Grow with the flow: Is phenotypic plasticity across hydrodynamic gradients common in seaweeds?

Seaweeds are widely assumed to be phenotypically plastic across hydrodynamic gradients, yet while many marine macroalgae exhibit intraspecific phenotypic variation that correlates with flow, researchers often fail to test whether such variation is due to plasticity or another mechanism, such as local adaptation. In this minireview, we considered mechanisms for sensing flow in seaweeds that could facilitate adaptive phenotypic plasticity across hydrodynamic gradients. We then reviewed the literature from 1900 to 2024 to see how often phenotypic variation and plasticity across hydrodynamic gradients had been observed and demonstrated in different groups of seaweeds. In the last 124 years, phenotypic variation and plasticity in response to flow have been well documented in brown algae but scarcely documented in red and green algae. This could suggest that brown algae are better able to sense and respond to flow than red and green algae, perhaps due to the intercalary meristem of many brown algae, including most kelps. However, this skewed distribution could also be the result of publication bias, as most studies involving flow have been conducted on brown algae. Only 30% of 141 papers specifically investigated if observations of phenotypic variation along hydrodynamic gradients were due to plasticity. To date, phenotypic plasticity in response to flow has been demonstrated in 20 brown algal species, five red algal species, and two green algal species. Thus, the assumption that phenotypic plasticity to flow is common across seaweeds is not particularly well supported by the literature. Mechanisms underlying plasticity to flow are poorly understood and remain a critical avenue for future research.

Functional ecological traits in young and adult thalli of canopy-forming brown macroalga Gongolaria barbata (Phaeophyta) from a transitional water system.

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.

Novel high-throughput oxygen saturation measurements for quantifying the physiological performance of macroalgal early life stages.

Understanding how macroalgal forests will respond to environmental change is critical for predicting future impacts on coastal ecosystems. Although measures of adult macroalgae physiological responses to environmental stress are advancing, measures of early life-stage physiology are rare, in part due to the methodological difficulties associated with their small size. Here we tested a novel, high-throughput method (rate of oxygen consumption and production; V ̇ O 2 $$ \dot{V}{\mathrm{O}}_2 $$ ) via a sensor dish reader microplate system to rapidly measure physiological rates of the early life stages of three habitat-forming macroalgae, the kelp Ecklonia radiata and the fucoids Hormosira banksii and Phyllospora comosa. We measured the rate of O2 consumption (respiration) and O2 production (net primary production) to then calculate gross primary production (GPP) under temperatures representing their natural thermal range. The V ̇ O 2 $$ \dot{V}{\mathrm{O}}_2 $$ microplate system was suitable for rapidly measuring physiological rates over a temperature gradient to establish thermal performance curves for all species. The V ̇ O 2 $$ \dot{V}{\mathrm{O}}_2 $$ microplate system proved efficient for measures of early life stages of macroalgae ranging in size from approximately 50 µm up to 150 mm. This method has the potential for measuring responses of early life stages across a range of environmental factors, species, populations, and developmental stages, vastly increasing the speed, precision, and efficacy of macroalgal physiological measures under future ocean change scenarios.

Parallel loss of sexual reproduction in field populations of a brown alga sheds light on the mechanisms underlying the emergence of asexuality.

Sexual reproduction is widespread, but asexual lineages have repeatedly arisen from sexual ancestors across a wide range of eukaryotic taxa. The molecular changes underpinning the switch to asexuality remain elusive, particularly in organisms with haploid sexual systems. Here we explore independent events of loss of sex in the brown alga Scytosiphon, examine the proximate and evolutionary mechanisms involved, and test the importance of sexual conflict on gene expression changes following loss of sex. We find that asexual females ('Amazons') lose ability to produce sex pheromone and, consequently, are incapable of attracting males, whereas they gain rapid parthenogenic development from large, unfertilized eggs. These phenotypic changes are accompanied by convergent changes in gene expression. Decay of female functions, rather than relaxation of sexual antagonism, may be a dominant force at play during the emergence of asexuality in haploid sexual systems. Moreover, we show that haploid purifying selection plays a key role in limiting the accumulation of deleterious alleles in Amazons, and we identify an autosomal locus associated with the Amazon phenotype. The sex chromosome, together with this autosomal locus, may underlie the switch to obligate asexuality in the Amazon populations.

Various extracts of the brown seaweed Cystoseira barbata with different compositions exert biostimulant effects on seedling growth of wheat.

Worldwide, where the demand for novel and greener solutions for sustainable agricultural production is increasing, the use of eco-friendly products such as seaweed-derived biostimulants as pre-sowing treatment represent a promising and important approach for the future. Cystoseira barbata, a brown seaweed species abundant in the Mediterranean Region, was collected from the Marmara Sea and subjected to water, alkali, and acidic extractions, and the biostimulant activity of these extracts was tested on wheat (Triticum durum cv. Saricanak-98) using different rates through application to the seeds or germination medium (substrate) applications. The different extracts were characterized by mineral, total phenolic, free amino acid, mannitol, polysaccharide, antioxidant concentrations and hormone-like activity. The effects of the extracts on growth parameters, root morphology, esterase activity, and mineral nutrient concentrations of wheat seedlings were investigated. Our results suggest that the substrate application was more effective in enhancing the seedling performance compared to the seed treatment. High rates of seaweed extracts applied to substrates increased the shoot length and fresh weight of wheat seedlings by up to 20 and 25%, respectively. The substrate applications enhanced the root fresh weights of wheat seedlings by up to 25% when compared to control plants. Among the biostimulant extract applications, the water extract at the highest rate yielded the most promising results in terms of the measured parameters. Cystoseira barbata extracts with different compositions can be used as effective biostimulants to boost seedling growth. The local seaweed biomass affected by mucilage problems, has great potential as a bioeconomy resource and can contribute to sustainable practices for agriculture.

Chromatin landscape associated with sexual differentiation in a UV sex determination system.

In many eukaryotes, such as dioicous mosses and many algae, sex is determined by UV sex chromosomes and is expressed during the haploid phase of the life cycle. In these species, the male and female developmental programs are initiated by the presence of the U- or V-specific regions of the sex chromosomes but, as in XY and ZW systems, sexual differentiation is largely driven by autosomal sex-biased gene expression. The mechanisms underlying the regulation of sex-biased expression of genes during sexual differentiation remain elusive. Here, we investigated the extent and nature of epigenomic changes associated with UV sexual differentiation in the brown alga Ectocarpus, a model UV system. Six histone modifications were quantified in near-isogenic lines, leading to the identification of 16 chromatin signatures across the genome. Chromatin signatures correlated with levels of gene expression and histone PTMs changes in males versus females occurred preferentially at genes involved in sex-specific pathways. Despite the absence of chromosome scale dosage compensation and the fact that UV sex chromosomes recombine across most of their length, the chromatin landscape of these chromosomes was remarkably different to that of autosomes. Hotspots of evolutionary young genes in the pseudoautosomal regions appear to drive the exceptional chromatin features of UV sex chromosomes.

Parthenogenesis and the Evolution of Anisogamy.

Recently, it was pointed out that classic models for the evolution of anisogamy do not take into account the possibility of parthenogenetic reproduction, even though sex is facultative in many relevant taxa (e.g., algae) that harbour both anisogamous and isogamous species. Here, we complement this recent analysis with an approach where we assume that the relationship between progeny size and its survival may differ between parthenogenetically and sexually produced progeny, favouring either the former or the latter. We show that previous findings that parthenogenesis can stabilise isogamy relative to the obligate sex case, extend to our scenarios. We additionally investigate two different ways for one mating type to take over the entire population. First, parthenogenesis can lead to biased sex ratios that are sufficiently extreme that one type can displace the other, leading to de facto asexuality for the remaining type that now lacks partners to fuse with. This process involves positive feedback: microgametes, being numerous, lack opportunities for syngamy, and should they proliferate parthenogenetically, the next generation makes this asexual route even more prominent for microgametes. Second, we consider mutations to strict asexuality in producers of micro- or macrogametes, and show that the prospects of asexual invasion depend strongly on the mating type in which the mutation arises. Perhaps most interestingly, we also find scenarios in which parthenogens have an intrinsic survival advantage yet facultatively sexual isogamous populations are robust to the invasion of asexuals, despite us assuming no genetic benefits of recombination. Here, equal contribution from both mating types to zygotes that are sufficiently well provisioned can outweigh the additional costs associated with syngamy.

MiR8181 is involved in the cell growth regulation of Saccharina japonica.

Aureochrome, a blue-light receptor specifically found in photosynthetic stramenopiles, plays an important role in algal growth and development. It holds a reversed effector-sensor topology for the reception of blue light, acting as a candidate of optogenetic tool in transcriptional regulation. However, the inner regulatory mechanism of aureochrome is still unclear. In this study, we explored the potential regulatory relationship between microRNAs (miRNAs) and mRNAs by small RNA, transcriptome and degradome sequencing in Saccharina japonica. Through screening miRNA-mRNA interaction networks at the whole-genome level, we found that 18 miRNAs perfectly paired with aureochrome. Among these screened miRNAs, miR8181 was negatively correlated with aureochrome5 with high credibility, exhibiting tissue-specific expression in sporophyte of S. japonica. Degradome analysis further revealed the exact cleavage site of miR8181 on aureochrome5, confirming their targeting relationship. For the 54 target genes of miR8181, nine genes that exhibited similar expression to that of aureochrome5 competed for the same binding site, thus establishing a competing endogenous RNA network. Functional enrichment of the target genes revealed that miR8181 was involved in the regulation of cell differentiation and development in S. japonica. Moreover, overexpression of miR8181 resulted in significant decreases in the cell growth rates of Phaeodactylum tricornutum, suggesting negative roles of miR8181 in regulating cell growth. Our study revealed that miR8181, the targeting miRNA of aureochrome5, played negative roles in cell growth and development.

Histone modifications during the life cycle of the brown alga Ectocarpus.

BACKGROUND: Brown algae evolved complex multicellularity independently of the animal and land plant lineages and are the third most developmentally complex phylogenetic group on the planet. An understanding of developmental processes in this group is expected to provide important insights into the evolutionary events necessary for the emergence of complex multicellularity. Here, we focus on mechanisms of epigenetic regulation involving post-translational modifications of histone proteins. RESULTS: A total of 47 histone post-translational modifications are identified, including a novel mark H2AZR38me1, but Ectocarpus lacks both H3K27me3 and the major polycomb complexes. ChIP-seq identifies modifications associated with transcription start sites and gene bodies of active genes and with transposons. H3K79me2 exhibits an unusual pattern, often marking large genomic regions spanning several genes. Transcription start sites of closely spaced, divergently transcribed gene pairs share a common nucleosome-depleted region and exhibit shared histone modification peaks. Overall, patterns of histone modifications are stable through the life cycle. Analysis of histone modifications at generation-biased genes identifies a correlation between the presence of specific chromatin marks and the level of gene expression. CONCLUSIONS: The overview of histone post-translational modifications in the brown alga presented here will provide a foundation for future studies aimed at understanding the role of chromatin modifications in the regulation of brown algal genomes.

Effects of the brown algae Sargassum horneri and Saccharina japonica on survival, growth and resistance of small sea urchins Strongylocentrotus intermedius.

Mass mortality of the long line culture of the sea urchin Strongylocentrotus intermedius in summer, which is greatly associated with their disease, energy storage and resistant abilities, is the most serious problem for the development of the aquaculture. Here, a feeding experiment was conducted for ~ 9 weeks to investigate the survival, growth and gonadal development of small S. intermedius (~ 3 cm) fed either brown algae Sargassum horneri or Saccharina japonica. Subsequently, we assessed their resistant abilities via observing the behaviors of righting, tube feet extension and Aristotle's lantern reflex at both moderately elevated and acutely changed water temperatures. Sea urchins fed S. horneri showed significantly fewer diseased individuals and slower gonadal development than those fed S. japonica. Consistently, significantly greater Aristotle's lantern reflex occurred in sea urchins fed S. horneri at moderately elevated temperatures. These findings suggest that S. horneri has direct application potential as food for the long line culture of S. intermedius in summer because of the advantage in health, energy storage (avoid the energy loss caused by gonadal development at small body sizes) and resistance abilities. In comparison, sea urchins fed S. japonica outperformed those fed S. horneri for all experimental behaviors under the acutely changed water temperatures. These findings clearly suggest that S. intermedius fed S. japonica is more suitable for the areas with cold water mass in summer, because it can effectively avoid or reduce the negative impacts of acute changes of water temperature on sea urchins. The present study provides valuable information into the management of the long line culture of S. intermedius in summer.

Responses of the kelp Saccharina latissima (Phaeophyceae) to the warming Arctic: from physiology to transcriptomics.

The Arctic region is currently facing substantial environmental changes due to global warming. Melting glaciers cause reduced salinity environments in coastal Arctic habitats, which may be stressful for kelp beds. To investigate the responses of the kelp Saccharina latissima to the warming Arctic, we studied the transcriptomic changes of S. latissima from Kongsfjorden (Svalbard, Norway) over a 24-hour exposure to two salinities (Absolute Salinity [SA ] 20 and 30) after a 7-day pre-acclimation at three temperatures (0, 8 and 15°C). In addition, corresponding physiological data were assessed during an 11-days salinity/temperature experiment. Growth and maximal quantum yield for photosystem II fluorescence were positively affected by increased temperature during acclimation, whereas hyposalinity caused negative effects at the last day of treatment. In contrast, hyposalinity induced marked changes on the transcriptomic level. Compared to the control (8°C - SA 30), the 8°C - SA 20 exhibited the highest number of differentially expressed genes (DEGs), followed by the 0°C - SA 20. Comparisons indicate that S. latissima tends to convert its energy from primary metabolism (e.g. photosynthesis) to antioxidant activity under hyposaline stress. The increase in physiological performance at 15°C shows that S. latissima in the Arctic region can adjust and might even benefit from increased temperatures. However, in Arctic fjord environments its performance might become impaired by decreased salinity as a result of ice melting.

Alginates along the filament of the brown alga Ectocarpus help cells cope with stress.

Ectocarpus is a filamentous brown alga, which cell wall is composed mainly of alginates and fucans (80%), two non-crystalline polysaccharide classes. Alginates are linear chains of epimers of 1,4-linked uronic acids, ß-D-mannuronic acid (M) and α-L-guluronic acid (G). Previous physico-chemical studies showed that G-rich alginate gels are stiffer than M-rich alginate gels when prepared in vitro with calcium. In order to assess the possible role of alginates in Ectocarpus, we first immunolocalised M-rich or G-rich alginates using specific monoclonal antibodies along the filament. As a second step, we calculated the tensile stress experienced by the cell wall along the filament, and varied it with hypertonic or hypotonic solutions. As a third step, we measured the stiffness of the cell along the filament, using cell deformation measurements and atomic force microscopy. Overlapping of the three sets of data allowed to show that alginates co-localise with the stiffest and most stressed areas of the filament, namely the dome of the apical cell and the shanks of the central round cells. In addition, no major distinction between M-rich and G-rich alginate spatial patterns could be observed. Altogether, these results support that both M-rich and G-rich alginates play similar roles in stiffening the cell wall where the tensile stress is high and exposes cells to bursting, and that these roles are independent from cell growth and differentiation.

Immobilization of planktonic algal spores by inkjet printing.

The algal cell immobilization is a commonly used technique for treatment of waste water, production of useful metabolites and management of stock culture. However, control over the size of immobilized droplets, the population of microbes, and production rate in current techniques need to be improved. Here, we use drop-on-demand inkjet printing to immobilize spores of the alga Ecklonia cava within alginate microparticles for the first time. Microparticles with immobilized spores were generated by printing alginate-spore suspensions into a calcium chloride solution. We demonstrate that the inkjet technique can control the number of spores in an ejected droplet in the range of 0.23 to 1.87 by varying spore densities in bioink. After the printing-based spore encapsulation, we observe initial sprouting and continuous growth of thallus until 45 days of culture. Our study suggest that inkjet printing has a great potential to immobilize algae, and that the ability to control the number of encapsulated spores and their microenvironments can facilitate research into microscopic interactions of encapsulated spores.

High potassium seawater inhibits ascidian sperm chemotaxis, but does not affect the male gamete chemotaxis of a brown alga.

Male gamete chemotaxis towards the female gamete is a general strategy to facilitate the sexual reproduction in many marine eukaryotes. Biochemical studies of chemoattractants for male gametes of brown algae have advanced in the 1970s and 1980s, but the molecular mechanism of male gamete responses to the attractants remains elusive. In sea urchin, a K+ channel called the tetraKCNG channel plays a fundamental role in sperm chemotaxis and inhibition of K+ efflux through this channel by high K+ seawater blocks almost all cell responses to the chemoattractant. This signalling mechanism could be conserved in marine invertebrates as tetraKCNG channels are conserved in the marine invertebrates that exhibit sperm chemotaxis. We confirmed that high K+ seawater also inhibited sperm chemotaxis in ascidian, Ciona intestinalis (robusta), in this study. Conversely, the male gamete chemotaxis towards the female gamete of a brown alga, Mutimo cylindricus, was preserved even in high K+ seawater. This result indicates that none of the K+ channels is essential for male gamete chemotaxis in the brown alga, suggesting that the signalling mechanism for chemotaxis in this brown alga is quite different from that of marine invertebrates. Correlated to this result, we revealed that the channels previously proposed as homologues of tetraKCNG in brown algae have a distinct domain composition from that of the tetraKCNG. Namely, one of them possesses two repeats of the six transmembrane segments (diKCNG) instead of four. The structural analysis suggests that diKCNG is a cyclic nucleotide-modulated and/or voltage-gated K+ channel.

Diverse responses of sporophytic photochemical efficiency and gametophytic growth for two edible kelps, Saccharina japonica and Undaria pinnatifida, to ocean acidification and warming.

Ocean acidification and warming represent major environmental threats to kelp mariculture. In this study, sporophytic photochemical efficiency and gametophytic growth of Saccharina japonica and Undaria pinnatifida were evaluated under different pCO2 levels (360, 720, and 980 ppmv) and temperatures (5, 10, 15, and 20 °C for sporophytes; 15 and 20 °C for gametophytes). Sporophytic photochemical efficiencies of both kelps were significantly greater at 720 ppmv than at 360 and 980 ppmv. Female gametophytes of both kelps grew significantly better at 360 ppmv than at higher pCO2 levels. The growth of U. pinnatifida gametophytes was significantly greater at 20 °C than at 15 °C, while no significant difference was observed for the growth of S. japonica. These results indicate that increased pCO2 stimulated sporophytic photochemical efficiency while inhibited gametophytic growth of these kelps, which might negatively affect their seedling cultivation. U. pinnatifida exhibited higher productivity in warmer ocean than S. japonica.

Circular spectropolarimetric sensing of higher plant and algal chloroplast structural variations.

Photosynthetic eukaryotes show a remarkable variability in photosynthesis, including large differences in light-harvesting proteins and pigment composition. In vivo circular spectropolarimetry enables us to probe the molecular architecture of photosynthesis in a non-invasive and non-destructive way and, as such, can offer a wealth of physiological and structural information. In the present study, we have measured the circular polarizance of several multicellular green, red, and brown algae and higher plants, which show large variations in circular spectropolarimetric signals with differences in both spectral shape and magnitude. Many of the algae display spectral characteristics not previously reported, indicating a larger variation in molecular organization than previously assumed. As the strengths of these signals vary by three orders of magnitude, these results also have important implications in terms of detectability for the use of circular polarization as a signature of life.

Increased temperature but not pCO2 levels affect early developmental and reproductive traits of the economically important habitat-forming kelp Lessonia trabeculata.

The effects of ocean warming and ocean acidification on developmental and reproductive traits of Lessonia trabeculata were evaluated. Meiospores were cultured for 35 days in an experimental mesocosm where temperature (~15 and 19 °C) and partial CO2 pressure (pCO2, ~400 and 1300 µatm) were controlled. The results indicate that germination was reduced at 19 °C, whereas the increase of pCO2 only had effects at 15 °C. Likewise, the increase in temperature significantly affected the vegetative growth of female gametophytes. Sex ratio was not affected significantly by any of the variables studied. Fertility and reproductive success decreased by about 50% at 19 °C. The pCO2 levels had no significant effects on most early developmental traits. The results suggest that ocean warming or periodic warming events (e.g. an El Niño event) might affect the recruiting capacity of this or other similar species by affecting their early developmental stages.

UV Chromosomes and Haploid Sexual Systems.

The evolution of sex determination continues to pose major questions in biology. Sex-determination mechanisms control reproductive cell differentiation and development of sexual characteristics in all organisms, from algae to animals and plants. While the underlying processes defining sex (meiosis and recombination) are conserved, sex-determination mechanisms are highly labile. In particular, a flow of new discoveries has highlighted several fascinating features of the previously understudied haploid UV sex determination and related mating systems found in diverse photosynthetic taxa including green algae, bryophytes, and brown algae. Analyses integrating information from these systems and contrasting them with classical XY and ZW systems are providing exciting insights into both the universality and the diversity of sex-determining chromosomes across eukaryotes.

Physiological responses to variations in grazing and light conditions in native and invasive fucoids.

Poor physiological acclimatization to climate change has led to shifts in the distributional ranges of various species and to biodiversity loss. However, evidence also suggests the relevance of non-climatic physical factors, such as light, and biotic factors, which may act in interactive or additive way. We used a mechanistic approach to evaluate the ecophysiological responses of four seaweed species (three dominant intertidal fucoids, Fucus serratus, Ascophyllum nodosum, Bifurcaria bifurcata, and the invasive Sargassum muticum) to different conditions of grazing, light irradiance and ultraviolet (UV) radiation. We performed a large-scale mesocosm experiment with a total of 800 individual thalli of macroalgae. The factorial experimental design included major algal traits, photoacclimation, nutrient stoichiometry and chemical defence as response variables. Few significant effects of the factors acting alone or in combination were observed, suggesting a good capacity for acclimatization in all four species. The significant effects were generally additive and there were no potentially deleterious synergistic effects between factors. Fucus serratus, a species currently undergoing a drastic contraction of its southern distribution limit in Europe, was the most strongly affected species, showing overall lower photosynthetic efficiency than the other species. The growth rate of F. serratus decreased when UV radiation was filtered out, but only in the presence of grazers. Moreover, more individuals of this species tended to reach maturity in the absence of grazers, and the nitrogen content of tissues decreased under full-spectrum light. Only the phlorotannin content of tissues of B. bifurcata and of exudates of A. nodosum, both slow-growing species, were positively affected by respectively removal of UVB radiation and the presence of grazers. The findings for S. muticum, a well-established invasive seaweed across European coasts, suggested similar physiological response of this fast-growing species to different levels of grazing activity and light quality/intensity. As expected, this species grew faster than the other species. Bifurcaria bifurcata and A. nodosum only showed minor effects of light quality and grazing on phlorotannins content, which suggests good resistance of these two long-lived species to the experimental conditions. Mechanistic approaches that are designed to analyse interactive effects of physical and biotic factors provide an understanding of physiological responses of species and help to improve the confidence of predictive distribution models.