Experimental evolution reveals the synergistic genomic mechanisms of adaptation to ocean warming and acidification in a marine copepod.

Metazoan adaptation to global change relies on selection of standing genetic variation. Determining the extent to which this variation exists in natural populations, particularly for responses to simultaneous stressors, is essential to make accurate predictions for persistence in future conditions. Here, we identified the genetic variation enabling the copepod Acartia tonsa to adapt to experimental ocean warming, acidification, and combined ocean warming and acidification (OWA) over 25 generations of continual selection. Replicate populations showed a consistent polygenic response to each condition, targeting an array of adaptive mechanisms including cellular homeostasis, development, and stress response. We used a genome-wide covariance approach to partition the allelic changes into three categories: selection, drift and replicate-specific selection, and laboratory adaptation responses. The majority of allele frequency change in warming (57%) and OWA (63%) was driven by shared selection pressures across replicates, but this effect was weaker under acidification alone (20%). OWA and warming shared 37% of their response to selection but OWA and acidification shared just 1%, indicating that warming is the dominant driver of selection in OWA. Despite the dominance of warming, the interaction with acidification was still critical as the OWA selection response was highly synergistic with 47% of the allelic selection response unique from either individual treatment. These results disentangle how genomic targets of selection differ between single and multiple stressors and demonstrate the complexity that nonadditive multiple stressors will contribute to predictions of adaptation to complex environmental shifts caused by global change.

Whole-Genome Sequencing Reveals That Regulatory and Low Pleiotropy Variants Underlie Local Adaptation to Environmental Variability in Purple Sea Urchins.

AbstractOrganisms experience environments that vary across both space and time. Such environmental heterogeneity shapes standing genetic variation and may influence species' capacity to adapt to rapid environmental change. However, we know little about the kind of genetic variation that is involved in local adaptation to environmental variability. To address this gap, we sequenced the whole genomes of 140 purple sea urchins (Strongylocentrotus purpuratus) from seven populations that vary in their degree of pH variability. Despite no evidence of global population structure, we found a suite of single-nucleotide polymorphisms (SNPs) tightly correlated with local pH variability (outlier SNPs), which were overrepresented in regions putatively involved in gene regulation (long noncoding RNA and enhancers), supporting the idea that variation in regulatory regions is important for local adaptation to variability. In addition, outliers in genes were found to be (i) enriched for biomineralization and ion homeostasis functions related to low pH response, (ii) less central to the protein-protein interaction network, and (iii) underrepresented among genes highly expressed during early development. Taken together, these results suggest that loci that underlie local adaptation to pH variability in purple sea urchins fall in regions with potentially low pleiotropic effects (based on analyses involving regulatory regions, network centrality, and expression time) involved in low pH response (based on functional enrichment).

Simultaneous warming and acidification limit population fitness and reveal phenotype costs for a marine copepod.

Phenotypic plasticity and evolutionary adaptation allow populations to cope with global change, but limits and costs to adaptation under multiple stressors are insufficiently understood. We reared a foundational copepod species, Acartia hudsonica, under ambient (AM), ocean warming (OW), ocean acidification (OA), and combined ocean warming and acidification (OWA) conditions for 11 generations (approx. 1 year) and measured population fitness (net reproductive rate) derived from six life-history traits (egg production, hatching success, survival, development time, body size and sex ratio). Copepods under OW and OWA exhibited an initial approximately 40% fitness decline relative to AM, but fully recovered within four generations, consistent with an adaptive response and demonstrating synergy between stressors. At generation 11, however, fitness was approximately 24% lower for OWA compared with the AM lineage, consistent with the cost of producing OWA-adapted phenotypes. Fitness of the OWA lineage was not affected by reversal to AM or low food environments, indicating sustained phenotypic plasticity. These results mimic those of a congener, Acartia tonsa, while additionally suggesting that synergistic effects of simultaneous stressors exert costs that limit fitness recovery but can sustain plasticity. Thus, even when closely related species experience similar stressors, species-specific costs shape their unique adaptive responses.

Evidence of prezygotic isolation, but not assortative mating, between locally adapted populations of Fundulus heteroclitus across a salinity gradient.

Selection along environmental gradients can drive reproductive isolation and speciation. Among fishes, salinity is a major factor limiting species distributions, and despite its importance in generating species diversity, speciation events between marine and freshwater are rare. Here, we tested for mechanisms of reproductive isolation between locally adapted freshwater and brackish water-native populations of killifish, Fundulus heteroclitus, from either side of a hybrid zone along a salinity gradient. There was evidence for pre-zygotic endogenous reproductive isolation with reduced fertilization success between crosses of freshwater-native males and brackish water-native females. Exogenous pre-zygotic isolation was also present where females had highest fertilization in their native salinity. We used a replicated mass spawning design to test for mate choice in both brackish and fresh water. After genotyping 187 parents and 2523 offspring at 2347 SNPs across the genome, 85% of offspring were successfully assign to their parents. However, no reinforcing mate choice was observed. These results therefore demonstrate emerging, yet limited, reproductive isolation and incipient speciation across a marine to freshwater salinity gradient and suggest that both endogenous and exogenous mechanisms, but not assortative mating, contribute to divergence.

Integrative Population and Physiological Genomics Reveals Mechanisms of Adaptation in Killifish.

Adaptive divergence between marine and freshwater (FW) environments is important in generating phyletic diversity within fishes, but the genetic basis of this process remains poorly understood. Genome selection scans can identify adaptive loci, but incomplete knowledge of genotype-phenotype connections makes interpreting their significance difficult. In contrast, association mapping (genome-wide association mapping [GWAS], random forest [RF] analyses) links genotype to phenotype, but offer limited insight into the evolutionary forces shaping variation. Here, we combined GWAS, RF, and selection scans to identify loci important in adaptation to FW environments. We utilized FW-native and brackish water (BW)-native populations of Atlantic killifish (Fundulus heteroclitus) as well as a naturally admixed population between the two. We measured morphology and multiple physiological traits that differ between populations and may contribute to osmotic adaptation (salinity tolerance, hypoxia tolerance, metabolic rate, body shape) and used a reduced representation approach for genome-wide genotyping. Our results show patterns of population divergence in physiological capabilities that are consistent with local adaptation. Population genomic scans between BW-native and FW-native populations identified genomic regions evolving by natural selection, whereas association mapping revealed loci that contribute to variation for each trait. There was substantial overlap in the genomic regions putatively under selection and loci associated with phenotypic traits, particularly for salinity tolerance, suggesting that these regions and genes are important for adaptive divergence between BW and FW environments. Together, these data provide insight into the mechanisms that enable diversification of fishes across osmotic boundaries.

Rare genetic variation and balanced polymorphisms are important for survival in global change conditions.

Standing genetic variation is important for population persistence in extreme environmental conditions. While some species may have the capacity to adapt to predicted average future global change conditions, the ability to survive extreme events is largely unknown. We used single-generation selection experiments on hundreds of thousands of Strongylocentrotus purpuratus sea urchin larvae generated from wild-caught adults to identify adaptive genetic variation responsive to moderate (pH 8.0) and extreme (pH 7.5) low-pH conditions. Sequencing genomic DNA from pools of larvae, we identified consistent changes in allele frequencies across replicate cultures for each pH condition and observed increased linkage disequilibrium around selected loci, revealing selection on recombined standing genetic variation. We found that loci responding uniquely to either selection regime were at low starting allele frequencies while variants that responded to both pH conditions (11.6% of selected variants) started at high frequencies. Loci under selection performed functions related to energetics, pH tolerance, cell growth and actin/cytoskeleton dynamics. These results highlight that persistence in future conditions will require two classes of genetic variation: common, pH-responsive variants maintained by balancing selection in a heterogeneous environment, and rare variants, particularly for extreme conditions, that must be maintained by large population sizes.

Mitochondria, sex and variation in routine metabolic rate.

Variation in the metabolic costs associated with organismal maintenance may play a key role in determining fitness, and thus these differences among individuals are likely to be subject to natural selection. Although the evolvability of maintenance metabolism depends on its underlying genetic architecture, relatively little is known about the nature of genetic variation that underlies this trait. To address this, we measured variation in routine metabolic rate (MO2routine ), an index of maintenance metabolism, within and among three populations of Atlantic killifish, Fundulus heteroclitus, including a population from a region of genetic admixture between two subspecies. Polygenic association tests among individuals from the admixed population identified 54 single nucleotide polymorphisms (SNPs) that were associated with MO2routine , and these SNPs accounted for 43% of interindividual variation in this trait. However, genetic associations with MO2routine involved different SNPs if females and males were analysed separately, and there was a sex-dependent effect of mitochondrial genotype on variation in routine metabolism. These results imply that there are sex-specific genetic mechanisms, and potential mitonuclear interactions, that underlie variation in MO2routine . Additionally, there was evidence for epistatic interactions between 17% of the possible pairs of trait-associated SNPs, suggesting that epistatic effects on MO2routine are common. These data demonstrate not only that phenotypic variation in this ecologically important trait has a polygenic basis with considerable epistasis among loci, but also that these underlying genetic mechanisms, and particularly the role of mitochondrial genotype, may be sex-specific.

Tolerance traits related to climate change resilience are independent and polygenic.

The resilience of organisms to climate change through adaptive evolution is dependent on the extent of genetically based variation in key phenotypic traits and the nature of genetic associations between them. For aquatic animals, upper thermal tolerance and hypoxia tolerance are likely to be a important determinants of sensitivity to climate change. To determine the genetic basis of these traits and to detect associations between them, we compared naturally occurring populations of two subspecies of Atlantic killifish, Fundulus heteroclitus, that differ in both thermal and hypoxia tolerance. Multilocus association mapping demonstrated that 47 and 35 single nucleotide polymorphisms (SNPs) explained 43.4% and 51.9% of variation in thermal and hypoxia tolerance, respectively, suggesting that genetic mechanisms underlie a substantial proportion of variation in each trait. However, no explanatory SNPs were shared between traits, and upper thermal tolerance varied approximately linearly with latitude, whereas hypoxia tolerance exhibited a steep phenotypic break across the contact zone between the subspecies. These results suggest that upper thermal tolerance and hypoxia tolerance are neither phenotypically correlated nor genetically associated, and thus that rates of adaptive change in these traits can be independently fine-tuned by natural selection. This modularity of important traits can underpin the evolvability of organisms to complex future environmental change.

Local adaptation to osmotic environment in killifish, Fundulus heteroclitus, is supported by divergence in swimming performance but not by differences in excess post-exercise oxygen consumption or aerobic scope.

Regulation of internal ion homeostasis is essential for fishes inhabiting environments where salinities differ from their internal concentrations. It is hypothesized that selection will reduce energetic costs of osmoregulation in a population's native osmotic habitat, producing patterns of local adaptation. Killifish, Fundulus heteroclitus, occupy estuarine habitats where salinities range from fresh to seawater. Populations inhabiting an environmental salinity gradient differ in physiological traits associated with acclimation to acute salinity stress, consistent with local adaptation. Similarly, metabolic rates differ in populations adapted to different temperatures, but have not been studied in regard to salinity. We investigated evidence for local adaptation between populations of killifish native to fresh and brackish water habitats. Aerobic scope (the difference between minimum and maximum metabolic rates), excess post-exercise oxygen consumption, and swimming performance (time and distance to reach exhaustion) were used as proxies for fitness in fresh and brackish water treatments. Swimming performance results supported local adaptation; fish native to brackish water habitats performed significantly better than freshwater-native fish at high salinity while low salinity performance was similar between populations. However, results from metabolic measures did not support this conclusion; both populations showed an increase in resting metabolic rate and a decrease of aerobic scope in fresh water. Similarly, excess post-exercise oxygen consumption was higher for both populations in fresh than in brackish water. While swimming results suggest that environmentally dependent performance differences may be a result of selection in divergent osmotic environments, the differences between populations are not coupled with divergence in metabolic performance.

Reciprocal osmotic challenges reveal mechanisms of divergence in phenotypic plasticity in the killifish Fundulus heteroclitus.

The killifish Fundulus heteroclitus is an estuarine species with broad physiological plasticity, enabling acclimation to diverse stressors. Previous work suggests that freshwater populations expanded their physiology to accommodate low salinity environments; however, it is unknown whether this compromises their tolerance to high salinity. We used a comparative approach to investigate the mechanisms of a derived freshwater phenotype and the fate of an ancestral euryhaline phenotype after invasion of a freshwater environment. We compared physiological and transcriptomic responses to high- and low-salinity stress in fresh and brackish water populations and found an enhanced plasticity to low salinity in the freshwater population coupled with a reduced ability to acclimate to high salinity. Transcriptomic data identified genes with a conserved common response, a conserved salinity-dependent response and responses associated with population divergence. Conserved common acclimation responses revealed stress responses and alterations in cell-cycle regulation as important mechanisms in the general osmotic response. Salinity-specific responses included the regulation of genes involved in ion transport, intracellular calcium, energetic processes and cellular remodeling. Genes diverged between populations were primarily those showing salinity-specific expression and included those regulating polyamine homeostasis and the cell cycle. Additionally, when populations were matched with their native salinity, expression patterns were consistent with the concept of 'transcriptomic resilience', suggesting local adaptation. These findings provide insight into the fate of a plastic phenotype after a shift in environmental salinity and help to reveal mechanisms allowing for euryhalinity.

Model selection as a tool for phylogeographic inference: an example from the willow Salix melanopsis.

Phylogeographic inference has typically relied on analyses of data from one or a few genes to provide estimates of demography and population histories. While much has been learned from these studies, all phylogeographic analysis is conditioned on the data, and thus, inferences derived from data that represent a small sample of the genome are unavoidably tenuous. Here, we demonstrate one approach for moving beyond classic phylogeographic research. We use sequence capture probes and Illumina sequencing to generate data from >400 loci in order to infer the phylogeographic history of Salix melanopsis, a riparian willow with a disjunct distribution in coastal and the inland Pacific Northwest. We evaluate a priori phylogeographic hypotheses using coalescent models for parameter estimation, and the results support earlier findings that identified post-Pleistocene dispersal as the cause of the disjunction in S. melanopsis. We also conduct a series of model selection exercises using IMa2, Migrate-n and ∂a∂i. The resulting ranking of models indicates that refugial dynamics were complex, with multiple regions in the inland regions serving as the source for postglacial colonization. Our results demonstrate that new sources of data and new approaches to data analysis can rejuvenate phylogeographic research by allowing for the identification of complex models that enable researchers to both identify and estimate the most relevant parameters for a given system.

Phenotypic Plasticity Drives Seasonal Thermal Tolerance in a Baltic Copepod.

Seasonal changes in environmental conditions require substantial physiological responses for population persistence. Phenotypic plasticity is a common mechanism to tolerate these changes, but for organisms with short generation times rapid adaptation may also be a contributing factor. Here, we aimed to disentangle the impacts of adaptation from phenotypic plasticity on thermal tolerance of the calanoid copepod Acartia hudsonica collected throughout spring and summer of a single year. We used a common garden (11 °C and 18 °C) design to determine the relative impacts of plasticity versus adaptation. Acartia hudsonica were collected from five time points across the season and thermal tolerance was determined using critical thermal maximum (CTmax) followed by additional measurements after one generation of common garden. As sea surface temperature increased through the season, field collected individuals showed corresponding increases in thermal tolerance but decreases in body size. Despite different thermal tolerances of wild collections, common garden animals did not differ in CTmax within thermal treatments. Instead, there was evidence of phenotypic plasticity where higher temperatures were tolerated by the 18 °C versus the 11 °C treatment animals across all collections. Acclimation also had significant effects on body size, with higher temperatures resulting in smaller individuals, consistent with the temperature size rule. Therefore, the differences in thermal tolerance and body size observed in field collected A. hudsonica were likely driven by plasticity rather than adaptation. However, the observed decrease in body size suggests that nutrient availability and ecosystem functioning could be impacted if temperatures consistently increase with no change in copepod abundance. This is the first record of A. hudsonica in the Baltic Sea known to the authors.

Loss of transcriptional plasticity but sustained adaptive capacity after adaptation to global change conditions in a marine copepod.

Adaptive evolution and phenotypic plasticity will fuel resilience in the geologically unprecedented warming and acidification of the earth's oceans, however, we have much to learn about the interactions and costs of these mechanisms of resilience. Here, using 20 generations of experimental evolution followed by three generations of reciprocal transplants, we investigated the relationship between adaptation and plasticity in the marine copepod, Acartia tonsa, in future global change conditions (high temperature and high CO2). We found parallel adaptation to global change conditions in genes related to stress response, gene expression regulation, actin regulation, developmental processes, and energy production. However, reciprocal transplantation showed that adaptation resulted in a loss of transcriptional plasticity, reduced fecundity, and reduced population growth when global change-adapted animals were returned to ambient conditions or reared in low food conditions. However, after three successive transplant generations, global change-adapted animals were able to match the ambient-adaptive transcriptional profile. Concurrent changes in allele frequencies and erosion of nucleotide diversity suggest that this recovery occurred via adaptation back to ancestral conditions. These results demonstrate that while plasticity facilitated initial survival in global change conditions, it eroded after 20 generations as populations adapted, limiting resilience to new stressors and previously benign environments.

Unique Genomic and Phenotypic Responses to Extreme and Variable pH Conditions in Purple Urchin Larvae.

Environmental variation experienced by a species across space and time can promote the maintenance of genetic diversity that may be adaptive in future global change conditions. Selection experiments have shown that purple sea urchin, Strongylocentrotus purpuratus, populations have adaptive genetic variation for surviving pH conditions at the "edge" (pH 7.5) of conditions experienced in nature. However, little is known about whether populations have genetic variation for surviving low-pH events beyond those currently experienced in nature or how variation in pH conditions affects organismal and genetic responses. Here, we quantified survival, growth, and allele frequency shifts in experimentally selected developing purple sea urchin larvae in static and variable conditions at three pH levels: pH 8.1 (control), pH 7.5 (edge-of-range), and pH 7.0 (extreme). Variable treatments recovered body size relative to static treatments, but resulted in higher mortality, suggesting a potential tradeoff between survival and growth under pH stress. However, within each pH level, allele frequency changes were overlapping between static and variable conditions, suggesting a shared genetic basis underlying survival to mean pH regardless of variability. In contrast, genetic responses to pH 7.5 (edge) versus pH 7.0 (extreme) conditions were distinct, indicating a unique genetic basis of survival. In addition, loci under selection were more likely to be in exonic regions than regulatory, indicating that selection targeted protein-coding variation. Loci under selection in variable pH 7.5 conditions, more similar to conditions periodically experienced in nature, performed functions related to lipid biosynthesis and metabolism, while loci under selection in static pH 7.0 conditions performed functions related to transmembrane and mitochondrial processes. While these results are promising in that purple sea urchin populations possess genetic variation for surviving extreme pH conditions not currently experienced in nature, they caution that increased acidification does not result in a linear response but elicits unique physiological stresses and survival mechanisms.

Mitochondrial Ecophysiology: Assessing the Evolutionary Forces That Shape Mitochondrial Variation.

The mitonuclear species concept hypothesizes that incompatibilities between interacting gene products of the nuclear and mitochondrial genomes are a major factor establishing and maintaining species boundaries. However, most of the data available to test this concept come from studies of genetic variation in mitochondrial DNA, and clines in the mitochondrial genome across contact zones can be produced by a variety of forces. Here, we show that using a combination of population genomic analyses of the nuclear and mitochondrial genomes and studies of mitochondrial function can provide insight into the relative roles of neutral processes, adaptive evolution, and mitonuclear incompatibility in establishing and maintaining mitochondrial clines, using Atlantic killifish (Fundulus heteroclitus) as a case study. There is strong evidence for a role of secondary contact following the last glaciation in shaping a steep mitochondrial cline across a contact zone between northern and southern subspecies of killifish, but there is also evidence for a role of adaptive evolution in driving differentiation between the subspecies in a variety of traits from the level of the whole organism to the level of mitochondrial function. In addition, studies are beginning to address the potential for mitonuclear incompatibilities in admixed populations. However, population genomic studies have failed to detect evidence for a strong and pervasive influence of mitonuclear incompatibilities, and we suggest that polygenic selection may be responsible for the complex patterns observed. This case study demonstrates that multiple forces can act together in shaping mitochondrial clines, and illustrates the challenge of disentangling their relative roles.

Functional and population genomic divergence within and between two species of killifish adapted to different osmotic niches.

Adaptation to salinity affects species distributions, promotes speciation, and guides many evolutionary patterns in fishes. To uncover the basis of a complex trait like osmoregulation, genome-level analyses are sensible. We combine population genomic scans with genome expression profiling to discover candidate genes and pathways associated with divergence between osmotic environments. We compared transcriptome sequence divergence between multiple freshwater and saltwater populations of the rainwater killifish, Lucania parva. We also compared sequence divergence between L. parva and its sister species, Lucania goodei, a freshwater specialist. We found highly differentiated single nucleotide polymorphisms (SNPs) between freshwater and saltwater L. parva populations in cell junction and ion transport genes, including V-type H(+) ATPase. Between species, we found divergence in reproduction and osmotic stress genes. Genes that were differentially expressed between species during osmotic acclimation included genes involved in ion transport and cell volume regulation. Gene sets that were divergent in coding sequence and divergent in expression did not overlap, although they did converge in function. Like many studies using genomic scans, our approach may miss some loci that contribute to adaptation but have complicated patterns of allelic variation. Our study suggests that gene expression and coding sequence may evolve independently as populations adapt to a complex physiological challenge.