BACKGROUND
understanding the molecular basis of adaptation to environmental change is an important goal in environmental biology.
animal populations adapt to a variety of natural environmental stressors through genetic and epigenetic changes that affect gene expression or protein structure and/or function.
anthropogenic stressors, including toxic chemicals, can also drive selection in natural populations.
for example, evolved resistance of insects to the acute neurotoxicity of insecticides is well known and occurs through a variety of mechanisms involving reduced target site sensitivity or enhanced expression of proteins involved in biotransformation and excretion of the chemicals  <cit> .
thus, we have learned a great deal about adaptation to chemicals designed to be toxic to their target organisms.
however, field examples are less frequent and adaptive mechanisms are not as well understood for broadly distributed industrial pollutants that produce unintended consequences in non-target organisms.
recent studies  have provided strong evidence for adaptation of fish populations to aromatic hydrocarbons such as polychlorinated biphenyls , polychlorinated dibenzo-p-dioxins , and polycyclic aromatic hydrocarbons  that cause toxicity similar to that caused by  <dig> , <dig> -tetrachlorodibenzo-p-dioxin .
these “dioxin-like compounds”  are capable of interfering with embryonic development and eliciting acute and chronic effects on reproduction, immune function, and other essential processes  <cit>  with population-level consequences  <cit> .
populations of the non-migratory atlantic killifish fundulus heteroclitus that persist in highly contaminated environments may provide insight into the molecular mechanisms by which natural populations adapt to long-term, multi-generational exposure to dlcs.
f. heteroclitus is widely used as an environmental model  <cit>  for studying adaptations to natural environmental variables such as temperature  <cit>  and evolved tolerance to anthropogenic chemicals  <cit> .
several distinct and geographically distant populations of f. heteroclitus inhabiting highly contaminated superfund sites have been demonstrated to possess enhanced tolerance or resistance to one or more dlcs as compared to reference populations.
the most well-studied populations are found in superfund sites at newark bay, nj   <cit> , the elizabeth river, va   <cit> , and the acushnet river estuary , ma, contaminated with pcbs  <cit> )  <cit> .
adaptation also has been demonstrated in f. heteroclitus inhabiting more moderately contaminated sites  <cit> .
the molecular mechanism underlying the dlc resistance are not known for any of these populations.
however, the characteristics of the resistant phenotype provide important clues, which can be illustrated using the nbh population as an example.
first, killifish embryos from nbh are less sensitive to the developmental toxicity of  <dig> ′, <dig> ′,5-pentachlorobiphenyl  than embryos from a reference site  <cit> .
second, when exposed to dlcs, nbh larvae display poor inducibility of the well-known biomarker cytochrome p <dig> 1a   <cit> .
similar insensitivity to dlcs is found in adult killifish from nbh, in which the resistance to altered cyp1a gene expression occurs in all tissues and at the level of gene transcription  <cit> .
third, the altered sensitivity of nbh fish to the toxic and biochemical effects of dlcs is heritable through at least  <dig> generations, consistent with genetic adaptation rather than physiological acclimation  <cit> .
most of these phenotypic characteristics are shared among independent dlc-resistant populations, suggesting that similar mechanisms of dlc tolerance have evolved in parallel at multiple sites  <cit> .
theoretical considerations suggest that adaptation to extreme pollution such as that found in nbh and other contaminated sites is more likely to result from major gene effects rather than polygenic adaptation  <cit> .
killifish populations at nbh and other highly polluted sites experience strong selection intensity , exhibit a large phenotypic shift , have large population sizes  <cit> , and have gene flow from neighboring populations  <cit> —all features that favor adaptation via single genes with large effects  <cit> .
consistent with this, previous studies have shown that the pcb-resistant nbh population has similar levels of overall genetic diversity compared with nearby populations of pcb-sensitive fish  <cit> .
in light of these theoretical and empirical considerations and in keeping with a desire to employ a mechanistic perspective  <cit> , we have taken a candidate gene approach to investigate the molecular basis of adaptation to dlcs in killifish.
the most likely candidates for major genes affecting sensitivity to dlcs are those encoding proteins in the aryl hydrocarbon receptor -dependent signaling pathway, the master regulator of responses to many of the most toxic dlcs, including tcdd and the pcbs with tcdd-like effects.
the ahr is a ligand-activated transcription factor that exhibits high affinity for tcdd and other dlcs, regulates expression of a large set of genes in response to dlc exposure, and is required for tcdd or pcb toxicity in mammals  <cit>  and fish  <cit> .
previously, we identified and cloned multiple components of the f. heteroclitus ahr pathway, including two ahr paralogs , an ahr nuclear translocator , and ahr repressor   <cit> .
we hypothesized that allelic variation at one or more of these loci resulted in proteins with reduced sensitivity to activation by dlcs.
this hypothesis is consistent with results from other vertebrate species, where differences in the ahr pathway underlie many of the differences among species, strains, or cell lines in the sensitivity to dlc effects  <cit> .
in some mammalian systems, reduced sensitivity to ahr agonists results from allelic variation at the ahr locus  <cit> .
to identify common genetic loci associated with tolerance, we compared variation in ahr-related genes within and among fish populations resident to highly contaminated sites and nearby, less-contaminated reference sites.
in an earlier study, we identified multiple ahr <dig> alleles and observed differences in the frequency of these alleles between the pcb-sensitive  and -resistant  populations  <cit> .
here, we continue the focus on the nbh population and expand those studies to include additional populations of pcb-sensitive and –resistant fish as well as additional loci .
the contaminated sites included two of the most well-studied dlc-tolerant populations of f. heteroclitus, nbh, ma, and newark bay, nj  .
we also included fish from two other highly contaminated sites suspected of tolerance to dlcs or other contaminants: piles creek, nj   <cit> , and jamaica bay, ny   <cit> , as well as three reference sites: scorton creek, ma , flax pond, ny , and sandy hook, nj  .
a companion study  <cit>  used a ‘candidate gene scan’ approach to investigate associations between dlc tolerance and snp markers at  <dig> loci in four pairs of sensitive and tolerant populations of f. heteroclitus, including some of the same populations examined in the present study.
we hypothesized that there would be selection in favor of ahr variants that conferred reduced sensitivity to pcbs.
this might be detected as purifying selection reducing diversity at one or more of the ahr loci, or as positive selection for certain snps or haplotypes in all polluted sites versus reference sites.
alternatively, evidence for selection might be population-specific.
by sequencing the three ahr-related loci from individuals collected at these reference and polluted sites along the atlantic coast of north america, we compare genetic and haplotype diversity and haplotype distribution, and use multiple methods to assess signatures of positive selection at particular nucleotides.
together, our data support previous studies indicating no loss of genetic diversity in populations at polluted sites, but suggest that particular nucleotides in each gene have a signature of selection that may underlie the differences in phenotype in killifish populations.
RESULTS
ahr <dig>  ahr <dig>  and ahrr polymorphisms
ahr1—previously, twenty-five single nucleotide polymorphisms , nine of which were non-synonymous, were identified in killifish from sc and nbh  <cit> .
four of these nonsynonymous snps  occurred in the highly conserved and functionally important bhlh and pas domains, but not at positions that are highly conserved among ahrs from different species.
in the present study, full-length ahr <dig> cdnas  were sequenced from  <dig> individuals from five new locations  and these data were combined with the exon  <dig> sequences of  <dig> individuals from sc and nbh determined earlier  <cit> .
overall,  <dig> snps, including 20 ns-snps, were identified.
considering only exon  <dig> , there were  <dig> snps,  <dig> of which were non-synonymous.
twelve of these ns-snps were clustered in the c-terminal half of the coding sequence, surrounding the q-rich region that is involved in transactivation ; the resulting amino acid replacements included a mixture of conservative and non-conservative changes.
in addition to the snps, a 6-nucleotide deletion was also identified in some fish from jb, fp, pc, sh, and yc; none of the fish from sc or nbh had the deletion.
only sequences from exon  <dig>  which contained most of the snps , were used in subsequent population comparisons, because for some of the fish only exon  <dig> sequences were available.
ahr2—although fish have multiple ahrs, we have observed that ahr <dig> is the predominant  form in many fishes, suggesting that ahr <dig> may have an important role in adaptive and toxic responses  in fish  <cit> .
sequencing of  <dig> alleles  from  <dig> fish at  <dig> locations revealed  <dig> snps, including 9 ns-snps.
as observed for ahr <dig>  the ahr <dig> ns-snps occurred primarily in the c-terminal half of the coding sequence, including within the q-rich region ; all of the c-terminal ns-snps resulted in non-conservative amino acid changes.
two ns-snps were found in the n-terminal half: a ns-snp in the n-terminal basic region results in a conservative lys to arg change, and a ns-snp in the pas domain results in a non-conservative ile to ser change between the pas-a and pas-b repeats, in a region that is poorly conserved among species, but may have functional importance  <cit> .
ahrr—ahrr sequences  were obtained from  <dig> fish from the five hudson river estuary sites.
thirty-eight snps, including 10 ns-snps, were found in the  <dig> sequenced fish .
the ns-snps occur in the c-terminal region of the coding sequence , in a region after the pas domain , and in the region between the hlh and pas domains  .
f. heteroclitus
ahr <dig>  ahr <dig>  and ahrr from seven locations along the atlantic coast of the united states
note: n = number of alleles sequenced.
π = nucleotide diversity.
hap = number of haplotypes found.
hd = haplotype diversity.
snps = number of single nucleotide polymorphisms found.
no ahrr sequences were obtained from nbh or sc fish.
the “summary” line  includes the total number of distinct haplotypes and snps across all populations.
snp and haplotype counts for ahr <dig> include sequences from exon  <dig> only.
the unique haplotypes for each ahr locus are available in the dryad repository, at doi: <dig> /dryad.t <dig>
ahr <dig>  ahr <dig>  and ahrr haplotypes and genetic diversity
ahr1—the  <dig> ahr <dig> snps were arranged in  <dig> distinct haplotypes .
many of the haplotypes occurred only once or a few times and frequently at only one location.
the percentage of site-specific haplotypes  frequently exceeded that of haplotypes shared with other populations.
the highest percentage of private haplotypes was from jb, where fish contained  <dig> total haplotypes of which  <dig> were unique to this location.
a few haplotypes occurred at high frequency, and at multiple sites .
overall genetic diversity for exon  <dig> of ahr <dig> among the sampled fishes was high ; however, there was no significant difference in nucleotide or haplotype diversity between polluted and reference locations .
the categorization of fish from jb as reference or polluted did not affect these results.
f. heteroclitus
from polluted and reference sites
for these comparisons jb was grouped with other polluted populations.
however, when jb was considered a reference site, the relationships of nucleotide and haplotypes diversity remained unchanged.
values shown are the mean and .
pairwise fst comparisons of ahr <dig> among all populations identified approximately half  with significant differences between sites .
fst values varied from zero  to  <dig>  .
there were no consistent patterns for genetic differentiation between reference and polluted sites.
nbh and sc were significantly different when compared with all other populations  but not significantly different from one another.
st
values between all populations for ahr <dig>  ahr <dig>  and ahrr
ahr1
ahr2
ahrr
bold values indicate statistically significant differences after correcting for multiple comparisons.
yc was omitted from the ahr <dig> analysis because of insufficient data.
similarly, ahrr sequences were not available from nbh and sc; see methods for details.
ahr2—the  <dig> ahr <dig> snps were arranged in  <dig> haplotypes, many occurring only once or 2– <dig> times at a single location.
as with ahr <dig>  haplotypes unique to single locations accounted for more than half of the total, indicating a high proportion of private alleles .
there were also a few shared, high-frequency haplotypes  .
compared to ahr <dig>  overall genetic diversity for ahr <dig> among the sampled fishes was lower .
similar to ahr <dig>  there was no significant difference in nucleotide or haplotype diversity between polluted and reference locations overall .
the categorization of fish from jb as reference or polluted did not affect the results.
pairwise fst comparisons among all populations revealed a similar number of significant relationships as for ahr <dig> , but with some different populations exhibiting significant genetic differentiation .
fst values varied from  <dig>   to  <dig>  .
there were no consistent patterns for genetic differentiation between reference and polluted sites when considered as a group.
however, nbh  had significant fst values in comparison to both sc and fp , while sc did not differ significantly from fp .
ahrr—the  <dig> ahrr snps occurred in  <dig> haplotypes, most of which were low-frequency and site-specific .
some higher-frequency ahrr haplotypes were present in most populations but these showed similar distribution among the contaminated and reference sites.
overall genetic diversity for ahrr was intermediate between ahr <dig> and ahr <dig> .
there was no significant difference in nucleotide or haplotype diversity between polluted and reference locations .
as observed for ahr <dig> and ahr <dig>  the categorization of fish from jamaica bay as reference or polluted did not affect the results.
for ahrr, pairwise fst comparisons among all populations identified half  that were significantly different .
fst values varied from zero  to  <dig>  .
similar to ahr <dig> and ahr <dig>  there were no consistent patterns for genetic differentiation between reference and polluted sites.
tests for selection
we used three methods for detecting candidate nucleotides undergoing selection: tajima’s d and fu and li’s f*, likelihood ratio tests, and position-specific f-statistics.
tajima’s d was not significant for any of the loci .
however, fu and li’s f* test was significant for ahr <dig>  but not for ahr <dig>  or ahrr .
an f-statistic significantly greater than zero for ahr <dig> indicated an excess of intermediate frequency alleles.
using the sliding window approach for ahr <dig>  three nucleotide positions, clustered in the q-rich region of the transactivation domain , were significant .
the likelihood ratio tests implemented with codeml in the paml software suite provided evidence for positive selection shaping the frequency of nonsynonymous polymorphisms in all three loci.
both tests  were highly significant  for each locus.
for both comparisons, the bayes empirical bayes  method identified the same set of nucleotides as under positive selection; these included  <dig> residues in ahr <dig>   <dig> in ahr <dig>  and  <dig> in ahrr .
each of these residues was inferred with high probability  to be under strong selection, with ratios of nonsynonymous substitutions to synonymous substitution  greater than  <dig>  the  <dig> residues identified in ahr <dig> represented 80% of the ns-snps sequenced from these populations and  <dig> % of the total residues.
these sites are dispersed throughout the sequenced region of exon  <dig> with intervening sites showing no evidence of positive selection.
for ahr <dig>  one identified residue was in the region between the two pas domains and the remaining residues were in the tad or further in the c-terminus; four of these were successive substitutions in the c-terminus.
the six positively selected sites for ahrr were scattered throughout this locus, representing 60% of ns-snps, and included two residues in the region between the bhlh and pas domains.
over all three loci,  <dig> % of codons and more than 70% of nonsynonymous substitutions were identified as being under positive selection.
locus-by-locus amova was used to test for significant differences in snp frequencies between populations classified as polluted versus reference .
these tests did not identify any ns-snps in ahr <dig> with significant differences in these two habitat types.
these results were not affected by classification of jb as a polluted or reference site.
however, nucleotide frequencies were significantly different among populations for  <dig> of 15 ns-snps when all geographic locations were included  or for  <dig> sites within reference or polluted  .
synonymous substitutions, like the ns-snps, did not show significant variation between reference and polluted populations , but they did show some significant fst and fsc values.
as observed for ahr <dig>  f-statistics for ahr <dig> and ahrr identified no ns-snps as having significant differences in frequencies between populations classified as polluted versus reference.
four ahr <dig> ns-snps had significant fst values  and a single ns-snp located in the pas domain region of ahr <dig>  had a significant fsc value .
for ahrr, no ns-snps had significant fsc or fst values.
synonymous substitutions in both ahr <dig> and ahrr were also not significant when populations were grouped as reference or polluted, but two synonymous snps showed significant fsc and six showed significant fst .
comparing results of these different tests for ns-snps under selection, three ns-snps in the q-rich domain of ahr <dig> were identified by three tests .
an additional seven ns-snps in ahr <dig> and four ns-snps in ahr <dig> were identified by two tests.
no ns-snps in ahrr were identified by more than one test.
we compared the base composition between reference and contaminated sites for each of the ns-snps in all three loci .
for the three ns-snps identified by three tests for selection , we compared the frequency of each nucleotide in the sc and nbh populations.
despite overall similarity in base frequency between reference and polluted sites, the base frequencies varied considerably between sc and nbh for two of these ns-snps in ahr1: ns-snps  <dig> and  <dig>  two other positions  also show base frequency differences between sc and nbh and in the comparisons of all populations were identified as under selection by both likelihood ratio tests and amova-fst.
three positions in ahr <dig> showed dramatic differences in base composition between sc and nbh fish: ns-snp  <dig> , ns-snp  <dig> , and ns-snp  <dig>  .
two of these  were also identified by both likelihood ratio tests and amova-fst as under selection in the comparisons of all populations.
isolation by distance
the sampled populations in this analysis spanned a total geographic distance of more than 600 km along the atlantic coastline of the united states .
statistical tests for isolation-by-distance resulted in a significant regression when ahr <dig> diversity was compared among all sampled fishes , a relationship consistent with a neutral expectation, although distance explains only part of the difference in genetic diversity.
the regressions for ahr <dig> remained positive when fish were separated by type of habitat ; however these statistical tests were not significant .
in contrast to the results for ahr <dig>  results for ahr <dig> and ahrr indicated non-significant relationships  both when comparing among all estuaries  and when sites were separated by habitat type .
an alternative possibility, perhaps likely for ahrr, is that sample sizes were too small to detect isolation by distance.
discussion
the repeated evolution of resistance to dlcs in widely separated populations of f. heteroclitus along the u.s. east coast provides an opportunity to understand the mechanistic basis for rapid adaptation to anthropogenic environmental change.
there is strong evidence—initially from the widespread loss of inducibility of ahr-regulated cyp1a  and subsequently confirmed by gene expression profiling  <cit> —that this adaptation involves altered sensitivity of the ahr-dependent signaling pathway.
thus, we used a candidate gene approach and focused on three known ahr-related genes in seven populations of f. heteroclitus.
our analysis of the sequence data from all seven locations reveals a complex pattern of selection at the three loci.
because our primary focus has been on the nbh superfund site  <cit> , we also examined the patterns of variation at nbh and its two nearest reference sites, sc and fp.
comparisons of seven populations from polluted and reference sites
three ahr-related loci  from f. heteroclitus inhabiting seven estuaries along the u.s. east coast contain a large number of polymorphisms, many of which result in changes in the encoded amino acids.
overall,  <dig> % of the nucleotide positions were variable among the sequences analyzed in this study, and 38% of the snps were nonsynonymous.
in contrast, ahr <dig> in tomcod sampled from three sites  showed very low nucleotide variability   <cit> .
for comparison,  <dig> % of the nucleotide positions were variable in ahr coding sequences from  <dig> inbred strains of mice   <cit> .
by contrast, the human ahr  exhibits much less variability than either mouse or killifish ahrs  <cit> .
in previous studies examining inter-specific and intra-specific variability in ahr sequences  <cit> , the most highly conserved region is the basic-helix-loop-helix  domain, which is involved in dna binding and protein dimerization  <cit> .
the per-arnt-sim  domains, required for ligand-binding and protein-protein interactions  <cit> , are also well conserved, whereas the c-terminal half of the protein that harbors transactivation domains  <cit>  is more variable  <cit> .
our results showing that the majority of ns-snps are in the c-terminal half of the sequences or between bhlh and pas domains  are consistent with these earlier results.
the genetic diversity of these f. heteroclitus populations at these three loci is strongly partitioned among locations, but there are no significant differences in nucleotide diversity between populations inhabiting polluted habitats versus those at relatively clean habitats.
similarly, each locus is represented by dozens of haplotypes that exhibit a high degree of location-specific distribution but, again, with no consistent differences in haplotype diversity in polluted versus reference habitats.
in previous studies, examination of other sequence-based markers, microsatellites, and anonymous markers has led to the conclusion that there is restricted gene flow among these populations  and that populations inhabiting pollutant-impacted sites show no strong signature for a genetic bottleneck   <cit> .
our results show that these conclusions also pertain to the three ahr-related gene loci.
thus, the populations exhibit strong genetic structure at these loci but no loss of nucleotide or haplotype diversity in populations classified as “polluted.”
despite an overall similarity in genetic diversity between reference and polluted populations, a suite of tests suggested that some loci and certain polymorphisms may be under selection.
the three statistical methods we used test for deviations from neutrality using different metrics.
tajima’s d and fu and li’s f* statistics test for a statistically significant excess or reduction of allele frequencies among sampled individuals.
the likelihood/beb method tests for an excess of nonsynonymous substitutions compared to synonymous substitutions along particular haplotype lineages, at each position.
the comparative f-statistics test for significant differences in base-frequencies among populations  or between groups of populations classified as reference or polluted .
these tests may identify different sets of nucleotides potentially under selection.
however, when the same nucleotide is identified by more than one test, it increases confidence that it has been shaped by selection.
the most consistently identified three snps, identified by three tests, were located in the q-rich region of ahr <dig>  in a region of exon  <dig> associated with the transcriptional activation function  <cit> .
other ns-snps in exon  <dig> of ahr <dig> and in ahr <dig> were identified by two tests .
the position-specific likelihood ratio tests identified more residues under selection when compared with the other tests.
generally, ratio comparisons of nonsynonymous and synonymous snps are considered conservative tests for positive selection  <cit> .
additionally, our results were inferred through analysis of haplotypes in a phylogenetic framework, thus allowing a more accurate representation of the evolution of particular nucleotides within the lineage of an allele.
whether all identified snps represent true positives is uncertain, in part due to the limitations of our data set.
one limitation is that recombination within loci can hamper interpretation of residues under selection in tree-based analyses by increasing the proportion of false positives  <cit> .
an initial test for recombination at each of the three loci using the gard test  <cit>  suggests potential recombination events in the sampled individuals from this study.
these analyses indicate that one  or two  recombination events likely have occurred in our sampled sequences.
such a low frequency of recombination is unlikely to cause false positives in nucleotide-specific tests for selection  <cit> .
likelihood ratio tests are also sensitive to data sets in which polymorphic sites are not independent, and because of linkage the beb analysis may over-represent residues under selection  <cit> .
thus, some snps identified as undergoing selection may represent snps in linkage disequilibrium with neighboring nucleotides for which selection was operating.
discerning among these possibilities would be assisted by sequencing additional loci from these individuals.
more broadly, our results provide a mixed assessment of which ahr locus may represent the best candidate for explaining evolved resistance in natural populations of f. heteroclitus.
similar to population genetics studies of other f. heteroclitus loci  <cit> , at each ahr locus we observed a high number of polymorphisms that segregate among populations, with many haplotypes restricted to individual locations.
such a large proportion of geographically restricted genetic diversity reflects this species’ large population sizes and relatively limited migration between adjacent locations  <cit> .
ahr <dig> and ahr <dig> are possible candidates for explaining the mechanism of molecular adaptation by populations to polluted environments.
for the statistical tests for selection, ahr <dig> had proportionally larger numbers of ns-snps with evidence of selection.
however, ahr <dig> diversity showed a significant  relationship with geographic distance, a result consistent with either neutral evolution  or selection pressure that correlates with latitude   <cit> .
however, dlc contamination is not correlated with latitude, i.e., the polluted sites in this portion of f. heteroclitus’ range, as well as along the atlantic coast, are interspersed among clean sites.
thus, the mantel test result for ahr <dig>  showing the lack of a relationship of genetic diversity with geographic distance, is more consistent with adaptation to local environments.
on the other hand, we found fewer ahr <dig> ns-snps to be under selection, although some of these variable positions showed significant differences when comparing all populations  and in comparison of nbh and sc .
experimental tests to empirically determine functional characteristics  of the diverse ahr <dig> and ahr <dig> allelic variants would help to discern the role of snps in adaptation in these populations and to develop hypotheses about the role of particular haplotypes in polluted and reference populations of f. heteroclitus.
nbh versus reference population comparisons
examination of ahr diversity in multiple populations, including several exhibiting resistance to dlcs  <cit> , revealed evidence for ahr loci and specific snps under selection, but the population genetic data are complex and their interpretation is not straightforward.
a limitation of this multi-population approach is that resistance is likely to have evolved independently in the different resistant populations and may involve different loci or different snps or haplotypes under selection.
in addition, our classification of locations as “polluted” combined locations with very different types of pollution , and included a population  for which dlc resistance has not yet been assessed.
it is useful, then, to also take a more focused look at the population of greatest interest in our studies, the one inhabiting the nbh superfund site, and the two nearest reference populations, sc and fp, thus minimizing effects of geographic distance on the genetic data.
consistent with the mantel test showing isolation-by-distance for ahr <dig> across all populations, the nbh and sc populations did not show strong genetic differentiation at this locus, but each had significant pairwise fst values in comparison to all of the more distant populations .
by contrast, for the ahr <dig> locus nbh had significant fst values in pairwise comparisons to sc and fp , while the two reference sites did not differ significantly from each other, despite the fact that they are farther apart from each other than either is from nbh.
these results were supported by the distinct pattern of haplotype frequencies at nbh as compared with sc or fp  and by the identification of three ahr <dig> ns-snps for which nbh and sc differ substantially .
thus, examination of these three populations points to specific ahr <dig> snps and haplotypes as being associated with the pcb-resistant phenotype.
one interesting result is that, for both ahr <dig> and ahr <dig>  neither specific haplotypes nor the snps exhibiting evidence for selection were fixed in dlc-resistant fish populations, raising questions about their contribution to the resistant phenotype.
one possibility is that these loci individually have relatively small effect and are part of a larger polygenic adaptation response  <cit> .
alternatively, there could be multiple haplotypes at one of these loci  that confer resistance.
such a situation could arise from selection on pre-existing  genetic variation, in which one or more snps conferring reduced ahr function exists in multiple haplotypes in the population prior to environmental change, and selection leads to fixation of multiple alleles .
population genomic studies will help to distinguish between these possibilities.
role of ahr <dig> in controlling susceptibility of fish to dlc effects
fish have multiple ahr genes, classified in two clades, ahr <dig> and ahr <dig>  <cit> .
the functions of ahr <dig> and ahr <dig> are not completely understood, but ahr <dig> is the most likely candidate for a resistance locus, based on several lines of evidence.
first, studies using gene-specific knock-down in zebrafish embryos have shown that ahr <dig> controls the induction of cyp1a and sensitivity to developmental toxicity of tcdd, pcbs, and pahs in this species  <cit> .
second, ahr <dig> was one of the candidate genes emerging from a genome-wide qtl screen for genes controlling pcb cardiotoxicity in zebrafish embryos  <cit> .
third, and more directly relevant to the species of interest in the current study, knock-down of ahr <dig> in embryos of f. heteroclitus provided partial protection against the teratogenic effects of pahs and pcbs  <cit> .
in addition to the experimental studies cited above, two recent population-level studies suggest ahr <dig> as a resistance locus.
in an independent analysis being published as a companion paper in this journal  <cit> , a ‘candidate gene scan’ investigation of associations between dlc resistance and snp markers at  <dig> loci in four pairs of sensitive and tolerant populations of f. heteroclitus identified ahr <dig> as one of two loci under selection   <cit> .
there is partial overlap in the populations studied by proestou et al.
<cit>  and in the present paper  but the other populations examined were specific to each study .
in proestou et al.
<cit> , snps in both ahr <dig>  and ahr <dig>  exhibited evidence of selection  in  <dig> of  <dig> population pairs, including nbh and its reference site.
in our study, ahr1_ <dig> also had a significant fst value in a locus-by-locus amova and it is located just downstream from three ns-snps also exhibiting evidence for selection .
although the ahr2_ <dig> snp did not have a significant fst value in our study , it was near a ns-snp that did .
additional evidence for selection at the ahr2_ <dig> snp in proestou et al.
<cit>  came from patterns of minor allele frequencies between pairs of populations and the identification of this snp as the only outlier after fst modeling of pooled sensitive and tolerant populations  <cit> .
a second study, in another fish species showing population-specific evolution of pcb resistance, also implicated the ahr <dig> locus.
atlantic tomcod  inhabiting the pcb-contaminated hudson river were nearly monomorphic for an ahr <dig> variant with reduced capacity to bind and be activated by halogenated ahr ligands such as tcdd or pcb- <dig>  <cit> .
the ahr <dig> variant in hudson river fish was characterized by a 2-amino acid deletion, just downstream from the pas domain, that was proposed to alter the ligand-binding affinity or stability of the ahr <dig> protein in these fish.
a similar deletion was not found in the ahr <dig> variants of nbh killifish in our study, but a snp within the pas domain and several near the c-terminal transactivation domain emerged as potentially under selection and with distinct patterns in nbh fish as compared to the reference sites .
based on our results and those described above  <cit> , we suggest that evolution of resistance to pcbs in fish may converge on a common target gene, ahr <dig>  but that the specific molecular changes may differ between species, and perhaps also within a species among populations that have independently evolved the resistant phenotype .
nevertheless, changes in other loci—including paralogous ahr loci  as well as other loci encoding proteins involved in the mechanism of dioxin toxicity—may also play a role in conferring the resistant phenotype.
population genomic surveys currently underway will help illuminate such possibilities.
since completion of this work, through transcriptome sequencing  <cit> , we have identified two additional ahr loci in f. heteroclitus, paralogs of the ahr loci studied here.
multiple ahrs, often occurring as pairs of paralogous ahr <dig> and/or ahr <dig> forms, have been identified in other species of fish including danio rerio , takifugu rubripes and tetraodon nigroviridis , oryzias latipes , and salmonids .
consistent with phylogenetic relationships  and the nomenclature we have used for other fish ahrs  <cit> , the original killifish ahr genes  have been designated ahr1a and ahr2a; the novel ahr genes are ahr1b and ahr2b.
the function and expression patterns of these new ahrs are not known, but are under active investigation in our laboratory.
sequencing and assembly of the f. heteroclitus genome has revealed that ahr1a and ahr2a occur in tandem , as do ahr1b and ahr2b , as we have described for other fish ahr1/ahr <dig> pairs  <cit> .
linkage of ahr1a and ahr2a may have influenced the patterns of diversity and evidence for selection obtained in our study and that of proestou et al.
<cit> , for example by causing both ahr <dig> and ahr <dig> to display evidence for selection even if only one of these genes may be involved in the mechanism of resistance.
clearly, additional research will be needed to determine the function of the new ahrs and the possible role of all four ahr genes in evolved resistance to pcbs and related chemicals.
CONCLUSIONS
the data presented here suggest that f. heteroclitus populations in reference and polluted sites have similar genetic diversity, with no evidence for genetic bottlenecks in populations inhabiting polluted locations.
however, the populations exhibit strong genetic structure at all three ahr-related loci, and for ahr <dig> the nbh population exhibits significant genetic differentiation from its two nearby reference sites.
in addition, the data revealed positive selection at specific nucleotides in ahr <dig> and ahr <dig>  and specific ahr <dig> snps and haplotypes that are associated with the pcb-resistant phenotype in the nbh population.
the results suggest that ahrs, and especially ahr <dig>  may be recurring targets for selection during local adaptation of fish to dioxin-like aromatic hydrocarbon contaminants, although the specific molecular changes may vary among independently adapting populations or species.

 understanding the molecular basis of adaptation to environmental change is an important goal in environmental biology. animal populations adapt to a variety of natural environmental stressors through genetic and epigenetic changes that affect gene expression or protein structure and/or function. anthropogenic stressors, including toxic chemicals, can also drive selection in natural populations. for example, evolved resistance of insects to the acute neurotoxicity of insecticides is well known and occurs through a variety of mechanisms involving reduced target site sensitivity or enhanced expression of proteins involved in biotransformation and excretion of the chemicals. thus, we have learned a great deal about adaptation to chemicals designed to be toxic to their target organisms. however, field examples are less frequent and adaptive mechanisms are not as well understood for broadly distributed industrial pollutants that produce unintended consequences in non-target organisms. recent studies have provided strong evidence for adaptation of fish populations to aromatic hydrocarbons such as polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins, and polycyclic aromatic hydrocarbons that cause toxicity similar to that caused by, -tetrachlorodibenzo-p-dioxin. these “dioxin-like compounds” are capable of interfering with embryonic development and eliciting acute and chronic effects on reproduction, immune function, and other essential processes with population-level consequences. populations of the non-migratory atlantic killifish fundulus heteroclitus that persist in highly contaminated environments may provide insight into the molecular mechanisms by which natural populations adapt to long-term, multi-generational exposure to dlcs. f. heteroclitus is widely used as an environmental model for studying adaptations to natural environmental variables such as temperature and evolved tolerance to anthropogenic chemicals. several distinct and geographically distant populations of f. heteroclitus inhabiting highly contaminated superfund sites have been demonstrated to possess enhanced tolerance or resistance to one or more dlcs as compared to reference populations. the most well-studied populations are found in superfund sites at newark bay, nj, the elizabeth river, va, and the acushnet river estuary, ma, contaminated with pcbs ). adaptation also has been demonstrated in f. heteroclitus inhabiting more moderately contaminated sites. the molecular mechanism underlying the dlc resistance are not known for any of these populations. however, the characteristics of the resistant phenotype provide important clues, which can be illustrated using the nbh population as an example. first, killifish embryos from nbh are less sensitive to the developmental toxicity of ′, ′,5-pentachlorobiphenyl than embryos from a reference site. second, when exposed to dlcs, nbh larvae display poor inducibility of the well-known biomarker cytochrome p 1a. similar insensitivity to dlcs is found in adult killifish from nbh, in which the resistance to altered cyp1a gene expression occurs in all tissues and at the level of gene transcription. third, the altered sensitivity of nbh fish to the toxic and biochemical effects of dlcs is heritable through at least generations, consistent with genetic adaptation rather than physiological acclimation. most of these phenotypic characteristics are shared among independent dlc-resistant populations, suggesting that similar mechanisms of dlc tolerance have evolved in parallel at multiple sites. theoretical considerations suggest that adaptation to extreme pollution such as that found in nbh and other contaminated sites is more likely to result from major gene effects rather than polygenic adaptation. killifish populations at nbh and other highly polluted sites experience strong selection intensity, exhibit a large phenotypic shift, have large population sizes, and have gene flow from neighboring populations —all features that favor adaptation via single genes with large effects. consistent with this, previous studies have shown that the pcb-resistant nbh population has similar levels of overall genetic diversity compared with nearby populations of pcb-sensitive fish. in light of these theoretical and empirical considerations and in keeping with a desire to employ a mechanistic perspective, we have taken a candidate gene approach to investigate the molecular basis of adaptation to dlcs in killifish. the most likely candidates for major genes affecting sensitivity to dlcs are those encoding proteins in the aryl hydrocarbon receptor -dependent signaling pathway, the master regulator of responses to many of the most toxic dlcs, including tcdd and the pcbs with tcdd-like effects. the ahr is a ligand-activated transcription factor that exhibits high affinity for tcdd and other dlcs, regulates expression of a large set of genes in response to dlc exposure, and is required for tcdd or pcb toxicity in mammals and fish. previously, we identified and cloned multiple components of the f. heteroclitus ahr pathway, including two ahr paralogs, an ahr nuclear translocator, and ahr repressor. we hypothesized that allelic variation at one or more of these loci resulted in proteins with reduced sensitivity to activation by dlcs. this hypothesis is consistent with results from other vertebrate species, where differences in the ahr pathway underlie many of the differences among species, strains, or cell lines in the sensitivity to dlc effects. in some mammalian systems, reduced sensitivity to ahr agonists results from allelic variation at the ahr locus. to identify common genetic loci associated with tolerance, we compared variation in ahr-related genes within and among fish populations resident to highly contaminated sites and nearby, less-contaminated reference sites. in an earlier study, we identified multiple ahr alleles and observed differences in the frequency of these alleles between the pcb-sensitive and -resistant populations. here, we continue the focus on the nbh population and expand those studies to include additional populations of pcb-sensitive and –resistant fish as well as additional loci. the contaminated sites included two of the most well-studied dlc-tolerant populations of f. heteroclitus, nbh, ma, and newark bay, nj. we also included fish from two other highly contaminated sites suspected of tolerance to dlcs or other contaminants: piles creek, nj, and jamaica bay, ny, as well as three reference sites: scorton creek, ma, flax pond, ny, and sandy hook, nj. a companion study used a ‘candidate gene scan’ approach to investigate associations between dlc tolerance and snp markers at loci in four pairs of sensitive and tolerant populations of f. heteroclitus, including some of the same populations examined in the present study. we hypothesized that there would be selection in favor of ahr variants that conferred reduced sensitivity to pcbs. this might be detected as purifying selection reducing diversity at one or more of the ahr loci, or as positive selection for certain snps or haplotypes in all polluted sites versus reference sites. alternatively, evidence for selection might be population-specific. by sequencing the three ahr-related loci from individuals collected at these reference and polluted sites along the atlantic coast of north america, we compare genetic and haplotype diversity and haplotype distribution, and use multiple methods to assess signatures of positive selection at particular nucleotides. together, our data support previous studies indicating no loss of genetic diversity in populations at polluted sites, but suggest that particular nucleotides in each gene have a signature of selection that may underlie the differences in phenotype in killifish populations.  ahr ahr and ahrr polymorphisms ahr1—previously, twenty-five single nucleotide polymorphisms, nine of which were non-synonymous, were identified in killifish from sc and nbh. four of these nonsynonymous snps occurred in the highly conserved and functionally important bhlh and pas domains, but not at positions that are highly conserved among ahrs from different species. in the present study, full-length ahr cdnas were sequenced from individuals from five new locations and these data were combined with the exon sequences of individuals from sc and nbh determined earlier. overall, snps, including 20 ns-snps, were identified. considering only exon, there were snps, of which were non-synonymous. twelve of these ns-snps were clustered in the c-terminal half of the coding sequence, surrounding the q-rich region that is involved in transactivation; the resulting amino acid replacements included a mixture of conservative and non-conservative changes. in addition to the snps, a 6-nucleotide deletion was also identified in some fish from jb, fp, pc, sh, and yc; none of the fish from sc or nbh had the deletion. only sequences from exon which contained most of the snps, were used in subsequent population comparisons, because for some of the fish only exon sequences were available. ahr2—although fish have multiple ahrs, we have observed that ahr is the predominant form in many fishes, suggesting that ahr may have an important role in adaptive and toxic responses in fish. sequencing of alleles from fish at locations revealed snps, including 9 ns-snps. as observed for ahr the ahr ns-snps occurred primarily in the c-terminal half of the coding sequence, including within the q-rich region; all of the c-terminal ns-snps resulted in non-conservative amino acid changes. two ns-snps were found in the n-terminal half: a ns-snp in the n-terminal basic region results in a conservative lys to arg change, and a ns-snp in the pas domain results in a non-conservative ile to ser change between the pas-a and pas-b repeats, in a region that is poorly conserved among species, but may have functional importance. ahrr—ahrr sequences were obtained from fish from the five hudson river estuary sites. thirty-eight snps, including 10 ns-snps, were found in the sequenced fish. the ns-snps occur in the c-terminal region of the coding sequence, in a region after the pas domain, and in the region between the hlh and pas domains. f. heteroclitus ahr ahr and ahrr from seven locations along the atlantic coast of the united states note: n = number of alleles sequenced. π = nucleotide diversity. hap = number of haplotypes found. hd = haplotype diversity. snps = number of single nucleotide polymorphisms found. no ahrr sequences were obtained from nbh or sc fish. the “summary” line includes the total number of distinct haplotypes and snps across all populations. snp and haplotype counts for ahr include sequences from exon only. the unique haplotypes for each ahr locus are available in the dryad repository, at doi: /dryad.t ahr ahr and ahrr haplotypes and genetic diversity ahr1—the ahr snps were arranged in distinct haplotypes. many of the haplotypes occurred only once or a few times and frequently at only one location. the percentage of site-specific haplotypes frequently exceeded that of haplotypes shared with other populations. the highest percentage of private haplotypes was from jb, where fish contained total haplotypes of which were unique to this location. a few haplotypes occurred at high frequency, and at multiple sites. overall genetic diversity for exon of ahr among the sampled fishes was high; however, there was no significant difference in nucleotide or haplotype diversity between polluted and reference locations. the categorization of fish from jb as reference or polluted did not affect these results. f. heteroclitus from polluted and reference sites for these comparisons jb was grouped with other polluted populations. however, when jb was considered a reference site, the relationships of nucleotide and haplotypes diversity remained unchanged. values shown are the mean and. pairwise fst comparisons of ahr among all populations identified approximately half with significant differences between sites. fst values varied from zero to. there were no consistent patterns for genetic differentiation between reference and polluted sites. nbh and sc were significantly different when compared with all other populations but not significantly different from one another. st values between all populations for ahr ahr and ahrr ahr1 ahr2 ahrr bold values indicate statistically significant differences after correcting for multiple comparisons. yc was omitted from the ahr analysis because of insufficient data. similarly, ahrr sequences were not available from nbh and sc; see methods for details. ahr2—the ahr snps were arranged in haplotypes, many occurring only once or 2– times at a single location. as with ahr haplotypes unique to single locations accounted for more than half of the total, indicating a high proportion of private alleles. there were also a few shared, high-frequency haplotypes. compared to ahr overall genetic diversity for ahr among the sampled fishes was lower. similar to ahr there was no significant difference in nucleotide or haplotype diversity between polluted and reference locations overall. the categorization of fish from jb as reference or polluted did not affect the results. pairwise fst comparisons among all populations revealed a similar number of significant relationships as for ahr, but with some different populations exhibiting significant genetic differentiation. fst values varied from to. there were no consistent patterns for genetic differentiation between reference and polluted sites when considered as a group. however, nbh had significant fst values in comparison to both sc and fp, while sc did not differ significantly from fp. ahrr—the ahrr snps occurred in haplotypes, most of which were low-frequency and site-specific. some higher-frequency ahrr haplotypes were present in most populations but these showed similar distribution among the contaminated and reference sites. overall genetic diversity for ahrr was intermediate between ahr and ahr. there was no significant difference in nucleotide or haplotype diversity between polluted and reference locations. as observed for ahr and ahr the categorization of fish from jamaica bay as reference or polluted did not affect the results. for ahrr, pairwise fst comparisons among all populations identified half that were significantly different. fst values varied from zero to. similar to ahr and ahr there were no consistent patterns for genetic differentiation between reference and polluted sites. tests for selection we used three methods for detecting candidate nucleotides undergoing selection: tajima’s d and fu and li’s f*, likelihood ratio tests, and position-specific f-statistics. tajima’s d was not significant for any of the loci. however, fu and li’s f* test was significant for ahr but not for ahr or ahrr. an f-statistic significantly greater than zero for ahr indicated an excess of intermediate frequency alleles. using the sliding window approach for ahr three nucleotide positions, clustered in the q-rich region of the transactivation domain, were significant. the likelihood ratio tests implemented with codeml in the paml software suite provided evidence for positive selection shaping the frequency of nonsynonymous polymorphisms in all three loci. both tests were highly significant for each locus. for both comparisons, the bayes empirical bayes method identified the same set of nucleotides as under positive selection; these included residues in ahr in ahr and in ahrr. each of these residues was inferred with high probability to be under strong selection, with ratios of nonsynonymous substitutions to synonymous substitution greater than the residues identified in ahr represented 80% of the ns-snps sequenced from these populations and % of the total residues. these sites are dispersed throughout the sequenced region of exon with intervening sites showing no evidence of positive selection. for ahr one identified residue was in the region between the two pas domains and the remaining residues were in the tad or further in the c-terminus; four of these were successive substitutions in the c-terminus. the six positively selected sites for ahrr were scattered throughout this locus, representing 60% of ns-snps, and included two residues in the region between the bhlh and pas domains. over all three loci, % of codons and more than 70% of nonsynonymous substitutions were identified as being under positive selection. locus-by-locus amova was used to test for significant differences in snp frequencies between populations classified as polluted versus reference. these tests did not identify any ns-snps in ahr with significant differences in these two habitat types. these results were not affected by classification of jb as a polluted or reference site. however, nucleotide frequencies were significantly different among populations for of 15 ns-snps when all geographic locations were included or for sites within reference or polluted. synonymous substitutions, like the ns-snps, did not show significant variation between reference and polluted populations, but they did show some significant fst and fsc values. as observed for ahr f-statistics for ahr and ahrr identified no ns-snps as having significant differences in frequencies between populations classified as polluted versus reference. four ahr ns-snps had significant fst values and a single ns-snp located in the pas domain region of ahr had a significant fsc value. for ahrr, no ns-snps had significant fsc or fst values. synonymous substitutions in both ahr and ahrr were also not significant when populations were grouped as reference or polluted, but two synonymous snps showed significant fsc and six showed significant fst. comparing results of these different tests for ns-snps under selection, three ns-snps in the q-rich domain of ahr were identified by three tests. an additional seven ns-snps in ahr and four ns-snps in ahr were identified by two tests. no ns-snps in ahrr were identified by more than one test. we compared the base composition between reference and contaminated sites for each of the ns-snps in all three loci. for the three ns-snps identified by three tests for selection, we compared the frequency of each nucleotide in the sc and nbh populations. despite overall similarity in base frequency between reference and polluted sites, the base frequencies varied considerably between sc and nbh for two of these ns-snps in ahr1: ns-snps and two other positions also show base frequency differences between sc and nbh and in the comparisons of all populations were identified as under selection by both likelihood ratio tests and amova-fst. three positions in ahr showed dramatic differences in base composition between sc and nbh fish: ns-snp, ns-snp, and ns-snp. two of these were also identified by both likelihood ratio tests and amova-fst as under selection in the comparisons of all populations. isolation by distance the sampled populations in this analysis spanned a total geographic distance of more than 600 km along the atlantic coastline of the united states. statistical tests for isolation-by-distance resulted in a significant regression when ahr diversity was compared among all sampled fishes, a relationship consistent with a neutral expectation, although distance explains only part of the difference in genetic diversity. the regressions for ahr remained positive when fish were separated by type of habitat; however these statistical tests were not significant. in contrast to the results for ahr results for ahr and ahrr indicated non-significant relationships both when comparing among all estuaries and when sites were separated by habitat type. an alternative possibility, perhaps likely for ahrr, is that sample sizes were too small to detect isolation by distance. discussion the repeated evolution of resistance to dlcs in widely separated populations of f. heteroclitus along the u.s. east coast provides an opportunity to understand the mechanistic basis for rapid adaptation to anthropogenic environmental change. there is strong evidence—initially from the widespread loss of inducibility of ahr-regulated cyp1a and subsequently confirmed by gene expression profiling —that this adaptation involves altered sensitivity of the ahr-dependent signaling pathway. thus, we used a candidate gene approach and focused on three known ahr-related genes in seven populations of f. heteroclitus. our analysis of the sequence data from all seven locations reveals a complex pattern of selection at the three loci. because our primary focus has been on the nbh superfund site, we also examined the patterns of variation at nbh and its two nearest reference sites, sc and fp. comparisons of seven populations from polluted and reference sites three ahr-related loci from f. heteroclitus inhabiting seven estuaries along the u.s. east coast contain a large number of polymorphisms, many of which result in changes in the encoded amino acids. overall, % of the nucleotide positions were variable among the sequences analyzed in this study, and 38% of the snps were nonsynonymous. in contrast, ahr in tomcod sampled from three sites showed very low nucleotide variability. for comparison, % of the nucleotide positions were variable in ahr coding sequences from inbred strains of mice. by contrast, the human ahr exhibits much less variability than either mouse or killifish ahrs. in previous studies examining inter-specific and intra-specific variability in ahr sequences, the most highly conserved region is the basic-helix-loop-helix domain, which is involved in dna binding and protein dimerization. the per-arnt-sim domains, required for ligand-binding and protein-protein interactions, are also well conserved, whereas the c-terminal half of the protein that harbors transactivation domains is more variable. our results showing that the majority of ns-snps are in the c-terminal half of the sequences or between bhlh and pas domains are consistent with these earlier results. the genetic diversity of these f. heteroclitus populations at these three loci is strongly partitioned among locations, but there are no significant differences in nucleotide diversity between populations inhabiting polluted habitats versus those at relatively clean habitats. similarly, each locus is represented by dozens of haplotypes that exhibit a high degree of location-specific distribution but, again, with no consistent differences in haplotype diversity in polluted versus reference habitats. in previous studies, examination of other sequence-based markers, microsatellites, and anonymous markers has led to the conclusion that there is restricted gene flow among these populations and that populations inhabiting pollutant-impacted sites show no strong signature for a genetic bottleneck. our results show that these conclusions also pertain to the three ahr-related gene loci. thus, the populations exhibit strong genetic structure at these loci but no loss of nucleotide or haplotype diversity in populations classified as “polluted.” despite an overall similarity in genetic diversity between reference and polluted populations, a suite of tests suggested that some loci and certain polymorphisms may be under selection. the three statistical methods we used test for deviations from neutrality using different metrics. tajima’s d and fu and li’s f* statistics test for a statistically significant excess or reduction of allele frequencies among sampled individuals. the likelihood/beb method tests for an excess of nonsynonymous substitutions compared to synonymous substitutions along particular haplotype lineages, at each position. the comparative f-statistics test for significant differences in base-frequencies among populations or between groups of populations classified as reference or polluted. these tests may identify different sets of nucleotides potentially under selection. however, when the same nucleotide is identified by more than one test, it increases confidence that it has been shaped by selection. the most consistently identified three snps, identified by three tests, were located in the q-rich region of ahr in a region of exon associated with the transcriptional activation function. other ns-snps in exon of ahr and in ahr were identified by two tests. the position-specific likelihood ratio tests identified more residues under selection when compared with the other tests. generally, ratio comparisons of nonsynonymous and synonymous snps are considered conservative tests for positive selection. additionally, our results were inferred through analysis of haplotypes in a phylogenetic framework, thus allowing a more accurate representation of the evolution of particular nucleotides within the lineage of an allele. whether all identified snps represent true positives is uncertain, in part due to the limitations of our data set. one limitation is that recombination within loci can hamper interpretation of residues under selection in tree-based analyses by increasing the proportion of false positives. an initial test for recombination at each of the three loci using the gard test suggests potential recombination events in the sampled individuals from this study. these analyses indicate that one or two recombination events likely have occurred in our sampled sequences. such a low frequency of recombination is unlikely to cause false positives in nucleotide-specific tests for selection. likelihood ratio tests are also sensitive to data sets in which polymorphic sites are not independent, and because of linkage the beb analysis may over-represent residues under selection. thus, some snps identified as undergoing selection may represent snps in linkage disequilibrium with neighboring nucleotides for which selection was operating. discerning among these possibilities would be assisted by sequencing additional loci from these individuals. more broadly, our results provide a mixed assessment of which ahr locus may represent the best candidate for explaining evolved resistance in natural populations of f. heteroclitus. similar to population genetics studies of other f. heteroclitus loci, at each ahr locus we observed a high number of polymorphisms that segregate among populations, with many haplotypes restricted to individual locations. such a large proportion of geographically restricted genetic diversity reflects this species’ large population sizes and relatively limited migration between adjacent locations. ahr and ahr are possible candidates for explaining the mechanism of molecular adaptation by populations to polluted environments. for the statistical tests for selection, ahr had proportionally larger numbers of ns-snps with evidence of selection. however, ahr diversity showed a significant relationship with geographic distance, a result consistent with either neutral evolution or selection pressure that correlates with latitude. however, dlc contamination is not correlated with latitude, i.e., the polluted sites in this portion of f. heteroclitus’ range, as well as along the atlantic coast, are interspersed among clean sites. thus, the mantel test result for ahr showing the lack of a relationship of genetic diversity with geographic distance, is more consistent with adaptation to local environments. on the other hand, we found fewer ahr ns-snps to be under selection, although some of these variable positions showed significant differences when comparing all populations and in comparison of nbh and sc. experimental tests to empirically determine functional characteristics of the diverse ahr and ahr allelic variants would help to discern the role of snps in adaptation in these populations and to develop hypotheses about the role of particular haplotypes in polluted and reference populations of f. heteroclitus. nbh versus reference population comparisons examination of ahr diversity in multiple populations, including several exhibiting resistance to dlcs, revealed evidence for ahr loci and specific snps under selection, but the population genetic data are complex and their interpretation is not straightforward. a limitation of this multi-population approach is that resistance is likely to have evolved independently in the different resistant populations and may involve different loci or different snps or haplotypes under selection. in addition, our classification of locations as “polluted” combined locations with very different types of pollution, and included a population for which dlc resistance has not yet been assessed. it is useful, then, to also take a more focused look at the population of greatest interest in our studies, the one inhabiting the nbh superfund site, and the two nearest reference populations, sc and fp, thus minimizing effects of geographic distance on the genetic data. consistent with the mantel test showing isolation-by-distance for ahr across all populations, the nbh and sc populations did not show strong genetic differentiation at this locus, but each had significant pairwise fst values in comparison to all of the more distant populations. by contrast, for the ahr locus nbh had significant fst values in pairwise comparisons to sc and fp, while the two reference sites did not differ significantly from each other, despite the fact that they are farther apart from each other than either is from nbh. these results were supported by the distinct pattern of haplotype frequencies at nbh as compared with sc or fp and by the identification of three ahr ns-snps for which nbh and sc differ substantially. thus, examination of these three populations points to specific ahr snps and haplotypes as being associated with the pcb-resistant phenotype. one interesting result is that, for both ahr and ahr neither specific haplotypes nor the snps exhibiting evidence for selection were fixed in dlc-resistant fish populations, raising questions about their contribution to the resistant phenotype. one possibility is that these loci individually have relatively small effect and are part of a larger polygenic adaptation response. alternatively, there could be multiple haplotypes at one of these loci that confer resistance. such a situation could arise from selection on pre-existing genetic variation, in which one or more snps conferring reduced ahr function exists in multiple haplotypes in the population prior to environmental change, and selection leads to fixation of multiple alleles. population genomic studies will help to distinguish between these possibilities. role of ahr in controlling susceptibility of fish to dlc effects fish have multiple ahr genes, classified in two clades, ahr and ahr. the functions of ahr and ahr are not completely understood, but ahr is the most likely candidate for a resistance locus, based on several lines of evidence. first, studies using gene-specific knock-down in zebrafish embryos have shown that ahr controls the induction of cyp1a and sensitivity to developmental toxicity of tcdd, pcbs, and pahs in this species. second, ahr was one of the candidate genes emerging from a genome-wide qtl screen for genes controlling pcb cardiotoxicity in zebrafish embryos. third, and more directly relevant to the species of interest in the current study, knock-down of ahr in embryos of f. heteroclitus provided partial protection against the teratogenic effects of pahs and pcbs. in addition to the experimental studies cited above, two recent population-level studies suggest ahr as a resistance locus. in an independent analysis being published as a companion paper in this journal, a ‘candidate gene scan’ investigation of associations between dlc resistance and snp markers at loci in four pairs of sensitive and tolerant populations of f. heteroclitus identified ahr as one of two loci under selection. there is partial overlap in the populations studied by proestou et al. and in the present paper but the other populations examined were specific to each study. in proestou et al., snps in both ahr and ahr exhibited evidence of selection in of population pairs, including nbh and its reference site. in our study, ahr1_ also had a significant fst value in a locus-by-locus amova and it is located just downstream from three ns-snps also exhibiting evidence for selection. although the ahr2_ snp did not have a significant fst value in our study, it was near a ns-snp that did. additional evidence for selection at the ahr2_ snp in proestou et al. came from patterns of minor allele frequencies between pairs of populations and the identification of this snp as the only outlier after fst modeling of pooled sensitive and tolerant populations. a second study, in another fish species showing population-specific evolution of pcb resistance, also implicated the ahr locus. atlantic tomcod inhabiting the pcb-contaminated hudson river were nearly monomorphic for an ahr variant with reduced capacity to bind and be activated by halogenated ahr ligands such as tcdd or pcb-. the ahr variant in hudson river fish was characterized by a 2-amino acid deletion, just downstream from the pas domain, that was proposed to alter the ligand-binding affinity or stability of the ahr protein in these fish. a similar deletion was not found in the ahr variants of nbh killifish in our study, but a snp within the pas domain and several near the c-terminal transactivation domain emerged as potentially under selection and with distinct patterns in nbh fish as compared to the reference sites. based on our results and those described above, we suggest that evolution of resistance to pcbs in fish may converge on a common target gene, ahr but that the specific molecular changes may differ between species, and perhaps also within a species among populations that have independently evolved the resistant phenotype. nevertheless, changes in other loci—including paralogous ahr loci as well as other loci encoding proteins involved in the mechanism of dioxin toxicity—may also play a role in conferring the resistant phenotype. population genomic surveys currently underway will help illuminate such possibilities. since completion of this work, through transcriptome sequencing, we have identified two additional ahr loci in f. heteroclitus, paralogs of the ahr loci studied here. multiple ahrs, often occurring as pairs of paralogous ahr and/or ahr forms, have been identified in other species of fish including danio rerio, takifugu rubripes and tetraodon nigroviridis, oryzias latipes, and salmonids. consistent with phylogenetic relationships and the nomenclature we have used for other fish ahrs, the original killifish ahr genes have been designated ahr1a and ahr2a; the novel ahr genes are ahr1b and ahr2b. the function and expression patterns of these new ahrs are not known, but are under active investigation in our laboratory. sequencing and assembly of the f. heteroclitus genome has revealed that ahr1a and ahr2a occur in tandem, as do ahr1b and ahr2b, as we have described for other fish ahr1/ahr pairs. linkage of ahr1a and ahr2a may have influenced the patterns of diversity and evidence for selection obtained in our study and that of proestou et al., for example by causing both ahr and ahr to display evidence for selection even if only one of these genes may be involved in the mechanism of resistance. clearly, additional research will be needed to determine the function of the new ahrs and the possible role of all four ahr genes in evolved resistance to pcbs and related chemicals.  the data presented here suggest that f. heteroclitus populations in reference and polluted sites have similar genetic diversity, with no evidence for genetic bottlenecks in populations inhabiting polluted locations. however, the populations exhibit strong genetic structure at all three ahr-related loci, and for ahr the nbh population exhibits significant genetic differentiation from its two nearby reference sites. in addition, the data revealed positive selection at specific nucleotides in ahr and ahr and specific ahr snps and haplotypes that are associated with the pcb-resistant phenotype in the nbh population. the results suggest that ahrs, and especially ahr may be recurring targets for selection during local adaptation of fish to dioxin-like aromatic hydrocarbon contaminants, although the specific molecular changes may vary among independently adapting populations or species. 
we identified single nucleotide polymorphisms within three ahr-related loci among all populations, including synonymous and nonsynonymous substitutions. genetic diversity at these three loci was not significantly different in contaminated sites as compared to reference sites. together, these data suggest that f. heteroclitus populations in reference and polluted sites have similar genetic diversity, providing no evidence for strong genetic bottlenecks for populations in polluted locations. here we investigated gene diversity and evidence for positive selection at three ahr-related loci in f. heteroclitus by comparing alleles from seven locations ranging over 600 km along the northeastern us, including extremely polluted and reference estuaries, with a focus on new bedford harbor, a pcb superfund site, and nearby reference sites. tests for positive selection revealed ten nonsynonymous polymorphisms in ahr and four in ahr four nonsynonymous snps in ahr and three in ahr showed large differences in base frequency between new bedford harbor and its reference site. the results suggest that ahrs, and especially ahr may be important, recurring targets for selection in local adaptation to dioxin-like aromatic hydrocarbon contaminants. however, the data provide evidence for genetic differentiation among sites, selection at specific nucleotides in ahr and ahr and specific ahr snps and haplotypes that are associated with the pcb-resistant phenotype in the new bedford harbor population. the non-migratory killifish fundulus heteroclitus inhabits clean and polluted environments interspersed throughout its range along the atlantic coast of north america. however, for ahr the new bedford harbor population had significant fst values in comparison to the nearest reference populations. haplotype distributions were spatially segregated and f-statistics suggested strong population genetic structure at these loci, consistent with previous studies showing strong population genetic structure at other f. heteroclitus loci. previous studies suggest that the mechanism of resistance to these and other “dioxin-like compounds” may involve reduced signaling through the aryl hydrocarbon receptor pathway.
 the non-migratory killifish fundulus heteroclitus inhabits clean and polluted environments interspersed throughout its range along the atlantic coast of north america. several populations of this species have successfully adapted to environments contaminated with toxic aromatic hydrocarbon pollutants such as polychlorinated biphenyls. previous studies suggest that the mechanism of resistance to these and other “dioxin-like compounds” may involve reduced signaling through the aryl hydrocarbon receptor pathway. here we investigated gene diversity and evidence for positive selection at three ahr-related loci in f. heteroclitus by comparing alleles from seven locations ranging over 600 km along the northeastern us, including extremely polluted and reference estuaries, with a focus on new bedford harbor, a pcb superfund site, and nearby reference sites.  we identified single nucleotide polymorphisms within three ahr-related loci among all populations, including synonymous and nonsynonymous substitutions. haplotype distributions were spatially segregated and f-statistics suggested strong population genetic structure at these loci, consistent with previous studies showing strong population genetic structure at other f. heteroclitus loci. genetic diversity at these three loci was not significantly different in contaminated sites as compared to reference sites. however, for ahr the new bedford harbor population had significant fst values in comparison to the nearest reference populations. tests for positive selection revealed ten nonsynonymous polymorphisms in ahr and four in ahr four nonsynonymous snps in ahr and three in ahr showed large differences in base frequency between new bedford harbor and its reference site. tests for isolation-by-distance revealed evidence for non-neutral change at the ahr locus.  together, these data suggest that f. heteroclitus populations in reference and polluted sites have similar genetic diversity, providing no evidence for strong genetic bottlenecks for populations in polluted locations. however, the data provide evidence for genetic differentiation among sites, selection at specific nucleotides in ahr and ahr and specific ahr snps and haplotypes that are associated with the pcb-resistant phenotype in the new bedford harbor population. the results suggest that ahrs, and especially ahr may be important, recurring targets for selection in local adaptation to dioxin-like aromatic hydrocarbon contaminants. local adaptationpollutionmolecular mechanismresistancetoleranceconvergent evolutionpopulation genetics 