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.

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.

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 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.

Prospects for genomic surveillance for selection in schistosome parasites.

Schistosomiasis is a neglected tropical disease caused by multiple parasitic Schistosoma species, and which impacts over 200 million people globally, mainly in low- and middle-income countries. Genomic surveillance to detect evidence for natural selection in schistosome populations represents an emerging and promising approach to identify and interpret schistosome responses to ongoing control efforts or other environmental factors. Here we review how genomic variation is used to detect selection, how these approaches have been applied to schistosomes, and how future studies to detect selection may be improved. We discuss the theory of genomic analyses to detect selection, identify experimental designs for such analyses, and review studies that have applied these approaches to schistosomes. We then consider the biological characteristics of schistosomes that are expected to respond to selection, particularly those that may be impacted by control programs. Examples include drug resistance, host specificity, and life history traits, and we review our current understanding of specific genes that underlie them in schistosomes. We also discuss how inherent features of schistosome reproduction and demography pose substantial challenges for effective identification of these traits and their genomic bases. We conclude by discussing how genomic surveillance for selection should be designed to improve understanding of schistosome biology, and how the parasite changes in response to selection.

Fifty million years of beetle evolution along the Antarctic Polar Front.

Global cooling and glacial-interglacial cycles since Antarctica's isolation have been responsible for the diversification of the region's marine fauna. By contrast, these same Earth system processes are thought to have played little role terrestrially, other than driving widespread extinctions. Here, we show that on islands along the Antarctic Polar Front, paleoclimatic processes have been key to diversification of one of the world's most geographically isolated and unique groups of herbivorous beetles-Ectemnorhinini weevils. Combining phylogenomic, phylogenetic, and phylogeographic approaches, we demonstrate that these weevils colonized the sub-Antarctic islands from Africa at least 50 Ma ago and repeatedly dispersed among them. As the climate cooled from the mid-Miocene, diversification of the beetles accelerated, resulting in two species-rich clades. One of these clades specialized to feed on cryptogams, typical of the polar habitats that came to prevail under Miocene conditions yet remarkable as a food source for any beetle. This clade's most unusual representative is a marine weevil currently undergoing further speciation. The other clade retained the more common weevil habit of feeding on angiosperms, which likely survived glaciation in isolated refugia. Diversification of Ectemnorhinini weevils occurred in synchrony with many other Antarctic radiations, including penguins and notothenioid fishes, and coincided with major environmental changes. Our results thus indicate that geo-climatically driven diversification has progressed similarly for Antarctic marine and terrestrial organisms since the Miocene, potentially constituting a general biodiversity paradigm that should be sought broadly for the region's taxa.

Of Traits and Trees: Probabilistic Distances under Continuous Trait Models for Dissecting the Interplay among Phylogeny, Model, and Data.

Stochastic models of character trait evolution have become a cornerstone of evolutionary biology in an array of contexts. While probabilistic models have been used extensively for statistical inference, they have largely been ignored for the purpose of measuring distances between phylogeny-aware models. Recent contributions to the problem of phylogenetic distance computation have highlighted the importance of explicitly considering evolutionary model parameters and their impacts on molecular sequence data when quantifying dissimilarity between trees. By comparing two phylogenies in terms of their induced probability distributions that are functions of many model parameters, these distances can be more informative than traditional approaches that rely strictly on differences in topology or branch lengths alone. Currently, however, these approaches are designed for comparing models of nucleotide substitution and gene tree distributions, and thus, are unable to address other classes of traits and associated models that may be of interest to evolutionary biologists. Here, we expand the principles of probabilistic phylogenetic distances to compute tree distances under models of continuous trait evolution along a phylogeny. By explicitly considering both the degree of relatedness among species and the evolutionary processes that collectively give rise to character traits, these distances provide a foundation for comparing models and their predictions, and for quantifying the impacts of assuming one phylogenetic background over another while studying the evolution of a particular trait. We demonstrate the properties of these approaches using theory, simulations, and several empirical data sets that highlight potential uses of probabilistic distances in many scenarios. We also introduce an open-source R package named PRDATR for easy application by the scientific community for computing phylogenetic distances under models of character trait evolution.[Brownian motion; comparative methods; phylogeny; quantitative traits.].

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.

PhyloWGA: chromosome-aware phylogenetic interrogation of whole genome alignments.

SUMMARY: Here, we present PhyloWGA, an open source R package for conducting phylogenetic analysis and investigation of whole genome data. AVAILABILITYAND IMPLEMENTATION: Available at Github (https://github.com/radamsRHA/PhyloWGA). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Genome-wide SNPs clarify lineage diversity confused by coloration in coralsnakes of the Micrurus diastema species complex (Serpentes: Elapidae).

New world coralsnakes of the genus Micrurus are a diverse radiation of highly venomous and brightly colored snakes that range from North Carolina to Argentina. Species in this group have played central roles in developing and testing hypotheses about the evolution of mimicry and aposematism. Despite their diversity and prominence as model systems, surprisingly little is known about species boundaries and phylogenetic relationships within Micrurus, which has substantially hindered meaningful analyses of their evolutionary history. Here we use mitochondrial genes together with thousands of nuclear genomic loci obtained via ddRADseq to study the phylogenetic relationships and population genomics of a subclade of the genus Micrurus: The M. diastema species complex. Our results indicate that prior species and species-group inferences based on morphology and color pattern have grossly misguided taxonomy, and that the M. diastema complex is not monophyletic. Based on our analyses of molecular data, we infer the phylogenetic relationships among species and populations, and provide a revised taxonomy for the group. Two non-sister species-complexes with similar color patterns are recognized, the M. distans and the M. diastema complexes, the first being basal to the monadal Micrurus and the second encompassing most North American monadal taxa. We examined all 13 species, and their respective subspecies, for a total of 24 recognized taxa in the M. diastema species complex. Our analyses suggest a reduction to 10 species, with no subspecific designations warranted, to be a more likely estimate of species diversity, namely, M. apiatus, M. browni, M. diastema, M. distans, M. ephippifer, M. fulvius, M. michoacanensis, M. oliveri, M. tener, and one undescribed species.

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.

Probabilistic Species Tree Distances: Implementing the Multispecies Coalescent to Compare Species Trees Within the Same Model-Based Framework Used to Estimate Them.

Despite the ubiquitous use of statistical models for phylogenomic and population genomic inferences, this model-based rigor is rarely applied to post hoc comparison of trees. In a recent study, Garba et al. derived new methods for measuring the distance between two gene trees computed as the difference in their site pattern probability distributions. Unlike traditional metrics that compare trees solely in terms of geometry, these measures consider gene trees and associated parameters as probabilistic models that can be compared using standard information theoretic approaches. Consequently, probabilistic measures of phylogenetic tree distance can be far more informative than simply comparisons of topology and/or branch lengths alone. However, in their current form, these distance measures are not suitable for the comparison of species tree models in the presence of gene tree heterogeneity. Here, we demonstrate an approach for how the theory of Garba et al. (2018), which is based on gene tree distances, can be extended naturally to the comparison of species tree models. Multispecies coalescent (MSC) models parameterize the discrete probability distribution of gene trees conditioned upon a species tree with a particular topology and set of divergence times (in coalescent units), and thus provide a framework for measuring distances between species tree models in terms of their corresponding gene tree topology probabilities. We describe the computation of probabilistic species tree distances in the context of standard MSC models, which assume complete genetic isolation postspeciation, as well as recent theoretical extensions to the MSC in the form of network-based MSC models that relax this assumption and permit hybridization among taxa. We demonstrate these metrics using simulations and empirical species tree estimates and discuss both the benefits and limitations of these approaches. We make our species tree distance approach available as an R package called pSTDistanceR, for open use by the community.

Supergene validation: A model-based protocol for assessing the accuracy of non-model-based supergene methods.

Genome-scale species tree inference is largely restricted to heuristic approaches that use estimated gene trees to reconstruct species-level relationships. Central to these heuristic species tree methods is the assumption that the gene trees are estimated without error. To increase the accuracy of input gene trees used to infer species trees, several techniques have recently been developed for constructing longer "supergenes" that represent sets of loci inferred to share the same genealogical history. While these supergene methods are designed to increase the amount of data for gene tree estimation by concatenating several loci into "supergenes" to increase gene tree accuracy, no formal protocols have been proposed to validate this key "supergene" concatenation step. In a recent study, we developed several supergene validation strategies for assessing the accuracy of a popular supergene method: the so-called "statistical binning" pipeline. In this article, we describe a more generalizable and model-based "supergene validation" protocol for assessing the accuracy of supergenes and supergene methods using model-based tests of phylogenetic congruency. •Supergenes are validated by adopting model-based tests of topological congruence•These model-based procedures out preform non-model based methods for supergene construction•The results of this protocol can be used to assess the overall performance of a supergene method across a phylogenomic dataset.

Genomic Basis of Convergent Island Phenotypes in Boa Constrictors.

Convergent evolution is often documented in organisms inhabiting isolated environments with distinct ecological conditions and similar selective regimes. Several Central America islands harbor dwarf Boa populations that are characterized by distinct differences in growth, mass, and craniofacial morphology, which are linked to the shared arboreal and feast-famine ecology of these island populations. Using high-density RADseq data, we inferred three dwarf island populations with independent origins and demonstrate that selection, along with genetic drift, has produced both divergent and convergent molecular evolution across island populations. Leveraging whole-genome resequencing data for 20 individuals and a newly annotated Boa genome, we identify four genes with evidence of phenotypically relevant protein-coding variation that differentiate island and mainland populations. The known roles of these genes involved in body growth (PTPRS, DMGDH, and ARSB), circulating fat and cholesterol levels (MYLIP), and craniofacial development (DMGDH and ARSB) in mammals link patterns of molecular evolution with the unique phenotypes of these island forms. Our results provide an important genome-wide example for quantifying expectations of selection and convergence in closely related populations. We also find evidence at several genomic loci that selection may be a prominent force of evolutionary change-even for small island populations for which drift is predicted to dominate. Overall, while phenotypically convergent island populations show relatively few loci under strong selection, infrequent patterns of molecular convergence are still apparent and implicate genes with strong connections to convergent phenotypes.

A database of amphibian karyotypes.

One of the first characteristics that we learn about the genome of many species is the number of chromosomes it is divided among. Despite this, many questions regarding the evolution of chromosome number remain unanswered. Testing hypotheses of chromosome number evolution using comparative approaches requires trait data to be readily accessible and associated with currently accepted taxonomy. The lack of accessible karyotype data that can be linked to phylogenies has limited the application of comparative approaches that could help us understand the evolution of genome structure. Furthermore, for taxonomists, the significance of new karyotype data can only be determined with reference to records for other species. Here, we describe a curated database (karyotype.org) developed to facilitate access to chromosome number and sex chromosome system data for amphibians. The open web interface for this database allows users to generate customized exploratory plots and tables of selected clades, as well as downloading CSV files for offline analyses.

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.

Statistical binning leads to profound model violation due to gene tree error incurred by trying to avoid gene tree error.

Fundamental to all phylogenomic studies is the notion that increasing the amount of data - to entire genomes when possible - will increase the accuracy of phylogenetic inference. Simply adding more data does not, however, guarantee phylogenomic inferences will be more accurate. Even genome-scale reconstructions of species histories can suffer the effects of both incomplete lineage sorting (ILS) and gene tree estimation error (GTEE). Weighted statistical binning was originally proposed as a technique to assist the avian phylogenomics project in solving the bird tree of life, which has long eluded resolution as a result of both ILS and GTEE. These so-called "statistical binning procedures" seek to overcome GTEE by concatenating loci into longer multi-locus "supergenes" that are used to reconstruct a species tree under the assumption that the supergene tree set is an accurate estimate of the true underlying gene tree distribution. Here we evaluate the performance of the method using the original avian phylogenomics dataset. Our results suggest that statistical binning constructs false supergenes that concatenate loci with different coalescent histories more often than not: >92% of supergenes comprise discordant loci. Our results underscore a major logical inconsistency: GTEE - the sole justification for using statistical binning instead of standard concatenation - also makes these methods unreliable. These findings underscore the need for developing new robust frameworks for phylogenomic inference that more appropriately accommodate GTEE and ILS at a genome-wide scale.

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.

Molecular Adaptations for Sensing and Securing Prey and Insight into Amniote Genome Diversity from the Garter Snake Genome.

Colubridae represents the most phenotypically diverse and speciose family of snakes, yet no well-assembled and annotated genome exists for this lineage. Here, we report and analyze the genome of the garter snake, Thamnophis sirtalis, a colubrid snake that is an important model species for research in evolutionary biology, physiology, genomics, behavior, and the evolution of toxin resistance. Using the garter snake genome, we show how snakes have evolved numerous adaptations for sensing and securing prey, and identify features of snake genome structure that provide insight into the evolution of amniote genomes. Analyses of the garter snake and other squamate reptile genomes highlight shifts in repeat element abundance and expansion within snakes, uncover evidence of genes under positive selection, and provide revised neutral substitution rate estimates for squamates. Our identification of Z and W sex chromosome-specific scaffolds provides evidence for multiple origins of sex chromosome systems in snakes and demonstrates the value of this genome for studying sex chromosome evolution. Analysis of gene duplication and loss in visual and olfactory gene families supports a dim-light ancestral condition in snakes and indicates that olfactory receptor repertoires underwent an expansion early in snake evolution. Additionally, we provide some of the first links between secreted venom proteins, the genes that encode them, and their evolutionary origins in a rear-fanged colubrid snake, together with new genomic insight into the coevolutionary arms race between garter snakes and highly toxic newt prey that led to toxin resistance in garter snakes.

Squamate reptiles challenge paradigms of genomic repeat element evolution set by birds and mammals.

Broad paradigms of vertebrate genomic repeat element evolution have been largely shaped by analyses of mammalian and avian genomes. Here, based on analyses of genomes sequenced from over 60 squamate reptiles (lizards and snakes), we show that patterns of genomic repeat landscape evolution in squamates challenge such paradigms. Despite low variance in genome size, squamate genomes exhibit surprisingly high variation among species in abundance (ca. 25-73% of the genome) and composition of identifiable repeat elements. We also demonstrate that snake genomes have experienced microsatellite seeding by transposable elements at a scale unparalleled among eukaryotes, leading to some snake genomes containing the highest microsatellite content of any known eukaryote. Our analyses of transposable element evolution across squamates also suggest that lineage-specific variation in mechanisms of transposable element activity and silencing, rather than variation in species-specific demography, may play a dominant role in driving variation in repeat element landscapes across squamate phylogeny.