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.

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.

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.

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.

Consequences of Whole-Genome Triplication as Revealed by Comparative Genomic Analyses of the Wild Radish Raphanus raphanistrum and Three Other Brassicaceae Species.

Polyploidization events are frequent among flowering plants, and the duplicate genes produced via such events contribute significantly to plant evolution. We sequenced the genome of wild radish (Raphanus raphanistrum), a Brassicaceae species that experienced a whole-genome triplication event prior to diverging from Brassica rapa. Despite substantial gene gains in these two species compared with Arabidopsis thaliana and Arabidopsis lyrata, ∼70% of the orthologous groups experienced gene losses in R. raphanistrum and B. rapa, with most of the losses occurring prior to their divergence. The retained duplicates show substantial divergence in sequence and expression. Based on comparison of A. thaliana and R. raphanistrum ortholog floral expression levels, retained radish duplicates diverged primarily via maintenance of ancestral expression level in one copy and reduction of expression level in others. In addition, retained duplicates differed significantly from genes that reverted to singleton state in function, sequence composition, expression patterns, network connectivity, and rates of evolution. Using these properties, we established a statistical learning model for predicting whether a duplicate would be retained postpolyploidization. Overall, our study provides new insights into the processes of plant duplicate loss, retention, and functional divergence and highlights the need for further understanding factors controlling duplicate gene fate.

Identification and functional analyses of sex determination genes in the sexually dimorphic stag beetle Cyclommatus metallifer.

BACKGROUND: Genes in the sex determination pathway are important regulators of sexually dimorphic animal traits, including the elaborate and exaggerated male ornaments and weapons of sexual selection. In this study, we identified and functionally analyzed members of the sex determination gene family in the golden metallic stag beetle Cyclommatus metallifer, which exhibits extreme differences in mandible size between males and females. RESULTS: We constructed a C. metallifer transcriptomic database from larval and prepupal developmental stages and tissues of both males and females. Using Roche 454 pyrosequencing, we generated a de novo assembled database from a total of 1,223,516 raw reads, which resulted in 14,565 isotigs (putative transcript isoforms) contained in 10,794 isogroups (putative identified genes). We queried this database for C. metallifer conserved sex determination genes and identified 14 candidate sex determination pathway genes. We then characterized the roles of several of these genes in development of extreme sexual dimorphic traits in this species. We performed molecular expression analyses with RT-PCR and functional analyses using RNAi on three C. metallifer candidate genes--Sex-lethal (CmSxl), transformer-2 (Cmtra2), and intersex (Cmix). No differences in expression pattern were found between the sexes for any of these three genes. In the RNAi gene-knockdown experiments, we found that only the Cmix had any effect on sexually dimorphic morphology, and these mimicked the effects of Cmdsx knockdown in females. Knockdown of CmSxl had no measurable effects on stag beetle phenotype, while knockdown of Cmtra2 resulted in complete lethality at the prepupal period. These results indicate that the roles of CmSxl and Cmtra2 in the sex determination cascade are likely to have diverged in stag beetles when compared to Drosophila. Our results also suggest that Cmix has a conserved role in this pathway. In addition to those three genes, we also performed a more complete functional analysis of the C. metallifer dsx gene (Cmdsx) to identify the isoforms that regulate dimorphism more fully using exon-specific RNAi. We identified a total of 16 alternative splice variants of the Cmdsx gene that code for up to 14 separate exons. Despite the variation in RNA splice products of the Cmdsx gene, only four protein isoforms are predicted. The results of our exon-specific RNAi indicated that the essential CmDsx isoform for postembryonic male differentiation is CmDsxB, whereas postembryonic female specific differentiation is mainly regulated by CmDsxD. CONCLUSIONS: Taken together, our results highlight the importance of studying the function of highly conserved sex determination pathways in numerous insect species, especially those with dramatic and exaggerated sexual dimorphism, because conservation in protein structure does not always translate into conservation in downstream function.

The sex-limited effects of mutations in the EGFR and TGF-ß signaling pathways on shape and size sexual dimorphism and allometry in the Drosophila wing.

Much of the morphological diversity in nature-including among sexes within a species-is a direct consequence of variation in size and shape. However, disentangling variation in sexual dimorphism for both shape (SShD), size (SSD), and their relationship with one another remains complex. Understanding how genetic variation influences both size and shape together, and how this in turn influences SSD and SShD, is challenging. In this study, we utilize Drosophila wing size and shape as a model system to investigate how mutations influence size and shape as modulated by sex. Previous work has demonstrated that mutations in epidermal growth factor receptor (EGFR) and transforming growth factor-ß (TGF-ß) signaling components can influence both wing size and shape. In this study, we re-analyze this data to specifically address how they impact the relationship between size and shape in a sex-specific manner, in turn altering the pattern of sexual dimorphism. While most mutations influence shape overall, only a subset have a genotypic specific effect that influences SShD. Furthermore, while we observe sex-specific patterns of allometric shape variation, the effects of most mutations on allometry tend to be small. We discuss this within the context of using mutational analysis to understand sexual size and shape dimorphism.

Does your gene need a background check? How genetic background impacts the analysis of mutations, genes, and evolution.

The premise of genetic analysis is that a causal link exists between phenotypic and allelic variation. However, it has long been documented that mutant phenotypes are not a simple result of a single DNA lesion, but are instead due to interactions of the focal allele with other genes and the environment. Although an experimentally rigorous approach focused on individual mutations and isogenic control strains has facilitated amazing progress within genetics and related fields, a glimpse back suggests that a vast complexity has been omitted from our current understanding of allelic effects. Armed with traditional genetic analyses and the foundational knowledge they have provided, we argue that the time and tools are ripe to return to the underexplored aspects of gene function and embrace the context-dependent nature of genetic effects. We assert that a broad understanding of genetic effects and the evolutionary dynamics of alleles requires identifying how mutational outcomes depend upon the 'wild type' genetic background. Furthermore, we discuss how best to exploit genetic background effects to broaden genetic research programs.

Experimental evidence for within- and cross-seasonal effects of fear on survival and reproduction.

Fear of predation can have non-lethal effects on individuals within a season but whether, and to what extent, these effects carry over into subsequent seasons is not known. Using a replicated seasonal population of the common fruit fly, Drosophila melanogaster, we examined both within- and cross-seasonal effects of fear on survival and reproductive output. Compared to controls, flies exposed to the scent of mantid (Tenodera sinensis) predators in the non-breeding season had 64% higher mortality, and lost 60% more mass by the end of the non-breeding season and, in the subsequent breeding season, produced 20% fewer offspring that weighed 9% less at maturity. Flies exposed to the scent of mantids in the breeding season did not produce fewer offspring, but their offspring developed faster and weighed less as adults compared to the controls. Our results demonstrate how effects of fear can be manifested both within and across seasons and emphasize the importance of understanding how events throughout the annual cycle influence individual success of animals living in seasonal environments.

The conditional nature of genetic interactions: the consequences of wild-type backgrounds on mutational interactions in a genome-wide modifier screen.

The phenotypic outcome of a mutation cannot be simply mapped onto the underlying DNA variant. Instead, the phenotype is a function of the allele, the genetic background in which it occurs and the environment where the mutational effects are expressed. While the influence of genetic background on the expressivity of individual mutations is recognized, its consequences on the interactions between genes, or the genetic network they form, is largely unknown. The description of genetic networks is essential for much of biology; yet if, and how, the topologies of such networks are influenced by background is unknown. Furthermore, a comprehensive examination of the background dependent nature of genetic interactions may lead to identification of novel modifiers of biological processes. Previous work in Drosophila melanogaster demonstrated that wild-type genetic background influences the effects of an allele of scalloped (sd), with respect to both its principal consequence on wing development and its interactions with a mutation in optomotor blind. In this study we address whether the background dependence of mutational interactions is a general property of genetic systems by performing a genome wide dominant modifier screen of the sd(E3) allele in two wild-type genetic backgrounds using molecularly defined deletions. We demonstrate that ~74% of all modifiers of the sd(E3) phenotype are background-dependent due in part to differential sensitivity to genetic perturbation. These background dependent interactions include some with qualitative differences in the phenotypic outcome, as well as instances of sign epistasis. This suggests that genetic interactions are often contingent on genetic background, with flexibility in genetic networks due to segregating variation in populations. Such background dependent effects can substantially alter conclusions about how genes influence biological processes, the potential for genetic screens in alternative wild-type backgrounds identifying new loci that contribute to trait expression, and the inferences of the topology of genetic networks.

Exaggerated trait growth in insects.

Animal structures occasionally attain extreme proportions, eclipsing in size the surrounding body parts. We review insect examples of exaggerated traits, such as the mandibles of stag beetles (Lucanidae), the claspers of praying mantids (Mantidae), the elongated hindlimbs of grasshoppers (Orthoptera: Caelifera), and the giant heads of soldier ants (Formicidae) and termites (Isoptera). Developmentally, disproportionate growth can arise through trait-specific modifications to the activity of at least four pathways: the sex determination pathway, the appendage patterning pathway, the insulin/IGF signaling pathway, and the juvenile hormone/ecdysteroid pathway. Although most exaggerated traits have not been studied mechanistically, it is already apparent that distinct developmental mechanisms underlie the evolution of the different types of exaggerated traits. We suggest this reflects the nature of selection in each instance, revealing an exciting link between mechanism, form, and function. We use this information to make explicit predictions for the types of regulatory pathways likely to underlie each type of exaggerated trait.

The significance and scope of evolutionary developmental biology: a vision for the 21st century.

Evolutionary developmental biology (evo-devo) has undergone dramatic transformations since its emergence as a distinct discipline. This paper aims to highlight the scope, power, and future promise of evo-devo to transform and unify diverse aspects of biology. We articulate key questions at the core of eleven biological disciplines-from Evolution, Development, Paleontology, and Neurobiology to Cellular and Molecular Biology, Quantitative Genetics, Human Diseases, Ecology, Agriculture and Science Education, and lastly, Evolutionary Developmental Biology itself-and discuss why evo-devo is uniquely situated to substantially improve our ability to find meaningful answers to these fundamental questions. We posit that the tools, concepts, and ways of thinking developed by evo-devo have profound potential to advance, integrate, and unify biological sciences as well as inform policy decisions and illuminate science education. We look to the next generation of evolutionary developmental biologists to help shape this process as we confront the scientific challenges of the 21st century.

A general mechanism for conditional expression of exaggerated sexually-selected traits.

Sexually-selected exaggerated traits tend to be unusually reliable signals of individual condition, as their expression tends to be more sensitive to nutritional history and physiological circumstance than that of other phenotypes. As such, these traits are the foundation for many models of sexual selection and animal communication, such as "handicap" and "good genes" models. Exactly how expression of these traits is linked to the bearer's condition has been a central yet unresolved question, in part because the underlying physiological mechanisms regulating their development have remained largely unknown. Recent discoveries across animals as diverse as deer, beetles, and flies now implicate the widely conserved insulin-like signaling pathway, as a common physiological mechanism regulating condition-sensitive structures with extreme growth. This raises the exciting possibility that one highly conserved pathway may underlie the evolution of trait exaggeration in a multitude of sexually-selected signal traits across the animal kingdom.

A pipeline for the de novo assembly of the Themira biloba (Sepsidae: Diptera) transcriptome using a multiple k-mer length approach.

BACKGROUND: The Sepsidae family of flies is a model for investigating how sexual selection shapes courtship and sexual dimorphism in a comparative framework. However, like many non-model systems, there are few molecular resources available. Large-scale sequencing and assembly have not been performed in any sepsid, and the lack of a closely related genome makes investigation of gene expression challenging. Our goal was to develop an automated pipeline for de novo transcriptome assembly, and to use that pipeline to assemble and analyze the transcriptome of the sepsid Themira biloba. RESULTS: Our bioinformatics pipeline uses cloud computing services to assemble and analyze the transcriptome with off-site data management, processing, and backup. It uses a multiple k-mer length approach combined with a second meta-assembly to extend transcripts and recover more bases of transcript sequences than standard single k-mer assembly. We used 454 sequencing to generate 1.48 million reads from cDNA generated from embryo, larva, and pupae of T. biloba and assembled a transcriptome consisting of 24,495 contigs. Annotation identified 16,705 transcripts, including those involved in embryogenesis and limb patterning. We assembled transcriptomes from an additional three non-model organisms to demonstrate that our pipeline assembled a higher-quality transcriptome than single k-mer approaches across multiple species. CONCLUSIONS: The pipeline we have developed for assembly and analysis increases contig length, recovers unique transcripts, and assembles more base pairs than other methods through the use of a meta-assembly. The T. biloba transcriptome is a critical resource for performing large-scale RNA-Seq investigations of gene expression patterns, and is the first transcriptome sequenced in this Dipteran family.

Daily blood feeding rhythms of laboratory-reared North American Culex pipiens.

BACKGROUND: Blood feeding by free-living insect vectors of disease is rhythmic and can be used to predict when infectious bites will occur. These daily rhythms can also be targeted by control measures, as in insecticide-treated nets. Culex pipiens form pipiens and C.p. f. molestus are two members of the Culex pipiens assemblage and vectors of West Nile Virus throughout North America. Although Culex species vector human pathogens and parasites, the daily blood feeding rhythms of C.p. f. molestus, to our knowledge, have not been studied. We described and compared the daily blood feeding rhythms of three laboratory-reared populations of Culex pipiens, one of which has confirmed molestus ancestry. We also examined the plasticity of blood feeding time for these three populations. RESULTS: For most (>70%) C.p. f. pipiens and C.p. f. molestus collected from metropolitan Chicago, IL, blood feeding took place during scotophase. Peak blood feeding occurred in mid-scotophase, 3-6 hours after lights off. For C.p. f. pipiens originating from Pennsylvania, most mosquitoes (> 90%) blood fed during late photophase and early scotophase. C.p. f. molestus denied a blood meal during scotophase were less likely to blood feed during early photophase (< 20%) than were C.p. f. pipiens from Chicago (> 50%). C.p. f. pipiens from Pennsylvania were capable of feeding readily at any hour of photo- or scotophase. CONCLUSIONS: Daily blood feeding rhythms of C.p. f. molestus are similar to those of C.p. f. pipiens, particularly when populations originate from the same geographic region. However, the timing of blood feeding is more flexible for C.p. f. pipiens populations relative to C.p. f. molestus.

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 (SD), 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 SD. 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.

Cloning and characterization of an mRNA encoding an insulin receptor from the horned scarab beetle Onthophagus nigriventris (Coleoptera: Scarabaeidae).

The insulin signaling pathway is the primary signaling pathway coupling growth with nutritional condition in all animals. Sensitivity to circulating levels of insulin has been shown to regulate the growth of specific traits in a dose-dependent manner in response to environmental conditions in a diversity of insect species. Alternative phenotypes in insects manifest in a variety of morphologies such as the sexually dimorphic and male dimorphic horned beetles. Large males of the sexually dimorphic dung beetle Onthophagus nigriventris develop a thoracic horn up to twice the length of the body whereas small males and females never develop this horn. The regulation of this dimorphism is known to be nutrition dependent for males. We focused on the insulin signaling pathway as a potential regulator of this dimorphism. We sequenced a full-length gene transcript encoding the O. nigriventris insulin receptor (OnInR), which is the receptor for circulating insulin and insulin-like peptides in animals. We show that the predicted OnInR protein is similar in overall amino acid identity to other insulin receptors (InRs) and is most closely related phylogenetically to insect InRs. Expression of the OnInR transcript was found during development of imaginal tissues in both males and females. However, expression of OnInR in the region where a horn would grow of small males and female was significantly higher than in the horn tissues of large males at the end of growth. This variation in OnInR expression between sexes and morphs indicates a role for the InR in polymorphic horn development.

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.

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.