Magnitude and Predictability of pH Fluctuations Shape Plastic Responses to Ocean Acidification.

AbstractPhenotypic plasticity is expected to facilitate the persistence of natural populations as global change progresses. The attributes of fluctuating environments that favor the evolution of plasticity have received extensive theoretical investigation, yet empirical validation of these findings is still in its infancy. Here, we combine high-resolution environmental data with a laboratory-based experiment to explore the influence of habitat pH fluctuation dynamics on the plasticity of gene expression in two populations of the Mediterranean mussel, Mytilus galloprovincialis. We linked differences in the magnitude and predictability of pH fluctuations in two habitats to population-specific gene expression profiles in ambient and stressful pH treatments. Our results demonstrate population-based differentiation in gene expression plasticity, whereby mussels native to a habitat exhibiting a large magnitude of pH fluctuations with low predictability display reduced phenotypic plasticity between experimentally imposed pH treatments. This work validates recent theoretical findings on evolution in fluctuating environments, suggesting that the predictability of fluctuating selection pressures may play a predominant role in shaping the phenotypic variation observed across natural populations.

A mineralogical record of ocean change: Decadal and centennial patterns in the California mussel.

Ocean acidification, a product of increasing atmospheric carbon dioxide, may already have affected calcified organisms in the coastal zone, such as bivalves and other shellfish. Understanding species' responses to climate change requires the context of long-term dynamics. This can be particularly difficult given the longevity of many important species in contrast with the relatively rapid onset of environmental changes. Here, we present a unique archival dataset of mussel shells from a locale with recent environmental monitoring and historical climate reconstructions. We compare shell structure and composition in modern mussels, mussels from the 1970s, and mussel shells dating back to 1000-2420 years BP. Shell mineralogy has changed dramatically over the past 15 years, despite evidence for consistent mineral structure in the California mussel, Mytilus californianus, over the prior 2500 years. We present evidence for increased disorder in the calcium carbonate shells of mussels and greater variability between individuals. These changes in the last decade contrast markedly from a background of consistent shell mineralogy for centuries. Our results use an archival record of natural specimens to provide centennial-scale context for altered minerology and variability in shell features as a response to acidification stress and illustrate the utility of long-term studies and archival records in global change ecology. Increased variability between individuals is an emerging pattern in climate change responses, which may equally expose the vulnerability of organisms and the potential of populations for resilience.

Climate drivers and animal host use determine kelp performance over decadal scales in the kelp Pleurophycus gardneri (Laminariales, Phaeophyceae).

Primary producers respond to climate directly and indirectly due to effects on their consumers. In the temperate coastal ocean, the highly productive brown algae known as kelp have both strong climate and grazer linkages. We analyzed the demographic response of the kelp Pleurophycus gardneri over a 25-year span to determine the interaction between ocean climate indicators and invertebrate infestation rates. Pleurophycus hosts amphipod species that burrow in the stipe, increasing mortality. Although kelp performance is generally greater with more negative values of the Pacific Decadal Oscillation (PDO) and colder seawater temperatures, Pleurophycus showed the opposite pattern. When we compared the 1990s, a period of positive values for the PDO and warmer sea surface temperatures, with the following decade, a period characterized by negative PDO values, we documented a contradictory outcome for proxies of kelp fitness. In the 1990s, Pleurophycus unexpectedly showed greater longevity, faster growth, greater reproductive effort, and a trend toward decreased amphipod infestation compared with the 2006-2012 period. In contrast, the period from 2006 to 2012 showed opposite kelp performance patterns and with a trend toward greater amphipod infestation. Pleurophycus performance metrics suggest that some coastal primary producers will respond differently to climate drivers, particularly if they interact strongly with grazers.

Ocean acidification affects competition for space: projections of community structure using cellular automata.

Historical ecological datasets from a coastal marine community of crustose coralline algae (CCA) enabled the documentation of ecological changes in this community over 30 years in the Northeast Pacific. Data on competitive interactions obtained from field surveys showed concordance between the 1980s and 2013, yet also revealed a reduction in how strongly species interact. Here, we extend these empirical findings with a cellular automaton model to forecast ecological dynamics. Our model suggests the emergence of a new dominant competitor in a global change scenario, with a reduced role of herbivory pressure, or trophic control, in regulating competition among CCA. Ocean acidification, due to its energetic demands, may now instead play this role in mediating competitive interactions and thereby promote species diversity within this guild.

Historical baselines and the future of shell calcification for a foundation species in a changing ocean.

Seawater pH and the availability of carbonate ions are decreasing due to anthropogenic carbon dioxide emissions, posing challenges for calcifying marine species. Marine mussels are of particular concern given their role as foundation species worldwide. Here, we document shell growth and calcification patterns in Mytilus californianus, the California mussel, over millennial and decadal scales. By comparing shell thickness across the largest modern shells, the largest mussels collected in the 1960s-1970s and shells from two Native American midden sites (∼1000-2420 years BP), we found that modern shells are thinner overall, thinner per age category and thinner per unit length. Thus, the largest individuals of this species are calcifying less now than in the past. Comparisons of shell thickness in smaller individuals over the past 10-40 years, however, do not show significant shell thinning. Given our sampling strategy, these results are unlikely to simply reflect within-site variability or preservation effects. Review of environmental and biotic drivers known to affect shell calcification suggests declining ocean pH as a likely explanation for the observed shell thinning. Further future decreases in shell thickness could have significant negative impacts on M. californianus survival and, in turn, negatively impact the species-rich complex that occupies mussel beds.

Detecting the unexpected: a research framework for ocean acidification.

The threat that ocean acidification (OA) poses to marine ecosystems is now recognized and U.S. funding agencies have designated specific funding for the study of OA. We present a research framework for studying OA that describes it as a biogeochemical event that impacts individual species and ecosystems in potentially unexpected ways. We draw upon specific lessons learned about ecosystem responses from research on acid rain, carbon dioxide enrichment in terrestrial plant communities, and nitrogen deposition. We further characterize the links between carbon chemistry changes and effects on individuals and ecosystems, and enumerate key hypotheses for testing. Finally, we quantify how U.S. research funding has been distributed among these linkages, concluding that there is an urgent need for research programs designed to anticipate how the effects of OA will reverberate throughout assemblages of species.

Ammonification by kelp associated microbes increases ammonium availability.

Microbes contribute biologically available nitrogen to the ocean by fixing nitrogen gas from the atmosphere and by mineralizing organic nitrogen into bioavailable dissolved inorganic nitrogen (DIN). Although the large concentration of plants and algae in marine coastal environments provides ample habitat and reliable resources for microbial communities, the role of the microbiome in host-microbe nitrogen cycling remains poorly understood. We tested whether ammonification by epiphytic microbes increased water column ammonium and improved host access to nitrogen resources by converting organic nitrogen into inorganic nitrogen that is available for assimilation by hosts. When bull kelp (Nereocystis luetkeana) in the northeast Pacific was incubated with 15N labelled amino acid tracers, there was accumulation of 15N in kelp tissue, as well as accumulation of 15NH4 in seawater, all consistent with the conversion of dissolved organic nitrogen to ammonium. Metagenomic analysis of surface microbes from two populations of Nereocystis indicated relative similarity in the percentage of genes related to ammonification between the two locations, though the stressed kelp population that had lower tissue nitrogen and a sparser microbiome had greater ammonification rates. Microbial communities on coastal macrophytes may contribute to the nitrogen requirements of their hosts through metabolisms that make ammonium available.

Bacteria on the foundational kelp in kelp forest ecosystems: Insights from culturing, whole genome sequencing and metabolic assays.

In coastal marine ecosystems, kelp forests serve as a vital habitat for numerous species and significantly influence local nutrient cycles. Bull kelp, or Nereocystis luetkeana, is a foundational species in the iconic kelp forests of the northeast Pacific Ocean and harbours a complex microbial community with potential implications for kelp health. Here, we report the isolation and functional characterisation of 16 Nereocystis-associated bacterial species, comprising 13 Gammaproteobacteria, 2 Flavobacteriia and 1 Actinomycetia. Genome analyses of these isolates highlight metabolisms potentially beneficial to the host, such as B vitamin synthesis and nitrogen retention. Assays revealed that kelp-associated bacteria thrive on amino acids found in high concentrations in the ocean and in the kelp (glutamine and asparagine), generating ammonium that may facilitate host nitrogen acquisition. Multiple isolates have genes indicative of interactions with key elemental cycles in the ocean, including carbon, nitrogen and sulphur. We thus report a collection of kelp-associated microbial isolates that provide functional insight for the future study of kelp-microbe interactions.

Experimental separation of genetic and demographic factors on extinction risk in wild populations.

When populations reach small size, an extinction risk vortex may arise from genetic (inbreeding depression, genetic drift) and ecological (demographic stochasticity, Allee effects, environmental fluctuation) processes. The relative contribution of these processes to extinction in wild populations is unknown, but important for conserving endangered species. In experimental field populations of a harvested kelp (Postelsia palmaeformis), in which we independently varied initial genetic diversity (completely inbred, control, outbred) and population size, ecological processes dominated the risk of extinction, whereas the contribution of genetic diversity was slight. Our results match theoretical predictions that demographic processes will generally doom small populations to extinction before genetic effects act strongly, prioritize detailed ecological analysis over descriptions of genetic structure in assessing conservation of at-risk species, and highlight the need for field experiments manipulating both demographics and genetic structure on long-term extinction risk.

Microbial associates of an endemic Mediterranean seagrass enhance the access of the host and the surrounding seawater to inorganic nitrogen under ocean acidification.

Seagrasses are important primary producers in oceans worldwide. They live in shallow coastal waters that are experiencing carbon dioxide enrichment and ocean acidification. Posidonia oceanica, an endemic seagrass species that dominates the Mediterranean Sea, achieves high abundances in seawater with relatively low concentrations of dissolved inorganic nitrogen. Here we tested whether microbial metabolisms associated with P. oceanica and surrounding seawater enhance seagrass access to nitrogen. Using stable isotope enrichments of intact seagrass with amino acids, we showed that ammonification by free-living and seagrass-associated microbes produce ammonium that is likely used by seagrass and surrounding particulate organic matter. Metagenomic analysis of the epiphytic biofilm on the blades and rhizomes support the ubiquity of microbial ammonification genes in this system. Further, we leveraged the presence of natural carbon dioxide vents and show that the presence of P. oceanica enhanced the uptake of nitrogen by water column particulate organic matter, increasing carbon fixation by a factor of 8.6-17.4 with the greatest effect at CO2 vent sites. However, microbial ammonification was reduced at lower pH, suggesting that future ocean climate change will compromise this microbial process. Thus, the seagrass holobiont enhances water column productivity, even in the context of ocean acidification.

Functional Insights into the Kelp Microbiome from Metagenome-Assembled Genomes.

Eukaryotic organisms evolved in a microbial world and often have intimate associations with diverse bacterial groups. Kelp, brown macroalgae in the order Laminariales, play a vital role in coastal ecosystems, yet we know little about the functional role of the microbial symbionts that cover their photosynthetic surfaces. Here, we reconstructed 79 bacterial metagenome-assembled genomes (MAGs) from blades of the bull kelp, Nereocystis luetkeana, allowing us to determine their metabolic potential and functional roles. Despite the annual life history of bull kelp, nearly half of the bacterial MAGs were detected across multiple years. Diverse members of the kelp microbiome, spanning 6 bacterial phyla, contained genes for transporting and assimilating dissolved organic matter (DOM), which is secreted by kelp in large quantities and likely fuels the metabolism of these heterotrophic bacteria. Bacterial genomes also contained alginate lyase and biosynthesis genes, involved in polysaccharide degradation and biofilm formation, respectively. Kelp-associated bacterial genomes contained genes for dissimilatory nitrate reduction and urea hydrolysis, likely providing a reduced source of nitrogen to the host kelp. The genome of the most abundant member of the kelp microbiome and common macroalgal symbiont, Granulosicoccus, contained a full suite of genes for synthesizing cobalamin (vitamin B12), suggesting that kelp-associated bacteria have the potential to provide their host kelp with vitamins. Finally, kelp-associated Granulosicoccus contained genes that typify the aerobic anoxygenic phototrophic bacteria, including genes for bacteriochlorophyll synthesis and photosystem II reaction center proteins, making them the first known photoheterotrophic representatives of this genus. IMPORTANCE Kelp (brown algae in the order Laminariales) are foundational species that create essential habitat in temperate and arctic coastal marine ecosystems. These photosynthetic giants host millions of microbial taxa whose functions are relatively unknown, despite their potential importance for host-microbe interactions and nutrient cycling in kelp forest ecosystems. We reconstructed bacterial genomes from metagenomic samples collected from blades of the bull kelp, Nereocystis luetkeana, allowing us to determine the functional gene content of specific members of the kelp microbiome. These bacterial genomes spanned 6 phyla and 19 families and included common alga-associated microbial symbionts such as Granulosicoccus. Key functions encoded in kelp-associated bacterial genomes included dissolved organic matter assimilation, alginate metabolism, vitamin B12 biosynthesis, and nitrogen reduction from nitrate and urea to ammonium, potentially providing the host kelp with vitamins and reduced nitrogen.

Conceptual Exchanges for Understanding Free-Living and Host-Associated Microbiomes.

Whether a microbe is free-living or associated with a host from across the tree of life, its existence depends on a limited number of elements and electron donors and acceptors. Yet divergent approaches have been used by investigators from different fields. The "environment first" research tradition emphasizes thermodynamics and biogeochemical principles, including the quantification of redox environments and elemental stoichiometry to identify transformations and thus an underlying microbe. The increasingly common "microbe first" research approach benefits from culturing and/or DNA sequencing methods to first identify a microbe and encoded metabolic functions. Here, the microbe itself serves as an indicator for environmental conditions and transformations. We illustrate the application of both approaches to the study of microbiomes and emphasize how both can reveal the selection of microbial metabolisms across diverse environments, anticipate alterations to microbiomes in host health, and understand the implications of a changing climate for microbial function.

Spatial organization of the kelp microbiome at micron scales.

BACKGROUND: Elucidating the spatial structure of host-associated microbial communities is essential for understanding taxon-taxon interactions within the microbiota and between microbiota and host. Macroalgae are colonized by complex microbial communities, suggesting intimate symbioses that likely play key roles in both macroalgal and bacterial biology, yet little is known about the spatial organization of microbes associated with macroalgae. Canopy-forming kelp are ecologically significant, fixing teragrams of carbon per year in coastal kelp forest ecosystems. We characterized the micron-scale spatial organization of bacterial communities on blades of the kelp Nereocystis luetkeana using fluorescence in situ hybridization and spectral imaging with a probe set combining phylum-, class-, and genus-level probes to localize and identify > 90% of the microbial community. RESULTS: We show that kelp blades host a dense microbial biofilm composed of disparate microbial taxa in close contact with one another. The biofilm is spatially differentiated, with clustered cells of the dominant symbiont Granulosicoccus sp. (Gammaproteobacteria) close to the kelp surface and filamentous Bacteroidetes and Alphaproteobacteria relatively more abundant near the biofilm-seawater interface. A community rich in Bacteroidetes colonized the interior of kelp tissues. Microbial cell density increased markedly along the length of the kelp blade, from sparse microbial colonization of newly produced tissues at the meristematic base of the blade to an abundant microbial biofilm on older tissues at the blade tip. Kelp from a declining population hosted fewer microbial cells compared to kelp from a stable population. CONCLUSIONS: Imaging revealed close association, at micrometer scales, of different microbial taxa with one another and with the host. This spatial organization creates the conditions necessary for metabolic exchange among microbes and between host and microbiota, such as provisioning of organic carbon to the microbiota and impacts of microbial nitrogen metabolisms on host kelp. The biofilm coating the surface of the kelp blade is well-positioned to mediate interactions between the host and surrounding organisms and to modulate the chemistry of the surrounding water column. The high density of microbial cells on kelp blades (105-107 cells/cm2), combined with the immense surface area of kelp forests, indicates that biogeochemical functions of the kelp microbiome may play an important role in coastal ecosystems. Video abstract.

The Diversity and Functional Capacity of Microbes Associated with Coastal Macrophytes.

Coastal marine macrophytes exhibit some of the highest rates of primary productivity in the world. They have been found to host a diverse set of microbes, many of which may impact the biology of their hosts through metabolisms that are unique to microbial taxa. Here, we characterized the metabolic functions of macrophyte-associated microbial communities using metagenomes collected from 2 species of kelp (Laminaria setchellii and Nereocystis luetkeana) and 3 marine angiosperms (Phyllospadix scouleri, P. serrulatus, and Zostera marina), including the rhizomes of two surfgrass species (Phyllospadix spp.), the seagrass Zostera marina, and the sediments surrounding P. scouleri and Z. marina. Using metagenomic sequencing, we describe 63 metagenome-assembled genomes (MAGs) that potentially benefit from being associated with macrophytes and may contribute to macrophyte fitness through their metabolic activity. Host-associated metagenomes contained genes for the use of dissolved organic matter from hosts and vitamin (B1, B2, B7, B12) biosynthesis in addition to a range of nitrogen and sulfur metabolisms that recycle dissolved inorganic nutrients into forms more available to the host. The rhizosphere of surfgrass and seagrass contained genes for anaerobic microbial metabolisms, including nifH genes associated with nitrogen fixation, despite residing in a well-mixed and oxygenated environment. The range of oxygen environments engineered by macrophytes likely explains the diversity of both oxidizing and reducing microbial metabolisms and contributes to the functional capabilities of microbes and their influences on carbon and nitrogen cycling in nearshore ecosystems. IMPORTANCE Kelps, seagrasses, and surfgrasses are ecosystem engineers on rocky shorelines, where they show remarkably high levels of primary production. Through analysis of their associated microbial communities, we found a variety of microbial metabolisms that may benefit the host, including nitrogen metabolisms, sulfur oxidation, and the production of B vitamins. In turn, these microbes have the genetic capabilities to assimilate the dissolved organic compounds released by their macrophyte hosts. We describe a range of oxygen environments associated with surfgrass, including low-oxygen microhabitats in their rhizomes that host genes for nitrogen fixation. The tremendous productivity of coastal seaweeds and seagrasses is likely due in part to the activities of associated microbes, and an increased understanding of these associations is needed.

The mixed mating system of the sea palm kelp Postelsia palmaeformis: few costs to selfing.

Naturally isolated populations have conflicting selection pressures for successful reproduction and inbreeding avoidance. These species with limited seasonal reproductive opportunities may use selfing as a means of reproductive assurance. We quantified the frequency of selfing and the fitness consequences for inbred versus outcrossed progeny of an annual kelp, the sea palm (Postelsia palmaeformis). Using experimentally established populations and microsatellite markers to assess the extent of selfing in progeny from six founding parents, we found the frequency of selfing was higher than expected in every population, and few fitness costs were detected in selfed offspring. Despite a decline in heterozygosity of 30 per cent in the first generation of selfing, self-fertilization did not affect individual size or reproduction, and correlated only with a marginally significant decline in survival. Our results suggest both that purging of deleterious recessive alleles may have already occurred and that selfing may be key to reproductive assurance in this species with limited dispersal. Postelsia has an alteration of a free-living diploid and haploid stage, where the haploid stage may provide increased efficiency for purging the genetic load. This life history is shared by many seaweeds and may thus be an important component of mating system evolution in the sea.

Dynamic patterns and ecological impacts of declining ocean pH in a high-resolution multi-year dataset.

Increasing global concentrations of atmospheric CO(2) are predicted to decrease ocean pH, with potentially severe impacts on marine food webs, but empirical data documenting ocean pH over time are limited. In a high-resolution dataset spanning 8 years, pH at a north-temperate coastal site declined with increasing atmospheric CO(2) levels and varied substantially in response to biological processes and physical conditions that fluctuate over multiple time scales. Applying a method to link environmental change to species dynamics via multispecies Markov chain models reveals strong links between in situ benthic species dynamics and variation in ocean pH, with calcareous species generally performing more poorly than noncalcareous species in years with low pH. The models project the long-term consequences of these dynamic changes, which predict substantial shifts in the species dominating the habitat as a consequence of both direct effects of reduced calcification and indirect effects arising from the web of species interactions. Our results indicate that pH decline is proceeding at a more rapid rate than previously predicted in some areas, and that this decline has ecological consequences for near shore benthic ecosystems.

Oxygen metabolism shapes microbial settlement on photosynthetic kelp blades compared to artificial kelp substrates.

We examined factors shaping community assembly of the bull kelp (Nereocystis luetkeana) microbiome by comparing microbial biofilm formation on photosynthetic kelp blade tissues and artificial kelp substrates ('agar substrates') deployed into a kelp forest. New kelp blade tissues were colonized by markedly distinct microbial taxa relative to agar substrates during the same time interval, even when agar substrates were infused with N. luetkeana blades, suggesting that microbial settlement onto kelp surfaces is more than just attraction to a polysaccharide-rich surface. Further, common seawater taxa such as Colwellia sp. and Psychromonas sp. became abundant on agar substrates but avoided new kelp blade tissues, indicating that host-specific factors may deter certain surface-associated marine microbial taxa. Over two-thirds of the bacterial taxa in the kelp microbiome were associated with strictly aerobic metabolisms; thus, photosynthetic production of O2 may favour aerobic microbial metabolisms. While living kelp blades primarily recruited aerobic microbes, including the obligate aerobe Granulosicoccus sp., microbes that colonized agar substrates were predominantly facultative anaerobes. We also found that infusion of kelp tissues into agar substrates altered microbial community composition and lowered taxonomic diversity relative to control agar substrates, suggesting that non-living components of the kelp blade also impact microbial community assembly.

The dynamics and stoichiometry of dissolved organic carbon release by kelp.

Canopy-forming kelps are foundational species in coastal ecosystems, fixing tremendous amounts of carbon, yet we know little about the ecological and physiological determinants of dissolved organic carbon (DOC) release by kelps. We examined DOC release by the bull kelp, Nereocystis luetkeana, in relation to carbon fixation, nutrient uptake, tissue nitrogen content, and light availability. DOC release was approximately 3.5 times greater during the day than at night. During the day, N. luetkeana blades released an average of 16.2% of fixed carbon as DOC. Carbon fixation increased with light availability but DOC release did not, leading to a lower proportion of fixed carbon released as DOC at high light levels. We found no relationship between carbon fixation and DOC release rates measured concurrently. Rather, DOC release by N. luetkeana blades declined with marginal significance as blade tissue nitrogen content increased and with experimental nitrate addition, supporting the role of stoichiometric relationships in DOC release. Using a stable isotope (13 C) tracer method, we demonstrated that inorganic carbon is rapidly fixed and released by N. luetkeana blades as 13 DOC, within hours. However, recently fixed carbon (13 DOC) comprised less than 20% of the total DOC released, indicating that isotope studies that rely on tracer production alone may underestimate total DOC release, as it is decoupled from recent kelp productivity. Comparing carbon and nitrogen assimilation dynamics of the annual kelp N. luetkeana with the perennial kelp Macrocystis pyrifera revealed that N. luetkeana had significantly higher carbon fixation, DOC production and nitrogen uptake rates per unit dry mass. Both kelp species were able to perform light-independent carbon fixation at night. Carbon fixation by the annual kelp N. luetkeana is as high as 2.35 kg C·m-2 ·yr-1 , but an average of 16% of this carbon (376 g C·m-2 ·yr-1 ) is released as DOC. As kelp forests are increasingly viewed as vehicles for carbon sequestration, it is important to consider the fate of this substantial quantity of DOC released by canopy-forming kelps.