Andrew Whitehead

Variation within and among species in gene expression: raw material for evolution

Heritable variation in regulatory or coding regions is the raw material for evolutionary processes. The advent of microarrays has recently promoted examination of the extent of variation in gene expression within and among taxa and examination of the evolutionary processes affecting variation. This review examines these issues. We find: (i) microarray‐based measures of gene expression are precise given appropriate experimental design; (ii) there is large inter‐individual variation, which is composed of a minor nongenetic component and a large heritable component; (iii) variation among populations and species appears to be affected primarily by neutral drift and stabilizing selection, and to a lesser degree by directional selection; and (iv) neutral evolutionary divergence in gene expression becomes nonlinear with greater divergence times due to functional constraint. Evolutionary analyses of gene expression reviewed here provide unique insights into partitioning of regulatory variation in nature. However, common limitations of these studies include the tendency to assume a linear relationship between expression divergence and species divergence, and failure to test explicit hypotheses that involve the ecological context of evolutionary divergence.

Variation in tissue-specific gene expression among natural populations

BackgroundVariation in gene expression is extensive among tissues, individuals, strains, populations and species. The interactions among these sources of variation are relevant for physiological studies such as disease or toxic stress; for example, it is common for pathologies such as cancer, heart failure and metabolic disease to be associated with changes in tissue-specific gene expression or changes in metabolic gene expression. But how conserved these differences are among outbred individuals and among populations has not been well documented. To address this we examined the expression of a selected suite of 192 metabolic genes in brain, heart and liver in three populations of the teleost fish Fundulus heteroclitus using a highly replicated experimental design.ResultsHalf of the genes (48%) were differentially expressed among individuals within a population-tissue group and 76% were differentially expressed among tissues. Differences among tissues reflected well established tissue-specific metabolic requirements, suggesting that these measures of gene expression accurately reflect changes in proteins and their phenotypic effects. Remarkably, only a small subset (31%) of tissue-specific differences was consistent in all three populations.ConclusionsThese data indicate that many tissue-specific differences in gene expression are unique to one population and thus are unlikely to contribute to fundamental differences between tissue types. We suggest that those subsets of treatment-specific gene expression patterns that are conserved between taxa are most likely to be functionally related to the physiological state in question.

Transcriptomic variation and plasticity in rufous-collared sparrows (Zonotrichia capensis) along an altitudinal gradient

As modern genomic tools are developed for ecologically compelling models, field manipulation experiments will become important for establishing the role of functional genomic variation in physiological acclimation and evolutionary adaptation along environmental clines. High‐altitude habitats expose individuals to hypoxic and thermal stress, necessitating physiological acclimation, which may result in evolutionary adaptation. We assayed skeletal muscle transcriptomic profiles of rufous‐collared sparrows (Zonotrichia capensis) distributed along an altitudinal gradient on the Pacific slope of the Peruvian Andes. Nearly 200 unique transcripts were differentially expressed between high‐altitude [4100 m above sea level (a.s.l.)] and low‐altitude (2000 m a.s.l.) populations in their native habitats. Gene ontology and network analyses revealed that these transcripts are primarily involved in oxidative phosphorylation, protein biosynthesis, signal transduction and oxidative stress response pathways. To assess the plasticity in gene expression differences between populations, we performed a ‘common garden’ experiment in which high‐ and low‐altitude individuals were transferred to a common low‐altitude site (~150 m). None of the genes that were differentially expressed between populations at the native altitudes remained significantly different between populations in the common garden. The role of gene expression variation in adaptation and acclimation to environmental stress is largely unexplored in natural populations of birds. These results demonstrate substantial plasticity in the biochemical pathways that underpin cold and hypoxia compensation in Z. capensis, which may mechanistically contribute to enabling the broad altitudinal distribution of the species.

Comparative transcriptomics implicates mechanisms of evolved pollution tolerance in a killifish population.

Wild populations of the killifish Fundulus heteroclitus resident in heavily contaminated North American Atlantic coast estuaries have recently and independently evolved dramatic, heritable, and adaptive pollution tolerance. We compared physiological and transcriptome responses to embryonic polychlorinated biphenyl (PCB) exposures between one tolerant population and a nearby sensitive population to gain insight into genomic, physiological and biochemical mechanisms of evolved tolerance in killifish, which are currently unknown. The PCB exposure concentrations at which developmental toxicity emerged, the range of developmental abnormalities exhibited, and global as well as specific gene expression patterns were profoundly different between populations. In the sensitive population, PCB exposures produced dramatic, dose‐dependent toxic effects, concurrent with the alterations in the expression of many genes. For example, PCB‐mediated cardiovascular system failure was associated with the altered expression of cardiomyocyte genes, consistent with sarcomere mis‐assembly. In contrast, genome‐wide expression was comparatively refractory to PCB induction in the tolerant population. Tolerance was associated with the global blockade of the aryl hydrocarbon receptor (AHR) signalling pathway, the key mediator of PCB toxicity, in contrast to the strong dose‐dependent up‐regulation of AHR pathway elements observed in the sensitive population. Altered regulation of signalling pathways that cross‐talk with AHR was implicated as one candidate mechanism for the adaptive AHR signalling repression and the pollution tolerance that it affords. In addition to revealing mechanisms of PCB toxicity and tolerance, this study demonstrates the value of comparative transcriptomics to explore molecular mechanisms of stress response and evolved adaptive differences among wild populations.

Relative influences of historical and contemporary forces shaping the distribution of genetic variation in the Atlantic killifish, Fundulus heteroclitus

A major goal of population genetics research is to identify the relative influences of historical and contemporary processes that serve to structure genetic variation. Most population genetic models assume that populations exist in a state of migration‐drift equilibrium. However, in the past this assumption has rarely been verified, and is likely rarely achieved in natural populations. We assessed the equilibrium status at both local and regional scales of the Atlantic killifish, Fundulus heteroclitus. This species is a model organism for the study of adaptive clinal variation, but has also experienced a complicated history of range expansion and secondary contact following allopatric divergence, potentially obscuring the influence of contemporary evolutionary processes. Presumptively neutral genetic markers (microsatellites) demonstrated zones of secondary intergradation among coastal populations centred around northern New Jersey and the Chesapeake Bay region. Analysis of genetic variation indicated isolation by distance among some populations and provided supporting evidence that the Delaware Bay, but not the Chesapeake Bay, has acted as a barrier to dispersal among coastal populations. Bayesian estimates indicated large effective population sizes and low migration rates, and were in good agreement with empirically derived estimates of population and neighbourhood size from mark–recapture studies. These data indicate that populations are not in migration‐drift equilibrium at a regional scale, and suggest that contributing factors include large population size combined with relatively low migration rates. These conditions should be considered when interpreting the evolutionary significance of the distribution of genetic variation among F. heteroclitus populations.

Functional Genomics of Physiological Plasticity and Local Adaptation in Killifish

Evolutionary solutions to the physiological challenges of life in highly variable habitats can span the continuum from evolution of a cosmopolitan plastic phenotype to the evolution of locally adapted phenotypes. Killifish (Fundulus sp.) have evolved both highly plastic and locally adapted phenotypes within different selective contexts, providing a comparative system in which to explore the genomic underpinnings of physiological plasticity and adaptive variation. Importantly, extensive variation exists among populations and species for tolerance to a variety of stressors, and we exploit this variation in comparative studies to yield insights into the genomic basis of evolved phenotypic variation. Notably, species of Fundulus occupy the continuum of osmotic habitats from freshwater to marine and populations within Fundulus heteroclitus span far greater variation in pollution tolerance than across all species of fish. Here, we explore how transcriptome regulation underpins extreme physiological plasticity on osmotic shock and how genomic and transcriptomic variation is associated with locally evolved pollution tolerance. We show that F. heteroclitus quickly acclimate to extreme osmotic shock by mounting a dramatic rapid transcriptomic response including an early crisis control phase followed by a tissue remodeling phase involving many regulatory pathways. We also show that convergent evolution of locally adapted pollution tolerance involves complex patterns of gene expression and genome sequence variation, which is confounded with body-weight dependence for some genes. Similarly, exploiting the natural phenotypic variation associated with other established and emerging model organisms is likely to greatly accelerate the pace of discovery of the genomic basis of phenotypic variation.

THE EVOLUTIONARY RADIATION OF DIVERSE OSMOTOLERANT PHYSIOLOGIES IN KILLIFISH (FUNDULUS SP.)

Fish have radiated to exploit diverse habitats, but little is known about the evolutionary lability and directionality of associated physiological specialization. Killifish of the genus Fundulus present a compelling system to explore the evolution of osmotic tolerance because closely related species have evolved to occupy most osmotic niches, and physiological osmotic tolerance data are available for most species. This study seeks to determine the number of times, and the rate at which, alternate osmotic tolerance physiologies have evolved, and to determine the directionality of physiological transitions, by mapping comparative physiology data to a molecular phylogeny for the genus. Character mapping and phylogeographic inference indicate that freshwater tolerance is derived, can evolve rapidly, has evolved several times within the genus, and that variation in osmotic tolerance contributes to defining species distributions. The derivation of alternate physiologies within Fundulus appears associated with contraction of physiological plasticity rather than shifts in tolerance ranges, and the degree of contraction is surprisingly similar across convergent physiological types. The rate of physiological transition is relatively high within Fundulus compared to other taxa, but directionality from high salt tolerance to intolerance appears to be the rule. Together, these comparative physiology and phylogenetic data yield insight into the patterns of evolution of ecological specialization.

Simultaneous Extraction of High-Quality RNA and DNA from Small Tissue Samples

Purification of high-quality DNA and RNA from a single sample is becoming increasingly important for studies seeking both genomic and transcriptomic data. We compare different methods for isolating DNA and RNA from fish embryos (Gulf killifish; Fundulus grandis) and describe an optimal technique to extract high-quality DNA and RNA from a single embryo. The optimal method utilizes a chaotropic buffer and spin column technology. From embryos weighing approximately 4 mg, we were able to isolate an average of 6.1 microg of DNA and 1.1 microg of RNA per sample. Relative amounts of DNA and RNA can be adjusted as needed per study. Although these extraction trials were conducted on fish embryos, they can be potentially applied to small samples that typically do not yield high concentrations of nucleic acids.

The effects of mitochondrial genotype on hypoxic survival and gene expression in a hybrid population of the killifish, Fundulus heteroclitus.

The physiological link between oxygen availability and mitochondrial function is well established. However, whether or not fitness variation is associated with mitochondrial genotypes in the field remains a contested topic in evolutionary biology. In this study, we draw on a population of the teleost fish, Fundulus heteroclitus, where functionally distinct subspecies hybridize, likely as a result of past glacial events. We had two specific aims: (i) to determine the effect of mtDNA genotype on survivorship of male and female fish under hypoxic stress and (ii) to determine the effect of hypoxic stress, sex and mtDNA genotype on gene expression. We found an unexpected and highly significant effect of sex on survivorship under hypoxic conditions, but no significant effect of mtDNA genotype. Gene expression analyses revealed hundreds of transcripts differentially regulated by sex and hypoxia. Mitochondrial transcripts and other predicted pathways were among those influenced by hypoxic stress, and a transcript corresponding to the mtDNA control region was the most highly suppressed transcript under the conditions of hypoxia. An RT–PCR experiment on the control region was consistent with microarray results. Effects of mtDNA sequence variation on genome expression were limited; however, a potentially important epistasis between mtDNA sequence and expression of a nuclear‐encoded mitochondrial translation protein was discovered. Overall, these results confirm that mitochondrial regulation is a major component of hypoxia tolerance and further suggest that purifying selection has been the predominant selective force on mitochondrial genomes in these two subspecies.