A Critical Gap in Seagrass Protection: Impact of Anthropogenic Off-Shore Nutrient Discharges on Deep Posidonia oceanica Meadows.

In the Mediterranean, anthropogenic pressures (specifically those involving nutrient loads) have been progressively moved to deeper off-shore areas to meet current policies dealing with the protection of marine biodiversity (e.g., European Directives). However, conservation efforts devoted to protecting Posidonia oceanica and other vulnerable marine habitats against anthropogenic pressures have dedicated very little attention to the deepest areas of these habitats. We studied the remote influence of off-shore nutrient discharge on the physiology and structure of deep P. oceanica meadows located nearest to an urban sewage outfall (WW; 1 km) and an aquaculture facility (FF; 2.5 km). Light reduction and elevated external nutrient availability (as indicated by high δ15N, total N and P content and N uptake rates of seagrass tissues) were consistent with physiological responses to light and nutrient stress. This was particularly evident in the sites located up to 2.5 km from the WW source, where carbon budget imbalances and structural alterations were more evident. These results provide evidence that anthropogenic nutrient inputs can surpass critical thresholds for the species, even in off-shore waters at distances within the km scale. Therefore, the critical distances between this priority habitat and nutrient discharge points have been underestimated and should be corrected to achieve a good conservation status.

Factors structuring microbial communities in highly impacted coastal marine sediments (Mar Menor lagoon, SE Spain).

Coastal marine lagoons are environments highly vulnerable to anthropogenic pressures such as agriculture nutrient loading or runoff from metalliferous mining. Sediment microorganisms, which are key components in the biogeochemical cycles, can help attenuate these impacts by accumulating nutrients and pollutants. The Mar Menor, located in the southeast of Spain, is an example of a coastal lagoon strongly altered by anthropic pressures, but the microbial community inhabiting its sediments remains unknown. Here, we describe the sediment prokaryotic communities along a wide range of environmental conditions in the lagoon, revealing that microbial communities were highly heterogeneous among stations, although a core microbiome was detected. The microbiota was dominated by Delta- and Gammaproteobacteria and members of the Bacteroidia class. Additionally, several uncultured groups such as Asgardarchaeota were detected in relatively high proportions. Sediment texture, the presence of Caulerpa or Cymodocea, depth, and geographic location were among the most important factors structuring microbial assemblages. Furthermore, microbial communities in the stations with the highest concentrations of potentially toxic elements (Fe, Pb, As, Zn, and Cd) were less stable than those in the non-contaminated stations. This finding suggests that bacteria colonizing heavily contaminated stations are specialists sensitive to change.

Thermo-priming increases heat-stress tolerance in seedlings of the Mediterranean seagrass P. oceanica.

Seawater warming and increased incidence of marine heatwaves (MHW) are threatening the integrity of coastal marine habitats including seagrasses, which are particularly vulnerable to climate changes. Novel stress tolerance-enhancing strategies, including thermo-priming, have been extensively applied in terrestrial plants for enhancing resilience capacity under the re-occurrence of a stress event. We applied, for the first time in seedlings of the Mediterranean seagrass Posidonia oceanica, a thermo-priming treatment through the exposure to a simulated warming event. We analyzed the photo-physiological and growth performance of primed and non-primed seedlings, and the gene expression responses of selected genes (i.e. stress-, photosynthesis- and epigenetic-related genes). Results revealed that during the re-occurring stress event, primed seedlings performed better than unprimed showing unaltered photo-physiology supported by high expression levels of genes related to stress response, photosynthesis, and epigenetic modifications. These findings offer new opportunities to improve conservation and restoration efforts in a future scenario of environmental changes.

The role of competition and herbivory in biotic resistance against invaders: a synergistic effect.

Invasive species pose a major threat to global diversity, and once they are well established their eradication typically becomes unfeasible. However, certain natural mechanisms can increase the resistance of native communities to invaders and can be used to guide effective management policies. Both competition and herbivory have been identified as potential biotic resistance mechanisms that can limit plant invasiveness, but it is still under debate to what extent they might be effective against well-established invaders. Surprisingly, whereas biotic mechanisms are known to interact strongly, most studies to date have examined single biotic mechanisms separately, which likely influences our understanding of the strength and effectiveness of biotic resistance against invaders. Here we use long-term field data, benthic assemblage sampling, and exclusion experiments to assess the effect of native assemblage complexity and herbivory on the invasion dynamics of a successful invasive species, the alga Caulerpa cylindracea. A higher complexity of the native algal assemblage limited C. cylindracea invasion, probably through competition by canopy-forming and erect algae. Additionally, high herbivory pressure by the fish Sarpa salpa reduced C. cylindracea abundance by more than four times. However, long-term data of the invasion reflects that biotic resistance strength can vary across the invasion process and it is only where high assemblage complexity is concomitant with high herbivory pressure, that the most significant limitation is observed (synergistic effect). Overall, the findings reported in this study highlight that neglecting the interactions between biotic mechanisms during invasive processes and restricting the studied time scales may lead to underestimations of the true capacity of native assemblages to develop resistance to invaders.

Interactive effects of global warming and eutrophication on a fast-growing Mediterranean seagrass.

Coastal ecosystems, such as seagrasses, are subjected to local (e.g. eutrophication) and global (e.g. warming) stressors. While the separate effects of warming and eutrophication on seagrasses are relatively well known, their joint effects remain largely unstudied. In order to fill this gap, and using Cymodocea nodosa as a model species, we assessed the joint effects of warming (three temperatures, 20 °C, 30 °C and 35 °C) with two potential outcomes of eutrophication: (i) increase in nutrients concentration in the water column (30 and 300 µM), and (ii) organic enrichment in the sediment). Our results confirm that temperature in isolation clearly affects plant performance; while plants exposed to 30 °C performed better than control plants, plants exposed to 35 °C showed clear symptoms of deterioration (e.g. decline of photosynthetic capacity, increase of incidence of necrotic tissue). Plants were unaffected by high ammonium concentrations; however, organic enrichment of sediment had deleterious effects on plant function (photosynthesis, growth, demographic balance). Interestingly, these negative effects were exacerbated by increased temperature. Our findings indicate that in addition to the possibility of the persistence of C. nodosa being directly jeopardized by temperature increase, the joint effects of warming and eutrophication may further curtail its survival. This should be taken into consideration in both predictions of climate change consequences and in local planning.

Effects of an experimental heat wave on fatty acid composition in two Mediterranean seagrass species.

Global warming is emerging as one of the most critical threats to terrestrial and marine species worldwide. This study assessed the effects of simulated warming events in culture on two seagrass species, Posidonia oceanica and Cymodocea nodosa, which play a key role in coastal ecosystems of the Mediterranean Sea. Changes in fatty acids as key metabolic indicators were assessed in specimens from two geographical populations of each species adapted to different in situ temperature regimes. Total fatty acid (TFA) content and composition were compared in C. nodosa and P. oceanica from natural populations and following exposure to heat stress in culture. After heat exposure, individuals of C. nodosa and P. oceanica adapted to colder temperatures in situ accumulated significantly more TFA than controls. For both species, the proportion of polyunsaturated fatty acids (PUFA) decreased, and the percentage of saturated fatty acids (SFA) increased significantly after the heat treatment. These results highlight that populations of both species living at warmest temperatures in situ were more thermo-tolerant and exhibited a greater capacity to cope with heat stress by readjusting their lipid composition faster. Finally, exposure of seagrasses to warmer conditions may induce a decrease in PUFA/SFA ratio which could negatively affect their nutritional value and generate important consequences in the healthy state of next trophic levels.

The modulation of leaf metabolism plays a role in salt tolerance of Cymodocea nodosa exposed to hypersaline stress in mesocosms.

Applying proteomics, we tested the physiological responses of the euryhaline seagrass Cymodocea nodosa to deliberate manipulation of salinity in a mesocosm system. Plants were subjected to a chronic hypersaline condition (43 psu) to compare protein expression and plant photochemistry responses after 15 and 30 days of exposure with those of plants cultured under normal/ambient saline conditions (37 psu). Results showed a general decline in the expression level of leaf proteins in hypersaline stressed plants, with more intense reductions after long-lasting exposure. Specifically, the carbon-fixing enzyme RuBisCo displayed a lower accumulation level in stressed plants relative to controls. In contrast, the key enzymes involved in the regulation of glycolysis, cytosolic glyceraldehyde-3-phosphate dehydrogenase, enolase 2 and triose-phosphate isomerase, showed significantly higher accumulation levels. These responses suggested a shift in carbon metabolism in stressed plants. Hypersaline stress also induced a significant alteration of the photosynthetic physiology of C. nodosa by means of a down-regulation in structural proteins and enzymes of both PSII and PSI. However we found an over-expression of the cytochrome b559 alpha subunit of the PSII initial complex, which is a receptor for the PSII core proteins involved in biogenesis or repair processes and therefore potentially involved in the absence of effects at the photochemical level of stressed plants. As expected hypersalinity also affects vacuolar metabolism by increasing the leaf cell turgor pressure and enhancing the up-take of Na(+) by over-accumulating the tonoplast specific intrinsic protein pyrophosphate-energized inorganic pyrophosphatase (H(+)-PPase) coupled to the Na(+)/H(+)-antiporter. The modulation of carbon metabolism and the enhancement of vacuole capacity in Na(+) sequestration and osmolarity changes are discussed in relation to salt tolerance of C. nodosa.

Ecophysiological plasticity of shallow and deep populations of the Mediterranean seagrasses Posidonia oceanica and Cymodocea nodosa in response to hypersaline stress.

The differential expression of the plant phenotypic plasticity due to inter- and intraspecific divergences can determine the plant physiological tolerance under stress. In this work, we examined the interspecific ecophysiological plasticity that the main Mediterranean seagrass species with distinct marine environmental distribution (Posidonia oceanica and Cymodocea nodosa) can exhibit in response to hypersaline stress. We also tested the potential implication of ecotypic intraspecific divergences in the development of such plasticities. To this end, plants from shallow (5-7 m) and deep (18-20 m) meadows of both were maintained under two salinity treatments (natural salinity level of 37, and hypersaline treatment of 43; Practical Salinity Scale) during a long-term experiment (i.e. 62 days) developed in a highly controlled mesocosm system. Hypersaline stress caused notable plastic physiological alterations in P. oceanica and C. nodosa, with appreciable inter- and intraspecific differences. Although both species were similarly able to osmoregulate by means of organic solute accumulation (proline and sugars) in response to hypersalinity stress, higher carbon balance reductions were detected in P. oceanica plants from the deep meadow and in shallower C. nodosa plants, due to both photosynthetic inhibition and enhancement of respiration. None of these deleterious effects were found in C. nodosa plants form the deeper meadow. Leaf photosynthetic pigments generally increased in P. oceanica from both depths, but light absorbance capacities by leaves and photosynthetic efficiency followed contrasting patterns, increasing and decreasing in plants from the deep and the shallow meadows, respectively, indicating distinct strategies to cope with photosynthetic dysfunctions. Despite the significant reduction of pigments in the shallower C. nodosa plants, their leaves were able to increase their light capture capacities under hypersaline stress, by means of particular leaf optics adjustments (pigment packaging reduction). The metabolic costs as a consequence of the physiological plasticity integration seemed to compromise the vitality of P. oceanica, but not in the case of C. nodosa. These results confirm that both the inter- and intraspecific divergences play a key role in the responses which both Mediterranean seagrasses could develop under hypersaline stress conditions, and that these were consistent with their distinct ecological strategies and salinity tolerance ranges.

Responses of the Mediterranean seagrass Posidonia oceanica to hypersaline stress duration and recovery.

We studied the hypersaline stress responses of the Mediterranean seagrass Posidonia oceanica to determine if the species was tolerant to salinity increases that occur in coastal waters by the desalination industry. Water relations, amino acids, carbohydrates, ions, photosynthesis, respiration, chlorophyll a fluorescence, leaf growth and morphology, and plant mortality were analysed after exposing the mesocosm P. oceanica to a salinity level of 43 for one and three months followed by a month for recovery. One-month saline-stressed plants exhibited sub-lethal effects, including a leaf cell turgor pressure reduction, loss of ionic equilibrium and decreased leaf growth. There were also changes in photoprotective mechanisms, increased concentrations of organic osmolytes in leaves and reduced leaf ageing. All these dysfunctions recovered after removing the stress. After the longer exposure of three months, stress symptoms were much more acute and plants showed an excessive ionic exclusion capacity, increased leaf cell turgor, reduced plant carbon balance, increased leaf aging and leaf decay and increased plant mortality, which indicated that the plant had entered a stage of severe physiological stress. In addition, the long-term saline-stressed plants were not able to recover, still showing sustained injury after the one-month recovery period as reflected by unbalanced leaf ionic content, persistently impaired photosynthesis, decline in internal carbon resources and decreased leaf growth that resulted in undersized plants. In conclusion, P. oceanica was not able to acclimate to the saline conditions tested since it could not reach a new physiological equilibrium or recover after a chronic exposure of 3 months.