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SummaryToxic cardiotonic steroids (CTS) act as a defense mechanism in many firefly species (Lampyridae) by inhibiting a crucial enzyme called Na+,K+-ATPase (NKA). While most fireflies produce these toxins internally, species of the genusPhoturisacquire them from a surprising source: predation on other fireflies. The contrasting physiology of toxin metabolism betweenPhoturisand other firefly genera suggests the possibility of distinct strategies evolving to prevent self-intoxication. Our study demonstrates that bothPhoturisand their firefly prey have evolved highly-resistant NKAs. Using an evolutionary analysis of the specific target of CTS (ATPα) in fireflies, and gene-editing inDrosophila, we find that the initial steps towards resistance were shared amongPhoturisand other firefly lineages. However, thePhoturislineage subsequently underwent multiple rounds of gene duplication and neofunctionalization, resulting in the development of ATPα paralogs that are differentially expressed and exhibit increasing resistance to CTS. In contrast, other firefly species have maintained a single copy. Our results implicate gene duplication as a facilitator in the transition ofPhoturisto its distinct ecological role as predator of toxic firefly prey.One-Sentence SummaryGene duplication is associated with distinct ecological roles for predatory fireflies and their firefly prey.

The recurrent evolution of resistance to cardiotonic steroids (CTS) across diverse animals most frequently involves convergent amino-acid substitutions to the H1-H2 extracellular loop of Na+,K+-ATPase (NKA). Previous work established that hystricognath rodents (e.g. chinchilla) and pterocliform birds (sandgrouse) have convergently evolved amino-acid insertions in the H1-H2 loop, but their functional significance is not known. Using protein engineering, we show that these insertions have distinct effects on CTS resistance of NKA in the two lineages that strongly depend on intramolecular interactions with other residues. Removing the insertion in the chinchilla lineage unexpectedly increases CTS resistance and decreases NKA activity. In the sandgrouse lineage, the insertion works in concert with the substitution Q111R to increase CTS resistance while maintaining wild-type ATPase activity levels. Molecular docking simulations provide additional insight into the biophysical mechanisms responsible for the context-specific CTS insensitivity of the enzyme. Our results highlight the diversity of genetic substrates that underlie CTS insensitivity in vertebrate NKA and reveal how amino-acid insertions can alter the phenotypic effects of point mutations at key sites in the same protein domain.

The domesticated silkworm, Bombyx mori, and its wild progenitor, B. mandarina, are extensively studied as a model case of the evolutionary process of domestication. A conspicuous difference between these species is the dramatic reduction in melanin pigmentation in both larval and adult B. mori. Here we evaluate the efficiency of CRISPR/Cas9-targeted knockouts of pigment-related genes as a tool to understand their potential contributions to domestication-associated melanin pigmentation loss in B. mori. To demonstrate the efficacy of targeted knockouts in B. mandarina, we generated a homozygous CRISPR/Cas9-targeted knockout of yellow-y. In yellow-y knockout mutants, black body colour became lighter throughout the larval, pupal and adult stages, confirming a role for this gene in melanin pigment formation. Further, we performed allele-specific CRISPR/Cas9-targeted knockouts of the pigment-related transcription factor, apontic-like (apt-like) in B. mori × B. mandarina F

The domesticated silkworm, Bombyx mori, and its wild progenitor, B. mandarina, are extensively studied as a model case of the evolutionary process of domestication. A conspicuous difference between these species is the dramatic reduction in pigmentation in both larval and adult B. mori. Here we evaluate the efficiency of CRISPR/Cas9-targeted knockouts of pigment-related genes as a tool to understand their potential contributions to domestication-associated pigmentation loss in B. mori. To demonstrate the efficacy of targeted knockouts in B. mandarina, we generated a homozygous CRISPR/Cas9-targeted knockout of yellow-y. In yellow-y knockout mutants, black body color became lighter throughout the larval, pupal and adult stages, confirming a role for this gene in pigment formation. Further, we performed allele-specific CRISPR/Cas9-targeted knockouts of the pigment-related transcription factor, apontic-like (apt-like) in B. mori × B. mandarina F1 hybrid individuals. Knockout of the B. mandarina allele of apt-like in F1 embryos results in depigmented patches on the dorsal integument of larvae, whereas corresponding knockouts of the B. mori allele consistently exhibit normal F1 larval pigmentation. These results demonstrate a contribution of apt-like to the evolution of reduced pigmentation in B. mori. Together, our results demonstrate the feasibility of CRISPR/Cas9-targeted knockouts as a tool for understanding the genetic basis of traits associated with B. mori domestication.Brief abstractBombyx mori and its wild progenitor are an important model for the study of phenotypic evolution associated with domestication. As proof-of-principle, we used CRISPR/Cas9 to generate targeted knockouts of two pigmentation-related genes. By generating a homozygous knockout of yellow-y in B. mandarina, we confirmed this gene”s role in pigment formation. Further, by generating allele-specific knockouts of apontic-like (apt-like) in B. mori × B. mandarina F1 hybrids, we establish that evolution of apt-like contributed to reduced pigmentation during B. mori domestication.Graphical TOC/Abstract

The recurrent evolution of resistance to cardiotonic steroids (CTS) across diverse animals most frequently involves convergent amino acid substitutions in the H1-H2 extracellular loop of Na+,K+-ATPase (NKA). Previous work revealed that hystricognath rodents (e.g., chinchilla) and pterocliform birds (sandgrouse) have convergently evolved amino acid insertions in the H1-H2 loop, but their functional significance was not known. Using protein engineering, we show that these insertions have distinct effects on CTS resistance in homologs of each of the two species that strongly depend on intramolecular interactions with other residues. Removing the insertion in the chinchilla NKA unexpectedly increases CTS resistance and decreases NKA activity. In the sandgrouse NKA, the amino acid insertion and substitution Q111R both contribute to an augmented CTS resistance without compromising ATPase activity levels. Molecular docking simulations provide additional insight into the biophysical mechanisms responsible for the context-specific mutational effects on CTS insensitivity of the enzyme. Our results highlight the diversity of genetic substrates that underlie CTS insensitivity in vertebrate NKA and reveal how amino acid insertions can alter the phenotypic effects of point mutations at key sites in the same protein domain.

A growing body of theoretical and experimental evidence suggests that intramolecular epistasis is a major determinant of rates and patterns of protein evolution and imposes a substantial constraint on the evolution of novel protein functions. Here, we examine the role of intramolecular epistasis in the recurrent evolution of resistance to cardiotonic steroids (CTS) across tetrapods, which occurs via specific amino acid substitutions to the α-subunit family of Na,K-ATPases (ATP1A). After identifying a series of recurrent substitutions at two key sites of ATP1A that are predicted to confer CTS resistance in diverse tetrapods, we then performed protein engineering experiments to test the functional consequences of introducing these substitutions onto divergent species backgrounds. In line with previous results, we find that substitutions at these sites can have substantial background-dependent effects on CTS resistance. Globally, however, these substitutions also have pleiotropic effects that are consistent with additive rather than background-dependent effects. Moreover, the magnitude of a substitution’s effect on activity does not depend on the overall extent of ATP1A sequence divergence between species. Our results suggest that epistatic constraints on the evolution of CTS-resistant forms of Na,K-ATPase likely depend on a small number of sites, with little dependence on overall levels of protein divergence. We propose that dependence on a limited number sites may account for the observation of convergent CTS resistance substitutions observed among taxa with highly divergent Na,K-ATPases.Significance StatementIndividual amino acid residues within a protein work in concert to produce a functionally coherent structure that must be maintained even as orthologous proteins in different species diverge over time. Given this dependence, we expect identical mutations to have more similar effects on protein function in more closely related species. We tested this hypothesis by performing protein-engineering experiments on ATP1A, an enzyme mediating target-site insensitivity to cardiotonic steroids (CTS) in diverse animals. These experiments reveal that the phenotypic effects of substitutions can sometimes be background-dependent, but also that the magnitude of these phenotypic effects does not correlate with overall levels of ATP1A sequence divergence. Our results suggest that epistatic constraints are determined by states at a small number of sites, potentially explaining the frequent convergent CTS resistance substitutions among Na,K-ATPases of highly divergent taxa.

Evolutionary theory suggests that lifespan-reducing alleles should be purged from the gene pool, and yet decades of genome-wide association and model organism studies have shown that they persist. One potential explanation is that alleles that regulate lifespan do so only in certain environmental contexts. We exposed outbred Drosophila to control and high-sugar diets and genotyped more than 10,000 adult flies to track allele frequency changes over the course of a single adult lifespan. We identified thousands of lifespan-associated alleles associated with early versus late-life trade-offs, late-onset effects and genotype-by-environment interactions. Remarkably, a third of lifespan-associated genetic variation had environmentally dependent effects on lifespan. We find that lifespan-reducing alleles are often recently derived, have stronger effects on a high-sugar diet and show signatures of selection in wild Drosophila populations, consistent with the evolutionary mismatch hypothesis. Our results provide insight into the highly polygenic and context-dependent genetic architecture of lifespan variation and the evolutionary processes that shape this key trait.

Although gene duplication is an important source of evolutionary innovation, the functional divergence of duplicates can be opposed by ongoing gene conversion between them. Here we report on the evolution of a tandem duplication of Na(+),K(+)-ATPase subunit α1 (ATP1A1) shared by frogs in the genus Leptodactylus, a group of species that feeds on toxic toads. One ATP1A1 paralog evolved resistance to toad toxins while the other retained ancestral susceptibility. Within species, frequent non-allelic gene conversion homogenized most of the sequence between the two copies, but was counteracted by strong selection on 12 amino acid substitutions that distinguish the two paralogs. Protein-engineering experiments show that two of these substitutions substantially increase toxin resistance, whereas the additional 10 mitigate their deleterious effects on ATPase activity. Our results reveal how examination of neo-functionalized gene duplicate evolution can help pinpoint key functional substitutions and interactions with the genetic backgrounds on which they arise.

Understanding how evolutionary forces interact to drive patterns of selection and distribute genetic variation across a species' range is of great interest in ecology and evolution, especially in an era of global change. While theory predicts how and when populations at range margins are likely to undergo local adaptation, empirical evidence testing these models remains sparse. Here, we address this knowledge gap by investigating the relationship between selection, gene flow and genetic drift in the yellowtail clownfish, Amphiprion clarkii, from the core to the northern periphery of the species range. Analyses reveal low genetic diversity at the range edge, gene flow from the core to the edge and genomic signatures of local adaptation at 56 single nucleotide polymorphisms in 25 candidate genes, most of which are significantly correlated with minimum annual sea surface temperature. Several of these candidate genes play a role in functions that are upregulated during cold stress, including protein turnover, metabolism and translation. Our results illustrate how spatially divergent selection spanning the range core to the periphery can occur despite the potential for strong genetic drift at the range edge and moderate gene flow from the core populations.

Despite the development of effective vaccines against SARS-CoV-2, epidemiological control of the virus is still challenging due to slow vaccine rollouts, incomplete vaccine protection to current and emerging variants, and unwillingness to get vaccinated. Therefore, frequent testing of individuals to identify early SARS-CoV-2 infections, contact-tracing and isolation strategies remain crucial to mitigate viral spread. Here, we describe WHotLAMP, a rapid molecular test to detect SARS-CoV-2 in saliva. WHotLAMP is simple to use, highly sensitive (3.6 viral RNA copies per microliter of saliva) and specific, as well as inexpensive, making it ideal for frequent screening. Moreover, WHotLAMP does not require toxic chemicals or specialized equipment and thus can be performed in point-of-care settings, and may also be adapted for resource-limited environments or home use. While applied here to SARS-CoV-2, WHotLAMP can be modified to detect other pathogens, making it adaptable for other diagnostic assays, including for use in future outbreaks.

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 aphidHormaphis cornu, which makes distinctive “cone” galls on leaves of witch hazelHamamelis virginiana. We found that derived genetic variants in the aphid genedeterminant of gall color(dgc) are associated with strong downregulation ofdgctranscription 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.Dgcis 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.Bicyclegenes are most strongly expressed in the salivary glands specifically of galling aphid generations, suggesting that they may regulate many aspects of gall development.Bicyclegenes 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.One Sentence SummaryAphidbicyclegenes, which encode diverse secreted proteins, contribute to plant gall development.

Evolutionary theory suggests that lifespan-associated alleles should be purged from the gene pool, and yet decades of GWAS and model organism studies have shown they persist. Here, we address one potential explanation, the idea that the alleles that regulate lifespan do so only in certain contexts. We exposed thousands of outbred Drosophila to a standard and a high sugar diet. We then sequenced over 10,000 individuals and track genome-wide allele frequency changes over time, as these populations aged. We mapped thousands of lifespan-altering alleles, some associated with early vs late life tradeoffs, late-onset effects, and genotype-by-environment interactions. We find that lifespan-reducing alleles are most likely to be recently derived, have stronger effects on a high-sugar diet, consistent with the hypothesis that historically neutral or beneficial alleles can become detrimental in novel conditions. We also show that the gene midway, a regulator of lipid storage and ortholog of the lifespan-associated gene DGAT1 in mice, also regulates lifespan in Drosophila. Our results provide insight into the highly polygenic and context-dependent genetic architecture of lifespan, as well as the evolutionary processes that shape this key trait.

The causes of rate variation among sites within proteins are as yet poorly understood. Here, we compare the spatial autocorrelation of non-synonymous substitutions among species within diverse phylogenetic groups:Saccharomyces, Drosophila, Arabidopsis, and primates. Across these taxa, we find that amino acid substitutions exhibit excess clustering that extends over a 20-30 codon length (10-20 Angstrom distance) scale. We show that these substitutions cluster more strongly and exhibit compensatory dynamics within species lineages but exhibit patterns of convergent evolution between lineages. We evaluate a simple model of thermodynamic constraints on protein folding and conclude that it is unable to recapitulate the observed spatial clustering of substitutions. While pairs of substitutions with the strongest epistasis tend to spatially cluster in these simulations, the magnitude and length scale are smaller than that observed in real data. Additionally, we show that the pattern of convergent substitution is also not expected under this model, suggesting it is likely caused by factors other than these simple thermodynamic constraints. Our results support a prevalent role for epistasis and convergent evolution in shaping protein evolution across the tree of life.

Gene duplication is an important source of evolutionary innovation, but the adaptive division-of-labor between duplicates can be opposed by ongoing gene conversion between them. Here we document a tandem duplication of Na+,K+-ATPase subunit α1 (ATP1A1) shared by frogs in the genusLeptodactylus, a group of species that feeds on toxic toads. One ATP1A1 paralog evolved resistance to toad toxins while the other paralog retained ancestral susceptibility. We show that the twoLeptodactylusparalogs are distinguished by 12 amino acid substitutions that were maintained by strong selection that counteracted the homogenizing effect of gene conversion. Protein-engineering experiments show that two major-effect substitutions confer toxin resistance, whereas the 10 additional substitutions mitigate deleterious pleiotropic effects on enzyme function. Our results highlight how trans-specific, neofunctionalized gene duplicates can provide unique insights into interactions between adaptive substitutions and the genetic backgrounds on which they arise.One Sentence SummarySelection counteracts gene conversion to maintain an adaptive division-of-labor between tandemly duplicated genes.

Biologists since Darwin have been fascinated by the evolution of sexually selected ornaments, particularly those that reduce viability. Uncovering the genetic architecture of these traits is key to understanding how they evolve and are maintained. Here, we investigate the genetic architecture of a sexually selected ornament, the “sword” fin extension that characterizes many species of swordtail fish (Xiphophorus). Using sworded and swordless sister species ofXiphophorus, we generated a mapping population and show that the sword ornament is polygenic – with ancestry across the genome explaining substantial variation in the trait. After accounting for the impacts of genome-wide ancestry, we identify one major effect QTL that explains ∼5% of the overall variation in the trait. Using a series of approaches, we narrow this large QTL interval to a handful of likely candidate genes, including the genesp8. Notably,sp8plays a regulatory role in fin regeneration and harbors several derived substitutions that are predicted to impact protein function in the species that has lost the sword ornament. Furthermore, we find evidence of selection on ancestry atsp8in four natural hybrid populations, consistent with selection against the sword in these populations.

Conservation help from genomics Corals worldwide are under threat from rising sea temperatures and pollution. One response to heat stress is coral bleaching—the loss of photosynthetic endosymbionts that provide energy for the coral. Fuller et al. present a high-resolution genome of the coral Acropora millepora (see the Perspective by Bay and Guerrero). They were able to perform population genetic analyses with samples sequenced at lower coverage and conduct genome-wide association studies. These data were combined to generate a polygenic risk score for bleaching that can be used in coral conservation. Science this issue p. eaba4674 ; see also p. 249

Mapping vertebrate incompatibility alleles Deleterious gene interactions may underlie the observed hybrid incompatibilities. However, few genes underlying hybrid incompatibilities have been identified, and most of these involve species that do not hybridize in natural conditions. Powell et al. used genome sequencing to map genes likely responsible for incompatibilities that reduce fitness in naturally occurring hybrid swordtail fish. These gene combinations result in malignant melanoma, which is found in naturally hybridizing populations but is not present in the parental populations (see the Perspective by Dagilis and Matute). Using genome and population resequencing, the authors performed a genome-wide association study to identify potentially causative mutations. Using an admixture mapping approach that assessed introgression between multiple swordtail fish species, the authors suggest that lineages carry different genes that interact with the same candidate gene, resulting in the observed melanomas and providing insight into convergent hybrid incompatibles that arise between species. Science , this issue p. 731 ; see also p. 710

Although reef-building corals are rapidly declining worldwide, responses to bleaching vary both within and among species. Because these inter-individual differences are partly heritable, they should in principle be predictable from genomic data. Towards that goal, we generated a chromosome-scale genome assembly for the coralAcropora millepora. We then obtained whole genome sequences for 237 phenotyped samples collected at 12 reefs distributed along the Great Barrier Reef, among which we inferred very little population structure. Scanning the genome for evidence of local adaptation, we detected signatures of long-term balancing selection in the heat-shock co-chaperonesacsin. We further used 213 of the samples to conduct a genome-wide association study of visual bleaching score, incorporating the polygenic score derived from it into a predictive model for bleaching in the wild. These results set the stage for the use of genomics-based approaches in conservation strategies.

Although gene duplication is an important source of evolutionary innovation, the functional divergence of duplicates can be opposed by ongoing gene conversion between them. Here, we report on the evolution of a tandem duplication of Na+,K+-ATPase subunit α1 (ATP1A1) shared by frogs in the genus Leptodactylus, a group of species that feeds on toxic toads. One ATP1A1 paralog evolved resistance to toad toxins although the other retained ancestral susceptibility. Within species, frequent non-allelic gene conversion homogenized most of the sequence between the two copies but was counteracted by strong selection on 12 amino acid substitutions that distinguish the two paralogs. Protein-engineering experiments show that two of these substitutions substantially increase toxin resistance, whereas the additional 10 mitigate their deleterious effects on ATPase activity. Our results reveal how examination of neo-functionalized gene duplicate evolution can help pinpoint key functional substitutions and interactions with the genetic backgrounds on which they arise.

Numerous animal lineages have expanded and diversified the opsin-based photoreceptors in their eyes underlying color vision behavior. However, the selective pressures giving rise to new photoreceptors and their spectral tuning remain mostly obscure. Previously, we identified a violet receptor (UV2) that is the result of a UV opsin gene duplication specific to Heliconius butterflies. At the same time the violet receptor evolved, Heliconius evolved UV-yellow coloration on their wings, due to the pigment 3-hydroxykynurenine (3-OHK) and the nanostructure architecture of the scale cells. In order to better understand the selective pressures giving rise to the violet receptor, we characterized opsin expression patterns using immunostaining (14 species) and RNA-Seq (18 species), and reconstructed evolutionary histories of visual traits in five major lineages within Heliconius and one species from the genus Eueides. Opsin expression patterns are hyperdiverse within Heliconius. We identified six unique retinal mosaics and three distinct forms of sexual dimorphism based on ommatidial types within the genus Heliconius. Additionally, phylogenetic analysis revealed independent losses of opsin expression, pseudogenization events, and relaxation of selection on UVRh2 in one lineage. Despite this diversity, the newly evolved violet receptor is retained across most species and sexes surveyed. Discriminability modeling of behaviorally preferred 3-OHK yellow wing coloration suggests that the violet receptor may facilitate Heliconius color vision in the context of conspecific recognition. Our observations give insights into the selective pressures underlying the origins of new visual receptors.

Chromatin boundary elements subdivide chromosomes in multicellular organisms into physically independent domains. In addition to this architectural function, these elements also play a critical role in gene regulation. Here we investigated the evolution of a Drosophila Bithorax complex boundary element called Fab-7, which is required for the proper parasegment specific expression of the homeotic Abd-B gene. Using a “gene” replacement strategy, we show that Fab-7 boundaries from two closely related species, D. erecta and D. yakuba, and a more distant species, D. pseudoobscura, are able to substitute for the melanogaster boundary. Consistent with this functional conservation, the two known Fab-7 boundary factors, Elba and LBC, have recognition sequences in the boundaries from all species. However, the strategies used for maintaining binding and function in the face of sequence divergence is different. The first is conventional, and depends upon conservation of the 8 bp Elba recognition sequence. The second is unconventional, and takes advantage of the unusually large and flexible sequence recognition properties of the LBC boundary factor, and the deployment of multiple LBC recognition elements in each boundary. In the former case, binding is lost when the recognition sequence is altered. In the latter case, sequence divergence is accompanied by changes in the number, relative affinity, and location of the LBC recognition elements.

Summary Mechanisms of sex chromosome dosage compensation (SCDC) differ strikingly among animals. In Drosophila flies, chromosome-wide transcription is doubled from the single X chromosome in hemizygous (XY) males, whereas in Caenorhabditis nematodes, expression is halved for both X copies in homozygous (XX) females [ 1 , 2 ]. Unlike other female-heterogametic (WZ female and ZZ male) animals, moths and butterflies exhibit sex chromosome dosage compensation patterns typically seen only in male-heterogametic species [ 3 ]. The monarch butterfly carries a newly derived Z chromosome segment that arose from an autosomal fusion with the ancestral Z [ 4 ]. Using a highly contiguous genome assembly, we show that gene expression is balanced between sexes along the entire Z chromosome but with distinct modes of compensation on the two segments. On the ancestral Z segment, depletion of H4K16ac corresponds to nearly halving of biallelic transcription in males, a pattern convergent to nematodes. Conversely, the newly derived Z segment shows a Drosophila-like mode of compensation, with enriched H4K16ac levels corresponding to doubled monoallelic transcription in females. Our work reveals that, contrary to the expectation of co-opting regulatory mechanisms readily in place, the evolution of plural modes of dosage compensation is also possible along a single sex chromosome within a species.

Predicting how species will respond to selection pressures requires understanding the factors that constrain their evolution. We use genome engineering of Drosophila to investigate constraints on the repeated evolution of unrelated herbivorous insects to toxic cardiac glycosides, which primarily occurs via a small subset of possible functionally-relevant substitutions to Na+,K+-ATPase. Surprisingly, we find that frequently observed adaptive substitutions at two sites, 111 and 122, are lethal when homozygous and adult heterozygotes exhibit dominant neural dysfunction. We identify a phylogenetically correlated substitution, A119S, that partially ameliorates the deleterious effects of substitutions at 111 and 122. Despite contributing little to cardiac glycoside-insensitivity in vitro, A119S, like substitutions at 111 and 122, substantially increases adult survivorship upon cardiac glycoside exposure. Our results demonstrate the importance of epistasis in constraining adaptive paths. Moreover, by revealing distinct effects of substitutions in vitro and in vivo, our results underscore the importance of evaluating the fitness of adaptive substitutions and their interactions in whole organisms.

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.

Biologists since Darwin have been fascinated by the evolution of sexually selected ornaments, particularly those that reduce viability. Uncovering the genetic architecture of these traits is key to understanding how they evolve and are maintained. Here, we investigate the genetic architecture and evolutionary loss of a sexually selected ornament, the "sword" fin extension that characterizes many species of swordtail fish (Xiphophorus). Using sworded and swordless sister species of Xiphophorus, we generated a mapping population and show that the sword ornament is polygenic-with ancestry across the genome explaining substantial variation in the trait. After accounting for the impacts of genome-wide ancestry, we identify one major-effect quantitative trait locus (QTL) that explains ~5% of the overall variation in the trait. Using a series of approaches, we narrow this large QTL interval to several likely candidate genes, including genes involved in fin regeneration and growth. Furthermore, we find evidence of selection on ancestry at one of these candidates in four natural hybrid populations, consistent with selection against the sword in these populations.

Many distantly related insect species are specialized feeders of cardenolide-containing host plants such as milkweed (Asclepias spp.). Previous studies have revealed frequent, parallel substitution of a functionally important amino acid substitution (N122H) in the alpha subunit of Na(+) ,K(+) -ATPase in a number of these species. This substitution facilitates the ability of these insects to feed on their toxic hosts and sequester cardenolides for their own use in defense. Among milkweed butterflies of the genus Danaus, the previously established phylogeny for this group suggests that N122H arose independently and fixed in two distinct lineages. We reevaluate this conclusion by examining Danaus phylogenetic relationships using >400 orthologous gene sequences assembled from transcriptome data. Our results indicate that the three Danaus species known to harbor the N122H substitution are more closely related than previously thought, consistent with a single, common origin for N122H. However, we also find evidence of both incomplete lineage sorting and post-speciation genetic exchange among these butterfly species, raising the possibility of collateral evolution of cardenolide-insensitivity in this species group.

In a fascinating study, Barrera-Guzman et al. (1) demonstrate a hybrid origin for the golden-crowned manakin and describe a sexually dimorphic plumage trait derived in the hybrid lineage. However, their work stops short of showing that “the evolution of this unique crown-color signal likely culminated in premating isolation of the hybrid species from both parental species” (1), and indeed, this statement runs counter to theoretical expectations about the role of mate choice in speciation. For a novel mating signal to facilitate reproductive isolation, it has to promote mating between genetically similar individuals by being more attractive to hybrid choosers and/or less attractive to parentals (2, 3 … [↵][1]1To whom correspondence should be addressed. Email: grosenthal{at}bio.tamu.edu. [1]: #xref-corresp-1-1

Selection, recombination, and hybrid evolution Hybridization is an important force in evolution. The effects of hybridization across the whole genome are not understood. Using a fine-scale genetic map, Schumer et al. examined local ancestry in replicate natural hybrid populations of swordtail fish. Each parental species contributes different proportions of genetic material to the genomes of their descendants. Genes from the “minor” (less well-represented) parent occur in regions of the genome that are subject to higher recombination rates and where there are fewer potentially deleterious genes. Neanderthal ancestry in human genomes shows similar patterns. Science , this issue p. 656

Individuals within a species vary in their responses to a wide range of stimuli, partly as a result of differences in their genetic makeup. Relatively little is known about the genetic and neuronal mechanisms contributing to diversity of behavior in natural populations. By studying intraspecies variation in innate avoidance behavior to thermal stimuli in the nematode Caenorhabditis elegans, we uncovered genetic principles of how different components of a behavioral response can be altered in nature to generate behavioral diversity. Using a thermal pulse assay, we uncovered heritable variation in responses to a transient temperature increase. Quantitative trait locus mapping revealed that separate components of this response were controlled by distinct genomic loci. The loci we identified contributed to variation in components of thermal pulse avoidance behavior in an additive fashion. Our results show that the escape behavior induced by thermal stimuli is composed of simpler behavioral components that are influenced by at least six distinct genetic loci. The loci that decouple components of the escape behavior reveal a genetic system that allows independent modification of behavioral parameters. Our work sets the foundation for future studies of evolution of innate behaviors at the molecular and neuronal level.

Although freshwater (FW) is the ancestral habitat for larval mosquitoes, multiple species independently evolved the ability to survive in saltwater (SW). Here, we use quantitative trait locus (QTL) mapping to investigate the genetic architecture of osmoregulation in Anopheles mosquitoes, vectors of human malaria. We analyzed 1134 backcross progeny from a cross between the obligate FW species An. coluzzii, and its closely related euryhaline sibling species An. merus. Tests of 2387 markers with Bayesian interval mapping and machine learning (random forests) yielded six genomic regions associated with SW tolerance. Overlap in QTL regions from both approaches enhances confidence in QTL identification. Evidence exists for synergistic as well as disruptive epistasis among loci. Intriguingly, one QTL region containing ion transporters spans the 2Rop chromosomal inversion that distinguishes these species. Rather than a simple trait controlled by one or a few loci, our data are most consistent with a complex, polygenic mode of inheritance.

While hybridization between species is increasingly appreciated to be a common occurrence, little is known about the forces that govern the subsequent evolution of hybrid genomes. We considered this question in three independent, naturally-occurring hybrid populations formed between swordtail fish speciesXiphophorus birchmanniandX. malinche.To this end, we built a fine-scale genetic map and inferred patterns of local ancestry along the genomes of 690 individuals sampled from the three populations. In all three cases, we found hybrid ancestry to be more common in regions of high recombination and where there is linkage to fewer putative targets of selection. These same patterns are also apparent in a reanalysis of human-Neanderthal admixture. Our results lend support to models in which ancestry from the “minor” parental species persists only where it is rapidly uncoupled from alleles that are deleterious in hybrids, and show the retention of hybrid ancestry to be at least in part predictable from genomic features. Our analyses further indicate that in swordtail fish, the dominant source of selection on hybrids stems from deleterious combinations of epistatically-interacting alleles.One sentence summaryThe persistence of hybrid ancestry is predictable from local recombination rates, in three replicate hybrid populations as well as in humans.

The emergence of new species is driven by the establishment of mechanisms that limit gene flow between populations. A major challenge is reconciling the theoretical and empirical importance of assortative mating in speciation with the ease with which it can fail. Swordtail fish have an evolutionary history of hybridization and fragile prezygotic isolating mechanisms. Hybridization between two swordtail species likely arose via pollution-mediated breakdown of assortative mating in the 1990s. Here we track unusual genetic patterns in one hybrid population over the past decade using whole-genome sequencing. Hybrids in this population formed separate genetic clusters by 2003, and maintained near-perfect isolation over 25 generations through strong ancestry-assortative mating. However, we also find that assortative mating was plastic, varying in strength over time and disappearing under manipulated conditions. In addition, a nearby population did not show evidence of assortative mating. Thus, our findings suggest that assortative mating may constitute an intermittent and unpredictable barrier to gene flow, but that variation in its strength can have a major effect on how hybrid populations evolve. Understanding how reproductive isolation varies across populations and through time is critical to understanding speciation and hybridization, as well as their dependence on disturbance.

Evolutionary shifts in plant–herbivore interactions provide a model for understanding the link among the evolution of behaviour, ecological specialization and incipient speciation. Drosophila mojavensis uses different host cacti across its range, and volatile chemicals emitted by the host are the primary cue for host plant identification. In this study, we show that changes in host plant use between distinct D. mojavensis populations are accompanied by changes in the olfactory system. Specifically, we observe differences in olfactory receptor neuron specificity and sensitivity, as well as changes in sensillar subtype abundance, between populations. Additionally, RNA-seq analyses reveal differential gene expression between populations for members of the odorant receptor gene family. Hence, alterations in host preference are associated with changes in development, regulation and function at the olfactory periphery.