Ecophysiological responses to ammonium enrichment in seagrasses

Anthropogenic nutrient loading in coastal areas is one of the major causes of seagrass decline worldwide. One of the main negative effects is indirectly caused by the proliferation of fast-growing species promoting light attenuation, sediment anoxia and sulphide intrusion risks. However, the parallel direct toxic effect caused by high ammonium (NH4+) concentrations on seagrasses has received little attention, in spite of some pioneer works. Adverse effects of NH4+ enrichment have been traditionally explained by internal accumulation of NH4+. To prevent toxic effects, plants must assimilate this nutrient into amino acids and other non-toxic organic compounds, generating strong internal demand for ATP and organic carbon skeletons, which must be provided by photosynthesis or from C-reserves. Thus, any factor affecting NH4+ uptake and/or assimilation could intensify or ameliorate NH4+ toxicity in seagrasses. This PhD Thesis studies the interactive effect of NH4+ enrichment and different environmental factors in the genus Zostera. In the first two chapters, the interaction between NH4+ and phosphate uptake rates and the interactive effect of light, hydrodynamics and NH4+ enrichment was studied in Z. noltei. In these assays ammonium was taken up following a diffusive trend, and while a shortcoming of phosphate uptake was found in leaves of Z. noltei when NH4+ was present, NH4+ uptake was unaffected by phosphate presence (chapter 1). As well, a non-linear response to NH4+ enrichment with flow velocity was recorded, with the strongest negative effect at intermediate flow (chapter 2). The effects of light and NH4+ on morphology, physiology, nitrogen metabolism and carbon reserves were studied in Z. marina (chapter 3). Light reduction had negative and synergistic effects with NH4+ enrichment, which were related with a drastic drop in carbon reserves and a remarkable increase in amino acid concentrations, indicating a tight coupling between carbon an nitrogen metabolisms. The response to the interaction between NH4+ loading and hyposaline stress was analyzed on different physiological and morphological properties of Z. marina (chapter 4). The negative and interactive effect between high NH4+ concentrations and low salinity was correlated with an increase in intracellular NH4+ content and a decrease in photosynthetic rates and non-structural carbohydrates, causing a drop in growth and survival of plants. Thus, hyposaline stress enforced the toxic effect of NH4+ by increasing the competition between ammonium assimilation and osmotic regulation for energy and C-skeletons. In summary, this Thesis highlights that toxic effects promoted by NH4+ on seagrasses can be intensified or alleviated depending in the presence of other natural stressors. In nature, organisms are rarely stressed by only one factor, and the interaction among multiple stressors may drive synergistic, antagonist or even non- linear responses. The understanding of nature of such interactions is important for developing better predictions and managing policies for seagrass ecosystems, since NH4+ load is expected to increase in the near future in coastal areas, jointly with the modification of a broad range of environmental factors as a consequence of the global change.La eutrofización constituye una seria amenaza para las zonas costeras y se postula como una de las principales causas del declive de las praderas de angiospermas marinas a nivel global. De un modo indirecto, una elevada carga de nutrientes favorece la proliferación de especies de rápido crecimiento causando una reducción en los niveles de luz o un incremento de la materia orgánica, aumentando el riesgo de anoxia en el sedimento y la intrusión de sulfuro en las plantas. Asimismo, las propias concentraciones elevadas de amonio (NH4+) pueden resultar tóxicas. A pesar de la existencia de algunos estudios pioneros, la toxicidad del NH4+ en angiospermas marinas es aún un fenómeno poco estudiado. El efecto tóxico del NH4+ está ligado al incremento de dicho nutriente en el interior celular. Para evitar su acumulación el amonio ha de ser asimilado rápidamente en aminoácidos u otros compuestos orgánicos, generando una fuerte demanda de energía en forma de ATP e hidratos de carbono no estructurales que son proporcionados a través de fotosíntesis o bien suministrados a partir de las reservas internas. Por lo tanto, cualquier factor que afecte tanto a los procesos de incorporación como a la asimilación de NH4+, podría potenciar o aliviar su toxicidad. El objetivo principal de esta tesis doctoral es el estudio de la interacción entre diferentes variables ambientales y elevadas dosis de NH4+ en dos especies de fanerógamas del genero Zostera (Z. noltei y Z. marina). El capítulo 1 se centró en el estudio de la interacción entre las tasas incorporación de fosfato y amonio en hojas de Z. noltei. Bajo elevadas concentraciones de NH4+ se produjo una disminución en las tasas de incorporación de fosfato; a su vez, la incorporación de NH4+ mostró un claro comportamiento difusivo y no fue afectada por la presencia de fosfato en el medio. El capítulo 2 abordó el estudio de los efectos de una alta disponibilidad de NH4+ bajo diferentes condiciones hidrodinámicas y lumínicas en Z. noltei. La interacción entre estos factores dio lugar a una respuesta no lineal, registrándose una mayor toxicidad en velocidades intermedias. En el capítulo 3, se estudiaron los efectos producidos por diferentes niveles de luz y concentraciones de NH4+ en diversas propiedades morfológicas y fisiológicas, y las implicaciones sobre el metabolismo del nitrógeno y del carbono en Z. marina. Se observó un efecto sinérgico negativo entre las altas concentraciones de NH4+ y las condiciones de luz limitantes, que se correspondieron con una drástica disminución en el contenido de hidratos de carbono no estructurales y con un notable incremento en las concentraciones de aminoácidos libres, lo que puso de manifiesto la estrecha relación existente entre el metabolismo del carbono y del nitrógeno. En el capítulo 4 se evaluó la interacción entre las condiciones de enriquecimiento de NH4+ y el estrés hiposalino, sobre diferentes variables fisiológicas y morfológicas en Z. marina. El hallazgo más relevante fue el efecto negativo y aditivo entre las altas concentraciones de NH4+ y las condiciones de baja salinidad. Esto se relacionó con un incremento en el contenido intracelular de NH4+ y una disminución en las tasas fotosintéticas y en las concentraciones de hidratos de carbono no estructurales, provocando una reducción en el crecimiento y la supervivencia de Z. marina. Por lo tanto, las condiciones hiposalinas potenciaron el efecto tóxico del NH4+, debido a que los procesos de osmorregulación compiten por los mismos recursos que los requeridos para la asimilación del NH4+ (ATP e hidratos de carbono no estructurales). En conclusión, los resultados de esta tesis mostraron que los efectos tóxicos producidos por el NH4+ sobre las angiospermas marinas podrían intensificarse o aminorarse en función de las condiciones ambientales que coocurren de un modo natural en las zonas costeras. En las próximas décadas, se prevé tanto un incremento en la carga de NH4+, como cambios en las condiciones ambientales a consecuencia del cambio global. La interacción entre múltiples factores pueden conducir a respuestas sinérgicas, antagónicas o incluso no lineales en sistemas dominados por angiospermas marinas; la comprensión de cómo múltiples factores ambientales interactúan entre sí resulta por tanto trascendental para el desarrollo de políticas que conduzcan a una correcta gestión de los ecosistemas dominados por estas especies tan carismáticas.Número de páginas 16

Differential ecophysiological responses to inorganic nitrogen sources (ammonium versus nitrate) and light levels in the seagrass Zostera noltei

eagrasses can use both ammonium (NH4+) and nitrate (NO3−) as inorganic nitrogen (N) sources. However, NO3− uptake and assimilation are energetically more expensive and tightly regulated than NH4+ uptake. The objective of this study was to test the complex interactive effects between different forms of N enrichment (NH4+ and NO3−) and light levels on the morphological and physiological traits in the intertidal seagrass Zostera noltei. Plants were cultured over 40 d under 2 levels of light (low and high) with 2 inorganic N concentrations supplied at the same dose, NO3− (25 μM) and NH4+ (25 μM), and a control, following a 2-factorial design. Results showed a differential response in Z. noltei depending on the inorganic N source and light dose. NH4+ enrichment negatively affected almost all morphometric and dynamic variables analyzed, both in isolation and combined with low light conditions. In contrast, NO3− enrichment had a positive effect on Z. noltei survival compared with the control treatment in terms of net growth rate and rhizomatic growth, mainly under high light conditions. Therefore, our study demonstrated that the effects promoted by nutrient enrichment largely depend on the source of N used. Light levels play a crucial role in this response by potentially shifting the effects from toxic (under low light) to beneficial (under high light) when NO3− is the main N source. Our findings highlight that N form in eutrophication events should be considered when evaluating the potential impacts of nutrient enrichment and light reduction on seagrass communities14 página

The morphometric acclimation to depth explains the long-term resilience of the seagrass Cymodocea nodosa in a shallow tidal lagoon

Cadiz Bay is a shallow mesotidal lagoon with extensive populations of the seagrass Cymodocea nodosa at intertidal and shallow subtidal elevations. This work aims to understand the mechanisms behind the resilience of this species to gradual sea level rise by studying its acclimation capacity to depth along the shallow littoral, and therefore, to gradual variations in the light environment. To address this objective, these populations have been monitored seasonally over a 10 year period, representing the longest seasonal database available in the literature for this species. The monitoring included populations at 0.4, -0.08 and -0.5 m LAT. The results show that C. nodosa has a strong seasonality for demographic and shoot dynamic properties - with longer shoots and larger growth in summer (high temperature) than in winter (low temperature), but also some losses. Moreover, shoots have different leaf morphometry depending on depth, with small and dense shoots in the intertidal areas (0.4 m) and sparse large shoots in the subtidal ones (-0.08 and 0.5 m). These differences in morphometry and shoot dynamic properties, combined with the differences in shoot density, explain the lack of differences in meadow production balance (i.e. meadow growth - meadow losses) between the intertidal (0.4 m) and the deepest population (-0.5 m), supporting the long term resilience of Cymodocea nodosa in Cadiz Bay. This study contributes to the understanding of the mechanisms behind seagrass stability and resilience, which is particularly important towards predicting the effects of climate change on these key coastal ecosystems, and also highlights the value of continuous long-term monitoring efforts to evaluate seagrass trajectories

Stones in the road prevents effective implementation of eDNA-based freshwater quality monitoring in the Iberian Peninsula

The Water Framework Directive (WFD) monitoring requires the use of stressor-specific Multimetric Indices, intercalibrated and validated at river basin level, such as the IBMWP, which is one of the most widely used indices for the biomonitoring of rivers in the Iberian Peninsula. This qualitative index is based on the identification mainly at family level of 125 groups of macroinvertebrates. Currently, molecular biomonitoring systems (biomonitoring 2.0) is widely considered as a good alternative to conduct water quality assessments. The problem comes with the requirement of having complete DNA sequences databases for the targeted species within the indicator groups. In order to know the percentage of sequence coverage of the different taxonomic groups, is needed to know the number of freshwater species in each group. The fact that currently it is only necessary to identify them morphologically at family level to calculate the index means that there are no exhaustive lists of lower taxonomic ranges. In this study, we have studied the available taxonomic databases at species level for river macroinvertebrates in the Iberian Peninsula. For this purpose, species geographically referred to the Iberian Peninsula were extracted from three sources: two European (Freshwaterecology.info database and Weigand et al. (2019)) and one Iberian database/list (Múrria et al. 2020). The resultant species list was compared with GBIF (Global Biodiversity Information Facility) using its tool “Species Matching” to find synonyms and fuzzy names. The similarities and differences between databases were studied (considering the synonyms but not the fuzzy names) and a new list of 3586 species was constructed. Our analysis revealed that from 125 taxonomic groups covered by the IBMWP, there are no species collected for 11 of them (9%) and for another 34 groups (27%) there are less than 5 species collected in the databases. Then, using the BAGS software, all sequences of the mitochondrial gene COX1 were mined from BOLD database. From the 3586 species in the list, only 1900 (53%) have at least one sequence in BOLD. Therefore, conducting biomonitoring 2.0 in Iberian freshwater ecosystems is far from effective today. We need to overcome two big obstacles to reach the goal of using molecular biomonitoring in the Iberian Peninsula: a complete taxonomic list of quality indicators species by groups, and to sequence DNA barcodes from all relevant species