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.

Redox signaling is important for embryogenesis, guiding pathways that govern processes crucial for embryo patterning, including cell polarization, proliferation, and apoptosis. Exposure to pro-oxidants during this period can be deleterious, resulting in altered physiology, teratogenesis, later-life diseases, or lethality. We previously reported that the glutathione antioxidant defense system becomes increasingly robust, including a doubling of total glutathione and dynamic shifts in the glutathione redox potential at specific stages during embryonic development in the zebrafish, Danio rerio. However, the mechanisms underlying these changes are unclear, as is the effectiveness of the glutathione system in ameliorating oxidative insults to the embryo at different stages. Here, we examine how the glutathione system responds to the model pro-oxidants tert-butylhydroperoxide and tert-butylhydroquinone at different developmental stages, and the role of Nuclear factor erythroid 2-related factor (Nrf) proteins in regulating developmental glutathione redox status. Embryos became increasingly sensitive to pro-oxidants after 72h post-fertilization (hpf), after which the duration of the recovery period for the glutathione redox potential was increased. To determine whether the doubling of glutathione or the dynamic changes in glutathione redox potential are mediated by zebrafish paralogs of Nrf transcription factors, morpholino oligonucleotides were used to knock down translation of Nrf1 and Nrf2 (nrf1a, nrf1b, nrf2a, nrf2b). Knockdown of Nrf1a or Nrf1b perturbed glutathione redox state until 72 hpf. Knockdown of Nrf2 paralogs also perturbed glutathione redox state but did not significantly affect the response of glutathione to pro-oxidants. Nrf1b morphants had decreased gene expression of glutathione synthesis enzymes, while hsp70 increased in Nrf2b morphants. This work demonstrates that despite having a more robust glutathione system, embryos become more sensitive to oxidative stress later in development, and that neither Nrf1 nor Nrf2 alone appear to be essential for the response and recovery of glutathione to oxidative insults.

Populations of the teleost fish Fundulus heteroclitus appear to flourish in heavily polluted and geographically separated Superfund sites. Populations from three Superfund sites (New Bedford Harbor, MA, Newark Bay, NJ, and Elizabeth River, VA) have independently evolved adaptive resistance to chemical pollutants. In these polluted populations, natural selection likely has altered allele frequencies of loci that affect fitness or that are linked to these loci. The aim of this study was to identify loci that exhibit non-neutral behavior in the F. heteroclitus genome in polluted populations versus clean reference populations.

Background: By targeting SNPs contained in both coding and non-coding areas of the genome, we are able to identify genetic differences and characterize genome-wide patterns of variation among individuals, populations and species. We investigated the utility of 454 sequencing and MassARRAY genotyping for population genetics in natural populations of the teleost, Fundulus heteroclitus as well as closely related Fundulus species (F. grandis, F. majalis and F. similis). Results: We used 454 pyrosequencing and MassARRAY genotyping technology to identify and type 458 genome-wide SNPs and determine genetic differentiation within and between populations and species of Fundulus. Specifically, pyrosequencing identified 96 putative SNPs across coding and non-coding regions of the F. heteroclitus genome: 88.8% were verified as true SNPs with MassARRAY. Additionally, putative SNPs identified in F. heteroclitus EST sequences were verified in most (86.5%) F. heteroclitus individuals; fewer were genotyped in F. grandis (74.4%), F. majalis (72.9%), and F. similis (60.7%) individuals. SNPs were polymorphic and showed latitudinal clinal variation separating northern and southern populations and established isolation by distance in F. heteroclitus populations. In F. grandis, SNPs were less polymorphic but still established isolation by distance. Markers differentiated species and populations. Conclusions: In total, these approaches were used to quickly determine differences within the Fundulus genome and provide markers for population genetic studies.

Evolution by natural selection acts on natural populations amidst migration, gene-by-environmental interactions, constraints, and tradeoffs, which affect the rate and frequency of adaptive change. We asked how many and how rapidly loci change in populations subject to severe, recent environmental changes. To address these questions, we used genomic approaches to identify randomly selected single nucleotide polymorphisms (SNPs) with evolutionarily significant patterns in three natural populations of Fundulus heteroclitus that inhabit and have adapted to highly polluted Superfund sites. Three statistical tests identified 1.4-2.5% of SNPs that were significantly different from the neutral model in each polluted population. These nonneutral patterns in populations adapted to highly polluted environments suggest that these loci or closely linked loci are evolving by natural selection. One SNP identified in all polluted populations using all tests is in the gene for the xenobiotic metabolizing enzyme, cytochrome P4501A (CYP1A), which has been identified previously as being refractory to induction in the three highly polluted populations. Extrapolating across the genome, these data suggest that rapid evolutionary change in natural populations can involve hundreds of loci, a few of which will be shared in independent events.

Transcription factors in the CNC-bZIP family (NFE2, NRF1, NRF2 and NRF3) regulate genes with a wide range of functions in response to both physiological and exogenous signals, including those indicating changes in cellular redox status. Given their role in helping to maintain cellular homeostasis, it is imperative to understand the expression, regulation, and function of CNC-bZIP genes during embryonic development. We explored the expression and function of six nrf genes (nfe2, nrf1a, nrf1b, nrf2a, nrf2b, and nrf3) using zebrafish embryos as a model system. Analysis by microarray and quantitative RT-PCR showed that genes in the nrf family were expressed throughout development from oocytes to larvae. The spatial expression of nrf3 suggested a role in regulating the development of the brain, brachia and pectoral fins. Knock-down by morpholino anti-sense oligonucleotides suggested that none of the genes were necessary for embryonic viability, but nfe2 was required for proper cellular organization in the pneumatic duct and subsequent swim bladder function, as well as for proper formation of the otic vesicles. nrf genes were induced by the oxidant tert-butylhydroperoxide, and some of this response was regulated through family members Nrf2a and Nrf2b. Our results provide a foundation for understanding the role of nrf genes in normal development and in regulating the response to oxidative stress in vertebrate embryos.