Pleiotropic Constraints Research Articles

Rapid Gene Family Evolution of a Nematode Sperm Protein Despite Sequence Hyper-conservation.

Reproductive proteins are often observed to be the most rapidly evolving elements within eukaryotic genomes. The major sperm protein (MSP) is unique to the phylum Nematoda and is required for proper sperm locomotion and fertilization. Here, we annotate the MSP gene family and analyze their molecular evolution in 10 representative species across Nematoda. We show that MSPs are hyper-conserved across the phylum, having maintained an amino acid sequence identity of 83.5–97.7% for over 500 million years. This extremely slow rate of evolution makes MSPs some of the most highly conserved genes yet identified. However, at the gene family level, we show hyper-variability in both gene copy number and genomic position within species, suggesting rapid, lineage-specific gene family evolution. Additionally, we find evidence that extensive gene conversion contributes to the maintenance of sequence identity within chromosome-level clusters of MSP genes. Thus, while not conforming to the standard expectation for the evolution of reproductive proteins, our analysis of the molecular evolution of the MSP gene family is nonetheless consistent with the widely repeatable observation that reproductive proteins evolve rapidly, in this case in terms of the genomic properties of gene structure, copy number, and genomic organization. This unusual evolutionary pattern is likely generated by strong pleiotropic constraints acting on these genes at the sequence level, balanced against expansion at the level of the whole gene family.

Regulatory and evolutionary signatures of sex-biased genes on both the X chromosome and the autosomes

BackgroundSex is an important but understudied factor in the genetics of human diseases. Analyses using a combination of gene expression data, ENCODE data, and evolutionary data of sex-biased gene expression in human tissues can give insight into the regulatory and evolutionary forces acting on sex-biased genes.MethodsIn this study, we analyzed the differentially expressed genes between males and females. On the X chromosome, we used a novel method and investigated the status of genes that escape X-chromosome inactivation (escape genes), taking into account the clonality of lymphoblastoid cell lines (LCLs). To investigate the regulation of sex-biased differentially expressed genes (sDEG), we conducted pathway and transcription factor enrichment analyses on the sDEGs, as well as analyses on the genomic distribution of sDEGs. Evolutionary analyses were also conducted on both sDEGs and escape genes.ResultsGenome-wide, we characterized differential gene expression between sexes in 462 RNA-seq samples and identified 587 sex-biased genes, or 3.2% of the genes surveyed. On the X chromosome, sDEGs were distributed in evolutionary strata in a similar pattern as escape genes. We found a trend of negative correlation between the gene expression breadth and nonsynonymous over synonymous mutation (dN/dS) ratios, showing a possible pleiotropic constraint on evolution of genes. Genome-wide, nine transcription factors were found enriched in binding to the regions surrounding the transcription start sites of female-biased genes. Many pathways and protein domains were enriched in sex-biased genes, some of which hint at sex-biased physiological processes.ConclusionsThese findings lend insight into the regulatory and evolutionary forces shaping sex-biased gene expression and their involvement in the physiological and pathological processes in human health and diseases.

Constrained vertebrate evolution by pleiotropic genes.

Despite morphological diversification of chordates over 550 million years of evolution, their shared basic anatomical pattern (or 'bodyplan') remains conserved by unknown mechanisms. The developmental hourglass model attributes this to phylum-wide conserved, constrained organogenesis stages that pattern the bodyplan (the phylotype hypothesis); however, there has been no quantitative testing of this idea with a phylum-wide comparison of species. Here, based on data from early-to-late embryonic transcriptomes collected from eight chordates, we suggest that the phylotype hypothesis would be better applied to vertebrates than chordates. Furthermore, we found that vertebrates' conserved mid-embryonic developmental programmes are intensively recruited to other developmental processes, and the degree of the recruitment positively correlates with their evolutionary conservation and essentiality for normal development. Thus, we propose that the intensively recruited genetic system during vertebrates' organogenesis period imposed constraints on its diversification through pleiotropic constraints, which ultimately led to the common anatomical pattern observed in vertebrates.

Transposable Element Exaptation into Regulatory Regions Is Rare, Influenced by Evolutionary Age, and Subject to Pleiotropic Constraints.

Transposable element (TE)-derived sequences make up approximately half of most mammalian genomes, and many TEs have been co-opted into gene regulatory elements. However, we lack a comprehensive tissue- and genome-wide understanding of how and when TEs gain regulatory activity in their hosts. We evaluated the prevalence of TE-derived DNA in enhancers and promoters across hundreds of human and mouse cell lines and primary tissues. Promoters are significantly depleted of TEs in all tissues compared with their overall prevalence in the genome (P < 0.001); enhancers are also depleted of TEs, though not as strongly as promoters. The degree of enhancer depletion also varies across contexts (1.5-3×), with reproductive and immune cells showing the highest levels of TE regulatory activity in humans. Overall, in spite of the regulatory potential of many TE sequences, they are significantly less active in gene regulation than expected from their prevalence. TE age is predictive of the likelihood of enhancer activity; TEs originating before the divergence of amniotes are 9.2 times more likely to have enhancer activity than TEs that integrated in great apes. Context-specific enhancers are more likely to be TE-derived than enhancers active in multiple tissues, and young TEs are more likely to overlap context-specific enhancers than old TEs (86% vs. 47%). Once TEs obtain enhancer activity in the host, they have similar functional dynamics to one another and non-TE-derived enhancers, likely driven by pleiotropic constraints. However, a few TE families, most notably endogenous retroviruses, have greater regulatory potential. Our observations suggest a model of regulatory co-option in which TE-derived sequences are initially repressed, after which a small fraction obtains context-specific enhancer activity, with further gains subject to pleiotropic constraints.

Genotypic Complexity of Fisher’s Geometric Model

Fisher's geometric model was originally introduced to argue that complex adaptations must occur in small steps because of pleiotropic constraints. When supplemented with the assumption of additivity of mutational effects on phenotypic traits, it provides a simple mechanism for the emergence of genotypic epistasis from the nonlinear mapping of phenotypes to fitness. Of particular interest is the occurrence of reciprocal sign epistasis, which is a necessary condition for multipeaked genotypic fitness landscapes. Here we compute the probability that a pair of randomly chosen mutations interacts sign epistatically, which is found to decrease with increasing phenotypic dimension n, and varies nonmonotonically with the distance from the phenotypic optimum. We then derive expressions for the mean number of fitness maxima in genotypic landscapes comprised of all combinations of L random mutations. This number increases exponentially with L, and the corresponding growth rate is used as a measure of the complexity of the landscape. The dependence of the complexity on the model parameters is found to be surprisingly rich, and three distinct phases characterized by different landscape structures are identified. Our analysis shows that the phenotypic dimension, which is often referred to as phenotypic complexity, does not generally correlate with the complexity of fitness landscapes and that even organisms with a single phenotypic trait can have complex landscapes. Our results further inform the interpretation of experiments where the parameters of Fisher's model have been inferred from data, and help to elucidate which features of empirical fitness landscapes can be described by this model.

Evolutionary constraints shape caste-specific gene expression across 15 ant species.

Development of polymorphic phenotypes from similar genomes requires gene expression differences. However, little is known about how morph-specific gene expression patterns vary on a broad phylogenetic scale. We hypothesize that evolution of morph-specific gene expression, and consequently morph-specific phenotypic evolution, may be constrained by gene essentiality and the amount of pleiotropic constraints. Here, we use comparative transcriptomics of queen and worker morphs, that is, castes, from 15 ant species to understand the constraints of morph-biased gene expression. In particular, we investigate how measures of evolutionary constraints at the sequence level (expression level, connectivity, and number of gene ontology [GO] terms) correlate with morph-biased expression. Our results show that genes indeed vary in their potential to become morph-biased. The existence of genes that are constrained in becoming caste-biased potentially limits the evolutionary decoupling of the caste phenotypes, that is, it might result in "caste load" occasioning from antagonistic fitness variation, similarly to sexually antagonistic fitness variation between males and females. On the other hand, we suggest that genes under low constraints are released from antagonistic variation and thus more likely to be co-opted for morph specific use. Overall, our results suggest that the factors that affect sequence evolutionary rates and evolution of plastic expression may largely overlap.

Maternal vernalization and vernalization-pathway genes influence progeny seed germination.

Different life stages frequently respond to the same environmental cue to regulate development so that each life stage is matched to its appropriate season. We investigated how independently each life stage can respond to shared environmental cues, focusing on vernalization, in Arabidopsis thaliana plants. We first tested whether effects of rosette vernalization persisted to influence seed germination. To test whether genes in the vernalization flowering pathway also influence germination, we assessed germination of functional and nonfunctional alleles of these genes and measured their level of expression at different life stages in response to rosette vernalization. Rosette vernalization increased seed germination in diverse ecotypes. Genes in the vernalization flowering pathway also influenced seed germination. In the Columbia accession, functional alleles of most of these genes opposed the germination response observed in the ecotypes. Some genes influenced germination in a manner consistent with their known effects on FLOWERING LOCUS C gene regulation during the transition to flowering. Others did not, suggesting functional divergence across life stages. Despite persistent effects of environmental conditions across life stages, and despite pleiotropy of genes that affect both flowering and germination, the function of these genes can differ across life stages, potentially mitigating pleiotropic constraints and enabling independent environmental regulation of different life stages.

The landscape of extreme genomic variation in the highly adaptable Atlantic killifish.

Understanding and predicting the fate of populations in changing environments require knowledge about the mechanisms that support phenotypic plasticity and the adaptive value and evolutionary fate of genetic variation within populations. Atlantic killifish (Fundulus heteroclitus) exhibit extensive phenotypic plasticity that supports large population sizes in highly fluctuating estuarine environments. Populations have also evolved diverse local adaptations. To yield insights into the genomic variation that supports their adaptability, we sequenced a reference genome and 48 additional whole genomes from a wild population. Evolution of genes associated with cell cycle regulation and apoptosis is accelerated along the killifish lineage, which is likely tied to adaptations for life in highly variable estuarine environments. Genome-wide standing genetic variation, including nucleotide diversity and copy number variation, is extremely high. The highest diversity genes are those associated with immune function and olfaction, whereas genes under greatest evolutionary constraint are those associated with neurological, developmental, and cytoskeletal functions. Reduced genetic variation is detected for tight junction proteins, which in killifish regulate paracellular permeability that supports their extreme physiological flexibility. Low-diversity genes engage in more regulatory interactions than high-diversity genes, consistent with the influence of pleiotropic constraint on molecular evolution. High genetic variation is crucial for continued persistence of species given the pace of contemporary environmental change. Killifish populations harbor among the highest levels of nucleotide diversity yet reported for a vertebrate species, and thus may serve as a useful model system for studying evolutionary potential in variable and changing environments.

Why Do We Care for Old Parents? Evolutionary Genetic Model of Elderly Caring

From the standpoint of evolution, caring for old parents may be maladaptive, because they have ceased reproduction, so that the benefit for inclusive fitness may not be expected. Then why do we care for old parents? In this study, the evolution of care for the elderly was examined, by using an evolutionary genetic model, in which pleiotropic constraints between behaviors expressed in different social contexts among family members were assumed. It was suggested that establishing a solid relationship with parents during infancy should be selectively favorable, even though old parents have to be cared for in the future, but that caring for old parents may be excluded from the population if this behavior imposes high costs on reproduction of the younger generation. Despite the diminishing numbers of individuals within the population, care for the elderly may not be readily selected against, but at the same time this may not contribute to the rate of increase in population size. The significance of discussing the behavior of elderly caring from the standpoint of evolutionary genetics was emphasized.

Bacterial Quorum Sensing Stabilizes Cooperation by Optimizing Growth Strategies.

Cooperation is a fundamental problem for evolutionary biology to explain. Conditional participation through phenotypic plasticity driven by communication is a potential solution to this dilemma. Thus, among bacteria, QS has been proposed to be a proximate stabilizing mechanism for cooperative behaviors. Here, we empirically demonstrate that QS in V. harveyi prevents cheating and subsequent invasion by nonproducing defectors by maximizing the growth rate at low cell densities and the growth yield at high cell densities, whereas an unconditional cooperator is rapidly driven to extinction by defectors. Our findings provide experimental evidence that QS regulation prevents the invasion of cooperative populations by QS defectors even under unstructured conditions, and they strongly support the role of communication in bacteria as a mechanism that stabilizes cooperative traits.

Sexually antagonistic selection on genetic variation underlying both male and female same-sex sexual behavior.

BackgroundIntralocus sexual conflict, arising from selection for different alleles at the same locus in males and females, imposes a constraint on sex-specific adaptation. Intralocus sexual conflict can be alleviated by the evolution of sex-limited genetic architectures and phenotypic expression, but pleiotropic constraints may hinder this process. Here, we explored putative intralocus sexual conflict and genetic (co)variance in a poorly understood behavior with near male-limited expression. Same-sex sexual behaviors (SSBs) generally do not conform to classic evolutionary models of adaptation but are common in male animals and have been hypothesized to result from perception errors and selection for high male mating rates. However, perspectives incorporating sex-specific selection on genes shared by males and females to explain the expression and evolution of SSBs have largely been neglected.ResultsWe performed two parallel sex-limited artificial selection experiments on SSB in male and female seed beetles, followed by sex-specific assays of locomotor activity and male sex recognition (two traits hypothesized to be functionally related to SSB) and adult reproductive success (allowing us to assess fitness consequences of genetic variance in SSB and its correlated components). Our experiments reveal both shared and sex-limited genetic variance for SSB. Strikingly, genetically correlated responses in locomotor activity and male sex-recognition were associated with sexually antagonistic fitness effects, but these effects differed qualitatively between male and female selection lines, implicating intralocus sexual conflict at both male- and female-specific genetic components underlying SSB.ConclusionsOur study provides experimental support for the hypothesis that widespread pleiotropy generates pervasive intralocus sexual conflict governing the expression of SSBs, suggesting that SSB in one sex can occur due to the expression of genes that carry benefits in the other sex.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-016-0658-4) contains supplementary material, which is available to authorized users.

Divergence in gene expression within and between two closely related flycatcher species.

Relatively little is known about the character of gene expression evolution as species diverge. It is for instance unclear if gene expression generally evolves in a clock‐like manner (by stabilizing selection or neutral evolution) or if there are frequent episodes of directional selection. To gain insights into the evolutionary divergence of gene expression, we sequenced and compared the transcriptomes of multiple organs from population samples of collared (Ficedula albicollis) and pied flycatchers (F. hypoleuca), two species which diverged less than one million years ago. Ordination analysis separated samples by organ rather than by species. Organs differed in their degrees of expression variance within species and expression divergence between species. Variance was negatively correlated with expression breadth and protein interactivity, suggesting that pleiotropic constraints reduce gene expression variance within species. Variance was correlated with between‐species divergence, consistent with a pattern expected from stabilizing selection and neutral evolution. Using an expression PST approach, we identified genes differentially expressed between species and found 16 genes uniquely expressed in one of the species. For one of these, DPP7, uniquely expressed in collared flycatcher, the absence of expression in pied flycatcher could be associated with a ≈20‐kb deletion including 11 of 13 exons. This study of a young vertebrate speciation model system expands our knowledge of how gene expression evolves as natural populations become reproductively isolated.

Contrasting effects of intralocus sexual conflict on sexually antagonistic coevolution

Evolutionary conflict between the sexes can induce arms races in which males evolve traits that are detrimental to the fitness of their female partners, and vice versa. This interlocus sexual conflict (IRSC) has been proposed as a cause of perpetual intersexual antagonistic coevolution with wide-ranging evolutionary consequences. However, theory suggests that the scope for perpetual coevolution is limited, if traits involved in IRSC are subject to pleiotropic constraints. Here, we consider a biologically plausible form of pleiotropy that has hitherto been ignored in treatments of IRSC and arrive at drastically different conclusions. Our analysis is based on a quantitative genetic model of sexual conflict, in which genes controlling IRSC traits have side effects in the other sex, due to incompletely sex-limited gene expression. As a result, the genes are exposed to intralocus sexual conflict (IASC), a tug-of-war between opposing male- and female-specific selection pressures. We find that the interaction between the two forms of sexual conflict has contrasting effects on antagonistic coevolution: Pleiotropic constraints stabilize the dynamics of arms races if the mating traits are close to evolutionary equilibrium but can prevent populations from ever reaching such a state. Instead, the sexes are drawn into a continuous cycle of arms races, causing the buildup of IASC, alternated by phases of IASC resolution that trigger the next arms race. These results encourage an integrative perspective on the biology of sexual conflict and generally caution against relying exclusively on equilibrium stability analysis.

Natural variation, differentiation, and genetic trade-offs of ecophysiological traits in response to water limitation in Brachypodium distachyon and its descendent allotetraploid B. hybridum (Poaceae).

Differences in tolerance to water stress may underlie ecological divergence of closely related ploidy lineages. However, the mechanistic basis of physiological variation governing ecogeographical cytotype segregation is not well understood. Here, using Brachypodium distachyon and its derived allotetraploid B. hybridum as model, we test the hypothesis that, for heteroploid annuals, ecological divergence of polyploids in drier environments is based on trait differentiation enabling drought escape. We demonstrate that under water limitation allotetraploids maintain higher photosynthesis and stomatal conductance and show earlier flowering than diploids, concordant with a drought-escape strategy to cope with water stress. Increased heterozygosity and greater genetic variability and plasticity of polyploids could confer a superior adaptive capability. Consistent with these predictions, we document (1) greater standing within-population genetic variation in water-use efficiency (WUE) and flowering time in allotetraploids, and (2) the existence of (nonlinear) environmental clines in physiology across allotetraploid populations. Increased gas exchange and diminished WUE occurred at the driest end of the gradient, consistent with a drought-escape strategy. Finally, we found that allotetraploids showed weaker genetic correlations than diploids congruous with the expectation of relaxed pleiotropic constraints in polyploids. Our results suggest evolutionary divergence of ecophysiological traits in each ploidy lineage.

Complex constraints on allometry revealed by artificial selection on the wing of Drosophila melanogaster

Precise exponential scaling with size is a fundamental aspect of phenotypic variation. These allometric power laws are often invariant across taxa and have long been hypothesized to reflect developmental constraints. Here we test this hypothesis by investigating the evolutionary potential of an allometric scaling relationship in drosophilid wing shape that is nearly invariant across 111 species separated by at least 50 million years of evolution. In only 26 generations of artificial selection in a population of Drosophila melanogaster, we were able to drive the allometric slope to the outer range of those found among the 111 sampled species. This response was rapidly lost when selection was suspended. Only a small proportion of this reversal could be explained by breakup of linkage disequilibrium, and direct selection on wing shape is also unlikely to explain the reversal, because the more divergent wing shapes produced by selection on the allometric intercept did not revert. We hypothesize that the reversal was instead caused by internal selection arising from pleiotropic links to unknown traits. Our results also suggest that the observed selection response in the allometric slope was due to a component expressed late in larval development and that variation in earlier development did not respond to selection. Together, these results are consistent with a role for pleiotropic constraints in explaining the remarkable evolutionary stability of allometric scaling.

Hybrid incompatibility despite pleiotropic constraint in a sequence-based bioenergetic model of transcription factor binding.

Hybrid incompatibility can result from gene misregulation produced by divergence in trans-acting regulatory factors and their cis-regulatory targets. However, change in trans-acting factors may be constrained by pleiotropy, which would in turn limit the evolution of incompatibility. We employed a mechanistically explicit bioenergetic model of gene expression wherein parameter combinations (number of transcription factor molecules, energetic properties of binding to the regulatory site, and genomic background size) determine the shape of the genotype-phenotype (G-P) map, and interacting allelic variants of mutable cis and trans sites determine the phenotype along that map. Misregulation occurs when the phenotype differs from its optimal value. We simulated a pleiotropic regulatory pathway involving a positively selected and a conserved trait regulated by a shared transcription factor (TF), with two populations evolving in parallel. Pleiotropic constraints shifted evolution in the positively selected trait to its cis-regulatory locus. We nevertheless found that the TF genotypes often evolved, accompanied by compensatory evolution in the conserved trait, and both traits contributed to hybrid misregulation. Compensatory evolution resulted in "developmental system drift," whereby the regulatory basis of the conserved phenotype changed although the phenotype itself did not. Pleiotropic constraints became stronger and in some cases prohibitive when the bioenergetic properties of the molecular interaction produced a G-P map that was too steep. Likewise, compensatory evolution slowed and hybrid misregulation was not evident when the G-P map was too shallow. A broad pleiotropic "sweet spot" nevertheless existed where evolutionary constraints were moderate to weak, permitting substantial hybrid misregulation in both traits. None of these pleiotropic constraints manifested when the TF contained nonrecombining domains independently regulating the respective traits.

Role of pleiotropy during adaptation of TEM-1 β-lactamase to two novel antibiotics.

Pleiotropy is a key feature of the genotype–phenotype map, and its form and extent have many evolutionary implications, including for the dynamics of adaptation and the evolution of specialization. Similarly, pleiotropic effects of antibiotic resistance mutations may affect the evolution of antibiotic resistance in the simultaneous or fluctuating presence of different antibiotics. Here, we study the role of pleiotropy during the in vitro adaptation of the enzyme TEM-1 β-lactamase to two novel antibiotics, cefotaxime (CTX) and ceftazidime (CAZ). We subject replicate lines for four rounds of evolution to selection with CTX and CAZ alone, and in their combined and fluctuating presence. Evolved alleles show positive correlated responses when selecting with single antibiotics. Nevertheless, pleiotropic constraints are apparent from the effects of single mutations and from selected alleles showing smaller correlated than direct responses and smaller responses after simultaneous and fluctuating selection with both than with single antibiotics. We speculate that these constraints result from structural changes in the oxyanion pocket surrounding the active site, where accommodation of CTX and the larger CAZ is balanced against their positioning with respect to the active site. Our findings suggest limited benefits from the combined or fluctuating application of these related cephalosporins for containing antibiotic resistance.

The Ontogeny and Evolution of Sex-Biased Gene Expression in Drosophila melanogaster

Sexually dimorphic phenotypes are thought to largely result from sex differences in gene expression, and genes with sex-biased expression have been well characterized in adults of many species. Although most sexual dimorphisms manifest in adults, many result from sex-specific developmental trajectories, implying that juveniles may exhibit significant levels of sex-biased expression. However, it is unclear how much sex-biased expression occurs before reproductive maturity and whether preadult sex-biased genes should exhibit the same evolutionary dynamics observed for adult sex-biased genes. In order to understand the continuity, or lack thereof, and evolutionary dynamics of sex-biased expression throughout the life cycle, we examined sex-biased genes in pre-gonad tissue of two preadult stages and compared them with the adult gonad of Drosophila melanogaster. We found that the majority of the genome is sex-biased at some point in the life cycle, with some genes exhibiting conserved sex-biased expression and others displaying stage-specific sex bias. Our results also reveal a far more complex pattern of evolution for sex-biased genes throughout development. The most rapid evolutionary divergence occurred in genes expressed only in larvae within each sex, compared with continuously expressed genes. In females—but not males—this pattern appeared to be due to relaxed purifying selection in larva-limited genes. Furthermore, genes that retained male bias throughout life evolved more rapidly than stage-specific male-biased genes, due to stronger purifying selection in stage-specific genes. However, female-biased genes that were specific to larvae evolved most rapidly, a pattern that could not be definitively attributed to differences in adaptive evolution or purifying selection, suggesting that pleiotropic constraints on protein-coding sequences can arise when genes are broadly expressed across developmental stages. These results indicate that the signature of sex-specific selection can be detected well before reproductive maturity and is strongest during development.

A comparative study indicates both positive and purifying selection within ryanodine receptor (RyR) genes, as well as correlated evolution

Ryanodine receptors are Ca(2+) ion channels that allow Ca(2+) to flow into the cytosol, from an internal store, in the form of transients. RyRs form a small gene family and vertebrates have three major isoforms, RyR1, RyR2, and RyR3, which are mixed and matched in different combinations in different tissues resulting in different Ca(2+) transients for each tissue. In this study, we characterized the interspecies evolution of RyRs within vertebrates. First, we compared the nucleotide divergence of key gene regions including divergent regions (DRs), which are believed to be responsible for the functional divergence between RyRs, and mutation hot-spot regions, which are responsible for RyR-related pathologies. We found evidence that DRs undergo positive selection and mutation hot-spot regions undergo purifying selection. Second, we estimated the extent of purifying selection for RyR1, RyR2, and RyR3 by estimating dN/dS ratios. We found all three genes to be under strong purifying selection, overall. This is consistent with RyRs being used in a diverse set of physiological contexts and therefore under potentially high pleiotropic constraint. Third, we tested for the correlated evolution of dN/dS ratios between RyR genes. We found that RyR2 and RyR3, and RyR3 and a skeletal form of dihydropyridine receptor (DHPR) have correlated rates of evolution. We propose that compensatory effects may explain their correlated evolution. We tested for compensatory function by simulating mutations via a physiological model of RyR function, but did not find evidence for compensation, which indicates that the correlation is likely a result of another process.

Pleiotropic Constraints, Expression Level, and the Evolution of miRNA Sequences

Post-transcriptional gene regulation mediated by microRNAs (miRNAs) plays critical roles during development by modulating gene expression and conferring robustness to stochastic errors. Phylogenetic analyses suggest that miRNA acquisition could play a role in phenotypic innovation. Moreover, miRNA-induced regulation strongly impacts genome evolution, increasing selective constraints on 3'UTRs, protein sequences, and expression level divergence. Thus, it is essential to understand the factors governing sequence evolution for this important class of regulatory molecules. Investigation of the patterns of molecular evolution at miRNA loci have been limited in Caenorhabditis elegans because of the lack of a close outgroup. Instead, I used Caenorhabditis briggsae as the focus point of this study because of its close relationship to Caenorhabditis sp. 9. I also corroborated the patterns of sequence evolution in Caenorhabditis using published orthologous relationships among miRNAs in Drosophila. In nematodes and in flies, miRNA sequence divergence is not influenced by the genomic neighborhood (i.e., intronic or intergenic) but is nevertheless affected by the genomic context because X-linked miRNAs evolve faster than autosomal miRNAs. However, this effect of chromosomal linkage can be explained by differential expression levels rather than a fast-X effect. The results presented here support a universal negative relationship between rates of molecular evolution and expression level, and suggest that mutations in highly expressed miRNAs are more likely to be deleterious because they potentially affect a larger number of target genes. Finally, I show that many single family member miRNAs evolve faster than miRNAs from multigene families and have limited functional scope, suggesting that they are not strongly integrated in gene regulatory networks.