Despite considerable progress in recent decades in dissecting the genetic causes of natural morphological variation, there is limited understanding of how variation within species ultimately contributes to species differences. We have studied patterning of the non-sensory hairs, commonly known as "trichomes," on the dorsal cuticle of first-instar larvae of Drosophila. Most Drosophila species produce a dense lawn of dorsal trichomes, but a subset of these trichomes were lost in D. sechellia and D. ezoana due entirely to regulatory evolution of the shavenbaby (svb) gene. Here, we describe intraspecific variation in dorsal trichome patterns of first-instar larvae of D. virilis that is similar to the trichome pattern variation identified previously between species. We found that a single large effect QTL, which includes svb, explains most of the trichome number difference between two D. virilis strains and that svb expression correlates with the trichome difference between strains. This QTL does not explain the entire difference between strains, implying that additional loci contribute to variation in trichome numbers. Thus, the genetic architecture of intraspecific variation exhibits similarities and differences with interspecific variation that may reflect differences in long-term and short-term evolutionary processes.

Pigmentation divergence between Drosophila species has emerged as a model trait for studying the genetic basis of phenotypic evolution, with genetic changes contributing to pigmentation differences often mapping to genes in the pigment synthesis pathway and their regulators. These studies of Drosophila pigmentation have tended to focus on pigmentation changes in one body part for a particular pair of species, but changes in pigmentation are often observed in multiple body parts between the same pair of species. The similarities and differences of genetic changes responsible for divergent pigmentation in different body parts of the same species thus remain largely unknown. Here we compare the genetic basis of pigmentation divergence between Drosophila elegans and D. gunungcola in the wing, legs, and thorax. Prior work has shown that regions of the genome containing the pigmentation genes yellow and ebony influence the size of divergent male-specific wing spots between these two species. We find that these same two regions of the genome underlie differences in leg and thorax pigmentation; however, divergent alleles in these regions show differences in allelic dominance and epistasis among the three body parts. These complex patterns of inheritance can be explained by a model of evolution involving tissue-specific changes in the expression of Yellow and Ebony between D. elegans and D. gunungcola.

Many genomes contain rapidly evolving and highly divergent genes whose homology to genes of known function often cannot be determined from sequence similarity alone. However, coding sequence-independent features of genes, such as intron-exon boundaries, often evolve more slowly than coding sequences and can provide complementary evidence for homology. We found that a linear logistic regression classifier using only structural features of rapidly evolving bicycle aphid effector genes identified many putative bicycle homologs in aphids, phylloxerids, and scale insects, whereas sequence similarity search methods yielded few homologs in most aphids and no homologs in phylloxerids and scale insects. Subsequent examination of sequence features and intron locations supported homology assignments. Differential expression studies of newly-identified bicycle homologs, together with prior proteomic studies, support the hypothesis that BICYCLE proteins act as plant effector proteins in many aphid species and perhaps also in phylloxerids and scale insects.

Although different animal species often exhibit extensive variation in many behaviors, typically scientists examine one or a small number of behaviors in any single study. Here, we propose a new framework to simultaneously study the evolution of many behaviors. We measured the behavioral repertoire of individuals from six species of fruit flies using unsupervised techniques and identified all stereotyped movements exhibited by each species. We then fit a Generalized Linear Mixed Model to estimate the intra- and inter-species behavioral covariances, and, by using the known phylogenetic relationships among species, we estimated the (unobserved) behaviors exhibited by ancestral species. We found that much of intra-specific behavioral variation has a similar covariance structure to previously described long-time scale variation in an individual's behavior, suggesting that much of the measured variation between individuals of a single species in our assay reflects differences in the status of neural networks, rather than genetic or developmental differences between individuals. We then propose a method to identify groups of behaviors that appear to have evolved in a correlated manner, illustrating how sets of behaviors, rather than individual behaviors, likely evolved. Our approach provides a new framework for identifying co-evolving behaviors and may provide new opportunities to study the mechanistic basis of behavioral evolution.

In an elaborate form of inter-species exploitation, many insects hijack plant development to induce novel plant organs called galls that provide the insect with a source of nutrition and a temporary home. Galls result from dramatic reprogramming of plant cell biology driven by insect molecules, but the roles of specific insect molecules in gall development have not yet been determined. Here, we study the aphid Hormaphis cornu, which makes distinctive "cone" galls on leaves of witch hazel Hamamelis virginiana. We found that derived genetic variants in the aphid gene determinant of gall color (dgc) are associated with strong downregulation of dgc transcription in aphid salivary glands, upregulation in galls of seven genes involved in anthocyanin synthesis, and deposition of two red anthocyanins in galls. We hypothesize that aphids inject DGC protein into galls and that this results in differential expression of a small number of plant genes. dgc is a member of a large, diverse family of novel predicted secreted proteins characterized by a pair of widely spaced cysteine-tyrosine-cysteine (CYC) residues, which we named BICYCLE proteins. bicycle genes are most strongly expressed in the salivary glands specifically of galling aphid generations, suggesting that they may regulate many aspects of gall development. bicycle genes have experienced unusually frequent diversifying selection, consistent with their potential role controlling gall development in a molecular arms race between aphids and their host plants.

Changes in gene regulation underlie much of phenotypic evolution. However, our understanding of the potential for regulatory evolution is biased, because most evidence comes from either natural variation or limited experimental perturbations. Using an automated robotics pipeline, we surveyed an unbiased mutation library for a developmental enhancer in Drosophila melanogaster. We found that almost all mutations altered gene expression and that parameters of gene expression-levels, location, and state-were convolved. The widespread pleiotropic effects of most mutations may constrain the evolvability of developmental enhancers. Consistent with these observations, comparisons of diverse Drosophila larvae revealed apparent biases in the phenotypes influenced by the enhancer. Developmental enhancers may encode a higher density of regulatory information than has been appreciated previously, imposing constraints on regulatory evolution.

The evolution of sexual traits often involves correlated changes in morphology and behavior. For example, in Drosophila, divergent mating displays are often accompanied by divergent pigment patterns. To better understand how such traits co-evolve, we investigated the genetic basis of correlated divergence in wing pigmentation and mating display between the sibling species Drosophila elegans and D. gunungcola. Drosophila elegans males have an area of black pigment on their wings known as a wing spot and appear to display this spot to females by extending their wings laterally during courtship. By contrast, D. gunungcola lost both of these traits. Using Multiplexed Shotgun Genotyping (MSG), we identified a ∼440 kb region on the X chromosome that behaves like a genetic switch controlling the presence or absence of male-specific wing spots. This region includes the candidate gene optomotor-blind (omb), which plays a critical role in patterning the Drosophila wing. The genetic basis of divergent wing display is more complex, with at least two loci on the X chromosome and two loci on autosomes contributing to its evolution. Introgressing the X-linked region affecting wing spot development from D. gunungcola into D. elegans reduced pigmentation in the wing spots but did not affect the wing display, indicating that these are genetically separable traits. Consistent with this observation, broader sampling of wild D. gunungcola populations confirmed the wing spot and wing display are evolving independently: some D. gunungcola males performed wing displays similar to D. elegans despite lacking wing spots. These data suggest that correlated selection pressures rather than physical linkage or pleiotropy are responsible for the coevolution of these morphological and behavioral traits. They also suggest that the change in morphology evolved prior to the change in behavior. This article is protected by copyright. All rights reserved.

Wing dimorphisms have long served as models for examining the ecological and evolutionary tradeoffs associated with alternative phenotypes. Here, we investigated the genetic cause of the pea aphid () male wing dimorphism, wherein males exhibit one of two morphologies that differ in correlated traits that include the presence or absence of wings. We mapped this trait difference to a single genomic region and, using third generation, long-read sequencing, we identified a 120 kb insertion in the wingless allele. This insertion includes a duplicated gene, which is a strong candidate gene in the minimal mapped interval to cause the dimorphism. We found that both alleles were present prior to pea aphid biotype lineage diversification, we estimated that the insertion occurred millions of years ago, and we propose that both alleles have been maintained in the species, likely due to balancing selection.

males perform a series of courtship behaviors that, when successful, result in copulation with a female. For over a century, mutations in the gene, named for its effects on pigmentation, have been known to reduce male mating success. Prior work has suggested that influences mating behavior through effects on wing extension, song, and/or courtship vigor. Here, we rule out these explanations, as well as effects on the nervous system more generally, and find instead that the effects of on male mating success are mediated by its effects on pigmentation of male-specific leg structures called sex combs. Loss of expression in these modified bristles reduces their melanization, which changes their structure and causes difficulty grasping females prior to copulation. These data illustrate why the mechanical properties of anatomy, not just neural circuitry, must be considered to fully understand the development and evolution of behavior.