Denise Champlin

Population genetic diversity and fitness in multiple environments

BackgroundWhen a large number of alleles are lost from a population, increases in individual homozygosity may reduce individual fitness through inbreeding depression. Modest losses of allelic diversity may also negatively impact long-term population viability by reducing the capacity of populations to adapt to altered environments. However, it is not clear how much genetic diversity within populations may be lost before populations are put at significant risk. Development of tools to evaluate this relationship would be a valuable contribution to conservation biology. To address these issues, we have created an experimental system that uses laboratory populations of an estuarine crustacean, Americamysis bahia with experimentally manipulated levels of genetic diversity. We created replicate cultures with five distinct levels of genetic diversity and monitored them for 16 weeks in both permissive (ambient seawater) and stressful conditions (diluted seawater). The relationship between molecular genetic diversity at presumptive neutral loci and population vulnerability was assessed by AFLP analysis.ResultsPopulations with very low genetic diversity demonstrated reduced fitness relative to high diversity populations even under permissive conditions. Population performance decreased in the stressful environment for all levels of genetic diversity relative to performance in the permissive environment. Twenty percent of the lowest diversity populations went extinct before the end of the study in permissive conditions, whereas 73% of the low diversity lines went extinct in the stressful environment. All high genetic diversity populations persisted for the duration of the study, although population sizes and reproduction were reduced under stressful environmental conditions. Levels of fitness varied more among replicate low diversity populations than among replicate populations with high genetic diversity. There was a significant correlation between AFLP diversity and population fitness overall; however, AFLP markers performed poorly at detecting modest but consequential losses of genetic diversity. High diversity lines in the stressful environment showed some evidence of relative improvement as the experiment progressed while the low diversity lines did not.ConclusionsThe combined effects of reduced average fitness and increased variability contributed to increased extinction rates for very low diversity populations. More modest losses of genetic diversity resulted in measurable decreases in population fitness; AFLP markers did not always detect these losses. However when AFLP markers indicated lost genetic diversity, these losses were associated with reduced population fitness.

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

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

Common mechanism underlies repeated evolution of extreme pollution tolerance

Human alterations to the environment can exert strong evolutionary pressures, yet contemporary adaptation to human-mediated stressors is rarely documented in wildlife populations. A common-garden experimental design was coupled with comparative transcriptomics to discover evolved mechanisms enabling three populations of killifish resident in urban estuaries to survive normally lethal pollution exposure during development, and to test whether mechanisms are unique or common across populations. We show that killifish populations from these polluted sites have independently converged on a common adaptive mechanism, despite variation in contaminant profiles among sites. These populations are united by a similarly profound desensitization of aryl-hydrocarbon receptor-mediated transcriptional activation, which is associated with extreme tolerance to the lethal effects of toxic dioxin-like pollutants. The rapid, repeated, heritable and convergent nature of evolved tolerance suggests that ancestral killifish populations harboured genotypes that enabled adaptation to twentieth-century industrial pollutants.

A RAPID, NON-DESTRUCTIVE METHOD FOR ESTIMATING ABOVEGROUND BIOMASS OF SALT MARSH GRASSES

Understanding the primary productivity of salt marshes requires accurate estimates of biomass. Unfortunately, these estimates vary enough within and among salt marshes to require large numbers of replicates if the averages are to be statistically meaningful. Large numbers of replicates are rarely taken, however, because they involve too much labor. Here, we present data on a fast, non-destructive method for measuring aboveground biomass of Spartina alterniflora and Phragmites australis that uses only the average height of the five tallest shoots and the total density of shoots over 10 cm tall. Collecting the data takes only a few minutes per replicate, and calculated values for biomass compare favorably with destructive measurements on harvested samples.

Influence of larval exposure to salinity and cadmium stress on juvenile performance of two marine invertebrates (Capitella sp. I and Crepidula fornicata)

Delayed metamorphosis and short-term food limitation reduce juvenile or adult fitness in a number of marine invertebrate species. In this study, we tested the ability of pollutant and salinity stress to bring about similar effects on juvenile or adult performance. Larvae of the polychaete Capitella sp. I were exposed to sublethal cadmium stress (up to 2000 μg l−1) or salinity stress (down to 10‰) for 24 and 48 h at 23 °C. Following exposure, we induced surviving larvae to metamorphose and monitored the subsequent survival, growth, and reproductive output of juveniles reared under control conditions (no added cadmium, 32‰ salinity). Similarly, larvae of the gastropod Crepidula fornicata were exposed for 24 and 48 h to cadmium in seawater (up to a nominal concentration of 20,000 μg l−1). Surviving larvae were reared to metamorphic competence in the absence of cadmium, induced to metamorphose, and maintained under control conditions for an additional 5 days to monitor juvenile growth rates and survival. Exposing larvae of Capitella sp. I to low salinity (10–12‰) for 48 h generally did not affect adult fecundity, but stressing the larvae for as little as 24 h significantly reduced post-settlement survival and juvenile growth rates (P<0.05). In contrast, exposing larvae of this species to cadmium for even 48 h had no significant effects on post-settlement survival or fecundity, and no consistent effect on mean juvenile growth rate. Similarly, cadmium exposure did not significantly affect mean juvenile growth rates for C. fornicata, even when larvae were severely stressed (i.e., when larval mortality exceeded 50% during exposures). We suggest that heavy metal stressors do not act through the same mechanism as the stresses of inadequate food supply, reduced salinity, and delayed metamorphosis.

Targeted approach to identify genetic loci associated with evolved dioxin tolerance in Atlantic Killifish (Fundulus heteroclitus)

BackgroundThe most toxic aromatic hydrocarbon pollutants are categorized as dioxin-like compounds (DLCs) to which extreme tolerance has evolved independently and contemporaneously in (at least) four populations of Atlantic killifish (Fundulus heteroclitus). Surprisingly, the magnitude and phenotype of DLC tolerance is similar among these killifish populations that have adapted to varied, but highly aromatic hydrocarbon-contaminated urban/industrialized estuaries of the US Atlantic coast. Multiple tolerant and neighboring sensitive killifish populations were compared with the expectation that genetic loci associated with DLC tolerance would be revealed.ResultsSince the aryl hydrocarbon receptor (AHR) pathway partly or fully mediates DLC toxicity in vertebrates, single nucleotide polymorphisms (SNPs) from 42 genes associated with the AHR pathway were identified to serve as targeted markers. Wild fish (N = 36/37) from four highly tolerant killifish populations and four nearby sensitive populations were genotyped using 59 SNP markers. Similar to other killifish population genetic analyses, strong genetic differentiation among populations was detected, consistent with isolation by distance models. When DLC-sensitive populations were pooled and compared to pooled DLC-tolerant populations, multi-locus analyses did not distinguish the two groups. However, pairwise comparisons of nearby tolerant and sensitive populations revealed high differentiation among sensitive and tolerant populations at these specific loci: AHR 1 and 2, cathepsin Z, the cytochrome P450s (CYP1A and 3A30), and the NADH dehydrogenase subunits. In addition, significant shifts in minor allele frequency were observed at AHR2 and CYP1A loci across most sensitive/tolerant pairs, but only AHR2 exhibited shifts in the same direction across all pairs.ConclusionsThe observed differences in allelic composition at the AHR2 and CYP1A SNP loci were identified as significant among paired sensitive/tolerant populations of Atlantic killifish with multiple statistical tests. The genetic patterns reported here lend support to the argument that AHR2 and CYP1A play a role in the adaptive response to extreme DLC contamination. Additional functional assays are required to isolate the exact mechanism of DLC tolerance.

Evolution of tolerance to PCBs and susceptibility to a bacterial pathogen (Vibrio harveyi) in Atlantic killifish (Fundulus heteroclitus) from New Bedford (MA, USA) harbor.

A population of the non-migratory estuarine fish Fundulus heteroclitus (Atlantic killifish) resident to New Bedford (NB), Massachusetts, USA, an urban harbor highly contaminated with polychlorinated biphenyls (PCBs), demonstrates recently evolved tolerance to some aspects of PCB toxicity. PCB toxicology, ecological theory, and some precedence supported expectations of increased susceptibility to pathogens in NB killifish. However, laboratory bacterial challenges of the marine pathogen Vibrio harveyi to wild fish throughout the reproductive season and to their mature laboratory-raised progeny demonstrated comparable survival by NB and reference killifish, and improved survival by NB males. These results are inconsistent with hypothesized trade-offs of adaptation, and suggest that evolved tolerance in NB killifish may include mechanisms that minimize the immunosuppressive effects of PCBs. Compensatory strategies of populations persisting in highly contaminated environments provide a unique perspective for understanding the long-term ecological effects of toxic chemicals.

Targeted mutagenesis of aryl hydrocarbon receptor 2a and 2b genes in Atlantic killifish (Fundulus heteroclitus).

Understanding molecular mechanisms of toxicity is facilitated by experimental manipulations, such as disruption of function by gene targeting, that are especially challenging in non-standard model species with limited genomic resources. While loss-of-function approaches have included gene knock-down using morpholino-modified oligonucleotides and random mutagenesis using mutagens or retroviruses, more recent approaches include targeted mutagenesis using zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology. These latter methods provide more accessible opportunities to explore gene function in non-traditional model species. To facilitate evaluation of toxic mechanisms for important categories of aryl hydrocarbon pollutants, whose actions are known to be receptor mediated, we used ZFN and CRISPR-Cas9 approaches to generate aryl hydrocarbon receptor 2a (AHR2a) and AHR2b gene mutations in Atlantic killifish (Fundulus heteroclitus) embryos. This killifish is a particularly valuable non-traditional model, with multiple paralogs of AHR whose functions are not well characterized. In addition, some populations of this species have evolved resistance to toxicants such as halogenated aromatic hydrocarbons. AHR-null killifish will be valuable for characterizing the role of the individual AHR paralogs in evolved resistance, as well as in normal development. We first used five-finger ZFNs targeting exons 1 and 3 of AHR2a. Subsequently, CRISPR-Cas9 guide RNAs were designed to target regions in exon 2 and 3 of AHR2a and AHR2b. We successfully induced frameshift mutations in AHR2a exon 3 with ZFN and CRISPR-Cas9 guide RNAs, with mutation frequencies of 10% and 16%, respectively. In AHR2b, mutations were induced using CRISPR-Cas9 guide RNAs targeting sites in both exon 2 (17%) and exon 3 (63%). We screened AHR2b exon 2 CRISPR-Cas9-injected embryos for off-target effects in AHR paralogs. No mutations were observed in closely related AHR genes (AHR1a, AHR1b, AHR2a, AHRR) in the CRISPR-Cas9-injected embryos. Overall, our results demonstrate that targeted genome-editing methods are efficient in inducing mutations at specific loci in embryos of a non-traditional model species, without detectable off-target effects in paralogous genes.

Regulation of pregnane-X-receptor, CYP3A and P-glycoprotein genes in the PCB-resistant killifish (Fundulus heteroclitus) population from New Bedford Harbor

Killifish survive and reproduce in the New Bedford Harbor (NBH) in Massachusetts (MA), USA, a site severely contaminated with polychlorinated biphenyls (PCBs) for decades. Levels of 22 different PCB congeners were analyzed in liver from killifish collected in 2008. Concentrations of dioxin-like PCBs in liver of NBH killifish were ∼400 times higher, and the levels of non-dioxin-like PCBs ∼3000 times higher than in killifish from a reference site, Scorton Creek (SC), MA. The NBH killifish are known to be resistant to the toxicity of dioxin-like compounds and to have a reduced aryl hydrocarbon receptor (AhR) signaling response. Little is known about the responses of these fish to non-dioxin-like PCBs, which are at extraordinarily high levels in NBH fish. In mammals, some non-dioxin-like PCB congeners act through nuclear receptor 1I2, the pregnane-X-receptor (PXR). To explore this pathway in killifish, a PXR cDNA was sequenced and its molecular phylogenetic relationship to other vertebrate PXRs was determined. Killifish were also collected in 2009 from NBH and SC, and after four months in the laboratory they were injected with a single dose of either the dioxin-like PCB 126 (an AhR agonist) or the non-dioxin-like PCB 153 (a mammalian PXR agonist). Gills and liver were sampled three days after injection and transcript levels of genes encoding PXR, cytochrome P450 3A (CYP3A), P-glycoprotein (Pgp), AhR2 and cytochrome P450 1A (CYP1A) were measured by quantitative PCR. As expected, there was little effect of PCB exposure on mRNA expression of AhR2 or CYP1A in liver and gills of NBH fish. In NBH fish, but not in SC fish, there was increased mRNA expression of hepatic PXR, CYP3A and Pgp upon exposure to either of the two PCB congeners. However, basal PXR and Pgp mRNA levels in liver of NBH fish were significantly lower than in SC fish. A different pattern was seen in gills, where there were no differences in basal mRNA expression of these genes between the two populations. In SC fish, but not in NBH fish, there was increased mRNA expression of branchial PXR and CYP3A upon exposure to PCB126 and of CYP3A upon exposure to PCB153. The results suggest a difference between the two populations in non-AhR transcription factor signaling in liver and gills, and that this could involve killifish PXR. It also implies possible cross-regulatory interactions between that factor (presumably PXR) and AhR2 in liver of these fish.

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

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