Mapping the microscale variability of microphytobenthos: Development of a hyperspectral imaging method

Microphytobenthos (MPB) is a grouping of microbial benthic phototrophs that inhabit the sediments of coastal regions. There are clear indications that MPB distributions exhibit profuse spatio-temporal variability on the scale of milli- to centi-meters, which corresponds to the spatial span of their proximal habitats. This microscale variability in MPB distributions escapes detection by most traditional measurement techniques. Analysis of the spatio-temporal aspects of MPB ecology is limited due to the inability of current methods to capture the MPB distributions with high spatial and temporal resolution. This doctoral study identifies a methodological gap in our ability to measure in situ MPB distributions at the microscale and attempts to rectify it through the development of a field instrument and measurement protocol that utilize hyperspectral imaging technology. The ecological applications of the novel ability to measure and visualize full-field patterns of MPB distribution with a high temporal resolution are explored in subsequent studies

Underwater Hyperspectral Imaging (UHI): a review of systems and applications for proximal seafloor ecosystem studies

Marine ecosystem monitoring requires observations of its attributes at different spatial and temporal scales that traditional sampling methods (e.g., RGB imaging, sediment cores) struggle to efficiently provide. Proximal optical sensing methods can fill this observational gap by providing observations of, and tracking changes in, the functional features of marine ecosystems non-invasively. Underwater hyperspectral imaging (UHI) employed in proximity to the seafloor has shown a further potential to monitor pigmentation in benthic and sympagic phototrophic organisms at small spatial scales (mm–cm) and for the identification of minerals and taxa through their finely resolved spectral signatures. Despite the increasing number of studies applying UHI, a review of its applications, capabilities, and challenges for seafloor ecosystem research is overdue. In this review, we first detail how the limited band availability inherent to standard underwater cameras has led to a data analysis “bottleneck” in seafloor ecosystem research, in part due to the widespread implementation of underwater imaging platforms (e.g., remotely operated vehicles, time-lapse stations, towed cameras) that can acquire large image datasets. We discuss how hyperspectral technology brings unique opportunities to address the known limitations of RGB cameras for surveying marine environments. The review concludes by comparing how different studies harness the capacities of hyperspectral imaging, the types of methods required to validate observations, and the current challenges for accurate and replicable UHI research

Photosynthetic metabolism in microphytobenthos : modulation by temperature and dissolved inorganic carbon

Tese de doutoramento, Biologia (Ecofisiologia), Universidade de Lisboa, Faculdade de Ciências, 2017Main primary producers of estuarine systems include benthic microalgae, predominantly diatoms, and cyanobacteria that colonize subtidal and intertidal zones, commonly referred to as microphytobenthos (MPB). MPB communities account for about 50% of the primary productivity of estuarine systems (e.g. MacIntyre et al., 1996; Barranguet et al., 1998; Underwood and Kromkamp, 1999; Serôdio and Catarino, 2000). This is highly relevant considering that estuarine systems are among the most productive ecosystems in the biosphere (Constanza et al., 1997). Despite the extreme variability of environmental parameters (irradiance, temperature and salinity) that characterizes the estuarine habitat, these communities are able to display high photosynthetic rates. It is common to distinguish two types of MPB communities: epipelic communities (typical of muddy sediments) composed of motile species and epipsammic communities (typical of sandy sediments) composed largely by non-motile species attached to sand grains. Contrary to epipsammic, epipelic diatoms exhibit circadian and circatidal migratory rhythms (Palmer and Round, 1967; Palmer, 1973; Admiraal et al., 1982; Serôdio and Catarino, 2000). Being MPB critical for the function of estuarine and coastal ecosystems, understanding the impact of climate change on its structure and productivity is mandatory. In particular, the increase of temperature and inorganic carbon availability can markedly affect photosynthesis, diversity and productivity of estuarine MPB communities. However, studies of the impact of climate changes on MPB are still scarce, and in particular the effects of the interaction between increased temperature and inorganic carbon availability are, to a large extent, still unknown. Thus, in this project we studied the effects of temperature and dissolved inorganic carbon (DIC) availability in two MPB communities of the Tagus estuary, in central Portugal, applying new methods of remote sensing, namely laser induced fluorescence (LIF) and modulated imaging fluorescence (Imaging-PAM). The LIF methodology (chapter 2) was used to trace migratory rhythms of benthic epipelic microalgae typical of mud sediments. Indeed, it was confirmed that, during the diurnal low tide these microalgae migrate to the surface of sediment to drive photosynthesis and then migrate in depth before the arrival of the tide. Furthermore, epipelic diatoms show photophobic migration when exposed to high light levels, a process known as behavioral photoprotection. This technique proved to be adequate to the study of MPB communities, both for the determination of surface biomass and the study of migratory rhythms. The possibility of using LIF for middle-range remote sensing under natural light conditions opens good perspectives for its use in environmental monitoring of estuarine systems. In the last decades, conventional pulse amplitude modulated fluorescence (PAM) has been increasingly applied to estuarine MPB communities (Serôdio et al., 1997; Kromkamp et al., 1998; Paterson et al., 1998; Serôdio et al., 2001; Honeywill et al., 2002; Perkins et al., 2002; Serôdio, 2003; Stephens et al., 2003; Forster and Kromkamp, 2004; Murphy et al., 2004; Serôdio, 2004; Forster and Jesus, 2005; Murphy et al., 2005). Lower resolution imaging systems allow the mapping of fluorescence parameters over large areas, making it a unique technique to study the spatial heterogeneity of the photosynthetic activity across an autotrophic surface (Scholes and Rolfe, 1996; Hill et al., 2004. This can be particularly relevant in the study of MPB biofilms, characterized by a “patchy,” heterogeneous distribution and can provide useful information about the spatial distribution of biomass and productivity in sediments. Yet, important hardware differences exist between conventional and imaging systems and therefore a comparative study of the results obtained with these technologies in MPB was required (chapter 3). In fact, we observed significant differences between the results obtained with conventional and imaging systems, especially in what refers to the analysis of rapid fluorescence curves as a function of irradiance (RLCs, Rapid Light Curves). Lower values of α (initial slope of the rETR vs. E curve), ETRmax (maximum relative ETR), Ek (light saturation parameter) and Fv/Fm (maximum quantum efficiency of photosystem II of dark-adapted samples) were obtained using the Imaging- PAM system. The discrepancy between instruments was dependent on sample type, being more pronounced in mud than in sandy MPB sediments. The differences may be largely explained by the interplay between the different depth-integration of the fluorescence signal, dependent on the thickness of the photosynthetic layer, and the different attenuation coefficients of the light sources used by the two types of instrument. It is therefore important to take into account the specificity of each fluorescence system when interpreting and comparing chlorophyll fluorescence data of MPB communities, taking full advantage of the application of imaging fluorescence to the MPB communities, in particular in what refers to its unique ability to study the spatial heterogeneity of their photosynthetic activity. The in-depth understanding of the primary production processes of MPB is a prerequisite to assess the impacts of climate change on estuarine systems. Among the global change variables, increased temperature and atmospheric CO2 (and concomitantly dissolved inorganic carbon, DIC) are expected to have the most significant impact over the primary productivity in estuaries. Therefore, we studied their effect, as well as their interaction, on the photosynthesis of MPB biofilms. The short-term effect of increasing temperature in the photosynthetic activity of two communities of MPB (Alcochete and Trancão) was studied (chapter 4). The Trancão MPB community had higher photosynthetic electron transport capacity (higher ETRmax) being photoacclimated at higher irradiance (higher EK) and had lower energy conversion efficiency at limiting irradiance (lower α) .The differences in the species composition and size class may explain the results obtained for the two sites, since smaller cells are generally more metabolically active. However, for the both MPB communities photosynthetic capacity increased with temperature up to 35 °C and decreased with extreme temperature of 42 °C. Photosynthetic efficiencies at low irradiance (α) were not affected by the temperature in the 15-35 °C range, and decreased at 42 °C. These MPB estuarine communities were able to increase the photosynthetic capacity under exposure to short-term high temperatures sediments, similar to happens during the summer midday in the diurnal low tides. We also studied the effect of dissolved inorganic carbon availability (DIC) in photosynthesis, on the two intertidal MPB communities, taking as reference the model diatom species Phaeodactylum tricornutum, using fluorescence imaging (on intact MPB communities) and polarographic oxygen measurement (in cell suspensions) (chapter 5). The parameters of DIC acquisition kinetics (Vmax and Km (DIC)) estimated for MPB communities were higher ((Km (DIC) = 0.31 mM; Vmax = 7.78 nmol min-1 μg (Chl a)-1), than those obtained for Phaeodactylum tricornutum (Km (DIC) = 0.23 mM; Vmax = 4.64 nmol min-1 μg (Chl a)-1), showing that these communities have species with low affinity for DIC and high photosynthetic capacity. The net photosynthesis of MPB suspensions reached saturation at DIC concentrations of 1-1.5 mM, 17 lower than those found in the sediment top 5 mm interstitial water, suggesting that the photosynthesis of these communities is not limited by DIC availability. However, when NaHCO3 was added to intact MPB samples, an increase in the maximum photosynthetic electron transport rate (ETRmax) was observed. These results suggest that the local depletion of DIC in the photic layer of the sediment, where the microalgae accumulate during diurnal low tide, limits the photosynthetic activity of MPB, providing the first experimental evidence of DIC limitation of the productivity of intertidal MPB communities. The interactive effects of temperature and DIC in biomass, species composition and photosynthetic performance of MPB was studied using a microcosm ELSS ('experimental life support system') with tide regime (chapter 6). The longer term effect of increased temperature (24 ° C) had a negative effect on MPB biomass and photosynthetic performance under both simulated DIC conditions (pH = 7.4 and pH = 8.0). Furthermore, at elevated temperatures there was a change in the relative abundance of the major species of diatoms and an increase of cyanobacteria. The long term effect of increasing DIC ( pH = 7.4) under low temperature (18 °C) had a positive effect on biomass MPB, possibly due to decreased local depletion of dissolved inorganic carbon. No significant effects were found on the relative abundance of the major groups of microalgae. Our results suggest that the interactive effects of increased temperature and DIC availability of estuarine MPB communities can have a negative impact on the structure, diversity and productivity of these communities, eventually affecting the entire estuarine ecosystems. Therefore, a deepen understanding of the potential impacts of climate change in these complex ecosystems is required

Sentinel-2 remote sensing of Zostera noltei-dominated intertidal seagrass meadows

Accurate habitat mapping methods are urgently required for the monitoring, conservation, and management of blue carbon ecosystems and their associated services. This study focuses on exposed intertidal seagrass meadows, which play a major role in the functioning of nearshore ecosystems. Using Sentinel-2 (S2) data, we demonstrate that satellite remote sensing can be used to map seagrass percent cover (SPC) and leaf biomass (SB), and to characterize its seasonal dynamics. In situ radiometric and biological data were acquired from three intertidal meadows of Zostera noltei along the European Atlantic coast in the summers of 2018 and 2019. This information allowed algorithms to estimate SPC and SB from a vegetation index to be developed and assessed. Importantly, a single SPC algorithm could consistently be used to study Z. noltei-dominated meadows at several sites along the European Atlantic coast. To analyze the seagrass seasonal cycle and to select images corresponding to its maximal development, a two-year S2 dataset was acquired for a French study site in Bourgneuf Bay. The po-tential of S2 to characterize the Z. noltei seasonal cycle was demonstrated for exposed intertidal meadows. The SPC map that best represented seagrass growth annual maximum was validated using in situ measurements, resulting in a root mean square difference of 14%. The SPC and SB maps displayed a patchy distribution, influenced by emersion time, mudflat topology, and seagrass growth pattern. The ability of S2 to measure the surface area of different classes of seagrass cover was investigated, and surface metrics based on seagrass areas with SPC >= 50% and SPC >= 80% were computed to estimate the interannual variation in the areal extent of the meadow. Due to the high spatial resolution (pixel size of 10 m), frequent revisit time (<= 5 days), and long-term objective of the S2 mission, S2-derived seagrass time-series are expected to contribute to current coastal ecosystem management, such as the European Water Framework Directive, but to also guide future adaptation plans to face global change in coastal areas. Finally, recommendations for future intertidal seagrass studies are proposed

Phénologie des spectres de réflectance de la végétation littorale d'un marais intertidal de l'estuaire du Saint-Laurent (Isle-Verte, QC)

RÉSUMÉ : Les écosystèmes possédant un climat tempéré froid sont grandement affectés par une variabilité saisonnière engendrant ainsi une évolution de la végétation (phénologie). Vu la courte saison de croissance de la végétation, ces changements consistent à la germination, croissance, sénescence, et la mort des plants. La nécessité de documenter ces changements est une priorité afin de quantifier l'état de l'écosystème, réaliser un suivi saisonnier et pour cartographier la répartition de la végétation. L'objectif du mémoire était de documenter et caractériser le cycle saisonnier de la végétation intertidale de la mi-mai à la fin octobre. Les propriétés biophysiques, de spectres de réflectance in situ ont été acquises pour quatre espèces végétales du marais de L'Isle-Verte (zostère, macroalgues, spartine alterniflore et l'arroche hastée) et une surface vaseuse dénudée de végétation. En parallèle, des séries temporelles d'images multispectrales ont été analysées. Bien que l'on observe des changements bio-physiques chez la végétation, l'évolution des spectres de réflectance in situ ne varie pas chez toutes les espèces. Selon le moment de la collecte des données tel qu'en juin et juillet, les spectres de toutes les espèces végétales présenteront des formes relativement similaires. Des indices de végétation, dont l'indice de végétation par différence normalisée (NDVI), ont été calculés à partir des réflectances in situ et des images multispectrales provenant de deux capteurs (Sentinel-2 MSI et PlanetScope), lesquels étaient fortement corrélés. L'indice NDVI a permis de bien identifier la phénologie pour l'arroche hastée et la spartine alterniflore, mais pas pour la zostère et les macroalgues qui montraient peu de variation saisonnière. Le potentiel de la série temporelle Sentinel-2 pour cartographier les types de végétation a été démontré en appliquant un algorithme de classification par arbre de décisions aléatoires (XGBoost). La classification a été validée à l'aide de mesures in situ et de photos aériennes. Cette classification a montré que la meilleure représentation de la réalité du terrain était obtenue avec l'image acquise à la fin septembre. La compréhension de la variabilité saisonnière a permis d'identifier les moments clés de la végétation, sa répartition et son impact sur chaque espèce du littoral. Enfin, des recommandations pour de futures études sur la végétation intertidale sont proposées. -- Mot(s) clé(s) en français : phénologie de la végétation, signature spectrale, écosystème côtier, télédétection, capteurs multispectraux. -- ABSTRACT : Ecosystems with a cold temperate climate are greatly affected by seasonal variability resulting in changes in vegetation (phenology). Given the short growing season of vegetation, these changes consist of germination, growth, senescence, and death of plants. The need to document these changes is a priority in order to quantify the state of the ecosystem, conduct seasonal monitoring, and map the distribution of vegetation. The objective of the dissertation was to document and characterize the seasonal cycle of intertidal vegetation in a cold temperate saltmarsh from mid-May to late October. The biophysical properties of in situ reflectance spectra were acquired for four plant species of the L'Isle-Verte (eelgrass, macroalgae, cordgrass, and creeping saltbush) and a muddy surface devoid of vegetation. In parallel, time series of multispectral images were analyzed. Although bio-physical changes in vegetation are observed, the evolution of in situ reflectance spectra does not vary in all species. Depending on the time of data collection such as June and July, the spectra of all plant species will show relatively similar shapes. Vegetation indices, including the normalized difference vegetation index (NDVI), were calculated from in situ reflectances and multispectral images from two sensors (Sentinel-2 MSI and PlanetScope), which were highly corroletated. The NDVI provided a good identification of phenology for both creeping saltbush and cordgrass, but not for eelgrass and macroalgae that remained relatively stable. The potential of the Sentinel-2 time series to map vegetation types was demonstrated by applying a random decision tree classification algorithm (XGBoost). The classification was validated using in situ measurements and aerial photos. This classification showed that the best representation of the terrain reality was obtained with the image acquired at the end of September. The understanding of seasonal variability allowed the identification of the key moments of vegetation, its distribution and its impact on each species of the intertidal zone. Finally, recommendations for future studies on intertidal vegetation are proposed. -- Mot(s) clé(s) en anglais : vegetation phenology, spectral signature, coastal ecosystem, remote sensing, multispectral sensors

Os estuários como sumidouros decarbono: fotossíntese, fotoinibição e resiliência da produtividade primária pelágica e bêntica

Estuaries are recognized amongst the most productive ecosystems on Earth. Their high primary productivity is largely due to the photosynthetic carbon fixation by phytoplankton and microphytobenthos, the communities of microalgae and cyanobacteria that inhabit the water column and subtidal or intertidal sediments, respectively. In comparison with the phytoplankton, the microphytobenthos has been much less studied regarding the photophysiological processes affecting primary productivity, and their relative role as contributors to estuarine-level production. One of these processes is photoinhibition, the high light-induced decrease in photosynthetic activity, considered a major limiting factor of growth and primary productivity in the variable and extreme estuarine environment. The detrimental impact of photoinhibition on photosynthesis depends on the balance between the photoinactivation and repair of photosystem II (PSII). By successfully adapting to microphytobenthos a recently-developed methodology based on multi-actinic imaging of chlorophyll fluorescence, this work evaluated their photoacclimation and photoprotective capacity, as measured by the reduction in PSII photoinactivation. PSII photoinactivation and repair was found to vary between different communities, pointing to a trade-off between cellular motility-based and physiological photoprotective mechanisms. Epipelic (motile) species showed a reduced physiological capacity for preventing photodamage, while epipsammic (non-motile) forms appeared less susceptible to photoinactivation and more dependent on physiological photoprotection. This work further investigated an overlooked aspect of microphytobenthos ecology, related to the presence of substantial amounts of microalgal biomass in subsurface sediments. By studying samples from intertidal areas of the Ria de Aveiro (Portugal), this work found that buried cells can quickly regain photosynthetic activity when exposed to surface conditions. Potential viable subsurface (0.5-10 cm) microalgal biomass was found to represent 2-3 times the amount of biomass present at the surface layers (0.0-0.5 cm). These results support the hypothesis that subsurface biomass may play an important ecological role as a source of photosynthetically competent cells capable of ‘reinoculating’ the surface, contributing to the high productivity of intertidal areas. The relative importance of phytoplankton and microphytobenthos as contributors to ecosystem-level primary productivity was evaluated in the Ria de Aveiro, by comparing the spatio-temporal variability of biomass and productivity of different communities. This study made use of a new type of fluorometer allowing the measurement of absolute rates of PSII electron transport rates and the estimation of carbon fixation rates. Biomass-specific productivity rates for phytoplankton and microphytobenthos were found to reach 68.0 and 19.1 mg C mg Chl a-1 d-1, respectively. Annual areal production rates were higher for the microphytobenthos, reaching 105.2 g C m-2 yr-1, as opposed to 49.9 g C m-2 yr-1 for the phytoplankton. The annual rates upscaled for the whole Ria de Aveiro highlight the importance of the intertidal areas as significant carbon sinks and reservoirs of active ‘blue carbon’, and as main sites of primary productivity, found to contribute with more than 60% of the total ecosystem-level budget 12428.3 t C yr-1.Os estuários são reconhecidos como um dos tipos de ecossistemas mais produtivos na Terra. A sua elevada produtividade primária é devida em larga medida à fixação fotossintética de carbono pelo fitoplâncton e microfitobentos, as comunidades de microalgas e cianobactérias que habitam a coluna de àgua e os sedimentos subtidais e intertidais, respetivamente. Em comparação com o fitoplâncton, o microfitobentos tem sido muito menos estudado relativamente aos processos fotofisiológicos que controlam a sua produtividade, bem como à sua contribuição para a produção primária global do estuário. Um destes processos é a fotoinibição, a diminuição da atividade fotossintética causada pela luz, considerada como um importante fator limitante da produtividade primária no ambiente estuarino. O impacto negativo da fotoinibição na fotossíntese depende do balanço entre a fotoinativação e a reparação do fotossistema II (PSII). Baseado num recente método de imagiologia multi-actínica de fluorescência da clorofila, este trabalho avaliou a fotoaclimatação e capacidade de fotoproteção contra a fotoinibição, medida pela redução da fotoinativação do PSII. A fotoinativação e reparação do PSII variou entre diferentes tipos de comunidades, indicando a existência de um balanço entre a fotoproteção baseada na motilidade celular e em mecanismos fisiológicos. Espécies epipélicas (móveis) mostraram uma menor capacidade fisiológica de prevenir danos, enquanto as formas epipsâmicas (imóveis) aparentaram ser menos suscetíveis à fotoinibição e mais dependentes de fotoproteção fisiológica. Este trabalho investigou ainda um aspeto pouco estudado, relacionado com a presença de quantidades substanciais de biomass de microalgas em sedimentos subsuperficias. Pela análise de sedimentos intertidais da Ria de Aveiro (Portugal), foi descoberto que as células enterradas conseguem recuperar rapidamente a sua atividade fotossintética quando expostas a condições da superfície. Foi também concluído que a biomassa subsuperficial (0.5-10 cm) potencialmente viável representa 2-3 vezes a biomassa presente nas camadas superficiais (0.0-0.5 cm). Estes resultados suportam a hipótese de que a biomassa subsuperficial desempenha um papel ecológico importante enquanto fonte de células fotossinteticamente competentes capazes de ‘reinocular’ a superfície, contribuindo para a elevada produtividade das áreas intertidais. A importância relativa da contribuição do fitoplâncton e do microfitobentos para a produtividade primária ao nível do ecossistema foi avaliada para a Ria de Aveiro, comparando a variabilidade espacio-temporal da biomass e produtividade de diferentes comunidades. Este estudo baseou-se na medição de taxas absolutas de transporte de electrões no PSII e a estimação de taxas de fixação de carbono. Por unidade de biomass, estas atingiram 68,0 e 19,1 mg C mg Chl a-1 d-1, para o fitoplâncton e o microfitobentos, respectivamente. Por unidade de área, a produtividade anual foi mais elevada no caso do microfitobentos, atingindo 105,2 g C m-2 yr-1, por oposição a 49,9 g C m-2 yr-1, para o fitoplâncton. Considerando a totalidade da área da Ria de Aveiro, os resultados salientam a importância das áreas intertidais enquanto sumidouros de carbono e reservatórios de “carbono azul”, e locais de elevada produtividade primária, contribuindo com mais de 60% do total anual de 12428,3 t C yr-1.Programa Doutoral em Biologi

The importance of weather and tides on the resuspension and deposition of microphytobenthos (MPB) on intertidal mudflats

Abiotic variables, such as weather and tidal forces, are potentially as important as biotic factors (growth, predation, competition) in driving the variability of microphytobenthic (MPB) biomass on intertidal flats. Patterns of spatial distribution and temporal variability in MPB Chl. a, sediment Extracellular Polymeric Substances (EPS) and benthic diatom species composition were investigated during daily sampling spanning neap to spring tide periods on intertidal mudflats in the Colne Estuary, U.K., in three different seasons, with a particular focus on the influence of wind, rainfall, sun hours in the days prior to sampling, and tidal range. Spatial distribution (at < 1 m and <5 m scales) made the greatest contribution to biomass variability, followed by temporal (inter-monthly) variability. MPB Chl. a and EPS concentrations were positively correlated with sun-hours and tidal range, and negatively with rainfall and wind speed. Higher benthic MPB biomass was associated with lower suspended solid and Chl a loads, indicating biostabilisation of surface sediment. Suspended sediment loads and suspended Chl. a concentrations were positively correlated, and were significantly higher during neap rather than spring tides. Sediment settlement rates were higher during neap tides and related to suspended sediment load. The percent similarity in the benthic and suspended diatom assemblages (species relative abundance, RA) increased linearly with suspended solid load, with highest similarity during neap tides, with pennate benthic diatom taxa (Gyrosigma balticum, G. scalproides and Pleurosigma angulatum) dominant, indicating local sediment resuspension. During Spring tides, species similarity was lower, with a higher RA of planktonic centric diatoms (Actinoptychus, Coscinodiscus and Odontella) and lower sediment loads. Despite greater volumes of water movement during high tidal range periods, the highest levels of localised resuspension and remobilisation of MPB biomass across the mudflats occurred during low tidal range neap tide periods, when wind-induced waves were a key factor, particularly with shallower water depths over the intertidal mudflats