The genetic regulatory architecture and epigenomic basis for age-related changes in rattlesnake venom.

Developmental phenotypic changes can evolve under selection imposed by age- and size-related ecological differences. Many of these changes occur through programmed alterations to gene expression patterns, but the molecular mechanisms and gene-regulatory networks underlying these adaptive changes remain poorly understood. Many venomous snakes, including the eastern diamondback rattlesnake (Crotalus adamanteus), undergo correlated changes in diet and venom expression as snakes grow larger with age, providing models for identifying mechanisms of timed expression changes that underlie adaptive life history traits. By combining a highly contiguous, chromosome-level genome assembly with measures of expression, chromatin accessibility, and histone modifications, we identified cis-regulatory elements and trans-regulatory factors controlling venom ontogeny in the venom glands of C. adamanteus. Ontogenetic expression changes were significantly correlated with epigenomic changes within genes, immediately adjacent to genes (e.g., promoters), and more distant from genes (e.g., enhancers). We identified 37 candidate transcription factors (TFs), with the vast majority being up-regulated in adults. The ontogenetic change is largely driven by an increase in the expression of TFs associated with growth signaling, transcriptional activation, and circadian rhythm/biological timing systems in adults with corresponding epigenomic changes near the differentially expressed venom genes. However, both expression activation and repression contributed to the composition of both adult and juvenile venoms, demonstrating the complexity and potential evolvability of gene regulation for this trait. Overall, given that age-based trait variation is common across the tree of life, we provide a framework for understanding gene-regulatory-network-driven life-history evolution more broadly.

The Tiger Rattlesnake genome reveals a complex genotype underlying a simple venom phenotype.

Variation in gene regulation is ubiquitous, yet identifying the mechanisms producing such variation, especially for complex traits, is challenging. Snake venoms provide a model system for studying the phenotypic impacts of regulatory variation in complex traits because of their genetic tractability. Here, we sequence the genome of the Tiger Rattlesnake, which possesses the simplest and most toxic venom of any rattlesnake species, to determine whether the simple venom phenotype is the result of a simple genotype through gene loss or a complex genotype mediated through regulatory mechanisms. We generate the most contiguous snake-genome assembly to date and use this genome to show that gene loss, chromatin accessibility, and methylation levels all contribute to the production of the simplest, most toxic rattlesnake venom. We provide the most complete characterization of the venom gene-regulatory network to date and identify key mechanisms mediating phenotypic variation across a polygenic regulatory network.

Phylogenetically diverse diets favor more complex venoms in North American pitvipers.

The role of natural selection in the evolution of trait complexity can be characterized by testing hypothesized links between complex forms and their functions across species. Predatory venoms are composed of multiple proteins that collectively function to incapacitate prey. Venom complexity fluctuates over evolutionary timescales, with apparent increases and decreases in complexity, and yet the causes of this variation are unclear. We tested alternative hypotheses linking venom complexity and ecological sources of selection from diet in the largest clade of front-fanged venomous snakes in North America: the rattlesnakes, copperheads, cantils, and cottonmouths. We generated independent transcriptomic and proteomic measures of venom complexity and collated several natural history studies to quantify dietary variation. We then constructed genome-scale phylogenies for these snakes for comparative analyses. Strikingly, prey phylogenetic diversity was more strongly correlated to venom complexity than was overall prey species diversity, specifically implicating prey species' divergence, rather than the number of lineages alone, in the evolution of complexity. Prey phylogenetic diversity further predicted transcriptomic complexity of three of the four largest gene families in viper venom, showing that complexity evolution is a concerted response among many independent gene families. We suggest that the phylogenetic diversity of prey measures functionally relevant divergence in the targets of venom, a claim supported by sequence diversity in the coagulation cascade targets of venom. Our results support the general concept that the diversity of species in an ecological community is more important than their overall number in determining evolutionary patterns in predator trait complexity.

Venom Gene Sequence Diversity and Expression Jointly Shape Diet Adaptation in Pitvipers.

Understanding the joint roles of protein sequence variation and differential expression during adaptive evolution is a fundamental, yet largely unrealized goal of evolutionary biology. Here, we use phylogenetic path analysis to analyze a comprehensive venom-gland transcriptome dataset spanning three genera of pitvipers to identify the functional genetic basis of a key adaptation (venom complexity) linked to diet breadth (DB). The analysis of gene-family-specific patterns reveals that, for genes encoding two of the most important venom proteins (snake venom metalloproteases and snake venom serine proteases), there are direct, positive relationships between sequence diversity (SD), expression diversity (ED), and increased DB. Further analysis of gene-family diversification for these proteins showed no constraint on how individual lineages achieved toxin gene SD in terms of the patterns of paralog diversification. In contrast, another major venom protein family (PLA2s) showed no relationship between venom molecular diversity and DB. Additional analyses suggest that other molecular mechanisms-such as higher absolute levels of expression-are responsible for diet adaptation involving these venom proteins. Broadly, our findings argue that functional diversity generated through sequence and expression variations jointly determine adaptation in the key components of pitviper venoms, which mediate complex molecular interactions between the snakes and their prey.

Varying Modes of Selection Among Toxin Families in the Venoms of the Giant Desert Hairy Scorpions (Hadrurus).

Venoms are primarily believed to evolve under strong diversifying selection resulting from persistent coevolution between predator and prey. Recent research has challenged this hypothesis, proposing that venoms from younger venomous lineages (e.g., snakes and cone snails) are governed predominantly by diversifying selection, while venoms from older venomous lineages (e.g., centipedes, scorpions, and spiders) are under stronger purifying selection. However, most research in older lineages has tested selection at more diverse phylogenetic scales. Although these tests are important for evaluating broad macroevolutionary trends underlying venom evolution, they are less equipped to detect species-level evolutionary trends, which likely have large impacts on venom variation seen at more diverse phylogenetic scales. To test for selection among closely related species from an older venomous lineage, we generated high-throughput venom-gland transcriptomes and venom proteomes for four populations of Giant Desert Hairy Scorpions (Hadrurus), including three Hadrurus arizonensis populations and one Hadrurus spadix population. We detected significant episodic and pervasive diversifying selection across a highly abundant toxin family that likely has a major role in venom function ([Formula: see text]KTxs), providing a contrast to the stronger purifying selection identified from other studies on scorpion venoms. Conversely, we detected weak episodic diversifying and/or stronger purifying selection in four toxin families (non-disulfide bridged peptides, phospholipase A2s, scorpine-like antimicrobial peptides, and serine proteases), most of which were less abundant and likely have ancillary functional roles. Finally, although we detected several major toxin families at disproportionate transcriptomic and/or proteomic abundances, we did not identify significant sex-based variation in Hadrurus venoms.

Sequence Divergence in Venom Genes Within and Between Montane Pitviper (Viperidae: Crotalinae: Cerrophidion) Species is Driven by Mutation-Drift Equilibrium.

Snake venom can vary both among and within species. While some groups of New World pitvipers-such as rattlesnakes-have been well studied, very little is known about the venom of montane pitvipers (Cerrophidion) found across the Mesoamerican highlands. Compared to most well-studied rattlesnakes, which are widely distributed, the isolated montane populations of Cerrophidion may facilitate unique evolutionary trajectories and venom differentiation. Here, we describe the venom gland transcriptomes for populations of C. petlalcalensis, C. tzotzilorum, and C. godmani from Mexico, and a single individual of C. sasai from Costa Rica. We explore gene expression variation in Cerrophidion and sequence evolution of toxins within C. godmani specifically. Cerrophidion venom gland transcriptomes are composed primarily of snake venom metalloproteinases, phospholipase A[Formula: see text]s (PLA[Formula: see text]s), and snake venom serine proteases. Cerrophidion petlalcalensis shows little intraspecific variation; however, C. godmani and C. tzotzilorum differ significantly between geographically isolated populations. Interestingly, intraspecific variation was mostly attributed to expression variation as we did not detect signals of selection within C. godmani toxins. Additionally, we found PLA[Formula: see text]-like myotoxins in all species except C. petlalcalensis, and crotoxin-like PLA[Formula: see text]s in the southern population of C. godmani. Our results demonstrate significant intraspecific venom variation within C. godmani and C. tzotzilorum. The toxins of C. godmani show little evidence of directional selection where variation in toxin sequence is consistent with evolution under a model of mutation-drift equilibrium. Cerrophidion godmani individuals from the southern population may exhibit neurotoxic venom activity given the presence of crotoxin-like PLA[Formula: see text]s; however, further research is required to confirm this hypothesis.

RESUMEN: El veneno de las serpientes puede variar entre y dentro de las especies. Mientras algunos grupos de viperidos del Nuevo Mundo­como las cascabeles­han sido bien estudiadas, muy poco se sabe acerca del veneno de las nauyacas de frío (Cerrophidion) que se encuentran en las zonas altas de Mesoamérica. Comparadas con las extensamente estudiadas cascabeles, que estan ampliamente distribuidas, las poblaciones de Cerrophidion, aisladas en montañas, pueden poseer trayectorias evolutivas y diferenciación en su veneno unicos. En el presente trabajo, describimos el transcriptoma de las glándulas de veneno de poblaciones de C. petlalcalensis, C. tzotzilorum, y C. godmani de México, y un individuo de C. sasai de Costa Rica. Exploramos la variación en la expresión de toxinas en Cerrophidion y la evolución en las secuencias geneticas en C. godmani específicamente. El transcriptoma de la glándula de veneno de Cerrophidion esta compuesto principalmente de Metaloproteinasas de Veneno de Serpiente, Fosfolipasas A[Formula: see text] (PLA[Formula: see text]s), y Serin Proteasas de Veneno de Serpiente. Cerrophidion petlalcalensis presenta poca variación intraespecífica; sin embargo, los transcriptomas de la glandula de veneno de C. godmani y C. tzotzilorum difieren significativamente entre poblaciones geográficamente aisladas. Curiosamente, la variación intraespecífica estuvo atribuida principalmente a la expresión de las toxinas ya que no encontramos señales de selección en las toxinas de C. godmani. Adicionalmente, encontramos miotoxinas similares a PLA[Formula: see text] en todas las especies excepto C. petlalcalensis, y PLA[Formula: see text]s similares a crotoxina en la población sureña de C. godmani. Nuestros resultados demuestran la presencia de variacion intraespecífica presente en el veneno de C. godmani y C. tzotzilorum. Las toxinas de Cerrophidion godmani muestran poca evidencia de selección direccional, y la variación en la secuencias de las toxinas es consistente con evolucion bajo un modelo de equilibrio de mutación-deriva. Algunos individuos de C. godmani de la población del sur potencialmente tienen un veneno neurotóxico dada la presencia de PLA[Formula: see text]s similares a la crotoxina, sin embargo, se necesita más evidencia para corroborar esta hipótesis.

ToxCodAn: a new toxin annotator and guide to venom gland transcriptomics.

MOTIVATION: Next-generation sequencing has become exceedingly common and has transformed our ability to explore nonmodel systems. In particular, transcriptomics has facilitated the study of venom and evolution of toxins in venomous lineages; however, many challenges remain. Primarily, annotation of toxins in the transcriptome is a laborious and time-consuming task. Current annotation software often fails to predict the correct coding sequence and overestimates the number of toxins present in the transcriptome. Here, we present ToxCodAn, a python script designed to perform precise annotation of snake venom gland transcriptomes. We test ToxCodAn with a set of previously curated transcriptomes and compare the results to other annotators. In addition, we provide a guide for venom gland transcriptomics to facilitate future research and use Bothrops alternatus as a case study for ToxCodAn and our guide. RESULTS: Our analysis reveals that ToxCodAn provides precise annotation of toxins present in the transcriptome of venom glands of snakes. Comparison with other annotators demonstrates that ToxCodAn has better performance with regard to run time ($>20x$ faster), coding sequence prediction ($>3x$ more accurate) and the number of toxins predicted (generating $>4x$ less false positives). In this sense, ToxCodAn is a valuable resource for toxin annotation. The ToxCodAn framework can be expanded in the future to work with other venomous lineages and detect novel toxins.

Evolutionary allometry and ecological correlates of fang length evolution in vipers.

Traits for prey acquisition form the phenotypic interface of predator-prey interactions. In venomous predators, morphological variation in venom delivery apparatus like fangs and stingers may be optimized for dispatching prey. Here, we determine how a single dimension of venom injection systems evolves in response to variation in the size, climatic conditions and dietary ecology of viperid snakes. We measured fang length in more than 1900 museum specimens representing 199 viper species (55% of recognized species). We find both phylogenetic signal and within-clade variation in relative fang length across vipers suggesting both general taxonomic trends and potential adaptive divergence in fang length. We recover positive evolutionary allometry and little static allometry in fang length. Proportionally longer fangs have evolved in larger species, which may facilitate venom injection in more voluminous prey. Finally, we leverage climatic and diet data to assess the global correlates of fang length. We find that models of fang length evolution are improved through the inclusion of both temperature and diet, particularly the extent to which diets are mammal-heavy diets. These findings demonstrate how adaptive variation can emerge among components of complex prey capture systems.

The Chemosensory Repertoire of the Eastern Diamondback Rattlesnake (Crotalus adamanteus) Reveals Complementary Genetics of Olfactory and Vomeronasal-Type Receptors.

Pitviper sensory perception incorporates diverse stimuli through the integration of trichromatic color vision, bifocal heat-sensing, and dual-system chemoperception. Chemoperception, or olfaction, is mediated by chemoreceptors in the olfactory bulb and the vomeronasal organ, but the true genomic complexity of the gene families and their relative contributions is unknown. A full genomic accounting of pitviper chemoperception directly complements our current understanding of their venoms by generating a more complete polyphenic representation of their predatory arsenal. To characterize the genetic repertoire of pitviper chemoperception, we analyzed a full-genome assembly for Crotalus adamanteus, the eastern diamondback rattlesnake. We identified hundreds of genes encoding both olfactory receptors (ORs; 362 full-length genes) and type-2 vomeronasal receptors (V2Rs; 430 full-length genes). Many chemoreceptor genes are organized into large tandem repeat arrays. Comparative analysis of V2R orthologs across squamates demonstrates how gene array expansion and contraction underlies the evolution of the chemoreceptor repertoire, which likely reflects shifts in life history traits. Chromosomal assignments of chemosensory genes identified sex chromosome specific chemoreceptor genes, providing gene candidates underlying observed sex-specific chemosensory-based behaviors. We detected widespread episodic evolution in the extracellular, ligand-binding domains of both ORs and V2Rs, suggesting the diversification of chemoreceptors is driven by transient periods of positive selection. We provide a robust genetic framework for studying pitviper chemosensory ecology and evolution.

Trait differentiation and modular toxin expression in palm-pitvipers.

BACKGROUND: Modularity is the tendency for systems to organize into semi-independent units and can be a key to the evolution and diversification of complex biological systems. Snake venoms are highly variable modular systems that exhibit extreme diversification even across very short time scales. One well-studied venom phenotype dichotomy is a trade-off between neurotoxicity versus hemotoxicity that occurs through the high expression of a heterodimeric neurotoxic phospholipase A2 (PLA2) or snake venom metalloproteinases (SVMPs). We tested whether the variation in these venom phenotypes could occur via variation in regulatory sub-modules through comparative venom gland transcriptomics of representative Black-Speckled Palm-Pitvipers (Bothriechis nigroviridis) and Talamancan Palm-Pitvipers (B. nubestris). RESULTS: We assembled 1517 coding sequences, including 43 toxins for B. nigroviridis and 1787 coding sequences including 42 toxins for B. nubestris. The venom gland transcriptomes were extremely divergent between these two species with one B. nigroviridis exhibiting a primarily neurotoxic pattern of expression, both B. nubestris expressing primarily hemorrhagic toxins, and a second B. nigroviridis exhibiting a mixed expression phenotype. Weighted gene coexpression analyses identified six submodules of transcript expression variation, one of which was highly associated with SVMPs and a second which contained both subunits of the neurotoxic PLA2 complex. The sub-module association of these toxins suggest common regulatory pathways underlie the variation in their expression and is consistent with known patterns of inheritance of similar haplotypes in other species. We also find evidence that module associated toxin families show fewer gene duplications and transcript losses between species, but module association did not appear to affect sequence diversification. CONCLUSION: Sub-modular regulation of expression likely contributes to the diversification of venom phenotypes within and among species and underscores the role of modularity in facilitating rapid evolution of complex traits.

Tipping the Scales: The Migration-Selection Balance Leans toward Selection in Snake Venoms.

The migration-selection interaction is the strongest determinant of whether a beneficial allele increases in frequency within a population. Results of empirical studies examining the role of gene flow in an adaptive context, however, have largely been equivocal, with examples of opposing outcomes being repeatedly documented (e.g., local adaptation with high levels of gene flow vs. gene swamping). We compared neutral genomic and venom expression divergence for three sympatric pit vipers with differing ecologies to determine if and how migration-selection disequilibria result in local adaptation. We specifically tested whether neutral differentiation predicted phenotypic differentiation within an isolation-by-distance framework. The decoupling of neutral and phenotypic differentiation would indicate selection led to adaptive divergence irrespective of migration, whereas a significant relationship between neutral and venom expression differentiation would provide evidence in favor of the constraining force of gene flow. Neutral differentiation and geographic distance predicted phenotypic differentiation only in the generalist species, indicating that selection was the predominant mechanism in the migration-selection balance underlying adaptive venom evolution in both specialists. Dispersal is thought to be a stronger influence on genetic differentiation than specialization, but our results suggest the opposite. Greater specialization may lead to greater diversification rates, and extreme spatial and temporal variation in selective pressures can favor generalist phenotypes evolving under strong stabilizing selection. Our results are consistent with these expectations, suggesting that the equivocal findings of studies examining the role of gene flow in an adaptive context may be explained by ecological specialization theory.

Drift, selection and adaptive variation in small populations of a threatened rattlesnake.

An important goal of conservation genetics is to determine if the viability of small populations is reduced by a loss of adaptive variation due to genetic drift. Here, we assessed the impact of drift and selection on direct measures of adaptive variation (toxin loci encoding venom proteins) in the eastern massasauga rattlesnake (Sistrurus catenatus), a threatened reptile that exists in small isolated populations. We estimated levels of individual polymorphism in 46 toxin loci and 1,467 control loci across 12 populations of this species, and compared the results with patterns of selection on the same loci following speciation of S. catenatus and its closest relative, the western massasauga (S. tergeminus). Multiple lines of evidence suggest that both drift and selection have had observable impacts on standing adaptive variation. In support of drift effects, we found little evidence for selection on toxin variation within populations and a significant positive relationship between current levels of adaptive variation and long- and short-term estimates of effective population size. However, we also observed levels of directional selection on toxin loci among populations that are broadly similar to patterns predicted from interspecific selection analyses that pre-date the effects of recent drift, and that functional variation in these loci persists despite small short-term effective sizes. This suggests that much of the adaptive variation present in populations may represent an example of "drift debt," a nonequilibrium state where present-day levels of variation overestimate the amount of functional genetic diversity present in future populations.

A Single Mutation Unlocks Cascading Exaptations in the Origin of a Potent Pitviper Neurotoxin.

Evolutionary innovations and complex phenotypes seemingly require an improbable amount of genetic change to evolve. Rattlesnakes display two dramatically different venom phenotypes. Type I venoms are hemorrhagic with low systemic toxicity and high expression of tissue-destroying snake venom metalloproteinases. Type II venoms are highly neurotoxic and lack snake venom metalloproteinase expression and associated hemorrhagic activity. This dichotomy hinges on Mojave toxin (MTx), a phospholipase A2 (PLA2) based ß-neurotoxin expressed in Type II venoms. MTx is comprised of a nontoxic acidic subunit that undergoes extensive proteolytic processing and allosterically regulates activity of a neurotoxic basic subunit. Evolution of the acidic subunit presents an evolutionary challenge because the need for high expression of a nontoxic venom component and the proteolytic machinery required for processing suggests genetic changes of seemingly little immediate benefit to fitness. We showed that MTx evolved through a cascading series of exaptations unlocked by a single nucleotide change. The evolution of one new cleavage site in the acidic subunit unmasked buried cleavage sites already present in ancestral PLA2s, enabling proteolytic processing. Snake venom serine proteases, already present in the venom to disrupt prey hemostasis, possess the requisite specificities for MTx acidic subunit proteolysis. The dimerization interface between MTx subunits evolved by exploiting a latent, but masked, hydrophobic interaction between ancestral PLA2s. The evolution of MTx through exaptation of existing functional and structural features suggests complex phenotypes that depend on evolutionary innovations can arise from minimal genetic change enabled by prior evolution.

Biophysical Spandrels form a Hot-Spot for Kosmotropic Mutations in Bacteriophage Thermal Adaptation.

Temperature plays a dominating role in protein structure and function, and life has evolved myriad strategies to adapt proteins to environmental thermal stress. Cellular systems can utilize kosmotropic osmolytes, the products of complex biochemical pathways, to act as chemical chaperones. These extrinsic molecules, e.g., trehalose, alter local water structure to modulate the strength of the hydrophobic effect and increase protein stability. In contrast, simpler genetic systems must rely on intrinsic mutation to affect protein stability. In naturally occurring microvirid bacteriophages of the subfamily Bullavirinae, capsid stability is randomly distributed across the phylogeny, suggesting it is not phylogenetically linked and could be altered through adaptive mutation. We hypothesized that these phages could utilize an adaptive mechanism that mimics the stabilizing effects of the kosmotrope trehalose through mutation. Kinetic stability of wild-type ID8, a relative of ΦX174, displays a saturable response to trehalose. Thermal adaptation mutations in ID8 improve capsid stability and reduce responsiveness to trehalose suggesting the mutations move stability closer to the kosmotropic saturation point, mimicking the kosmotropic effect of trehalose. These mutations localize to and modulate the hydrophobicity of a cavern formation at the interface of phage coat and spike proteins-an evolutionary spandrel. Across a series of genetically distinct phages, responsiveness to trehalose correlates positively with cavern hydrophobicity suggesting that the level of hydrophobicity of the cavern may provide a biophysical gating mechanism constraining or permitting adaptation in a lineage-specific manner. Our results demonstrate that a single mutation can exploit pre-existing, non-adaptive structural features to mimic the adaptive effects of complex biochemical pathways.

Intraspecific sequence and gene expression variation contribute little to venom diversity in sidewinder rattlesnakes ( Crotalus cerastes).

Traits can evolve rapidly through changes in gene expression or protein-coding sequences. However, these forms of genetic variation can be correlated and changes to one can influence the other. As a result, we might expect traits lacking differential expression to preferentially evolve through changes in protein sequences or morphological adaptation. Given the lack of differential expression across the distribution of sidewinder rattlesnakes ( Crotalus cerastes), we tested this hypothesis by comparing the coding regions of genes expressed in the venom gland transcriptomes and fang morphology. We calculated Tajima's D and FST across four populations comparing toxin and nontoxin loci. Overall, we found little evidence of directional selection or differentiation between populations, suggesting that changes to protein sequences do not underlie the evolution of sidewinder venom or that toxins are under extremely variant selection pressures. Although low-expression toxins do not have higher sequence divergence between populations, they do have more standing variation on which selection can act. Additionally, we found significant differences in fang length among populations. The lack of differential expression and sequence divergence suggests sidewinders-given their generalist diet, moderate gene flow and environmental variation-are under stabilizing selection which functions to maintain a generalist phenotype. Overall, we demonstrate the importance of examining the relationship between gene expression and protein-coding changes to understand the evolution of complex traits.

Mutation-Driven Parallel Evolution during Viral Adaptation.

Convergent evolution has been demonstrated across all levels of biological organization, from parallel nucleotide substitutions to convergent evolution of complex phenotypes, but whether instances of convergence are the result of selection repeatedly finding the same optimal solution to a recurring problem or are the product of mutational biases remains unsettled. We generated 20 replicate lineages allowed to fix a single mutation from each of four bacteriophage genotypes under identical selective regimes to test for parallel changes within and across genotypes at the levels of mutational effect distributions and gene, protein, amino acid, and nucleotide changes. All four genotypes shared a distribution of beneficial mutational effects best approximated by a distribution with a finite upper bound. Parallel adaptation was high at the protein, gene, amino acid, and nucleotide levels, both within and among phage genotypes, with the most common first-step mutation in each background fixing on an average in 7 of 20 replicates and half of the substitutions in two of the four genotypes occurring at shared sites. Remarkably, the mutation of largest beneficial effect that fixed for each genotype was never the most common, as would be expected if parallelism were driven by selection. In fact, the mutation of smallest benefit for each genotype fixed in a total of 7 of 80 lineages, equally as often as the mutation of largest benefit, leading us to conclude that adaptation was largely mutation-driven, such that mutational biases led to frequent parallel fixation of mutations of suboptimal effect.

Quantity, Not Quality: Rapid Adaptation in a Polygenic Trait Proceeded Exclusively through Expression Differentiation.

A trait's genomic architecture can affect the rate and mechanism of adaptation, and although many ecologically-important traits are polygenic, most studies connecting genotype, phenotype, and fitness in natural populations have focused on traits with relatively simple genetic bases. To understand the genetic basis of polygenic adaptation, we must integrate genomics, phenotypic data, ecology, and fitness effects for a genetically tractable, polygenic trait; snake venoms provide such a system for studying polygenic adaptation because of their genetic tractability and vital ecological role in feeding and defense. We used a venom transcriptome-proteome map, quantitative proteomics, genomics, and fitness assays in sympatric prey to construct a genotype-phenotype-fitness map for the venoms of an island-mainland pair of rattlesnake populations. Reciprocal fitness experiments demonstrated that each population was locally adapted to sympatric prey. We identified significant expression differentiation with little to no coding-sequence variation across populations, demonstrating that expression differentiation was exclusively the genetic basis of polygenic adaptation. Previous research on the genetics of adaptation, however, has largely been biased toward investigating protein-coding regions because of the complexity of gene regulation. Our results showed that biases at the molecular level can be in the opposite direction, highlighting the need for more systematic comparisons of different molecular mechanisms underlying rapid, adaptive evolution in polygenic traits.

Local prey community composition and genetic distance predict venom divergence among populations of the northern Pacific rattlesnake (Crotalus oreganus).

Identifying the environmental correlates of divergence in functional traits between populations can provide insights into the evolutionary mechanisms that generate local adaptation. Here, we assess patterns of population differentiation in expressed venom proteins in Northern Pacific rattlesnakes (Crotalus oreganus) from 13 locations across California. We evaluate the relative importance of major biotic (prey species community composition), abiotic (temperature, precipitation and elevation) and genetic factors (genetic distance based on RAD-seq loci) as correlates of population divergence in venom phenotypes. We found that over half of the variation in venom composition is associated with among-population differentiation for genetic and environmental variables and that this variation occurred along axes defining previously observed functional trade-offs between venom proteins that have neurotoxic, myotoxic and hemorrhagic effects. Surprisingly, genetic differentiation among populations was the best predictor of venom divergence, accounting for 46% of overall variation, whereas differences in prey community composition and abiotic factors explained smaller amounts of variation (23% and 19%, respectively). The association between genetic differentiation and venom composition could be due to an isolation-by-distance effect or, more likely, an isolation-by-environment effect where selection against recent migrants is strong, producing a correlation between neutral genetic differentiation and venom differentiation. Our findings suggest that even coarse estimates of prey community composition can be useful in understanding the selection pressures acting on patterns of venom protein expression. Additionally, our results suggest that factors other than adaptation to spatial variation in prey need to be considered when explaining population divergence in venom.

Payoffs, not tradeoffs, in the adaptation of a virus to ostensibly conflicting selective pressures.

The genetic architecture of many phenotypic traits is such that genes often contribute to multiple traits, and mutations in these genes can therefore affect multiple phenotypes. These pleiotropic interactions often manifest as tradeoffs between traits where improvement in one property entails a cost in another. The life cycles of many pathogens include periods of growth within a host punctuated with transmission events, such as passage through a digestive tract or a passive stage of exposure in the environment. Populations exposed to such fluctuating selective pressures are expected to acquire mutations showing tradeoffs between reproduction within and survival outside of a host. We selected for individual mutations under fluctuating selective pressures for a ssDNA microvirid bacteriophage by alternating selection for increased growth rate with selection on biophysical properties of the phage capsid in high-temperature or low-pH conditions. Surprisingly, none of the seven unique mutations identified showed a pleiotropic cost; they all improved both growth rate and pH or temperature stability, suggesting that single mutations even in a simple genetic system can simultaneously improve two distinct traits. Selection on growth rate alone revealed tradeoffs, but some mutations still benefited both traits. Tradeoffs were therefore prevalent when selection acted on a single trait, but payoffs resulted when multiple traits were selected for simultaneously. We employed a molecular-dynamics simulation method to determine the mechanisms underlying beneficial effects for three heat-shock mutations. All three mutations significantly enhanced the affinities of protein-protein interfacial bindings, thereby improving capsid stability. The ancestral residues at the mutation sites did not contribute to protein-protein interfacial binding, indicating that these sites acquired a new function. Computational models, such as those used here, may be used in future work not only as predictive tools for mutational effects on protein stability but, ultimately, for evolution.

Phenotypic integration in the feeding system of the eastern diamondback rattlesnake (Crotalus adamanteus).

Selection can vary geographically across environments and temporally over the lifetime of an individual. Unlike geographic contexts, where different selective regimes can act on different alleles, age-specific selection is constrained to act on the same genome by altering age-specific expression. Snake venoms are exceptional traits for studying ontogeny because toxin expression variation directly changes the phenotype; relative amounts of venom components determine, in part, venom efficacy. Phenotypic integration is the dependent relationship between different traits that collectively produce a complex phenotype and, in venomous snakes, may include traits as diverse as venom, head shape and fang length. We examined the feeding system of the eastern diamondback rattlesnake (Crotalus adamanteus) across environments and over the lifetime of individuals and used a genotype-phenotype map approach, protein expression data and morphological data to demonstrate that: (i) ontogenetic effects explained more of the variation in toxin expression variation than geographic effects, (ii) both juveniles and adults varied geographically, (iii) toxin expression variation was a result of directional selection and (iv) different venom phenotypes covaried with morphological traits also associated with feeding in temporal (ontogenetic) and geographic (functional) contexts. These data are the first to demonstrate, to our knowledge, phenotypic integration between multiple morphological characters and a biochemical phenotype across populations and age classes. We identified copy number variation as the mechanism driving the difference in the venom phenotype associated with these morphological differences, and the parallel mitochondrial, venom and morphological divergence between northern and southern clades suggests that each clade may warrant classification as a separate evolutionarily significant unit.