Ecophysiological and biochemical responses to cold and heat waves of native Spartina maritima, alien S. densifora and their reciprocal hybrids

Vol.:(0123456789) Planta (2025) 261:99 https://doi.org/10.1007/s00425-025-04675-4 ORIGINAL ARTICLE Ecophysiological andbiochemical responses tocold andheat waves ofnative Spartina maritima, alien S. densiflora andtheir reciprocal hybrids RosarioÁlvarez1· SalvadorA.Fernandez‑Gonzalez1· AdriánPerera‑Bonaño1· AlfonsoDeCires1· JesúsM.Castillo1· BlancaGallego‑Tévar1 Received: 14 December 2024 / Accepted: 15 March 2025 / Published online: 1 April 2025 © The Author(s) 2025 Abstract Main conclusion Spartina hybrids outperform parental species, showing transgressive acclimation to extreme climates. Native S. maritima demonstrates high seasonal adaptability and invasive S. densiflora low physiological impact, suggesting resilience under climate change. Abstract Extreme climatic events, such as cold and heat waves, are becoming more frequent, intense, and prolonged due to climate change. Simultaneously, invasive alien plant species are altering the composition of plant communities. Both climate change and the introduction of alien species pose significant threats to biodiversity. We studied the responses of 25 biochemical and physiological functional traits for native Spartina maritima, alien invasive S. densiflora and their reciprocal hybrids to changing environmental conditions during a cold snap in winter and a heat wave in summer in Guadiana Marshes (Southwest Iberian Peninsula). These four closely related taxa responded differently to seasonal environmental fluctuations. Both hybrid taxa, particularly S. maritima × densiflora, exhibited transgressive responses, allowing them to display a wider range of acclimation responses to air temperature compared to their parental species. Native S. maritima also demonstrated a relatively high acclimation capacity to seasonal meteorological changes. In contrast, alien S. densiflora presented few acclimation responses to seasonal environmental changes, responding primarily to sediment salinity rather than to air temperature. Even so, all four studied Spartina taxa appear to be well-adapted to the occurrence of cold and heat waves in the Gulf of Cadiz. These findings underscore the complexity of plant acclimation strategies in response to extreme climatic events and highlight the potential for hybrid taxa to face the future dynamics of salt marshes under climate change. Keywords Anthocyanins· Climate change· Cordgrass· Hybridization· Mediterranean climate· Thermal stress Abbreviations EC Electrical conductivity Eh Redox potential F0, Fm, Fv Initial, maximal, and variable fluorescence Fv/Fm Maximum quantum efficiency of PSII MDA Malondialdehyde NPQ Non-photochemical quenching WC Water content Vmax Maximal rate of photosynthetic oxygen evolution ΦPSII Effective quantum efficiency of PSII Introduction Extreme climatic events, such as cold and heat waves, are increasing in frequency, intensity and duration in the present scenario of climate change (IPCC 2022). These climatic changes are especially relevant for the Mediterranean Basin which has been identified as a climate change hotspot (Noto etal. 2023). Cold and heat waves expose plants to high levels of thermal stress that can diminish photosynthesis, a highly sensitive process to temperature changes (Chovancek etal. 2019; Grossman 2023). Communicated by Dorothea Bartels. * Blanca Gallego-Tévar [email protected] 1 Departamento de Biología Vegetal y Ecología, Universidad de Sevilla, Ap 1095, 41080Seville, Spain Planta (2025) 261:9999 Page 2 of 17 Thermal stress may reduce the efficiency of photosynthetic electron transport in both photosystems and alter pigment accumulation and chlorophyll (Chl) fluorescence quenchings (Teskey etal. 2015; Nievola etal. 2017). Diminished photosynthesis as a result of thermal stress can reduce biomass production and the capacity to face environmental stresses that, in turn, may cause broad biogeographic shifts and regional changes in distribution (Aagesen etal. 2016; Liancourt etal. 2020). Together with climate change, invasive alien plant species are changing species composition of plant communities and constitute also a severe threat to biodiversity (Fried etal. 2014; van Kleunen etal. 2018). Some alien plant species can suffer from non-reversible photodamage and reduced photosynthesis due to thermal stress (Zhu etal. 2019). In contrast, many alien species may exhibit enhanced responses, at biochemical and photochemical levels, to thermal stress than their native congeners (Duarte etal. 2016; Kunert etal. 2022). The physiological challenges posed by climate change for native and alien plant species can be faced by phenotypic plasticity and local adaptation. In general, invasive species present higher phenotypic plasticity in their photosynthesis performance than non-invasive species (Davidson etal. 2011). Moreover, some alien plant species have adapted a wide range of biochemical and physiological traits to the climate in their introduced range, facilitating extensive invasions (Fenollosa and Munne-Bosch 2019). In this context, rapid global warming has enabled alien species to expand into regions in which they previously could not survive and reproduce (Walther etal. 2009; Kathiresan and Gualbert 2016). Liu etal. (2017) found that elevated temperature and CO2 enrichment favoured invasive alien plants more strongly than native plants. Nevertheless, in general, both invasive alien plants and their native counterparts usually undergo similar adaptive evolution responses of physiological performance to global warming (Gianoli etal. 2021). Alien plant species can hybridize with native species and the upcoming hybrids can show increased fitness in responses to different environmental stresses due to biochemical and physiological transgressive traits (GallegoTévar etal. 2019a, 2020a, b; Zhang etal. 2020). In this context, Sun etal. (2015) found that hybridization slightly catalyzed the tolerance of a hybrid between native and alien Sphagneticola species to low temperature and weak light conditions, recorded as changes in photosynthetic and antioxidant functional traits. Maternal effects also play a relevant role in the functional trait expression of interspecific hybrids facing environmental stress (Filipe and Montesinos 2016), including the expression of photosynthetic traits (Ji and Jiao 1999). Moreover, the formation of these hybrids can be modulated by increasing temperatures in the present scenario of global warming (Gallego-Tévar etal. 2019b). Therefore, hybridizations between native and alien species are of great concern to evolutionary biologists and ecologists interested in quick evolutionary processes. Understanding the biochemical and physiological responses of native and alien plant congener species and their hybrids to seasonal meteorological changes and extreme climatic events is crucial for predicting how these taxa might cope with future environments in the context of climate change. To our knowledge, none study has analyzed the biochemical and physiological responses of native and invasive congeners and their hybrids to cold and heat waves under field conditions. Field studies are especially important since they capture the intricate interplay of environmental factors influencing plant performance and resilience. For example, plant growth-promoting bacteria found in natural conditions can improve the tolerance of the photosynthetic apparatus to thermal stress (Duarte etal. 2023). Our study system was the native cordgrass Spartina maritima (Curtis) Fernald (2n = 60), the alien invasive Spartina densiflora Brongn. (2n = 70) and their reciprocal hybrids growing in Guadiana Marshes (Gulf of Cadiz, southwest Iberian Peninsula). Spartina maritima is a foundation species and a marsh-building halophyte that dominates Iberian and other European and South African low salt marsh zones (Castellanos etal. 1994). Alien Spartina densiflora is a neophyte with high phenotypic plasticity (Castillo etal. 2018) that is invading contrasting habitats along the tidal gradient in the Gulf of Cadiz (Nieva etal. 2001). The hybrids S. maritima × densiflora (2n = c. 95) and S. densiflora × maritima (2n = 65) are sterile hybrids that show some transgressive traits in the field (eg. taller shoots and higher growth rates than the parental species) (Castillo etal. 2010) and in response to salt stress under controlled conditions (GallegoTévar etal. 2018a, 2019a). We studied the responses of 23 biochemical and physiological functional traits of our four focal Spartina taxa to changing environmental conditions during a cold snap in winter and a heat wave in summer. We hypothesized that both parental species would show high tolerance to changing environmental conditions through acclimation of different functional traits and that both hybrids would present transgressive traits enabling them to show higher acclimation capacity than parental species. Materials andmethods Study area Our study was carried out in the San Bruno Marshes (37º10ʹ−37º16ʹN, 7º28ʹ−7º16ʹW), located in the southwest of the Iberian Peninsula, in the Guadiana River Marshes (Fig.S1). The Guadiana Estuary is a mesotidal open estuary with semidiurnal tides, a mean range of 2.10m and a mean spring tidal range of 2.97m (Castellanos etal. 1994). The Planta (2025) 261:99 Page 3 of 17 99 study area presents a gentle slope along a wide tidal gradient, showing a clear plant zonation pattern with low marshes dominated by Spartina maritima and Sarcocornia perennis (Mill.) A.J. Scott, middle marshes by Halimione portulacoides (L.) Aellen, Sarcocornia fruticosa (L.) A.J. Scott and exotic S. densiflora, and high marshes by Arthrocnemum macrostachyum (Moric.) C. Koch and Limoniastrum monopetalum (L.) Boiss. (Gallego-Tévar etal. 2018b). The study area has a Mediterranean climate with Atlantic influence with mild and relatively wet winters (mean air temperature is ca. + 11 ºC in January; average annual precipitation is c. 506mm), and hot and dry summers (mean air temperature is + 25 ºC in August with almost no rainfall). The studied Mediterranean salt marshes are exposed to broad daily and seasonal changes in temperature (> 30°C) and very high temperatures during summertime (> + 40°C) (Boughalleb etal. 2022). Sediment salinity levels peak near seawater concentration during summer droughts (Contreras-Cruzado etal. 2017). Additionally, the studied Mediterranean salt marshes are exposed to sea level rise (+ 4.02mm yr−1 and accelerating) and reduced rainfalls due to climate change (Kovats etal. 2014; NOAA 2018). Environmental matrix Meteorological data (minimum, average and maximum air temperature, ºC) for the sampling days and previous 9days in winter and summer were collected from Ayamonte meteorological station, located 2km away from the studied marshes (code 4549Y; AEMET 2024). The sedimentary environment was characterized at the same time that leaf samples were collected for laboratory analyses. We recorded sediment redox potential (Eh; mV), pH, electrical conductivity (EC; mS cm−1) and water content (WC; %) in the root zone, between 0 and 10cm deep, of every marked Spartina clump (n = 10 samples per taxon). Eh was recorded insitu using an electrode system (Crison Instruments pH/ mV p-506, Hach Lange Spain, S.L.U., Barcelona, Spain) (Castillo etal. 2000). Sediments (250ml) were sampled in sealed plastic containers and transported to the laboratory. Sediment pore-water pH and EC were recorded in the unfiltered supernatant after adding distilled water to the sediment samples (1:1, v/v) using a pHmeter (pH/redox Crison PH25 with the probe Crison 5052) and a conductivity meter (Crison CM35), respectively, in the laboratory. Sediment WC was recorded by weighting ca. 100g of sediments before and after drying to constant weight at + 80 ºC in a forced-air stove (Curado etal. 2014). Plant material Field measurements and sample collections took place during low tidal level around solar noon on sunny days and low tide conditions in January 2021 during a cold snap associated with Storm Filomena and in July 2021 during a heat wave (defined by the Spanish Meteorological State Agency (AEMET; acronym in Spanish) as ≥ 3days with temperatures above the 95th percentile for July–August 1971–2000). All measurements were carried out on the first totally expanded adult leaf from adult shoots of ten randomly selected clumps of each taxon: native Spartina maritima, alien invasive S. densiflora and their reciprocal hybrids S. maritima × densiflora and S. densiflora × maritima. Each taxon was sampled along the tidal gradient there where it was more abundant. Spartina maritima and S. maritima × densiflora were sampled in low marshes, and S. densiflora and S. densiflora × maritima in middle marshes. Sampled clumps were separated at least 2m from each other to ensure they were different individual plants that were permanently marked. Crude extract forlaboratory analyses First, totally developed adult leaves (flag leaves) were collected from 5 randomly selected plants from the 10 marked plant of each taxa (n = 5 samples per taxon), immediately stored in zip-lock plastic bags with silica gel in the field and stored at −20 ºC once in the laboratory. To obtain the crude extract, 0.2g of leaves were weighed and processed in mortar during 10–15min with 10ml of pure methanol (using sand to help extraction). To eliminate any solid residue, samples were centrifuged at 9981g, 15min at 10 ºC. Supernatant was used as a crude extract for the determinations of pigments, total antioxidant capacity, malondialdehyde (MDA) and polyphenol concentrations. Pigments determination The concentrations of photosynthetic pigments and anthocyanins are modified in response to thermal stress (Vetoshkina etal. 2023). To assess chlorophylls (Chl) and carotenoids (Car) concentrations from the crude extract (previously diluted 10 times in pure methanol), absorbance (Abs) at 665.2nm, 652.4nm and 470nm was measured. Chl a, b and carotenoids were calculated using the following equations (Lichtenthaler and Buschmann 2001): Anthocyanin concentration was measured diluting the crude extract with HCl until the final concentration reached 1% (v/v). Samples were kept at 4 ºC in darkness. After 24h, Chl a( 𝜇 g∕ml)=16.72 Abs665.2 −9.16 Abs652.4 Chl b( 𝜇 g∕ml)=34.09 Abs652.4 −15.28 Abs665.2 Car (𝜇g∕ml)= ( 1000 Abs470 −1.63 Chl a−104.96 Chl b ) ∕ 221 Planta (2025) 261:9999 Page 4 of 17 absorbance was measured at 530nm and 653nm. Anthocyanin concentration was determined according to Mancinelli etal. (1974). These and all other UV/visible spectrophotometric assays described below were carried out using a UV-3100PC spectrophotometer (VWR). Polyphenols Many plant species synthesize and accumulate phenolic compounds as a defense mechanism against environmental stressors (Yang etal. 2018). Phenolic compounds were assayed using the Folin-Ciocalteu reagent following Singleton and Rossi (1965). To assess polyphenol concentrations, 0.5ml of Folin–Ciocalteau and 4.5ml of distilled water were added to 1ml of crude extract (previously diluted in distilled water in a 7:3 ratio). Samples were put in darkness at room temperature. After 8min, 4ml of 7.5% (w/v) sodium carbonate (Na2CO3) was added to each sample. After 1-h incubation under the same conditions absorbance at 765nm was measured. Polyphenol concentrations were expressed in mg of gallic acid (GA) g DW−1 (Slama etal. 2017). GA was used as a standard compound. GA stock solution (20mg/100ml methanol) was prepared and various dilutions were obtained for the standard calibration curve. Total antioxidant capacity Reduction of Mo (VI) to Mo (V) at acid pH produces a turquoise compound that can be measured to assess total antioxidant capacity. To record total antioxidant capacity 1ml of reagent (0.6M sulfuric acid, 28mM sodium phosphate, 4mM ammonium molybdate) was added to 0.1ml of crude extract (previously diluted in distilled water in a 7:3 ratio). Samples were kept at 95 ºC for 90min. After cooling for 20min at room temperature, absorbance at 695nm was measured against a blank. Total antioxidant capacity was expressed in mg GA/g DW−1 (Prieto etal. 1999; Slama etal. 2017). Malondialdehyde determination MDA, a biomarker of lipid peroxidation, indicates oxidative damage resulting from abiotic stresses (Kim etal. 2017). To assess MDA concentration as a lipid peroxidation index, we followed the protocol described by Hodges etal. (1999) with some modifications (Taulavuori etal. 2001). A total of 2.5ml of crude extract was divided into two test tubes, each containing 1.25ml. We added 1.5ml of 20% (w/v) trichloroacetic acid (TCA) to the first tube, while the second tube received 1.5ml of a solution containing 0.5% (w/v) thiobarbituric acid (TBA) in 20% (w/v) TCA. Then, 0.25ml of pure methanol was added to each tube. After a 15-min incubation at 95°C, samples were placed on ice to terminate the reaction. After centrifuging samples at 9981g for 10min, the supernatant's absorbance was measured at 440nm, 532nm, and 600nm. MDA concentration was calculated using the following equations: Apical leaf growth Apical leaf growth serves as an effective measure for quantifying stress responses in Spartina taxa in field conditions (Castillo etal. 2014). Apical leaf growth was measured marking 3–5 leaves per clump with permanent sealant at their base, and measuring the distance from the sealant to the leaf base 2days later for the ten marked clumps per taxon (n = 10 samples per taxon). Maximal rate ofphotosynthetic oxygen evolution Maximal rate of photosynthetic oxygen evolution (Vmax) has been previously recorded to quantify foliar thermal stress in halophytes (Figueroa-Luque etal. 2024). We measured Vmax to obtain information on the state of the photosynthetic apparatus under forced conditions of CO2 saturation and high radiation level. Leaves were sampled from the ten marked clumps per taxon in the evening when the tide was raising and just before the study plants were being inundated (n = 10 samples per taxon). Then leaves were stored in a water vapor-saturated atmosphere at 25°C at darkness and 4 ºC until measurements were carried out early in the morning. Vmax measurements were carried out using an oxygen electrode type Clark (Hansatech LD2, Pentney, UK) at 25°C and photosynthetic photon flux density (PPFD) 1400µmol m– 2 s−1 in a CO2-saturated atmosphere obtained with a 1M carbonate/bicarbonate buffer (pH = 9). Vmax was recorded as a release of O2 per unit of time and g FW (µmolO2 s−1 gFW−1) (n = 10 measurements per taxon) (Farquhar etal. 2001; Popova etal. 2019). Chlorophyll fluorescence Chlorophyll a fluorescence is a useful tool to assess the effects of thermal stress on the photosynthetic apparatus (Kunert 2024). Light and dark-adapted Chl fluorescence were measured in one leaf per each of the 10 marked clumps per taxon (n = 10 samples per taxon) at sunrise (PPFD ca. 200µmol m−2 s−1) and at noon (PPFD A =Abs 532 −Abs 600 − ( Abs 532 OnlyTCA −Abs 600 OnlyTCA) B=(Abs 440 −Abs 600)∗0.0571 MDA(nmol mL ) = (A−B 157000 ) ∗10 6 Planta (2025) 261:99 Page 5 of 17 99 of 1100–2100µmol m−2 s−1) with a portable modulated fluorimeter (FMS-2, Hansatech Instruments) using leaf clips for dark adaptation for 30min. Noon measurements inform about the levels of total (dynamic and permanent) photoinhibition, while sunrise measurements record the permanent photoinhibition levels that has not been able to recover during the night (Fernández-Baco etal. 1998). Chl fluorescence parameters were measured according to Maxwell and Johnson (2000). Initial fluorescence (F0) in the dark-adapted state was measured using a PPFD < 0.05µmol m–2 s−1 for 1.8µs, too small to induce significant physiological changes in the plant. Maximal fluorescence (Fm) was recorded after a saturating light pulse of 15.000µmol photons m−2 s−1. Variable fluorescence (Fv = Fm − F0) and maximum quantum efficiency of photosystem II (PSII) photochemistry (Fv/Fm) were calculated to quantify photoinhibition. Using the same leaf section, light-adapted parameters were measured before assessing dark-adapted Chl fluorescence in leaves acclimated to full solar radiation. Steady-state fluorescence yield (Fs) was recorded after adapting plants to ambient light conditions (with full sunlight of 1150µmol photons m−2 s−1). A saturating actinic light pulse of 15,000µmol photons m−2 s−1 for 0.7s was then used to produce the maximum fluorescence yield (Fm´) by temporarily inhibiting PSII photochemistry. Effective quantum efficiency of PSII (ΦPSII = (Fm´—Fs)/Fm´) was calculated. Non-photochemical quenching (NPQ) was calculated from parameters obtained in both dark and light-adapted states. Data analyses Inheritance mechanisms To investigate the inheritance mechanisms underlying salt stress responses, the above-described plant traits were analyzed in the reciprocal hybrids of S. maritima and S. densiflora. The following inheritance mechanisms were identified, as described by Favre and Karrenberg (2011). (1) Dominant inheritance, when a hybrid exhibited a trait similar to one of its parents. This was denoted as “D-Sm” for S. maritima or “D-Sd” for S. densiflora. (2) Parental codominance when a hybrid's trait was not different from the two parents, represented as “D-Sm,Sd”. (3) Parental additivity, when a hybrid trait fell within the range of its parents but was significantly different from both. (4) Transgressive segregation, when hybrids exhibited traits that exceeded, by at least 5%, the phenotypic range of both parents. These inheritance mechanisms were quantified for each plant trait in both hybrid populations and the differences between species were based on the significant differences exerted for each variable (see next section). Statistical analyses All statistical analyses were carried out using R software (R-core team 2024). We applied a significance level (α) of 0.05 for every analysis. Deviations of all data were calculated as the standard error of the mean (SE). Plant traits were classified into two functional groups: (1) biochemical stress responses (anthocyanins, Chl a, Chl b, carotenoids, MDA, polyphenols and antioxidant capacity), and (2) ecophysiological and growth responses (F0, Fm, Fv, Fv/Fm, Fs, Fm´, ΦPSII and NPQ at sunrise and noon, Vmax and apical leaf growth). To protect analyses from type I error, the means of the dependent variables of each trait group were compared using multivariate analysis of variance (MANOVA) and Wilks’ Lambda to evaluate the significance of the factors taxon (S. maritima, S. densiflora, S. maritima × densiflora and S. densiflora × S. maritima) and season (winter and summer) (Scheiner 2001). Redundant, highly correlated variables (r > 0.90) were identified prior to MANOVA analysis and were omitted from the statistical models (Fm at sunrise and noon). Once multivariate significance was confirmed via MANOVA, the main univariate differences of plant traits were evaluated for each plant trait with General Lineal Models (GLMs) and Bonferroni-Dunn's test as a post hoc analysis. Environmental variables were also analyzed using GLMs with taxa and season as fixed factors and Bonferroni-Dunn's test as a post hoc analysis. The adequacy of model assumptions for the GLMs, including checking for uniformity and residual diagnostics, was assessed using the DHARMa package (Hartig 2022). Environmental variables were also compared between seasons and taxa using GLMs and Bonferroni-Dunn's test as a post hoc analysis. We performed Canonical Correspondence Analyses (CCA), using the vegan package (Oksanen etal. 2024), to identify the environmental variables that most greatly influenced plant traits in the studied Spartina taxa. CCA was conducted using a full model to test the significance of the relationships between the environmental variables measured in winter and summer and the plant traits matrix for each taxon. Monte-Carlo permutation tests (999 permutations) were performed for assessing the significance of the canonical correlation coefficients. Average daily maximum and minimum air temperatures were removed during the analysis due to their multicollinearity with average daily mean air temperature. Results Environmental matrix The study area presented marked seasonal air temperatures and rainfall was concentrated mainly from Planta (2025) 261:9999 Page 6 of 17 September to February during the sampling year (Fig. S2). Mean minimum daily air temperature was 21 ºC lower during the winter cold snap than in the summer heat wave. This seasonal difference was 10 ºC for average daily temperature and 19 ºC for maximum daily temperature. Mean sediment Eh was always higher than 95mV for every taxon in both seasons, being higher in winter than in summer. Sediment WC did not present seasonal differences, and varied between 45 ± 1% for S. densiflora × maritima in winter and 72 ± 2% for S. maritima × densiflora in summer. Sediment EC was higher in summer than in winter, and varied between 8.7 ± 0.5 mS cm−1 for S. densiflora in winter and 16.7 ± 0.5 mS cm−1 for S. densiflora × maritima in summer. Mean sediment pH was higher in winter than in summer and ranged between 6.7 and 7.5 (Tables1 and 2). Plant responses Spartina maritima, S. densiflora and both reciprocal hybrids exhibited seasonal variations in different biochemical, ecophysiological and growth traits (Tables3 and 4). Pigments The four Spartina taxa exhibited higher anthocyanin concentration in winter than in summer, with S. densiflora showing a lower increase than the hybrids (Fig.1A). Spartina densiflora and S. densiflora × maritima presented lower anthocyanin concentrations than S. maritima and S. maritima × densiflora (TableS1). Spartina maritima presented higher Chl a concentration and lower carotenoids concentration in summer than in winter (Fig.1B, D). Spartina maritima accumulated more carotenoids in winter than S. Table 1 F-statistic and P-values of General Linear Models with taxon (T), season (S) (winter and summer) and their interaction (T x S) as fixed factors, for environmental variables measured in individuals of Spartina maritima, Spartina densiflora and their reciprocal hybrids in the Guadiana Marshes (Southwest Iberian Peninsula) Values for F-statistics with degrees of freedom (n) as subscripts are displayed. Significant differences are highlighted in bold Environmental variables Taxon (T) Season (S) T × S F3,72 PF1,18–72 PF3,72 P Minimum daily air temperature (ºC) – – 294.63 < 0.0001 – – Average daily air temperature (ºC) – – 873.15 < 0.0001 – – Maximum daily air temperature (ºC) – – 536.83 < 0.0001 – – Sediment redox potential (mV) 3.888 0.012 4.657 0.034 0.267 0.849 Sediment water content (%) 37.139 < 0.0001 1.105 0.297 4.534 0.006 Sediment electrical conductivity (mS cm−1)16.204 < 0.0001 11.921 0.001 0.017 0.997 Sediment pH 52.956 < 0.0001 50.579 < 0.0001 2.403 0.075 Table 2 Meteorological and sedimentary environmental factors for native Spartina maritima, introduced S. densiflora and their reciprocal hybrids in the Guadiana Marshes (Southwest Iberian Peninsula) Values are mean ± SE (n = 10). Different letters indicate significant seasonal differences for a given taxon (General Lineal Models and Bonferroni-Dunn's test as a post hoc analysis; see Table1) Winter Summer S. maritima S. maritima × densiflora S. densiflora S. densiflora × maritima S. maritima S. maritima × densiflora S. densiflora S. densiflora × maritima Minimum daily air temperature (ºC) 4.0 ± 0.6a25.2 ± 0.4b Average daily air temperature (ºC) 8.8 ± 0.4a18.7 ± 0.6b Maximum daily air temperature (ºC) 13.3 ± 0.6a31.7 ± 0.5b Sediment redox potential (mV) 167 ± 22 175 ± 24 189 ± 18 125 ± 11 124 ± 21 168 ± 28 149 ± 18 96 ± 15 Sediment water content (%) 70 ± 2 70 ± 2 71 ± 2 45 ± 1 67 ± 2 72 ± 2 68 ± 3 56 ± 2 Sediment pH 7.5 ± 0.0 7.4 ± 0.0 7.5 ± 0.0 6.9 ± 0.0 7.1 ± 0.1 7.3 ± 0.1 7.2 ± 0.1 6.7 ± 0.0 Sediment electrical conductivity (mS cm−1) 9.1 ± 0.7 9.0 ± 0.9 8.7 ± 0.5 14.5 ± 1.0 11.3 ± 1.1 11.4 ± 1.3 11.2 ± 1.2 16.7 ± 0.5 Planta (2025) 261:99 Page 7 of 17 99 densiflora and S. densiflora × maritima (TableS1). Chl b concentration did not change significantly between seasons for any taxa (Fig.1C). MDA, antioxidant capacity andpolyphenols The hybrid S. maritima × densiflora was the only taxon that presented a higher (+ 68%) MDA concentration in summer than in winter (Fig.1E). Spartina maritima × densiflora accumulated less MDA than S. maritima in winter, with S. densiflora and S. densiflora × maritima presenting intermediate values (TableS1). Both hybrids produced a higher (+ 35%) amount of polyphenols (Fig.1F) and had greater (+ 47%) total antioxidant capacity in winter compared to summer. Spartina maritima also exhibited higher (+ 24%) total antioxidant capacity in winter than in summer (Fig.1G). In winter, S. maritima accumulated more polyphenols than S. densiflora × maritima and S. densiflora. Total antioxidant capacity was lower for S. densiflora in winter and for S. maritima × densiflora in summer than for the other three taxa (TableS1). Growth andphotosynthesis Apical leaf growth was 38% higher in summer than in winter. This seasonal change was recorded for every taxon, being significant for S. densiflora and S. Table 3 Wilks’ lambda, F-statistic, degrees of freedom and P-values from MANOVAs for the two trait response groups for the factors season (winter and summer), taxon (S. maritima, S. densiflora, S. maritima × densiflora and S. densiflora × maritima) and their interaction The higher Wilks’ lambda is, the stronger the evidence that the independent variables (factors) have a statistically significant effect on the dependent variable Factors Wilks’ Lambda F df P Biochemistry Season 0.165 18.832 7 < 0.001 Taxon 0.023 0.984 21 < 0.001 Season*Taxon 0.057 6.120 21 < 0.001 Ecophysiology and growth Season 0.042 81.726 16 < 0.001 Taxon 0.094 4.308 48 < 0.001 Season*Taxon 0.233 2.240 48 < 0.001 Table 4 F-statistic and P-values of General Linear Models with taxon (T), season (S) (winter and summer) and their interaction (T x S) as fixed factors, for biochemical and ecophysiological and growth-related traits measured in individuals of Spartina maritima, Spartina densiflora and their reciprocal hybrids in the Guadiana Marshes (Southwest Iberian Peninsula) Significant differences are highlighted in bold Plant traits Taxon (T) Season (S) T × S F3,32 PF1,32 PF3,32 P Biochemistry Anthocyanins (µg g DW−1)9.28 < 0.001 49.33 < 0.001 2.05 0.126 Chl a (µg g DW−1)12.45 < 0.001 12.00 < 0.01 2.17 0.111 Chl b (µg g DW−1)4.82 < 0.001 2.29 0.140 0.68 0.572 Carotenoids (µg g DW−1)8.17 < 0.001 17.69 < 0.001 10.90 < 0.001 MDA (nmol g DW−1) 2.263 0.100 4.58 < 0.05 6.13 < 0.01 Polyphenols (mg g DW−1)66.12 < 0.001 15.81 < 0.001 6.08 < 0.01 Antioxidant capacity (mg g DW−1)17.75 < 0.001 71.53 < 0.001 20.62 < 0.001 F3,72 PF1,72 PF3,72 P Ecophysiology and growth Apical leaf growth (cm) 21.37 < 0.001 35.68 < 0.001 0.54 0.653 Vmax (μmol O2 g−1 FW−1)6.20 < 0.001 26.16 < 0.001 0.61 0.608 F0 sunrise 2.77 < 0.05 1.49 0.226 2.00 0.121 Fv sunrise 6.57 < 0.001 22.83 < 0.001 5.78 < 0.01 Fv/Fm sunrise 6.22 < 0.001 13.91 < 0.001 1.29 0.284 Fs sunrise 2.90 < 0.05 9.28 < 0.01 4.31 < 0.01 Fm’ sunrise 3.09 < 0.05 103.30 < 0.001 6.89 < 0.001 ΦPSII sunrise 6.36 < 0.01 726.75 < 0.001 3.39 < 0.05 NPQ sunrise 1.44 0.238 25.19 < 0.001 2.50 0.066 F0 noon 0.85 0.470 7.53 < 0.01 0.89 0.451 Fv noon 4.32 < 0.01 21.16 < 0.001 4.64 < 0.01 Fv/Fm noon 2.63 0.056 4.12 < 0.05 2.38 0.077 Fs noon 2.21 0.094 1.63 0.205 5.22 < 0.01 Fm’ noon 2.07 0.112 0.55 0.459 4.80 < 0.01 ΦPSII noon 1.01 0.392 15.62 < 0.001 1.47 0.229 NPQ noon 1.13 0.341 3.32 0.0725 3.80 < 0.05 Planta (2025) 261:9999 Page 8 of 17 maritima × densiflora (Fig.2A). Vmax tended to be higher in winter than in summer for every taxon, although this seasonal variation was only significant for S. densiflora × maritima (Fig.2B). Spartina maritima presented higher Vmax, but grew less than the other three taxa (TableS1). Chlorophyll fluorescence Spartina maritima × densiflora was the only taxon showing higher (+ 9%) Fv/Fm at sunrise in summer than in winter (Fig.2E), through a marked increase (+ 46%) in Fv (Fig.2D). ΦPSII at sunrise was markedly higher (+ 67%) Fig. 1 Seasonal differences in biochemical traits for Spartina maritima (Sm, black column), Spartina densiflora (Sd, white column) and their two reciprocal hybrids, S. maritima × densiflora (Smxd, dark grey column) and S. densiflora × maritima (Sdxm, light grey column). Values are means ± SE (n = 5). Negative values indicate higher magnitudes in winter and positive values indicate higher magnitudes in summer. Asterisks denote seasonal significance at P < 0.05 (GLM) Planta (2025) 261:99 Page 9 of 17 99 in summer than in winter for all taxa (Fig.2H) due to higher Fm’ (Fig.2G), except for S. densiflora that showed higher (+ 107%) Fs in winter than summer (Fig.2F). Both hybrids presented higher ΦPSII at sunrise than S. maritima in summer. Additionally, the two hybrids showed higher NPQ at sunrise in winter than in summer, with S. maritima × densiflora exhibiting the greatest (+ 355%) seasonal increase (Fig.2I) (TableS1). When Chl fluorescence measurements were recorded at noon, S. maritima × densiflora exhibited the majority of the seasonal variations. This hybrid showed higher (+ 9%) Fv/Fm [associated with increased (+ 46%) Fv], and lower (−229%) ΦPSII (with increased Fs and Fm’ values) in summer than in winter (Fig.3B–F). 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