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.

Sea stars resist wasting through active immune and collagen systems.

Epidemics are becoming more common and severe, however, pinpointing the causes can be challenging, particularly in marine environments. The cause of sea star wasting (SSW) disease, the ongoing, largest known panzootic of marine wildlife, is unresolved. Here, we measured gene expression longitudinally of 24 adult Pisaster ochraceus sea stars, collected from a recovered site, as they remained asymptomatic (8 individuals) or naturally progressed through SSW (16 individuals) in individual aquaria. Immune, tissue integrity and pro-collagen genes were more highly expressed in asymptomatic relative to wasting individuals, while hypoxia-inducible factor 1-α and RNA processing genes were more highly expressed in wasting relative to asymptomatic individuals. Integrating microbiome data from the same tissue samples, we identified genes and microbes whose abundance/growth was associated with disease status. Importantly, sea stars that remained visibly healthy showed that laboratory conditions had little effect on microbiome composition. Lastly, considering genotypes at 98 145 single-nucleotide polymorphism, we found no variants associated with final health status. These findings suggest that animals exposed to the cause(s) of SSW remain asymptomatic with an active immune response and sustained control of their collagen system while animals that succumb to wasting show evidence of responding to hypoxia and dysregulation of RNA processing systems.

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.

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.

Four plastic additives reduce larval growth and survival in the sea urchin Strongylocentrotus purpuratus.

Plastic additives are utilized during the production of plastic to modify the attributes and stability of the polymer. As oceanic plastic waste degrades, these additives can leach, and are harmful to global marine ecosystems. Despite the high abundance of additives leached into the marine environment, little is known about their direct impact on marine zooplankton. Here we test for impacts of four plastic additives, UV-327, Irganox 1010, DEHP, and methylparaben, all commonly used in plastic manufacturing, on purple sea urchin (Strongylocentrotus purpuratus) larval growth and survival in a serial dose response for 4 days. Methylparaben, UV-327, and Irganox 1010 significantly reduced larval body length by about 5% for at least one dose. In contrast, all compounds reduced larval survival by 20-70% with strongest effects at intermediate rather than high doses. Our results highlight that plastic additives should be tested for their effects on marine organisms.

Microplastics reduce net population growth and fecal pellet sinking rates for the marine copepod, Acartia tonsa.

Microplastics (<5 mm) are ubiquitous in the global environment and are increasingly recognized as a biological hazard, particularly in the oceans. Zooplankton, at the base of the marine food web, have been known to consume microplastics. However, we know little about the impacts of microplastics across life history stages and on carbon settling. Here, we investigated the effects of ingestion of neutrally buoyant polystyrene beads (6.68 µm) by the copepod Acartia tonsa on (1) growth and survival across life history stages, (2) fecundity and egg quality, (3) and fecal characteristics. We found that microplastic exposure reduced body length and survival for nauplii and resulted in smaller eggs when copepods were exposed during oogenesis. Combining these life history impacts, our models estimate a 15% decrease in population growth leading to a projected 30-fold decrease in abundance over 1 year or 20 generations with microplastic exposure. In addition, microplastic-contaminated fecal pellets were 2.29-fold smaller and sinking rates were calculated to be 1.76-fold slower, resulting in an estimated 4.03-fold reduction in fecal volume settling to the benthos per day. Taken together, declines in population sizes and fecal sinking rates suggest that microplastic consumption by zooplankton could have cascading ecosystem impacts via reduced trophic energy transfer and slower carbon settling.

Signals of selection beyond bottlenecks between exotic populations of the bull-headed dung beetle, Onthophagus taurus.

Colonization of new environments can lead to population bottlenecks and rapid phenotypic evolution that could be due to neutral and selective processes. Exotic populations of the bull-headed dung beetle (Onthophagus taurus) have differentiated in opposite directions from native beetles in male horn-to-body size allometry and female fecundity. Here we test for genetic and transcriptional differences among two exotic and one native O. taurus populations after three generations in common garden conditions. We sequenced RNA from 24 individuals for each of the three populations including both sexes, and spanning four developmental stages for the two exotic, differentiated populations. Identifying 270,400 high-quality single nucleotide polymorphisms, we revealed a strong signal of genetic differentiation between the three populations, and evidence of recent bottlenecks within and an excess of outlier loci between exotic populations. Differences in gene expression between populations were greatest in prepupae and early adult life stages, stages during which differences in male horn development and female fecundity manifest. Finally, genes differentially expressed between exotic populations also had greater genetic differentiation and performed functions related to chitin biosynthesis and nutrient sensing, possibly underlying allometry and fecundity trait divergences. Our results suggest that beyond bottlenecks, recent introductions have led to genetic and transcriptional differences in genes correlated with observed phenotypic differences.

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.

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.

Microbiome shifts with onset and progression of Sea Star Wasting Disease revealed through time course sampling.

The recent outbreak of Sea Star Wasting Disease (SSWD) is one of the largest marine epizootics in history, but the host-associated microbial community changes specific to disease progression have not been characterized. Here, we sampled the microbiomes of ochre sea stars, Pisaster ochraceus, through time as animals stayed healthy or became sick and died with SSWD. We found community-wide differences in the microbiomes of sick and healthy sea stars, changes in microbial community composition through disease progression, and a decrease in species richness of the microbiome in late stages of SSWD. Known beneficial taxa (Pseudoalteromonas spp.) decreased in abundance at symptom onset and through disease progression, while known pathogenic (Tenacibaculum spp.) and putatively opportunistic bacteria (Polaribacter spp. and Phaeobacter spp.) increased in abundance in early and late disease stages. Functional profiling revealed microbes more abundant in healthy animals performed functions that inhibit growth of other microbes, including pathogen detection, biosynthesis of secondary metabolites, and degradation of xenobiotics. Changes in microbial composition with disease onset and progression suggest that a microbial imbalance of the host could lead to SSWD or be a consequence of infection by another pathogen. This work highlights the importance of the microbiome in SSWD and also suggests that a healthy microbiome may help confer resistance to SSWD.

Transcriptomic responses to extreme low salinity among locally adapted populations of Olympia oyster (Ostrea lurida).

The Olympia oyster (Ostrea lurida) is a foundation species inhabiting estuaries along the North American west coast. In California estuaries, O. lurida is adapted to local salinity regimes and populations differ in low salinity tolerance. In this study, oysters from three California populations were reared for two generations in a laboratory common garden and subsequently exposed to low salinity seawater. Comparative transcriptomics was then used to understand species-level responses to hyposmotic stress and population-level mechanisms underlying divergent salinity tolerances. Gene expression patterns indicate Olympia oysters are sensitive to hyposmotic stress: All populations respond to low salinity by up-regulating transcripts indicative of protein unfolding, DNA damage and cell cycle arrest after sub-lethal exposure. Among O. lurida populations, transcriptomic profiles differed constitutively and in response to low salinity. Despite two generations in common-garden conditions, transcripts encoding apoptosis modulators were constitutively expressed at significantly different levels in the most tolerant population. Expression of cell death regulators may facilitate cell fate decisions when salinity declines. Following low salinity exposure, oysters from the more tolerant population expressed a small number of mRNAs at significantly higher levels than less tolerant populations. Proteins encoded by these transcripts regulate ciliary activity within the mantle cavity and may function to prolong valve closure and reduce mortality in low salinity seawater. Collectively, gene expression patterns suggest sub-lethal impacts of hyposmotic stress in Olympia oysters are considerable and that even oysters with greater low salinity tolerance may be vulnerable to future freshwater flooding events.

Gene expression across tissues, sex, and life stages in the sea urchin Tripneustes gratilla [Toxopneustidae, Odontophora, Camarodonta].

The pan-tropical sea urchin Tripneustes gratilla is an ecologically and economically important shallow water algal grazer. The aquaculture of T. gratilla has spurred growing interest in the population biology of the species, and by extension the generation of more molecular resources. To this purpose, de novo transcriptomes of T. gratilla were generated for two adults, a male and a female, as well as for a cohort of approximately 1000 plutei larvae. Gene expression profiles of three adult tissue samples were quantified and compared. These samples were of gonadal tissue, the neural ring, and pooled tube feet and pedicellariae. Levels of shared and different gene expression between sexes, as well as across functional categories of interest, including the immune system, toxins, genes involved in fertilization, and sensory genes are highlighted. Differences in expression of isoforms between the sexes and Sex determining Region Y-related High Mobility Group box groups is observed. Additionally an expansion of the tumor suppressor DMBT1 is observed in T. gratilla when compared to the annotated genome of the sea urchin Strongylocentrotus purpuratus. The draft transcriptome of T. gratilla is presented here in order to facilitate more genomic level analysis of emerging model sea urchin systems.

Transcriptomic responses to seawater acidification among sea urchin populations inhabiting a natural pH mosaic.

Increasing awareness of spatial and temporal variation in ocean pH suggests some marine populations may be adapted to local pH regimes and will therefore respond differently to present-day pH variation and to long-term ocean acidification. In the Northeast Pacific Ocean, differences in the strength of coastal upwelling cause latitudinal variation in prevailing pH regimes that are hypothesized to promote local adaptation and unequal pH tolerance among resident populations. In this study, responses to experimental seawater acidification were compared among embryos and larvae from six populations of purple sea urchins (Strongylocentrotus purpuratus) inhabiting areas that differ in their frequency of low pH exposure and that prior research suggests are locally adapted to seawater pH. Transcriptomic analyses demonstrate urchin populations most frequently exposed to low pH seawater responded to experimental acidification by expressing genes within major ATP-producing pathways at greater levels than populations encountering low pH less often. Multiple genes within the tricarboxylic acid cycle, electron transport chain and fatty acid beta oxidation pathways were upregulated in urchin populations experiencing low pH conditions most frequently. These same metabolic pathways were significantly over-represented among genes both expressed in a population-specific manner and putatively under selection to enhance low pH tolerance. Collectively, these data suggest natural selection is acting on metabolic gene networks to redirect ATP toward maintaining acid-base homeostasis and enhance tolerance of seawater acidification. As a trade-off, marine populations more tolerant of low pH may have less energy to put towards other aspects of fitness and to respond to additional ocean change.

Loss of genetic diversity as a consequence of selection in response to high pCO2.

Standing genetic variation may allow for rapid evolutionary response to the geologically unprecedented changes in global conditions. However, there is little known about the consequences of such rapid evolutionary change. Here, we measure genetic responses to experimental low and high pCO 2 levels in purple sea urchin larvae, Strongylocentrotus purpuratus. We found greater loss of nucleotide diversity in high pCO 2 levels (18.61%; 900 µatm) compared to low pCO 2 levels (10.12%; 400 µatm). In the wild, this loss could limit the evolutionary capacity of future generations. In contrast, we found minimal evidence that purple sea urchin larvae physiologically respond to high pCO 2 through alternative splicing of transcripts (11 genes), despite a strong signal of alternative splicing between different developmental stages (1193 genes). However, in response to high pCO 2, four of the 11 alternatively spliced transcripts encoded ribosomal proteins, suggesting the regulation of translation as a potential response mechanism. The results of this study indicate that while the purple urchin presently may have enough standing genetic variation in response to rapid environmental change, this reservoir of resilience is a finite resource and could quickly diminish.

SNP genotyping and population genomics from expressed sequences - current advances and future possibilities.

With the rapid increase in production of genetic data from new sequencing technologies, a myriad of new ways to study genomic patterns in nonmodel organisms are currently possible. Because genome assembly still remains a complicated procedure, and because the functional role of much of the genome is unclear, focusing on SNP genotyping from expressed sequences provides a cost-effective way to reduce complexity while still retaining functionally relevant information. This review summarizes current methods, identifies ways that using expressed sequence data benefits population genomic inference and explores how current practitioners evaluate and overcome challenges that are commonly encountered. We focus particularly on the additional power of functional analysis provided by expressed sequence data and how these analyses push beyond allele pattern data available from nonfunction genomic approaches. The massive data sets generated by these approaches create opportunities and problems as well - especially false positives. We discuss methods available to validate results from expressed SNP genotyping assays, new approaches that sidestep use of mRNA and review follow-up experiments that can focus on evolutionary mechanisms acting across the genome.

Ocean acidification research in the 'post-genomic' era: Roadmaps from the purple sea urchin Strongylocentrotus purpuratus.

Advances in nucleic acid sequencing technology are removing obstacles that historically prevented use of genomics within ocean change biology. As one of the first marine calcifiers to have its genome sequenced, purple sea urchins (Strongylocentrotus purpuratus) have been the subject of early research exploring genomic responses to ocean acidification, work that points to future experiments and illustrates the value of expanding genomic resources to other marine organisms in this new 'post-genomic' era. This review presents case studies of S. purpuratus demonstrating the ability of genomic experiments to address major knowledge gaps within ocean acidification. Ocean acidification research has focused largely on species vulnerability, and studies exploring mechanistic bases of tolerance toward low pH seawater are comparatively few. Transcriptomic responses to high pCO2 seawater in a population of urchins already encountering low pH conditions have cast light on traits required for success in future oceans. Secondly, there is relatively little information on whether marine organisms possess the capacity to adapt to oceans progressively decreasing in pH. Genomics offers powerful methods to investigate evolutionary responses to ocean acidification and recent work in S. purpuratus has identified genes under selection in acidified seawater. Finally, relatively few ocean acidification experiments investigate how shifts in seawater pH combine with other environmental factors to influence organism performance. In S. purpuratus, transcriptomics has provided insight into physiological responses of urchins exposed simultaneously to warmer and more acidic seawater. Collectively, these data support that similar breakthroughs will occur as genomic resources are developed for other marine species.

The nutritionally responsive transcriptome of the polyphenic beetle Onthophagus taurus and the importance of sexual dimorphism and body region.

Developmental responses to nutritional variation represent one of the ecologically most important classes of adaptive plasticity. However, knowledge of genome-wide patterns of nutrition-responsive gene expression is limited. Here, we studied genome-wide transcriptional responses to nutritional variation and their dependency on trait and sex in the beetle Onthophagus taurus. We find that averaged across the transcriptome, nutrition contributes less to overall variation in gene expression than do sex or body region, but that for a modest subset of genes nutrition is by far the most important determinant of expression variation. Furthermore, our results reject the hypothesis that a common machinery may underlie nutrition-sensitive development across body regions. Instead, we find that magnitude (measured by number of differentially expressed contigs), composition (measured by functional enrichment) and evolutionary consequences (measured by patterns of sequence variation) are heavily dependent on exactly which body region is considered and the degree of sexual dimorphism observed on a morphological level. More generally, our findings illustrate that studies into the developmental mechanisms and evolutionary consequences of nutrition-biased gene expression must take into account the dynamics and complexities imposed by other sources of variation in gene expression such as sexual dimorphism and trait type.

Differences in the regulation of growth and biomineralization genes revealed through long-term common-garden acclimation and experimental genomics in the purple sea urchin.

Across heterogeneous landscapes, populations may have adaptive differences in gene regulation that adjust their physiologies to match local environments. Such differences could have origins in acclimation or in genetically fixed variation between habitats. Here we use common-garden experiments to evaluate differences in gene expression between populations of the purple sea urchin, Strongylocentrotus purpuratus, spanning 1700 km and average temperature differences of 5°C to 8°C. Across expression profiles from 18,883 genes after 3 years of common conditions, we find highly correlated expression patterns (Pearson's r = 0.992) among most genes. However, 66 genes were differentially expressed, including many ribosomal protein and biomineralization genes, which had higher expression in urchins originally from the southern population. Gene function analyses revealed slight but pervasive expression differences in genes related to ribosomal function, metabolism, transport, "bone" development, and response to stimuli. In accord with gene expression patterns, a post-hoc spine regrowth experiment revealed that urchins of southern origin regrew spines at a faster rate than northern urchins. These results suggest that there may be genetically controlled, potentially adaptive differences in gene regulation across habitats and that gene expression differences may be under strong enough selection to overcome high, dispersal-mediated gene flow in this marine species.

Signals of selection in outlier loci in a widely dispersing species across an environmental mosaic.

Local adaptation reflects a balance between natural selection and gene flow and is classically thought to require the retention of locally adapted alleles. However, organisms with high dispersal potential across a spatially or temporally heterogeneous landscape pose an interesting challenge to this view requiring local selection every generation or when environmental conditions change to generate adaptation in adults. Here, we test for geographical and sequence-based signals of selection in five putatively adaptive and two putatively neutral genes identified in a previous genome scan of the highly dispersing purple sea urchin, Strongylocentrotus purpuratus. Comparing six populations spanning the species' wide latitudinal range from Canada to Baja California, Mexico, we find positive tests for selection in the putative adaptive genes and not in the putative neutral genes. Specifically, we find an excess of low-frequency and nonsynonymous polymorphisms in two transcription factors and a transporter protein, and an excess of common amino acid polymorphisms in the two transcription factors, suggestive of spatially balancing selection. We test for a genetic correlation with temperature, a dominant environmental variable in this coastal ecosystem. We find mild clines and a stronger association of genetic variation with temperature than latitude in four of the five putative adaptive loci and a signal of local adaptation in the Southern California Bight. Overall, patterns of genetic variation match predictions based on spatially or temporally balancing selection in a heterogeneous landscape and illustrate the value of geographical and coalescent tests on candidate loci identified in a genome-wide scan for selection.