Natural variation, and the capacity to adapt to ocean acidification in the keystone sea urchin Strongylocentrotus purpuratus

A rapidly growing body of literature documents the potential negative effects of CO₂-driven ocean acidification (OA) on marine organisms. However, nearly all of this work has focused on the effects of future conditions on modern populations, neglecting the role of adaptation. Rapid evolution can alter demographic responses to environmental change, ultimately affecting the likelihood of population persistence, but the capacity for adaptation will differ among populations and species. Here, we measure the capacity of the ecologically important purple sea urchin Strongylocentrotus purpuratus to adapt to OA, using a breeding experiment to estimate additive genetic variance for larval size (an important component of fitness) under future high pCO₂/low pH conditions. Although larvae reared under future conditions were smaller than those reared under present-day conditions, we show that there is also abundant genetic variation for body size under elevated pCO₂, indicating that this trait can evolve. The observed heritability of size was 0.40±0.32 (95% CI) under low pCO₂, and 0.50±0.30 under high pCO₂ conditions. Accounting for the observed genetic variation in models of future larval size and demographic rates substantially alters projections of performance for this species in the future ocean. Importantly, our model shows that after incorporating the effects of adaptation, the OA-driven decrease in population growth rate is up to 50% smaller, than that predicted by the “no-adaptation” scenario. Adults used in the experiment were collected from two sites on the coast of the Northeast Pacific that are characterized by different pH regimes, as measured by autonomous sensors. Comparing results between sites, we also found subtle differences in larval size under high pCO₂ rearing conditions, consistent with local adaptation to carbonate chemistry in the field. These results suggest that spatially varying selection may help to maintain genetic variation necessary for adaptation to future ocean acidification.

Ocean acidification effects on marine microbial communities

Anthropogenic CO2 emissions are causing an acidification of the world’s oceans. The consequences for marine organisms and especially heterotrophic bacteria remain under debate, and almost nothing is known concerning marine fungi. Both microbial groups are important players in organic matter decomposition and nutrient cycling, and their pH tolerance is known to be broad in relation to the predicted acidification. So far, ocean acidification effects on marine bacterial communities have mainly been investigated in large-scale mesocosm studies. In these systems, indirect effects mediated through complex food web interactions come into play. Until now, these experiments were not carried out in sufficient replication. In this thesis, we chose an alternative approach and investigated bacterial and fungal communities in highly replicated microcosm experiments (1-1.6 L). The duration of the experiments was four weeks. We incubated the natural microbial community from Helgoland Roads (North Sea) at in situ seawater pH, pH 7.82 and pH 7.67. These pH levels represent the present-day situation and acidification at atmospheric CO2 of 700 or 1000 ppm, projected for the southern North Sea for the year 2100. For the bacterial community, different dilution approaches were used to select for different ecological groups. Seasonality was accounted for by repeating the experiment four times (spring, summer, autumn, winter). In a second experiment repeated in two consecutive years, we investigated direct pH effects on marine fungal communities. We additionally isolated marine yeasts and identified them by Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and partial sequencing of the large subunit (LSU) rRNA gene. To reveal changes in community structure, we applied the culture-independent fingerprint method automated ribosomal intergenic spacer analysis (ARISA) for both bacteria and fungi. Bacterial communities were furthermore analyzed by 16S ribosomal amplicon pyrosequencing. Abundances were determined by flow cytometry (bacteria) and colony forming unit counts (fungi). To be able to interpret results comprehensively, we determined the natural variability of the carbonate system at Helgoland Roads over a yearly cycle. We found that from September 2010 to September 2011, pH at Helgoland Roads ranged from 8.06 to 8.43, corresponding to partial pressures of carbon dioxide (pCO2) of 215-526 µatm. The acidification predicted for the year 2100 consequently represents a strong perturbation of the system. Bacterial communities developing in the microcosms were primarily influenced by season and dilution, demonstrating that diverse communities had been generated. We predominantly found pH-dependent shifts in bacterial community structure already at pH 7.82. Groups involved in these shifts were different members of Gammaproteobacteria, Flavobacteriaceae, Rhodobacteraceae, Campylobacteraceae and further less abundant groups. While Rhodobacteraceae were consistently less characteristic for reduced pH, Campylobacteraceae profited from pH reduction. For most other bacterial groups however, pH effects were context-dependent, i.e. dependent on season, dilution or an interaction of effects. Regarding bacterial abundance, no pH effect was found. Fungal community structure was significantly different between both years of the experiment, hinting at inter-annual variability. Shifts in response to pH occurred predominantly only at pH 7.67. In contrast, a strong pH effect was observed on fungal abundance. In comparison to in situ pH, fungal numbers were on average 9 times higher at pH 7.82 and 34 times higher at pH 7.67. Concerning marine yeasts, Leucosporidium scottii, Rhodotorula mucilaginosa and related species, as well as Cryptococcus sp. and Debaromyces hansenii reacted positively to low pH. Our findings demonstrate that already small reductions in pH have direct effects on both bacterial and fungal communities. A tipping point for community shifts appears to be reached earlier for bacteria than for fungi. Regarding bacteria and yeasts, both naturally abundant groups and rare species were affected by pH reductions. The strong increase in fungal numbers at reduced pH suggests that with ocean acidification, marine fungi may reach higher importance in marine biogeochemical cycles and as infectious agents. Using a microcosm approach, a robust analysis of direct ocean acidification effects on marine bacterial and fungal communities was accomplished. Results yield valuable hypotheses to test in future large-scale and long-term studies.

Interactive effects of global climate change and pollution on marine microbes: the way ahead

Global climate change has the potential to seriously and adversely affect marine ecosystem functioning. Numerous experimental and modeling studies have demonstrated how predicted ocean acidification and increased ultraviolet radiation (UVR) can affect marine microbes. However, researchers have largely ignored interactions between ocean acidification, increased UVR and anthropogenic pollutants in marine environments. Such interactions can alter chemical speciation and the bioavailability of several organic and inorganic pollutants with potentially deleterious effects, such as modifying microbial-mediated detoxification processes. Microbes mediate major biogeochemical cycles, providing fundamental ecosystems services such as environmental detoxification and recovery. It is, therefore, important that we understand how predicted changes to oceanic pH, UVR, and temperature will affect microbial pollutant detoxification processes in marine ecosystems. The intrinsic characteristics of microbes, such as their short generation time, small size, and functional role in biogeochemical cycles combined with recent advances in molecular techniques (e.g., metagenomics and metatranscriptomics) make microbes excellent models to evaluate the consequences of various climate change scenarios on detoxification processes in marine ecosystems. In this review, we highlight the importance of microbial microcosm experiments, coupled with high-resolution molecular biology techniques, to provide a critical experimental framework to start understanding how climate change, anthropogenic pollution, and microbiological interactions may affect marine ecosystems in the future.

Evolutionary change during experimental ocean acidification

Rising atmospheric carbon dioxide (CO₂) conditions are driving unprecedented changes in seawater chemistry, resulting in reduced pH and carbonate ion concentrations in the Earth’s oceans. This ocean acidification has negative but variable impacts on individual performance in many marine species. However, little is known about the adaptive capacity of species to respond to an acidified ocean, and, as a result, predictions regarding future ecosystem responses remain incomplete. Here we demonstrate that ocean acidification generates striking patterns of genome-wide selection in purple sea urchins (Strongylocentrotus purpuratus) cultured under different CO₂ levels. We examined genetic change at 19,493 loci in larvae from seven adult populations cultured under realistic future CO₂ levels. Although larval development and morphology showed little response to elevated CO₂, we found substantial allelic change in 40 functional classes of proteins involving hundreds of loci. Pronounced genetic changes, including excess amino acid replacements, were detected in all populations and occurred in genes for biomineralization, lipid metabolism, and ion homeostasis—gene classes that build skeletons and interact in pH regulation. Such genetic change represents a neglected and important impact of ocean acidification that may influence populations that show few outward signs of response to acidification. Our results demonstrate the capacity for rapid evolution in the face of ocean acidification and show that standing genetic variation could be a reservoir of resilience to climate change in this coastal upwelling ecosystem. However, effective response to strong natural selection demands large population sizes and may be limited in species impacted by other environmental stressors.

Short- and long-term consequences of larval stage exposure to constantly and ephemerally elevated carbon dioxide for marine bivalve populations (update)

While larval bivalves are highly sensitive to ocean acidification, the basis for this sensitivity and the longer-term implications of this sensitivity are unclear. Experiments were performed to assess the short-term (days) and long-term (months) consequences of larval stage exposure to varying CO₂ concentrations for calcifying bivalves. Higher CO₂ concentrations depressed both calcification rates assessed using ⁴⁵Ca uptake and RNA : DNA ratios in Mercenaria mercenaria and Argopecten irradians larvae with RNA : DNA ratios being highly correlated with larval growth rates (r²>0.9). These findings suggested that high CO₂ has a cascading negative physiological impact on bivalve larvae stemming in part from lower calcification rates. Exposure to elevated CO₂ during the first four days of larval development significantly depressed A. irradians larval survival rates, while a 10-day exposure later in larval development did not, demonstrating the extreme CO₂ sensitivity of bivalve larvae during first days of development. Short- (weeks) and long-term (10 month) experiments revealed that individuals surviving exposure to high CO₂ during larval development grew faster when exposed to normal CO₂ as juveniles compared to individuals reared under ambient CO₂ as larvae. These increased growth rates could not, however, overcome size differences established during larval development, as size deficits of individuals exposed to even moderate levels of CO₂ as larvae were evident even after 10 months of growth under normal CO₂ concentrations. This “legacy effect” emphasizes the central role larval stage CO₂ exposure can play in shaping the success of modern-day bivalve populations.

Genes related to ion-transport and energy production are upregulated in response to CO2-driven pH decrease in corals: new insights from transcriptome analysis

Since the preindustrial era, the average surface ocean pH has declined by 0.1 pH units and is predicted to decline by an additional 0.3 units by the year 2100. Although subtle, this decreasing pH has profound effects on the seawater saturation state of carbonate minerals and is thus predicted to impact on calcifying organisms. Among these are the scleractinian corals, which are the main builders of tropical coral reefs. Several recent studies have evaluated the physiological impact of low pH, particularly in relation to coral growth and calcification. However, very few studies have focused on the impact of low pH at the global molecular level. In this context we investigated global transcriptomic modifications in a scleractinian coral (Pocillopora damicornis) exposed to pH 7.4 compared to pH 8.1 during a 3-week period. The RNAseq approach shows that 16% of our transcriptome was affected by the treatment with 6% of upregulations and 10% of downregulations. A more detailed analysis suggests that the downregulations are less coordinated than the upregulations and allowed the identification of several biological functions of interest. In order to better understand the links between these functions and the pH, transcript abundance of 48 candidate genes was quantified by q-RT-PCR (corals exposed at pH 7.2 and 7.8 for 3 weeks). The combined results of these two approaches suggest that pH≥7.4 induces an upregulation of genes coding for proteins involved in calcium and carbonate transport, conversion of CO₂ into HCO₃⁻ and organic matrix that may sustain calcification. Concomitantly, genes coding for heterotrophic and autotrophic related proteins are upregulated. This can reflect that low pH may increase the coral energy requirements, leading to an increase of energetic metabolism with the mobilization of energy reserves. In addition, the uncoordinated downregulations measured can reflect a general trade-off mechanism that may enable energy reallocation.

Temperature and CO2 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.

Evolutionary responses of a coccolithophorid Gephyrocapsa oceanica to ocean acidification

The ongoing ocean acidification associated with a changing carbonate system may impose profound effects on marine planktonic calcifiers. Here, we show that a coccolithophore, Gephyrocapsa oceanica, evolved in response to an elevated CO2 concentration of 1000 μatm (pH reduced to 7.8) in a long term (∼ 670 generations) selection experiment. The high CO2 selected cells showed increases in photosynthetic carbon fixation, growth rate, cellular particulate organic carbon (POC) or nitrogen (PON) production and a decrease in C:N elemental ratio, indicating a greater up-regulation of PON than of POC production under the ocean acidification condition. Cells from the low CO2 selection process shifted to high CO2 exposure showed an enhanced cellular POC and PON production rates. Our data suggest that the coccolithophorid could adapt to ocean acidification with enhanced assimilations of carbon and nitrogen but decreased C:N ratios.

Dissecting the impact of CO2 and pH on the mechanisms of photosynthesis and calcification in the coccolithophore Emiliania huxleyi

Summary

• Coccolithophores are important calcifying phytoplankton predicted to be impacted by changes in ocean carbonate chemistry caused by the absorption of anthropogenic CO₂. However, it is difficult to disentangle the effects of the simultaneously changing carbonate system parameters (CO₂, bicarbonate, carbonate and protons) on the physiological responses to elevated CO₂.
• Here, we adopted a multifactorial approach at constant pH or CO₂ whilst varying dissolved inorganic carbon (DIC) to determine physiological and transcriptional responses to individual carbonate system parameters.
• We show that Emiliania huxleyi is sensitive to low CO₂ (growth and photosynthesis) and low bicarbonate (calcification) as well as low pH beyond a limited tolerance range, but is much less sensitive to elevated CO₂ and bicarbonate. Multiple up-regulated genes at low DIC bear the hallmarks of a carbon-concentrating mechanism (CCM) that is responsive to CO₂ and bicarbonate but not to pH.
• Emiliania huxleyi appears to have evolved mechanisms to respond to limiting rather than elevated CO₂. Calcification does not function as a CCM, but is inhibited at low DIC to allow the redistribution of DIC from calcification to photosynthesis. The presented data provides a significant step in understanding how E. huxleyi will respond to changing carbonate chemistry at a cellular level.

Response of Nodularia spumigena to pCO2 – Part 3: Turnover of phosphorus compounds (update)

Diazotrophic cyanobacteria form extensive summer blooms in the Baltic Sea driving the surrounding surface waters into phosphate limitation. One of the main bloom-forming species is the heterocystous cyanobacterium Nodularia spumigena. N. spumigena exhibits accelerated uptake of phosphate through the release of the extracellular enzyme alkaline phosphatase whose activity also serves as an indicator of the hydrolysis of dissolved organic phosphorus (DOP). The present study investigated the utilisation of DOP and its compounds (e.g., ATP) by N. spumigena during growth under different CO₂ concentrations, in order to estimate potential consequences of ocean acidification on the cell’s supply with phosphorus (P). Cell growth, the phosphorus pool, and four DOP compounds (ATP, DNA, RNA, and phospholipids) were determined in three setups with different CO₂ concentrations (average 341 μatm, 399 μatm, and 508 μatm) during a 15-day batch experiment. The results showed stimulated growth of N. spumigena and a rapid depletion of dissolved inorganic phosphorus (DIP) in all pCO₂ treatments. DOP uptake was enhanced by a factor of 1.32 at 399 μatm and of 2.25 at 508 μatm compared to the lowest CO₂ concentration. Among the measured DOP compounds, none was found to accumulate preferentially during the incubation or in response to a specific pCO₂ treatment. However, at the beginning 61.9 ± 4.3% of total DOP were not characterised but comprised the most utilised fraction. This is demonstrated by the decrement of this fraction to 27.4 ± 9.9% of total DOP during the growth phase with a preference at high pCO₂. Our results indicate a stimulated growth of diazotrophic cyanobacteria at increasing CO₂ concentrations which is accompanied by increasing utilisation of DOP as an alternative P source.