
==== Front
PeerJ
PeerJ
peerj
PeerJ
2167-8359
PeerJ Inc. San Diego, USA

17959
10.7717/peerj.17959
Biochemistry
Ecology
Ecosystem Science
Marine Biology
Functional ecological traits in young and adult thalli of canopy-forming brown macroalga Gongolaria barbata (Phaeophyta) from a transitional water system
Pica Maria Luisa 1
Vitale Ermenegilda 12
Donadio Rosa 1
Costanzo Giulia 1
Munari Marco 34
Fabbrizzi Erika 125
Fraschetti Simonetta 125
Arena Carmen c.arena@unina.it
12
1 Department of Biology, University of Naples Federico II, Naples, Italy
2 NBFC, National Biodiversity Future Center, Palermo, Italy
3 Department of Integrative Marine Ecology, Stazione Zoologica Anton Dohrn, Napoli, Italy
4 Department of Biology, Stazione Idrobiologica ‘Umberto d’Ancona’, University of Padova, Padova, Italy
5 Consorzio Nazionale Interuniversitario per le Scienze del Mare, Roma, Italy
Banaszak Anastazia
12 9 2024
2024
12 e1795926 1 2024
31 7 2024
©2024 Pica et al.
2024
Pica et al.
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited.

Background

Gongolaria barbata is a canopy-forming brown macroalga that thrives in the intertidal and subtidal habitats of the warm-temperate Mediterranean Sea, which is particularly exposed to environmental changes due to its peculiar geographical location and exposure to both global and local stressors. Testing whether this species is featured by specific functional, eco-physiological and biochemical traits allowing an efficient use of habitat resources and adaptation to environmental stress, and whether this potential might change with population growth, is essential for predicting the performance of the algae under different environmental abiotic variables (e.g., temperature, nutrient availability, light) and biotic interactions (such as grazing).

Methods

Young (juveniles) and adult thalli of G. barbata were sampled in the winter season from the Venice Lagoon, Italy, featured by high environmental changes (temperature, salinity) and analyzed for thallus dry matter content (TDMC), photosynthetic activity, photosynthetic pigment and protein content, and antioxidant capacity to assess if thallus age may be considered a significant driver in determining the ecological responses of this species to environmental changes.

Results

Our results showed that TDMC was higher in adults than juveniles. At the functional level, rapid light curves indicated an elevated photosynthetic efficiency in juveniles compared to adults highlighted by the higher quantum yield of PSII electron transport, electron transport rate, and Rubisco content observed in juveniles. On the contrary, adults exhibited a higher non-photochemical quenching and total pigment concentration. No difference in maximum PSII photochemical efficiency and D1 protein content between the two thalli groups was found. Along with better photosynthesis, juveniles also displayed a higher amount of total polyphenols, flavonoids, and tannins, and a stronger antioxidant capacity compared to adults.

Conclusions

Our findings revealed significant differences in the eco-physiological characteristics of G. barbata at different growth stages. It was observed that young thalli, allocate more energy to photosynthesis and chemical defenses by increasing the production of antioxidant compounds, such as polyphenols, flavonoids, and tannins. With growth, thalli likely adopt a more conservative strategy, reducing photosynthesis and promoting structural biomass accumulation to mitigate the potential risks associated with prolonged exposure to environmental stressors, such as the wavy way. Although our study focused on a single phase of G. barbata life cycle under winter settings, it offers preliminary insights into this species eco-physiological traits and auto-ecology. Future research could explore the potential implications of these findings, evaluating the species’ resilience to environmental changes at the population level.

Antioxidants
Adaptation
Brown seaweed
Environmental stress
Photosynthesis
Thallus age
National Biodiversity Future Center—NBFCMUR: CN00000033 CUP UNINA: E63C22000990007 This research received funding by National Biodiversity Future Center—NBFC, project code MUR: CN00000033—CUP UNINA: E63C22000990007. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
==== Body
pmcIntroduction

Canopy-forming brown macroalgae (i.e., kelps, fucoids), are well-known for their crucial role as habitat formers in both intertidal and subtidal habitats of cold-temperate latitudes. They increase the three-dimensional complexity and spatial heterogeneity of the substrate they colonize (Verdura et al., 2018). Their vertical and branched canopies increase coastal primary production, offer shelter to smaller epiphytic algae and many meiofaunal invertebrates, represent nursery areas for juvenile fish, and protect them from predators and hydrodynamics (Krumhansl et al., 2016; Verdura et al., 2018; Orlando-Bonaca, Pitacco & Lipej, 2021; Gran et al., 2022; Manca et al., 2022). The conservation status of these long-living species is indicative of habitat loss, environmental degradation (Orlando-Bonaca, Pitacco & Lipej, 2021) and quality of Mediterranean coastal waters (Ballesteros et al., 2007; Orlando-Bonaca et al., 2013). Finally, they also contribute to many ecosystem services by providing foraging and preserving species of commercial interest, sustaining coastal fisheries, absorbing pollutants and filtering water, reoxygenating sediments and acting as an important sink for carbon through its sequestration to the seafloor; hence, they are known to be one of the most productive ecosystems on Earth (Gran et al., 2022; Manca et al., 2022). Regardless of all benefits, these communities are exposed to multiple stressors and threatened by human activities, including eutrophication, pollution, outbreaks of grazers caused by overfishing, invasive species introduction, increasing sediment resuspension and climate change-driven consequences (Ballesteros et al., 2007; Orlando-Bonaca et al., 2013; Orlando-Bonaca, Pitacco & Lipej, 2021). Species belonging to the genus Cystoseira sensu lato (s.l.) (Molinari-Novoa & Guiry, 2020) are endemic of the Mediterranean, classified as threatened (except for C. compressa) under the Barcelona Convention (Annex II of the Barcelona Convention, COM/2009/0585/FIN), and protected by local regulations. Despite the reduction of impacts imposed by legislation, Cystoseira s.l. forests experienced regression events at the basin scale that led to habitat loss (Cebrian et al., 2021; Verdura et al., 2023), and, in some cases, to regime shift to algal turfs, which are less complex and poorly productive communities inhibiting recolonization by canopy-forming species (Benedetti-Cecchi et al., 2015).

In recent years, several studies have been conducted to assess the ecological strategies of marine plants and macroalgae in terms of resource-use strategies by evaluating physiological and structural attributes (Starko & Martone, 2016; Ishizawa et al., 2021; Sakanishi, Kasai & Tanaka, 2023). Understanding whether a species exhibits variations in functional traits, such as eco-physiological and biochemical characteristics across different growth stages is pivotal for evaluating its potential ecological adaptation or vulnerability to future changes in dynamic environmental conditions.

Macroalgae have developed specific eco-physiological, biochemical, morphological mechanisms for mitigating fluctuations in environmental factors, demonstrating spatial and temporal adaptations and species-specific responses to single or combined stressors (Hurd et al., 2014; Starko & Martone, 2016; Cappelatti, Mauffrey & Griffin, 2019; Ishizawa et al., 2021; Hanley, Firth & Foggo, 2024). The thallus age, as plant ontogeny in terrestrial ecosystems (Rusman et al., 2020), could represent a valuable feature in assessing growth-defense mechanisms against environmental stressors (Pellizzari, Oliveira & Yokoya, 2008), including unpredictable events, such as temperature rise, tidal variations, nutrient and salinity fluctuations, water acidification and grazing pressure. However, to date, the adaptation patterns of canopy-forming macroalgae across different ages are rarely investigated, limiting our potential to predict different vulnerability across life stages with consequences at population levels.

This study focused on juvenile and adult individuals of Gongolaria barbata (Stackhouse) Kuntze, a Mediterranean widespread canopy-forming macroalga. Gongolaria barbata is a well-known adapted species to both euryhaline and polyhaline environments (Sadogurska et al., 2021; Tursi et al., 2023), and even broad changes in salinity do not affect its growth (Baghdadli et al., 1990; Irving et al., 2009). Studies on Cystoseira s.l demonstrated good tolerance of this species to acidification, which promoted growth rate, photosynthesis, antioxidant activity, and photoprotection (Celis-Plá et al., 2017). Gongolaria barbata also showed adaptability to a wide range of temperatures (Orfanidis, 1991; Iveša et al., 2022; Fabbrizzi et al., 2023). However, while adults have been extensively studied, there is missing information about the ecophysiology of early life stages for this species. Adults in the vegetative phase can endure high temperatures up to 30–34 °C during summer and freezing temperatures, during winter (Iveša et al., 2022). Recruits develop optimally at 15 °C and sufficiently from 10 °C to 25 °C (Orfanidis, 1991) but are particularly sensitive to temperature-induced stress, experiencing high mortality rates (Lokovšek et al., 2024).

Grazing also may threaten the large-scale population and restoration interventions of G. barbata (Savonitto et al., 2021). Herbivores and thallus age deeply influence grazers’ feeding preferences, which depend on the different palatability resulting from chemical and morphological changes occurring during thalli development (Van Alstyne, Ehlig & Whitman, 1999).

The concentration of some compounds, such as polyphenols, varies not only in response to seasonal shifts, nutrient levels, acidification, temperature fluctuations, irradiance intensity or desiccation (Celis-Plá et al., 2014; Celis-Plá et al., 2016; Celis-Plá et al., 2017) but also with thallus growth, morphology and age. Indeed, adult thalli concentrate in longer, more complex, and degradable forms than younger individuals. At the same time, a higher amount of polyphenols and phlorotannins is generally produced for the protection of the zygotes or in the cell-wall hardening to cope with grazers (Mannino & Micheli, 2020; Monserrat et al., 2023). As age increases, tissues tend to thicken, providing more resistance to grazing and physical stresses (Mauffrey, Cappelatti & Griffin, 2020).

Our study explores essential eco-physiological, biochemical and functional traits, such as photosynthetic activity, antioxidant defenses, and thallus dry matter content, to investigate potential age-related variations in juvenile vs adult individuals of Gongolaria barbata from the transitional water system of the Venice Lagoon (Italy). By examining how these attributes may change with age and influence local adaptation mechanisms, this study may shed light on the trade-off between acquisition and conservation strategies employed by young and adult individuals of G. barbata populations. Our findings may offer valuable insights into the physiological cost associated with thalli growth and development, defense mechanisms, and overall primary production (Cappelatti, Mauffrey & Griffin, 2019; Sakanishi, Kasai & Tanaka, 2023).

Materials & Methods

Sampling and experimental design

Juveniles and adult individuals (n = 5 per group) of the species G. barbata were randomly sampled in winter, at the beginning of February 2023, before the reproductive phase (Bevilacqua et al., 2019; Savonitto et al., 2021). Sampling was performed in Italy (Fig. 1A), at the offshore location of Ca’ Roman (45°14′42.2″N 2°17′44.7″E) (Fig. 1B), recognized as natural reserve of regional interest under the Regional Law n. 40/1984 and situated within the Venice Lagoon (Natura 2000 site, IT 3250023) (Fig. 1). The permission for sampling was provided by Regione del Veneto (decree number 369, date 04.05.2023).

10.7717/peerj.17959/fig-1 Figure 1 Images of the sampling site identified by coordinates retrieved from Google Earth Pro Software.

(A) Sampling location identified in European geographical context; (B) sampling site in the natural reserve of Ca’ Roman within the Venice lagoon. The location is marked by coordinates and displayed from a satellite perspective. Images are retrieved from Google Earth Pro Software (Map data ©2023 Google: Image Landsat Copernicus; Map data ©2023 Google: Image ©2023 Terrametrics).

Juveniles and adult thalli were collected at a depth of two meters, in a sampling area of 10 m2, at 0.20–1.0 m from each other and at a sea-water temperature of 8 °C. Individuals were selected in situ on thallus length basis through direct observations and measurements. Thallus age was estimated according to the well-known relationship between age and thallus length reported by Khailov & Firsov (1976) for G. barbata from the Black Sea, and by Bianchelli et al. (2023) for G. barbata from Adriatic Sea. Therefore, we identified two groups of algae, each of five individuals: the adults, over one year old, with a thallus length in the range 40–50 cm, and the juveniles, less than one year, with a thallus length within 10–20 cm. Thalli were transported to the laboratory in tanks filled with marine water and maintained at the temperature of almost 8 °C ± 1, which corresponds to water temperature at the sampling time. Photosystem II (PSII) chlorophyll-a fluorescence measurements in vivo were performed at room temperature of 18 ± 1 °C, taking care to maintain each thallus submerged in sampling water, maintained at almost 8 °C ± 1, to avoid thermal shock to the algae. Before biochemical determinations, thalli were carefully cleaned from any epiphytes and debris with demineralized water. Juveniles and adult thalli were compared for thallus dry matter content (TDMC), photosynthetic pigments, PSII D1 and Rubisco proteins, and antioxidants.

To characterize the sampling site, we retrieved the environmental variables data from the Copernicus Marine Environment Monitoring Service (CMEMS) database (https://marine.copernicus.eu/), using the products Global Ocean Physics Analysis and Forecast and Global Ocean Biogeochemistry Analysis and Forecast (DOI: 10.48670/moi-00016, Accessed on 19-06-2023). We covered the period from October 2020 to February 2023 and selected the geographical coordinates of the sampling site. Data of sea water temperature (T), and water salinity (S) derived from the product Global Ocean Physics Analysis and Forecast (https://data.marine.copernicus.eu/product/GLOBAL_ANALYSISFORECAST_PHY_001_024/description).

This dataset provides gridded data with a spatial resolution of 0.083° × 0.083° (∼9 km × 9 km) and hourly temporal resolution aggregated with a weakly scale for the plot.

The sea water pH (pH), and molar concentration of nitrate (NO3−) and phosphate (PO43−) were derived from the product Global Ocean Biogeochemistry Analysis and Forecast (https://data.marine.copernicus.eu/product/GLOBAL_ANALYSISFORECAST_BGC_001_028/description), and were acquired with a spatial resolution of 0.25° × 0.25° (∼30 km × 30 km) and hourly temporal resolution aggregated with a weakly scale for the plot. Both datasets derived from the numerical resolution of a global ocean model and are available in the Hindcast format data (an assessment of the past state of the ocean variables made using numerical models with or without data assimilation from satellite and in situ observation). For data processing, in both cases, the grid points closer to our target site have been selected.

Photosynthetic efficiency of thalli

Photosystem II (PSII) chlorophyll-a fluorescence analysis in vivo was performed by means of pulse amplitude modulated fluorometer (Junior-PAM, Walz Gmbh, Effeltrich, Germany) on the apical part of juvenile and adult thalli of G. barbata to assess the photosynthetic performance. Photosynthetic activity was measured in response to increasing irradiance levels, to assess the light-use efficiency of thalli in photochemistry. Rapid light curves (RLCs) were performed on four individuals for each group (n = 4) considering eight actinic light steps at a Photosynthetic Photon Flux Densities (PPFD) of 125, 190, 285, 420, 625, 820, 1,150, 1,500 µmol photons m−2 s−1 and lasting 60 s each to allow the steady-state fluorescence in actinic light (Nielsen & Nielsen, 2008; Porzio et al., 2020). Thalli were disposed at 0.5 mm from an optic fibre of 1 mm diameter inclined at 45° respect to samples immerged in seawater suspension. To measure the PSII maximum photochemical efficiency, Fv/Fm, thalli were 15 min dark-adapted to allow full oxidation of the PSII reaction centres (Porzio et al., 2017). Basal fluorescence (F0) was achieved by applying a weak blue light signal (Alestra & Schiel, 2015; Fabbrizzi et al., 2023) of 1–2 µmol photons m−2 s−1, the maximum fluorescence level in the dark (Fm) was obtained by applying a saturating light pulse of 7,000 µmol photons m−2 s−1. The maximal photochemical efficiency of PSII, Fv/Fm, was calculated according to the formula: Fv/Fm = (Fm-F0)/Fm (Beer, Björk & Beardall, 2014). The quantum yield of the PSII electron transport (ΦPSII) was calculated according to Genty, Briantais & Baker (1989) following the equation: ΦPSII = (Fm’–Ft)/Fm’. The Electron Transport Rate (ETR) was evaluated as: ETR =ΦPSII*PAR*0.5*A FII (Schreiber, 2004), where the value of 0.5 represents the energy equally distributed between the two photosystems, while A FII is the fraction of light absorbed by Photosystem II, corresponding to the value 0.8 in Phaeophyta (Celis-Plá et al., 2016). Non-photochemical quenching (NPQ) was determined as: NPQ = (Fm −Fm’)/ Fm’ (Bilger & Björkman, 1990).

Thallus dry matter content

The thallus dry matter content (TDMC) consists of the proportion of structural compounds and water-filled tissues, which are mainly photosynthetically active. It represents an important functional trait often utilized to assess the adaptability of algae to its environment (Cappelatti, Mauffrey & Griffin, 2019). For the TDMC determination, single individuals (n = 5) were weighed soon after sampling to determine the fresh mass, successively samples were dried in an oven at 37 °C for 24 h and weighted up to constant dry mass. Finally, the ratio between dry and fresh mass was determined.

Photosystem II D1 protein and Rubisco determination

Algal samples were fine grounded with liquid nitrogen by a mortar and pestle. Samples were kept on ice in Eppendorf and mechanically homogenized using a pestle and 200 µl of 1x PEB (protein extraction buffer, product no AS08300, Agrisera, Vännäs, Sweeden). Then, samples were centrifuged at 14.000 rpm for 20 min at 4 °C and the supernatants transferred into new tubes. Protein extracts were quantified with the Bradford assay (Bradford, 1976), using the BioRad Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, Hercules, CA, USA) and the bovine serum albumin (BSA) has been used as a protein standard. The SDS-PAGE (10%) was carried out following Vitale et al. (2022) with slight modifications. Briefly, the western blot procedure started with the blocking solution (100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% Tween20, 5% Milk). To reveal the protein of interest, samples were incubated with the primary antibody (Agrisera, Vännäs, Sweeden) anti-PsbA (rabbit, 1:15,000 v/v, AS05 084) for D1 protein of PSII and anti-RbcL (rabbit, 1:10,000 v/v, AS03037) for Rubisco. Goat anti-Rabbit IgG (H&L), HRP conjugated (1:6,000 v/v, AS09 602) was used as the secondary antibody. Immuno-revelation was performed using the kit for chemiluminescence (Westar supernova, Cyanagen Srl, Bologna, Italy) via ChemiDoc System (Bio-Rad, Hercules, CA, USA). The software Image Lab version 5.2.1 (Bio-Rad Laboratories, Hercules, CA, USA) was utilized for the densitometric analysis: band signals were quantified, and the background values were subtracted to obtain and adjusted volume in counts for each band. The density value was expressed in arbitrary units and represented as a boxplot.

Photosynthetic pigment content analysis

Photosynthetic pigments content, namely total chlorophylls (a + c) and total carotenoids, was determined on five individuals per group, considering one individual as one replicate, and performing three pseudo-replicates per replicate (n = 15). The analysis was performed according to Jeffrey & Humphrey (1975) and Lichtenthaler (1987) following the procedure reported in Porzio et al. (2017). Samples from each thallus (0.040 g of dried powder) were mechanically extracted in 100% acetone inside glass test tubes and left to rest for half an hour in ice and total darkness, to avoid photo-oxidation phenomena. The extracts were centrifuged at 5,000 rpm for 5 min in a Labofuge GL (Heraeus Sepatech, Hanau, Germany). The sample absorbance was measured by a spectrophotometer (UV–VIS Cary 100; Agilent Technologies, Santa Clara, CA, USA) at wavelengths of 662 nm, 630 nm, and 470 nm for chlorophyll a, chlorophyll c and total carotenoids, respectively. Pigment concentration was expressed as µg g−1 of dried weight (µg g−1 DW).

Soluble antioxidants and antioxidant capacity determination

The polyphenol content was evaluated on five individuals per group, considering one individual as one replicate, and performing three pseudo-replicates each replicate (n = 15), through the Folin-Ciocalteu method following the procedure reported in Fabbrizzi et al. (2023). Methanolic extracts were made pestering 0.200 g of dried powder in 2 ml of cold methanol and were stored at 4 °C for 24 h to ultimate the extraction. Then, samples were centrifuged at 4 °C, 11,000 rpm for 10 min in a SL 16R centrifuge (Thermo Fisher Scientific™, Waltham, MA, USA). Then, the supernatant was mixed with 10% Folin–Ciocâlteu solution, 1:1 v/v, and after 3 min, 700 mM Na2CO3 solution was added to the resulting mixture (1:5, v/v). Samples were incubated for 45 min in total darkness, and the absorbance was measured at 765 nm by a spectrophotometer (UV–VIS Cary 100; Agilent Technologies, Santa Clara, CA, USA). The total polyphenol content was expressed as mg of Gallic Acid Equivalents g−1 DW (mg GAE g−1 DW) using a gallic acid standard curve. The total flavonoid content was assessed according to the procedure of Moulehi et al. (2012) and Sun, Ricardo-da Silva & Spranger (1998). Methanolic extracts were mixed with a solution of 5% NaNO2 (ratio 3:1 v/v); after 6 min, a 10% solution of AlCl3 and a 1M solution of NaOH were added, adjusting the volume with distilled water. Samples were left resting in darkness for 15 min to let the colorimetric reaction happen and finally the absorbance was measured at a wavelength of 510 nm. The total flavonoid content was estimated through a standard catechin curve and expressed as mg of catechin equivalent per gram of dried weight (mg CAT g−1 DW). Total condensed tannins were estimated by modifying the procedures described by Sun, Ricardo-da Silva & Spranger (1998) and Moulehi et al. (2012), as reported by Costanzo et al. (2022). Briefly, 2.5 mL of methanol-vanillin solution and 2.5 mL of 97% H2SO4 were mixed with one mL of sample methanolic extract. Then, the mixture was incubated for 15 min in total darkness, and the absorbance was measured at 500 nm. Tannins were quantified with a catechin standard curve and expressed as mg catechin equivalents per gram of dry weight (mg CAT g−1 DW). The antioxidant capacity was measured through the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay, where 0.067 mL of methanolic extracts were added to two mL of 6 × 10−5 M DPPH in methanol solution and heated at 37 °C for 20 min in a dry bath (Benchmark Scientific, My block™ Mini Dry Bath). Then, absorbance was measured at a wavelength of 515 nm. Antioxidant capacity was assessed using Trolox as positive control and expressed as percentage of radical inhibition using the formula: % inhibition = ((white Abs − sample Abs)/ white Abs) *100.

Statistical analysis

To assess the statistically significant differences between the groups (adults and juveniles), the PSII maximal photochemical efficiency (Fv/Fm), electron transport rate (ETR), non-photochemical quenching (NPQ), photosynthetic pigments content, photosynthetic protein amounts and antioxidant concentrations were compared performing t-test using the Sigma-Stat 12.0 software (Jandel Scientific, USA). Differences were considered statistically significant for P ≤ 0.05. The Shapiro–Wilk test was applied to check the normality of the data. Whenever the Shapiro–Wilk test failed, the Mann–Whitney Rank Sum Test was applied. The results reported correspond to the average ± standard error. Asterisks were used to indicate statistically significant differences (*** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05, ns P ≥ 0.05). Boxplots report interquartile range, mean line, whiskers, and outliers. For the RLCs, t-tests were performed on the whole data set of RLC-curves and at each PPFD value. All data, including environmental variables, were plotted and visualized by means of R environment software (version 4.2.2., R Core Team, 2022) using the ggplot2 package version 3.5.0.

Results

Environmental variables at the sampling site

The environment of the sampled specimens of G. barbata is classified as a transitional water system (TWS). It is part of the Venice Lagoon and is closely related to the Adriatic Sea. For these reasons the biogeochemical variables of TWS are strongly influenced by those of the seawater environment, and by human activities (Solidoro et al., 2010).

The analysis of biogeochemical variables at the sampling site indicated that seasonal variations of temperature (Fig. 2A) showed peaks of 25–26 °C during summer and of 8–9 °C during winter. The pH fluctuations, reported on the total scale, were in line with the general trend of the Adriatic Sea and ranged from 8.045 to 8.175 (Fig. 2B).

10.7717/peerj.17959/fig-2 Figure 2 Seasonal variations (date YYYY/MM/DD) of environmental conditions measured at sampling site.

Analysis of seasonal variations (date YYYY/MM/DD) of environmental conditions measured at sampling site. Straight black line marks the approximate start of juveniles growth. (A) Sea surface temperature (°C); (B) salinity; (C) pH reported on total scale; (D) nitrates (NO3) and phosphates (PO4) concentration (mmol m−3). All data are retrieved from Copernicus Marine Environment Monitoring Service (CMEMS) database using the products Global Ocean Physics Analysis and Forecast and Global Ocean Biogeochemistry Analysis and Forecast (DOI: 10.48670/moi-00016, Accessed on 19-06-2023) selecting the geographical coordinates of the sampling site (45°14′42.2″N 12°17′44.7″E).

The salinity showed seasonal variations slightly different between 2021 and 2022 spring-summer periods likely due to higher loads of freshwater from estuaries and abundant rainfalls in 2022 more than in 2021 (ARPAV, 2024) (Fig. 2C).

Events such as high nutrient loads, mainly nitrogen and phosphorous, and consequent eutrophication often occurred in the Venice Lagoon since the 1920s, along with the direct release of heavy metals and organic micropollutants (Morand & Briand, 1996; Caliceti et al., 2002; Pastres et al., 2004). The wastewater treatment plant processes and the total ban of phosphorous in detergents since 1989, contributed to reduce the nutrient loads (Acri, Braga & Aubry, 2020; Zirino et al., 2016). In the years 2020–2023, nitrogen loads were below the threshold of 18 µmol L−1 for water bodies with salinity >30 (Fig. 2D) set by the national legislation for the implementation of the Gazetta Officiale (2011), with peaks exceeding the limit only during winter. In contrast, phosphorous levels always exceeded the threshold of 0.48 µmol L−1, except during summer, when usually phytoplankton blooms occurred, and primary productivity was the highest (Fig. 2D). Even if the trophic status of the Venice Lagoon improved significantly (Çevirgen et al., 2020), the comparison between years 2017–2019 (Regione Veneto et al., 2021) and 2020–2023 (Copernicus Marine Environment Monitoring Service; https://marine.copernicus.eu/) evidenced a significant increase of nitrogen and phosphorous concentrations. To date a discrete environmental state was found utilizing the Trophic Index (TRIX) assessment (Vollenweider et al., 1998), for most of the lagoonal waters (Çevirgen et al., 2020; ARPAV TRIX, 2022).

Thallus dry matter and photosynthetic pigments content

TDMC showed a significant difference between the groups with 1.3-fold higher values (P = 0.019) for adult than juvenile thalli. Pigments differed significantly between adults and juveniles, showing values 1.8-fold higher in adults for total chlorophylls (P = 0.001) and 1.7-fold higher for carotenoids (P = 0.016) compared to juveniles (Table 1).

10.7717/peerj.17959/table-1 Table 1 Thallus dry matter content (TDMC) and photosynthetic pigments content in adults and juveniles.

Data are reported as mean ± SE (TDMC n = 5, pigments n = 15).

	Adults	Juveniles	
TDMC (g g−1 DW)	0.131 ± 0.005*	0.103 ± 0.008	
Total chlorophylls (µg g−1 DW)	609.34 ± 41.25**	340.77 ± 36.68	
Total carotenoids (µg g−1 DW)	166.17 ± 17.51*	99.72 ± 13.09	
Notes.

Asterisks indicate the statistically significant differences (∗∗P ≤ 0.01, ∗P ≤ 0.05) according to t-test.

Rapid light curves

The analysis of the quantum yield of PSII electron transport (Fig. 3A) evidenced higher values (P < 0.05) in juveniles. Specifically, up to 125 µmol photons m−2 s−1, the two groups did not show any statistical differences. Conversely, in the range of PPFD from 190 to 1,500 µmol photons m−2 s−1 juveniles showed higher ΦPSII values (P ≤ 0.01) compared to adults. The PSII electron transport rate statistically differs (P < 0.05) between adults and juveniles. In detail, starting from 285 µmol photons m−2 s−1, young thalli, compared to adults, showed a higher electron transport activity (P ≤ 0.01) (Fig. 3B). Conversely, the non-photochemical quenching (NPQ) exhibited statistically significant differences between young and adult individuals only in the range from 125 to 285 µmol photons m−2 s−1, reaching the highest (P ≤ 0.01) value for adult thalli (Fig. 3C). The maximum photochemical efficiency (Fv/Fm) was not statistically different (P ≥ 0.05) between the two groups with a mean value of 0.619 ± 0.041 in adults and 0.612 ± 0.013 in juveniles (Fig. 3D).

10.7717/peerj.17959/fig-3 Figure 3 Rapid light curves for the photochemical parameters.

RLCs for the photochemical parameters: (A) Quantum yield of PSII electron transport, Φ PSII; (B) Electron transport rate of PSII (ETR); (C) Non-Photochemical Quenching (NPQ); (D) maximum PSII photochemical efficiency, Fv/Fm. Data are reported as means ± SE (n = 4). Statistically significant differences were checked according to t-test.

Photosystem II D1 protein and Rubisco

The densitometric analysis of the D1 protein (Figs. 4A, 4C) showed no statistically significant difference between adults and juveniles. Conversely, the Rubisco protein amount was 1.8-fold higher in juveniles (P = 0.001) than in adults, exhibiting an increase of 44% (Figs. 3A, 3B).

10.7717/peerj.17959/fig-4 Figure 4 Western blot and densitometric analysis of Rubisco and D1 proteins.

(A) Western blot of Rubisco and D1 proteins with relative molecular weights; (B) densitometric analysis in arbitrary units of Rubisco protein; (C) densitometric analysis in arbitrary units of D1 protein. Asterisks indicate the statistically significant differences (∗∗∗P ≤ 0.001, ∗∗P ≤ 0.01, ∗P ≤ 0.05, ns P ≥ 0.05) according to t-test. The images of western blot were downloaded by ChemiDoc System (Bio-Rad).

Soluble antioxidants content

Total polyphenols content showed significant difference (P = 0.0003) in response to thallus age, with values 1.4-fold higher in juveniles (4.948 ± 0.206 mg GAE g−1 DW) than in adults (3.570 ± 0.102 mg GAE g−1 DW) (Fig. 5A). The same behavior was observed for total flavonoids, whose concentration was 2.0-times higher (P < 0.00001) in juveniles than in adults, with values of 13.373 ± 0.662 and 6.554 ± 0.279 mg CAT g−1 DW, respectively (Fig. 5B). Also, tannins were 1.36-fold higher (P = 0.012) in juveniles (74.880 ± 4.387 mg CAT g−1 DW) than in adults (55.075 ± 4.345 mg CAT g−1 DW) (Fig. 5C). Finally, the radical scavenging activity was 1.2-times higher (P < 0.00001) in juveniles (% inhibition = 73.463 ±  0.331) than in adults (% inhibition = 61.588 ± 1.164) (Fig. 5D).

10.7717/peerj.17959/fig-5 Figure 5 Antioxidant content in adult and juvenile G. barbata thalli.

(A) Total polyphenols; (B) flavonoids; (C) tannins; (D) antioxidant capacity, measures as radical scavenging activity. Data are reported as means ± SE (n = 15). Asterisks indicate the statistically significant differences (∗∗∗P ≤ 0.001, ∗∗P ≤ 0.01, ∗P ≤ 0.05) according to t-test.

Discussion

Our study explored if G. barbata is featured by specific functional, eco-physiological and biochemical traits, allowing an efficient use of habitat resources, and if this potential might change with individual growth. The results evidenced that juveniles and adults differed in their photophysiological traits, modulation of antioxidant production and biomass partitioning into photosynthetic and non-photosynthetic tissues.

Our study area, experiences seasonal fluctuations in salinity, temperature, and nutrients. These fluctuations are within the tolerance range documented for the G. barbata species (Orfanidis, 1991; Irving et al., 2009; Iveša et al., 2022), which is found in euhaline and polyhaline environments (Sadogurska et al., 2021; Tursi et al., 2023). The species seems potentially also tolerant to pH decrease (Celis-Plá et al., 2017).

Gongolaria barbata is also well adapted to unusually high as cold temperatures occurring throughout the northern Adriatic Sea (Iveša & Devescovi, 2014; Iveša, Djakovac & Devescovi, 2016; Iveša et al., 2022). Not only adults in the vegetative phase can overcame temperature up to 30–34 °C (Iveša et al., 2022), but also recruits can trigger physiological acclimation mechanisms to cope with marine heat waves (Fabbrizzi et al., 2023). During winter, eight and even two-month-old recruits survived at temperatures close to or below ‘zero’ without physical damage (Orfanidis, 1991; Iveša et al., 2022). The temperatures recorded during the period 2020–2023 fall within the temperature tolerance of the species (Orfanidis, 1991), well adapted to these specific environmental conditions and likely capable of withstanding extreme winter and summer events. Several studies (Iveša & Devescovi, 2014; Iveša, Djakovac & Devescovi, 2016; Iveša et al., 2022) also evidenced that populations of G. barbata inhabit lagoons, systems with not-limiting nutrient patterns, located in the northern Adriatic Sea. In sites such as the Venice Lagoon, the availability of nutrients and irradiance are driving factors in regulating the photosynthetic activity and growth of macroalgae. During thalli development, resource requirements and management may change depending on seasonal environmental fluctuations and individual age (Harrison & Hurd, 2001; Taylor, Fletcher & Raven, 2001; Delgado, Ballesteros & Vidal, 1994; Stengel & Dring, 1998; Stengel, McGrath & Morrison, 2005). The absorption of micronutrients, such as trace metals (copper, iron and manganese), is indispensable for several metabolic functions, like enzyme activation, photosynthetic electron transport and nitrogen metabolism. Their availability is regulated by water temperature, pH and salinity and accumulation rate in brown algae may differ depending on thallus age and species (Stengel, McGrath & Morrison, 2005). Macronutrients as nitrogen (N) and phosphorous (P) play a pivotal role in the regulation of photosynthesis and growth rate of seaweeds. In the investigation area, N and P in available forms of nitrates and phosphates, exhibited the lowest concentration during summer and the highest levels during winter. In particular, in winter both adult and juvenile thalli may accumulate nutrients as a reservoir for next periods of nutrient limitation (Harrison & Hurd, 2001; Delgado, Ballesteros & Vidal, 1994; Celis-Plá et al., 2016). N and P-storage capacity and utilization varied between juvenile and adult stages of G. barbata (Delgado, Ballesteros & Vidal, 1994; Harrison & Hurd, 2001; Ohtake et al., 2021). Generally, young tissues uptake more nutrients than older (Delgado, Ballesteros & Vidal, 1994; Harrison & Hurd, 2001; Ohtake et al., 2021) indicating a higher investment of N and P in the photochemical and assimilation reactions thus supporting the highest photosynthetic activity found in juveniles compared to adults.

However, regardless of the age, our results indicate no stress occurrence for photosynthetic apparatus and a fully functional PSII photochemistry in both juveniles and adult thalli. Indeed, we observed in both groups an Fv/Fm ratio close to 0.7 (Baker, 2008), consistent to values found in other Cystoseira spp. (Celis-Plá et al., 2016; Mancuso et al., 2019). Kaleb et al. (2023) demonstrated that juvenile thalli of G. barbata from sites close to Marano and Grado lagoon (Adriatic Sea), benefit from the nutrient enrichment of the habitat even at temperatures of 10 and 14 °C, suggesting that also in our case, G. barbata thalli irrespective of the age, manage well local available nutrients.

Consistent with no difference in Fv/Fm ratio, PSII D1 protein amount did not vary between young and adult thalli, highlighting that the photosynthetic efficiency reduction found in adults was due to a down-regulation of photochemistry rather than to an impairment of photosystems. It is well known that the D1 protein is the primary target of light-induced oxidative damages (Mulo, Sakurai & Aro, 2012), and that its decrease is often correlated with photoinhibition and oxidative stress of PSII, resulting in a reduction of Fv/Fm ratio (Schofield, Evens & Millie, 1998). Our results support the evidence that photosynthetic apparatus of both juveniles and adults perform, and the high photochemical efficiency is pivotal in providing the adaptive solid potential of G. barbata thalli in its habitat.

Conversely to Fv/Fm ratio, quantum yield of PSII electron rate (ΦPSII), electron transport rate (ETR), non-photochemical quenching (NPQ), as well as pigment and protein production were differently modulated in juveniles and adults suggesting that young individuals improve photosynthesis by allocating more nutrients into components of the electron transport chain and Rubisco synthesis, while the adult ones utilize nitrogen mainly to potentiate the pigment content synthesis (Harrison & Hurd, 2001; Gómez, Wiencke & Thomas, 1996; Stengel & Dring, 1998; Ohtake et al., 2021).

According to the higher photosynthetic efficiency found in younger than older individuals (Gómez, Wiencke & Thomas, 1996; Kim & Garbary, 2009), the rapid light curves evidenced how juveniles and adults differently used harvested light in the photosynthetic process at both unsaturated and saturated irradiances. More specifically, juveniles invest more light energy into photosynthesis, displaying 1.6-fold higher quantum yield of PSII electron transport (ΦPSII) and electron transport rate (ETR), along with 1.8-fold higher content of Rubisco. It is likely that young individuals invest more of the available P in ATP energy transfer during photosynthesis (Xu et al., 2017; Ohtake et al., 2021). This is because in young thalli, the higher activity of the electron transport chain is expected to produce more ATP and NADPH molecules, which are used in CO2 fixation by Rubisco. Rubisco concentration increases with high photochemical activity (Raven, 1997; Gylle et al., 2013). Our data align with other studies showing that the amount of Rubisco may decrease as thallus age increases. The age-related Rubisco and photosynthesis reduction is consistent with other studies on higher plants (Gómez, Wiencke & Thomas, 1996; Bertamini & Nedunchezhian, 2002).

As a result of a compensation mechanism, the higher investment of light energy in photochemical activity observed in young thalli implies the reduction of dissipation processes, explaining the lower NPQ values. Conversely, adult thalli, more exposed to unfavorable water surface conditions (i.e., light excess during tide events, higher UV levels, high or low temperature) than young, submerged thalli, showed higher NPQ values in the PPFD range from 100 to 500 µmol photons m−2 s−1, dissipating the excess light mainly as heat.

The higher levels of carotenoids in adult thalli, together to the NPQ rise (Lavaud & Goss, 2014), may suggest the activation of thermal dissipation mechanisms mediated by xanthophyll cycle for photoprotection purposes (Demmig-Adams & Adams III, 2006; Celis-Plá et al., 2016).

Beyond a photoprotective role, the higher photosynthetic pigment content found in adults (1.8 and 1.7 folds for chlorophylls and carotenoids, respectively) compared to juveniles may be also a way to compensate for the reduced photochemistry (Porzio et al., 2017). However, it cannot be excluded that the increase of chlorophyll a with age during winter, may be also due to the combination of low surface irradiances and shading produced by other individuals or self-shading linked to the thallus density and morphology (Stengel & Dring, 1998).

As regards the morpho-functional traits, thallus dry matter content (TDMC) significantly differed between young and mature individuals indicating a dissimilar distribution between structural compounds and water-filled and nutrient-rich photosynthetically active tissues (Cappelatti, Mauffrey & Griffin, 2019). TDMC modulation during growth is involved in organism survival (Elger & Willby, 2003) and its increase is generally associated with high resistance to wave damage and desiccation (Cappelatti, Mauffrey & Griffin, 2019).

Cystoseira s.l. is known to exhibit seasonal variations in TDMC during its development. This occurs as the thalli shift from primary growth in winter and spring to dormancy in summer and autumn. During dormancy, individuals shed many secondary branches, leading to lower water content (Orfanidis et al., 2017; Iveša et al., 2022).

In our study, even if TDMC was not measured seasonally, the increase of dry biomass found in adults compared to juveniles during winter, when temperature are low, suggests an investment of carbon in structural compounds with age. This response might provide an advantage for adult individuals coping with waves or grazing pressure. A positive correlation exists between thallus-size, life-stage and wave action (Thomsen, Wernberg & Kendrick, 2004). Considering the different size between juvenile and adult individuals of G. barbata, wave-generated forces may represent an environmental stimulus that elicits changes in the carbon metabolism, inducing adaptative variations in morphology, and structural and biomechanical properties of adult thalli. Such changes may consist in the dislodgement of carbohydrate polymers toward cell wall synthesis (Kraemer & Chapman, 1991; Dudgeon & Johnson, 1992), making adults more resistant and attached to the substrate than juveniles (Kraemer & Chapman, 1991; Cappelatti, Mauffrey & Griffin, 2019). The elevated dry matter content of macroalgae is not only an indicator of mechanical resistance but also a proxy to predict variations in palatability for grazers (Elger & Willby, 2003). Notably, the difference in TDMC between young and adult thalli was accompanied by a diverse regulation of photosynthetic activity. The reduced palatability and the lower photosynthetic rate suggest that adults prefer a resource conservation strategy and an investment into structural defenses (Elger & Willby, 2003; Mauffrey, Cappelatti & Griffin, 2020).

The availability of nitrogen in the growth environment and increased photosynthetic activity may explain why juveniles exhibited higher levels of antioxidants compared to adults. Nitrate enrichment has been reported to enhance the accumulation of phenolic compounds in C. tamariscifolia (Celis-Plá et al., 2014). There is a positive relationship between high photosynthetic rates and the accumulation of carbon with secondary metabolism in the form of phenolic compounds (Celis-Plá et al., 2016). The increased synthesis of such scavengers provides photoprotection for photosynthetic apparatus and an advantage against predation.

The photoprotection has been observed in C. tamariscifolia thalli habiting in oligo and ultra-oligotrophic transparent coastal waters where higher irradiance levels can be found and photodamage should be prevented (Celis-Plá et al., 2017). As this is not the case of the Venice lagoon, we hypothesize that young thalli promote secondary metabolism and antioxidant compounds as a defense against grazing (Mannino et al., 2016). Phenolics are particularly abundant in brown macroalgae (Phaeophyceae) due to their exclusive production of phlorotannins (Montero et al., 2019) and play structural, antibacterial, photoprotective, and herbivore deterrent roles (Li et al., 2011; Steevensz et al., 2012; Stiger et al., 2014; Mancuso et al., 2019).

The antioxidant level found in our study is comparable with that reported by other authors who analyzed specimens of G. barbata from natural environments (Cadar et al., 2019; Castillo et al., 2023). We observed higher concentrations of total phenolic compounds in juveniles supporting the hypothesis of the increase of resources allocation into chemical defenses.

Flavonoids are another class of phenolic compounds whose role, even if not thoroughly investigated in algae yet, is primarily to guarantee photoprotection by scavenging reactive oxygen species (ROS) (Fernando, Lee & Ahn, 2022). Tannins, involved in cell-wall hardening, exert a structural and protective purpose and, as polyphenols, provide a chemical defense against grazers (Mannino & Micheli, 2020). To support the general trend of antioxidant compounds, we found in juveniles a higher radical scavenging activity than in adults. It may be hypothesized that the elevated scavenging activity of young thalli was mainly due to tannins, polyphenols, and flavonoids, as observed in previous studies on other Phaeophyceae (Connan et al., 2006; Nunes et al., 2021; Ruiz-Medina, Sansón & González-Rodríguez, 2022).

Conclusions

The overall data suggests that during the winter season, young and adult thalli of G. barbata inhabiting the transitional water system of the Venice lagoon, adopt different growth strategies and show remarkable variations in their eco-physiological and biochemical traits (Fig. 6).

10.7717/peerj.17959/fig-6 Figure 6 Schema of main results obtained in the study.

Conceptual diagram summarizing the conclusions of the study. Image icons from Powerpoint.

Juvenile thalli preferentially utilize the available nutrients, potentiating the photosynthetic components involved in electron transport chain and Rubisco synthesis, thus determining a higher photosynthetic activity and production of secondary metabolites. The elevated production of flavonoids, tannins and polyphenols confers more resistance against potential grazers to juvenile thalli. Conversely, adult thalli, more exposed to unfavorable water surface conditions (i.e., light excess during tide events, higher UV levels, higher or lower temperature, shading phenomena), show a higher photosynthetic pigment content to compensate for the lower photochemistry and potentiate the thermal dissipation processes as a photoprotective mechanism. Furthermore, being more wave-exposed, adults also exhibit an increase of biomass allocation towards structural tissues, conferring thalli resistance against grazers and wave-generated forces. Despite further studies with an increased samples size in space and time are needed to corroborate our results, these preliminary findings propose ‘thallus age’ as a valuable and potential ecological trait to assess growth-defense strategies exploited by G. barbata during different seasons and against multiple environmental stressors.

Supplemental Information

10.7717/peerj.17959/supp-1 Supplemental Information 1 Raw data

All the measurements on the eco-physiological traits and western blot analyses on which graphs and tables were built.

10.7717/peerj.17959/supp-2 Supplemental Information 2 Western blot PSII-D1 protein

10.7717/peerj.17959/supp-3 Supplemental Information 3 Western blot of Rubisco

Additional Information and Declarations

Competing Interests

Author Contributions

Field Study Permissions

Data Availability

Carmen Arena is an Academic Editor for PeerJ.

Maria Luisa Pica performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.

Ermenegilda Vitale performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.

Rosa Donadio performed the experiments, analyzed the data, authored or reviewed drafts of the article, and approved the final draft.

Giulia Costanzo performed the experiments, analyzed the data, authored or reviewed drafts of the article, and approved the final draft.

Marco Munari performed the experiments, analyzed the data, authored or reviewed drafts of the article, and approved the final draft.

Erika Fabbrizzi analyzed the data, authored or reviewed drafts of the article, and approved the final draft.

Simonetta Fraschetti conceived and designed the experiments, authored or reviewed drafts of the article, contributed reagents, materials, and approved the final draft.

Carmen Arena conceived and designed the experiments, authored or reviewed drafts of the article, contributed reagents, materials, and approved the final draft.

The following information was supplied relating to field study approvals (i.e., approving body and any reference numbers):

Field experiments were authorised by Regione del Veneto with decree n. 369 (date 04.05.2023).

The following information was supplied regarding data availability:

The raw measurements are available in the Supplementary Files.
==== Refs
References

Acri, Braga & Aubry (2020) Acri F Braga F Aubry FB 2020 Long-term dynamics in nutrients, chlorophyll a and water quality parameters in the Lagoon of Venice Scientia Marina 84 199 309 10.3989/scimar.05035.08A
Alestra & Schiel (2015) Alestra T Schiel DR 2015 Impacts of local and global stressors in intertidal habitats: influence of altered nutrient, sediment and temperature levels on the early life history of three habitat-forming macroalgae Journal of Experimental Marine Biology and Ecology 468 29 36 10.1016/j.jembe.2015.03.017
ARPAV (2024) ARPAV Venetto weather 2024 https://wwwold.arpa.veneto.it/previsioni/it/html/
ARPAV TRIX (2022) ARPAV TRIX Trophic index for coastal marine waters 2022 https://www.arpa.veneto.it/arpavinforma/indicatori-ambientali/indicatori_ambientali/idrosfera/qualita-dei-corpi-idrici/indice-trofico-trix-per-le-acque-marino-costiere/2023
Baghdadli et al. (1990) Baghdadli D Tremblin G Pellegrini M Coudret A 1990 Effects of environmental parameters on net photosynthesis of a free-living brown seaweed, Cystoseira barbata formarepens: determination of optimal photosynthetic culture conditions Journal of Applied Phycology 2 281 287 10.1007/BF02179786
Baker (2008) Baker NR 2008 Chlorophyll fluorescence: a probe of photosynthesis in vivo Annual Review of Plant Biology 59 89 113 10.1146/annurev.arplant.59.032607.092759
Ballesteros et al. (2007) Ballesteros E Torras X Pinedo S García M Mangialajo L De Torres M 2007 A new methodology based on littoral community cartography dominated by macroalgae for the implementation of the European Water Framework Directive Marine Pollution Bulletin 55 172 180 10.1016/j.marpolbul.2006.08.038 17045303
Beer, Björk & Beardall (2014) Beer S Björk M Beardall J 2014 Photosynthesis in the marine environment United States Wiley-Blackwell
Benedetti-Cecchi et al. (2015) Benedetti-Cecchi L Tamburello L Maggi E Bulleri F 2015 Experimental perturbations modify the performance of early warning indicators of regime shift Current Biology 25 1867 1872 10.1016/j.cub.2015.05.035 26166776
Bertamini & Nedunchezhian (2002) Bertamini M Nedunchezhian N 2002 Leaf age effects on chlorophyll, Rubisco, photosynthetic electron transport activities and thylakoid membrane protein in field grown grapevine leaves Journal of Plant Physiology 59 7 799 803 10.1078/0176-1617-0597
Bevilacqua et al. (2019) Bevilacqua S Savonitto G Lipizer M Mancuso P Ciriaco S Srijemsi M Falace A 2019 Climatic anomalies may create a long-lasting ecological phase shift by altering the reproduction of a foundation species Ecology 100 12 e02838 10.1002/ecy.2838 31330045
Bianchelli et al. (2023) Bianchelli S Fraschetti S Martini F Lo Martire M Nepote E Ippoliti D Rindi F Danovaro R 2023 Macroalgal forest restoration: the effect of the foundation species Frontiers in Marine Science 10 1213184 10.3389/fmars.2023.1213184
Bilger & Björkman (1990) Bilger W Björkman O 1990 Role of the xanthophyll cycle in photoprotection elucidated by measurements of light-induced absorbance changes, fluorescence and photosynthesis in leaves of Hedera canariensis Photosynthesis Research 25 173 185 10.1007/BF00033159 24420348
Bradford (1976) Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Analytical Biochemistry 72 248 254 10.1016/0003-2697(76)90527-3 942051
Cadar et al. (2019) Cadar E Sirbu R Ibram A Ionescu AM 2019 Evaluation of total phenolic content in relation to antioxidant activity of brown algae Cystoseira barbata from Black Sea Revista de Chimie 70 2684 2689 10.37358/RC.19.7.7406
Caliceti et al. (2002) Caliceti M Argese E Sfriso A Pavoni B 2002 Heavy metal contamination in the seaweeds of the Venice lagoon Chemosphere 47 443 454 10.1016/s0045-6535(01)00292-2 11999620
Cappelatti, Mauffrey & Griffin (2019) Cappelatti L Mauffrey ARL Griffin JN 2019 Applying continuous functional traits to large brown macroalgae: variation across tidal emersion and wave exposure gradients Marine Biology 166 145 10.1007/s00227-019-3574-5
Castillo et al. (2023) Castillo A Celeiro M Lores M Grgić K Banožić M Jerković I Jokić S 2023 Bioprospecting of targeted phenolic compounds of Dictyota dichotoma, Gongolaria barbata, Ericaria amentacea, Sargassum hornschuchii and Ellisolandia elongata from the Adriatic Sea extracted by two green methods Marine Drugs 21 97 10.3390/md21020097 36827138
Cebrian et al. (2021) Cebrian E Tamburello L Verdura J Guarnieri G Medrano A Linares C Hereu B Garrabou J Cerrano C Galobart C Fraschetti S 2021 A roadmap for the restoration of Mediterranean Macroalgal Forests Frontiers in Marine Sciences 8 709219 10.3389/fmars.2021.709219
Celis-Plá et al. (2016) Celis-Plá PSM Bouzon ZL Hall-Spencer JM Schmidt EC Korbee N Figueroa FL 2016 Seasonal biochemical and photophysiological responses in the intertidal macroalga Cystoseira tamariscifolia (Ochrophyta) Marine Environmental Research 115 89 97 10.1016/j.marenvres.2015.11.014 26724873
Celis-Plá et al. (2017) Celis-Plá PSM Martínez B Korbee N Hall-Spencer JM Figueroa FL 2017 Ecophysiological responses to elevated CO2 and temperature in Cystoseira tamariscifolia (Phaeophyceae) Climatic Change 142 67 81 10.1007/s10584-017-1943-y
Celis-Plá et al. (2014) Celis-Plá PSM Martínez B Quintano E García-Sánchez M Pedersen A Navarro NP Copertino MS Mangaiyarkarasi N Mariath R Figueroa RL Korbee N 2014 Short-term ecophysiological and biochemical responses of Cystoseira tamariscifolia and Ellisolandia elongata to environmental changes Aquatic Biology 22 227 243 10.3354/ab00573
Çevirgen et al. (2020) Çevirgen S Elwany H Pesce M Zirino A 2020 Managing nutrient pollution in Venice Lagoon (Italy): a practical tool for assessment of water quality Sustainable Water Resources Management 6 33 10.1007/s40899-020-00390-y
Connan et al. (2006) Connan S Delisle F Deslandes E Ar Gall E 2006 Intra-thallus phlorotannin content and antioxidant activity in Phaeophyceae of temperate waters Botanica Marina 49 39 46 10.1515/BOT.2006.005
Costanzo et al. (2022) Costanzo G Vitale E Iesce MR Naviglio D Amoresano A Fontanarosa C Spinelli M Ciaravolo M Arena C 2022 Antioxidant properties of pulp, peel and seeds of Phlegrean Mandarin (Citrus reticulata Blanco) at different stages of fruit ripening Antioxidants 11 187 10.3390/antiox11020187 35204071
Delgado, Ballesteros & Vidal (1994) Delgado O Ballesteros E Vidal M 1994 Seasonal variation in tissue nitrogen and phosphorus of Cystoseira mediterranea Sauvageau (Fucales, Phaeophyceae) in the Northwestern Mediterranean Sea Botanica Marina 37 1 1 10 10.1515/botm.1994.37.1.1
Demmig-Adams & Adams III (2006) Demmig-Adams B Adams III WW 2006 Photoprotection in an ecological context: the remarkable complexity of thermal dissipation New Phytologist 172 11e21 10.1111/j.1469-8137.2006.01835.x 16945085
Dudgeon & Johnson (1992) Dudgeon SR Johnson AS 1992 Thick vs. thin: thallus morphology and tissue mechanics influence differential drag and dislodgement of two co-dominant seaweeds Journal of Experimental Marine Biology and Ecology 165 1 23 43 10.1016/0022-0981(92)90287-K
Elger & Willby (2003) Elger A Willby NJ 2003 Leaf dry matter content as an integrative expression of plant palatability: the case of freshwater macrophytes Functional Ecology 17 58 65 10.1046/j.1365-2435.2003.00700.x
Fabbrizzi et al. (2023) Fabbrizzi E Munari M Fraschetti S Arena C Chiarore A Cannavacciuolo A Colletti A Costanzo G Soler-Fajardo A Nannini M Savinelli B Silvestrini C Vitale E Tamburello L 2023 Canopy-forming macroalgae can adapt to marine heatwaves Environmental Research 238 117218 10.1016/j.envres.2023.117218 37778611
Fernando, Lee & Ahn (2022) Fernando IPS Lee W Ahn G 2022 Marine algal flavonoids and phlorotannins; an intriguing frontier of biofunctional secondary metabolites Critical Reviews Biotechnology 42 23 45 10.1080/07388551.2021.1922351
Gazetta Officiale (2011) Gazetta Officiale Ministry of the environment and protection of land and seadecree 8 November 2010, n. 260 https://www.gazzettaufficiale.it/eli/id/2011/02/07/011G0035/sg 2011
Genty, Briantais & Baker (1989) Genty B Briantais J-M Baker NR 1989 The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence Biochimica et Biophysica Acta (BBA) - General Subjects 990 87 92 10.1016/S0304-4165(89)80016-9
Gómez, Wiencke & Thomas (1996) Gómez I Wiencke C Thomas DN 1996 Variations in photosynthetic characteristics of the Antarctic marine brown alga Ascoseira mirabilis in relation to thallus age and size European Journal of Phycology 31 2 167 172 10.1080/09670269600651341
Gran et al. (2022) Gran A Movilla J Ballesteros E Sales M Bolado I Galobart C Cefalì ME 2022 Assessing the expansion and success of a restored population of Gongolaria barbata (Stackhouse) Kuntze (Fucales, Phaeophyceae) using high-precision positioning tools and size distribution frequencies Mediterranean Marine Science 23 907 916 10.12681/mms.30500
Gylle et al. (2013) Gylle AM Nygård CA Svan CI Pocock T Ekelud NGA 2013 Photosynthesis in relation to D1, PsaA and Rubisco in marine and brackish water ecotypes of Fucus vesiculosus and Fucus radicans (Phaeophyceae) Hydrobiologia 700 109 119 10.1007/s10750-012-1231-9
Hanley, Firth & Foggo (2024) Hanley ME Firth LB Foggo A 2024 Victim of changes? Marine macroalgae in a changing world Annals of Botany 133 1 16 10.1093/aob/mcad185 37996092
Harrison & Hurd (2001) Harrison PJ Hurd CL 2001 Nutrient physiology of seaweeds: application of concepts to aquaculture Cahiers de Biologie Marine 42 1 71 82
Hurd et al. (2014) Hurd CL Harrison PJ Bischof K Lobban CS 2014 Seaweed ecology and physiology Cambridge University Press Cambridge, UK
Irving et al. (2009) Irving AD Balata D Colosio F Ferrando GA Airoldi L 2009 Light, sediment, temperature, and the early life-history of the habitat-forming alga Cystoseira barbata Marine Biology 156 1223 1231 10.1007/s00227-009-1164-7
Ishizawa et al. (2021) Ishizawa H Onoda Y Kitajima K Kuroda M Inoue D Ike M 2021 Coordination of leaf economics traits within the family of the world’s fastest growing plants (Lemnaceae) Journal of Ecology 109 2950 2962 10.1111/1365-2745.13710
Iveša et al. (2022) Iveša L Bilajac A Gljušćić E Najdek M 2022 Gongolaria barbata forest in the shallow lagoon on the southern Istrian Coast (northern Adriatic Sea) Botanica Marina 65 255 268 10.1515/bot-2022-0021
Iveša & Devescovi (2014) Iveša L Devescovi M 2014 Distribution and composition of stands along the west Istrian coast (northern Adriatic, Croatia) and comparison with historical data Proceedings of the 5th Mediterranean Symposium on Marine Vegetation Slovenia, Portorož Langar H Bouafif C Ouerghi A 102 107
Iveša, Djakovac & Devescovi (2016) Iveša L Djakovac T Devescovi M 2016 Long-term fluctuation in Cystoseira population along the west Istrian Coast (Croatia) related to eutrophication patterns in the northern Adriatic Sea Marine Pollution Bullettin 106 162 173 10.1016/j.marpolbul.2016.03.010
Jeffrey & Humphrey (1975) Jeffrey SW Humphrey GF 1975 New spectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton Journal of Molecular Structure 29 379 382 10.1016/0022-2860(75)85046-0
Kaleb et al. (2023) Kaleb S Sánchez de Pedro R Bañares España E Alboresi A Savonitto G Natale S Bevilacqua S Falace A 2023 Cultivation of Gongolaria barbata (Fucales, Phaeophyceae) with a seaweed-derived biostimulant in order to improve photophysiological fitness and promote fertility to advance the restoration of marine macroalgal forests Journal of Applied Phycology 35 5 2337 2350 10.1007/s10811-023-02984-3
Khailov & Firsov (1976) Khailov KM Firsov YK 1976 The relationships between weight, length, age and intensity of photosynthesis and organotrophy in the thallus of Cystoseira barbata from the Black Sea Botanica Marina 19 329 334 10.1515/botm.1976.19.6.329
Kim & Garbary (2009) Kim KY Garbary DJ 2009 Form, function and longevity in fucoid thalli: chlorophyll a fluorescence differentiation of Ascophyllum nodosum, Fucus vesiculosus and F. distichus (Phaeophyceae) Algae 24 2 93 104 10.4490/algae.2009.24.2.093
Kraemer & Chapman (1991) Kraemer GP Chapman DJ 1991 Effects of tensile force and nutrient availability on carbon uptake and cell wall synthesis in blades of juvenile Egregia menziesii (Turn.) Aresch.(Phaeophyta) Journal of Experimental Marine Biology and Ecology 149 2 267 277 10.1016/0022-0981(91)90049-3
Krumhansl et al. (2016) Krumhansl KA Okamoto DK Rassweiler A Novak M Bolton JJ Cavanaugh KC Connell SD Johnson CR Konar B Ling SD Micheli F Norderhaug KM Pérez-Matus A Sousa-Pinto I Reed DC Salomon AK Shears NT Wernberg T Anderson RJ Barrett NS Buschmann AH Carr MH Caselle JE Derrien-Courtel S Edgar GJ Edwards M Estes JA Goodwin C Kenner MC Kushner DJ Moy FE Nunn J Steneck RS Vásquez J Watson J Witman JD Byrnes JEK 2016 Global patterns of kelp forest change over the past half-century Proceedings of the National Academy of Sciences of the United States of America 113 13785 13790 10.1073/pnas.1606102113 27849580
Lavaud & Goss (2014) Lavaud J Goss R 2014 The peculiar features of non-photochemical fluorescence quenching in diatoms and brown algae, non-photochemical quenching and energy dissipation in plants, algae and cyanobacteria Advances in photosynthesis and respiration Springer Dordrecht Demming-Adams B Garab G Adams III W Govindjee 421 443 10.1007/978-94-017-9032-1_20
Li et al. (2011) Li YX Wijesekara I Li Y Kim SK 2011 Phlorotannins as bioactive agents from brown algae Process Biochemistry 46 2219 2224 10.1016/j.procbio.2011.09.015
Lichtenthaler (1987) Lichtenthaler HK 1987 Chlorophylls and carotenoids: pigments of photosynthetic biomembranes Methods in Enzymology 148 350 382 10.1016/0076-6879(87)48036-1
Lokovšek et al. (2024) Lokovšek A Pitacco V Falace A Trkov D Orlando-Bonaca M 2024 Too hot to handle: effects of water temperature on the early life stages of Gongolaria barbata (Fucales) Journal of Marine Science and Engineering 12 514 10.3390/jmse12030514
Manca et al. (2022) Manca F Mulà C Gustafsson C Mauri A Roslin T Thomas DN Benedetti-Cecchi L Norkko A Strona G 2022 Unveiling the complexity and ecological function of aquatic macrophyte–animal networks in coastal ecosystems Biological Reviews 97 1306 1324 10.1111/brv.12842 35174616
Mancuso et al. (2019) Mancuso FP Messina CM Santulli A Laudicella VA Giommi C Sarà G Airoldi L 2019 Influence of ambient temperature on the photosynthetic activity and phenolic content of the intertidal Cystoseira compressa along the Italian coastline Journal of Applied Phycology 31 3069 3076 10.1007/s10811-019-01802-z
Mannino & Micheli (2020) Mannino AM Micheli C 2020 Ecological function of phenolic compounds from Mediterranean fucoid algae and seagrasses: an overview on the Genus Cystoseira sensu lato and Posidonia oceanica (L.) Delile Journal of Marine Science and Engineering 8 19 10.3390/jmse8010019
Mannino et al. (2016) Mannino AM Vagliaca C Cammarata M Oddo E 2016 Effects of temperature on total phenolic compounds in Cystoseira amentacea (C. Agardh) Bory (Fucales, Phaeophyceae) from southern Mediterranean Sea Plant Biosystems 150 1 152 160 10.1080/11263504.2014.941033
Mauffrey, Cappelatti & Griffin (2020) Mauffrey ARL Cappelatti L Griffin JN 2020 Seaweed functional diversity revisited: Confronting traditional groups with quantitative traits Journal of Ecology 108 2390 2405 10.1111/1365-2745.13460
Molinari-Novoa & Guiry (2020) Molinari-Novoa E Guiry M 2020 Reinstatement of the genera Gongolaria Boehmer and Ericaria stackhouse (sargassaceae, Phaeophyceae) Notulae Algarum 172 1 10
Monserrat et al. (2023) Monserrat M Verdura J Comeau S Cottalorda JM Priouzeau F Romero G Mangialajo L 2023 The role of grazers in early-life stages of Cystoseira sensu lato can be crucial in the restoration of marine forests Frontiers in Marine Science 10 1176780 10.3389/fmars.2023.1176780
Montero et al. (2019) Montero L Del Pilar Sánchez-Camargo A Ibáñez E Gilbert-López B 2019 Phenolic compounds from edible algae: bioactivity and health benefits Current Medical Chemistry 25 4808 4826 10.2174/0929867324666170523120101
Morand & Briand (1996) Morand P Briand X 1996 Excessive growth of macroalgae: a symptom of environmental disturbance Botanica Marina 39 491 516 10.1515/botm.1996.39.1-6.491
Moulehi et al. (2012) Moulehi I Bourgou S Ourghemmi I Tounsi M 2012 Variety and ripening impact on phenolic composition and antioxidant activity of mandarin (Citrus reticulate Blanco) and bitter orange (Citrus aurantium L.) seeds extracts Industrial Crops and Products 39 74 80 10.1016/j.indcrop.2012.02.013
Mulo, Sakurai & Aro (2012) Mulo P Sakurai I Aro EM 2012 Strategies for psbA gene expression in cyanobacteria, green algae and higher plants: from transcription to PSII repair Biochimica et Biophysica Acta (BBA) - Bioenergetics 1817 247 257 10.1016/j.bbabio.2011.04.011 21565160
Nielsen & Nielsen (2008) Nielsen HD Nielsen SL 2008 Evaluation of imaging and conventional PAM as a measure of photosynthesis in thin- and thick-leaved marine macroalgae Aquatic Biology 3 121 131 10.3354/ab00069
Nunes et al. (2021) Nunes D André R Ressaissi A Duarte B Melo R Serralheiro ML 2021 Influence of gender and age of brown seaweed (Fucus vesiculosus) on biochemical activities of its aqueous extracts Foods 11 1 39 10.3390/foods11010039 35010166
Ohtake et al. (2021) Ohtake M Kurita R Tsunogai M Nishihara G Toda T 2021 Storage capacity for phosphorus during growth and maturation in a brown alga Sargassum macrocarpum Science of the Total Environment 750 141221 10.1016/j.scitotenv.2020.141221 32846250
Orfanidis (1991) Orfanidis S 1991 Temperature responses and distribution of macroalgae belonging to the warm-temperate Mediterranean-Atlantic Distribution Group Botanica Marina 34 541 552 10.1515/botm.1991.34.6.541
Orfanidis et al. (2017) Orfanidis S Iveša L Gounaris S Tsioli S Devescovi M Papathanasiou V 2017 Cystoseira scale-based biometric relationships Botanica Marina 60 285 295 10.1515/bot-2017-0024
Orlando-Bonaca et al. (2013) Orlando-Bonaca M Mannoni P-A Poloniato D Falace A 2013 Assessment of Fucus virsoides distribution in the Gulf of Trieste (Adriatic Sea) and its relation to environmental variables Botanica Marina 56 451 459 10.1515/bot-2013-0027
Orlando-Bonaca, Pitacco & Lipej (2021) Orlando-Bonaca M Pitacco V Lipej L 2021 Loss of canopy-forming algal richness and coverage in the northern Adriatic Sea Ecological Indicators 125 107501 10.1016/j.ecolind.2021.107501
Pastres et al. (2004) Pastres R Solidoro C Ciavatta S Petrizzo A Cossarini G 2004 Long-term changes of inorganic nutrients in the Lagoon of Venice (Italy) Journal of Marine Systems 51 179 189 10.1016/j.jmarsys.2004.05.011
Pellizzari, Oliveira & Yokoya (2008) Pellizzari F Oliveira EC Yokoya NS 2008 Life-history, thallus ontogeny, and the effects of temperature, irradiance and salinity on growth of the edible green seaweed Gayralia spp. (Chlorophyta) from Southern Brazil Journal of Applied Phycology 20 75 82 10.1007/s10811-007-9183-6
Porzio et al. (2020) Porzio L Arena C Lorenti M De Maio A Buia MC 2020 Long-term response of Dictyota dichotoma var. intricata (C. Agardh) Greville (Phaeophyceae) to ocean acidification: Insights from high pCO2 vents Science of The Total Environment 731 138896 10.1016/j.scitotenv.2020.138896 32408206
Porzio et al. (2017) Porzio L Buia MC Lorenti M De Maio A Arena C 2017 Physiological responses of a population of Sargassum vulgare (Phaeophyceae) to high pCO2/low pH: implications for its long-term distribution Science of the Total Environment 576 917 925 10.1016/j.scitotenv.2016.10.096 27865121
Raven (1997) Raven J 1997 Putting the C in phycology European Journal of Phycology 32 319 333 10.1080/09670269710001737259
R Core Team (2022) R Core Team R: A language and environment for statistical computing 2022 R Foundation for Statistical Computing Vienna, Austria https://www.R-project.org/
Regione Veneto et al. (2021) Regione Veneto ARPAV ISPRA 2021 Monitoring plan of the Venice lagoon aimed at the definition of the ecological status under Directive 2000/60/EC (Legislative decree N. 152/2006 s.m.i.)
Ruiz-Medina, Sansón & González-Rodríguez (2022) Ruiz-Medina MA Sansón M González-Rodríguez AM 2022 Changes in antioxidant activity of fresh marine macroalgae from the Canary Islands during air-drying process Algal Research 66 102798 10.1016/j.algal.2022.102798
Rusman et al. (2020) Rusman Q Lucas-Barbosa D Hassan K Poelman EH 2020 Plant ontogeny determines strength and associated plant fitness consequences of plant-mediated interactions between herbivores and flower visitors Journal of Ecology 108 3 1046 1060 10.1111/1365-2745.13370 32421019
Sadogurska et al. (2021) Sadogurska S Neiva J Falace A Serrao E Israel Á 2021 The genus Cystoseira s.l. (Ochrophyta, Fucales, Sargassaceae) in the Black Sea: morphological variability and molecular taxonomy of Gongolaria barbata and endemic Ericaria crinita f. bosphorica comb. nov Phytotaxa 480 1 21 10.11646/phytotaxa.480.1.1
Sakanishi, Kasai & Tanaka (2023) Sakanishi Y Kasai H Tanaka J 2023 Physiological and structural trade-offs as a basis for the coordination of functional traits in marine macroalgae Fisheries Science 89 625 632 10.1007/s12562-023-01707-4
Savonitto et al. (2021) Savonitto G De La Fuente G Tordoni E Ciriaco S Srijemsiet M Bacaro G Chiantore M Falace A 2021 Addressing reproductive stochasticity and grazing impacts in the restoration of a canopy-forming brown alga by implementing mitigation solutions Aquatic Conservation: Marine and Freshwater Ecosystems 31 1611 1623 10.1002/aqc.3555
Schofield, Evens & Millie (1998) Schofield O Evens TJ Millie DF 1998 Photosystem II quantum yields and Xanthophyll-Cycle pigments of the Macroalga Sargassum Natans (phaeophyceae): responses under natural sunlight Journal of Phycology 34 104 112 10.1046/j.1529-8817.1998.340104.x
Schreiber (2004) Schreiber U 2004 Pulse-Amplitude-Modulation (PAM) fluorometry and saturation pulse method: an overview Chlorophyll a fluorescence: a signature of photosynthesis Springer Netherlands Dordrecht Papageorgiou GC Govindjee 279 319
Solidoro et al. (2010) Solidoro C Bandelj V Bernardi FA Camatti E Ciavatta S Cossarini G Facca C Franzoi P Libralato S Melaku Canu D Pastres R Pranovi F Raicevich S Socal G Sfriso A Sigovini M Tagliapietra D Torricelli P 2010 Response of the Venice Lagoon ecosystem to natural and anthropogenic pressures over the last 50 years Coastal lagoons–critical habitats of environmental change CRC Press Boca Raton Kennish MJ Paerl HW 483 511
Starko & Martone (2016) Starko S Martone PT 2016 Evidence of an evolutionary-developmental trade-off between drag avoidance and tolerance strategies in wave-swept intertidal kelps (Laminariales, Phaeophyceae) Journal of Phycology 52 54 63 10.1111/jpy.12368 26987088
Steevensz et al. (2012) Steevensz AJ Mackinnon SL Hankinson R Craft C Connan S Stengel DB Melanson JE 2012 Profiling phlorotannins in brown macroalgae by liquid chromatography-high resolution mass spectrometry Phytochemical Analysis 23 547 553 10.1002/pca.2354 22383068
Stengel & Dring (1998) Stengel DB Dring MJ 1998 Seasonal variation in the pigment content and photosynthesis of different thallus regions of Ascophyllum nodosum (Fucales, Phaeophyta) in relation to position in the canopy Phycologia 37 4 259 268 10.2216/i0031-8884-37-4-259.1
Stengel, McGrath & Morrison (2005) Stengel DB McGrath H Morrison LJ 2005 Tissue Cu, Fe and Mn concentrations in different-aged and different functional thallus regions of three brown algae from western Ireland Estuarine, Coastal and Shelf Science 65 4 687 696 10.1016/j.ecss.2005.07.003
Stiger et al. (2014) Stiger V Jégou C Cérantola S Guérard F Le Lann K 2014 Phlorotannins in Sargassaceae species from Brittany (France): interesting molecules for ecophysiological and valorisation purposes Advances in Botanical Research 71 379 411 10.1016/B978-0-12-408062-1.00013-5
Sun, Ricardo-da Silva & Spranger (1998) Sun B Ricardo-da Silva JM Spranger I 1998 Critical factors of vanillin assay for catechins and proanthocyanidins Journal of Agricultural and Food Chemistry 46 10 4267 4274 10.1021/jf980366j
Taylor, Fletcher & Raven (2001) Taylor R Fletcher RL Raven JA 2001 Preliminary studies on the growth of selected ‘Green Tide’ algae in laboratory culture: effects of irradiance, temperature, salinity and nutrients on growth rate Botanica marina 44 4 327 336 10.1515/BOT.2001.042
Thomsen, Wernberg & Kendrick (2004) Thomsen MS Wernberg T Kendrick GA 2004 The effect of thallus size, life stage, aggregation, wave exposure and substratum conditions on the forces required to break or dislodge the small kelp Ecklonia radiata Botanica Marina 47 454 460 10.1515/BOT.2004.068
Tursi et al. (2023) Tursi A Lisco A Chimienti G Mastrototaro F Ungaro N Bottalico A 2023 Salinity as a key factor in structuring macrophyte assemblages in transitional water bodies: the case of the Apulian coastal lagoons (Southern Italy) Diversity 15 615 10.3390/d15050615
United Nations Environment Programme (2019) United Nations Environment Programme 2019 Convention for the protection of the marine environment and the Coastal Region of the Mediterranean and its protocols https://planbleu.org/sites/default/files/upload/files/Barcelona_convention_and_protocols_2005_eng.pdf
Van Alstyne, Ehlig & Whitman (1999) Van Alstyne KL Ehlig JM Whitman SH 1999 Feeding preferences for juvenile and adult algae depend on algal stage and herbivore species Marine Ecology Progress Series 180 179 185 10.3354/meps180179
Verdura et al. (2023) Verdura J Rehues L Mangialajo L Fraschetti S Belattmania Z Bianchelli S Blanfuné A Sabour B Chiarore A Danovaro R Fabbrizzi E Giakoumi S Iveša I Katsanevakis S Kytinou E Nasto I Nikolaou A Orfanidis S Rilov G Rindi G Sales M Sini M Tamburello L Thibaut T Tsirintanis K Cebrian E 2023 Distribution, health and threats to Mediterranean macroalgal forests: defining the baselines for their conservation and restoration Frontiers in Marine Science 10 1258842 10.3389/fmars.2023.1258842
Verdura et al. (2018) Verdura J Sales M Ballesteros E Cefalì ME Cebrian E 2018 Restoration of a canopy-forming alga based on recruitment enhancement: methods and long-term success assessment Frontiers in Plant Sciences 9 1832 10.3389/fpls.2018.01832
Vitale et al. (2022) Vitale E Velikova V Tsonev T Costanzo G Paradiso R Arena C 2022 Manipulation of light quality is an effective tool to regulate photosynthetic capacity and fruit antioxidant properties of Solanum lycopersicum L. cv. ‘Microtom’ in a controlled environment PeerJ 1 10 e13677 10.7717/peerj.13677
Vollenweider et al. (1998) Vollenweider RA Giovanardi F Montanari G Rinaldi A 1998 Characterization of the trophic conditions of marine coastal waters with special reference to the NW Adriatic Sea: proposal for a trophic scale, turbidity and generalized water quality index Environmetrics 9 329 357 10.1002/(SICI)1099-095X(199805/06)9:3<329::AID-ENV308>3.0.CO;2-9
Xu et al. (2017) Xu Z Gao G Xu J Wu H 2017 Physiological response of a golden tide alga (Sargassum muticum) to the interaction of ocean acidification and phosphorus enrichment Biogeosciences 14 671 681
Zirino et al. (2016) Zirino A Elwany H Facca C Maicu’ F Neira C Mendoza G 2016 Nitrogen to phosphorus ratio in the Venice (Italy) Lagoon (2001–2010) and its relation to macroalgae Marine Chemistry 180 33 41 10.1016/j.marchem.2016.01.002
