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  • Effectiveness and consistency of a suite of descriptors to assess the ecological status of seagrass meadows (Posidonia oceanica L. Delile)
    Publication . Rotini, Alice; Belmonte, A.; Barrote, Isabel; Micheli, C.; Peirano, A.; Santos, Rui; Silva, João
    The increasing rate of human-induced environmental changes on coastal marine ecosystems has created a demand for effective descriptors, in particular for those suitable for monitoring the status of seagrass meadows. Growing evidence has supported the useful application of biochemical and genetic descriptors such as secondary metabolite synthesis, photosynthetic activity and genetic diversity. In the present study, we have investigated the effectiveness of different descriptors (traditional, biochemical and genetic) in monitoring seagrass meadow conservation status. The Posidonia oceanica meadow of Monterosso al Mare (Ligurian sea, NW Mediterranean) was subjected to the measurement of bed density, leaf biometry, total phenols, soluble protein and photosynthetic pigment content as well as to RAPD marker analysis. This suite of descriptors provided evidence of their effectiveness and convenient application as markers of the conservation status of P. oceanica and/or other seagrasses. Biochemical/genetic descriptors and those obtained by traditional methods depicted a well conserved meadow with seasonal variability and, particularly in summer, indicated a healthier condition in a portion of the bed (station C), which was in agreement with the physical and sedimentological features of the station. Our results support the usefulness of introducing biochemical and genetic approaches to seagrass monitoring programs since they are effective indicators of plant physiological stress and environmental disturbance.
  • Response of key stress-related genes of the seagrass Posidonia oceanica in the vicinity of submarine volcanic vents
    Publication . Lauritano, C.; Ruocco, M.; Dattolo, E.; Buia, M. C.; Silva, João; Santos, Rui; Olivé, Irene; Costa, M. M.; Procaccini, G.
    Submarine volcanic vents are being used as natural laboratories to assess the effects of increased ocean acidity and carbon dioxide (CO2) concentration on marine organisms and communities. However, in the vicinity of volcanic vents other factors in addition to CO2, which is the main gaseous component of the emissions, may directly or indirectly confound the biota responses to high CO2. Here we used for the first time the expression of antioxidant and stress-related genes of the seagrass Posidonia oceanica to assess the stress levels of the species. Our hypothesis is that unknown factors are causing metabolic stress that may confound the putative effects attributed to CO2 enrichment only. We analyzed the expression of 35 antioxidant and stress-related genes of P. oceanica in the vicinity of submerged volcanic vents located in the islands of Ischia and Panarea, Italy, and compared them with those from control sites away from the influence of vents. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) was used to characterize gene expression patterns. Fifty-one percent of genes analyzed showed significant expression changes. Metal detoxification genes were mostly down-regulated in relation to controls at both Ischia and Panarea, indicating that P. oceanica does not increase the synthesis of heavy metal detoxification proteins in response to the environmental conditions present at the two vents. The up-regulation of genes involved in the free radical detoxification response (e.g., CAPX, SODCP and GR) indicates that, in contrast with Ischia, P. oceanica at the Panarea site faces stressors that result in the production of reactive oxygen species, triggering antioxidant responses. In addition, heat shock proteins were also activated at Panarea and not at Ischia. These proteins are activated to adjust stress-accumulated misfolded proteins and prevent their aggregation as a response to some stressors, not necessarily high temperature. This is the first study analyzing the expression of target genes in marine plants living near natural CO2 vents. Our results call for contention to the general claim of seagrasses as "winners" in a high-CO2 world, based on observations near volcanic vents. Careful consideration of factors that are at play in natural vents sites other than CO2 and acidification is required. This study also constitutes a first step for using stress-related genes as indicators of environmental pressures in a changing ocean.
  • Measuring seagrass photosynthesis: methods and applications
    Publication . Silva, João; Sharon, Yoni; Santos, Rui; Beer, Sven
    This review originates from a keynote lecture given at the recent 8th Group for Aquatic Productivity (GAP) workshop held in Eilat, Israel. Here we examine the most important methodologies for photosynthetic measurements in seagrasses and evaluate their applications, advantages and disadvantages, and also point out the most relevant results. The most commonly used methodologies are based on oxygen (O(2)) evolution and chlorophyll fluorescence measurements. O(2)-based methodologies allowed for the first approaches to evaluate seagrass productivity, whereas chlorophyll a fluorescence has more recently become the choice method for in situ experiments, particularly in evaluating photosynthetic responses to light and assessing stress responses. New methodologies have also emerged, such as O(2) optodes, underwater CO(2) flux measurements, geo-acoustic inversion and the eddy correlation technique. However, these new methods still need calibration and validation. Our analysis of the literature also reveals several significant gaps in relevant topics concerning seagrass photosynthesis, namely the complete absence of studies on deep-growing populations that photosynthesise under extreme low light conditions and the uncertainties about the true degree of seagrass carbon limitation, which limits our ability to predict responses to global changes.
  • Seagrass can mitigate negative ocean acidification effects on calcifying algae
    Publication . Bergstrom, Ellie; Silva, João; Martins, Cíntia; Horta, Paulo
    The ultimate effect that ocean acidification (OA) and warming will have on the physiology of calcifying algae is still largely uncertain. Responses depend on the complex interactions between seawater chemistry, global/local stressors and species-specific physiologies. There is a significant gap regarding the effect that metabolic interactions between coexisting species may have on local seawater chemistry and the concurrent effect of OA. Here, we manipulated CO2 and temperature to evaluate the physiological responses of two common photoautotrophs from shallow tropical marine coastal ecosystems in Brazil: the calcifying alga Halimeda cuneata, and the seagrass Halodule wrightii. We tested whether or not seagrass presence can influence the calcification rate of a widespread and abundant species of Halimeda under OA and warming. Our results demonstrate that under elevated CO2, the high photosynthetic rates of H. wrightii contribute to raise H. cuneata calcification more than two-fold and thus we suggest that H. cuneata populations coexisting with H. wrightii may have a higher resilience to OA conditions. This conclusion supports the more general hypothesis that, in coastal and shallow reef environments, the metabolic interactions between calcifying and non-calcifying organisms are instrumental in providing refuge against OA effects and increasing the resilience of the more OA-susceptible species.
  • A 15-month survey of Dimethylsulfoniopropionate and Dimethylsulfoxide content in Posidonia oceanica
    Publication . Richir, Jonathan; Champenois, Willy; Engels, Guyliann; Abadie, Arnaud; Gobert, Sylvie; Lepoint, Gilles; Silva, João; Santos, Rui; Sirjacobs, Damien; V. Borges, Alberto
    Posidonia oceanica is the only reported seagrass to produce significant amount of dimethylsulfoniopropionate (DMSP). It is also the largest known producer of DMSP among coastal and inter-tidal higher plants. Here, we studied (i) the weekly to seasonal variability and the depth variability of DMSP and its related compound dimethylsulfoxide (DMSO) in P. oceanica leaves of a non-disturbed meadow in Corsica, France, (ii) the weekly to seasonal variability and the depth variability of DMSP to DMSO concentration to assess the potential of the DMSP:DMSO ratio as indicator of stress, and (iii) the relationships between DMSP, DMSO, and the DMSP:DMSO ratio with potential explanatory variables such as light, temperature, photosynthetic activity (effective quantum yield of photosystem II), and leaf size. The overall average concentrations of organosulfured compounds in P. oceanica leaves were 130 ± 39 µmol.g−1 fw for DMSP and 4.9 ± 2.1 µmol.g−1 fw for DMSO. Concentrations of DMSP and DMSO in P. oceanica were overall distinctly higher and exhibited a wider range of variations than other marine primary producers such as Spartina alterniflora, phytoplankton communities, epilithic Cyanobacteria and macroalgae. Concentrations of both DMSP and DMSO in P. oceanica leaves decreased from a maximum in autumn to a minimum in summer; they changed little with depth. Potential explanatory variables except the leaf size, i.e., the leaf age were little or not related to measured concentrations. To explain the seasonal pattern of decreasing concentrations with leaf aging, we hypothesized two putative protection functions of DMSP in young leaves: antioxidant against reactive oxygen species and predator-deterrent. The similar variation of the two molecule concentrations over time and with depth suggested that DMSO content in P. oceanica leaves results from oxidation of DMSP. The DMSP:DMSO ratio remained constant around a mean value of 29.2 ± 9.0 µmol:µmol for the non-disturbed harvested meadow regardless of the time of the year, the depth or the leaf size. As suggested for the salt march plant S. alterniflora, we hypothesized the DMSP:DMSO ratio could be considered as indicator of stress in seagrasses exposed to environmental or anthropogenic stressors. More research would now be needed to confirm the functions of DMSP and DMSO in seagrasses and how the DMSP:DMSO ratio will vary under various disturbances.
  • Light is more important than nutrient ratios of fertilization for cymodocea nodosa seedling development
    Publication . Alexandre, Ana; Silva, João; Santos, Rui
    Restoration of seagrass beds through seedlings is an alternative to the transplantation of adult plants that reduces the impact over donor areas and increases the genetic variability of restored meadows. To improve the use of Cymodocea nodosa seedlings, obtained from seeds germinated in vitro, in restoration programs, we investigated the ammonium and phosphate uptake rates of seedlings, and the synergistic effects of light levels (20 and 200 mu mol quanta m(-2) s(-1)) and different nitrogen to phosphorus molar ratios (40 mu M N:10 mu M P, 25 mu M N:25 mu M P, and 10 mu N:40 mu M P) on the photosynthetic activity and growth of seedlings. The nutrient content of seedlings was also compared to the seed nutrient reserves to assess the relative importance of external nutrient uptake for seedling development. Eighty two percent of the seeds germinated after 48 days at a mean rate of 1.5 seeds per day. All seedlings under all treatments survived and grew during the 4 weeks of the experiment. Seedlings of C. nodosa acquired ammonium and phosphate from the incubation media while still attached to the seed, at rates of about twice of adult plants. The relevance of external nutrient uptake was further highlighted by the observation that seedlings' tissues were richer in nitrogen and phosphorus than non-germinated seeds. The uptake of ammonium followed saturation kinetics with a half saturation constant of 32 mu M whereas the uptake of phosphate increased linearly with nutrient concentration within the range tested (5 - 100 mu M). Light was more important than the nutrient ratio of fertilization for the successful development of the young seedlings. The seedlings' photosynthetic and growth rates were about 20% higher in the high light treatment, whereas different nitrogen to phosphorus ratios did not significantly affect growth. The photosynthetic responses of the seedlings to changes in the light level and their capacity to use external nutrient sources showed that seedlings of C. nodosa have the ability to rapidly acclimate to the surrounding light and nutrient environment while still attached to the seeds. C. nodosa seedlings experiencing fertilization under low light levels showed slightly enhanced growth if nourished with a balanced formulation, whereas a slight increase in growth was also observed with unbalanced formulations under a higher light level. Our results highlight the importance of high light availability at the seedling restoration sites.
  • Nitrogen uptake in light versus darkness of the seagrass Zostera noltei: integration with carbon metabolism
    Publication . Alexandre, Ana; Silva, João; Santos, Rui
    We conducted a study that shows that light and dark conditions do not affect the uptake rates of ammonium and nitrate by the seagrass Zostera noltei. This is an important advantage over some seaweed species in which these rates are severely reduced at night. In the light, the ammonium uptake rates were initially higher (15 and 20molg(-1)h(-1)) and stabilized at a rate of 5molg(-1)h(-1) after 1h, whereas in the dark the rates remained constant at a rate of 10molg(-1)h(-1) over the first 180min of incubation. The rates of nitrate uptake in the light were high within the first 120min of incubation(7.2-11.1molg(-1)h(-1)) and decreased afterwards to lower values (0.8-3.9molg(-1)h(-1)), whereas in the dark the rates fluctuated around 0.0-11.1molg(-1)h(-1) throughout the whole incubation time (7h). The soluble sugar content of Z.noltei leaves increased significantly with both ammonium and nitrate incubations in the light, indicating the metabolic outcome of photosynthesis. In the dark, there was no significant variation in either the soluble sugar or in the starch content of leaves, rhizomes or roots in either the ammonium or nitrate incubations. However, the total starch content of plants decreased at night whereas the total soluble sugars increased, suggesting a process of starch catabolism to generate energy with the consequent production of smaller monosaccharide products. The starch content of rhizomes decreased significantly during the light incubations with nitrate but not with ammonium. These results suggest that carbohydrate mobilization is necessary for Z.noltei to account for extra energetic costs needed for the uptake and assimilation of nitrate. Furthermore, our results suggest that nitrate uptake, at least during the day, requires the mobilization of starch whereas the uptake of ammonium does not.
  • Physiological stress indicators in seagrasses
    Publication . Barrote, Isabel; Santos, Rui; Silva, João
    We investigated the combined effects of several environmental stressors in the biochemical defence mechanisms of the intertidal seagrass Zostera noltii and the subtidal seagrasses Zostera marina and Cymodocea nodosa in Ria Formosa coastal lagoon (southern Portugal). During one year Z. noltii was sampled for antioxidant enzymes activity, pigments, soluble protein and malondialdehyde (MDA) in both neap and spring tides. Intertidal seagrasses are periodically exposed to potentially stressful conditions such as desiccation, osmotic stress, high light and very high or freezing temperatures, depending on the season, latitude and tidal amplitude. In Ria Formosa Z. marina and C. nodosa appear frequently in the same meadow and compete for space, light and nutrients. These species are also exposed to stressful environmental conditions such as light or temperature stress and their survival depends on their relative ability to cope with all these stressors. In order to study the effects of low light in Z. marina and C. nodosa, four different levels of shading were imposed on field plots of both species during three weeks. P-I relationships were determined and samples were collected to determine photosynthetic pigments, non-structural carbohydrates, soluble protein, phenols and malondialdehyde concentration. Reactive oxygen species (ROS) are formed as a normal part of the plants metabolism. An increment on ROS formation is a common response to stress and can induce several types of damage, namely lipid peroxidation. MDA is a product of the peroxidation of membrane lipids and thus is commonly used as an indicator of oxidative stress, as is the concentration of phenolic compounds. Carotenoids and antioxidant enzymes are part of the plants’ antioxidative system. Higher content of carotenoids, besides amplifying the photosynthetic useful wavelengths, indicate higher photoprotection. Increased activities of ROS scavenging enzymes are correlated with stress tolerance. The relationships among experimental and environmental data will be discussed.
  • Contribution of the seagrass Syringodium isoetifolium to the metabolic functioning of a tropical reef lagoon
    Publication . Olivé, Irene; García-Robledo, Emilio; Silva, João; Pintado-Herrera, Marina G.; Santos, Rui; Kamenos, Nicholas A.; Cuet, Pascale; Frouin, Patrick
    Seagrasses are gaining attention thanks to their metabolism and potential major role as carbon sinks, with further implications as nature-based solutions against climate change. Despite their recognized importance and the growing number of studies published, there is still a striking paucity of information on seagrass metabolism and contribution to biogeochemical cycles for some seagrass species and ocean areas. In this study we assessed the metabolic balance and nutrient cycling contribution of seagrasses to the benthic compartment of a tropical reef lagoon in Reunion Island, providing original information on a barely studied seagrass species (Syringodium isoetifolium) and a poorly studied ocean region (West Indian Ocean). We measured the net productivity, respiration and the metabolic balance in different components of the lagoon benthic compartment (i.e. seagrass, sediment, and benthic community) and the water-sediment nutrient benthic fluxes at differently impacted sites within the lagoon. The biogeochemical environmental variability, including inorganic and organic indicators of anthropogenic contamination, was also assessed at each site.Large spatial variability was detected in the metabolic balance of each benthic component assessed, also associated with the natural and/or anthropic-driven environmental variability found in the lagoon. The seagrass S. isoetifolium was net autotrophic across the lagoon and contributed to the lagoon benthic metabolism with net plant productivity exceeding by one order of magnitude the plant respiration. The lowest seagrass metabolism was detected at the impacted site. The metabolic balance of the sediment was heterotrophic but the high productivity of S. isoetifolium contributed to reducing the heterotrophy of the whole benthic community. The lagoon-wide benthic metabolic balance was slightly heterotrophic, but the associated uncertainty ranged from autotrophy to heterotrophy. Nutrient concentrations in the lagoon were low and the benthic community capacity for nutrient retention (uptake) and removal (denitrification and anammox) indicated potential for buffering moderate nutrient inputs into the lagoon. Organic contaminants of emerging concern (CECs) were low but detectable in the lagoon, especially in highly frequented beach areas, arising as an environmental quality indicator of interest.
  • 2b-RAD Genotyping of the Seagrass Cymodocea nodosa Along a Latitudinal Cline Identifies Candidate Genes for Environmental Adaptation
    Publication . Ruocco, Miriam; Jahnke, Marlene; Silva, João; Procaccini, Gabriele; Dattolo, Emanuela
    Plant populations distributed along broad latitudinal gradients often show patterns of clinal variation in genotype and phenotype. Differences in photoperiod and temperature cues across latitudes influence major phenological events, such as timing of flowering or seed dormancy. Here, we used an array of 4,941 SNPs derived from 2b-RAD genotyping to characterize population differentiation and levels of genetic and genotypic diversity of three populations of the seagrass Cymodocea nodosa along a latitudinal gradient extending across the Atlantic-Mediterranean boundary (i.e., Gran Canaria-Canary Islands, Faro-Portugal, and Ebro Delta-Spain). Our main goal was to search for potential outlier loci that could underlie adaptive differentiation of populations across the latitudinal distribution of the species. We hypothesized that such polymorphisms could be related to variation in photoperiod-temperature regime occurring across latitudes. The three populations were clearly differentiated and exhibited diverse levels of clonality and genetic diversity. Cymodocea nodosa from the Mediterranean displayed the highest genotypic richness, while the Portuguese population had the highest clonality values. Gran Canaria exhibited the lowest genetic diversity (as observed heterozygosity). Nine SNPs were reliably identified as outliers across the three sites by two different methods (i.e., BayeScan and pcadapt), and three SNPs could be associated to specific protein-coding genes by screening available C. nodosa transcriptomes. Two SNPs-carrying contigs encoded for transcription factors, while the other one encoded for an enzyme specifically involved in the regulation of flowering time, namely Lysine-specific histone demethylase 1 homolog 2. When analyzing biological processes enriched within the whole dataset of outlier SNPs identified by at least one method, "regulation of transcription" and "signalling" were among the most represented. Our results highlight the fundamental importance signal integration and gene-regulatory networks, as well as epigenetic regulation via DNA (de)methylation, could have for enabling adaptation of seagrass populations along environmental gradients.