We present a de novo assembly of a eukaryote transcriptome using 454 pyrosequencing data. The Glanville fritillary butterfly (Melitaea cinxia; Lepidoptera: Nymphalidae) is a prominent species in population biology but had no previous genomic data. Sequencing runs using two normalized complementary DNA collections from a genetically diverse pool of larvae, pupae, and adults yielded 608,053 expressed sequence tags (mean length = 110 nucleotides), which assembled into 48,354 contigs (sets of overlapping DNA segments) and 59,943 singletons. BLAST comparisons confirmed the accuracy of the sequencing and assembly, and indicated the presence of c. 9000 unique genes, along with > 6000 additional microarray-confirmed unannotated contigs. Average depth of coverage was 6.5-fold for the longest 4800 contigs (348-2849 bp in length), sufficient for detecting large numbers of single nucleotide polymorphisms. Oligonucleotide microarray probes designed from the assembled sequences showed highly repeatable hybridization intensity and revealed biological differences among individuals. We conclude that 454 sequencing, when performed to provide sufficient coverage depth, allows de novo transcriptome assembly and a fast, cost-effective, and reliable method for development of functional genomic tools for nonmodel species. This development narrows the gap between approaches based on model organisms with rich genetic resources vs. species that are most tractable for ecological and evolutionary studies.

Evolution may depend more strongly on variation in gene expression than on differences between variant forms of proteins. Regions of DNA that affect gene expression are highly variable, containing 0.6% polymorphic sites. These naturally occurring polymorphic nucleotides can alter in vivo transcription rates. Thus, one might expect substantial variation in gene expression between individuals. But the natural variation in mRNA expression for a large number of genes has not been measured. Here we report microarray studies addressing the variation in gene expression within and between natural populations of teleost fish of the genus Fundulus. We observed statistically significant differences in expression between individuals within the same population for approximately 18% of 907 genes. Expression typically differed by a factor of 1.5, and often more than 2.0. Differences between populations increased the variation. Much of the variation between populations was a positive function of the variation within populations and thus is most parsimoniously described as random. Some genes showed unexpected patterns of expression--changes unrelated to evolutionary distance. These data suggest that substantial natural variation exists in gene expression and that this quantitative variation is important in evolution.

Atlantic killifish populations have rapidly adapted to normally lethal levels of pollution in four urban estuaries. Through analysis of 384 whole killifish genome sequences and comparative transcriptomics in four pairs of sensitive and tolerant populations, we identify the aryl hydrocarbon receptor-based signaling pathway as a shared target of selection. This suggests evolutionary constraint on adaptive solutions to complex toxicant mixtures at each site. However, distinct molecular variants apparently contribute to adaptive pathway modification among tolerant populations. Selection also targets other toxicity-mediating genes, and genes of connected signaling pathways, indicating complex tolerance phenotypes and potentially compensatory adaptations. Molecular changes are consistent with selection on standing genetic variation. In killifish high nucleotide diversity has likely been a crucial substrate for selective sweeps to propel rapid adaptation.

Variation among populations in gene expression should be related to the accumulation of random-neutral changes and evolution by natural selection. The following evolutionary analysis has general applicability to biological and medical science because it accounts for genetic relatedness and identifies patterns of expression variation that are affected by natural selection. To identify genes evolving by natural selection, we allocate the maximum amongpopulation variation to genetic distance and then examine the remaining variation relative to a hypothesized important ecological parameter (temperature). These analyses measure the expression of metabolic genes in common-gardened populations of the fish Fundulus heteroclitus whose habitat is distributed along a steep thermal gradient. Although much of the variation in gene expression fits a null model of neutral drift, the variation in expression for 22% of the genes that regress with habitat temperature was far greater than could be accounted for by genetic distance alone. The most parsimonious explanation for amongpopulation variation for these genes is evolution by natural selection. In addition, many metabolic genes have patterns of variation incongruent with neutral evolution: They have too much or too little variation. These patterns of biological variation in expression may reflect important physiological or ecological functions.evolutionary analysis ͉ Fundulus ͉ microarray ͉ phylogenetic comparative approach ͉ genomics G ene expression has been hypothesized to be of adaptive importance (1), and heritable variation that affects fitness is necessary for evolution by natural selection. Although adaptive differences in expression have been identified in single-gene studies (2-6; see ref. 7 for review), microarray approaches offer great promise to rigorously address this hypothesis because they assay many loci at once. Furthermore, it is generally agreed that much of variation in gene expression for a particular environmental condition has a genetic basis (8, 9) according to studies in yeast (10-13), Drosophila (14-18), mice (19), and humans (20)(21)(22).Widespread heritable variation combined with extensive natural variation in gene expression revealed by microarray studies (11,13,14,(23)(24)(25)(26) provides the substrates for evolution. However, two evolutionary forces govern the variance of traits among taxa: neutral drift and natural selection. Under a neutral drift model, the variation in a trait has little biological effect § and is a function of genetic distance: Traits will be more similar among closely related taxa than among more distantly related taxa (27)(28)(29). If natural selection has occurred, the variation in a trait affects an organism's fitness and is a function of the ecological setting: Traits are conserved or diverge depending on the specific ecological pressures (30). Recent studies suggest that much of the extensive variation in gene expression among individuals and taxa is simply random neutral divergence (31, 32), whereas others have fo...

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 interindividual 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.

A strong foundation of basic and applied research documents that the estuarine fish Fundulus heteroclitus and related species are unique laboratory and field models for understanding how individuals and populations interact with their environment. In this paper we summarize an extensive body of work examining the adaptive responses of Fundulus species to environmental conditions, and describe how this research has contributed importantly to our understanding of physiology, gene regulation, toxicology, and ecological and evolutionary genetics of teleosts and other vertebrates. These explorations have reached a critical juncture at which advancement is hindered by the lack of genomic resources for these species. We suggest that a more complete genomics toolbox for F. heteroclitus and related species will permit researchers to exploit the power of this model organism to rapidly advance our understanding of fundamental biological and pathological mechanisms among vertebrates, as well as ecological strategies and evolutionary processes common to all living organisms.

Individual variation in gene expression is important for evolutionary adaptation and susceptibility to diseases and pathologies. In this study, we address the functional importance of this variation by comparing cardiac metabolism to patterns of mRNA expression using microarrays. There is extensive variation in both cardiac metabolism and the expression of metabolic genes among individuals of the teleost fish Fundulus heteroclitus from natural outbred populations raised in a common environment: metabolism differed among individuals by a factor of more than 2, and expression levels of 94% of genes were significantly different (P < 0.01) between individuals in a population. This unexpectedly high variation in metabolic gene expression explains much of the variation in metabolism, suggesting that it is biologically relevant. The patterns of gene expression that are most important in explaining cardiac metabolism differ between groups of individuals. Apparently, the variation in metabolism seems to be related to different patterns of gene expression in the different groups of individuals. The magnitude of differences in gene expression in these groups is not important; large changes in expression have no greater predictive value than small changes. These data suggest that variation in physiological performance is related to the subtle variation in gene expression and that this relationship differs among individuals.

Fish offer important advantages for defining the organism-environment interface and responses to natural or anthropogenic stressors. Genomic approaches using fish promise increased investigative power, and have already provided insights into the mechanisms that underlie short-term and long-term environmental adaptations. The range of fish species for which genomic resources are available is increasing, but will require significant further expansion for the optimal application of fish environmental genomics.