Population-specific responses in eastern oysters exposed to low salinity in the northern Gulf of Mexico.

Eastern oysters, Crassostrea virginica, are facing rapid environmental changes in the northern Gulf of Mexico and can respond to these changes via plasticity or evolution. Plastic responses can immediately buffer against environmental changes, although this buffering may impact the organism's ability to evolve in subsequent generations. While plasticity and evolution are not mutually exclusive, the relative contribution and interaction between them remains unclear. In this study, we investigated the roles of plastic and evolved responses of C. virginica acclimated to low salinity using a common garden experiment with four populations exposed to two salinities. We used three transcriptomic analyses (edgeR, PERMANOVA and WGCNA) combined with physiology data to identify the effect of genotype (population), environment (salinity) and the genotype-environment interaction on both whole-organism and molecular phenotypes. We demonstrate that variation in gene expression is mainly driven by population, with relatively small changes in response to salinity. In contrast, the morphology and physiology data reveal that salinity has a larger influence on oyster performance than the population of origin. All analyses lacked signatures of the genotype×environment interaction and, in contrast to previous studies, we found no evidence for population-specific responses to low salinity. However, individuals from the highest salinity estuary displayed highly divergent gene expression from that of other populations, which could potentially drive population-specific responses to other stressors. Our findings suggest that C. virginica largely rely on plasticity in physiology to buffer the effects of low salinity, but that these changes in physiology do not rely on large persistent changes in gene expression.

An Experimental Test of Adaptive Introgression in Locally Adapted Populations of Splash Pool Copepods.

As species struggle to keep pace with the rapidly warming climate, adaptive introgression of beneficial alleles from closely related species or populations provides a possible avenue for rapid adaptation. We investigate the potential for adaptive introgression in the copepod, Tigriopus californicus, by hybridizing two populations with divergent heat tolerance limits. We subjected hybrids to strong heat selection for 15 generations followed by whole-genome resequencing. Utilizing a hybridize evolve and resequence (HER) technique, we can identify loci responding to heat selection via a change in allele frequency. We successfully increased the heat tolerance (measured as LT50) in selected lines, which was coupled with higher frequencies of alleles from the southern (heat tolerant) population. These repeatable changes in allele frequencies occurred on all 12 chromosomes across all independent selected lines, providing evidence that heat tolerance is polygenic. These loci contained genes with lower protein-coding sequence divergence than the genome-wide average, indicating that these loci are highly conserved between the two populations. In addition, these loci were enriched in genes that changed expression patterns between selected and control lines in response to a nonlethal heat shock. Therefore, we hypothesize that the mechanism of heat tolerance divergence is explained by differential gene expression of highly conserved genes. The HER approach offers a unique solution to identifying genetic variants contributing to polygenic traits, especially variants that might be missed through other population genomic approaches.

Comparative transcriptomics reveals altered species interaction between the bioeroding sponge Cliona varians and the coral Porites furcata under ocean acidification.

Bioeroding sponges interact and compete with corals on tropical reefs. Experimental studies have shown global change alters this biotic interaction, often in favour of the sponge. Ocean acidification in particular increases sponge bioerosion and reduces coral calcification, yet little is known about the molecular basis of these changes. We used RNA-Seq data to understand how acidification impacts the interaction between the bioeroding sponge, Cliona varians, and the coral, Porites furcata, at the transcriptomic level. Replicate sponge and coral genets were exposed to ambient (8.1 pH) and acidified (7.6 pH) conditions in isolation and in treatments where they were joined for 48 h. The coral had a small gene expression response (tens of transcripts) to the sponge, suggesting it does little at the transcriptomic level to deter sponge overgrowth. By contrast, the sponge differentially expressed 7320 transcripts in response to the coral under ambient conditions and 3707 transcripts in response to acidification. Overlap in the responses to acidification and the coral, 2500 transcripts expressed under both treatments, suggests a similar physiological response to both cues. The sponge expressed 50× fewer transcripts in response to the coral under acidification, suggesting energetic costs of bioerosion, and other cellular processes, are lower for sponges under acidification. Our results suggest how acidification drives ecosystem-level changes in the accretion/bioerosion balance on coral reefs. This shift is not only the result of changes to the thermodynamic balance of these chemical reactions but also the result of active physiological responses of organisms to each other and their abiotic environment.

Differential DNA methylation across environments has no effect on gene expression in the eastern oyster.

It has been hypothesized that environmentally induced changes to gene body methylation could facilitate adaptive transgenerational responses to changing environments. We compared patterns of global gene expression (Tag-seq) and gene body methylation (reduced representation bisulfite sequencing) in 80 eastern oysters Crassostrea virginica from six full-sib families, common gardened for 14 months at two sites in the northern Gulf of Mexico that differed in mean salinity. At the time of sampling, oysters from the two sites differed in mass by 60% and in parasite loads by nearly two orders of magnitude. They also differentially expressed 35% of measured transcripts. However, we observed differential methylation at only 1.4% of potentially methylated loci in comparisons between individuals from these different environments, and little correspondence between differential methylation and differential gene expression. Instead, methylation patterns were largely driven by genetic differences among families, with a PERMANOVA analysis indicating nearly a two orders of magnitude greater number of genes differentially methylated between families than between environments. An analysis of CpG observed/expected values (CpG O/E) across the C. virginica genome showed a distinct bimodal distribution, with genes from the first cluster showing the lower CpG O/E values, greater methylation and higher and more stable gene expression, while genes from the second cluster showed lower methylation, and lower and more variable gene expression. Taken together, the differential methylation results suggest that only a small portion of the C. virginica genome is affected by environmentally induced changes in methylation. At this point, there is little evidence to suggest that environmentally induced methylation states would play a leading role in regulating gene expression responses to new environments.

Transgenerational plasticity and the capacity to adapt to low salinity in the eastern oyster, Crassostrea virginica.

Salinity conditions in oyster breeding grounds in the Gulf of Mexico are expected to drastically change due to increased precipitation from climate change and anthropogenic changes to local hydrology. We determined the capacity of the eastern oyster, Crassostrea virginica, to adapt via standing genetic variation or acclimate through transgenerational plasticity (TGP). We outplanted oysters to either a low- or medium-salinity site in Louisiana for 2 years. We then crossed adult parents using a North Carolina II breeding design, and measured body size and survival of larvae 5 dpf raised under low or ambient salinity. We found that TGP is unlikely to significantly contribute to low-salinity tolerance since we did not observe increased growth or survival in offspring reared in low salinity when their parents were also acclimated at a low-salinity site. However, we detected genetic variation for body size, with an estimated heritability of 0.68 ± 0.25 (95% CI). This suggests there is ample genetic variation for this trait to evolve, and that evolutionary adaptation is a possible mechanism through which oysters will persist with future declines in salinity. The results of this experiment provide valuable insights into successfully breeding low-salinity tolerance in this commercially important species.

Lack of genotype-by-environment interaction suggests limited potential for evolutionary changes in plasticity in the eastern oyster, Crassostrea virginica.

Eastern oysters in the northern Gulf of Mexico are facing rapid environmental changes and can respond to this change via plasticity or evolution. Plasticity can act as an immediate buffer against environmental change, but this buffering could impact the organism's ability to evolve in subsequent generations. While plasticity and evolution are not mutually exclusive, the relative contribution and interaction between them remains unclear. In this study, we investigate the roles of plastic and evolved responses to environmental variation and Perkinsus marinus infection in Crassostrea virginica by using a common garden experiment with 80 oysters from six families outplanted at two field sites naturally differing in salinity. We use growth data, P. marinus infection intensities, 3' RNA sequencing (TagSeq) and low-coverage whole-genome sequencing to identify the effect of genotype, environment and genotype-by-environment interaction on the oyster's response to site. As one of first studies to characterize the joint effects of genotype and environment on transcriptomic and morphological profiles in a natural setting, we demonstrate that C. virginica has a highly plastic response to environment and that this response is parallel among genotypes. We also find that genes responding to genotype have distinct and opposing profiles compared to genes responding to environment with regard to expression levels, Ka/Ks ratios and nucleotide diversity. Our findings suggest that C. virginica may be able to buffer the immediate impacts of future environmental changes by altering gene expression and physiology, but the lack of genetic variation in plasticity suggests limited capacity for evolved responses.

Transcriptomic signatures of temperature adaptation in the eastern oyster Crassostrea virginica.

The large geographic distribution of the eastern oyster, Crassostrea virginica, makes it an ideal species to test how populations have adapted to latitudinal gradients in temperature. Despite inhabiting distinct thermal regimes, populations of C. virginica near the species' southern and northern geographic range show no population differences in their physiological response to temperature. In this study, we used comparative transcriptomics to understand how oysters from either end of the species' range maintain enantiostasis across three acclimation temperatures (10, 20, and 30°C). With this approach, we identified genes that were differentially expressed in response to temperature between individuals of C. virginica collected from New Brunswick, Canada and Louisiana, USA. We observed a core set of genes whose expression responded to temperature in both populations, but also an even larger set of genes with expression patterns that were unique to each population. Intriguingly, the genes with population-specific responses to temperature had elevated FST and Ka/Ks ratios compared to the genome-wide average. In contrast, genes showing only a response to temperature were found to only have elevated FST values suggesting that divergent FST may be due to selection on linked regulatory regions rather than positive selection on protein coding regions. Taken together, our results suggest that, despite coarse-scale physiological similarities, natural selection has shaped divergent gene expression responses to temperature in geographically separated populations of this broadly eurythermal marine invertebrate.

Tolerance of northern Gulf of Mexico eastern oysters to chronic warming at extreme salinities.

The eastern oyster, Crassostrea virginica, provides critical ecosystem services and supports valuable fishery and aquaculture industries in northern Gulf of Mexico (nGoM) subtropical estuaries where it is grown subtidally. Its upper critical thermal limit is not well defined, especially when combined with extreme salinities. The cumulative mortalities of the progenies of wild C. virginica from four nGoM estuaries differing in mean annual salinity, acclimated to low (4.0), moderate (20.0), and high (36.0) salinities at 28.9 °C (84 °F) and exposed to increasing target temperatures of 33.3 °C (92 °F), 35.6 °C (96 °F) or 37.8 °C (100 °F), were measured over a three-week period. Oysters of all stocks were the most sensitive to increasing temperatures at low salinity, dying quicker (i.e., lower median lethal time, LT50) than at the moderate and high salinities and resulting in high cumulative mortalities at all target temperatures. Oysters of all stocks at moderate salinity died the slowest with high cumulative mortalities only at the two highest temperatures. The F1 oysters from the more southern and hypersaline Upper Laguna Madre estuary were generally more tolerant to prolonged higher temperatures (higher LT50) than stocks originating from lower salinity estuaries, most notably at the highest salinity. Using the measured temperatures oysters were exposed to, 3-day median lethal Celsius degrees (LD50) were estimated for each stock at each salinity. The lowest 3-day LD50 (35.1-36.0 °C) for all stocks was calculated at a salinity of 4.0, while the highest 3-day LD50 (40.1-44.0 °C) was calculated at a salinity of 20.0.

Differential responses to ocean acidification between populations of Balanophyllia elegans corals from high and low upwelling environments.

Ocean acidification (OA), the global decrease in surface water pH from absorption of anthropogenic CO2 , may put many marine taxa at risk. However, populations that experience extreme localized conditions, and are adapted to these conditions predicted in the global ocean in 2,100, may be more tolerant to future OA. By identifying locally adapted populations, researchers can examine the mechanisms used to cope with decreasing pH. One oceanographic process that influences pH is wind-driven upwelling. Here we compare two Californian populations of the coral Balanophyllia elegans from distinct upwelling regimes, and test their physiological and transcriptomic responses to experimental seawater acidification. We measured respiration rates, protein and lipid content, and gene expression in corals from both populations exposed to pH levels of 7.8 and 7.4 for 29 days. Corals from the population that experiences lower pH due to high upwelling maintained the same respiration rate throughout the exposure. In contrast, corals from the low upwelling site had reduced respiration rates, protein content and lipid-class content at low pH exposure, suggesting they have depleted their energy reserves. Using RNA-Seq, we found that corals from the high upwelling site upregulated genes involved in calcium ion binding and ion transport, most likely related to pH homeostasis and calcification. In contrast, corals from the low upwelling site downregulated stress response genes at low pH exposure. Divergent population responses to low pH observed in B. elegans highlight the importance of multi-population studies for predicting a species' response to future OA.

Phenotypic and transcriptomic responses to salinity stress across genetically and geographically divergent Tigriopus californicus populations.

Species inhabiting the North American west coast intertidal must tolerate an extremely variable environment, with large fluctuations in both temperature and salinity. Uncovering the mechanisms for this tolerance is key to understanding species' persistence. We tested for differences in salinity tolerance between populations of Tigriopus californicus copepods from locations in northern (Bodega Reserve) and southern (San Diego) California known to differ in temperature, precipitation and humidity. We also tested for differences between populations in their transcriptomic responses to salinity. Although these two populations have ~20% mtDNA sequence divergence and differ strongly in other phenotypic traits, we observed similarities in their phenotypic and transcriptomic responses to low and high salinity stress. Salinity significantly affected respiration rate (increased under low salinity and reduced under high salinity), but we found no significant effect of population on respiration or a population by salinity interaction. Under high salinity, there was no population difference in knock-down response, but northern copepods had a smaller knock-down under low salinity stress, corroborating previous results for T. californicus. Northern and southern populations had a similar transcriptomic response to salinity based on a principle components analysis, although differential gene expression under high salinity stress was three times lower in the northern population compared to the southern population. Transcripts differentially regulated under salinity stress were enriched for "amino acid transport" and "ion transport" annotation categories, supporting previous work demonstrating that the accumulation of free amino acids is important for osmotic regulation in T. californicus.

Transcriptomics reveal transgenerational effects in purple sea urchin embryos: Adult acclimation to upwelling conditions alters the response of their progeny to differential pCO2 levels.

Understanding the mechanisms with which organisms can respond to a rapidly changing ocean is an important research priority in marine sciences, especially in the light of recent predictions regarding the pace of ocean change in the coming decades. Transgenerational effects, in which the experience of the parental generation can shape the phenotype of their offspring, may serve as such a mechanism. In this study, adult purple sea urchins, Strongylocentrotus purpuratus, were conditioned to regionally and ecologically relevant pCO2 levels and temperatures representative of upwelling (colder temperature and high pCO2 ) and nonupwelling (average temperature and low pCO2 ) conditions typical of coastal upwelling regions in the California Current System. Following 4.5 months of conditioning, adults were spawned and offspring were raised under either high or low pCO2 levels, to examine the role of maternal effects. Using RNA-seq and comparative transcriptomics, our results indicate that differential conditioning of the adults had an effect on the gene expression patterns of the progeny during the gastrula stage of early development. For example, maternal conditioning under upwelling conditions intensified the transcriptomic response of the progeny when they were raised under high versus low pCO2 conditions. Additionally, mothers that experienced upwelling conditions produced larger progeny. The overall findings of this study are complex, but do suggest that transgenerational plasticity in situ could act as an important mechanism by which populations might keep pace with rapid environmental change.

Plastic and Evolved Responses to Global Change: What Can We Learn from Comparative Transcriptomics?

Physiological plasticity and adaptive evolution may facilitate persistence in a changing environment. As a result, there is an interest in understanding species' capacities for plastic and evolved responses, and the mechanisms by which these responses occur. Transcriptome sequencing has become a powerful tool for addressing these questions, providing insight into otherwise unobserved effects of changing conditions on organismal physiology and variation in these effects among individuals and populations. Here, we review recent studies using comparative transcriptomics to understand plastic and evolutionary responses to changing environments. We focus on 2 areas where transcriptomics has played an important role: first, in understanding the genetic basis for local adaptation to current gradients as a proxy for future adaptation, and second, in understanding organismal responses to multiple stressors. We find most studies examining multiple stressors have tested the effects of each stressor individually; the few studies testing multiple stressors simultaneously have found synergistic effects on gene expression that would not have been predicted from single stressor studies. We discuss the importance of robust experimental design to allow for a more sophisticated characterization of transcriptomic responses and conclude by offering recommendations for future research, including integrating genomics with transcriptomics, testing gene regulatory networks, and comparing the equivalence of transcription to translation and the effects of environmental stress on the proteome.

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.

Trade-offs, geography, and limits to thermal adaptation in a tide pool copepod.

Antagonistic correlations among traits may slow the rate of adaptation to a changing environment. The tide pool copepod Tigriopus californicus is locally adapted to temperature, but within populations, the response to selection for increased heat tolerance plateaus rapidly, suggesting either limited variation within populations or costs of increased tolerance. To measure possible costs of thermal tolerance, we selected for increased upper lethal limits for 10 generations in 22 lines of T. californicus from six populations. Then, for each line, we measured six fitness-related traits. Selected lines showed an overall increase in male and female body sizes, fecundity, and starvation resistance, suggesting a small benefit from (rather than costs of) increased tolerance. The effect of selection on correlated traits also varied significantly by population for five traits, indicating that the genetic basis for the selection response differed among populations. Our results suggest that adaptation was limited by the presence of variation within isolated populations rather than by costs of increased tolerance.

Temperature and CO(2) additively regulate physiology, morphology and genomic responses of larval sea urchins, Strongylocentrotus purpuratus.

Ocean warming and ocean acidification, both consequences of anthropogenic production of CO2, will combine to influence the physiological performance of many species in the marine environment. In this study, we used an integrative approach to forecast the impact of future ocean conditions on larval purple sea urchins (Strongylocentrotus purpuratus) from the northeast Pacific Ocean. In laboratory experiments that simulated ocean warming and ocean acidification, we examined larval development, skeletal growth, metabolism and patterns of gene expression using an orthogonal comparison of two temperature (13°C and 18°C) and pCO2 (400 and 1100 µatm) conditions. Simultaneous exposure to increased temperature and pCO2 significantly reduced larval metabolism and triggered a widespread downregulation of histone encoding genes. pCO2 but not temperature impaired skeletal growth and reduced the expression of a major spicule matrix protein, suggesting that skeletal growth will not be further inhibited by ocean warming. Importantly, shifts in skeletal growth were not associated with developmental delay. Collectively, our results indicate that global change variables will have additive effects that exceed thresholds for optimized physiological performance in this keystone marine species.

Limited potential for adaptation to climate change in a broadly distributed marine crustacean.

The extent to which acclimation and genetic adaptation might buffer natural populations against climate change is largely unknown. Most models predicting biological responses to environmental change assume that species' climatic envelopes are homogeneous both in space and time. Although recent discussions have questioned this assumption, few empirical studies have characterized intraspecific patterns of genetic variation in traits directly related to environmental tolerance limits. We test the extent of such variation in the broadly distributed tidepool copepod Tigriopus californicus using laboratory rearing and selection experiments to quantify thermal tolerance and scope for adaptation in eight populations spanning more than 17° of latitude. Tigriopus californicus exhibit striking local adaptation to temperature, with less than 1 per cent of the total quantitative variance for thermal tolerance partitioned within populations. Moreover, heat-tolerant phenotypes observed in low-latitude populations cannot be achieved in high-latitude populations, either through acclimation or 10 generations of strong selection. Finally, in four populations there was no increase in thermal tolerance between generations 5 and 10 of selection, suggesting that standing variation had already been depleted. Thus, plasticity and adaptation appear to have limited capacity to buffer these isolated populations against further increases in temperature. Our results suggest that models assuming a uniform climatic envelope may greatly underestimate extinction risk in species with strong local adaptation.

Love the one you're with: proximity determines paternity success in the barnacle Tetraclita rubescens.

A species' mating system sets limits on the strength of sexual selection. Sexual selection is widespread in dioecious species, but is less well documented in hermaphrodites, and may be less important. We used four highly polymorphic microsatellite markers to assign paternity to broods of the hermaphroditic eastern Pacific volcano barnacle Tetraclita rubescens. These data were used to describe the species' mating system and to examine factors affecting male reproductive success. Tetraclita can sire broods over distances of 11.2 cm, but proximity to the sperm recipient had a highly significant effect on the probability of siring success. There was no effect of body size or the mass of male reproductive tissues on siring success. Broods showed relatively low frequencies of multiple paternity; even at high densities, 75% of broods had only one father. High frequencies of single-paternity broods imply either that this species does not compete via sperm displacement, or that sperm displacement is extremely effective, potentially explaining the lack of a positive relationship between male investment and paternity. In addition, there was low variance in siring success among individuals, suggesting a lack of strong sexual selection on male traits. Low variance among sires and the strong effect of proximity are probably driven by the unusual biology of a sessile copulating species.

Constitutive gene expression differs in three brain regions important for cognition in neophobic and non-neophobic house sparrows (Passer domesticus).

Neophobia (aversion to new objects, food, and environments) is a personality trait that affects the ability of wildlife to adapt to new challenges and opportunities. Despite the ubiquity and importance of this trait, the molecular mechanisms underlying repeatable individual differences in neophobia in wild animals are poorly understood. We evaluated wild-caught house sparrows (Passer domesticus) for neophobia in the lab using novel object tests. We then selected a subset of neophobic and non-neophobic individuals (n = 3 of each, all females) and extracted RNA from four brain regions involved in learning, memory, threat perception, and executive function: striatum, caudal dorsomedial hippocampus, medial ventral arcopallium, and caudolateral nidopallium (NCL). Our analysis of differentially expressed genes (DEGs) used 11,889 gene regions annotated in the house sparrow reference genome for which we had an average of 25.7 million mapped reads/sample. PERMANOVA identified significant effects of brain region, phenotype (neophobic vs. non-neophobic), and a brain region by phenotype interaction. Comparing neophobic and non-neophobic birds revealed constitutive differences in DEGs in three of the four brain regions examined: hippocampus (12% of the transcriptome significantly differentially expressed), striatum (4%) and NCL (3%). DEGs included important known neuroendocrine mediators of learning, memory, executive function, and anxiety behavior, including serotonin receptor 5A, dopamine receptors 1, 2 and 5 (downregulated in neophobic birds), and estrogen receptor beta (upregulated in neophobic birds). These results suggest that some of the behavioral differences between phenotypes may be due to underlying gene expression differences in the brain. The large number of DEGs in neophobic and non-neophobic birds also implies that there are major differences in neural function between the two phenotypes that could affect a wide variety of behavioral traits beyond neophobia.

Selection Experiments in the Sea: What Can Experimental Evolution Tell Us About How Marine Life Will Respond to Climate Change?

AbstractRapid evolution may provide a buffer against extinction risk for some species threatened by climate change; however, the capacity to evolve rapidly enough to keep pace with changing environments is unknown for most taxa. The ecosystem-level consequences of climate adaptation are likely to be the largest in marine ecosystems, where short-lived phytoplankton with large effective population sizes make up the bulk of primary production. However, there are substantial challenges to predicting climate-driven evolution in marine systems, including multiple simultaneous axes of change and considerable heterogeneity in rates of change, as well as the biphasic life cycles of many marine metazoans, which expose different life stages to disparate sources of selection. A critical tool for addressing these challenges is experimental evolution, where populations of organisms are directly exposed to controlled sources of selection to test evolutionary responses. We review the use of experimental evolution to test the capacity to adapt to climate change stressors in marine species. The application of experimental evolution in this context has grown dramatically in the past decade, shedding light on the capacity for evolution, associated trade-offs, and the genetic architecture of stress-tolerance traits. Our goal is to highlight the utility of this approach for investigating potential responses to climate change and point a way forward for future studies.