. The gene ontology terms neuropeptide hormone activity and serotonin receptor activity were significantly enriched in the set of significant genes. Of seven genes chosen for RNAi knockdown validation tests, knockdown of four genes showed the expected effect on FC behaviours. Taken together, our results provide important information about the apparently independent genetic architectures that underlie natural variation in sexual aggression due to evolution and plasticity.

Evolution of sociability by artificial selection.

There has been extensive research on the ecology and evolution of social life in animals that live in groups. Less attention, however, has been devoted to apparently solitary species, even though recent research indicates that they also possess complex social behaviors. To address this knowledge gap, we artificially selected on sociability, defined as the tendency to engage in nonaggressive activities with others, in fruit flies. Our goal was to quantify the factors that determine the level of sociability and the traits correlated with this feature. After 25 generations of selection, the high-sociability lineages showed sociability scores about 50% higher than did the low-sociability lineages. Experiments using the evolved lineages indicated that there were no differences in mating success between flies from the low and high lineages. Both males and females from the low lineages, however, were more aggressive than males and females from the high lineages. Finally, the evolved lineages maintained their sociability scores after 10 generations of relaxed selection, suggesting no costs to maintaining low and high sociability, at least under our settings. Sociability is a complex trait, which we currently assess through genomic work on the evolved lineages.

Sex chromosome degeneration, turnover, and sex-biased expression of sex-linked transcripts in African clawed frogs (Xenopus).

The tempo of sex chromosome evolution-how quickly, in what order, why and how their particular characteristics emerge during evolution-remains poorly understood. To understand this further, we studied three closely related species of African clawed frog (genus Xenopus), that each has independently evolved sex chromosomes. We identified population polymorphism in the extent of sex chromosome differentiation in wild-caught Xenopus borealis that corresponds to a large, previously identified region of recombination suppression. This large sex-linked region of X. borealis has an extreme concentration of genes that encode transcripts with sex-biased expression, and we recovered similar findings in the smaller sex-linked regions of Xenopus laevis and Xenopus tropicalis. In two of these species, strong skews in expression (mostly female-biased in X. borealis, mostly male-biased in X. tropicalis) are consistent with expectations associated with recombination suppression, and in X. borealis, we hypothesize that a degenerate ancestral Y-chromosome transitioned into its contemporary Z-chromosome. These findings indicate that Xenopus species are tolerant of differences between the sexes in dosage of the products of multiple genes, and offer insights into how evolutionary transformations of ancestral sex chromosomes carry forward to affect the function of new sex chromosomes. This article is part of the theme issue 'Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part I)'.

Spatial heterogeneity in resources alters selective dynamics in Drosophila melanogaster.

Environmental features can alter the behaviors and phenotypes of organisms, influencing the dynamics of natural and sexual selection. Experimental environmental manipulation, particularly when conducted in experiments where the dynamics of the purging of deleterious alleles are compared, has demonstrated both direct and indirect effects on the strength and direction of selection. However, many of these studies are conducted with fairly simplistic environments, where it is not always clear how or why particular forms of spatial heterogeneity influence behavior or selection. Using Drosophila melanogaster, we tested three different spatial environments designed to determine if spatial constraint of critical resources influences the efficiency of natural and sexual selection. We conducted two allele purging experiments to (1) assess effects of these spatial treatments on selective dynamics of six recessive mutations, and (2) determine how these dynamics changed when sexual selection was relaxed and spatial area reduced for two of the mutants. Allele purging dynamics depended on spatial environment, however the patterns of purging rates between the environments differed across distinct deleterious mutations. We also tested two of the mutant alleles, and demonstrate sexual selection increased the purging rate.

Genetic and environmental canalization are not associated among altitudinally varying populations of Drosophila melanogaster.

Organisms are exposed to environmental and mutational effects influencing both mean and variance of phenotypes. Potentially deleterious effects arising from this variation can be reduced by the evolution of buffering (canalizing) mechanisms, ultimately reducing phenotypic variability. There has been interest regarding the conditions enabling the evolution of canalization. Under some models, the circumstances under which genetic canalization evolves are limited despite apparent empirical evidence for it. It has been argued that genetic canalization evolves as a correlated response to environmental canalization (congruence model). Yet, empirical evidence has not consistently supported predictions of a correlation between genetic and environmental canalization. In a recent study, a population of Drosophila adapted to high altitude showed evidence of genetic decanalization relative to those from low altitudes. Using strains derived from these populations, we tested if they varied for multiple aspects of environmental canalization We observed the expected differences in wing size, shape, cell (trichome) density and mutational defects between high- and low-altitude populations. However, we observed little evidence for a relationship between measures of environmental canalization with population or with defect frequency. Our results do not support the predicted association between genetic and environmental canalization.

Sexual Selection Does Not Increase the Rate of Compensatory Adaptation to a Mutation Influencing a Secondary Sexual Trait in Drosophila melanogaster.

Theoretical work predicts that sexual selection can enhance natural selection, increasing the rate of adaptation to new environments and helping purge harmful mutations. While some experiments support these predictions, remarkably little work has addressed the role of sexual selection on compensatory adaptation-populations' ability to compensate for the costs of deleterious alleles that are already present. We tested whether sexual selection, as well as the degree of standing genetic variation, affect the rate of compensatory evolution via phenotypic suppression in experimental populations of Drosophila melanogaster These populations were fixed for a spontaneous mutation causing mild abnormalities in the male sex comb, a structure important for mating success. We fine-mapped this mutation to an ∼85 kb region on the X chromosome containing three candidate genes, showed that the mutation is deleterious, and that its phenotypic expression and penetrance vary by genetic background. We then performed experimental evolution, including a treatment where opportunity for mate choice was limited by experimentally enforced monogamy. Although evolved populations did show some phenotypic suppression of the morphological abnormalities in the sex comb, the amount of suppression did not depend on the opportunity for sexual selection. Sexual selection, therefore, may not always enhance natural selection; instead, the interaction between these two forces may depend on additional factors.

Individual Cryptic Scaling Relationships and the Evolution of Animal Form.

Artificial selection offers a powerful tool for the exploration of how selection and development shape the evolution of morphological scaling relationships. An emerging approach models the expression and evolution of morphological scaling relationships as a function of variation among individuals in the developmental mechanisms that regulate trait growth. These models posit the existence of genotype-specific morphological scaling relationships that are unseen or "cryptic." Within-population allelic variation at growth-regulating loci determines how these individual cryptic scaling relationships are distributed, and exposure to environmental factors that affect growth determines the size phenotype expressed by each individual on their cryptic, genotype-specific scaling relationship. These models reveal that evolution of the intercept and slope of the population-level static allometry is determined, often in counterintuitive ways, largely by the shape of the distribution of these underlying individual-level scaling relationships. Here we review this modeling framework and present the wing-body size individual cryptic scaling relationships from a population of Drosophila melanogaster. To determine how these models might inform interpretation of published work on scaling relationship evolution, we review studies where artificial selection was applied to alter the parameters of population-level static allometries. Finally, motivated by our review, we outline areas in need of empirical work and describe a research program to address these topics; the approach includes describing the distribution of individual cryptic scaling relationships across populations and environments, empirical testing of the model's predictions, and determining the effects of environmental heterogeneity on realized trait distributions and how this affects allometry evolution.

Chloroform and desflurane immobilization with recovery of viable Drosophila larvae for confocal imaging.

Imaging of living, intact Drosophila larvae is challenged if normal bodily function must be observed or when healthy larvae must be recovered for subsequent studies. Here, we describe a simple and short protocol that employs transient airborne chloroform or desflurane (1,2,2,2-tetrafluoroethyl difluoromethyl ether) to efficiently immobilize larvae without the use of manipulation devices, vaporizers or imaging chambers. This non-lethal method allows the use of anesthetics while allowing tracking of individual Drosophila into adulthood for follow-up experiments. At dosages sufficient to immobilize larvae, Desflurane, but not chloroform reduced the central nervous system response to auditory stimulus. Desflurane doses were sufficient to arrest the heart, however significant rapid recovery was observed. With our method, chloroform provided more rapid anesthesia but slower recovery than Desflurane. Without specialized hardware, this technique allows for repeated imaging of living Drosophila larvae.

The behavioral repertoire of Drosophila melanogaster in the presence of two predator species that differ in hunting mode.

The fruit fly, Drosophila melanogaster, has proven to be an excellent model organism for genetic, genomic and neurobiological studies. However, relatively little is known about the natural history of D. melanogaster. In particular, neither the natural predators faced by wild populations of D. melanogaster, nor the anti-predatory behaviors they may employ to escape and avoid their enemies have been documented. Here we observe and describe the influence of two predators that differ in their mode of hunting: zebra jumping spiders, Salticus scenicus (active hunters) and Chinese praying mantids, Tenodera sinensis (ambush predators) on the behavioral repertoire of Drosophila melanogaster. We documented three particularly interesting behaviors: abdominal lifting, stopping, and retreat-which were performed at higher frequency by D. melanogaster in the presence of predators. While mantids had only a modest influence on the locomotory activity of D. melanogaster, we observed a significant increase in the overall activity of D. melanogaster in the presence of jumping spiders. Finally, we observed considerable among-individual behavioral variation in response to both predators.

A Multivariate Genome-Wide Association Study of Wing Shape in Drosophila melanogaster.

Due to the complexity of genotype-phenotype relationships, simultaneous analyses of genomic associations with multiple traits will be more powerful and informative than a series of univariate analyses. However, in most cases, studies of genotype-phenotype relationships have been analyzed only one trait at a time. Here, we report the results of a fully integrated multivariate genome-wide association analysis of the shape of the Drosophila melanogaster wing in the Drosophila Genetic Reference Panel. Genotypic effects on wing shape were highly correlated between two different laboratories. We found 2396 significant SNPs using a 5% false discovery rate cutoff in the multivariate analyses, but just four significant SNPs in univariate analyses of scores on the first 20 principal component axes. One quarter of these initially significant SNPs retain their effects in regularized models that take into account population structure and linkage disequilibrium. A key advantage of multivariate analysis is that the direction of the estimated phenotypic effect is much more informative than a univariate one. We exploit this fact to show that the effects of knockdowns of genes implicated in the initial screen were on average more similar than expected under a null model. A subset of SNP effects were replicable in an unrelated panel of inbred lines. Association studies that take a phenomic approach, considering many traits simultaneously, are an important complement to the power of genomics.

Weed evolution: Genetic differentiation among wild, weedy, and crop radish.

Approximately 200 weed species are responsible for more than 90% of crop losses and these comprise less than one percent of all named plant species, suggesting that there are only a few evolutionary routes that lead to weediness. Agricultural weeds can evolve along three main paths: they can be escaped crops, wild species, or crop-wild hybrids. We tested these three hypotheses in weedy radish, a weed of small grains and an emerging model for investigating the evolution of agricultural weeds, using 21 CAPS and SSR markers scored on 338 individuals from 34 populations representing all major species and sub-species in the radish genus Raphanus. To test for adaptation of the weeds to the agricultural environment, we estimated genetic differentiation in flowering time in a series of common garden experiments with over 2,400 individuals from 43 populations (all but one of the genotyped populations plus 10 additional populations). Our findings suggest that the agricultural weed radish R. r. raphanistrum is most genetically similar to native populations of R. r. raphanistrum and is likely not a feral crop or crop hybrid. We also show that weedy radish flowers more rapidly than any other Raphanus population or cultivar, which is consistent with rapid adaptation to the frequent and severe disturbance that characterizes agricultural fields.

Sexual dimorphism and heightened conditional expression in a sexually selected weapon in the Asian rhinoceros beetle.

Among the most dramatic examples of sexual selection are the weapons used in battles between rival males over access to females. As with ornaments of female choice, the most "exaggerated" sexually selected weapons vary from male to male more widely than other body parts (hypervariability), and their growth tends to be more sensitive to nutritional state or physiological condition compared with growth of other body parts ("heightened" conditional expression). Here, we use RNAseq analysis to build on recent work exploring these mechanisms in the exaggerated weapons of beetles, by examining patterns of differential gene expression in exaggerated (head and thorax horns) and non-exaggerated (wings, genitalia) traits in the Asian rhinoceros beetle, Trypoxylus dichotomus. Our results suggest that sexually dimorphic expression of weaponry involves large-scale changes in gene expression, relative to other traits, while nutrition-driven changes in gene expression in these same weapons are less pronounced. However, although fewer genes overall were differentially expressed in high- vs. low-nutrition individuals, the number of differentially expressed genes varied predictably according to a trait's degree of condition dependence (head horn > thorax horn > wings > genitalia). Finally, we observed a high degree of similarity in direction of effects (vectors) for subsets of differentially expressed genes across both sexually dimorphic and nutritionally responsive growth. Our results are consistent with a common set of mechanisms governing sexual size dimorphism and condition dependence.

Sociability in Fruit Flies: Genetic Variation, Heritability and Plasticity.

Sociability, defined as individuals' propensity to participate in non-aggressive activities with conspecifics, is a fundamental feature of behavior in many animals including humans. However, we still have a limited knowledge of the mechanisms and evolutionary biology of sociability. To enhance our understanding, we developed a new protocol to quantify sociability in fruit flies (Drosophila melanogaster). In a series of experiments with 59 F1 hybrids derived from inbred lines, we documented, first, significant genetic variation in sociability in both males and females, with broad-sense heritabilities of 0.24 and 0.21 respectively. Second, we observed little genetic correlation in sociability between the sexes. Third, we found genetic variation in social plasticity among the hybrids, with a broad-sense heritability of ~0.24. That is, genotypes differed in the degree of sociability after experiencing the same relevant social experience. Our data pave the way for further research on the mechanisms that underlie sociability as well as its ecological and evolutionary consequences.

How well do you know your mutation? Complex effects of genetic background on expressivity, complementation, and ordering of allelic effects.

For a given gene, different mutations influence organismal phenotypes to varying degrees. However, the expressivity of these variants not only depends on the DNA lesion associated with the mutation, but also on factors including the genetic background and rearing environment. The degree to which these factors influence related alleles, genes, or pathways similarly, and whether similar developmental mechanisms underlie variation in the expressivity of a single allele across conditions and among alleles is poorly understood. Besides their fundamental biological significance, these questions have important implications for the interpretation of functional genetic analyses, for example, if these factors alter the ordering of allelic series or patterns of complementation. We examined the impact of genetic background and rearing environment for a series of mutations spanning the range of phenotypic effects for both the scalloped and vestigial genes, which influence wing development in Drosophila melanogaster. Genetic background and rearing environment influenced the phenotypic outcome of mutations, including intra-genic interactions, particularly for mutations of moderate expressivity. We examined whether cellular correlates (such as cell proliferation during development) of these phenotypic effects matched the observed phenotypic outcome. While cell proliferation decreased with mutations of increasingly severe effects, surprisingly it did not co-vary strongly with the degree of background dependence. We discuss these findings and propose a phenomenological model to aid in understanding the biology of genes, and how this influences our interpretation of allelic effects in genetic analysis.

Disintegrating the fly: A mutational perspective on phenotypic integration and covariation.

The structure of environmentally induced phenotypic covariation can influence the effective strength and magnitude of natural selection. Yet our understanding of the factors that contribute to and influence the evolutionary lability of such covariation is poor. Most studies have either examined environmental variation without accounting for covariation, or examined phenotypic and genetic covariation without distinguishing the environmental component. In this study, we examined the effect of mutational perturbations on different properties of environmental covariation, as well as mean shape. We use strains of Drosophila melanogaster bearing well-characterized mutations known to influence wing shape, as well as naturally derived strains, all reared under carefully controlled conditions and with the same genetic background. We find that mean shape changes more freely than the covariance structure, and that different properties of the covariance matrix change independently from each other. The perturbations affect matrix orientation more than they affect matrix eccentricity or total variance. Yet, mutational effects on matrix orientation do not cluster according to the developmental pathway that they target. These results suggest that it might be useful to consider a more general concept of "decanalization," involving all aspects of variation and covariation.