Seasonal Acclimation Modulates the Impacts of Simulated Warming and Light Reduction on Temperate Seagrass Productivity and Biochemical Composition

fmars-08-731152 September 23, 2021 Time: 17:37 # 1 ORIGINAL RESEARCH published: 28 September 2021 doi: 10.3389/fmars.2021.731152 Edited by: Gang Li, South China Sea Institute of Oceanology, Chinese Academy of Sciences (CAS), China Reviewed by: Zhijian Jiang, South China Sea Institute of Oceanology, Chinese Academy of Sciences (CAS), China Amrit Kumar Mishra, Indian Institute of Technology Bhubaneswar, India *Correspondence: Pedro Beca-carretero [email protected] Specialty section: This article was submitted to Global Change and the Future Ocean, a section of the journal Frontiers in Marine Science Received: 26 June 2021 Accepted: 16 August 2021 Published: 28 September 2021 Citation: Beca-Carretero P, Azcárate-García T, Julia-Miralles M, Stanschewski CS, Guihéneuf F and Stengel DB (2021) Seasonal Acclimation Modulates the Impacts of Simulated Warming and Light Reduction on Temperate Seagrass Productivity and Biochemical Composition. Front. Mar. Sci. 8:731152. doi: 10.3389/fmars.2021.731152 Seasonal Acclimation Modulates the Impacts of Simulated Warming and Light Reduction on Temperate Seagrass Productivity and Biochemical Composition Pedro Beca-Carretero1,2*, Tomás Azcárate-García1,3, Marc Julia-Miralles4, Clara S. Stanschewski5, Freddy Guihéneuf6and Dagmar B. Stengel1 1Botany and Plant Science, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland, 2Department of Oceanography, Instituto de Investigacións Mariñas (IIM-CSIC), Vigo, Spain, 3Departamento de Biología, Área de Ecología, Facultad de Ciencias del Mar y Ambientales, Universidad de Cádiz, Cádiz, Spain, 4Posgrado en Oceanografia Costera, Facultad de Ciencias Marinas, Universidad Autónoma de Baja California, Ensenada, Mexico, 5King Abdullah University of Science and Technology, Center for Desert Agriculture, Biological and Environmental Sciences and Engineering Division, Thuwal, Saudi Arabia, 6SAS Inalve, Villefranche-sur-Mer, France Increases in seawater temperature and reduction in light quality have emerged as some of the most important threats to marine coastal communities including seagrass ecosystems. Temperate seagrasses, including Zostera marina, typically have pronounced seasonal cycles which modulate seagrass growth, physiology and reproductive effort. These marked temporal patterns can affect experimental seagrass responses to climate change effects depending on the seasons of the year in which the experiments are conducted. This study aimed at evaluating how seasonal acclimatization modulates productivity and biochemical responses of Zostera marina to experimental warming and irradiance reduction. Seagrass shoots were exposed to different temperatures (6, 12, 16, 20, and 24◦C), combined with high (180 µmol photons m−2s−1) and low (60 µmol photons m−2s−1) light conditions across four seasons (spring: April, summer: July, and autumn: November 2015, and winter: January 2016). Plants exhibited similar temperature growth rates between 16 and 20◦C; at 24◦C, a drastic reduction in growth was observed; this was more accentuated in colder months and under low irradiance conditions. Higher leaf growth rates occurred in winter while the largest rhizomes were reached in experiments conducted in spring and summer. Increases in temperature induced a significant reduction in polyunsaturated fatty acids (PUFA), particularly omega-3 (n-3 PUFA). Our results highlight that temperate seagrass populations currently living under temperature limitation will be favored by future increases in sea surface temperature in terms of leaf and rhizome productivity. Together with results from this study on Z. marina from a temperate region, a wider review of the reported impacts of experimental warming indicates the likely reduction Frontiers in Marine Science | www.frontiersin.org 1September 2021 | Volume 8 | Article 731152 fmars-08-731152 September 23, 2021 Time: 17:37 # 2 Beca-Carretero et al. Seasonality Modulates Experimental Warming Responses in some compounds of nutritional importance for higher trophic levels in seagrass leaves. Our results further demonstrate that data derived from laboratory-based studies investigating environmental stress on seagrass growth and acclimation, and their subsequent interpretation, are strongly influenced by seasonality and in situ conditions that precede any experimental exposure. Keywords: seasonal acclimatization, temperature, irradiance, fatty acids, nutritional composition, Ireland, Zostera marina, reproductive effort HIGHLIGHTS – Seagrass responses to temperature and irradiance are partly modulated by seasonal acclimatization – 3–4◦C above in situ temperatures will favor temperate seagrass growth and production – Optimum growth temperature is similar across seasons and light levels – Light limitation enhances high temperature stress – Rises in temperature reduce the production and accumulation of omega-3 fatty acids. INTRODUCTION Seagrasses play a vital role in coastal zones in terms of productivity, support of biodiversity, constitution as a primary food source of nutrition for several marine organisms, protection of the coastline, carbon storage and nutrient retention (Hemminga and Duarte, 2000;Carruthers et al., 2007; Nordlund et al., 2018). However, over the last few decades, global seagrass distribution has been reduced by around 30%. Particularly Zostera marina is lost at a rate of 1.3% per year due to anthropogenic pressures and climate change effects (Orth et al., 2006;Waycott et al., 2009). Noteworthy, a recent study revealed signs of recovery of European seagrass meadows due to conservation efforts (de los Santos et al., 2019). Zostera marina L. is the dominant habitat-forming seagrass species in the northern hemisphere, currently distributed from subtropical regions in the Pacific coast of Mexico (24.3◦N) and in the Mediterranean Sea (35.1◦N), to sub-Arctic regions in Greenland (64.2◦N) and in the northern coast of Russia (74.3◦N) covering a latitudinal distribution of ∼50◦(Short et al., 2006). Reflecting its broad distribution, this species is adapted to a wide range of environmental conditions including annual average temperatures ranging from ∼1 to 25◦C (Tyberghein et al., 2012). Temperate seagrasses typically have pronounced seasonal cycles which modulate seagrass growth, physiology and flowering (e.g., Orth and Moore, 1986;Alcoverro et al., 2001). Temperate Z. marina populations usually grow faster and develop larger photosynthetic structures in summer months under more suitable climate conditions, storing high reserves of energetic compounds in the rhizomes such as carbohydrates. In less favorable environmental conditions, plants reduce their aboveground structures to reduce respiratory demands, and use the energetic reserves (e.g., Olesen and Sand-Jensen, 1993; Lee et al., 2005). Temperature is one the most important environmental factors controlling seagrass physiological, growth and reproductive processes (Lee et al., 2007;Qin et al., 2020b). Global warming has emerged as an important threat for marine coastal species including seagrass ecosystems (e.g., Duarte, 2002). Seagrass responses to predicted increases in temperature, including Z. marina, depends on population-specific thermal tolerance and their capacity to adapt to new local climate conditions (Short and Neckles, 1999). As a result, expected global warming may drive different effects within Z. marina populations across their latitudinal distribution range. For instance, some subarctic populations were reported to exhibit more favorable responses to temperature increases (Krause-Jensen et al., 2015; Olesen et al., 2015;Beca-Carretero et al., 2018a). On the contrary, warming may negatively affect southern Z. marina populations when summer temperatures can exceed their physiological optima (e.g., Reusch et al., 2005;Lopez-Calderon et al., 2016). Physiological and biochemical responses of centrally distributed populations to warming events remain more unclear (Beca-Carretero et al., 2018a;Aoki et al., 2020; Dubois et al., 2020). Irradiance is the most relevant environmental driver affecting photosynthetic activity and the vertical distribution of seagrasses (Ralph et al., 2007). The predicted increase in the frequency and intensity of storms alongside continuous coastal modifications will likely cause a reduction in underwater light and thus, cause low light stress in seagrass populations (Duarte, 2002; Silva et al., 2013;Bertelli and Unsworth, 2018). While some species can tolerate low light for short periods, longer exposure can cause chronic physiological stress and eventual seagrass loss (e.g., Short and Wyllie-Echeverria, 1996;Nguyen et al., 2021). Global warming is expected to enhance light limitation stress in seagrasses as minimum irradiance requirements, to maintain adequate growth and physiological performance, are increased at higher temperatures (Lee et al., 2007). Experimental studies of Z. marina populations distributed at their central distribution range reported that plants exposed to light-limited conditions (50 µmol photons m−2s−1) were highly vulnerable to temperatures above 20◦C, causing growth inhibition and negative carbon budgets (Beca-Carretero et al., 2018b). Recently, in situ studies provide evidence for largescale declines in Z. marina meadows due to combined effects of light reduction due to precipitation event sand sediment runoff and anomalous warming episodes of temperatures above 28◦C (Johnson et al., 2021). Warming episodes are well documented for summer seasons, however, with the substantial change in climate in northern Frontiers in Marine Science | www.frontiersin.org 2September 2021 | Volume 8 | Article 731152 fmars-08-731152 September 23, 2021 Time: 17:37 # 3 Beca-Carretero et al. Seasonality Modulates Experimental Warming Responses latitudes, they also occur during cold seasons in higher latitudes where Z. marina is present (Short et al., 2007; Bokhorst et al., 2009). Anomalous climate events throughout the year can alter seasonal rhythms, uncoupling plant internal clocks and, consequently, affecting their vegetative development, population dynamics and reproductive cycles (Alcoverro et al., 2001;Bokhorst et al., 2009;Bjerke et al., 2011). As a result, different growth and physiological seagrass responses to climate change effects can be expected depending on the seasons of the year in which these events occur. However, the majority of the controlled temperature exposure experiments using seagrasses were conducted within a single season (e.g., review in BecaCarretero et al., 2018a). Some studies reported differential sensitivities to temperature and irradiance treatments across seasons in the temperate species Z. marina (Staehr and Borum, 2011), which makes it highly relevant to assess the potential effects of warming during different seasons. While several studies have reported effects of warming or change in light climate on seagrass ecosystems (review in Koch et al., 2013; review in Roca et al., 2016; review in Nguyen et al., 2021), only few authors assessed the potential synergic effects of multiple stressors (e.g., Koch et al., 2007;Ontoria et al., 2019b). Irish coasts are characterized by the presence of Z. marina meadows in subtidal areas reaching maximum depths of 4–6 m, and in lesser extension in the lower intertidal (Madden et al., 1993;Beca-Carretero et al., 2019b). In Ireland, there is an overall lack of seagrass research including their distribution, ecology and healthy status (Dale et al., 2007; Wilkes et al., 2017). Recent studies reported that large subtidal Z. marina meadows remain undisturbed along the Irish coast (Beca-Carretero et al., 2019b;Cott et al., 2021), and it is expected that their distribution is wider than previously known (Beca-Carretero et al., 2020;Hastings et al., 2020). Ireland’s climate is defined as a temperate oceanic climate, characterized by defined climatic seasons with a lack of extreme cold or warm temperatures. Western Irish seagrasses are exposed to minimum temperatures of 4–5◦C in winter and a maximum of 16–17◦C reached in summer. With optimal temperature for growth of worldwide Z. marina populations ranging from 15.3 to 24◦C (review in Lee et al., 2007; review in Beca-Carretero et al., 2018b), it can be expected that an increase of 2–3◦C in Irish nearshore systems will favor seagrasses growth and production (Beca-Carretero et al., 2019b). Seagrasses constitute an important source of food for several marine organisms; important nutritional compounds include proteins, carbohydrates or lipids and fatty acids (review in Kim et al., 2021). Despite the ecological importance of seagrasses as a food source to higher tropic levels, only few studies have explicitly assessed the role of environmental changes on their nutritional value. Recently, studies of the seagrass species Z. marina,P. oceanica and Halophila stipulacea reported changes in essential nutritional compounds including reductions of polyunsaturated fatty acids (PUFAs) relative to saturated fatty acids (SFAs), lower leaf fiber and proteins, and an increase in leaf necrosis in response to increasing temperatures have been documented (e.g., Beca-Carretero et al., 2018b, 2020;Hernán et al., 2019;Nguyen et al., 2020a). Such biochemical changes render seagrasses exposed to stress conditions a less desirable food source for herbivores (Hernán et al., 2017) although it is currently not clear how warming can modify biochemical composition and associated nutritional value. Here, we assessed how combined temperature and irradiance exposure treatments affect centrally distributed Z. marina populations descriptors (morphological, production and biochemical composition), and how such responses are modulated by seasonal acclimatization. Specifically, we investigated, (i) the effects of a temperature increase by 3–4◦C above in situ temperatures on seagrass performance; (ii) the synergistic effects of thermal stress and light reduction on seagrass traits and survival; (iii) the extent to which any potential seasonal acclimatization affects the experimental optima for growth and productivity patterns and; finally (iv) the potential impact of experimental increases in temperature on the biochemical composition and nutritional value of seagrass leaves reported in the literature in comparison with results from this study. To address these questions, Z. marina shoots from Ireland were exposed to different temperature treatments (ranging from 6 to 24◦C) combined with high (180 µmol photons m−2s−1) versus low irradiance (60 µmol photons m−2s−1) levels across four seasons (spring: April, summer: July, and autumn: November 2015, and winter: January 2016). We hypothesize that, (i) predicted warming will favor growth of centrally distributed Z. marina populations, such as Irish meadows, likely growing under temperature limitation, (ii) plants exposed to low irradiance are more vulnerable to thermal stress than plants exposed to high light, and (iii) plants incubated in different seasons display diverse thermal responses and distinct optimum of temperature for growth. Finally, (iv) we expect that warming will reduce PUFA contents and particularly omega-3, as high temperatures dismiss the requirements of unsaturation levels in photosynthetic structures. MATERIALS AND METHODS Plant Collection Plants were collected from a monospecific Zostera marina L. meadow situated in western Ireland at Finavarra (FV) (53.149437N, −9.133993W), southern Galway Bay (Figure 1). This area is classified as a Special Area of Conservation (SAC), Natural Heritage Area (NHA), and described as an “unpolluted” area by the Irish Environmental Protective Area (EPA) (NPWS, 2014) (more details in Beca-Carretero et al., 2019a, 2020). Mature apical shoots were manually harvested by SCUBA diving or snorkeling at a shallow depth of 2–3 meters at intervals of 5–10 meters along a 50 m transect line to prevent potential resampling of the same genotypes. At this depth range, the meadow is denser and more homogenous than at shallower or deeper areas (Beca-Carretero et al., 2019a). We selected shoots with similar weights (average of 2.7 ±0.6 g DW shoot−1) and size (average of 32.3 ±8.3 cm shoot−1) and the number of mature rhizome segments (3–4). Shoots were transferred to cooling tanks filled with ambient seawater and directly transported to the laboratory at the National University of Ireland Galway campus (Supplementary Figure 1) within 1 h. The collected samples Frontiers in Marine Science | www.frontiersin.org 3September 2021 | Volume 8 | Article 731152 fmars-08-731152 September 23, 2021 Time: 17:37 # 4 Beca-Carretero et al. Seasonality Modulates Experimental Warming Responses FIGURE 1 | Map of Galway Bay (red rectangle) in western Ireland with Zostera marina meadows (green color; from Beca-Carretero et al., 2020) and the studied population at Finavarra (FV) (A). Data of daily sea surface temperature (SST) (B), and measurements of daily irradiance (mol photons m−2d−1) measured on the roof of the Martin Ryan Building, NUI Galway, Galway City (C). Red arrows represent the date of sample collection in April (spring), July (summer), November (autumn) (all 2015) and January (winter). were carefully cleaned, e.g., any remnants of sediment, epiphytic organisms or dead plant materials were removed from the shoots. Details of Experimental Set-Up We conducted laboratory-controlled experiments on Z. marina plants across four different times of year (April, July, November 2015 and January 2016), and each time, exposed samples to combined temperature (6, 12, 16, 20 and 24◦C) and irradiance (180 µmol photons m−2s−1and 60 µmol photons m−2s−1) treatments (Figure 2). The experimental temperature range represented the natural annual range of sea surface temperature (SST) in western Ireland (∼5–17◦C) (Supplementary Figure 1); the 20◦C treatment represented the predicted increases of 2.6–3.1◦C of SST for the study region by the end of 2100 (IPCC, 2014;Hobday et al., 2016;Tinker and Howes, 2020); 24◦C temperature represented an elevation by +3–4◦C above maximum in situ SST at the collection site (∼19−20◦C, Stengel et al., 1999) that may be reached during a summer heat-wave events in the Atlantic Ocean (Feudale and Shukla, 2011). The duration of the temperature treatment was selected to simulate the duration of previous warming episodes in temperate regions (Hobday et al., 2016). For each temperature treatment, two irradiance levels (180 µmol photons m−2s−1and 60 µmol photons m−2s−1) were tested. Light levels were chosen based on in situ values recorded in September 2014 (maximum annual temperatures of 16 –17◦C) during (typical) cloudy days at the location where plants were collected for the experiment. It is noteworthy that in western Ireland 63% of days have some precipitation and more than 96% days have more than 20% cloud cover1. Therefore, we considered that using light levels recorded during cloudy days were ecologically more relevant for Irish coastal ecosystems than those during sunny days. Irradiances measured using a DIVINGPAM-II2along a 50 m transect long were 174.7 ±42.4 µmol photons m−2s−1[n= 8]) at intermediate depths of Irish seagrass meadows (2–2.5 m), which is ∼70% higher than at deeper regions of the meadow (4–5 m; 57.6 ±25.4 µmol photons m−2 s−1[n= 8]) (Beca-Carretero et al., 2019b). Similar light levels were also previously used in previous Z. marina experiments (e.g., Evans et al., 1986;Höffle et al., 2011;Staehr and Borum, 2011;Beca-Carretero et al., 2018a). A cycle of 12 h: 12 h light (L): dark (D) was chosen to represent an intermediate step between winter and summer daylengths (Table 1 and Supplementary Figure 1). After collection, and prior to each experimental treatment, 120–150 plants were kept for 5 days in 20 L tanks at temperatures measured in situ at the time of collection (April: 11◦C; July: 17◦C; November: 11◦C; January: 6◦C), at a salinity of ∼35 PSU, and at an intermediate experimental irradiance of 120 µmol photons m−2s−1in a L: D cycle of 12 h: 12 h (Beca-Carretero et al., 2018a; Figure 2;Table 1). Before starting the experiment, shoots were progressively acclimated from in situ temperature to the target experimental temperature (6, 12, 16, 20, and 24◦C) by gradually increasing or reducing chamber temperatures by 1◦C every 24–48 h (Figure 2). Also, plants were progressively acclimated to the target irradiance applied (60 or 180 µmol photons m−2s−1) by increasing or reducing ∼30 µmol photons m−2s−1every 5–7 days. The experiment was started when all treatments reached their target temperatures and irradiance to ensure that all plants were pre-acclimated to lab-conditions for the same number of days (10–14 days). For each temperature and irradiance treatment, three individual plants were incubated in each of the three individual transparent cylinders (n= 3) consisting of cylindrical perspex bottles (ID 10 cm, height 35 cm) with a volume of ∼3.5 L. Shoots were loosely tied to a weighed-down plastic net at the bottom of each container to maintain them in a vertical orientation. Air was supplied through pumps ensuring constant mixing and CO2supply. 80% of the seawater in the cylinders was replaced every two to 3 days. 1https://www.meteoblue.com/ 2https://www.walz.com/ Frontiers in Marine Science | www.frontiersin.org 4September 2021 | Volume 8 | Article 731152 fmars-08-731152 September 23, 2021 Time: 17:37 # 5 Beca-Carretero et al. Seasonality Modulates Experimental Warming Responses FIGURE 2 | Schematic of the experimental design. Plants of Zostera marina incubated at high (180 µmol photons m−2s−1) or low 60 µmol photons m−2s−1) light and 5 temperatures (6, 12, 16, 20 and 24◦C). Three cylinders (n= 3) containing three seagrass shoots were incubated in each treatment. Irradiance levels (180 and 60 µmol photons m−2s−1) were measured inside the cylinder at the upper position of the seagrass leaves; the light was provided by Lumilux cool daylight fluorescent lamps (OSRAM L18W/865, Germany) TABLE 1 | Geographic coordinates at Finavarra (FV), western Ireland, where Z. marina shoots were collected, water depth relative to the mean water level, in situ sea surface water temperature (SST) (◦C) measured at times when Z. marina biomass was collected, and averaged SST and daylength (h) (https://www.timeanddate.com) across 14 days at collection time (collection date 7 ±days). Latitude 53◦805500 N Longitude −9◦705700 W Depth (m) 2–2.5 Temperature (◦C) Temperature (◦C) Daylength (h) Month Collection time Collection date ±7 days Apr-15 11 9.9±0.3 9.8±0.7 Jul-15 17 16.1±1.1 16.1±1.1 Nov-15 12 11.5±0.2 13.5±0.8 Jan-16 6 7.7±0.1 9.6±0.4 Data obtained from Beca-Carretero et al. (2019b, 2020). (Beca-Carretero et al., 2018a). We continuously recorded water temperature using HOBO loggers (UA-002-64, Onset) installed in one cylinder per temperature and irradiance treatment. After 15–16 days, we measured a set of response parameters (see following). Morphological Descriptors Once the Z. marina shoots were acclimated to the target temperatures and before starting the experiment, all individual shoots were morphologically characterized. We measured: shoot fresh weight (g FW), total length (cm shoot−1), number of leaves (shoot−1) and the length of the rhizomes (cm). We standardized leaves and rhizome length across the all experiments to avoid potential impacts of morphology in plant responses by adjusting all shoots to two-three mature rhizome segments (2–3 cm total rhizome length) and 3–4 healthy leaves. At the end of each experimental treatment, the number of new leaves and new rhizome segments were counted, and rhizome and leaf elongation rates were assessed. To measure leaf growth (elongation) rates over time, two holes were pierced into leaves, one above the other with a distance of 2 mm above the basal meristem of the plant with a hypodermic needle (Sand-Jensen, 1975;Short and Duarte, 2001). We quantified the leaf formation rate (leaves shoot−1d−1) by identifying Frontiers in Marine Science | www.frontiersin.org 5September 2021 | Volume 8 | Article 731152 fmars-08-731152 September 23, 2021 Time: 17:37 # 6 Beca-Carretero et al. Seasonality Modulates Experimental Warming Responses the new number of leaves without punched holes, divided by the number of incubation days. Leaf elongation rate (cm shoot−1d−1) was calculated as the length of new leaf material produced during the incubation time (in days), measured (i) from the base of the meristem to the punched holes, and (ii) the length of the newly produced leaves (Short and Duarte, 2001). The relative growth rate (RGR) was calculated according to: RGR = −Ln(Bf Bi)/t(1) where Bi is the initial weight and Bf, the final weight of the shoot and tis the incubation period in days. We haphazardly selected a group of 10 Z. marina shoots with similar size at the time of collection from the same seagrass meadow to assess the ratio of initial fresh weight (FW): dry weight (DW). Moreover, we evaluated the shoot mortality rate by the following equation: Mortality rate = −Ln(Nf Ni)/t(2) Nf is the final shoot population (n= 9; 3 shoots per cylinder); Ni is the initial shoot population and tis the duration of the experiment (in days). Biochemical Responses After 15–16 days of incubation, only healthy tissues (avoiding epiphytes or damaged parts) of the youngest and second youngest leaves were selected for biochemical analysis. Selected biomass was cleaned with distilled water prior to processing. Samples were frozen at −20◦C and 48 h later freeze-dried. The samples were then kept at −20◦C. 24 h before conducting the experiments samples were again freeze-dried to remove the potential humidity. Fatty Acid Analysis We determined the fatty acid content and composition of Z. marina leaf biomass by applying the protocol previously used for macroalgae and seagrasses (Schmid et al., 2014; Beca-Carretero et al., 2018b, 2019a, 2020). Fatty acid methyl esters (FAME) were obtained by direct transmethylation of ∼20–30 mg of powdered leaf biomass with dry methanol containing 2% (v/v) H2SO4. To prevent oxidation, vials were closed with nitrogen gas before being heated at 80◦C for 2 h under continuous stirring conditions. After transmethylation, we added 1 mL of Milli-Q water and later extracted the FAME using 0.5 mL of n-hexane. Analysis of FAME was conducted using an Agilent 7890A/5975C Gas Chromatograph/mass selective detector (GC/MSD) Series (Agilent Technologies, United States) equipped with a flame ionization detector and a fused silica capillary column (DB-WAXETR, 0.25 mm ×30 m ×0.25 µm, Agilent Technologies, Catalog No.: 122-7332). Identification of FAME was achieved by co-chromatography with authentic commercially available FAME standard of fish oil (Menhaden Oil, catalog no. 47116, Supelco). Total and individual fatty acid contents were quantified by comparison with a known quantity of added pentadecanoic acid 15:0 (99%, catalog no. A14664-09, Alfa Aesar, United Kingdom) as an internal standard. We added the 15:0 standard (10 µl, 5 mg mL−1) before starting the direct transmethylation and expressed the results as the mean values of 3 replicates (n= 3) for each treatment. Fatty acids (FAs) content and composition were chosen as seagrass indicator due to their proved sensitivity to environmental fluctuations including temperature and irradiance alongside its importance as nutritional compound (e.g., Falcone et al., 2004;Sanina et al., 2008; Beca-Carretero et al., 2020). Pigment Extraction Chlorophylls and total carotenoids were determined following two consecutive extractions, using 5 mL of 80% acetone each time to extract pigments from ∼20–30 mg of powdered leaf biomass (Beca-Carretero et al., 2019a, 2020). The first extraction was conducted over 20 h, and the second over 4 h. To ensure optimal extraction and to avoid pigment oxidation, both extractions were performed in darkness at 4◦C and with continuous stirring, and the vials were closed under nitrogen gas. After the first extraction, samples were centrifuged (10,000 rpm) for 60 s, the supernatant (5 mL) kept in darkness at 4◦C, and the remaining biomass was used for the second extraction. The supernatants of both extractions were combined to a final volume of 10 mL, which was then used for pigment analysis (n= 3). We quantified chlorophyll a,band carotenoids by spectrophotometric absorbance (CARY 50 Scan UV-Visible Spectrophotometer), following the equations of Lichtenthaler and Wellburn (1983). Chl a(µg mL−1) = 12.21E663 −2.81E646 Chl b(µg mL−1) = 20.13E646 −5.03E663 Carotenoids (µg mL−1) = (1000 A470 −(3.27 Chl a)−(104 Chl b))/227 Where: Exxx = Absorbance at xxx nm −Absorbance at 725 nm Literature Review A thorough literature survey evaluated previous seagrass studies on biochemical components with a potential nutritional value in response to controlled temperature exposure. Any data from field experiments or in situ observations were excluded. For the literature review selected compounds include lipids, fatty acids, proteins, carbohydrates, pigments and carbon and nitrogen composition among others. We included reports on necrosis as a parameter that may affect nutritional value. Table 2 contains details of leaf compounds which varied more than ±5% when seagrasses were grown under predicted warming conditions compared to in situ summer temperature. In addition, Supplementary Tables 13–14 (Excel file) include all results regardless of the degree of variation reported. Statistics Data of morphological descriptors, growth rates, FA content and composition and photosynthetic pigments were Ln transformed, checked for homogeneity of variance using Bartlett’s test and for normality applying Kolmogorov–Smirnov Frontiers in Marine Science | www.frontiersin.org 6September 2021 | Volume 8 | Article 731152 fmars-08-731152 September 23, 2021 Time: 17:37 # 7 Beca-Carretero et al. Seasonality Modulates Experimental Warming Responses TABLE 2 | Literature review of the effects of temperature in the nutritional composition of seagrasses leaf. Parameter Species Location Summer T. Warming T. % of Source (◦C) (◦C) variation Carbohydrates (% DW) Non-structural C. Halophila ovalis Australia 24 30 −41.69 Ontoria et al., 2020 (mg g−1DW) Non-structural C. Posidonia oceanica Italy 26 30 −30.95 Pazzaglia et al., 2020 (mg g−1DW) Non-structural C. Posidonia oceanica Italy 26 30 5.78 Pazzaglia et al., 2020 (mg g−1DW) Soluble C. Thalassia testudinum United States 28–29 34–35 77.28 Koch et al., 2007 (mg g−1DW) Soluble C. Halodule wrightii United States 28–29 34–35 71.93 Koch et al., 2007 (mg g−1DW) Soluble C. Cymodocea nodosa Spain 25 29 −43.75 Marín-Guirao et al., 2018 (mg g−1DW) Soluble C. Cymodocea nodosa Spain 23 27 22.26 Marín-Guirao et al., 2018 (mg g−1DW) Soluble C. Posidonia oceanica Spain 25 29 9.00 Marín-Guirao et al., 2018 (mg g−1DW) Starch Thalassia testudinum United States 28–29 34–35 60.15 Koch et al., 2007 (mg g−1DW) Starch Halodule wrightii United States 28–29 34–35 70.63 Koch et al., 2007 (µmol g−1FW) Starch Zostera marina United States 10 20 47.62 Zimmerman et al., 1989 (mg g−1DW) Starch Cymodocea nodosa Spain 22 26 −28.21 Egea et al., 2018 (mg g−1DW) Starch Cymodocea nodosa Spain 25 29 9.00 Marín-Guirao et al., 2018 (mg g−1DW) Starch Posidonia oceanica Spain 25 29 7.11 Marín-Guirao et al., 2018 (mg g−1DW) Starch Enhalus acoroides Indonesia 26 31 65.01 Artika et al., 2020 (mg g−1DW) Starch Thalassia hemprichii Tanzania 26 31 23.00 Viana et al., 2020 (mg g−1DW) Starch Cymodocea serrulata Tanzania 26 31 11.26 Viana et al., 2020 (% DW) Sucrose Posidonia oceanica Spain 25 29 19.64 Hernán et al., 2017 (mg g−1DW) Sucrose Enhalus acoroides Indonesia 26 31 37.04 Artika et al., 2020 (mg g−1DW) Sucrose Cymodocea nodosa Spain 22 26 36.00 Egea et al., 2018 (mg g−1DW) Sucrose Thalassia hemprichii Tanzania 26 31 19.53 Viana et al., 2020 (mg g−1DW) Sucrose Cymodocea serrulata Tanzania 26 31 5.25 Viana et al., 2020 (mg g−1DW) Sucrose Halophila stipulacea Tanzania 26 31 16.96 Viana et al., 2020 (µmol g−1FW) Sugar Zostera marina United States 10 20 −14.61 Zimmerman et al., 1989 Chemical elements (% DW) Carbon Zostera marina United States 26 28–29 8.94 Touchette et al., 2003 (% DW) Carbon Cymodocea nodosa Spain 22 26 6.25 Egea et al., 2018 (% DW) Carbon Zostera capensis South Africa 24 30 −8.30 Mvungi and Pillay, 2019 (g g−1DW) Carbon Thalassia hemprichii Tanzania 26 31 −6.04 Viana et al., 2020 (% DW) Nitrogen Cymodocea nodosa Spain 22 26 −22.73 Egea et al., 2018 (% DW) Nitrogen Zostera capensis South Africa 24 30 −11.00 Mvungi and Pillay, 2019 (% DW) Nitrogen Cymodocea nodosa Spain 30 35 24.19 Ontoria et al., 2019b (% DW) Nitrogen Posidonia oceanica Spain 25 29 9.20 Pereda-Briones et al., 2019 (g g−1DW) Nitrogen Thalassia hemprichii Tanzania 26 31 −13.29 Viana et al., 2020 (g g−1DW) Nitrogen Cymodocea serrulata Tanzania 26 31 −20.35 Viana et al., 2020 (g g−1DW) Nitrogen Halophila stipulacea Tanzania 26 31 −19.05 Viana et al., 2020 (% DW) Nitrogen Posidonia oceanica Italy 26 30 25.21 Pazzaglia et al., 2020 (% DW) Nitrogen Posidonia oceanica Italy 26 30 22.80 Pazzaglia et al., 2020 (g g−1DW) Nitrogen Halophila stipulacea Israel 27 31 36.12 Beca-Carretero (pers. data) (g g−1DW) Nitrogen Halophila stipulacea Israel 27 31 47.69 Beca-Carretero (pers. data) (% DW) Phosphorus Posidonia oceanica Spain 25 29 9.09 Hernán et al., 2017 Ratio C:N Zostera marina United States 26 28–29 10.02 Touchette et al., 2003 Ratio C:N Posidonia oceanica Spain 25 29 −8.90 Hernán et al., 2017 Ratio C:N Thalassia hemprichii Tanzania 26 31 8.37 Viana et al., 2020 Ratio C:N Cymodocea serrulata Tanzania 26 31 21.72 Viana et al., 2020 Ratio C:N Halophila stipulacea Tanzania 26 31 18.67 Viana et al., 2020 Ratio C:N Posidonia oceanica Italy 26 30 −21.64 Pazzaglia et al., 2020 Ratio C:N Posidonia oceanica Italy 26 30 −17.03 Pazzaglia et al., 2020 Ratio C:P Zostera capensis South Africa 24 30 −11.00 Mvungi and Pillay, 2019 (% DW) Folin phenols Posidonia oceanica Spain 25 29 13.51 Hernán et al., 2017 Fatty acids (% of TFA) MUFA Cymodocea nodosa Spain 23 27 −15.38 Beca-Carretero et al., 2018a (% of TFA) MUFA Cymodocea nodosa Spain 25 29 −13.04 Beca-Carretero et al., 2018a (% of TFA) MUFA Posidonia oceanica Spain 23 27 23.08 Beca-Carretero et al., 2018a (% of TFA) MUFA Posidonia oceanica Spain 25 29 −40.74 Beca-Carretero et al., 2018a (% of TFA) MUFA Zostera marina Ireland 16 24 12.50 Our Study (% DW) PUFA Cymodocea nodosa Spain 23 27 −5.33 Beca-Carretero et al., 2018a (% DW) SFA Cymodocea nodosa Spain 23 27 13.39 Beca-Carretero et al., 2018a (% DW) SFA Posidonia oceanica Spain 25 29 −5.37 Beca-Carretero et al., 2018a (% DW) SFA Posidonia oceanica Spain 25 29 5.29 Beca-Carretero et al., 2018a (% of TFA) SFA Zostera marina Ireland 16 24 8.57 Our Study (Continued) Frontiers in Marine Science | www.frontiersin.org 7September 2021 | Volume 8 | Article 731152 fmars-08-731152 September 23, 2021 Time: 17:37 # 8 Beca-Carretero et al. Seasonality Modulates Experimental Warming Responses TABLE 2 | (Continued) Parameter Species Location Summer T. Warming T. % of Source (◦C) (◦C) variation (% DW) TFA Cymodocea nodosa Spain 25 29 7.41 Beca-Carretero et al., 2018a (% DW) TFA Posidonia oceanica Spain 23 27 7.89 Beca-Carretero et al., 2018a (% DW) TFA Posidonia oceanica Spain 25 29 −5.41 Beca-Carretero et al., 2018a (% DW) TFA Zostera marina Ireland 16 24 −8.33 Our Study Ratio PUFA/SFA Zostera marina Ireland 16 24 −10.00 Our Study (% of TFA) % Omega-3 Zostera marina Ireland 16 24 −13.87 Our Study Ratio Omega 3/6 Cymodocea nodosa Spain 23 27 −15.38 Beca-Carretero et al., 2018a Ratio Omega 3/6 Cymodocea nodosa Spain 25 29 −13.04 Beca-Carretero et al., 2018a Ratio Omega 3/6 Posidonia oceanica Spain 23 27 23.08 Beca-Carretero et al., 2018a Ratio Omega 3/6 Posidonia oceanica Spain 25 29 −40.74 Beca-Carretero et al., 2018a Ratio Omega 3/6 Zostera marina Ireland 16 24 −28.57 Our Study Proteins (mg g−1FW) Protein Halophila stipulacea Cyprus 26 32 42.86 Nguyen et al., 2020a (mg g−1FW) Protein Halophila stipulacea Israel 26 32 −36.67 Nguyen et al., 2020a Pigments (µg g−1FW) Carotenoids Zostera noltei Portugal 18 22 55.93 Repolho et al., 2017 (mg g−1DW) Carotenoids Zostera marina Ireland 16 24 −25.00 Our Study (mg g−1FW) Chl. a Zostera marina United States 10 20 48.48 Zimmerman et al., 1989 (mg g−1FW) Chl. a Zostera marina China 20 25 −9.09 Niu et al., 2012 (µg cm−2) Chl. a Zostera marina China 20 32 −27.63 Gao et al., 2017 (µg g−1FW) Chl. a Zostera noltei Portugal 18 22 −35.79 Repolho et al., 2017 (µg cm−2) Chl. a Halophila decipiens Australia 26 30 9.09 Chartrand et al., 2018 (µg cm−2) Chl. a Halophila spinulosa Australia 26 30 27.91 Chartrand et al., 2018 (mg g−1DW) Chl. a Posidonia australis Australia 26 32 −19.00 Nguyen et al., 2020b (mg g−1DW) Chl. a Zostera marina Ireland 16 24 −23.53 Our Study (mg g−1FW) Chl. b Zostera marina United States 10 20 36.59 Zimmerman et al., 1989 (mg g−1FW) Chl. b Zostera marina China 20 25 −14.29 Niu et al., 2012 (µg g−1FW) Chl. b Zostera noltei Portugal 18 22 −15.31 Repolho et al., 2017 (µg cm−2) Chl. b Halophila decipiens Australia 26 30 20.00 Chartrand et al., 2018 (µg cm−2) Chl. b Halophila spinulosa Australia 26 30 36.36 Chartrand et al., 2018 (mg g−1DW) Chl. b Zostera marina Ireland 16 24 −21.43 Our Study (µg g−1FW) Phaeo. a Zostera noltei Portugal 18 22 355.62 Repolho et al., 2017 (µg g−1FW) Phaeo. b Zostera noltei Portugal 18 22 146.24 Repolho et al., 2017 (mg g−1FW) Chl. a+b Zostera marina United States 10 20 43.93 Zimmerman et al., 1989 (mg g−1FW) Chl. a+b Zostera marina China 20 25 −11.88 Niu et al., 2012 (µg g−1FW) Chl. a+b Zostera noltei Portugal 18 22 −29.80 Repolho et al., 2017 (mg g−1DW) Chl. a+b Zostera marina Greenland 20 28 13.51 Beca-Carretero et al., 2018b (µg cm−2) Chl. a+b Halophila decipiens Australia 26 30 15.09 Chartrand et al., 2018 (µg cm−2) Chl. a+b Halophila spinulosa Australia 26 30 34.38 Chartrand et al., 2018 (mg g−1DW) Chl. a+b Halophila stipulacea Israel 26 32 −45.45 Nguyen et al., 2020a (mg g−1DW) Chl. a+b Halophila stipulacea Cyprus 26 32 38.24 Nguyen et al., 2020a (mg g−1DW) Chl. a+b Zostera marina Ireland 16 24 −20.83 Our Study Ratio Chl. a/b Zostera marina China 20 32 −7.50 Gao et al., 2017 Ratio Chl. a/b Halophila decipiens Australia 26 30 −9.52 Chartrand et al., 2018 Ratio Chl. a/b Halophila spinulosa Australia 26 30 −8.59 Chartrand et al., 2018 Ratio Chl. a/b Zostera marina China 20 25 5.26 Niu et al., 2012 Ratio Chl. b/a Posidonia australis Australia 26 32 7.00 Nguyen et al., 2020b Ratio Carot.:Chl. Zostera noltei Portugal 18 22 88.46 Repolho et al., 2017 (mg g−1DW) Total content Zostera marina Ireland 16 24 −20.00 Our Study (µg cm−2) Total content Posidonia oceanica Italy 26 30 13.14 Pazzaglia et al., 2020 (µg g−1FW) Auroxanthin Zostera noltei Portugal 18 22 159.99 Repolho et al., 2017 (µg g−1FW) Antheraxanthin Zostera noltei Portugal 18 22 109.77 Repolho et al., 2017 (% DW) Violaxanthin Zostera muelleri Australia 27 30 −13.41 York et al., 2013 (µg g−1FW) Violaxanthin Zostera noltei Portugal 18 22 −16.71 Repolho et al., 2017 (mg mm−2) Zeaxanthin Zostera muelleri Australia 27 30 102.40 York et al., 2013 (µg g−1FW) Zeaxanthin Zostera noltei Portugal 18 22 24.34 Repolho et al., 2017 (µg g−1FW) β-carotene Zostera noltei Portugal 18 22 44.79 Repolho et al., 2017 (µg g−1FW) Luteín Zostera noltei Portugal 18 22 42.33 Repolho et al., 2017 Other indicators Ratio De-Epoxidation Zostera muelleri Australia 27 30 70.00 York et al., 2013 Ratio De-Epoxidation Zostera noltei Portugal 18 22 10.26 Repolho et al., 2017 (Continued) Frontiers in Marine Science | www.frontiersin.org 8September 2021 | Volume 8 | Article 731152 fmars-08-731152 September 23, 2021 Time: 17:37 # 9 Beca-Carretero et al. Seasonality Modulates Experimental Warming Responses TABLE 2 | (Continued) Parameter Species Location Summer T. Warming T. % of Source (◦C) (◦C) variation Necrosis (leaf surface %) Necrosis Cymodocea rotundata Australia 22.7 43 69.74 Collier and Waycott, 2014 (leaf surface %) Necrosis Halodule uninervis Australia 22.7 43 89.74 Collier and Waycott, 2014 (leaf surface %) Necrosis Thalassia hemprichii Australia 22.7 43 94.36 Collier and Waycott, 2014 (leaf surface %) Necrosis Posidonia oceanica Spain 20 32 59.30 Traboni et al., 2018 (leaf surface %) Necrosis Posidonia oceanica Spain 20 35 9.32 Ontoria et al., 2019a (leaf surface %) Necrosis Cymodocea nodosa Spain 30 35 16.94 Ontoria et al., 2019b (leaf surface %) Necrosis Cymodocea nodosa Spain 30 35 32.37 Ontoria et al., 2019b (cm2) Necrosis Posidonia oceanica Spain 25 29 575.71 Pereda-Briones et al., 2019 (leaf surface %) Necrosis Posidonia oceanica Italy 26 30 13.20 Pazzaglia et al., 2020 Carbohydrates; chemical elements; fatty acids; proteins; pigments; other compounds; necrosis. % of variation was calculated according to the relative percentage of change from the in situ summer temperature and the predicted warming temperature of the temperature experiments, C. = carbon. Chl. a/b = Chlorophyll a/b; Phaeo. a/b = Phaeophytin a/b. test. As data did not meet the criteria, the non-parametric test PERMANOVA analyses based on a similarity matrix created from the Euclidean distances were implemented. We performed two PERMANOVA models. The first model aimed at investigating differences in seagrass descriptors responses to month (April, July, November 2015 and January 2016), temperature (6, 12, 16, 20, and 24◦C) and irradiance levels (180 µmol photons m−2s−1vs. 60 µmol photons m−2s−1). We used “temperature,” “irradiance,” and “month” as fixed factors. A pairwise test was applied to identify the treatments that differed significantly (p<0.05). The second model was implemented to assess responses of seagrass traits to temperature and irradiance treatments each separate month. In this model “temperature” and “irradiance” were fixed factors. A pairwise test was applied to identify the treatments that differed significantly (p<0.05). All data treatments and statistical analysis were performed using the PRIMERandPERMANOVA 6 statistical package. Growth rates, FAs and pigment concentrations are reported as means and standard deviation (SD). Pearson correlation analysis was used to assess potential relationships between FA composition (e.g., PUFA, SFA) and experimental temperatures (6, 12, 16, 20, and 24◦C) including both irradiance levels (180 µmol photons m−2s−1vs. 60 µmol photons m−2s−1). RESULTS Effect of Temperature on Plant Traits Temperature treatment significantly affected growth of Z. marina plants in terms of leaf elongation, leaf formation, rhizome elongation, internode formation and RGR (PERMANOVA, p<0.01). Overall, most growth descriptors displayed bellshaped patterns, with 16–20◦C representing more favorable temperatures, and lowest rates reached at coldest (6◦C) and warmest temperatures (24◦C) at both irradiance levels (Figure 3 and Supplementary Tables 1–5). Most biochemical descriptors in Z. marina leaves including fatty acid content and composition and photosynthetic pigments were significantly affected by temperature treatments (Figure 4 and Supplementary Table 1). The average total fatty acid (TFA) content of Z. marina leaves was 1.41 ±0.32% of DW, however, no clear pattern in response to temperature increase was observed. The most abundant FAs were polyunsaturated fatty acids (PUFAs), which accounted for an average of 57.45 ±6.6% of TFA across all seasons (April, July, November and January). The most abundant PUFA was α-linolenic acid (ALA, 18:3 n-3), followed by linoleic acid (LA, 18:2 n−6) and hexadecatrienoic acid (HTA, 16:3 n−3). The second most abundant group of FAs were saturated fatty acids (SFAs) which accounted for an average value of 35.1 ±6.6% of TFA, where palmitic acid (16:0) was the most common SFA, followed by stearic acid (18:0). Finally, monounsaturated fatty acids (MUFAs) accounted for an average of 4.3 ±1.5% of TFA (Supplementary Tables 6–9). Common to all seasonal experiments, the observed reductions in PUFAs following high temperature exposure were mostly attributed to changes in n−3 PUFAs (18:3 n−3 and 16:3 n−3), and occurred at both high and low light; lowest percentages were detected at 24◦C (Figure 5). For example, in plants incubated at higher irradiances, a significant reduction (Pearson correlation, p<0.05, n= 30) of n−3 PUFAs from 6 to 24◦C was observed with a decrease of 0.9% by 1◦C increase. On the other hand, a significant increase in SFA and n−6 PUFA was observed with temperature (Figure 5). Effect of Low Light on Plant Traits Leaf and rhizome growth rates of plants in low light was significantly reduced in comparison to plants exposed to high light (PERMANOVA, p<0.01) (Figure 3 and Supplementary Table 2). For instance, plants acclimated at light-limited conditions produced new leaves and rhizome segments 21.1 and 18.7% slower than plant acclimated to high irradiances (PERMANOVA, p<0.05). At temperatures above the optimum range (16–20◦C), light limitation induced a significant, abrupt decline in both leaf and rhizome growth compared to plants grown at high light. Additionally, at high temperatures, mortality rates were higher in plants exposed to low light than in plants incubated at high light; at low light, 30% of the incubated shoots died when grown at 24◦C (Supplementary Table 12). Frontiers in Marine Science | www.frontiersin.org 9September 2021 | Volume 8 | Article 731152 fmars-08-731152 September 23, 2021 Time: 17:37 # 16 Beca-Carretero et al. Seasonality Modulates Experimental Warming Responses Lastly, our results demonstrated that warming will dismiss the capacity of temperate seagrasses to produce and accumulate the essential omega-3 PUFA. These observations can have critical biological consequences as omega-3 are essential FAs that cannot be synthetized by herbivores and are transferred throughout the trophic chain (Sargent et al., 1999;Tocher, 2003;Parrish, 2009). Besides the direct ecological impact on herbivores, a reduction in omega-3 PUFA in seagrasses in response to climate change may also reduce the nutritional quality of some marine food sources (fishes or crustaceans) for human consumption (e.g., Kang, 2011; Hixson and Arts, 2016). CONCLUSION Our results demonstrate that responses of temperate seagrasses to experimental temperature and light stress are partially modulated by the seasonal acclimatization during an annual cycle: the time of year at which experiments are conducted strongly influences the observed growth, physiological and survival responses. These finding have highly relevant implications for experimental design, and in the understanding and interpretation of the effects of experimental warming on seagrasses. Increases in sea surface temperature (SST) predicted for the end of this century are expected to favor the growth and production of temperate seagrasses growing under temperature-limited conditions; by contrast, light-limited plants will be more vulnerable to thermal stress. Additionally, warming will affect the biochemical composition and associated nutritional quality of seagrass leaves with a potential effect on herbivores and its associated trophic change. DATA AVAILABILITY STATEMENT The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s. AUTHOR CONTRIBUTIONS PB-C and DS developed the experimental design. PB-C, MJ-M, CS, and FG collected the samples and conducted the laboratory experiments at NUIG. TA-G, FG, CS, and PB-C performed the laboratory analysis. PB-C performed the statistical analysis, prepared a first draft of the manuscript. DS provided resources and funding for the project. All co-authors commented on and provided edits to finalize the original manuscript. All coauthors are in agreement with the submission of the final manuscript. All authors contributed to the article and approved the submitted version. FUNDING This project was supported by a College of Science (NUI Galway) Scholarship to PB-C. This research was additionally supported by the VOCAB project (Grant-Aid Agreement No. PBA/ME/16/01) funded by the Marine Institute under the Marine Research Programme by the Irish Government. ACKNOWLEDGMENTS We are very grateful to the Associate Editor and the reviewers for their valuable comments and constructive criticisms during the review process. 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Frontiers in Marine Science | www.frontiersin.org 20 September 2021 | Volume 8 | Article 731152