Varying Intensities of Introgression Obscure Incipient Venom-Associated Speciation in the Timber Rattlesnake (Crotalus horridus).

Ecologically divergent selection can lead to the evolution of reproductive isolation through the process of ecological speciation, but the balance of responsible evolutionary forces is often obscured by an inadequate assessment of demographic history and the genetics of traits under selection. Snake venoms have emerged as a system for studying the genetic basis of adaptation because of their genetic tractability and contributions to fitness, and speciation in venomous snakes can be associated with ecological diversification such as dietary shifts and corresponding venom changes. Here, we explored the neurotoxic (type A)-hemotoxic (type B) venom dichotomy and the potential for ecological speciation among Timber Rattlesnake (Crotalus horridus) populations. Previous work identified the genetic basis of this phenotypic difference, enabling us to characterize the roles geography, history, ecology, selection, and chance play in determining when and why new species emerge or are absorbed. We identified significant genetic, proteomic, morphological, and ecological/environmental differences at smaller spatial scales, suggestive of incipient ecological speciation between type A and type B C. horridus. Range-wide analyses, however, rejected the reciprocal monophyly of venom type, indicative of varying intensities of introgression and a lack of reproductive isolation across the range. Given that we have now established the phenotypic distributions and ecological niche models of type A and B populations, genome-wide data are needed and capable of determining whether type A and type B C. horridus represent distinct, reproductively isolated lineages due to incipient ecological speciation or differentiated populations within a single species.

Gradual and Discrete Ontogenetic Shifts in Rattlesnake Venom Composition and Assessment of Hormonal and Ecological Correlates.

Ontogenetic shifts in venom occur in many snakes but establishing their nature as gradual or discrete processes required additional study. We profiled shifts in venom expression from the neonate to adult sizes of two rattlesnake species, the eastern diamondback and the timber rattlesnake. We used serial sampling and venom chromatographic profiling to test if ontogenetic change occurs gradually or discretely. We found evidence for gradual shifts in overall venom composition in six of eight snakes, which sometimes spanned more than two years. Most chromatographic peaks shift gradually, but one quarter shift in a discrete fashion. Analysis of published diet data showed gradual shifts in overall diet composition across the range of body sizes attained by our eight study animals, while the shifts in abundance of different prey classes varied in form from gradual to discrete. Testosterone concentrations were correlated with the change in venom protein composition, but the relationship is not strong enough to suggest causation. Venom research employing simple juvenile versus adult size thresholds may be failing to account for continuous variation in venom composition lifespan. Our results imply that venom shifts represent adaptive matches to dietary shifts and highlight venom for studies of alternative gene regulatory mechanisms.

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.

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.

Expression Differentiation Is Constrained to Low-Expression Proteins over Ecological Timescales.

Protein expression level is one of the strongest predictors of protein sequence evolutionary rate, with high-expression protein sequences evolving at slower rates than low-expression protein sequences largely because of constraints on protein folding and function. Expression evolutionary rates also have been shown to be negatively correlated with expression level across human and mouse orthologs over relatively long divergence times (i.e., ∼100 million years). Long-term evolutionary patterns, however, often cannot be extrapolated to microevolutionary processes (and vice versa), and whether this relationship holds for traits evolving under directional selection within a single species over ecological timescales (i.e., <5000 years) is unknown and not necessarily expected. Expression is a metabolically costly process, and the expression level of a particular protein is predicted to be a tradeoff between the benefit of its function and the costs of its expression. Selection should drive the expression level of all proteins close to values that maximize fitness, particularly for high-expression proteins because of the increased energetic cost of production. Therefore, stabilizing selection may reduce the amount of standing expression variation for high-expression proteins, and in combination with physiological constraints that may place an upper bound on the range of beneficial expression variation, these constraints could severely limit the availability of beneficial expression variants. To determine whether rapid-expression evolution was restricted to low-expression proteins owing to these constraints on highly expressed proteins over ecological timescales, we compared venom protein expression levels across mainland and island populations for three species of pit vipers. We detected significant differentiation in protein expression levels in two of the three species and found that rapid-expression differentiation was restricted to low-expression proteins. Our results suggest that various constraints on high-expression proteins reduce the availability of beneficial expression variants relative to low-expression proteins, enabling low-expression proteins to evolve and potentially lead to more rapid adaptation.

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.

The transcriptomic and proteomic basis for the evolution of a novel venom phenotype within the Timber Rattlesnake (Crotalus horridus).

The genetics underlying adaptive trait evolution describes the intersection between the probability that particular types of mutation are beneficial and the rates they arise. Snake venoms can vary in a directly meaningful manner through coding mutations and regulatory mutations. The amounts of different components determine venom efficacy, but point mutations in coding sequences can also change efficacy and function. The Timber Rattlesnake (Crotalus horridus) has populations that have evolved neurotoxic venom from the typical hemorrhagic rattlesnake venom present throughout most of its range. We identified only a handful of nonsynonymous differences in just five loci between animals with each venom type, and these differences affected lower-abundance toxins. Expression of at least 18 loci encoding hemorrhagic toxins was severely reduced in the production of neurotoxic venom. The entire phospholipase A2 toxin family was completely replaced in the neurotoxic venom, possibly through intergeneric hybridization. Venom paedomorphosis could, at best, explain only some of the loss of expression of hemorrhagic toxins. The number of potential mechanisms for altering venom composition and the patterns observed for C. horridus suggest that rapid venom evolution should occur primarily through changes in venom composition, rather than point mutations affecting coding sequences.

Early significant ontogenetic changes in snake venoms.

Snake venom plays a critical role in food acquisition, digestion, and defense. Venoms are known to change throughout the life of some snake species, but nothing is known about the venom composition of hatchling/neonate snakes prior to and just after their first shedding cycle, despite this being a critical time in the life of the snake. Using a cohort of Crotalus horridus and two cohorts of Crotalus adamanteus, we showed for the first time that snakes undergo significant changes in venom composition after the postnatal shedding event. The number of changes among cohorts ranged widely and there was wide variation in the direction of protein regulation, which appeared to be on a locus-specific level rather than protein-family level. These significant venom composition changes that take place in the first few weeks of life most likely play critical roles in venom economy and resource conservation and may partially explain the rare, post-birth maternal care found in some venomous species.

Contrasting modes and tempos of venom expression evolution in two snake species.

Selection is predicted to drive diversification within species and lead to local adaptation, but understanding the mechanistic details underlying this process and thus the genetic basis of adaptive evolution requires the mapping of genotype to phenotype. Venom is complex and involves many genes, but the specialization of the venom gland toward toxin production allows specific transcripts to be correlated with specific toxic proteins, establishing a direct link from genotype to phenotype. To determine the extent of expression variation and identify the processes driving patterns of phenotypic diversity, we constructed genotype-phenotype maps and compared range-wide toxin-protein expression variation for two species of snake with nearly identical ranges: the eastern diamondback rattlesnake (Crotalus adamanteus) and the eastern coral snake (Micrurus fulvius). We detected significant expression variation in C. adamanteus, identified the specific loci associated with population differentiation, and found that loci expressed at all levels contributed to this divergence. Contrary to expectations, we found no expression variation in M. fulvius, suggesting that M. fulvius populations are not locally adapted. Our results not only linked expression variation at specific loci to divergence in a polygenic, complex trait but also have extensive conservation and biomedical implications. C. adamanteus is currently a candidate for federal listing under the Endangered Species Act, and the loss of any major population would result in the irrevocable loss of a unique venom phenotype. The lack of variation in M. fulvius has significant biomedical application because our data will assist in the development of effective antivenom for this species.

RNA-seq and high-definition mass spectrometry reveal the complex and divergent venoms of two rear-fanged colubrid snakes.

BACKGROUND: Largely because of their direct, negative impacts on human health, the venoms of front-fanged snakes of the families Viperidae and Elapidae have been extensively characterized proteomically, transcriptomically, and pharmacologically. However, relatively little is known about the molecular complexity and evolution of the venoms of rear-fanged colubrid snakes, which are, with a few notable exceptions, regarded as harmless to humans. Many of these snakes have venoms with major effects on their preferred prey, and their venoms are probably as critical to their survival as those of front-fanged elapids and viperids. RESULTS: We sequenced the venom-gland transcriptomes from a specimen of Hypsiglena (Desert Night Snake; family Colubridae, subfamily Dipsadinae) and of Boiga irregularis (Brown Treesnake; family Colubridae, subfamily Colubrinae) and verified the transcriptomic results proteomically by means of high-definition mass spectrometry. We identified nearly 3,000 nontoxin genes for each species. For B. irregularis, we found 108 putative toxin transcripts in 46 clusters with <1% nucleotide divergence, and for Hypsiglena we identified 79 toxin sequences that were grouped into 33 clusters. Comparisons of the venoms revealed divergent venom types, with Hypsiglena possessing a viper-like venom dominated by metalloproteinases, and B. irregularis having a more elapid-like venom, consisting primarily of three-finger toxins. CONCLUSIONS: Despite the difficulty of procuring venom from rear-fanged species, we were able to complete all analyses from a single specimen of each species without pooling venom samples or glands, demonstrating the power of high-definition transcriptomic and proteomic approaches. We found a high level of divergence in the venom types of two colubrids. These two venoms reflected the hemorrhagic/neurotoxic venom dichotomy that broadly characterizes the difference in venom strategies between elapids and viperids.

Linking the transcriptome and proteome to characterize the venom of the eastern diamondback rattlesnake (Crotalus adamanteus).

Understanding the molecular basis of the phenotype is key to understanding adaptation, and the relationship between genes and specific traits is represented by the genotype-phenotype map. The specialization of the venom-gland towards toxin production enables the use of transcriptomics to identify a large number of loci that contribute to a complex phenotype (i.e., venom), while proteomic techniques allow verification of the secretion of the proteins produced by these loci, creating a genotype-phenotype map. We used the extensive database of mRNA transcripts generated by the venom-gland transcriptome of Crotalus adamanteus along with proteomic techniques to complete the genotype-phenotype map for the C. adamanteus venom system. Nanospray LC/MS(E) analysis of a whole venom sample identified evidence for 52 of the 78 unique putative toxin transcript clusters, including 44 of the 50 most highly expressed transcripts. Tandem mass spectrometry and SDS-PAGE of reversed-phase high-performance liquid chromatography fractions identified 40 toxins which clustered into 20 groups and represented 10 toxin families, creating a genotype-phenotype map. By using the transcriptome to understand the proteome we were able to achieve locus-specific resolution and provide a detailed characterization of the C. adamanteus venom system. BIOLOGICAL SIGNIFICANCE: Identifying the mechanisms by which genetic variation presents itself to the sieve of selection at the phenotypic level is key to understanding the molecular basis of adaptation, and the first step in understanding this relationship is to identify the genetic basis of the phenotype through the construction of a genotype-phenotype map. We used the high-throughput venom-gland transcriptomic characterization of the eastern diamondback rattlesnake (C. adamanteus) and proteomic techniques to complete and confirm the genotype-phenotype map, providing a detailed characterization of the C. adamanteus venom system.

The genesis of an exceptionally lethal venom in the timber rattlesnake (Crotalus horridus) revealed through comparative venom-gland transcriptomics.

BACKGROUND: Snake venoms generally show sequence and quantitative variation within and between species, but some rattlesnakes have undergone exceptionally rapid, dramatic shifts in the composition, lethality, and pharmacological effects of their venoms. Such shifts have occurred within species, most notably in Mojave (Crotalus scutulatus), South American (C. durissus), and timber (C. horridus) rattlesnakes, resulting in some populations with extremely potent, neurotoxic venoms without the hemorrhagic effects typical of rattlesnake bites. RESULTS: To better understand the evolutionary changes that resulted in the potent venom of a population of C. horridus from northern Florida, we sequenced the venom-gland transcriptome of an animal from this population for comparison with the previously described transcriptome of the eastern diamondback rattlesnake (C. adamanteus), a congener with a more typical rattlesnake venom. Relative to the toxin transcription of C. adamanteus, which consisted primarily of snake-venom metalloproteinases, C-type lectins, snake-venom serine proteinases, and myotoxin-A, the toxin transcription of C. horridus was far simpler in composition and consisted almost entirely of snake-venom serine proteinases, phospholipases A2, and bradykinin-potentiating and C-type natriuretic peptides. Crotalus horridus lacked significant expression of the hemorrhagic snake-venom metalloproteinases and C-type lectins. Evolution of shared toxin families involved differential expansion and loss of toxin clades within each species and pronounced differences in the highly expressed toxin paralogs. Toxin genes showed significantly higher rates of nonsynonymous substitution than nontoxin genes. The expression patterns of nontoxin genes were conserved between species, despite the vast differences in toxin expression. CONCLUSIONS: Our results represent the first complete, sequence-based comparison between the venoms of closely related snake species and reveal in unprecedented detail the rapid evolution of snake venoms. We found that the difference in venom properties resulted from major changes in expression levels of toxin gene families, differential gene-family expansion and loss, changes in which paralogs within gene families were expressed at high levels, and higher nonsynonymous substitution rates in the toxin genes relative to nontoxins. These massive alterations in the genetics of the venom phenotype emphasize the evolutionary lability and flexibility of this ecologically critical trait.

Report from the first snake genomics and integrative biology meeting.

This report summarizes the proceedings of the 1st Snake Genomics and Integrative Biology Meeting held in Vail, CO USA, 5-8 October 2011. The meeting had over twenty registered participants, and was conducted as a single session of presentations. Goals of the meeting included coordination of genomic data collection and fostering collaborative interactions among researchers using snakes as model systems.

A high-throughput venom-gland transcriptome for the Eastern Diamondback Rattlesnake (Crotalus adamanteus) and evidence for pervasive positive selection across toxin classes.

Despite causing considerable human mortality and morbidity, animal toxins represent a valuable source of pharmacologically active macromolecules, a unique system for studying molecular adaptation, and a powerful framework for examining structure-function relationships in proteins. Snake venoms are particularly useful in the latter regard as they consist primarily of a moderate number of proteins and peptides that have been found to belong to just a handful of protein families. As these proteins and peptides are produced in dedicated glands, transcriptome sequencing has proven to be an effective approach to identifying the expressed toxin genes. We generated a venom-gland transcriptome for the Eastern Diamondback Rattlesnake (Crotalus adamanteus) using Roche 454 sequencing technology. In the current work, we focus on transcripts encoding toxins. We identified 40 unique toxin transcripts, 30 of which have full-length coding sequences, and 10 have only partial coding sequences. These toxins account for 24% of the total sequencing reads. We found toxins from 11 previously described families of snake-venom toxins and have discovered two putative, previously undescribed toxin classes. The most diverse and highly expressed toxin classes in the C. adamanteus venom-gland transcriptome are the serine proteinases, metalloproteinases, and C-type lectins. The serine proteinases are the most abundant class, accounting for 35% of the toxin sequencing reads. Metalloproteinases are the most diverse; 11 different forms have been identified. Using our sequences and those available in public databases, we detected positive selection in seven of the eight toxin families for which sufficient sequences were available for the analysis. We find that the vast majority of the genes that contribute directly to this vertebrate trait show evidence for a role for positive selection in their evolutionary history.