Diverse Gene Regulatory Mechanisms Alter Rattlesnake Venom Gene Expression at Fine Evolutionary Scales.

Understanding and predicting the relationships between genotype and phenotype is often challenging, largely due to the complex nature of eukaryotic gene regulation. A step towards this goal is to map how phenotypic diversity evolves through genomic changes that modify gene regulatory interactions. Using the Prairie Rattlesnake (Crotalus viridis) and related species, we integrate mRNA-seq, proteomic, ATAC-seq and whole-genome resequencing data to understand how specific evolutionary modifications to gene regulatory network components produce differences in venom gene expression. Through comparisons within and between species, we find a remarkably high degree of gene expression and regulatory network variation across even a shallow level of evolutionary divergence. We use these data to test hypotheses about the roles of specific trans-factors and cis-regulatory elements, how these roles may vary across venom genes and gene families, and how variation in regulatory systems drive diversity in venom phenotypes. Our results illustrate that differences in chromatin and genotype at regulatory elements play major roles in modulating expression. However, we also find that enhancer deletions, differences in transcription factor expression, and variation in activity of the insulator protein CTCF also likely impact venom phenotypes. Our findings provide insight into the diversity and gene-specificity of gene regulatory features and highlight the value of comparative studies to link gene regulatory network variation to phenotypic variation.

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.

Functional genomic diversity is correlated with neutral genomic diversity in populations of an endangered rattlesnake.

Theory predicts that genetic erosion in small, isolated populations of endangered species can be assessed using estimates of neutral genetic variation, yet this widely used approach has recently been questioned in the genomics era. Here, we leverage a chromosome-level genome assembly of an endangered rattlesnake (Sistrurus catenatus) combined with whole genome resequencing data (N = 110 individuals) to evaluate the relationship between levels of genome-wide neutral and functional diversity over historical and future timescales. As predicted, we found positive correlations between genome-wide estimates of neutral genetic diversity (π) and inferred levels of adaptive variation and an estimate of inbreeding mutation load, and a negative relationship between neutral diversity and an estimate of drift mutation load. However, these correlations were half as strong for projected future levels of neutral diversity based on contemporary effective population sizes. Broadly, our results confirm that estimates of neutral genetic diversity provide an accurate measure of genetic erosion in populations of a threatened vertebrate. They also provide nuance to the neutral-functional diversity controversy by suggesting that while these correlations exist, anthropogenetic impacts may have weakened these associations in the recent past and into the future.

The best of both worlds? Rattlesnake hybrid zones generate complex combinations of divergent venom phenotypes that retain high toxicity.

Studying the consequences of hybridization between closely related species with divergent traits can reveal patterns of evolution that shape and maintain extreme trophic adaptations. Snake venoms are an excellent model system for examining the evolutionary and ecological patterns that underlie highly selected polymorphic traits. Here we investigate hybrid venom phenotypes that result from natural introgression between two rattlesnake species that express highly divergent venom phenotypes: Crotalus o. concolor and C. v. viridis. Though not yet documented, interbreeding between these species may lead to novel venom phenotypes with unique activities that break the typical trends of venom composition in rattlesnakes. The characteristics of these unusual phenotypes could unveil the roles of introgression in maintaining patterns of venom composition and variation, including the near ubiquitous dichotomy between neurotoxic or degradative venoms observed across rattlesnakes. We use RADseq data to infer patterns of gene flow and hybrid ancestry between these diverged lineages and link these genetic data with analyses of venom composition, biological activity, and whole animal model toxicity tests to understand the impacts of introgression on venom composition. We find that introgressed populations express admixed venom phenotypes that do not sacrifice biological activity (lethal toxicity) or overall abundance of dominant toxins compared to parental venoms. These hybridized venoms therefore do not represent a trade-off in functionality between the typical phenotypic extremes but instead represent a unique combination of characters whose expression appears limited to the hybrid zone.

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.

Snakes on a plain: biotic and abiotic factors determine venom compositional variation in a wide-ranging generalist rattlesnake.

BACKGROUND: Snake venoms are trophic adaptations that represent an ideal model to examine the evolutionary factors that shape polymorphic traits under strong natural selection. Venom compositional variation is substantial within and among venomous snake species. However, the forces shaping this phenotypic complexity, as well as the potential integrated roles of biotic and abiotic factors, have received little attention. Here, we investigate geographic variation in venom composition in a wide-ranging rattlesnake (Crotalus viridis viridis) and contextualize this variation by investigating dietary, phylogenetic, and environmental variables that covary with venom. RESULTS: Using shotgun proteomics, venom biochemical profiling, and lethality assays, we identify 2 distinct divergent phenotypes that characterize major axes of venom variation in this species: a myotoxin-rich phenotype and a snake venom metalloprotease (SVMP)-rich phenotype. We find that dietary availability and temperature-related abiotic factors are correlated with geographic trends in venom composition. CONCLUSIONS: Our findings highlight the potential for snake venoms to vary extensively within species, for this variation to be driven by biotic and abiotic factors, and for the importance of integrating biotic and abiotic variation for understanding complex trait evolution. Links between venom variation and variation in biotic and abiotic factors indicate that venom variation likely results from substantial geographic variation in selection regimes that determine the efficacy of venom phenotypes across populations and snake species. Our results highlight the cascading influence of abiotic factors on biotic factors that ultimately shape venom phenotype, providing evidence for a central role of local selection as a key driver of venom variation.

Single-Cell Heterogeneity in Snake Venom Expression Is Hardwired by Co-Option of Regulators from Progressively Activated Pathways.

The ubiquitous cellular heterogeneity underlying many organism-level phenotypes raises questions about what factors drive this heterogeneity and how these complex heterogeneous systems evolve. Here, we use single-cell expression data from a Prairie rattlesnake (Crotalus viridis) venom gland to evaluate hypotheses for signaling networks underlying snake venom regulation and the degree to which different venom gene families have evolutionarily recruited distinct regulatory architectures. Our findings suggest that snake venom regulatory systems have evolutionarily co-opted trans-regulatory factors from extracellular signal-regulated kinase and unfolded protein response pathways that specifically coordinate expression of distinct venom toxins in a phased sequence across a single population of secretory cells. This pattern of co-option results in extensive cell-to-cell variation in venom gene expression, even between tandemly duplicated paralogs, suggesting this regulatory architecture has evolved to circumvent cellular constraints. While the exact nature of such constraints remains an open question, we propose that such regulatory heterogeneity may circumvent steric constraints on chromatin, cellular physiological constraints (e.g., endoplasmic reticulum stress or negative protein-protein interactions), or a combination of these. Regardless of the precise nature of these constraints, this example suggests that, in some cases, dynamic cellular constraints may impose previously unappreciated secondary constraints on the evolution of gene regulatory networks that favors heterogeneous expression.

A Promising Biomolecule Able to Degrade Neutrophil Extracellular Traps: CdcPDE, a Rattlesnake Phosphodiesterase.

Neutrophil extracellular traps (NETs) are an important mechanism for defense against pathogens. Their overproduction can be harmful since excessive NET formation promotes inflammation and tissue damage in several diseases. Nucleases are capable to degrade NET on basis of their DNA hydrolysis activity, including the CdcPDE, a nuclease isolated from Crotalus durissus collilineatus snake venom. Here, we report a new finding about CdcPDE activity, demonstrating its efficiency in degrading cell-free DNA from NETs, being a potential candidate to assist in therapies targeting inflammatory diseases.

Montane Rattlesnakes in México: Venoms of Crotalus tancitarensis and Related Species within the Crotalus intermedius Group.

The Crotalus intermedius group is a clade of rattlesnakes consisting of several species adapted to a high elevation habitat, primarily in México. Crotalus tancitarensis was previously classified as C. intermedius, until individuals occurring on Cerro Tancítaro in Michoacán, México, were reevaluated and classified as a new species (C. tancitarensis) based on scale pattern and geographic location. This study aimed to characterize the venom of C. tancitarensis and compare the venom profile to those of other species within the Crotalus intermedius group using gel electrophoresis, biochemical assays, reverse-phase high performance liquid chromatography, mass spectrometry, and lethal toxicity (LD50) assays. Results show that the venom profiles of species within the Crotalus intermedius group are similar, but with distinct differences in phospholipase A2 (PLA2), metalloproteinase PI (SVMP PI), and kallikrein-like serine proteinase (SVSP) activity and relative abundance. Proteomic analysis indicated that the highland forms produce venoms with 50-60 protein isoforms and a composition typical of type I rattlesnake venoms (abundant SVMPs, lack of presynaptic PLA2-based neurotoxins), as well as a diversity of typical Crotalus venom components such as serine proteinases, PLA2s, C-type lectins, and less abundant toxins (LAAOs, CRiSPs, etc.). The overall venom profile of C. tancitarensis appears most similar to C. transversus, which is consistent with a previous mitochondrial DNA analysis of the Crotalus intermedius group. These rattlesnakes of the Mexican highlands represent a radiation of high elevation specialists, and in spite of divergence of species in these Sky Island habitats, venom composition of species analyzed here has remained relatively conserved. The majority of protein family isoforms are conserved in all members of the clade, and as seen in other more broadly distributed rattlesnake species, differences in their venoms are largely due to relative concentrations of specific components.

Inferring population connectivity in eastern massasauga rattlesnakes (Sistrurus catenatus) using landscape genetics.

Assessing the environmental factors that influence the ability of a threatened species to move through a landscape can be used to identify conservation actions that connect isolated populations. However, direct observations of species' movement are often limited, making the development of alternate approaches necessary. Here we use landscape genetic analyses to assess the impact of landscape features on the movement of individuals between local populations of a threatened snake, the eastern massasauga rattlesnake (Sistrurus catenatus). We linked connectivity data with habitat information from two landscapes of similar size: a large region of unfragmented habitat and a previously studied fragmented landscape consisting of isolated patches of habitat. We used this analysis to identify features of the landscape where modification or acquisition would enhance population connectivity in the fragmented region. We found evidence that current connectivity was impacted by both contemporary land-cover features, especially roads, and inherent landscape features such as elevation. Next, we derived estimates of expected movement ability using a recently developed pedigree-based approach and least-cost paths through the unfragmented landscape. We then used our pedigree and resistance map to estimate resistance polygons of the potential extent for S. catenatus movement in the fragmented landscape. These polygons identify possible sites for future corridors connecting currently isolated populations in this landscape by linking the impact of future habitat modification or land acquisition to dispersal ability in this species. Overall, our study shows how modeling landscape resistance across differently fragmented landscapes can identify habitat features that affect contemporary movement in threatened species in fragmented landscapes and how this information can be used to guide mitigation actions whose goal is to connect isolated populations.

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

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

Evidence that genomic incompatibilities and other multilocus processes impact hybrid fitness in a rattlesnake hybrid zone.

Hybrid zones provide valuable opportunities to understand the genomic mechanisms that promote speciation by providing insight into factors involved in intermediate stages of speciation. Here, we investigate introgression in a hybrid zone between two rattlesnake species (Crotalus viridis and Crotalus oreganus concolor) that have undergone historical allopatric divergence and recent range expansion and secondary contact. We use Bayesian genomic cline models to characterize genomic patterns of introgression between these lineages and identify loci potentially subject to selection in hybrids. We find evidence for a large number of genomic regions with biased ancestry that deviate from the genomic background in hybrids (i.e., excess ancestry loci), which tend to be associated with genomic regions with higher recombination rates. We also identify suites of excess ancestry loci that show highly correlated allele frequencies (including conspecific and heterospecific combinations) across physically unlinked genomic regions in hybrids. Our findings provide evidence for multiple multilocus evolutionary processes impacting hybrid fitness in this system.

The Rattlesnake W Chromosome: A GC-Rich Retroelement Refugium with Retained Gene Function Across Ancient Evolutionary Strata.

Sex chromosomes diverge after the establishment of recombination suppression, resulting in differential sex-linkage of genes involved in genetic sex determination and dimorphic traits. This process produces systems of male or female heterogamety wherein the Y and W chromosomes are only present in one sex and are often highly degenerated. Sex-limited Y and W chromosomes contain valuable information about the evolutionary transition from autosomes to sex chromosomes, yet detailed characterizations of the structure, composition, and gene content of sex-limited chromosomes are lacking for many species. In this study, we characterize the female-specific W chromosome of the prairie rattlesnake (Crotalus viridis) and evaluate how recombination suppression and other processes have shaped sex chromosome evolution in ZW snakes. Our analyses indicate that the rattlesnake W chromosome is over 80% repetitive and that an abundance of GC-rich mdg4 elements has driven an overall high degree of GC-richness despite a lack of recombination. The W chromosome is also highly enriched for repeat sequences derived from endogenous retroviruses and likely acts as a "refugium" for these and other retroelements. We annotated 219 putatively functional W-linked genes across at least two evolutionary strata identified based on estimates of sequence divergence between Z and W gametologs. The youngest of these strata is relatively gene-rich, however gene expression across strata suggests retained gene function amidst a greater degree of degeneration following ancient recombination suppression. Functional annotation of W-linked genes indicates a specialization of the W chromosome for reproductive and developmental function since recombination suppression from the Z chromosome.

Origins, genomic structure and copy number variation of snake venom myotoxins.

Crotamine, myotoxin a and homologs are short peptides that often comprise major fractions of rattlesnake venoms and have been extensively studied for their bioactive properties. These toxins are thought to be important for rapidly immobilizing mammalian prey and are implicated in serious, and sometimes fatal, responses to envenomation in humans. While high quality reference genomes for multiple venomous snakes are available, the loci that encode myotoxins have not been successfully assembled in any existing genome assembly. Here, we integrate new and existing genomic and transcriptomic data from the Prairie Rattlesnake (Crotalus viridis viridis) to reconstruct, characterize, and infer the chromosomal locations of myotoxin-encoding loci. We integrate long-read transcriptomics (Pacific Bioscience's Iso-Seq) and short-read RNA-seq to infer gene sequence diversity and characterize patterns of myotoxin and paralogous ß-defensin expression across multiple tissues. We also identify two long non-coding RNA sequences which both encode functional myotoxins, demonstrating a newly discovered source of venom coding sequence diversity. We also integrate long-range mate-pair chromatin contact data and linked-read sequencing to infer the structure and chromosomal locations of the three myotoxin-like loci. Further, we conclude that the venom-associated myotoxin is located on chromosome 1 and is adjacent to non-venom paralogs. Consistent with this locus contributing to venom composition, we find evidence that the promoter of this gene is selectively open in venom gland tissue and contains transcription factor binding sites implicated in broad trans-regulatory pathways that regulate snake venoms. This study provides the best genomic reconstruction of myotoxin loci to date and raises questions about the physiological roles and interplay between myotoxin and related genes, as well as the genomic origins of snake venom variation.

The roles of balancing selection and recombination in the evolution of rattlesnake venom.

The origin of snake venom involved duplication and recruitment of non-venom genes into venom systems. Several studies have predicted that directional positive selection has governed this process. Venom composition varies substantially across snake species and venom phenotypes are locally adapted to prey, leading to coevolutionary interactions between predator and prey. Venom origins and contemporary snake venom evolution may therefore be driven by fundamentally different selection regimes, yet investigations of population-level patterns of selection have been limited. Here, we use whole-genome data from 68 rattlesnakes to test hypotheses about the factors that drive genomic diversity and differentiation in major venom gene regions. We show that selection has resulted in long-term maintenance of genetic diversity within and between species in multiple venom gene families. Our findings are inconsistent with a dominant role of directional positive selection and instead support a role of long-term balancing selection in shaping venom evolution. We also detect rapid decay of linkage disequilibrium due to high recombination rates in venom regions, suggesting that venom genes have reduced selective interference with nearby loci, including other venom paralogues. Our results provide an example of long-term balancing selection that drives trans-species polymorphism and help to explain how snake venom keeps pace with prey resistance.

Snake venom gene expression is coordinated by novel regulatory architecture and the integration of multiple co-opted vertebrate pathways.

Understanding how regulatory mechanisms evolve is critical for understanding the processes that give rise to novel phenotypes. Snake venom systems represent a valuable and tractable model for testing hypotheses related to the evolution of novel regulatory networks, yet the regulatory mechanisms underlying venom production remain poorly understood. Here, we use functional genomics approaches to investigate venom regulatory architecture in the prairie rattlesnake and identify cis-regulatory sequences (enhancers and promoters), trans-regulatory transcription factors, and integrated signaling cascades involved in the regulation of snake venom genes. We find evidence that two conserved vertebrate pathways, the extracellular signal-regulated kinase and unfolded protein response pathways, were co-opted to regulate snake venom. In one large venom gene family (snake venom serine proteases), this co-option was likely facilitated by the activity of transposable elements. Patterns of snake venom gene enhancer conservation, in some cases spanning 50 million yr of lineage divergence, highlight early origins and subsequent lineage-specific adaptations that have accompanied the evolution of venom regulatory architecture. We also identify features of chromatin structure involved in venom regulation, including topologically associated domains and CTCF loops that underscore the potential importance of novel chromatin structure to coevolve when duplicated genes evolve new regulatory control. Our findings provide a model for understanding how novel regulatory systems may evolve through a combination of genomic processes, including tandem duplication of genes and regulatory sequences, cis-regulatory sequence seeding by transposable elements, and diverse transcriptional regulatory proteins controlled by a co-opted regulatory cascade.

SEQUENCE SLIDER: integration of structural and genetic data to characterize isoforms from natural sources.

Proteins isolated from natural sources can be composed of a mixture of isoforms with similar physicochemical properties that coexist in the final steps of purification. Yet, even where unverified, the assumed sequence is enforced throughout the structural studies. Herein, we propose a novel perspective to address the usually neglected sequence heterogeneity of natural products by integrating biophysical, genetic and structural data in our program SEQUENCE SLIDER. The aim is to assess the evidence supporting chemical composition in structure determination. Locally, we interrogate the experimental map to establish which side chains are supported by the structural data, and the genetic information relating sequence conservation is integrated into this statistic. Hence, we build a constrained peptide database, containing most probable sequences to interpret mass spectrometry data (MS). In parallel, we perform MS de novo sequencing with genomic-based algorithms to detect point mutations. We calibrated SLIDER with Gallus gallus lysozyme, whose sequence is unequivocally established and numerous natural isoforms are reported. We used SLIDER to characterize a metalloproteinase and a phospholipase A2-like protein from the venom of Bothrops moojeni and a crotoxin from Crotalus durissus collilineatus. This integrated approach offers a more realistic structural descriptor to characterize macromolecules isolated from natural sources.

Insights on the inhibition properties of Jatromollistatin (a cyclic heptapeptide) against Crotalus adamanteus metalloendopeptidase using molecular docking analysis.

Jatropha mollissima is endemic to Brazil and is used for traditional medicinal purposes, including the treatment of snakebite. In this study, latex obtained from this plant was fractioned using reversed-phase chromatography, and the fractions were then screened for peptides. A 755 g/mol peptide was obtained, and MS/MS analyses indicated it had a cyclic sequence (Pro-Leu-Gly-Val-Leu-Leu-Tyr). This peptide sequence was present in the Jatropha genome database, and an identity value of 90.71%, an E-value of 0.0, and a score of 883 with NO-associated protein 1/chloroplastic/mitochondria of Jatropha curcas were obtained from the NCBI nonredundant protein sequence (nr) database. Molecular docking analyses performed with the peptide against a metalloendopeptidase belonging to Crotalus adamanteus snake venom suggested the cyclic peptide establishes favorable interactions with the catalytic site of the enzyme. Therefore, it could inhibit enzyme catalysis. This belief was corroborated by the formation of 6 hydrogen bonds with the linear form of the peptide. Tighter complexation of the cyclic form (41 kcal/mol more energetic) revealed better spatial blocking. The linear form outperformed the cyclic form in complexing the required energy, recruiting more catalytic residues (6/2), and in establishing more hydrogen bonds (6/3). However, cyclic folding provided a more significant spatial block within the catalytic site. The set of results suggests that the cycle peptide, here called Jatromollistatin, which was previously described as jatrophidin and pohlianin A in two other species of Jatropha, is a promising candidate to inhibit venom proteases. This belief is corroborated by the topical use of the latex for initial treatment of snakebites.

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.

Genome-wide data reveal extensive gene flow during the diversification of the western rattlesnakes (Viperidae: Crotalinae: Crotalus).

Hybridization and introgression are important, but often overlooked processes when inferring phylogenies. When these processes are not accounted for and a strictly diverging phylogenetic model is applied to groups with a history of hybridization, phylogenetic inference and parameter estimation can be inaccurate. Recent developments in phylogenetic network approaches coupled with the increasing availability of genomic data allow inferences of reticulate evolutionary histories across the tree of life. The western rattlesnake species group (C. viridis species complex, C. mitchellii species complex, C. scutulutas, and C. tigris) is an iconic snake lineage that is widespread across western North America. This group is composed of several species complexes with unclear species limits, likely the result of ongoing gene flow among nascent lineages. Here I generate reduced representation genomic data and test for a history of reticulation within this group. I demonstrate that all species have undergone hybridization with at least one other lineage, suggesting introgression is widespread in this group. Topologies differ between phylogenies estimated under the multispecies coalescent and multispecies network coalescent methods, indicating that gene flow has obscured phylogenetic relationships within this group. These past introgression events are predominantly restricted to species that co-occur geographically. However, within species that have a history of introgression, this signature is detected regardless of specimen sampling across geography. Overall, my results suggest the accumulation of reproductive isolating barriers occurs slowly in rattlesnakes which likely leads to the difficulty in delimiting species, furthermore, the results of this study have implications for trait evolution in this group.