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Abstract 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. 

Abstract 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. 

Abstract 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. 

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. 

Abstract 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.