Wright was right: leveraging old data and new methods to illustrate the critical role of epistasis in genetics and evolution.

Much of evolutionary theory is predicated on assumptions about the relative importance of simple additive versus complex epistatic genetic architectures. Previous work suggests traits strongly associated with fitness will lack additive genetic variation, whereas traits less strongly associated with fitness are expected to exhibit more additive genetic variation. We use a quantitative genetics method, line cross analysis, to infer genetic architectures that contribute to trait divergence. By parsing over 1,600 datasets by trait type, clade, and cross divergence, we estimated the relative importance of epistasis across the tree of life. In our comparison between life-history traits and morphological traits, we found greater epistatic contributions to life-history traits. Our comparison between plants and animals showed that animals have more epistatic contribution to trait divergence than plants. In our comparison of within-species versus between-species crosses, we found that only animals exhibit a greater epistatic contribution to trait divergence as divergence increases. While many scientists have argued that epistasis is ultimately of little importance, our results show that epistasis underlies much of trait divergence and must be accounted for in theory and practical applications like domestication, conservation breeding design, and understanding complex diseases.

Drift drives the evolution of chromosome number I: The impact of trait transitions on genome evolution in Coleoptera.

Chromosomal mutations such as fusions and fissions are often thought to be deleterious, especially in heterozygotes (underdominant), and consequently are unlikely to become fixed. Yet, many models of chromosomal speciation ascribe an important role to chromosomal mutations. When the effective population size (Ne) is small, the efficacy of selection is weakened, and the likelihood of fixing underdominant mutations by genetic drift is greater. Thus, it is possible that ecological and phenotypic transitions that modulate Ne facilitate the fixation of chromosome changes, increasing the rate of karyotype evolution. We synthesize all available chromosome number data in Coleoptera and estimate the impact of traits expected to change Ne on the rate of karyotype evolution in the family Carabidae and 12 disparate clades from across Coleoptera. Our analysis indicates that in Carabidae, wingless clades have faster rates of chromosome number increase. Additionally, our analysis indicates clades exhibiting multiple traits expected to reduce Ne, including strict inbreeding, oligophagy, winglessness, and island endemism, have high rates of karyotype evolution. Our results suggest that chromosome number changes are likely fixed by genetic drift despite an initial fitness cost and that chromosomal speciation models may be important to consider in clades with very small Ne.

Divergence time estimation of genus Tribolium by extensive sampling of highly conserved orthologs.

Tribolium castaneum, the red flour beetle, is among the most well-studied eukaryotic genetic model organisms. Tribolium often serves as a comparative bridge from highly derived Drosophila traits to other organisms. Simultaneously, as a member of the most diverse order of metazoans, Coleoptera, Tribolium informs us about innovations that accompany hyper diversity. However, understanding the tempo and mode of evolutionary innovation requires well-resolved, time-calibrated phylogenies, which are not available for Tribolium. The most recent effort to understand Tribolium phylogenetics used two mitochondrial and three nuclear markers. The study concluded that the genus may be paraphyletic and reported a broad range for divergence time estimates. Here we employ recent advances in Bayesian methods to estimate the relationships and divergence times among Tribolium castaneum, T. brevicornis, T. confusum, T. freemani, and Gnatocerus cornutus using 1368 orthologs conserved across all five species and an independent substitution rate estimate. We find that the most basal split within Tribolium occurred ~86 Mya [95% HPD 85.90-87.04 Mya] and that the most recent split was between T. freemani and T. castaneum at ~14 Mya [95% HPD 13.55-14.00]. Our results are consistent with broader phylogenetic analyses of insects and suggest that Cenozoic climate changes played a role in the Tribolium diversification.

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.

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.

The within-host dynamics of infection in trans-generationally primed flour beetles.

Many taxa exhibit plastic immune responses initiated after primary microbial exposure that provide increased protection against disease-induced mortality and the fitness costs of infection. In several arthropod species, this protection can even be passed from parents to offspring through a phenomenon called trans-generational immune priming. Here, we first demonstrate that trans-generational priming is a repeatable phenomenon in flour beetles (Tribolium castaneum) primed and infected with Bacillus thuringiensis (Bt). We then quantify the within-host dynamics of microbes and host physiological responses in infected offspring from primed and unprimed mothers by monitoring bacterial density and using mRNA-seq to profile host gene expression, respectively, over the acute infection period. We find that priming increases inducible resistance against Bt around a critical temporal juncture where host septicaemic trajectories, and consequently survival, may be determined in unprimed individuals. Our results identify a highly differentially expressed biomarker of priming, containing an EIF4-e domain, in uninfected individuals, as well as several other candidate genes. Moreover, the induction and decay dynamics of gene expression over time suggest a metabolic shift in primed individuals. The identified bacterial and gene expression dynamics are likely to influence patterns of bacterial fitness and disease transmission in natural populations.

Genome of the Asian longhorned beetle (Anoplophora glabripennis), a globally significant invasive species, reveals key functional and evolutionary innovations at the beetle-plant interface.

BACKGROUND: Relatively little is known about the genomic basis and evolution of wood-feeding in beetles. We undertook genome sequencing and annotation, gene expression assays, studies of plant cell wall degrading enzymes, and other functional and comparative studies of the Asian longhorned beetle, Anoplophora glabripennis, a globally significant invasive species capable of inflicting severe feeding damage on many important tree species. Complementary studies of genes encoding enzymes involved in digestion of woody plant tissues or detoxification of plant allelochemicals were undertaken with the genomes of 14 additional insects, including the newly sequenced emerald ash borer and bull-headed dung beetle. RESULTS: The Asian longhorned beetle genome encodes a uniquely diverse arsenal of enzymes that can degrade the main polysaccharide networks in plant cell walls, detoxify plant allelochemicals, and otherwise facilitate feeding on woody plants. It has the metabolic plasticity needed to feed on diverse plant species, contributing to its highly invasive nature. Large expansions of chemosensory genes involved in the reception of pheromones and plant kairomones are consistent with the complexity of chemical cues it uses to find host plants and mates. CONCLUSIONS: Amplification and functional divergence of genes associated with specialized feeding on plants, including genes originally obtained via horizontal gene transfer from fungi and bacteria, contributed to the addition, expansion, and enhancement of the metabolic repertoire of the Asian longhorned beetle, certain other phytophagous beetles, and to a lesser degree, other phytophagous insects. Our results thus begin to establish a genomic basis for the evolutionary success of beetles on plants.

Haldane's Rule: Genetic Bases and Their Empirical Support.

There are few patterns in evolution that are as rigidly held as Haldane's rule (HR), which states, "When in the first generation between hybrids between 2 species, 1 sex is absent, rare, or sterile, that sex is always the heterogametic sex." Yet despite considerable attention for almost a century, questions persist as to how many independent examples exist and what is (are) the underlying genetic cause(s). Here, we review recent evidence extending HR to plants, where previously it has only been documented in animals. We also discuss recent comparative analyses that show much more variation in sex-chromosome composition than previously recognized, thus increasing the number of potential independent origins of HR dramatically. Finally, we review the standing of genetic theories proposed to explain HR in light of the new examples and new molecular understanding.

An information-theoretic approach to estimating the composite genetic effects contributing to variation among generation means: Moving beyond the joint-scaling test for line cross analysis.

The pace and direction of evolution in response to selection, drift, and mutation are governed by the genetic architecture that underlies trait variation. Consequently, much of evolutionary theory is predicated on assumptions about whether genes can be considered to act in isolation, or in the context of their genetic background. Evolutionary biologists have disagreed, sometimes heatedly, over which assumptions best describe evolution in nature. Methods for estimating genetic architectures that favor simpler (i.e., additive) models contribute to this debate. Here we address one important source of bias, model selection in line cross analysis (LCA). LCA estimates genetic parameters conditional on the best model chosen from a vast model space using relatively few line means. Current LCA approaches often favor simple models and ignore uncertainty in model choice. To address these issues we introduce Software for Analysis of Genetic Architecture (SAGA), which comprehensively assesses the potential model space, quantifies model selection uncertainty, and uses model weighted averaging to accurately estimate composite genetic effects. Using simulated data and previously published LCA studies, we demonstrate the utility of SAGA to more accurately define the components of complex genetic architectures, and show that traditional approaches have underestimated the importance of epistasis.

The fragile Y hypothesis: Y chromosome aneuploidy as a selective pressure in sex chromosome and meiotic mechanism evolution.

Loss of the Y-chromosome is a common feature of species with chromosomal sex determination. However, our understanding of why some lineages frequently lose Y-chromosomes while others do not is limited. The fragile Y hypothesis proposes that in species with chiasmatic meiosis the rate of Y-chromosome aneuploidy and the size of the recombining region have a negative correlation. The fragile Y hypothesis provides a number of novel insights not possible under traditional models. Specifically, increased rates of Y aneuploidy may impose positive selection for (i) gene movement off the Y; (ii) translocations and fusions which expand the recombining region; and (iii) alternative meiotic segregation mechanisms (achiasmatic or asynaptic). These insights as well as existing evidence for the frequency of Y-chromosome aneuploidy raise doubt about the prospects for long-term retention of the human Y-chromosome despite recent evidence for stable gene content in older non-recombining regions.

Genomic origins of insect sex chromosomes.

Recent efforts to catalog the diversity of sex chromosome systems coupled with genome sequencing projects are adding a new level of resolution to our understanding of insect sex chromosome origins. Y-chromosome degeneration makes sequencing difficult and may erase homology so rapidly that their origins will often remain enigmatic. X-chromosome origins are better understood, but thus far prove to be remarkably labile, often lacking homology even among close relatives. Furthermore, evidence now suggests that differentiated X or Y-chromosomes may both revert to autosomal inheritance. Data for ZW systems is scarcer, but W and Y-chromosomes seem to share many characteristics. Limited evidence suggests that Z-chromosome homology is more conserved than X counterparts, but broader sampling of both sex chromosome systems is needed.

Estimating tempo and mode of Y chromosome turnover: explaining Y chromosome loss with the fragile Y hypothesis.

Chromosomal sex determination is phylogenetically widespread, having arisen independently in many lineages. Decades of theoretical work provide predictions about sex chromosome differentiation that are well supported by observations in both XY and ZW systems. However, the phylogenetic scope of previous work gives us a limited understanding of the pace of sex chromosome gain and loss and why Y or W chromosomes are more often lost in some lineages than others, creating XO or ZO systems. To gain phylogenetic breadth we therefore assembled a database of 4724 beetle species' karyotypes and found substantial variation in sex chromosome systems. We used the data to estimate rates of Y chromosome gain and loss across a phylogeny of 1126 taxa estimated from seven genes. Contrary to our initial expectations, we find that highly degenerated Y chromosomes of many members of the suborder Polyphaga are rarely lost, and that cases of Y chromosome loss are strongly associated with chiasmatic segregation during male meiosis. We propose the "fragile Y" hypothesis, that recurrent selection to reduce recombination between the X and Y chromosome leads to the evolution of a small pseudoautosomal region (PAR), which, in taxa that require XY chiasmata for proper segregation during meiosis, increases the probability of aneuploid gamete production, with Y chromosome loss. This hypothesis predicts that taxa that evolve achiasmatic segregation during male meiosis will rarely lose the Y chromosome. We discuss data from mammals, which are consistent with our prediction.

Genetic architecture of isolation between two species of Silene with sex chromosomes and Haldane's rule.

Examination of the genetic architecture of hybrid breakdown can provide insight into the genetic mechanisms of commonly observed isolating phenomena such as Haldane's rule. We used line-cross analysis to dissect the genetic architecture of divergence between two plant species that exhibit Haldane's rule for male sterility and rarity, Silene latifolia and Silene diclinis. We made 15 types of crosses, including reciprocal F1, F2, backcrosses, and later-generation crosses, grew the seeds to flowering, and measured the number of viable ovules, proportion of viable pollen, and sex ratio. Typically, Haldane's rule for male rarity in XY animal hybrids is explained by interactions involving recessive X-linked alleles that are deleterious when hemizygous (dominance theory), whereas sterility is explained by rapid evolution of spermatogenesis genes (faster-male evolution). In contrast, we found that the genetic mechanisms underlying Haldane's rule between the two Silene species did not follow these conventions. Dominance theory was sufficient to explain male sterility, but male rarity likely involved faster-male evolution. We also found an effect of the neo-sex chromosomes of S. diclinis on the extreme rarity of some hybrid males. Our findings suggest that the genetic architecture of Haldane's rule in dioecious plants may differ from those commonly found in animals.

Gene expression levels are correlated with synonymous codon usage, amino acid composition, and gene architecture in the red flour beetle, Tribolium castaneum.

Gene expression levels correlate with multiple aspects of gene sequence and gene structure in phylogenetically diverse taxa, suggesting an important role of gene expression levels in the evolution of protein-coding genes. Here we present results of a genome-wide study of the influence of gene expression on synonymous codon usage, amino acid composition, and gene structure in the red flour beetle, Tribolium castaneum. Consistent with the action of translational selection, we find that synonymous codon usage bias increases with gene expression. However, the correspondence between tRNA gene copy number and optimal codons is weak. At the amino acid level, translational selection is suggested by the positive correlation between tRNA gene numbers and amino acid usage, which is stronger for highly expressed genes. In addition, there is a clear trend for increased use of metabolically cheaper, less complex amino acids as gene expression increases. tRNA gene numbers also correlate negatively with amino acid size/complexity (S/C) score indicating the coupling between translational selection and selection to minimize the use of large/complex amino acids. Interestingly, the analysis of 10 additional genomes suggests that the correlation between tRNA gene numbers and amino acid S/C score is widespread and might be explained by selection against negative consequences of protein misfolding. At the level of gene structure, three major trends are detected: 1) complete coding region length increases across low and intermediate expression levels but decreases in highly expressed genes; 2) the average intron size shows the opposite trend, first decreasing with expression, followed by a slight increase in highly expressed genes; and 3) intron density remains nearly constant across all expression levels. These changes in gene architecture are only in partial agreement with selection favoring reduced cost of biosynthesis.

Why chromosome palindromes?

We look at sex-limited chromosome (Y or W) evolution with particular emphasis on the importance of palindromes. Y chromosome palindromes consist of inverted duplicates that allow for local recombination in an otherwise nonrecombining chromosome. Since palindromes enable intrachromosomal gene conversion that can help eliminate deleterious mutations, they are often highlighted as mechanisms to protect against Y degeneration. However, the adaptive significance of recombination resides in its ability to decouple the evolutionary fates of linked mutations, leading to both a decrease in degeneration rate and an increase in adaptation rate. Our paper emphasizes the latter, that palindromes may exist to accelerate adaptation by increasing the potential targets and fixation rates of incoming beneficial mutations. This hypothesis helps reconcile two enigmatic features of the "palindromes as protectors" view: (1) genes that are not located in palindromes have been retained under purifying selection for tens of millions of years, and (2) under models that only consider deleterious mutations, gene conversion benefits duplicate gene maintenance but not initial fixation. We conclude by looking at ways to test the hypothesis that palindromes enhance the rate of adaptive evolution of Y-linked genes and whether this effect can be extended to palindromes on other chromosomes.

Haldane's rule in marsupials: what happens when both sexes are functionally hemizygous?

During the process of speciation, diverging taxa often hybridize and produce offspring wherein the heterogametic sex (i.e., XY or ZW) is unfit (Haldane's rule). Dominance theory seeks to explain Haldane's rule in terms of the difference in X-linked dominance regimes experienced by the sexes. However, X inactivation in female mammals extends the effects of hemizygosity to both sexes. Here, we highlight where the assumptions of dominance theory are particularly problematic in marsupials, where X inactivation uniformly results in silencing the paternal X. We then present evidence of Haldane's rule for sterility but not for viability in marsupials, as well as the first violations of Haldane's rule for these traits among all mammals. Marsupials represent a large taxonomic group possessing heteromorphic sex chromosomes, where the dominance theory cannot explain Haldane's rule. In this light, we evaluate alternative explanations for the preponderance of male sterility in interspecific hybrids, including faster male evolution, X-Y interactions, and genomic conflict hypotheses.

Sex, war, and disease: the role of parasite infection on weapon development and mating success in a horned beetle (Gnatocerus cornutus).

While parasites and immunity are widely believed to play important roles in the evolution of male ornaments, their potential influence on systems where male weaponry is the object of sexual selection is poorly understood. We experimentally infect larval broad-horned flour beetles with a tapeworm and study the consequent effects on: 1) adult male morphology 2) male-male contests for mating opportunities, and 3) induction of the innate immune system. We find that infection significantly reduces adult male size in ways that are expected to reduce mating opportunities in nature. The sum of our morphological, competition, and immunological data indicate that during a life history stage where no new resources are acquired, males allocate their finite resources in a way that increases future mating potential.

Hyperexpression of the X chromosome in both sexes results in extensive female bias of X-linked genes in the flour beetle.

A genome's ability to produce two separate sexually dimorphic phenotypes is an intriguing biological mystery. Microarray-based studies of a handful of model systems suggest that much of the mystery can be explained by sex-biased gene expression evolved in response to sexually antagonistic selection. We present the first whole-genome study of sex-biased expression in the red flour beetle, Tribolium castaneum. Tribolium is a model for the largest eukaryotic order, Coleoptera, and we show that in whole-body adults, approximately 20% of the transcriptome is differentially regulated between the sexes. Among T. castaneum, Drosophila melanogaster, and Anopheles gambiae, we identify 416 1:1:1 orthologs with conserved sex-biased expression. Overrepresented functional categories among sex-biased genes are primarily those involved in gamete production and development. The genomic distribution of sex-biased genes in T. castaneum is distinctly nonrandom, with the strongest deficit of male-biased genes on the X chromosome (9 of 793) of any species studied to date. Tribolium also shows a significant enrichment of X-linked female-biased genes (408 of 793). Our analyses suggest that the extensive female bias of Tribolium X chromosome gene expression is due to hyperexpression of X-linked genes in both males and females. We propose that the overexpression of X chromosomes in females is an evolutionary side effect of the need to dosage compensate in males and that mechanisms to reduce female X chromosome gene expression to autosomal levels are sufficient but imperfect.

Adaptive evolution of young gene duplicates in mammals.

Duplicate genes act as a source of genetic material from which new functions arise. They exist in large numbers in every sequenced eukaryotic genome and may be responsible for many differences in phenotypes between species. However, recent work searching for the targets of positive selection in humans has largely ignored duplicated genes due to complications in orthology assignment. Here we find that a high proportion of young gene duplicates in the human, macaque, mouse, and rat genomes have experienced adaptive natural selection. Approximately 10% of all lineage-specific duplicates show evidence for positive selection on their protein sequences, larger than any reported amount of selection among single-copy genes in these lineages using similar methods. We also find that newly duplicated genes that have been transposed to new chromosomal locations are significantly more likely to have undergone positive selection than the ancestral copy. Human-specific duplicates evolving under adaptive natural selection include a surprising number of genes involved in neuronal and cognitive functions. Our results imply that genome scans for selection that ignore duplicated loci are missing a large fraction of all adaptive substitutions. The results are also in agreement with the classical model of evolution by gene duplication, supporting a common role for neofunctionalization in the long-term maintenance of gene duplicates.

The life and death of gene families.

One of the unique insights provided by the growing number of fully sequenced genomes is the pervasiveness of gene duplication and gene loss. Indeed, several metrics now suggest that rates of gene birth and death per gene are only 10-40% lower than nucleotide substitutions per site, and that per nucleotide, the consequent lineage-specific expansion and contraction of gene families may play at least as large a role in adaptation as changes in orthologous sequences. While gene family evolution is pervasive, it may be especially important in our own evolution since it appears that the "revolving door" of gene duplication and loss has undergone multiple accelerations in the lineage leading to humans. In this paper, we review current understanding of gene family evolution including: methods for inferring copy number change, evidence for adaptive expansion and adaptive contraction of gene families, the origins of new families and deaths of previously established ones, and finally we conclude with a perspective on challenges and promising directions for future research.