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.

Phylogenomic Discordance is Driven by Wide-Spread Introgression and Incomplete Lineage Sorting During Rapid Species Diversification Within Rattlesnakes (Viperidae: Crotalus and Sistrurus).

-Phylogenomics allows us to uncover the historical signal of evolutionary processes through time and estimate phylogenetic networks accounting for these signals. Insight from genome-wide data further allows us to pinpoint the contributions to phylogenetic signal from hybridization, introgression, and ancestral polymorphism across the genome. Here, we focus on how these processes have contributed to phylogenetic discordance among rattlesnakes (genera Crotalus and Sistrurus), a group for which there are numerous conflicting phylogenetic hypotheses based on a diverse array of molecular datasets and analytical methods. We address the instability of the rattlesnake phylogeny using genomic data generated from transcriptomes sampled from nearly all known species. These genomic data, analyzed with coalescent and network-based approaches, reveal numerous instances of rapid speciation where individual gene trees conflict with the species tree. Moreover, the evolutionary history of rattlesnakes is dominated by incomplete speciation and frequent hybridization, both of which have likely influenced past interpretations of phylogeny. We present a new framework in which the evolutionary relationships of this group can only be understood in light of genome-wide data and network-based analytical methods. Our data suggest that network radiations, like those seen within the rattlesnakes, can only be understood in a phylogenomic context, necessitating similar approaches in our attempts to understand evolutionary history in other rapidly radiating species.

Selection Across the Three-Dimensional Structure of Venom Proteins from North American Scolopendromorph Centipedes.

Gene duplication followed by nucleotide differentiation is one of the simplest mechanisms to develop new functions for genes. However, the evolutionary processes underlying the divergence of multigene families remain controversial. We used multigene families found within the diversity of toxic proteins in centipede venom to test two hypotheses related to venom evolution: the two-speed mode of venom evolution and the rapid accumulation of variation in exposed residues (RAVER) model. The two-speed mode of venom evolution proposes that different types of selection impact ancient and younger venomous lineages with negative selection being the predominant form in ancient lineages and positive selection being the dominant form in younger lineages. The RAVER hypothesis proposes that, instead of different types of selection acting on different ages of venomous lineages, the different types of selection will selectively contribute to amino acid variation based on whether the residue is exposed to the solvent where it can potentially interact directly with toxin targets. This hypothesis parallels the longstanding understanding of protein evolution that suggests that residues found within the structural or active regions of the protein will be under negative or purifying selection, and residues that do not form part of these areas will be more prone to positive selection. To test these two hypotheses, we compared the venom of 26 centipedes from the order Scolopendromorpha from six currently recognized species from across North America using both transcriptomics and proteomics. We first estimated their phylogenetic relationships and uncovered paraphyly among the genus Scolopendra and evidence for cryptic diversity among currently recognized species. Using our phylogeny, we then characterized the diverse venom components from across the identified clades using a combination of transcriptomics and proteomics. We conducted selection-based analyses in the context of predicted three-dimensional properties of the venom proteins and found support for both hypotheses. Consistent with the two-speed hypothesis, we found a prevalence of negative selection across all proteins. Consistent with the RAVER hypothesis, we found evidence of positive selection on solvent-exposed residues, with structural and less-exposed residues showing stronger signal for negative selection. Through the use of phylogenetics, transcriptomics, proteomics, and selection-based analyses, we were able to describe the evolution of venom from an ancient venomous lineage and support principles of protein evolution that directly relate to multigene family evolution.

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.

Emergence and influence of sequence bias in evolutionarily malleable, mammalian tandem arrays.

BACKGROUND: The radiation of mammals at the extinction of the dinosaurs produced a plethora of new forms-as diverse as bats, dolphins, and elephants-in only 10-20 million years. Behind the scenes, adaptation to new niches is accompanied by extensive innovation in large families of genes that allow animals to contact the environment, including chemosensors, xenobiotic enzymes, and immune and barrier proteins. Genes in these "outward-looking" families are allelically diverse among humans and exhibit tissue-specific and sometimes stochastic expression. RESULTS: Here, we show that these tandem arrays of outward-looking genes occupy AT-biased isochores and comprise the "tissue-specific" gene class that lack CpG islands in their promoters. Models of mammalian genome evolution have not incorporated the sharply different functions and transcriptional patterns of genes in AT- versus GC-biased regions. To examine the relationship between gene family expansion, sequence content, and allelic diversity, we use population genetic data and comparative analysis. First, we find that AT bias can emerge during evolutionary expansion of gene families in cis. Second, human genes in AT-biased isochores or with GC-poor promoters experience relatively low rates of de novo point mutation today but are enriched for non-synonymous variants. Finally, we find that isochores containing gene clusters exhibit low rates of recombination. CONCLUSIONS: Our analyses suggest that tolerance of non-synonymous variation and low recombination are two forces that have produced the depletion of GC bases in outward-facing gene arrays. In turn, high AT content exerts a profound effect on their chromatin organization and transcriptional regulation.

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.

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.

Genetic characterization of potential venom resistance proteins in California ground squirrels (Otospermophilus beecheyi) using transcriptome analyses.

Understanding the molecular basis of adaptations in coevolving species requires identifying the genes that underlie reciprocally selected phenotypes, such as those involved in venom in snakes and resistance to the venom in their prey. In this regard, California ground squirrels (CGS; Otospermophilus beecheyi) are eaten by northern Pacific rattlesnakes (Crotalus oreganus oreganus), but individual squirrels may still show substantial resistance to venom and survive bites. A recent study using proteomics identified venom interactive proteins (VIPs) in the blood serum of CGS. These VIPs represent possible resistance proteins, but the sequences of genes encoding them are unknown despite the value of such data to molecular studies of coevolution. To address this issue, we analyzed a de novo assembled transcriptome from CGS liver tissue-where many plasma proteins are synthesized-and other tissues from this species. We then examined VIP sequences in terms of three characteristics that identify them as possible resistance proteins: evidence for positive selection, high liver expression, and nonsynonymous variation across CGS populations. Based on these characteristics, we identified five VIPs (i.e., α-2-macroglobulin, α-1-antitrypsin-like protein GS55-LT, apolipoprotein A-II, hibernation-associated plasma protein HP-20, and hibernation-associated plasma protein HP-27) as the most likely candidates for resistance proteins among VIPs identified to date. Four of these proteins have been previously implicated in conferring resistance to the venom in mammals, validating our approach. When combined with the detailed information available for rattlesnake venom proteins, these results set the stage for future work focused on understanding coevolutionary interactions at the molecular level between these species.

A genome assembly for the southern Pacific rattlesnake, Crotalus oreganus helleri, in the western rattlesnake species complex.

Rattlesnakes play important roles in their ecosystems by regulating prey populations, are involved in complex coevolutionary dynamics with their prey, and exhibit a variety of unusual adaptations, including maternal care, heat-sensing pit organs, hinged fangs, and medically-significant venoms. The western rattlesnake (Crotalus oreganus) is one of the widest ranging rattlesnake species, with a distribution from British Columbia, where it is listed as threatened, to Baja California and east across the Great Basin to western Wyoming, Colorado and New Mexico. Here, we report a new reference genome assembly for one of six currently recognized subspecies, C. oreganus helleri, as part of the California Conservation Genomics Project (CCGP). Consistent with the reference genomic sequencing strategy of the CCGP, we used Pacific Biosciences HiFi long reads and Hi-C chromatin-proximity sequencing technology to produce a de novo assembled genome. The assembly comprises a total of 698 scaffolds spanning 1,564,812,557 base pairs, has a contig N50 of 64.7 Mb, a scaffold N50 of 110.8 Mb, and BUSCO complete score of 90.5%. This reference genome will be valuable for studies on the genomic basis of venom evolution and variation within Crotalus, in resolving the taxonomy of C. oreganus and its relatives, and for the conservation and management of rattlesnakes in general.

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.

Adaptive divergence despite low effective population size in a peripherally isolated population of the pygmy rabbit, Brachylagus idahoensis.

Local adaptation can occur when spatially separated populations are subjected to contrasting environmental conditions. Historically, understanding the genetic basis of adaptation has been difficult, but increased availability of genome-wide markers facilitates studies of local adaptation in non-model organisms of conservation concern. The pygmy rabbit (Brachylagus idahoensis) is an imperiled lagomorph that relies on sagebrush for forage and cover. This reliance has led to widespread population declines following reductions in the distribution of sagebrush, leading to geographic separation between populations. In this study, we used >20,000 single nucleotide polymorphisms, genotype-environment association methods, and demographic modeling to examine neutral genetic variation and local adaptation in the pygmy rabbit in Nevada and California. We identified 308 loci as outliers, many of which had functional annotations related to metabolism of plant secondary compounds. Likewise, patterns of spatial variation in outlier loci were correlated with landscape and climatic variables including proximity to streams, sagebrush cover, and precipitation. We found that populations in the Mono Basin of California probably diverged from other Great Basin populations during late Pleistocene climate oscillations, and that this region is adaptively differentiated from other regions in the southern Great Basin despite limited gene flow and low effective population size. Our results demonstrate that peripherally isolated populations can maintain adaptive divergence.

Phenotypic and functional variation in venom and venom resistance of two sympatric rattlesnakes and their prey.

Predator-prey interactions often lead to the coevolution of adaptations associated with avoiding predation and, for predators, overcoming those defences. Antagonistic coevolutionary relationships are often not simple interactions between a single predator and prey but rather a complex web of interactions between multiple coexisting species. Coevolution between venomous rattlesnakes and small mammals has led to physiological venom resistance in several mammalian taxa. In general, viperid venoms contain large quantities of snake venom metalloproteinase toxins (SVMPs), which are inactivated by SVMP inhibitors expressed in resistant mammals. We explored variation in venom chemistry, SVMP expression, and SVMP resistance across four co-distributed species (California Ground Squirrels, Bryant's Woodrats, Southern Pacific Rattlesnakes, and Red Diamond Rattlesnakes) collected from four different populations in Southern California. Our aim was to understand phenotypic and functional variation in venom and venom resistance in order to compare coevolutionary dynamics of a system involving two sympatric predator-prey pairs to past studies that have focused on single pairs. Proteomic analysis of venoms indicated that these rattlesnakes express different phenotypes when in sympatry, with Red Diamonds expressing more typical viperid venom (with a diversity of SVMPs) and Southern Pacifics expressing a more atypical venom with a broader range of non-enzymatic toxins. We also found that although blood sera from both mammals were generally able to inhibit SVMPs from both rattlesnake species, inhibition depended strongly on the snake population, with snakes from one geographic site expressing SVMPs to which few mammals were resistant. Additionally, we found that Red Diamond venom, rather than woodrat resistance, was locally adapted. Our findings highlight the complexity of coevolutionary relationships between multiple predators and prey that exhibit similar offensive and defensive strategies in sympatry.

The molecular basis of venom resistance in a rattlesnake-squirrel predator-prey system.

Understanding how interspecific interactions mould the molecular basis of adaptations in coevolving species is a long-sought goal of evolutionary biology. Venom in predators and venom resistance proteins in prey are coevolving molecular phenotypes, and while venoms are highly complex mixtures it is unclear if prey respond with equally complex resistance traits. Here, we use a novel molecular methodology based on protein affinity columns to capture and identify candidate blood serum resistance proteins ("venom interactive proteins" [VIPs]) in California Ground Squirrels (Otospermophilus beecheyi) that interact with venom proteins from their main predator, Northern Pacific Rattlesnakes (Crotalus o. oreganus). This assay showed that serum-based resistance is both population- and species-specific, with serum proteins from ground squirrels showing higher binding affinities for venom proteins of local snakes compared to allopatric individuals. Venom protein specificity assays identified numerous and diverse candidate prey resistance VIPs but also potential targets of venom in prey tissues. Many specific VIPs bind to multiple snake venom proteins and, conversely, single venom proteins bind multiple VIPs, demonstrating that a portion of the squirrel blood serum "resistome" involves broad-based inhibition of nonself proteins and suggests that resistance involves a toxin scavenging mechanism. Analyses of rates of evolution of VIP protein homologues in related mammals show that most of these proteins evolve under purifying selection possibly due to molecular constraints that limit the evolutionary responses of prey to rapidly evolving snake venom proteins. Our method represents a general approach to identify specific proteins involved in co-evolutionary interactions between species at the molecular level.

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.

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.

Coevolution of venom function and venom resistance in a rattlesnake predator and its squirrel prey.

Measuring local adaptation can provide insights into how coevolution occurs between predators and prey. Specifically, theory predicts that local adaptation in functionally matched traits of predators and prey will not be detected when coevolution is governed by escalating arms races, whereas it will be present when coevolution occurs through an alternate mechanism of phenotype matching. Here, we analyse local adaptation in venom activity and prey resistance across 12 populations of Northern Pacific rattlesnakes and California ground squirrels, an interaction that has often been described as an arms race. Assays of venom function and squirrel resistance show substantial geographical variation (influenced by site elevation) in both venom metalloproteinase activity and resistance factor effectiveness. We demonstrate local adaptation in the effectiveness of rattlesnake venom to overcoming present squirrel resistance, suggesting that phenotype matching plays a role in the coevolution of these molecular traits. Further, the predator was the locally adapted antagonist in this interaction, arguing that rattlesnakes are evolutionarily ahead of their squirrel prey. Phenotype matching needs to be considered as an important mechanism influencing coevolution between venomous animals and resistant prey.

Roads are associated with a blunted stress response in a North American pit viper.

Whereas numerous studies have examined roads as anthropogenic stressors in birds and mammals, comparatively few studies have been undertaken on reptiles. We investigated plasma corticosterone (CORT) levels at baseline and following 30min of restraint stress in free-ranging copperhead snakes (Agkistrodon contortrix) captured within the forest interior or while in contact with public roads. There was no difference in baseline CORT levels between snakes in the forest and on roads. Copperheads responded to restraint stress by increasing plasma levels of CORT; however snakes on roads exhibited a lower CORT stress response compared to forest snakes. Additionally, among snakes captured on roads there was a negative association between road traffic and baseline CORT, stressed CORT, and the magnitude of the CORT response. Our results suggest that roads are associated with a blunted stress response in copperheads. Reduced stress responses may be indicative of acclimation, the inhibited ability to mount a stress response in the face of prolonged chronic stress, or that road environments select for individuals with lower CORT responsiveness. Either scenario could result in increased road mortality if snakes do not perceive roads as a potential threat.

High-throughput amino acid-level characterization of the interactions of plasminogen activator inhibitor-1 with variably divergent proteases.

While members of large paralogous protein families share structural features, their functional niches often diverge significantly. Serine protease inhibitors (SERPINs), whose members typically function as covalent inhibitors of serine proteases, are one such family. Plasminogen activator inhibitor-1 (PAI-1) is a prototypic SERPIN, which canonically inhibits tissue-and urokinase-type plasminogen activators (tPA and uPA) to regulate fibrinolysis. PAI-1 has been shown to also inhibit other serine proteases, including coagulation factor XIIa (FXIIa) and transmembrane serine protease 2 (TMPRSS2). The structural determinants of PAI-1 inhibitory function toward these non-canonical protease targets, and the biological significance of these functions, are unknown. We applied deep mutational scanning (DMS) to assess the effects of ∼80% of all possible single amino acid substitutions in PAI-1 on its ability to inhibit three putative serine protease targets (uPA, FXIIa, and TMPRSS2). Selection with each target protease generated a unique PAI-1 mutational landscape, with the determinants of protease specificity distributed throughout PAI-1's primary sequence. Next, we conducted a comparative analysis of extant orthologous sequences, demonstrating that key residues modulating PAI-1 inhibition of uPA and FXIIa, but not TMPRSS2, are maintained by purifying selection. PAI-1's activity toward FXIIa may reflect how protease evolutionary relationships predict SERPIN functional divergence, which we support via a cophylogenetic analysis of all secreted SERPINs and their cognate serine proteases. This work provides insight into the functional diversification of SERPINs and lays the framework for extending these studies to other proteases and their regulators.

Experimentally altered navigational demands induce changes in the cortical forebrain of free-ranging northern pacific rattlesnakes (Crotalus o. oreganus).

The hippocampus of birds and mammals plays a crucial role in spatial memory and navigation. The hippocampus exhibits plasticity in adulthood in response to diverse environmental factors associated with spatial demands placed on an animal. The medial and dorsal cortices of the telencephalon of squamate reptiles have been implicated as functional homologues to the hippocampus. This study sought to experimentally manipulate the navigational demands placed on free-ranging northern Pacific rattlesnakes (Crotalus o. oreganus) to provide direct evidence of the relationship between spatial demands and neuroplasticity in the cortical telencephalon of the squamate brain. Adult male rattlesnakes were radio-tracked for 2 months, during which time 1 of 3 treatments was imposed weekly, namely 225-meter translocation in a random direction, 225-meter walk and release at that day's capture site (handling control) or undisturbed (control). Snakes were then sacrificed and the brains were removed and processed for histological analysis of cortical features. The activity range was larger in the translocated (Tr) group compared to the handled (Hd) and undisturbed control (Cn) groups when measured via 95% minimum convex polygon (MCP). At the 100% MCP level, Tr snakes had larger activity ranges than the Cn snakes only. The volume of the medial cortex (MC) was larger in the Tr group compared to the Cn group. The MC of Hd snakes was not significantly different from that of either of the other groups. No differences in dorsal cortex (DC) or lateral cortex volumes were detected among the groups. Numbers of 5-bromo-2'-deoxyuridine (BrdU)-labeled cells in the MC and DC 3 weeks after BrdU injection were not affected by treatment. This study establishes a causal relationship between navigational demands and greater MC volume in a free-ranging reptile.

Trio-binned genomes of the woodrats Neotoma bryanti and Neotoma lepida reveal novel gene islands and rapid copy number evolution of xenobiotic metabolizing genes.

The genomic architecture underlying the origins and maintenance of biodiversity is an increasingly accessible feature of species, due in large part to third-generation sequencing and novel analytical toolsets. Applying these techniques to woodrats (Neotoma spp.) provides a unique opportunity to study how herbivores respond to environmental change. Neotoma bryanti and N. lepida independently achieved a major dietary feat in the aftermath of a natural climate change event: switching to the novel, toxic food source creosote bush (Larrea tridentata). To better understand the genetic mechanisms underlying this ability, we employed a trio binning sequencing approach with a N. bryanti × N. lepida F1 hybrid, allowing the simultaneous assembly of genomes representing each parental species. The resulting phased, chromosome-level, highly complete haploid references enabled us to explore the genomic architecture of several gene families-cytochromes P450, UDP-glucuronosyltransferases (UGTs), and ATP-binding cassette (ABC) transporters-known to play key roles in the metabolism of naturally occurring toxic dietary compounds. In addition to duplication events in the ABCG and UGT2B subfamilies, we found expansions in three P450 gene families (2A, 2B, 3A), including the evolution of multiple novel gene islands within the 2B and 3A subfamilies, which may have provided the crucial substrate for dietary adaptation. Our assemblies demonstrate that trio binning from an F1 hybrid rodent effectively recovers parental genomes from species that diverged more than a million years ago.