Accelerated nitrogen cycling on Mediterranean seagrass leaves at volcanic CO2 vents.

Seagrass meadows form highly productive and diverse ecosystems in coastal areas worldwide, where they are increasingly exposed to ocean acidification (OA). Efficient nitrogen (N) cycling and uptake are essential to maintain plant productivity, but the effects of OA on N transformations in these systems are poorly understood. Here we show that complete N cycling occurs on leaves of the Mediterranean seagrass Posidonia oceanica at a volcanic CO2 vent near Ischia Island (Italy), with OA affecting both N gain and loss while the epiphytic microbial community structure remains largely unaffected. Daily leaf-associated N2 fixation contributes to 35% of the plant's N demand under ambient pH, while it contributes to 45% under OA. Nitrification potential is only detected under OA, and N-loss via N2 production increases, although the balance remains decisively in favor of enhanced N gain. Our work highlights the role of the N-cycling microbiome in seagrass adaptation to OA, with key N transformations accelerating towards increased N gain.

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.

Nested interactions between chemosynthetic lucinid bivalves and seagrass promote ecosystem functioning in contaminated sediments.

In seagrass sediments, lucinid bivalves and their chemoautotrophic bacterial symbionts consume H2S, relying indirectly on the plant productivity for the presence of the reduced chemical. Additionally, the role of lucinid bivalves in N provisioning to the plant (through N2 fixation by the symbionts) was hypothesized. Thus, lucinids may contribute to sediment detoxification and plant fitness. Seagrasses are subject to ever-increasing human pressure in coastal environments. Here, disentangling nested interactions between chemosynthetic lucinid bivalves and seagrass exposed to pollution may help to understand seagrass ecosystem dynamics and to develop successful seagrass restoration programs that consider the roles of animal-microbe symbioses. We evaluated the capacity of lucinid bivalves (Loripes orbiculatus) to promote nutrient cycling and seagrass (Cymodocea nodosa) growth during a 6-week mesocosm experiment. A fully crossed design was used to test for the effect of sediment contamination (metals, nutrients, and hydrocarbons) on plant and bivalve (alone or interacting) fitness, assessed by mortality, growth, and photosynthetic efficiency, and for the effect of their nested interaction on sediment biogeochemistry. Plants performed better in the contaminated sediment, where a larger pool of dissolved nitrogen combined with the presence of other trace elements allowed for an improved photosynthetic efficiency. In fact, pore water nitrogen accumulated during the experiment in the controls, while it was consumed in the contaminated sediment. This trend was accentuated when lucinids were present. Concurrently, the interaction between clams and plants benefitted both organisms and promoted plant growth irrespective of the sediment type. In particular, the interaction with lucinid clams resulted in higher aboveground biomass of C. nodosa in terms of leaf growth, leaf surface, and leaf biomass. Our results consolidate the notion that nested interactions involving animal-microbe associations promote ecosystem functioning, and potentially help designing unconventional seagrass restoration strategies that exploit chemosynthetic symbioses.

The role of epiphytes in seagrass productivity under ocean acidification.

Ocean Acidification (OA), due to rising atmospheric CO2, can affect the seagrass holobiont by changing the plant's ecophysiology and the composition and functioning of its epiphytic community. However, our knowledge of the role of epiphytes in the productivity of the seagrass holobiont in response to environmental changes is still very limited. CO2 vents off Ischia Island (Italy) naturally reduce seawater pH, allowing to investigate the adaptation of the seagrass Posidonia oceanica L. (Delile) to OA. Here, we analyzed the percent cover of different epiphytic groups and the epiphytic biomass of P. oceanica leaves, collected inside (pH 6.9-7.9) and outside (pH 8.1-8.2) the CO2 vents. We estimated the contribution of epiphytes to net primary production (NPP) and respiration (R) of leaf sections collected from the vent and ambient pH sites in laboratory incubations. Additionally, we quantified net community production (NCP) and community respiration (CR) of seagrass communities in situ at vent and ambient pH sites using benthic chambers. Leaves at ambient pH sites had a 25% higher total epiphytic cover with encrusting red algae (32%) dominating the community, while leaves at vent pH sites were dominated by hydrozoans (21%). Leaf sections with and without epiphytes from the vent pH site produced and respired significantly more oxygen than leaf sections from the ambient pH site, showing an average increase of 47 ± 21% (mean ± SE) in NPP and 50 ± 4% in R, respectively. Epiphytes contributed little to the increase in R; however, their contribution to NPP was important (56 ± 6% of the total flux). The increase in productivity of seagrass leaves adapted to OA was only marginally reflected by the results from the in situ benthic chambers, underlining the complexity of the seagrass community response to naturally occurring OA conditions.

Scaling up calcification, respiration, and photosynthesis rates of six prominent coral taxa.

Coral reefs provide a range of important services to humanity, which are underpinned by community-level ecological processes such as coral calcification. Estimating these processes relies on our knowledge of individual physiological rates and species-specific abundances in the field. For colonial animals such as reef-building corals, abundance is frequently expressed as the relative surface cover of coral colonies, a metric that does not account for demographic parameters such as coral size. This may be problematic because many physiological rates are directly related to organism size, and failure to account for linear scaling patterns may skew estimates of ecosystem functioning. In the present study, we characterize the scaling of three physiological rates - calcification, respiration, and photosynthesis - considering the colony size for six prominent, reef-building coral taxa in Mo'orea, French Polynesia. After a seven-day acclimation period in the laboratory, we quantified coral physiological rates for three hours during daylight (i.e., calcification and gross photosynthesis) and one hour during night light conditions (i.e., dark respiration). Our results indicate that area-specific calcification rates are higher for smaller colonies across all taxa. However, photosynthesis and respiration rates remain constant over the colony-size gradient. Furthermore, we revealed a correlation between the demographic dynamics of coral genera and the ratio between net primary production and calcification rates. Therefore, intraspecific scaling of reef-building coral physiology not only improves our understanding of community-level coral reef functioning but it may also explain species-specific responses to disturbances.

Unusually Warm Summer Temperatures Exacerbate Population and Plant Level Response of Posidonia oceanica to Anthropogenic Nutrient Stress.

Posidonia oceanica is a key foundation species in the Mediterranean providing valuable ecosystem services. However, this species is particularly vulnerable towards high coastal nutrient inputs and the rising frequency of intense summer heat waves, but their combined effect in situ has received little attention so far. Here, we investigated the effects of in situ nutrient addition during an unusually warm summer over a 4-month period, comparing different morphological, physiological and biochemical population metrics of seagrass meadows growing in protected areas (Ischia) with meadows already exposed to significant anthropogenic pressure (Baia - Gulf of Pozzuoli). Our study highlights that the effects of warmer than usual summer temperatures on the population level of seagrass meadows can be exacerbated if the plants are already exposed to higher anthropogenic pressures. Morphological and population level indicators mainly changed over time, possibly impacted by season and the warmer temperatures, and displayed more pronounced reductions in seagrasses from impacted sites. The additional nutrient supply had even more deleterious effects, as shown by a decrease in approximately 67% in cover in fertilized plots at high impacted sites and 33% at low impacted sites. Moreover, while rhizome starch concentration showed a seasonal increase in plants from low impacted sites it displayed a trend of a 27% decrease in fertilized plots of the high impacted sites. Epiphyte biomass was approximately four-fold higher on leaves of plants growing in impacted sites and even doubled with the additional nutrient input. Predicting and anticipating stress in P. oceanica is of crucial importance for conservation and management efforts, given the limited colonizing and reproductive ability and extremely slow growth of this ecosystem engineer. Our results suggest that monitoring efforts should focus especially on leaf area index (LAI), carbohydrate concentrations in the rhizomes, and epiphyte cover on leaves as indicators of the onset of stress in Posidonia oceanica, which can be used by decision makers to take appropriate measures before damage to the ecosystem becomes irreversible, minimize future human interference and strengthen the resilience of these important ecosystems.

Juvenile corals underpin coral reef carbonate production after disturbance.

Sea-level rise is predicted to cause major damage to tropical coastlines. While coral reefs can act as natural barriers for ocean waves, their protection hinges on the ability of scleractinian corals to produce enough calcium carbonate (CaCO3 ) to keep up with rising sea levels. As a consequence of intensifying disturbances, coral communities are changing rapidly, potentially reducing community-level CaCO3 production. By combining colony-level physiology and long-term monitoring data, we show that reefs recovering from major disturbances can produce 40% more CaCO3 than currently estimated due to the disproportionate contribution of juvenile corals. However, the buffering effect of highly productive juvenile corals is compromised by recruitment failures, which have been more frequently observed after large-scale, repeated bleaching events. While the size structure of corals can bolster a critical ecological function on reefs, climate change impacts on recruitment may undermine this buffering effect, thus further compromising the persistence of reefs and their provision of important ecosystem services.

A community perspective on the concept of marine holobionts: current status, challenges, and future directions.

Host-microbe interactions play crucial roles in marine ecosystems. However, we still have very little understanding of the mechanisms that govern these relationships, the evolutionary processes that shape them, and their ecological consequences. The holobiont concept is a renewed paradigm in biology that can help to describe and understand these complex systems. It posits that a host and its associated microbiota with which it interacts, form a holobiont, and have to be studied together as a coherent biological and functional unit to understand its biology, ecology, and evolution. Here we discuss critical concepts and opportunities in marine holobiont research and identify key challenges in the field. We highlight the potential economic, sociological, and environmental impacts of the holobiont concept in marine biological, evolutionary, and environmental sciences. Given the connectivity and the unexplored biodiversity specific to marine ecosystems, a deeper understanding of such complex systems requires further technological and conceptual advances, e.g., the development of controlled experimental model systems for holobionts from all major lineages and the modeling of (info)chemical-mediated interactions between organisms. Here we propose that one significant challenge is to bridge cross-disciplinary research on tractable model systems in order to address key ecological and evolutionary questions. This first step is crucial to decipher the main drivers of the dynamics and evolution of holobionts and to account for the holobiont concept in applied areas, such as the conservation, management, and exploitation of marine ecosystems and resources, where practical solutions to predict and mitigate the impact of human activities are more important than ever.

N2 fixation dominates nitrogen cycling in a mangrove fiddler crab holobiont.

Mangrove forests are among the most productive and diverse ecosystems on the planet, despite limited nitrogen (N) availability. Under such conditions, animal-microbe associations (holobionts) are often key to ecosystem functioning. Here, we investigated the role of fiddler crabs and their carapace-associated microbial biofilm as hotspots of microbial N transformations and sources of N within the mangrove ecosystem. 16S rRNA gene and metagenomic sequencing provided evidence of a microbial biofilm dominated by Cyanobacteria, Alphaproteobacteria, Actinobacteria, and Bacteroidota with a community encoding both aerobic and anaerobic pathways of the N cycle. Dinitrogen (N2) fixation was among the most commonly predicted process. Net N fluxes between the biofilm-covered crabs and the water and microbial N transformation rates in suspended biofilm slurries portray these holobionts as a net N2 sink, with N2 fixation exceeding N losses, and as a significant source of ammonium and dissolved organic N to the surrounding environment. N stable isotope natural abundances of fiddler crab carapace-associated biofilms were within the range expected for fixed N, further suggesting active microbial N2 fixation. These results extend our knowledge on the diversity of invertebrate-microbe associations, and provide a clear example of how animal microbiota can mediate a plethora of essential biogeochemical processes in mangrove ecosystems.

Zebra Mussel Holobionts Fix and Recycle Nitrogen in Lagoon Sediments.

Bivalves are ubiquitous filter-feeders able to alter ecosystems functions. Their impact on nitrogen (N) cycling is commonly related to their filter-feeding activity, biodeposition, and excretion. A so far understudied impact is linked to the metabolism of the associated microbiome that together with the host constitute the mussel's holobiont. Here we investigated how colonies of the invasive zebra mussel (Dreissena polymorpha) alter benthic N cycling in the shallow water sediment of the largest European lagoon (the Curonian Lagoon). A set of incubations was conducted to quantify the holobiont's impact and to quantitatively compare it with the indirect influence of the mussel on sedimentary N transformations. Zebra mussels primarily enhanced the recycling of N to the water column by releasing mineralized algal biomass in the form of ammonium and by stimulating dissimilatory nitrate reduction to ammonium (DNRA). Notably, however, not only denitrification and DNRA, but also dinitrogen (N2) fixation was measured in association with the holobiont. The diazotrophic community of the holobiont diverged substantially from that of the water column, suggesting a unique niche for N2 fixation associated with the mussels. At the densities reported in the lagoon, mussel-associated N2 fixation may account for a substantial (and so far, overlooked) source of bioavailable N. Our findings contribute to improve our understanding on the ecosystem-level impact of zebra mussel, and potentially, of its ability to adapt to and colonize oligotrophic environments.

Chemosymbiotic bivalves contribute to the nitrogen budget of seagrass ecosystems.

In many seagrass sediments, lucinid bivalves and their sulfur-oxidizing symbionts are thought to underpin key ecosystem functions, but little is known about their role in nutrient cycles, particularly nitrogen. We used natural stable isotopes, elemental analyses, and stable isotope probing to study the ecological stoichiometry of a lucinid symbiosis in spring and fall. Chemoautotrophy appeared to dominate in fall, when chemoautotrophic carbon fixation rates were up to one order of magnitude higher as compared with the spring, suggesting a flexible nutritional mutualism. In fall, an isotope pool dilution experiment revealed carbon limitation of the symbiosis and ammonium excretion rates up to tenfold higher compared with fluxes reported for nonsymbiotic marine bivalves. These results provide evidence that lucinid bivalves can contribute substantial amounts of ammonium to the ecosystem. Given the preference of seagrasses for this nitrogen source, lucinid bivalves' contribution may boost productivity of these important blue carbon ecosystems.

Effects of Water Column Mixing and Stratification on Planktonic Primary Production and Dinitrogen Fixation on a Northern Red Sea Coral Reef.

The northern Red Sea experiences strong annual differences in environmental conditions due to its relative high-latitude location for coral reefs. This allows the study of regulatory effects by key environmental parameters (i.e., temperature, inorganic nutrient, and organic matter concentrations) on reef primary production and dinitrogen (N2) fixation, but related knowledge is scarce. Therefore, this study measured environmental parameters, primary production and N2 fixation of phytoplankton groups in the water overlying a coral reef in the Gulf of Aqaba. To this end, we used a comparative approach between mixed and stratified water column scenarios in a full year of seasonal observations. Findings revealed that inorganic nutrient concentrations were significantly higher in the mixed compared to the stratified period. While gross photosynthesis and N2 fixation rates remained similar, net photosynthesis decreased from mixed to stratified period. Net heterotrophic activity of the planktonic community increased significantly during the stratified compared to the mixed period. While inorganic nitrogen (N) availability was correlated with net photosynthesis over the year, N2 fixation only correlated with N availability during the mixed period. This emphasizes the complexity of planktonic trophodynamics in northern Red Sea coral reefs. Comparing mixed and stratified planktonic N2 fixation rates with those of benthic organisms and substrates revealed a close seasonal activity similarity between free-living pelagic and benthic diazotrophs. During the mixed period, N2 fixation potentially contributed up to 3% of planktonic primary production N demand. This contribution increased by ca. one order of magnitude to 21% during the stratified period. Planktonic N2 fixation is likely a significant N source for phytoplankton to maintain high photosynthesis under oligotrophic conditions in coral reefs, especially during stratified conditions.

Contrasting seasonal responses in dinitrogen fixation between shallow and deep-water colonies of the model coral Stylophora pistillata in the northern Red Sea.

Tropical corals are often associated with dinitrogen (N2)-fixing bacteria (diazotrophs), and seasonal changes in key environmental parameters, such as dissolved inorganic nitrogen (DIN) availability and seawater temperature, are known to affect N2 fixation in coral-microbial holobionts. Despite, then, such potential for seasonal and depth-related changes in N2 fixation in reef corals, such variation has not yet been investigated. Therefore, this study quantified seasonal (winter vs. summer) N2 fixation rates associated with the reef-building coral Stylophora pistillata collected from depths of 5, 10 and 20 m in the northern Gulf of Aqaba (Red Sea). Findings revealed that corals from all depths exhibited the highest N2 fixation rates during the oligotrophic summer season, when up to 11% of their photo-metabolic nitrogen demand (CPND) could be met by N2 fixation. While N2 fixation remained seasonally stable for deep corals (20 m), it significantly decreased for the shallow corals (5 and 10 m) during the DIN-enriched winter season, accounting for less than 2% of the corals' CPND. This contrasting seasonal response in N2 fixation across corals of different depths could be driven by 1) release rates of coral-derived organic matter, 2) the community composition of the associated diazotrophs, and/or 3) nutrient acquisition by the Symbiodinium community.

Author Correction: Chemosynthetic symbionts of marine invertebrate animals are capable of nitrogen fixation.

In this Article, the completeness and number of contigs for draft genomes from two individuals of Laxus oneistus are incorrect in the main text, although the correct information is included in Table 1. The original and corrected versions of the relevant sentence are shown in the correction notice.

Chemosynthetic symbionts of marine invertebrate animals are capable of nitrogen fixation.

Chemosynthetic symbioses are partnerships between invertebrate animals and chemosynthetic bacteria. The latter are the primary producers, providing most of the organic carbon needed for the animal host's nutrition. We sequenced genomes of the chemosynthetic symbionts from the lucinid bivalve Loripes lucinalis and the stilbonematid nematode Laxus oneistus. The symbionts of both host species encoded nitrogen fixation genes. This is remarkable as no marine chemosynthetic symbiont was previously known to be capable of nitrogen fixation. We detected nitrogenase expression by the symbionts of lucinid clams at the transcriptomic and proteomic level. Mean stable nitrogen isotope values of Loripes lucinalis were within the range expected for fixed atmospheric nitrogen, further suggesting active nitrogen fixation by the symbionts. The ability to fix nitrogen may be widespread among chemosynthetic symbioses in oligotrophic habitats, where nitrogen availability often limits primary productivity.

Microbial dinitrogen fixation in coral holobionts exposed to thermal stress and bleaching.

Coral holobionts (i.e., coral-algal-prokaryote symbioses) exhibit dissimilar thermal sensitivities that may determine which coral species will adapt to global warming. Nonetheless, studies simultaneously investigating the effects of warming on all holobiont members are lacking. Here we show that exposure to increased temperature affects key physiological traits of all members (herein: animal host, zooxanthellae and diazotrophs) of both Stylophora pistillata and Acropora hemprichii during and after thermal stress. S. pistillata experienced severe loss of zooxanthellae (i.e., bleaching) with no net photosynthesis at the end of the experiment. Conversely, A. hemprichii was more resilient to thermal stress. Exposure to increased temperature (+ 6°C) resulted in a drastic increase in daylight dinitrogen (N2 ) fixation, particularly in A. hemprichii (threefold compared with controls). After the temperature was reduced again to in situ levels, diazotrophs exhibited a reversed diel pattern of activity, with increased N2 fixation rates recorded only in the dark, particularly in bleached S. pistillata (twofold compared to controls). Concurrently, both animal hosts, but particularly bleached S. pistillata, reduced both organic matter release and heterotrophic feeding on picoplankton. Our findings indicate that physiological plasticity by coral-associated diazotrophs may play an important role in determining the response of coral holobionts to ocean warming.

Functional significance of dinitrogen fixation in sustaining coral productivity under oligotrophic conditions.

Functional traits define species by their ecological role in the ecosystem. Animals themselves are host-microbe ecosystems (holobionts), and the application of ecophysiological approaches can help to understand their functioning. In hard coral holobionts, communities of dinitrogen (N2)-fixing prokaryotes (diazotrophs) may contribute a functional trait by providing bioavailable nitrogen (N) that could sustain coral productivity under oligotrophic conditions. This study quantified N2 fixation by diazotrophs associated with four genera of hermatypic corals on a northern Red Sea fringing reef exposed to high seasonality. We found N2 fixation activity to be 5- to 10-fold higher in summer, when inorganic nutrient concentrations were lowest and water temperature and light availability highest. Concurrently, coral gross primary productivity remained stable despite lower Symbiodinium densities and tissue chlorophyll a contents. In contrast, chlorophyll a content per Symbiodinium cell increased from spring to summer, suggesting that algal cells overcame limitation of N, an essential element for chlorophyll synthesis. In fact, N2 fixation was positively correlated with coral productivity in summer, when its contribution was estimated to meet 11% of the Symbiodinium N requirements. These results provide evidence of an important functional role of diazotrophs in sustaining coral productivity when alternative external N sources are scarce.

Benthic N2 fixation in coral reefs and the potential effects of human-induced environmental change.

Tropical coral reefs are among the most productive and diverse ecosystems, despite being surrounded by ocean waters where nutrients are in short supply. Benthic dinitrogen (N2) fixation is a significant internal source of "new" nitrogen (N) in reef ecosystems, but related information appears to be sparse. Here, we review the current state (and gaps) of knowledge on N2 fixation associated with coral reef organisms and their ecosystems. By summarizing the existing literature, we show that benthic N2 fixation is an omnipresent process in tropical reef environments. Highest N2 fixation rates are detected in reef-associated cyanobacterial mats and sea grass meadows, clearly showing the significance of these functional groups, if present, to the input of new N in reef ecosystems. Nonetheless, key benthic organisms such as hard corals also importantly contribute to benthic N2 fixation in the reef. Given the usually high coral coverage of healthy reef systems, these results indicate that benthic symbiotic associations may be more important than previously thought. In fact, mutualisms between carbon (C) and N2 fixers have likely evolved that may enable reef communities to mitigate N limitation. We then explore the potential effects of the increasing human interferences on the process of benthic reef N2 fixation via changes in diazotrophic populations, enzymatic activities, or availability of benthic substrates favorable to these microorganisms. Current knowledge indicates positive effects of ocean acidification, warming, and deoxygenation and negative effects of increased ultraviolet radiation on the amount of N fixed in coral reefs. Eutrophication may either boost or suppress N2 fixation, depending on the nutrient becoming limiting. As N2 fixation appears to play a fundamental role in nutrient-limited reef ecosystems, these assumptions need to be expanded and confirmed by future research efforts addressing the knowledge gaps identified in this review.

Red coral extinction risk enhanced by ocean acidification.

The red coral Corallium rubrum is a habitat-forming species with a prominent and structural role in mesophotic habitats, which sustains biodiversity hotspots. This precious coral is threatened by both over-exploitation and temperature driven mass mortality events. We report here that biocalcification, growth rates and polyps' (feeding) activity of Corallium rubrum are significantly reduced at pCO2 scenarios predicted for the end of this century (0.2 pH decrease). Since C. rubrum is a long-living species (>200 years), our results suggest that ocean acidification predicted for 2100 will significantly increases the risk of extinction of present populations. Given the functional role of these corals in the mesophotic zone, we predict that ocean acidification might have cascading effects on the functioning of these habitats worldwide.