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.

Phylogeny and historical biogeography of the swallow family (Hirundinidae) inferred from comparisons of thousands of UCE loci.

Swallows (Hirundinidae) are a globally distributed family of passerine birds that exhibit remarkable similarity in body shape but tremendous variation in plumage, sociality, nesting behavior, and migratory strategies. As a result, swallow species have become models for empirical behavioral ecology and evolutionary studies, and variation across the Hirundinidae presents an excellent opportunity for comparative analyses of trait evolution. Exploiting this potential requires a comprehensive and well-resolved phylogenetic tree of the family. To address this need, we estimated swallow phylogeny using genetic data from thousands of ultraconserved element (UCE) loci sampled from nearly all recognized swallow species. Maximum likelihood, coalescent-based, and Bayesian approaches yielded a well-resolved phylogenetic tree to the generic level, with minor disagreement among inferences at the species level, which likely reflect ongoing population genetic processes. The UCE data were particularly useful in helping to resolve deep nodes, which previously confounded phylogenetic reconstruction efforts. Divergence time estimates from the improved swallow tree support a Miocene origin of the family, roughly 13 million years ago, with subsequent diversification of major groups in the late Miocene and Pliocene. Our estimates of historical biogeography support the hypothesis that swallows originated in the Afrotropics and have subsequently expanded across the globe, with major in situ diversification in Africa and a secondary major radiation following colonization of the Neotropics. Initial examination of nesting and sociality indicates that the origin of mud nesting - a relatively rare nest construction phenotype in birds - was a major innovation coincident with the origin of a clade giving rise to over 40% of extant swallow diversity. In contrast, transitions between social and solitary nesting appear less important for explaining patterns of diversification among swallows.

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.

Signals of differential introgression in the genome of natural hybrids of Caribbean anoles.

Hybridization facilitates recombination between divergent genetic lineages and can be shaped by both neutral and selective processes. Upon hybridization, loci with no net fitness effects introgress randomly from parental species into the genomes of hybrid individuals. Conversely, alleles from one parental species at some loci may provide a selective advantage to hybrids, resulting in patterns of introgression that do not conform to random expectations. We investigated genomic patterns of differential introgression in natural hybrids of two species of Caribbean anoles, Anolis pulchellus and A. krugi in Puerto Rico. Hybrids exhibit A. pulchellus phenotypes but possess A. krugi mitochondrial DNA, originated from multiple, independent hybridization events, and appear to have replaced pure A. pulchellus across a large area in western Puerto Rico. Combining genome-wide SNP datasets with bioinformatic methods to identify signals of differential introgression in hybrids, we demonstrate that the genomes of hybrids are dominated by pulchellus-derived alleles and show only 10%-20% A. krugi ancestry. The majority of A. krugi loci in hybrids exhibit a signal of non-random differential introgression and include loci linked to genes involved in development and immune function. Three of these genes (delta like canonical notch ligand 1, jagged1 and notch receptor 1) affect cell differentiation and growth and interact with mitochondrial function. Our results suggest that differential non-random introgression for a subset of loci may be driven by selection favouring the inheritance of compatible mitochondrial and nuclear-encoded genes in hybrids.

Estimating phylogenies from genomes: A beginners review of commonly used genomic data in vertebrate phylogenomics.

Despite the increasing feasibility of sequencing whole genomes from diverse taxa, a persistent problem in phylogenomics is the selection of appropriate genetic markers or loci for a given taxonomic group or research question. In this review, we aim to streamline the decision-making process when selecting specific markers to use in phylogenomic studies by introducing commonly used types of genomic markers, their evolutionary characteristics, and their associated uses in phylogenomics. Specifically, we review the utilities of ultraconserved elements (including flanking regions), anchored hybrid enrichment loci, conserved nonexonic elements, untranslated regions, introns, exons, mitochondrial DNA, single nucleotide polymorphisms, and anonymous regions (nonspecific regions that are evenly or randomly distributed across the genome). These various genomic elements and regions differ in their substitution rates, likelihood of neutrality or of being strongly linked to loci under selection, and mode of inheritance, each of which are important considerations in phylogenomic reconstruction. These features may give each type of marker important advantages and disadvantages depending on the biological question, number of taxa sampled, evolutionary timescale, cost effectiveness, and analytical methods used. We provide a concise outline as a resource to efficiently consider key aspects of each type of genetic marker. There are many factors to consider when designing phylogenomic studies, and this review may serve as a primer when weighing options between multiple potential phylogenomic markers.

Identification of an integrated stress and growth response signaling switch that directs vertebrate intestinal regeneration.

BACKGROUND: Snakes exhibit extreme intestinal regeneration following months-long fasts that involves unparalleled increases in metabolism, function, and tissue growth, but the specific molecular control of this process is unknown. Understanding the mechanisms that coordinate these regenerative phenotypes provides valuable opportunities to understand critical pathways that may control vertebrate regeneration and novel perspectives on vertebrate regenerative capacities. RESULTS: Here, we integrate a comprehensive set of phenotypic, transcriptomic, proteomic, and phosphoproteomic data from boa constrictors to identify the mechanisms that orchestrate shifts in metabolism, nutrient uptake, and cellular stress to direct phases of the regenerative response. We identify specific temporal patterns of metabolic, stress response, and growth pathway activation that direct regeneration and provide evidence for multiple key central regulatory molecules kinases that integrate these signals, including major conserved pathways like mTOR signaling and the unfolded protein response. CONCLUSION: Collectively, our results identify a novel switch-like role of stress responses in intestinal regeneration that forms a primary regulatory hub facilitating organ regeneration and could point to potential pathways to understand regenerative capacity in vertebrates.

Microchromosomes Exhibit Distinct Features of Vertebrate Chromosome Structure and Function with Underappreciated Ramifications for Genome Evolution.

Microchromosomes are common yet poorly understood components of many vertebrate genomes. Recent studies have revealed that microchromosomes contain a high density of genes and possess other distinct characteristics compared with macrochromosomes. Whether distinctive characteristics of microchromosomes extend to features of genome structure and organization, however, remains an open question. Here, we analyze Hi-C sequencing data from multiple vertebrate lineages and show that microchromosomes exhibit consistently high degrees of interchromosomal interaction (particularly with other microchromosomes), appear to be colocalized to a common central nuclear territory, and are comprised of a higher proportion of open chromatin than macrochromosomes. These findings highlight an unappreciated level of diversity in vertebrate genome structure and function, and raise important questions regarding the evolutionary origins and ramifications of microchromosomes and the genes that they house.

The origins and evolution of chromosomes, dosage compensation, and mechanisms underlying venom regulation in snakes.

Here we use a chromosome-level genome assembly of a prairie rattlesnake (Crotalus viridis), together with Hi-C, RNA-seq, and whole-genome resequencing data, to study key features of genome biology and evolution in reptiles. We identify the rattlesnake Z Chromosome, including the recombining pseudoautosomal region, and find evidence for partial dosage compensation driven by an evolutionary accumulation of a female-biased up-regulation mechanism. Comparative analyses with other amniotes provide new insight into the origins, structure, and function of reptile microchromosomes, which we demonstrate have markedly different structure and function compared to macrochromosomes. Snake microchromosomes are also enriched for venom genes, which we show have evolved through multiple tandem duplication events in multiple gene families. By overlaying chromatin structure information and gene expression data, we find evidence for venom gene-specific chromatin contact domains and identify how chromatin structure guides precise expression of multiple venom gene families. Further, we find evidence for venom gland-specific transcription factor activity and characterize a complement of mechanisms underlying venom production and regulation. Our findings reveal novel and fundamental features of reptile genome biology, provide insight into the regulation of snake venom, and broadly highlight the biological insight enabled by chromosome-level genome assemblies.

Snake Recombination Landscapes Are Concentrated in Functional Regions despite PRDM9.

Meiotic recombination in vertebrates is concentrated in hotspots throughout the genome. The location and stability of hotspots have been linked to the presence or absence of PRDM9, leading to two primary models for hotspot evolution derived from mammals and birds. Species with PRDM9-directed recombination have rapid turnover of hotspots concentrated in intergenic regions (i.e., mammals), whereas hotspots in species lacking PRDM9 are concentrated in functional regions and have greater stability over time (i.e., birds). Snakes possess PRDM9, yet virtually nothing is known about snake recombination. Here, we examine the recombination landscape and test hypotheses about the roles of PRDM9 in rattlesnakes. We find substantial variation in recombination rate within and among snake chromosomes, and positive correlations between recombination rate and gene density, GC content, and genetic diversity. Like mammals, snakes appear to have a functional and active PRDM9, but rather than being directed away from genes, snake hotspots are concentrated in promoters and functional regions-a pattern previously associated only with species that lack a functional PRDM9. Snakes therefore provide a unique example of recombination landscapes in which PRDM9 is functional, yet recombination hotspots are associated with functional genic regions-a combination of features that defy existing paradigms for recombination landscapes in vertebrates. Our findings also provide evidence that high recombination rates are a shared feature of vertebrate microchromosomes. Our results challenge previous assumptions about the adaptive role of PRDM9 and highlight the diversity of recombination landscape features among vertebrate lineages.

The effects of climate and demographic history in shaping genomic variation across populations of the Desert Horned Lizard (Phrynosoma platyrhinos).

Species often experience spatial environmental heterogeneity across their range, and populations may exhibit signatures of adaptation to local environmental characteristics. Other population genetic processes, such as migration and genetic drift, can impede the effects of local adaptation. Genetic drift in particular can have a pronounced effect on population genetic structure during large-scale geographic expansions, where a series of founder effects leads to decreases in genetic variation in the direction of the expansion. Here, we explore the genetic diversity of a desert lizard that occupies a wide range of environmental conditions and that has experienced post-glacial expansion northwards along two colonization routes. Based on our analyses of a large SNP data set, we find evidence that both climate and demographic history have shaped the genetic structure of populations. Pronounced genetic differentiation was evident between populations occupying cold versus hot deserts, and we detected numerous loci with significant associations with climate. The genetic signal of founder effects, however, is still present in the genomes of the recently expanded populations, which comprise subsets of genetic variation found in the southern populations.

Sex-linked genetic diversity and differentiation in a globally distributed avian species complex.

Sex chromosomes often bear distinct patterns of genetic variation due to unique patterns of inheritance and demography. The processes of mutation, recombination, genetic drift and selection also influence rates of evolution on sex chromosomes differently than autosomes. Measuring such differences provides information about how these processes shape genomic variation and their roles in the origin of species. To test hypotheses and predictions about patterns of autosomal and sex-linked genomic diversity and differentiation, we measured population genetic statistics within and between populations and subspecies of the barn swallow (Hirundo rustica) and performed explicit comparisons between autosomal and Z-linked genomic regions. We first tested for evidence of low Z-linked genetic diversity and high Z-linked population differentiation relative to autosomes, then for evidence that the Z chromosome bears greater ancestry information due to faster lineage sorting. Finally, we investigated geographical clines across hybrid zones for evidence that the Z chromosome is resistant to introgression due to selection against hybrids. We found evidence that the barn swallow mating system, demographic history and linked selection each contribute to low Z-linked diversity and high Z-linked differentiation. While incomplete lineage sorting is rampant across the genome, our results indicate faster sorting of ancestral polymorphism on the Z. Finally, hybrid zone analyses indicate barriers to introgression on the Z chromosome, suggesting that sex-linked traits are important in reproductive isolation, especially in migratory divide regions. Our study highlights how selection, gene flow and demography shape sex-linked genetic diversity and underlines the relevance of the Z chromosome in speciation.

Population Genomic Analyses Confirm Male-Biased Mutation Rates in Snakes.

Male-biased mutation rates occur in a diverse array of organisms. The ratio of male-to-female mutation rate may have major ramifications for evolution across the genome, and for sex-linked genes in particular. In ZW species, the Z chromosome is carried by males two-thirds of the time, leading to the prediction that male-biased mutation rates will have a disproportionate effect on the evolution of Z-linked genes relative to autosomes and the W chromosome. Colubroid snakes (including colubrids, elapids, and viperids) have ZW sex determination, yet male-biased mutation rates have not been well studied in this group. Here we analyze a population genomic dataset from rattlesnakes to quantify genetic variation within and genetic divergence between species. We use a new method for unbiased estimation of population genetic summary statistics to compare variation between the Z chromosome and autosomes and to calculate net nucleotide differentiation between species. We find evidence for a 2.03-fold greater mutation rate in male rattlesnakes relative to females, corresponding to an average µZ/µA ratio of 1.1. Our results from snakes are quantitatively similar to birds, suggesting that male-biased mutation rates may be a common feature across vertebrate lineages with ZW sex determination.

Multi-species comparisons of snakes identify coordinated signalling networks underlying post-feeding intestinal regeneration.

Several snake species that feed infrequently in nature have evolved the ability to massively upregulate intestinal form and function with each meal. While fasting, these snakes downregulate intestinal form and function, and upon feeding restore intestinal structure and function through major increases in cell growth and proliferation, metabolism and upregulation of digestive function. Previous studies have identified changes in gene expression that underlie this regenerative growth of the python intestine, but the unique features that differentiate this extreme regenerative growth from non-regenerative post-feeding responses exhibited by snakes that feed more frequently remain unclear. Here, we leveraged variation in regenerative capacity across three snake species-two distantly related lineages ( Crotalus and Python) that experience regenerative growth, and one ( Nerodia) that does not-to infer molecular mechanisms underlying intestinal regeneration using transcriptomic and proteomic approaches. Using a comparative approach, we identify a suite of growth, stress response and DNA damage response signalling pathways with inferred activity specifically in regenerating species, and propose a hypothesis model of interactivity between these pathways that may drive regenerative intestinal growth in snakes.

ThetaMater: Bayesian estimation of population size parameter θ from genomic data.

Summary: We describe ThetaMater, an open source R package comprising a suite of functions for efficient and scalable Bayesian estimation of the population size parameter θ from genomic data. Availability and implementation: ThetaMater is available at GitHub (https://github.com/radamsRHA/ThetaMater). Contact: todd.castoe@uta.edu. Supplementary information: Supplementary data are available at Bioinformatics online.

Assessing the Impacts of Positive Selection on Coalescent-Based Species Tree Estimation and Species Delimitation.

The assumption of strictly neutral evolution is fundamental to the multispecies coalescent model and permits the derivation of gene tree distributions and coalescent times conditioned on a given species tree. In this study, we conduct computer simulations to explore the effects of violating this assumption in the form of species-specific positive selection when estimating species trees, species delimitations, and coalescent parameters under the model. We simulated data sets under an array of evolutionary scenarios that differ in both speciation parameters (i.e., divergence times, strength of selection) and experimental design (i.e., number of loci sampled) and incorporated species-specific positive selection occurring within branches of a species tree to identify the effects of selection on multispecies coalescent inferences. Our results highlight particular evolutionary scenarios and parameter combinations in which inferences may be more, or less, susceptible to the effects of positive selection. In some extreme cases, selection can decrease error in species delimitation and increase error in species tree estimation, yet these inferences appear to be largely robust to the effects of positive selection under many conditions likely to be encountered in empirical data sets.

GppFst: genomic posterior predictive simulations of FST and dXY for identifying outlier loci from population genomic data.

Summary: We introduce GppFst, an open source R package that generates posterior predictive distributions of FST and dx under a neutral coalescent model to identify putative targets of selection from genomic data. Availability and Implementation: GppFst is available at ( https://github.com/radamsRHA/GppFst ). Contact: todd.castoe@uta.edu. Supplementary information: Supplementary data are available at Bioinformatics online.

Plasticity and local adaptation explain lizard cold tolerance.

How does climate variation limit the range of species and what does it take for species to colonize new regions? In this issue of Molecular Ecology, Campbell-Staton et al. () address these broad questions by investigating cold tolerance adaptation in the green anole lizard (Anolis carolinensis) across a latitudinal transect. By integrating physiological data, gene expression data and acclimation experiments, the authors disentangle the mechanisms underlying cold adaptation. They first establish that cold tolerance adaptation in Anolis lizards follows the predictions of the oxygen- and capacity-limited thermal tolerance hypothesis, which states that organisms are limited by temperature thresholds at which oxygen supply cannot meet demand. They then explore the drivers of cold tolerance at a finer scale, finding evidence that northern populations are adapted to cooler thermal regimes and that both phenotypic plasticity and heritable genetic variation contribute to cold tolerance. The integration of physiological and gene expression data further highlights the varied mechanisms that drive cold tolerance adaptation in Anolis lizards, including both supply-side and demand-side adaptations that improve oxygen economy. Altogether, their work provides new insight into the physiological and genetic mechanisms underlying adaptation to new climatic niches and demonstrates that cold tolerance in northern lizard populations is achieved through the synergy of physiological plasticity and local genetic adaptation for thermal performance.

Novel ecological and climatic conditions drive rapid adaptation in invasive Florida Burmese pythons.

Invasive species provide powerful in situ experimental systems for studying evolution in response to selective pressures in novel habitats. While research has shown that phenotypic evolution can occur rapidly in nature, few examples exist of genomewide adaptation on short "ecological" timescales. Burmese pythons (Python molurus bivittatus) have become a successful and impactful invasive species in Florida over the last 30 years despite major freeze events that caused high python mortality. We sampled Florida Burmese pythons before and after a major freeze event in 2010 and found evidence for directional selection in genomic regions enriched for genes associated with thermosensation, behaviour and physiology. Several of these genes are linked to regenerative organ growth, an adaptive response that modulates organ size and function with feeding and fasting in pythons. Independent histological and functional genomic data sets provide additional layers of support for a contemporary shift in invasive Burmese python physiology. In the Florida population, a shift towards maintaining an active digestive system may be driven by the fitness benefits of maintaining higher metabolic rates and body temperature during freeze events. Our results suggest that a synergistic interaction between ecological and climatic selection pressures has driven adaptation in Florida Burmese pythons, demonstrating the often-overlooked potential of rapid adaptation to influence the success of invasive species.

Cryptic genetic diversity, population structure, and gene flow in the Mojave rattlesnake (Crotalus scutulatus).

The Mojave rattlesnake (Crotalus scutulatus) inhabits deserts and arid grasslands of the western United States and Mexico. Despite considerable interest in its highly toxic venom and the recognition of two subspecies, no molecular studies have characterized range-wide genetic diversity and population structure or tested species limits within C. scutulatus. We used mitochondrial DNA and thousands of nuclear loci from double-digest restriction site associated DNA sequencing to infer population genetic structure throughout the range of C. scutulatus, and to evaluate divergence times and gene flow between populations. We find strong support for several divergent mitochondrial and nuclear clades of C. scutulatus, including splits coincident with two major phylogeographic barriers: the Continental Divide and the elevational increase associated with the Central Mexican Plateau. We apply Bayesian clustering, phylogenetic inference, and coalescent-based species delimitation to our nuclear genetic data to test hypotheses of population structure. We also performed demographic analyses to test hypotheses relating to population divergence and gene flow. Collectively, our results support the existence of four distinct lineages within C. scutulatus, and genetically defined populations do not correspond with currently recognized subspecies ranges. Finally, we use approximate Bayesian computation to test hypotheses of divergence among multiple rattlesnake species groups distributed across the Continental Divide, and find evidence for co-divergence at this boundary during the mid-Pleistocene.

Growth and stress response mechanisms underlying post-feeding regenerative organ growth in the Burmese python.

BACKGROUND: Previous studies examining post-feeding organ regeneration in the Burmese python (Python molurus bivittatus) have identified thousands of genes that are significantly differentially regulated during this process. However, substantial gaps remain in our understanding of coherent mechanisms and specific growth pathways that underlie these rapid and extensive shifts in organ form and function. Here we addressed these gaps by comparing gene expression in the Burmese python heart, liver, kidney, and small intestine across pre- and post-feeding time points (fasted, one day post-feeding, and four days post-feeding), and by conducting detailed analyses of molecular pathways and predictions of upstream regulatory molecules across these organ systems. RESULTS: Identified enriched canonical pathways and upstream regulators indicate that while downstream transcriptional responses are fairly tissue specific, a suite of core pathways and upstream regulator molecules are shared among responsive tissues. Pathways such as mTOR signaling, PPAR/LXR/RXR signaling, and NRF2-mediated oxidative stress response are significantly differentially regulated in multiple tissues, indicative of cell growth and proliferation along with coordinated cell-protective stress responses. Upstream regulatory molecule analyses identify multiple growth factors, kinase receptors, and transmembrane receptors, both within individual organs and across separate tissues. Downstream transcription factors MYC and SREBF are induced in all tissues. CONCLUSIONS: These results suggest that largely divergent patterns of post-feeding gene regulation across tissues are mediated by a core set of higher-level signaling molecules. Consistent enrichment of the NRF2-mediated oxidative stress response indicates this pathway may be particularly important in mediating cellular stress during such extreme regenerative growth.