Signatures of selection in natural populations adapted to chronic pollution

<p>Abstract</p> <p>Background</p> <p>Populations of the teleost fish <it>Fundulus heteroclitus </it>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 <it>F. heteroclitus </it>genome in polluted populations <it>versus </it>clean reference populations.</p> <p>Results</p> <p>To detect signatures of natural selection and thus identify genetic bases for adaptation to anthropogenic stressors, we examined allele frequencies for many hundreds of amplified fragment length polymorphism markers among populations of <it>F. heteroclitus</it>. Specifically, we contrasted populations from three Superfund sites (New Bedford Harbor, MA, Newark Bay, NJ, and Elizabeth River, VA) to clean reference populations flanking the polluted sites. When empirical F_STvalues were compared to a simulated distribution of F_STvalues, 24 distinct outlier loci were identified among pairwise comparisons of pollutant impacted <it>F. heteroclitus </it>populations and both surrounding reference populations. Upon removal of all outlier loci, there was a strong correlation (R²= 0.79, p < 0.0001) between genetic and geographical distance. This apparently neutral evolutionary pattern was not evident when outlier loci were included (R²= 0.092, p = 0.0721). Two outlier loci were shared between New Bedford Harbor and Elizabeth River populations, and two different loci were shared between Newark Bay and Elizabeth River populations.</p> <p>Conclusion</p> <p>In total, 1% to 6% of loci are implicated as being under selection or linked to areas of the genome under selection in three <it>F. heteroclitus </it>populations that reside in polluted estuaries. Shared loci among polluted sites indicate that selection may be acting on multiple loci involved in adaptation, and loci shared between polluted sites potentially are involved in a generalized adaptive response.</p

Convergence and divergence in gene expression among natural populations exposed to pollution

<p>Abstract</p> <p>Background</p> <p>Natural populations of the teleost fish <it>Fundulus heteroclitus </it>tolerate a broad range of environmental conditions including temperature, salinity, hypoxia and chemical pollutants. Strikingly, populations of <it>Fundulus </it>inhabit and have adapted to highly polluted Superfund sites that are contaminated with persistent toxic chemicals. These natural populations provide a foundation to discover critical gene pathways that have evolved in a complex natural environment in response to environmental stressors.</p> <p>Results</p> <p>We used <it>Fundulus </it>cDNA arrays to compare metabolic gene expression patterns in the brains of individuals among nine populations: three independent, polluted Superfund populations and two genetically similar, reference populations for each Superfund population. We found that up to 17% of metabolic genes have evolved adaptive changes in gene expression in these Superfund populations. Among these genes, two (1.2%) show a conserved response among three polluted populations, suggesting common, independently evolved mechanisms for adaptation to environmental pollution in these natural populations.</p> <p>Conclusion</p> <p>Significant differences among individuals between polluted and reference populations, statistical analyses indicating shared adaptive changes among the Superfund populations, and lack of reduction in gene expression variation suggest that common mechanisms of adaptive resistance to anthropogenic pollutants have evolved independently in multiple <it>Fundulus </it>populations. Among three independent, Superfund populations, two genes have a common response indicating that high selective pressures may favor specific responses.</p

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 American Genetic Association. 2010. All rights reserved. For permissions, please email: [email protected] © The American Genetic Association. 2010. All rights reserved. For permissions, please email: [email protected]

Can diversifying selection be distinguished from history in geographic clines? A population genomic study of killifish (Fundulus heteroclitus)

© The Author(s), 2012. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS ONE 7 (2012): e45138, doi:10.1371/journal.pone.0045138.A common geographical pattern of genetic variation is the one-dimensional cline. Clines may be maintained by diversifying selection across a geographical gradient but can also reflect historical processes such as allopatry followed by secondary contact. To identify loci that may be undergoing diversifying selection, we examined the distribution of geographical variation patterns across the range of the killifish (Fundulus heteroclitus) in 310 loci, including microsatellites, allozymes, and single nucleotide polymorphisms. We employed two approaches to detect loci under strong diversifying selection. First, we developed an automated method to identify clinal variation on a per-locus basis and examined the distribution of clines to detect those that exhibited signifcantly steeper slopes. Second, we employed a classic -outlier method as a complementary approach. We also assessed performance of these techniques using simulations. Overall, latitudinal clines were detected in nearly half of all loci genotyped (i.e., all eight microsatellite loci, 12 of 16 allozyme loci and 44% of the 285 SNPs). With the exception of few outlier loci (notably mtDNA and malate dehydrogenase), the positions and slopes of Fundulus clines were statistically indistinguishable. The high frequency of latitudinal clines across the genome indicates that secondary contact plays a central role in the historical demography of this species. Our simulation results indicate that accurately detecting diversifying selection using genome scans is extremely difficult in species with a strong signal of secondary contact; neutral evolution under this history produces clines as steep as those expected under selection. Based on these results, we propose that demographic history can explain all clinal patterns observed in F. heteroclitus without invoking natural selection to either establish or maintain the pattern we observe today.This work was supported by the National Science Foundation (DEB-0919064 and IOS-105226

SNP identification, verification, and utility for population genetics in a non-model genus

<p>Abstract</p> <p>Background</p> <p>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, <it>Fundulus heteroclitus </it>as well as closely related <it>Fundulus </it>species (<it>F. grandis</it>, <it>F. majalis </it>and <it>F. similis</it>).</p> <p>Results</p> <p>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 <it>Fundulus</it>. Specifically, pyrosequencing identified 96 putative SNPs across coding and non-coding regions of the <it>F. heteroclitus </it>genome: 88.8% were verified as true SNPs with MassARRAY. Additionally, putative SNPs identified in <it>F. heteroclitus </it>EST sequences were verified in most (86.5%) <it>F. heteroclitus </it>individuals; fewer were genotyped in <it>F. grandis </it>(74.4%), <it>F. majalis </it>(72.9%), and <it>F. similis </it>(60.7%) individuals. SNPs were polymorphic and showed latitudinal clinal variation separating northern and southern populations and established isolation by distance in <it>F. heteroclitus </it>populations. In <it>F. grandis</it>, SNPs were less polymorphic but still established isolation by distance. Markers differentiated species and populations.</p> <p>Conclusions</p> <p>In total, these approaches were used to quickly determine differences within the <it>Fundulus </it>genome and provide markers for population genetic studies.</p

Genetic and Biophysical Modelling Evidence of Generational Connectivity in the Intensively Exploited, Western North Atlantic Red Grouper (Epinephelus morio)

Understanding the connectivity of reef organisms is important to assist in the conservation of biological diversity and to facilitate sustainable fisheries in these ecosystems. Common methods to assess reef connectivity include both population genetics and biophysical modelling. Individually, these techniques can offer insight into population structure; however, the information acquired by any singular analysis is often subject to limitations, underscoring the need for a multi-faceted approach. To assess the connectivity dynamics of the red grouper (Epinephelus morio), an economically important reef fish species found throughout the Gulf of Mexico and USA western Atlantic, we utilized two sets of genetic markers (12 microsatellite loci and 632 single nucleotide polymorphisms) to resolve this species’ population genetic structure, along with biophysical modelling to deliver a spatial forecast of potential larval “sources” and “sinks” across these same regions and spatial scale. Our genetic survey indicates little, if any, evidence of population genetic structure and modelling efforts indicate the potential for ecological connectivity between sampled regions over multiple generations. We offer that using a dual empirical and theoretical approach lessens the error associated with the use of any single method and provides an important step towards the validation of either of these methodologies

Transcriptomic assessment of resistance to effects of an aryl hydrocarbon receptor (AHR) agonist in embryos of Atlantic killifish (Fundulus heteroclitus) from a marine Superfund site

© The Author(s), 2011. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in BMC Genomics 12 (2011): 263, doi:10.1186/1471-2164-12-263.Populations of Atlantic killifish (Fundulus heteroclitus) have evolved resistance to the embryotoxic effects of polychlorinated biphenyls (PCBs) and other halogenated and nonhalogenated aromatic hydrocarbons that act through an aryl hydrocarbon receptor (AHR)-dependent signaling pathway. The resistance is accompanied by reduced sensitivity to induction of cytochrome P450 1A (CYP1A), a widely used biomarker of aromatic hydrocarbon exposure and effect, but whether the reduced sensitivity is specific to CYP1A or reflects a genome-wide reduction in responsiveness to all AHR-mediated changes in gene expression is unknown. We compared gene expression profiles and the response to 3,3',4,4',5-pentachlorobiphenyl (PCB-126) exposure in embryos (5 and 10 dpf) and larvae (15 dpf) from F. heteroclitus populations inhabiting the New Bedford Harbor, Massachusetts (NBH) Superfund site (PCB-resistant) and a reference site, Scorton Creek, Massachusetts (SC; PCB-sensitive). Analysis using a 7,000-gene cDNA array revealed striking differences in responsiveness to PCB-126 between the populations; the differences occur at all three stages examined. There was a sizeable set of PCB-responsive genes in the sensitive SC population, a much smaller set of PCB-responsive genes in NBH fish, and few similarities in PCB-responsive genes between the two populations. Most of the array results were confirmed, and additional PCB-regulated genes identified, by RNA-Seq (deep pyrosequencing). The results suggest that NBH fish possess a gene regulatory defect that is not specific to one target gene such as CYP1A but rather lies in a regulatory pathway that controls the transcriptional response of multiple genes to PCB exposure. The results are consistent with genome-wide disruption of AHR-dependent signaling in NBH fish.This work was supported in part by National Institutes of Health grants P42ES007381 (Superfund Basic Research Program at Boston University) and by a grant from the WHOI Ocean Life Institute

FunnyBase: a systems level functional annotation of Fundulus ESTs for the analysis of gene expression

BACKGROUND: While studies of non-model organisms are critical for many research areas, such as evolution, development, and environmental biology, they present particular challenges for both experimental and computational genomic level research. Resources such as mass-produced microarrays and the computational tools linking these data to functional annotation at the system and pathway level are rarely available for non-model species. This type of "systems-level" analysis is critical to the understanding of patterns of gene expression that underlie biological processes. RESULTS: We describe a bioinformatics pipeline known as FunnyBase that has been used to store, annotate, and analyze 40,363 expressed sequence tags (ESTs) from the heart and liver of the fish, Fundulus heteroclitus. Primary annotations based on sequence similarity are linked to networks of systematic annotation in Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) and can be queried and computationally utilized in downstream analyses. Steps are taken to ensure that the annotation is self-consistent and that the structure of GO is used to identify higher level functions that may not be annotated directly. An integrated framework for cDNA library production, sequencing, quality control, expression data generation, and systems-level analysis is presented and utilized. In a case study, a set of genes, that had statistically significant regression between gene expression levels and environmental temperature along the Atlantic Coast, shows a statistically significant (P < 0.001) enrichment in genes associated with amine metabolism. CONCLUSION: The methods described have application for functional genomics studies, particularly among non-model organisms. The web interface for FunnyBase can be accessed at . Data and source code are available by request at [email protected]

ExCyto PCR Amplification

ExCyto PCR cells provide a novel and cost effective means to amplify DNA transformed into competent bacterial cells. ExCyto PCR uses host E. coli with a chromosomally integrated gene encoding a thermostable DNA polymerase to accomplish robust, hot-start PCR amplification of cloned sequences without addition of exogenous enzyme.Because the thermostable DNA polymerase is stably integrated into the bacterial chromosome, ExCyto cells can be transformed with a single plasmid or complex library, and then the expressed thermostable DNA polymerase can be used for PCR amplification. We demonstrate that ExCyto cells can be used to amplify DNA from different templates, plasmids with different copy numbers, and master mixes left on ice for up to two hours. Further, PCR amplification with ExCyto cells is comparable to amplification using commercial DNA polymerases. The ability to transform a bacterial strain and use the endogenously expressed protein for PCR has not previously been demonstrated.ExCyto PCR reduces pipetting and greatly increases throughput for screening EST, genomic, BAC, cDNA, or SNP libraries. This technique is also more economical than traditional PCR and thus broadly useful to scientists who utilize analysis of cloned DNAs in their research