Ecoengineering Solutions for the Impairment of Spreading and Growth of Invasive Spartina patens in Mediterranean Salt Marshes

fmars-08-699528 September 1, 2021 Time: 9:1 # 1 ORIGINAL RESEARCH published: 30 August 2021 doi: 10.3389/fmars.2021.699528 Edited by: Stelios Katsanevakis, University of the Aegean, Greece Reviewed by: César Costa, Federal University of Rio Grande, Brazil Jenneke Visser, University of Louisiana at Lafayette, United States *Correspondence: Ricardo Cruz de Carvalho [email protected] Specialty section: This article was submitted to Marine Ecosystem Ecology, a section of the journal Frontiers in Marine Science Received: 23 April 2021 Accepted: 09 August 2021 Published: 30 August 2021 Citation: Cruz de Carvalho R, Feijão E, Duarte I, Pinto V, Silva M, Matos AR, da Silva AB, Caçador I, Reis-Santos P, Fonseca VF and Duarte B (2021) Ecoengineering Solutions for the Impairment of Spreading and Growth of Invasive Spartina patens in Mediterranean Salt Marshes. Front. Mar. Sci. 8:699528. doi: 10.3389/fmars.2021.699528 Ecoengineering Solutions for the Impairment of Spreading and Growth of Invasive Spartina patens in Mediterranean Salt Marshes Ricardo Cruz de Carvalho1,2*, Eduardo Feijão1, Irina Duarte1, Vanessa Pinto1, Marisa Silva1, Ana Rita Matos3,4, Anabela Bernardes da Silva3,4, Isabel Caçador1,3, Patrick Reis-Santos1,5, Vanessa F. Fonseca1,6 and Bernardo Duarte1,3 1MARE – Marine and Environmental Sciences Centre, Faculdade de Ciências da Universidade de Lisboa, Lisbon, Portugal, 2cE3c – Centre for Ecology, Evolution and Environmental Changes, Faculdade de Ciências da Universidade de Lisboa, Lisbon, Portugal, 3Departamento de Biologia Vegetal, Faculdade de Ciências, Universidade de Lisboa, Lisbon, Portugal, 4Biosystems and Applied Sciences Institute, Faculdade de Ciências da Universidade de Lisboa, Lisbon, Portugal, 5Southern Seas Ecology Laboratories, School of Biological Sciences, The University of Adelaide, Adelaide, SA, Australia, 6Departamento de Biologia Animal da Faculdade de Ciências da Universidade de Lisboa, Lisbon, Portugal The invasion of natural communities by non-indigenous species represents one of the most serious threats to biodiversity. Understanding the ecophysiology of invasive species can provide insights into potential physiological handicaps relative to native species. By doing so, we can leverage the development of ecoengineering solutions for the removal of non-indigenous species, preferably using non-chemical methods. Spartina patens is a known invasive species of cordgrass aggressively proliferating in Mediterranean salt marshes, producing impenetrable monospecific stands. As its occurrence is delimited by the upper high tide water level, we hypothesized that S. patens is intolerant to waterlogging. Therefore, we developed a field experiment where strands of S. patens were kept waterlogged over the entire tidal cycle for 30 days. At the end of the experimental period, plants in the trial plots exhibited severe stress symptoms at different physiological levels compared with control plots (no intervention). At the photobiological level, intervened plants exhibited lower efficiency in producing chemical energy from light, whilst at the biochemical level waterlogging impaired the antioxidant system and increased lipid peroxidation products. Furthermore, the application of chlorophyll apulse amplitude modulated (PAM) fluorometry, a non-invasive technique, allowed us to evaluate the effectiveness of the implemented measures, being the tool that provided the best separation between the control and intervened population. Considering the physiological traits observed here, ecoengineering solutions Frontiers in Marine Science | www.frontiersin.org 1August 2021 | Volume 8 | Article 699528 fmars-08-699528 September 1, 2021 Time: 9:1 # 2 Cruz de Carvalho et al. Ecoengineering for Impairing Spartina patens based on increased waterlogging of S. patens stands, can be a low-cost and efficient measure to reduce the spreading and growth of this invasive species in the Mediterranean and other salt marshes worldwide with little disturbance. Keywords: ecological restoration, invasive species, salt marsh, remote sensing, halophytes INTRODUCTION Salt marshes provide a wide range of ecological services, including nursery habitats for many animals, protection against coastal erosion, water purification, having the considerable capacity to store and sequester carbon, and are key players in the ecosystem natural remediation capacity (Couto et al., 2013; Teixeira et al., 2014;Duarte et al., 2018, 2021), being these services valuated in several millions of euros per year (Duarte et al., 2021). Being preferred locations for human settlement and a profusion of anthropogenic activities, coastal, and transitional areas have been severely impacted in their health and functioning worldwide. Habitat loss and degradation, climate change and the introduction of invasive species are amongst the major threats to salt marsh ecosystems (Duarte et al., 2015, 2018;Repolho et al., 2017;Pérez-Romero et al., 2018). Therefore, in the context of increasing degradation rates, the need to restore salt marsh ecosystems has been recognized as a priority by managers, scientists, and general society. This is well-emphasized in the EU Biodiversity Strategy for 2030 and its EU Nature Restoration Plan (EU, 2020) and reinforced by the UN Decade on Ecosystem Restoration (UN, 2020). Salt marshes have been widely affected by non-indigenous species (NIS) being a serious threat to wetland biodiversity (Heywood, 1989). Although many NIS plants were introduced long ago (more than a century), recent arrivals are of much concern (Aguiar and Ferreira, 2013;Ainouche and Gray, 2016; Martínez-Jauregui et al., 2018). The Spartina genus is highly successful amongst the halophyte plant group, being widespread across the globe. These plants have C4-type photosynthesis, in which a CO2concentration mechanism at ribulose-1,5bisphosphate carboxylase-oxygenase (Rubisco) level, involving the fixation of atmospheric CO2by phosphoenol-pyruvate carboxylase (PEPC), increases Rubisco carboxylase activity and allows a faster growth rate (von Caemmerer, 2020). Spartina patens (Ait.) Muhl. (Gramineae) is a perennial grass distributed along a wide range of coastal habitats, aggressively competing with native species (Duarte et al., 2015). It was introduced in the Mediterranean Sea probably due to ship traffic from America, being used as packing material in ships boxes and Abbreviations: APx, ascorbate peroxidase; CAP, canonical analysis of principal coordinates; CAT, catalase; DBI, double bond index; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; ETC, electron transport chain; FAME, fatty acids methyl esters; GOPx, guaiacol peroxidase; GR, glutathione reductase; LCPUFA, polyunsaturated fatty acids; MDA, malondialdehyde; NIS, non-indigenous species; PAM, pulse amplitude modulated; PCO, principal coordinates analysis; PEP, phosphoenol-pyruvate; PEPC, phosphoenol-pyruvate carboxylase; PMSF, phenylmethylsulfonyl fluoride; PS I, photosystem I; PS II, photosystem II; PVC, polyvinyl chloride; PVP, polyvinylpyrrolidone; RC, reaction centers; ROS, reactive oxygen species; Rubisco, ribulose-1,5-bisphosphate carboxylase-oxygenase; SOD, superoxide dismutase; TBA, thiobarbituric acid; TCA, trichloroacetic acid; TPF, triphenyl formazan; TTC, triphenyl-tetrazolium chloride. crates (Hultén, 1958). Therefore, although being present for some time along the Western Mediterranean coasts, it had not been recorded on the Eastern Iberian coast until recently (Baumel et al., 2016). Controlling invasive species is relevant for restoration efforts, and several approaches have been developed to address this issue, ranging from herbicide application to mowing and physical removal (Kerr et al., 2016). While the first appears as a simple and cost-effective method (Major et al., 2003;Patten et al., 2017), the long-term effects of herbicides may have serious impacts on the ecosystem (Cruz de Carvalho et al., 2020b). As for the other two methods, they will only prevent further spread needing to be constantly applied and being very timeand cost-consuming (Hedge et al., 2003). Therefore, we considered an alternative method underpinned on the physiology of the species, in particular the fact that S. patens has a low tolerance to waterlogging, due to poor aeration of the rhizosphere which consequently impairs its growth (Burdick, 1989;Bertness, 1991; Curado et al., 2020), and naturally limits this species distribution to the high tide border of salt marshes. Thus, in the present work, we tested the application of a physical barrier that would prolong waterlogging around the plants and evaluated the impact on its physiology. MATERIALS AND METHODS Plant Material and Collection Site Sampling occurred before the start of the growing season (February 2020) in the Hortas salt marsh (Alcochete; 38◦ 45.661’ N, 8◦56.116’ W), located in the middle estuary, adjacent to the Tagus Estuary Natural Reserve (Figure 1). This salt marsh is flooded twice per day, being dominated by the halophyte species Spartina maritima in the lower marsh (circa 12% coverage), Halimione portulacoides in the mid-upper marsh (circa 35% coverage), and Sarcocornia fruticosa in the upper marsh (circa 20% coverage) (Caçador et al., 2013). More recently, the halophyte invasive species S. patens has managed to establish itself in the margins, justifying the importance of the current study. Applied Treatments Spartina patens plants were subjected to two treatments: (1) control, where the plants did not undergo any intervention, and (2) the waterlogging treatment (waterlogged) where plant turfs filled the inside of a 50 cm long and 10 cm diameter PVC tube buried down into the sediments, leaving an 8 cm margin outside, which allowed the tidal water to enter the tube and remain longer inside and, thus, increase the waterlogging of the plants (n= 5 for each treatment). The experiment lasted for 30 days. Several Frontiers in Marine Science | www.frontiersin.org 2August 2021 | Volume 8 | Article 699528 fmars-08-699528 September 1, 2021 Time: 9:1 # 3 Cruz de Carvalho et al. Ecoengineering for Impairing Spartina patens FIGURE 1 | Map of the location of the sampling site in the Hortas salt marsh (Alcochete) in the Tagus estuary. field measurements were made, namely, in vivo chlorophyll a pulse amplitude modulated (PAM) fluorometry measurements. Moreover, 20 leaves from each treatment were also collected directly into individual tubes with liquid nitrogen (see sections below). Finally, whole plants of S. patens from both treatments were also collected, transported to the lab, washed, removed from excess water, weighted (fresh weight), and oven-dried at 60◦C to constant weight (dry weight). Chlorophyll aPulse Amplitude Modulated Fluorometry Ten leaves from plants from each treatment were dark-adapted for 15 min and PAM measurements were performed using a FluoroPen FP100 (Photo System Instruments, Czechia). For the analysis of chlorophyll transient light curves (Kautsky plot), and the derived fluorometric parameters (Table 1), the JIP-test was used (Duarte et al., 2017). Leaf Infrared Thermography Thermal images were obtained with a FLIR E50bx infrared camera (FLIR Systems, Inc., Wilsonville, OR, United States) producing images of 320 ×240 resolution with an accuracy of ±0.045◦C. Ten leaves were randomly selected from plants of both treatments, having a water bottle at ambient temperature near the leaves as reference. The average temperature of each leaf was calculated on each image. All image processing and analysis were performed in FLIR Tools software (version 6.4.18039.1003, FLIR Systems, Inc.). Root Respiration Fresh fine roots of each treatment weighing approximately 100 mg were transferred to 10-mL reaction tubes, and 6 mL of TTC-solution [0.6% (w/v) triphenyl-tetrazolium chloride in 0.06 M Na2HPO4–KH2PO4and 0.05% (v/v) Tween 20] was added to each tube (n= 5) (Brunner et al., 2002). In duplicate tubes, 0.15 mM KCN was added to determine the inhibited respiration. The samples were then incubated for 24 h at 25◦C. After incubation, the TTC solution was decanted and triphenyl formazan (TPF) extraction was made by adding 2 mL of ethanol and boiling at 80◦C for 15 min (Ruf and Brunner, 2003). After collecting the supernatant in new tubes, the absorbance of 1 mL was measured at 520 nm with a spectrophotometer (UV500 UVVisible Spectrometer, Unicam, Waltham, MA, United States). The root residues in the test tubes were dried at 80◦C for 72 h and weighed. Reduction of TTC was calculated as µg of TPF produced per hour per g dry weight (DW). Proline Quantification Proline content was determined according to Bates et al. (1973). For each treatment, plant leaves (n= 5) were homogenized in 3% aqueous sulfosalicylic acid and the homogenate centrifuged a 9,000 g for 15 min at 0◦C (Sigma Frontiers in Marine Science | www.frontiersin.org 3August 2021 | Volume 8 | Article 699528 fmars-08-699528 September 1, 2021 Time: 9:1 # 4 Cruz de Carvalho et al. Ecoengineering for Impairing Spartina patens TABLE 1 | Fluorometric analysis parameters and their description. JIP-test Area Corresponds to the oxidized quinone pool size available for reduction and is a function of the area above the Kautsky plot N Reaction center turnover rate SMCorresponds to the energy needed to close all reaction centers M0Net rate of PS II RC closure γRC Probability that a PS II chlorophyll molecule function as a RC 9Eo Probability that an absorbed photon will move an electron into the ETC ϕ0Probability that a trapped excitation moves an electron into the ETC beyond QA δRo Efficiency of the transfer of an electron from PQH2to final PS I acceptors RE0/RC Flux of electrons transferred from PQH2to final PSI acceptors per active PS II ABS/CS Absorbed energy flux per cross-section TR0/CS Trapped energy flux per cross-section ET0/CS Electron transport energy flux per cross-section DI0/CS Dissipated energy flux per cross-section RC/CS Number of available reaction centers per cross-section PGGrouping probability of the connectivity between the two PS II units δRo/(1-δRo) Contribution of PSI, reducing its end acceptors 90/(190) Contribution of the dark reactions from QA−to PC 9Eo/(19Eo) Equilibrium constant for the redox reactions between PS II and PS I RC/ABS Reaction center II density within the antenna chlorophyll bed of PS II TR0/DI0Contribution or partial performance due to the light reactions for primary photochemistry SFI Structure functional index for photosynthesis SFI (NO) Non-photosynthetic or dissipation structure functional index 2-16K, SIGMA Laborzentrifugen GmbH, Osterode am Harz, Germany). The supernatant was collected, and the reaction consisted of 2 mL of extract combined with 2 mL of glacial acetic acid and 2 mL of acid ninhydrin. The reaction occurred for 1 h at 100◦C, after which the reaction was stopped in an ice bath. The reaction mixture was extracted with 4 mL of toluene and its absorbance read at 520 nm with a spectrophotometer (UV500 UV-Visible Spectrometer, Unicam, Waltham, MA, United States) and compared with a standard curve of proline, expressed in µmol g−1fresh weight (FW). C4-Photosynthetic Carboxylating Enzymes Activity and Pigment Analysis Carboxylating enzymes, PEPC and Rubisco, were extracted from frozen leaf samples according to Carmo-Silva et al. (2008), except that 50 mM HEPES-KOH pH 7.3 was used and 0.5% (v/v) Triton X-100 added. Briefly, approximately 50 mg FW were extracted in a cold mortar containing quartz sand, 1% (w/v) insoluble polyvinylpyrrolidone (PVP) and 1 mL of ice-cold extraction medium [50 mM HEPES-KOH pH 7.3, 1 mM EDTA, 5% (w/v) PVP25000, 6% (w/v) polyethylene glycol (PEG4000), 10 mM dithiothreitol (DTT), 1% (v/v) protease cocktail inhibitor (Sigma, St Louis, MO, United States) and 0.5% (v/v) Triton X-100]. After taking aliquots for pigment analysis, the homogenate was centrifuged for 3 min at 16,800 g at 4◦C (Sigma 2-16K, SIGMA Laborzentrifugen GmbH, Osterode am Harz, Germany) and the supernatant (crude extract) was kept at 4◦C and immediately used for measuring the activities of Rubisco (EC 4.1.1.39) and PEPC (EC 4.1.1.31). The activities of Rubisco were assayed at 25◦C by 14CO2 incorporation into acid-stable products according to Parry et al. (1997) to with modifications (Correia et al., 2020). The assay medium (1 mL per sample) contained 50 mM Bicine-KOH pH 8.2, 40 mM MgCl2, 10 mM NaH14CO3(7.4 kBq µM−1) and 0.4 mM ribulose-1,5-bisphosphate (RuBP). To measure Rubisco initial activity (Vi), 25 µL of crude extract was added to assay medium, and the reaction stopped after 1 min with the addition of 100 µL of 1 M HCl. To determine Rubisco total activity (Vt), 25 µL of crude extract was added to assay medium without RuBP for 3 min, to allow the carbamylation of enzyme catalytic sites. Rubisco Vtreaction was started by adding RuBP and stopped after 1 min with the addition of 100 µL of 1 M HCl. The mixture was completely dried at 60◦C after which the residue was resuspended in 0.5 mL of distilled water and mixed with 5 mL of scintillation liquid (BioSafe LS Cocktail, Beckman, United States). Radioactivity of the 14C incorporated in the acidstable products was measured by scintillation counting (LS 7800 spectrophotometer, Beckmann Instruments Inc., Fullerton, CA, United States). Rubisco activation state (%) was determined by the Vi/Vtratio. PEPC physiological (Vphysiol) and maximum (Vmax) activities were measured in a continuous assay at 340 nm and 25◦C (UV500 UV-Visible spectrophotometer, Unicam, Cambridge, United Kingdom) according to Bakrim et al. (1992) with some modifications (Carmo-Silva et al., 2007). The reaction mixture for Vphysiol (1 mL) consisted of 50 mM HEPES-KOH pH 7.2, 10 mM MgCl2, 10 mM NaHCO3, 2.5 mM PEP (Sigma), 12 units of MDH (Sigma) and 20 µL of crude extract. For Vmax, the reaction mixture consisted of 50 mM HEPES-KOH pH 8.0, 10 mM MgCl2, 10 mM NaHCO3, 10 mM PEP (Sigma), 12 units of MDH (Sigma) and 20 µL of extract. In both cases, the reaction was started by the addition of 0.2 mM (final concentration) NADH (Sigma). Each measured activity is the mean of three replicate on the same extract. PEPC activation state (%) was calculated as Vphysiol/Vmax ratio. For pigment analysis, each 20 µL aliquot previously retrieved from the leaf extract was diluted in 980 µL of methanol. After mixing in the vortex, the samples were left in the dark at 4◦C overnight. After centrifuging for 1 min at 13,000 g at 4◦C (Sigma 2-16K, SIGMA Laborzentrifugen GmbH, Osterode am Harz, Germany), the absorbance at 470, 652.4, 665.2, and 700 nm were measured in an EpochTM 2 Microplate Spectrophotometer (BioTek, Winooski, VT, United States). Pigment concentrations were determined according to the equations in Lichtenthaler and Buschmann (2001). Antioxidant Enzyme Assays To extract the soluble protein fraction, leaf samples were grinded in a cooled mortar with 0.5 mL of 50 mM sodium/potassium phosphate extraction buffer (with 0.1 mM Na-EDTA, 2 mM PVP, 10 mM DTT, 0.1 mM PMSF, and 24 µM NADP, pH 7.6). Frontiers in Marine Science | www.frontiersin.org 4August 2021 | Volume 8 | Article 699528 fmars-08-699528 September 1, 2021 Time: 9:1 # 5 Cruz de Carvalho et al. Ecoengineering for Impairing Spartina patens The homogenate was centrifuged at 13,000 g for 10 min at 4◦C (Sigma 2-16K, SIGMA Laborzentrifugen GmbH, Osterode am Harz, Germany) and the supernatant was collected to a new tube. Protein concentration was determined according to Bradford (1976) in an EpochTM 2 Microplate Spectrophotometer (BioTek, Winooski, VT, United States). Catalase (CAT; EC 1.11.1.6) activity was measured according to Teranishi et al. (1974), through H2O2consumption monitoring and the decrease in absorbance at 240 nm (ε= 39.4 mM−1cm−1). The reaction mixture contained 50 mM of sodium/potassium phosphate buffer (pH 7.0), and 30 mM of H2O2with the reaction being started by the addition of 5 µL of extract. Ascorbate peroxidase (APx; EC 1.11.1.11) was assayed according to Tiryakioglu et al. (2006). The reaction mixture contained 50 mM of sodium/potassium phosphate buffer (pH 7.0), 0.1 mM of H2O2, and 0.25 mM L-ascorbate, and the reaction was also initiated with the addition of 5 µL of the extract. The activity was recorded as the decrease in absorbance at 290 nm and the amount of ascorbate oxidized calculated from the molar extinction coefficient (ε= 2.8 mM−1cm−1). Guaiacol peroxidase (GOPx; EC 1.11.1.7) activity was assayed according to Mika and Lüthje (2003) through the monitorization of guaiacol oxidation at 470 nm (ε= 26.6 mM−1cm−1). The reaction mixture contained 50 mM of sodium/potassium phosphate buffer (pH 7.0), 10 mM of H2O2, and 8 mM guaiacol, and the reaction was initiated with the addition of 5 µL of the extract. Superoxide dismutase (SOD; EC 1.15.1.1) activity was assayed according to Marklund and Marklund (1974) by measuring the reduction of pyrogallol at 325 nm. The reaction mixture contained 30 mM of sodium/potassium phosphate buffer (pH 7.0) and 0.24 mM of pyrogallol, with the reaction being started by the addition of 5 µL of extract. Glutathione reductase (GR; EC 1.8.1.7) activity was assayed according to Edwards et al. (1990) by measuring the fall in absorbance at 340 nm as NADPH was oxidized (ε= 6.22 mM−1cm−1). The reaction mixture contained 25 mM of sodium/potassium phosphate extraction buffer (pH 7.6), 0.5 mM of oxidized glutathione and 0.2 mM NADPH, starting the reaction by the addition of 5 µL of extract. Control assays were done in the absence of substrate to evaluate the autoxidation of the substrates. All assays were performed in a total volume of 200 µL per well at 25◦C in an EpochTM 2 Microplate Spectrophotometer (BioTek, Winooski, VT, United States). Lipid Peroxidation Analysis Lipid peroxidation products were determined as previously described (Heath and Packer, 1968). Leaves were homogenized briefly in 1.5 mL of 10% (v/v) trichloroacetic acid (TCA), containing 0.4% (w/v) thiobarbituric acid (TBA). The reaction was conducted at 100◦C for 30 min, being halted through placement in ice. After centrifugation at 15,000 g for 10 min at 4◦C (Sigma 2-16K, SIGMA Laborzentrifugen GmbH, Osterode am Harz, Germany), 1 mL of the supernatant was collected and mixed with 1 mL of 0.4% TBA and incubated again under the same conditions. The absorbance of the supernatant was recorded at 532 and 600 nm by spectrophotometry (UV500 UVVisible Spectrometer, Unicam, Waltham, MA, United States). The concentration of malondialdehyde (MDA) was determined using the molar extinction coefficient (ε= 155 mM−1cm−1). Fatty Acid Profiles The analysis of fatty acid was performed by direct transesterification of leaf samples, in freshly prepared methanol sulphuric acid (97.5:2.5, v/v), at 70◦C for 60 min, using the internal standard pentadecanoic acid (C15:0) (Feijão et al., 2018). Fatty acid methyl esters (FAME) were recovered using petroleum ether, dried with an N2flow, and re-suspended in an adequate amount of hexane. The FAME solution was analyzed through gas chromatography (Varian 430-GC gas chromatograph equipped with a hydrogen flame ionization detector set at 300◦C, Middelburg, Netherlands), by addition of 1 µL, setting the injector temperature to 270◦C, with a split ratio of 50. The fused-silica capillary column (50 m ×0.25 mm; WCOT Fused Silica, CP-Sil 88 for FAME; Varian, Middelburg, Netherlands) was maintained at a constant nitrogen flow of 2.0 mL min−1and the oven set to 190◦C. Fatty acids identification was performed by comparison of retention times with standards (Sigma-Aldrich) and chromatograms were analyzed by the peak surface method, using the Galaxy software. The double bond index (DBI) was calculated, to determine the membrane saturation levels, as previously described (Feijão et al., 2018): DBI =2×% monoenes +2×% dienes +3×% trienes 100 (1) Statistical Analysis Since the data lacked normality and homogeneity, the statistical analysis was based on Mann-Whitney non-parametric tests (GraphPad Prism 8.4.2 for Windows, GraphPad Software, San Diego, CA, United States). Multivariate statistical analyses [SIMPER and Canonical Analysis of Principal Coordinates (CAP)] were performed using Primer 6 software (Clarke and Gorley, 2006). The data obtained from the Kautsky plots, thermography data, oxidative stress and fatty acids were used FIGURE 2 | Chlorophyll transient kinetics (Kautsky plots) in leaves of control plants (dark gray) and waterlogged plants (light gray) of Spartina patens after 30 days (mean ±s.d., n= 10). Frontiers in Marine Science | www.frontiersin.org 5August 2021 | Volume 8 | Article 699528 fmars-08-699528 September 1, 2021 Time: 9:1 # 6 Cruz de Carvalho et al. Ecoengineering for Impairing Spartina patens FIGURE 3 | Boxplots of leaf energy fluxes [A, absorbed (ABS/CS); B, trapped (TR0/CS); C, transported (ET0/CS); D, dissipated (DI0/CS)] and (E) the number of available reaction centers per cross-section (RC/CS) in leaves of control plants (white boxes) and waterlogged plants (gray boxes) of Spartina patens after 30 days (n= 10, different letters indicate significant differences at p<0.05). as the basis for the construction of the respective resemblance matrixes based on the Euclidean distances between samples. To evaluate the different metabolic datasets obtained as a whole (in opposition to univariate analysis), statistical multivariate models based on Kautsky plots, thermography data, oxidative stress and fatty acids were generated using Principal Coordinates Analysis (PCO) (Clarke and Gorley, 2006). RESULTS Chlorophyll aPAM Analysis Observing the Kautsky plots resultant from the in vivo PAM fluorometric analysis, lower fluorescence values could be observed in plants subjected to the treatment when compared with control plants (Figure 2). Frontiers in Marine Science | www.frontiersin.org 6August 2021 | Volume 8 | Article 699528 fmars-08-699528 September 1, 2021 Time: 9:1 # 7 Cruz de Carvalho et al. Ecoengineering for Impairing Spartina patens FIGURE 4 | Boxplots of the photosystem II and ETC related photochemical traits [A, oxidized quinone pool; B, reaction center turnover rate (N); C, the energy needed to close all reaction centers (SM); D, the probability that a PSII chlorophyll molecule function as a RC (γRC); E, net rate of PS II RC closure (M0)], in leaves of control plants (white boxes) and waterlogged plants (gray boxes) of Spartina patens after 30 days (n= 10, different letters indicate significant differences at p<0.05). The four energy fluxes [Figure 3: A, energy absorbed by the photosystem II (PS II) antennae (ABS/CS); B, energy trapped inside the PS II (TR0/CS); C, energy transported within the electron transport chain (ETC) (ET0/CS); and D, the energy dissipation flux (DI0/CS)] showed the same pattern presenting lower values in the Frontiers in Marine Science | www.frontiersin.org 7August 2021 | Volume 8 | Article 699528 fmars-08-699528 September 1, 2021 Time: 9:1 # 8 Cruz de Carvalho et al. Ecoengineering for Impairing Spartina patens FIGURE 5 | Boxplots of the photosystems I (PS I) and II (PS II) photochemical traits. (A) Active oxygen-evolving complexes (OECs); (B) grouping probability between the two PS II units (PG); (C) the contribution of the dark reactions from quinone A to plastoquinone [ψ0/(1 - ψ0)]; (D) the equilibrium constant for the redox reactions between PS II and PS I [ψE0/(1 - ψE0)]; (E) electron transport from PQH2to the reduction of PS I end electron acceptors (RE0/RC); (F) the contribution of PS I reducing its end acceptors [δR0/(1δR0)]; (G) reaction center II density within the antenna chlorophyll bed of PS II (RC/ABS); (H) contribution or partial performance due to the light reactions for primary photochemistry (TR0/DI0), in leaves of control plants (white boxes) and waterlogged plants (gray boxes) of Spartina patens after 30 days (n= 10, different letters indicate significant differences at p<0.05). Frontiers in Marine Science | www.frontiersin.org 8August 2021 | Volume 8 | Article 699528 fmars-08-699528 September 1, 2021 Time: 9:1 # 9 Cruz de Carvalho et al. Ecoengineering for Impairing Spartina patens FIGURE 6 | Boxplots of the structure functional indexes for photosynthesis (A, SFI) and non-photosynthetic or dissipation processes [B, SFI (NO)] in leaves of control plants (white boxes) and waterlogged plants (gray boxes) of Spartina patens after 30 days (n= 10, different letters indicate significant differences at p<0.05). FIGURE 7 | Boxplots of the leaf surface temperature measured through infrared thermography in leaves of control plants (white box) and waterlogged plants (gray box) of Spartina patens after 30 days (n= 10, different letters indicate significant differences at p<0.05). waterlogged plants, although that decrease was only statistically significant for TR0/CS and ET0/CS. It was also a similar reduction in the number of oxidized PS II reaction centers (RC/CS) (Figure 3E). Further analysis of the functioning of different components of the photosystems and ETC (Figure 4) showed a decrease in the oxidized quinone pool size in the waterlogged plants, followed by an enhancement in the number of QA redox turnovers until maximum fluorescence was reached (N). Although no significant changes were observed in the energy needed to close all RCs (SM), there was a decrease in the probability of a PS II chlorophyll molecule functioning as a RC (γRC) in the treated plants. However, no significant differences were observed in the QA reduction rate (M0). Although the active oxygen-evolving complexes (OEC) showed no differences between control and waterlogged plants (Figure 5A), the PG, the grouping probability that correlates with the disconnection between the two PS II units, increased in the later plant group (Figure 5B). Regarding PS II and PS I, waterlogged plants presented a significant decrease in photochemical processes, both in the contribution of light (TR0/DI0;Figure 5H) and dark [ψ0/(1 -ψ0); Figure 5C] reactions of the photochemical cycle. On the other hand, at the PS I level there was a significant increase in the activity of this photosystem [δR0/(1 - δR0); Figure 5F] in the intervened plants, although there was a decrease in the equilibrium constant for the redox reaction between both photosystems toward the PS II [ψE0/(1 - ψE0); Figure 5D]. Nevertheless, there were no significant changes in the reaction center density within the PS II antenna chlorophyll bed (RC/ABS; Figure 5G) or in the electron transport from PQH2to the reduction of the PS I end acceptors (RE0/RC; Figure 5E). In summary and observing the structure functional indexes, there was a decrease of the photochemical processes (Figure 6A) and an increase of the non-photochemical or dissipative processes (Figure 6B) in the waterlogged plants. Leaf Thermography Regarding leaf surface temperature measured through infrared thermography, there was a statistically significant increase in temperature in the treated plants (11.06◦C) relatively to control ones (10.77◦C) (Figure 7). Frontiers in Marine Science | www.frontiersin.org 9August 2021 | Volume 8 | Article 699528 fmars-08-699528 September 1, 2021 Time: 9:1 # 16 Cruz de Carvalho et al. Ecoengineering for Impairing Spartina patens S. patens. Although long-term studies need to be performed, this ecoengineering solution has the potential to control and eliminate this species from salt marshes and other intertidal systems in future ecosystem restoration programs. Since S. patens turfs are easily identified, the upscaling of this technique could involve the application of tubes with different diameters according to plant turfs density, allowing waterlogging to be prolonged in space and time. Furthermore, the application of a simple bio-optical tool will allow the stakeholders to easily follow the process of suppressing the species without the interference of the process and allowing repeated measures over the intervention period. DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. AUTHOR CONTRIBUTIONS BD, VF, and PR-S: conceptualization. BD: methodology, supervision, project administration, and funding acquisition. RC and BD: formal analysis. RC, EF, ID, VP, MS, AS, and AM: investigation. RC: data curation and writing—original draft preparation. EF, ID, VP, MS, AM, AS, IC, PR-S, VF, and BD: writing—review and editing. All authors have read and agreed to the published version of the manuscript. FUNDING BD and VF were supported by investigation contracts (CEECIND/00511/2017 and DL57/2016/CP1479/CT0024). PR-S was supported by FCT through a postdoctoral grant (SFRH/BPD/95784/2013). We would like to thank the Fundação para a Ciência e a Tecnologia (FCT) for funding the research via project grants PTDC/CTA-AMB/30056/2017 (OPTOX), UID/MAR/04292/2019, and UIDB/04046/2020. We would also like to thank the MAR2020 program through the project RESTAURA2020 (16-01-04-FMP-0014). SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmars. 2021.699528/full#supplementary-material REFERENCES Aguiar, F. C. F., and Ferreira, M. T. (2013). Plant invasions in the rivers of the Iberian Peninsula, south-western Europe: A review. Plant Biol. 147, 1107–1119. doi: 10.1080/11263504.2013.861539 Ainouche, M., and Gray, A. (2016). 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