A novel broad spectrum venom metalloproteinase autoinhibitor in the rattlesnake Crotalus atrox evolved via a shift in paralog function.

The complexity of snake venom composition reflects adaptation to the diversity of prey and may be driven at times by a coevolutionary arms race between snakes and venom-resistant prey. However, many snakes are also resistant to their own venom due to serum-borne inhibitors of venom toxins, which raises the question of how snake autoinhibitors maintain their efficacy as venom proteins evolve. To investigate this potential three-way arms race among venom, prey, and autoinhibitors, we have identified and traced the evolutionary origin of serum inhibitors of snake venom metalloproteinases (SVMPs) in the Western Diamondback rattlesnake Crotalus atrox which possesses the largest known battery of SVMP genes among crotalids examined. We found that C. atrox expresses five members of a Fetuin A-related metalloproteinase inhibitor family but that one family member, FETUA-3, is the major SVMP inhibitor that binds to approximately 20 different C. atrox SVMPs and inhibits activities of all three SVMP classes. We show that the fetua-3 gene arose deep within crotalid evolution before the origin of New World species but, surprisingly, fetua-3 belongs to a different paralog group than previously identified SVMP inhibitors in Asian and South American crotalids. Conversely, the C. atrox FETUA-2 ortholog of previously characterized crotalid SVMP inhibitors shows limited activity against C. atrox SVMPs. These results reveal that there has been a functional evolutionary shift in the major SVMP inhibitor in the C. atrox lineage as the SVMP family expanded and diversified in the Crotalus lineage. This broad-spectrum inhibitor may be of potential therapeutic interest.

Evolution of the tan locus contributed to pigment loss in Drosophila santomea: a response to Matute et al.

We have shown previously that the loss of abdominal pigmentation in D. santomea relative to its sister species D. yakuba resulted, in part, from cis-regulatory mutations at the tan locus. Matute et al. claim, based solely upon extrapolation from genetic crosses of D. santomea and D. melanogaster, a much more divergent species, that at least four X chromosome regions but not tan are responsible for pigmentation differences. Here, we provide additional evidence from introgressions of D. yakuba genes into D. santomea that support a causative role for tan in the loss of pigmentation and present analyses that contradict Matute et al.'s claims. We discuss how the choice of parental species and other factors affect the ability to identify loci responsible for species divergence, and we affirm that all of our previously reported results and conclusions stand.

Evo-devo and an expanding evolutionary synthesis: a genetic theory of morphological evolution.

Biologists have long sought to understand which genes and what kinds of changes in their sequences are responsible for the evolution of morphological diversity. Here, I outline eight principles derived from molecular and evolutionary developmental biology and review recent studies of species divergence that have led to a genetic theory of morphological evolution, which states that (1) form evolves largely by altering the expression of functionally conserved proteins, and (2) such changes largely occur through mutations in the cis-regulatory sequences of pleiotropic developmental regulatory loci and of the target genes within the vast networks they control.

The evolution of gene regulation underlies a morphological difference between two Drosophila sister species.

Understanding the mechanisms underlying the morphological divergence of species is one of the central goals of evolutionary biology. Here, we analyze the genetic and molecular bases of the divergence of body pigmentation patterns between Drosophila yakuba and its sister species Drosophila santomea. We found that loss of pigmentation in D. santomea involved the selective loss of expression of the tan and yellow pigmentation genes. We demonstrate that tan gene expression was eliminated through the mutational inactivation of one specific tan cis-regulatory element (CRE) whereas the Tan protein sequence remained unchanged. Surprisingly, we identify three independent loss-of-function alleles of the tan CRE in the young D. santomea lineage. We submit that there is sufficient empirical evidence to support the general prediction that functional evolutionary changes at pleiotropic loci will most often involve mutations in their discrete, modular cis-regulatory elements.

The regulation and evolution of a genetic switch controlling sexually dimorphic traits in Drosophila.

Sexually dimorphic traits play key roles in animal evolution and behavior. Little is known, however, about the mechanisms governing their development and evolution. One recently evolved dimorphic trait is the male-specific abdominal pigmentation of Drosophila melanogaster, which is repressed in females by the Bric-à-brac (Bab) proteins. To understand the regulation and origin of this trait, we have identified and traced the evolution of the genetic switch controlling dimorphic bab expression. We show that the HOX protein Abdominal-B (ABD-B) and the sex-specific isoforms of Doublesex (DSX) directly regulate a bab cis-regulatory element (CRE). In females, ABD-B and DSX(F) activate bab expression whereas in males DSX(M) directly represses bab, which allows for pigmentation. A new domain of dimorphic bab expression evolved through multiple fine-scale changes within this CRE, whose ancestral role was to regulate other dimorphic features. These findings reveal how new dimorphic characters can emerge from genetic networks regulating pre-existing dimorphic traits.

The origin and diversification of a novel protein family in venomous snakes.

The genetic origins of novelty are a central interest of evolutionary biology. Most new proteins evolve from preexisting proteins but the evolutionary path from ancestral gene to novel protein is challenging to trace, and therefore the requirements for and order of coding sequence changes, expression changes, or gene duplication are not clear. Snake venoms are important novel traits that are comprised of toxins derived from several distinct protein families, but the genomic and evolutionary origins of most venom components are not understood. Here, we have traced the origin and diversification of one prominent family, the snake venom metalloproteinases (SVMPs) that play key roles in subduing prey in many vipers. Genomic analyses of several rattlesnake (Crotalus) species revealed the SVMP family massively expanded from a single, deeply conserved adam28 disintegrin and metalloproteinase gene, to as many as 31 tandem genes in the Western Diamondback rattlesnake (Crotalus atrox) through a number of single gene and multigene duplication events. Furthermore, we identified a series of stepwise intragenic deletions that occurred at different times in the course of gene family expansion and gave rise to the three major classes of secreted SVMP toxins by sequential removal of a membrane-tethering domain, the cysteine-rich domain, and a disintegrin domain, respectively. Finally, we show that gene deletion has further shaped the SVMP complex within rattlesnakes, creating both fusion genes and substantially reduced gene complexes. These results indicate that gene duplication and intragenic deletion played essential roles in the origin and diversification of these novel biochemical weapons.

A major role for noncoding regulatory mutations in the evolution of enzyme activity.

The quantitative evolution of protein activity is a common phenomenon, yet we know little about any general mechanistic tendencies that underlie it. For example, an increase (or decrease) in enzyme activity may evolve from changes in protein sequence that alter specific activity, or from changes in gene expression that alter the amount of protein produced. The latter in turn could arise via mutations that affect gene transcription, posttranscriptional processes, or copy number. Here, to determine the types of genetic changes underlying the quantitative evolution of protein activity, we dissected the basis of ecologically relevant differences in Alcohol dehydrogenase (Adh) enzyme activity between and within several Drosophila species. By using recombinant Adh transgenes to map the functional divergence of ADH enzyme activity in vivo, we find that amino acid substitutions explain only a minority (0 to 25%) of between- and within-species differences in enzyme activity. Instead, noncoding substitutions that occur across many parts of the gene (enhancer, promoter, and 5' and 3' untranslated regions) account for the majority of activity differences. Surprisingly, one substitution in a transcriptional Initiator element has occurred in parallel in two species, indicating that core promoters can be an important natural source of the tuning of gene activity. Furthermore, we show that both regulatory and coding substitutions contribute to fitness (resistance to ethanol toxicity). Although qualitative changes in protein specificity necessarily derive from coding mutations, these results suggest that regulatory mutations may be the primary source of quantitative changes in protein activity, a possibility overlooked in most analyses of protein evolution.

Expression of tandem gene duplicates is often greater than twofold.

Tandem gene duplication is an important mutational process in evolutionary adaptation and human disease. Hypothetically, two tandem gene copies should produce twice the output of a single gene, but this expectation has not been rigorously investigated. Here, we show that tandem duplication often results in more than double the gene activity. A naturally occurring tandem duplication of the Alcohol dehydrogenase (Adh) gene exhibits 2.6-fold greater expression than the single-copy gene in transgenic Drosophila This tandem duplication also exhibits greater activity than two copies of the gene in trans, demonstrating that it is the tandem arrangement and not copy number that is the cause of overactivity. We also show that tandem duplication of an unrelated synthetic reporter gene is overactive (2.3- to 5.1-fold) at all sites in the genome that we tested, suggesting that overactivity could be a general property of tandem gene duplicates. Overactivity occurs at the level of RNA transcription, and therefore tandem duplicate overactivity appears to be a previously unidentified form of position effect. The increment of surplus gene expression observed is comparable to many regulatory mutations fixed in nature and, if typical of other genomes, would shape the fate of tandem duplicates in evolution.

Gain of cis-regulatory activities underlies novel domains of wingless gene expression in Drosophila.

Changes in gene expression during animal development are largely responsible for the evolution of morphological diversity. However, the genetic and molecular mechanisms responsible for the origins of new gene-expression domains have been difficult to elucidate. Here, we sought to identify molecular events underlying the origins of three novel features of wingless (wg) gene expression that are associated with distinct pigmentation patterns in Drosophila guttifera. We compared the activity of cis-regulatory sequences (enhancers) across the wg locus in D. guttifera and Drosophila melanogaster and found strong functional conservation among the enhancers that control similar patterns of wg expression in larval imaginal discs that are essential for appendage development. For pupal tissues, however, we found three novel wg enhancer activities in D. guttifera associated with novel domains of wg expression, including two enhancers located surprisingly far away in an intron of the distant Wnt10 gene. Detailed analysis of one enhancer (the vein-tip enhancer) revealed that it overlapped with a region controlling wg expression in wing crossveins (crossvein enhancer) in D. guttifera and other species. Our results indicate that one novel domain of wg expression in D. guttifera wings evolved by co-opting pre-existing regulatory sequences governing gene activity in the developing wing. We suggest that the modification of existing enhancers is a common path to the evolution of new gene-expression domains and enhancers.

Wax, sex and the origin of species: Dual roles of insect cuticular hydrocarbons in adaptation and mating.

Evolutionary changes in traits that affect both ecological divergence and mating signals could lead to reproductive isolation and the formation of new species. Insect cuticular hydrocarbons (CHCs) are potential examples of such dual traits. They form a waxy layer on the cuticle of the insect to maintain water balance and prevent desiccation, while also acting as signaling molecules in mate recognition and chemical communication. Because the synthesis of these hydrocarbons in insect oenocytes occurs through a common biochemical pathway, natural or sexual selection on one role may affect the other. In this review, we explore how ecological divergence in insect CHCs can lead to divergence in mating signals and reproductive isolation. We suggest that the evolution of insect CHCs may be ripe models for understanding ecological speciation.

Generation of a novel wing colour pattern by the Wingless morphogen.

The complex, geometric colour patterns of many animal bodies have important roles in behaviour and ecology. The generation of certain patterns has been the subject of considerable theoretical exploration, however, very little is known about the actual mechanisms underlying colour pattern formation or evolution. Here we have investigated the generation and evolution of the complex, spotted wing pattern of Drosophila guttifera. We show that wing spots are induced by the Wingless morphogen, which is expressed at many discrete sites that are specified by pre-existing positional information that governs the development of wing structures. Furthermore, we demonstrate that the elaborate spot pattern evolved from simpler schemes by co-option of Wingless expression at new sites. This example of a complex design developing and evolving by the layering of new patterns on pre-patterns is likely to be a general theme in other animals.

Genetic and molecular insights into the development and evolution of sexual dimorphism.

Sexual dimorphism is common throughout the animal kingdom. However, a molecular understanding of how sex-specific traits develop and evolve has been elusive. Recently, substantial progress has been made in elucidating how diverse sex-determination systems are integrated into developmental gene networks. One common theme from these studies is that sex-limited traits and gene expression are produced by the combined action of transcriptional effectors of sex-determination pathways and other transcription factors on target gene cis-regulatory elements. Sex-specific traits evolve by the gain, loss or modification of linkages in the genetic networks regulated by sex-determination transcription factors.

Sexually dimorphic regulation of the Wingless morphogen controls sex-specific segment number in Drosophila.

Sexual dimorphism is widespread throughout the metazoa and plays important roles in mate recognition and preference, sex-based niche partitioning, and sex-specific coadaptation. One notable example of sex-specific differences in insect body morphology is presented by the higher diptera, such as Drosophila, in which males develop fewer abdominal segments than females. Because diversity in segment number is a distinguishing feature of major arthropod clades, it is of fundamental interest to understand how different numbers of segments can be generated within the same species. Here we show that sex-specific and segment-specific regulation of the Wingless (Wg) morphogen underlies the development of sexually dimorphic adult segment number in Drosophila. Wg expression is repressed in the developing terminal male abdominal segment by the combination of the Hox protein Abdominal-B (Abd-B) and the sex-determination regulator Doublesex (Dsx). The subsequent loss of the terminal male abdominal segment during pupation occurs through a combination of developmental processes including segment compartmental transformation, apoptosis, and suppression of cell proliferation. Furthermore, we show that ectopic expression of Wg is sufficient to rescue this loss. We propose that dimorphic Wg regulation, in concert with monomorphic segment-specific programmed cell death, are the principal mechanisms of sculpting the sexually dimorphic abdomen of Drosophila.

Evolutionary origin of a novel gene expression pattern through co-option of the latent activities of existing regulatory sequences.

Spatiotemporal changes in gene expression underlie many evolutionary novelties in nature. However, the evolutionary origins of novel expression patterns, and the transcriptional control elements ("enhancers") that govern them, remain unclear. Here, we sought to explore the molecular genetic mechanisms by which new enhancers arise. We undertook a survey of closely related Drosophila species to identify recently evolved novel gene expression patterns and traced their evolutionary history. Analyses of gene expression in a variety of developing tissues of the Drosophila melanogaster species subgroup revealed high rates of expression pattern divergence, including numerous evolutionary losses, heterochronic shifts, and expansions or contractions of expression domains. However, gains of novel expression patterns were much less frequent. One gain was observed for the Neprilysin-1 (Nep1) gene, which has evolved a unique expression pattern in optic lobe neuroblasts of Drosophila santomea. Dissection of the Nep1 cis-regulatory region localized a newly derived optic lobe enhancer activity to a region of an intron that has accumulated a small number of mutations. The Nep1 optic lobe enhancer overlaps with other enhancer activities, from which the novel activity was co-opted. We suggest that the novel optic lobe enhancer evolved by exploiting the cryptic activity of extant regulatory sequences, and this may reflect a general mechanism whereby new enhancers evolve.

Gene duplication and the adaptive evolution of a classic genetic switch.

How gene duplication and divergence contribute to genetic novelty and adaptation has been of intense interest, but experimental evidence has been limited. The genetic switch controlling the yeast galactose use pathway includes two paralogous genes in Saccharomyces cerevisiae that encode a co-inducer (GAL3) and a galactokinase (GAL1). These paralogues arose from a single bifunctional ancestral gene as is still present in Kluyveromyces lactis. To determine which evolutionary processes shaped the evolution of the two paralogues, here we assess the effects of precise replacement of coding and non-coding sequences on organismal fitness. We suggest that duplication of the ancestral bifunctional gene allowed for the resolution of an adaptive conflict between the transcriptional regulation of the two gene functions. After duplication, previously disfavoured binding site configurations evolved that divided the regulation of the ancestral gene into two specialized genes, one of which ultimately became one of the most tightly regulated genes in the genome.

Rapid evolution of sex pheromone-producing enzyme expression in Drosophila.

A wide range of organisms use sex pheromones to communicate with each other and to identify appropriate mating partners. While the evolution of chemical communication has been suggested to cause sexual isolation and speciation, the mechanisms that govern evolutionary transitions in sex pheromone production are poorly understood. Here, we decipher the molecular mechanisms underlying the rapid evolution in the expression of a gene involved in sex pheromone production in Drosophilid flies. Long-chain cuticular hydrocarbons (e.g., dienes) are produced female-specifically, notably via the activity of the desaturase DESAT-F, and are potent pheromones for male courtship behavior in Drosophila melanogaster. We show that across the genus Drosophila, the expression of this enzyme is correlated with long-chain diene production and has undergone an extraordinary number of evolutionary transitions, including six independent gene inactivations, three losses of expression without gene loss, and two transitions in sex-specificity. Furthermore, we show that evolutionary transitions from monomorphism to dimorphism (and its reversion) in desatF expression involved the gain (and the inactivation) of a binding-site for the sex-determination transcription factor, DOUBLESEX. In addition, we documented a surprising example of the gain of particular cis-regulatory motifs of the desatF locus via a set of small deletions. Together, our results suggest that frequent changes in the expression of pheromone-producing enzymes underlie evolutionary transitions in chemical communication, and reflect changing regimes of sexual selection, which may have contributed to speciation among Drosophila.

Repeated morphological evolution through cis-regulatory changes in a pleiotropic gene.

The independent evolution of morphological similarities is widespread. For simple traits, such as overall body colour, repeated transitions by means of mutations in the same gene may be common. However, for more complex traits, the possible genetic paths may be more numerous; the molecular mechanisms underlying their independent origins and the extent to which they are constrained to follow certain genetic paths are largely unknown. Here we show that a male wing pigmentation pattern involved in courtship display has been gained and lost multiple times in a Drosophila clade. Each of the cases we have analysed (two gains and two losses) involved regulatory changes at the pleiotropic pigmentation gene yellow. Losses involved the parallel inactivation of the same cis-regulatory element (CRE), with changes at a few nucleotides sufficient to account for the functional divergence of one element between two sibling species. Surprisingly, two independent gains of wing spots resulted from the co-option of distinct ancestral CREs. These results demonstrate how the functional diversification of the modular CREs of pleiotropic genes contributes to evolutionary novelty and the independent evolution of morphological similarities.

Chance caught on the wing: cis-regulatory evolution and the origin of pigment patterns in Drosophila.

The gain, loss or modification of morphological traits is generally associated with changes in gene regulation during development. However, the molecular bases underlying these evolutionary changes have remained elusive. Here we identify one of the molecular mechanisms that contributes to the evolutionary gain of a male-specific wing pigmentation spot in Drosophila biarmipes, a species closely related to Drosophila melanogaster. We show that the evolution of this spot involved modifications of an ancestral cis-regulatory element of the yellow pigmentation gene. This element has gained multiple binding sites for transcription factors that are deeply conserved components of the regulatory landscape controlling wing development, including the selector protein Engrailed. The evolutionary stability of components of regulatory landscapes, which can be co-opted by chance mutations in cis-regulatory elements, might explain the repeated evolution of similar morphological patterns, such as wing pigmentation patterns in flies.