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. N ATURAL selection can be a powerful force driving rapid diversification within species and is predicted to lead to local adaptation through the increase in frequency of mutations in gene-regulatory or protein-coding regions (Stern 2000;Hoekstra and Coyne 2007;Carroll 2008;Muller 2007). Expression variation at single loci has produced adaptive phenotypic divergence in the beaks of Darwin's finches (Abzhanov et al. 2004), coat color in mice (Manceau et al. 2011), and mimicry in butterflies (Reed et al. 2011). Most traits, however, are products of poorly characterized developmental pathways involving many loci. Many of the fundamental features of evolving systems, such as evolvability, epistasis, pleiotropy, and basic variational properties (Rokyta et al. 2008(Rokyta et al. , 2009(Rokyta et al. , 2011bWager 2008;Chou et al. 2011;Woods et al. 2011;Hill and Zhang 2012), result from the relationship between genotype and phenotype (Stadler et al. 2001;Hansen 2006), but the ability to study this relationship directly in polygenic traits is rare. Therefore, linking gene-regulatory changes to adaptive evolution in polygenic, complex phenotypes remains a challenge (Romero et al. 2012;Savolainen et al. 2013).Snake venoms are complex cocktails of 40-100 proteinaceous toxins (Boldrini-França et ...

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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.

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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.

BackgroundLargely 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.ResultsWe 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.ConclusionsDespite 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.

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 lowexpression proteins, enabling low-expression proteins to evolve and potentially lead to more rapid adaptation.KEYWORDS protein expression; selective constraints; evolutionary rates; adaptation T HE expression level of a protein is one of the strongest predictors of protein sequence evolutionary rate; sequences of highly expressed proteins evolve more slowly than low-expression proteins (Duret and Mouchiroud 1999;Pal et al. 2001;Gout et al. 2010;Yang et al. 2012;Nabholz et al. 2013;Park et al. 2013). This relationship may be a function of specific selective constraints on sequences to avoid protein misfolding (Drummond et al. 2005;Geiler-Samerotte et al. 2011), protein misinteractions (Yang et al. 2012, a decrease in protein function (Cherry 2010;Gout et al. 2010), and/or messenger RNA (mRNA) misfolding (Park et al. 2013). Analyses of microarray data have shown that expression evolutionary rate is also negatively...

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.