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Polyphenols from Stypopodium zonale (Phaeophyceae): Intrapopulational variation, induction by simulated herbivory, and epibiosis effects
Aquatic Botany 111 (2013) 125–129
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Polyphenols from Stypopodium zonale (Phaeophyceae): Intrapopulational variation, induction by simulated herbivory, and epibiosis effects Glaucia Ank, Bernardo Antonio Perez da Gama, Renato Crespo Pereira ∗ Universidade Federal Fluminense, Departamento de Biologia Marinha, 24020-140, Outeiro de São João Batista, s/n◦ , Centro, Niterói, Rio de Janeiro, Brazil
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Article history: Received 21 August 2012 Received in revised form 14 June 2013 Accepted 20 June 2013 Available online 10 July 2013 Keywords: Chemical defense Phlorotannins Stypopodium zonale Tropical macroalga
a b s t r a c t Phlorotannins are exclusive chemical components of brown macroalgae active against herbivores and fouling, but several dynamic aspects of these chemicals remain underexplored or unexplored, such as intra-populational variability, response to wounding damage in tropical macroalgae, and correlation to levels of coverage by epibionts on the macroalgal surface. In this study, we investigated (i) the variation of phlorotannin content in response to artificial damage (induction experiments), (ii) the relationship between phlorotannin contents and epibiosis levels (low, medium, or high percentage of cover on the macroalga Stypopodium zonale), and (iii) the variation of phlorotannins content within-population of this tropical macroalga. Clipping elicited an increase in phlorotannin concentration only after 2 days. In addition, we detected higher phlorotannin concentrations in individuals exposed to low and medium epibiosis coverage and a lower concentration of these chemicals in individuals with a high level of epibiosis. We detected a broad variation in phlorotannin content among S. zonale individuals, from 0.63 to 3.24% (dry weight). These results suggest that the varying levels of phlorotannins found in the tropical macroalga S. zonale may result from different levels of exposure to herbivores in nature. Similarly, our results suggest that S. zonale phlorotannins may be effective antifoulants. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Several species of macroalgae are more or less susceptible to herbivory due to the production of a diverse array of defensive chemicals, encompassing species from Antarctic polar waters (Amsler et al., 2008) to those from the tropics (Pereira and Da Gama, 2008). Among brown macroalgae (Phaeophyceae), quantitative phlorotannin levels, large polymers of phloroglucinol (1,3,5-tryhydroxybenzene) are often associated with a defensive property against herbivores (Targett and Arnold, 1998, 2001). Although most frequently associated with its concentration (Steinberg and Van Altena, 1992; Pereira and Yoneshigue-Valentin, 1999), the extent to which brown algal phlorotannins affect palatability to herbivores also seems to depend upon compound specificity or molecular structure (Boettcher and Targett, 1992). However, the effectiveness of phlorotannins as feeding inhibitors is still controversial (see Amsler and Fairhead, 2006), but some studies have demonstrated grazing reduction or inhibition (e.g., Geiselmann and McConnell, 1981; Pavia and Toth, 2000). Others
∗ Corresponding author. E-mail addresses: [email protected], [email protected] (R.C. Pereira). 0304-3770/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquabot.2013.06.007
have shown negligible responses or even increased herbivory (Steinberg and Van Altena, 1992; Kubanek et al., 2004). In addition to a defensive property against herbivores, phlorotannins from brown macroalgae play a role also as an antifouling agent. For example, phlorotannins from Fucus sp. have the potential to inhibit settlement of the barnacle Balanus improvisus (Wikström and Pavia, 2004), and those from Sargassum tenerrimum may inhibit larval settlement of the polychaete Hydroides elegans (Lau and Qian, 1997). Those from Sargassum vulgare inhibit the attachment of the mussel Perna perna (Plouguerné et al., 2010). In fact, macroalgal phlorotannins may be multifunctional or multiecological chemicals with putative roles in herbivore deterrence (Pereira and Yoneshigue-Valentin, 1999), antifouling (Plouguerné et al., 2012), as antioxidants (Cruces et al., 2012), and in UV protection (Maschek and Baker, 2008); they also have a primary function as cell wall components (Schoenwaelder and Clayton, 1999). The amount of phlorotannins may be variable, constituting up to 15% of the dry weight of brown macroalgae (Ragan and Glombitza, 1986), which can be influenced by a number of environmental factors. For example, these chemicals may be regulated by temporal variability in physical factors such as salinity (Ragan and Glombitza, 1986), nutrient regime (Van Alstyne and Pelletreau,
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2000), UV exposure (Swanson and Druehl, 2002), and water movement (Dayton, 1985). In addition, grazers or mechanical wounding may also increase phlorotannins levels as part of defense or healing mechanisms (Hammerstrom et al., 1998; Pavia and Toth, 2000; Hemmi et al., 2004). The evaluation of phlorotannins from brown macroalgae as a defense mechanism by simulating single wounding events has yielded conflicting or mixed results. In the order Fucales, wounding simulations induced (Yates and Peckol, 1993) or did not induce (Steinberg, 1994; Pavia et al., 1997) responses in phlorotannin content. For kelps, rapid induction of phlorotannins was detected in 4 out of 5 species from Washington, San Juan Island (Hammerstrom et al., 1998), and Australian Ecklonia radiata (Lüder and Clayton, 2004), but did not induce a response in other kelps (Steinberg, 1994; Martínez, 1996; Toth and Pavia, 2002). In general, phlorotannins occur in high concentrations (>2% DW) in temperate brown macroalgae from Australasia (Estes and Steinberg, 1988), but they are found in smaller amounts in North American species belonging to the same order (Steinberg, 1989; Hay and Steinberg, 1992). Studies on the ecological roles of these compounds are therefore better explored in temperate rather than tropical environments. To our knowledge, no previous papers studied either the variation in contents of these compounds or the wounding effects in tropical brown macroalgae, where grazing pressure is constant and intense, and where strongly selecting species are chemically defended (Pereira and Da Gama, 2008). The hypotheses tested in the present study are as follows: (1) Artificial damage simulating herbivory induces a higher phlorotannin concentration in this brown macroalga; (2) phlorotannins in Stypopodium zonale vary with epibiosis level; and (3) there is a natural variation of phlorotannins in different individuals of S. zonale from the same population. 2. Materials and methods 2.1. Study site and organisms The tropical brown macroalga S. zonale (J. V. Lamouroux) Papenfuss is found in abundance along the sublittoral of the Brazilian coast (Menezes-Széchy and de Paula, 2010). Specimens of this macroalga were collected at Praia do Forno, Armac¸ão de Búzios (22◦ 45 S, 41◦ 52 W, Southeastern Brazil, Rio de Janeiro), in shallow waters about 3 m deep. In February 2011, 40 adult S. zonale individuals with no signs of herbivore attack or excessive epibiosis were collected by hand during free diving and transported to the Chemical Ecology Laboratory at Universidade Federal Fluminense. All individuals were collected with thallus intact, including holdfast, and there was no physical damage on them. From these 40 individuals, 10 were carefully cleaned to remove epibionts and kept in aquaria for 3 days to acclimatize to laboratory conditions for further use in the simulated herbivory experiment. The remaining 30 individuals were separated by their distinct levels of epibiosis, then cleaned and frozen for later lyophilization. During another collection event in January 2012, 32 adult S. zonale individuals were collected at the same site for evaluation of the intra-populational phlorotannin contents. The same collection procedure was used to avoid damage to the thallus. The samples from this collection were transported to the laboratory, where they were carefully cleaned and immediately frozen for further lyophilization. 2.2. Extraction After lyophilization, S. zonale specimens were ground into powder and submitted to extraction of phlorotannins. To maximize the extraction of these chemicals, lipids were removed from the samples prior to the extraction using 1 mL hexane for 3 min, 3 times, as
described previously (Koivikko et al., 2007). The extraction used 10 mL acetone:water (7:3) for 100 mg of alga (dry weight) over 2 h under magnetic agitation, centrifuged for 10 min at 3500 rpm and filtered. After removal of the acetone at room temperature, an aqueous residue that was centrifuged for 10 min at 3500 rpm was obtained; volume was measured, and the residue was frozen for analysis of phlorotannin content. 2.3. Quantification of phlorotannins In order to quantify the phlorotannin content in S. zonale, we used the Folin–Ciocalteau (FC) method, where the FC reagent 1 N (Sigma–Aldrich) was added to an aliquot of diluted extract, and after 3 min, sodium carbonate 20% and water were added to the solution. The reaction was kept in the dark for 45 min, after which phlorotannins were quantified in a spectrophotometer Schimadzu UV1800 at 750 nm using a standard calibration curve obtained with phloroglucinol (r2 = 0.99). Three aliquots of each extract were prepared and analyzed. 2.4. Induction of phenolic production by simulated herbivory To understand the effect of herbivory on the phlorotannin content, we simulate herbivory in 10 S. zonale individuals kept in aquaria after collection for acclimatization to lab conditions (salinity = 35 + 1; temperature = 20 + 1 ◦ C). Artificial damage simulated attacks of herbivores. After 3 days, each S. zonale individual was divided in two fragments, each possessing about 15.0 g wet weight – one control fragment and one treatment. Both the control and treatment suffered one cut each, so any possible effect of this damage was the same in both. Using the same individual in treatment and control allows us to affirm that observed differences in phlorotannin contents would be only due to the treatment as there would be no genetic variation in phlorotannin content or tissue growth. The treatment fragments were submitted to simulate herbivory by removing 10 small circles (5 mm diameter) with a metal tube from the thallus of S. zonale. The injury was as similar as possible to a grazer’s damage (e.g., gastropods). After 2 and 4 days from the clipping event, one piece of both the control and treatment algae were removed for quantification of phlorotannins. 2.5. Epibiosis and phlorotannin content S. zonale individuals were separated according to level of epibionts living on the thallus. We visually established three distinct levels of epibiosis: low (less than 10% of the thallus covered by epibionts), medium (between 10 and 30%), and high (above 30%). Epibionts were not identified at the species level, but they were primarily determined as bacterial biofilm, polychaetes, algae, bryozoans, and hydrozoans. The S. zonale samples were cleaned to remove epibionts, lyophilized, and ground into powder. The powder from the individuals exposed to the same level of epibiosis were joined, homogenized, and extracted (n = 6 extracts per level). Thus, the phlorotannin content corresponded to algal tissue from both thallus parts (holdfasts and blades) of the different S. zonale individuals. 2.6. Statistical analysis The intrapopulational variability was analyzed by histogram distribution of the phlorotannin concentration in the different S. zonale individuals. The results of the simulated herbivory experiments were compared day-by-day with dependent t-tests as control and treatment fragments were obtained from the same individual, characterizing dependence within treatments. Content
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0,7
0,8 0,7 0,6 0,5
Unclipped Clipped
0,4 0,3 0,2 0,1 Day 2
Day 4
Fig. 1. Total phlorotannin content (expressed as % per dry weight) of S. zonale. unclipped (gray bars) and clipped (black bars) in the simulated herbivory experiment (mean + standard deviation). On day 2, “*” refers to significant differences between clipped and unclipped (n = 10; p = 0.038; dependent t-test). On day 4, no significant differences were observed (n = 10; p = 0.25; dependent t-test).
Total phenolic content (% DW)
Total phenolic content (% DW)
0,9
0
127
Low 0,6
Medium High
0,5 0,4 0,3 0,2 0,1 0
Levels of epibiosis
Fig. 2. Total phlorotannin content (expressed as % per dry weight) of S. zonale. possessing low (light gray bar), medium (dark gray bar), and high (black bar) levels of epibiosis (mean + standard deviation). The “*” refers to significant difference (n = 6; p = 0.0006; ANOVA).
of phlorotannins and levels of epibiosis were compared by one-way ANOVA. We considered the level of significance to be 5% (˛ = 0.05). 3. Results 3.1. Induction of phlorotannin content in S. zonale by simulated herbivory After 2 days of the clipping procedure, we observed a significant difference in phlorotannin content among control and treatment S. zonale individuals; specimens submitted to damage had significantly higher phlorotannin content than corresponding control individuals (n = 10; p = 0.038; t = −2.31; dependent t-test, Fig. 1). However, we observed an opposite non-significant trend (0.65% in control individuals and 0.50% in treatment, Fig. 1) on day 4. 3.2. Levels of epibiosis and phlorotannin content in S. zonale When the phlorotannin content of S. zonale with different levels of epibiosis were compared, we observed that the specimens of these algae exhibiting a high level of epibiosis had significantly lower amounts of these chemicals (0.368 + 0.022%; p < 0.0001; F = 12.79; ANOVA). However, S. zonale individuals possessing low and medium levels of epibionts were not significantly different in phlorotannin contents (0.540 + 0.051% and 0.526 + 0.098%, respectively; Fig. 2). 3.3. Intrapopulational content of phlorotannins in S. zonale The mean concentration of phlorotannins from 32 S. zonale individuals analyzed was 1.74%, but it varied from 0.63 to 3.24% of algal dry weight (Fig. 3). Despite the wide range of phlorotannin concentration, more than half of the individuals analyzed had values of this chemical around the mean (within 1.4–1.8% of phenolic DW). 4. Discussion 4.1. Induction of phlorotannin content in S. zonale by simulated herbivory Several simulated herbivory experiments have already investigated the effects on phlorotannin concentration (e.g., Yates and
Fig. 3. Intrapopulation variability of phlorotannin concentrations in S. zonale individuals from Praia do Forno, Armac¸ão dos Búzios, RJ, Brazil. Values are expressed in % per dry weight.
Peckol, 1993; Halm et al., 2011), but none involved tropical macroalgae. In this study, we detected higher phlorotannin content in specimens of S. zonale submitted to simulate herbivory when compared with uninjured algae 2 days after wounding. Rapid phlorotannin induction was already described for Agarum fibriatum, Pleurophycys gardneri, Laminaria complanata, and L. groenlandica, which showed higher phenolic content 1–3 days after wounding (Hammerstrom et al., 1998). However, 4 days after the clipping effect, we observed a drastic reduction of phlorotannin content in injured S. zonale. Hammerstrom et al. (1998) also reported rapid relaxation of phlorotannin induction after 7 days, so the relaxation in S. zonale can be considered even faster. This seems an interesting result because faster relaxation after an herbivore attack may be an advantage in tropical environments, where herbivore activity is not seasonally predictable. Mechanical or artificial damage have been used as simulations or substitutes for natural herbivory, but sometimes it fails as only the damage to the tissue itself is represented (e.g., Pavia and Toth, 2000), or produces contradictory results, as observed in Fucus vesiculosus, by decreasing palatability (Hemmi et al., 2004) rather than changing it (Rohde et al., 2004), or even increasing the
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susceptibility to herbivory (Haavisto et al., 2010). However, in this experiment, clipping was sufficient to evidence induced defense in the tropical macroalga S. zonale. S. zonale is known by the production of mixed-origin diterpenes (compounds exhibiting terpenoidic and phenol portions) as a chemical defense against herbivores (e.g., Littler et al., 1986; Hay et al., 1988), including specimens from Brazilian littoral (Pereira et al., 2004). Why does S. zonale produce terpenes as constitutive and polyphenols as inducible defenses? One explanation for the phlorotannin induction is that it is related to the healing process of the wound, and not an anti-herbivore response, as showed by Lüder and Clayton (2004). Further studies will be necessary to investigate the existence of a possible trade-off between biogenetically related compounds (phenolics and mixed-origin diterpenes). This process or change may be also affected by known metabolic turnover (Arnold and Targett, 2000). 4.2. Levels of epibiosis and phlorotannin content in S. zonale The S. zonale individuals exhibiting a high degree of epibiosis – i.e., with more than 30% of the thalli covered by epibionts – had a lower phlorotannin concentration than individuals exhibiting fewer epibionts. At first, this relationship between phlorotannin content and epibiosis on S. zonale could reinforce the experimental evidence that phenolics from brown macroalgae exhibit high antifouling activity. As UV-radiation can also be an inducing factor for phlorotannins (see Cruces et al., 2013), our results could be also due to higher UV absorption on the thallus of individuals with low and medium epibiosis levels, as the high level of epibiosis could decrease the absorption of UV radiation. Although phlorotannins of brown macroalgae have been previously proposed as antifoulants (Sieburth and Conover, 1965), they are water-soluble compounds. Unlike lipophylic terpenoids, they would be less effective defenses against epiphytes (Jennings and Steinberg, 1997). Indeed, the responses of phlorotannin concentration to epibiosis level conflict. For example, a decrease in growth and increase in fouling were observed in F. vesiculosus, but levels of epibiosis were not correlated to phenolic concentrations (Honkanen and Jormalainen, 2005). Phlorotannin concentration and fouling resistance were positively correlated for F. vesiculosus (Jormalainen et al., 2008). However, no such correlation was found for S. vulgare (Plouguerné et al., 2012); instead, there was a rise in antifouling activity accompanied by a drop in total phenolic content. 4.3. Intrapopulational content of phlorotannins in S. zonale We observed broad intrapopulational variability in phlorotannin content in S. zonale individuals. In general, the concentration of phlorotannins in brown macroalgae is extremely variable based on scales, such as time-days to months (see Jormalainen and Honkanen, 2008), between cell layers to within-thallus (e.g., Pereira and Yoneshigue-Valentin, 1999), and geographic (Steinberg, 1989; Targett et al., 1992; Van Alstyne et al., 1999). Because the efficacy of phlorotannins from brown macroalgae as a defensive compound is often concentration-dependent (Pereira and Yoneshigue-Valentin, 1999), and higher concentrations (more than 2.0% of dry weight) are active against herbivores (Ragan and Glombitza, 1986), it is likely that only 25% of the studied S. zonale population could be chemically defended from consumers (i.e., only those individuals possessing more than 2.0% of phlorotannins). But why do S. zonale individuals belonging to the same population produce distinct chemical defense concentrations? Why do all populations not produce large amounts of defensive chemicals against herbivory? For plants, the diversity of chemical phenotypes has been explained by a combination of genetic (Berembaum and Zangerl, 1992), developmental (Bowers and Stamp, 1993),
and environmental (Agrell et al., 2000) factors. However, due to peculiar characteristics of the macroalgae, we can presuppose that life history (Vergés et al., 2008), age structure (Paul and Van Alstyne, 1988b), environmental heterogeneity (Matlock et al., 1999), and limitation on gene flow among the natural population of macroalgae (Faugeraon et al., 2004) can determine the variability and the spatial structure of the secondary chemistry. Despite the known possibility of ontogenetic variability in the production of defensive chemicals in macroalgae (e.g., Paul and Van Alstyne, 1988a), we believe that this is not the reason for the high variability in phlorotannin contents found in the S. zonale individuals studied because only adult specimens were analyzed. However, S. zonale possesses alternating isomorphic life-stages that can be found simultaneously in the field, and this aspect remains a possible interference or explanation of the variable contents of phlorotannins since we may have collected both gametophytes and sporophytes as they are morphologically equal. A new study analyzing individual conditions in the field, like sun exposure, neighboring organisms, and depth could elucidate the reasons for such high intrapopulational variability. 5. Conclusions Phlorotannin content in S. zonale showed intra-populational variability and was higher in individuals submitted to simulated herbivory 2 days after the damage, but after 4 days, there was no difference. Phlorotannins also varied according to epibiosis cover, occurring in lower concentrations in individuals with higher level of epibiosis. Further studies are needed to cast more light on the causes of intra-populational variability in chemical defense; why S. zonale, a known producer of defensive terpenoids, would also produce polyphenols as induced defense after mechanical damage; and the effect of epibiosis on phlorotannins. Acknowledgments The authors thank CNPq and FAPERJ for research grants. GA also thanks CAPES for a doctoral scholarship. We also thank Daneila B. Sudatti, Leonardo M.S. Lima and Ricardo Rogers for help in algae collecting and designing experiments. References Agrell, J., Mcdonald, E.P., Lindroth, R.L., 2000. Effects of CO2 and light on tree phytochemistry and insect performance. Oikos 88, 259–272. Amsler, C.D., Fairhead, V.A., 2006. Defensive and sensory chemical ecology of brown algae. Adv. Bot. Res. 43, 1–91. Amsler, C.D., McClintock, J.B., Baker, B.J., 2008. Macroalgal chemical defenses in polar marine communities. In: Amsler, C.D. (Ed.), Algal chemical ecology. SpringerVerlag, Berlin/Heidelberg, pp. 91–103. Arnold, T.M., Targett, N.M., 2000. Evidence for metabolic turnover of polyphenolics in tropical brown algae. J. Chem. Ecol. 26, 1393–1410. Berembaum, M.R., Zangerl, A.R., 1992. Genetics of physiological and behavioral resistance to host furanocoumarins in Parsnip webworm. Evolution 46, 1373–1384. Boettcher, A.A., Targett, N.M., 1992. Role of polyphenolic molecular size in reduction of assimilation efficiency in Xiphister mucosus. Ecology 74, 891–903. Bowers, M.D., Stamp, N.E., 1993. Effects of plant age, genotype, and herbivory on Plantago performance and chemistry. Ecology 74, 1778–1791. Cruces, E., Huovinen, P., Gómez, I., 2012. Phlorotannin and antioxidant responses upon short-term exposure to UV radiation and elevated temperature in three South Pacific kelps. Photochem. Photobiol. 88, 58–66. Cruces, E., Huovinen, P., Gómez, I., 2013. Interactive effects of UV radiation and enhanced temperature on photosyntesis, phlorotannin induction and antioxidant activities of two sub-Antartic brown algae. Mar. Biol. 160, 1–13. Dayton, P.K., 1985. Ecology of kelp communities. Ann. Rev. Ecol. Syst. 16, 215–245. Estes, J.A., Steinberg, P.D., 1988. Predation, herbivory, and kelp evolution. Paleobiology 14, 19–36. Faugeraon, S., Martinez, E.A., Correa, J.A., Cardenas, L., Destombe, C., Valero, M., 2004. Reduced genetic diversity and increased population differentiation in peripheral and overharvested populations of Gigartina skottsbergii (Rhodophyta, Gigartinales) in southern Chile. J. Phycol. 40, 454–462.
G. Ank et al. / Aquatic Botany 111 (2013) 125–129 Geiselmann, J.A., McConnell, O.J., 1981. Polyphenols in brown algae Fucus vesiculosus and Ascophyllum nodosum: Chemical defenses against the marine herbivorous snail, Littorina littorea. J. Chem. Ecol. 6, 1115–1133. Haavisto, F., Välikangas, T., Jormalainen, V., 2010. Induced resistance in a brown alga: phlorotannins, genotypic variation and fitness costs for the crustacean herbivore. Oecologia 162, 685–695. Halm, H., Lüder, U.H., Wiencke, C., 2011. Induction of phlorotannins through mechanical wounding and radiation conditions in the brown macroalga Laminaria hyperborea. Eur. J. Phycol. 46, 16–26. Hammerstrom, K., Dethier, M.N., Duggins, D.O., 1998. Rapid phlorotannin induction and relaxation in five Washington kelps. Mar. Ecol. Prog. Ser. 165, 293–305. Hay, M.E., Steinberg, P.D., 1992. The chemical ecology of plant–herbivore interactions in marine versus terrestrial communities. In: Rosenthal, G., Berembaum, M. (Eds.), Herbivores: their interaction with secondary plant metabolits: ecological and evolutionary processes. Academic Press, New York, pp. 372–414. Hay, M.E., Duffy, J.E., Fenical, W., 1988. Seaweed chemical defenses: among compound and among-herbivore variance. In: Proc. 6th Int. Coral Reef Congr., vol. 3, pp. 43–48. Hemmi, A., Honkanen, T., Jormalainen, V., 2004. Inducible resistance to herbivory in Fucus vesiculosus – duration, spreading and variation with nutrient availability. Mar. Ecol. Prog. Ser. 273, 109–120. Honkanen, T., Jormalainen, V., 2005. Genotypic variation in tolerance and resistance to fouling in the brown alga Fucus vesiculosus. Oecologia 144, 196–205. Jennings, J.G., Steinberg, P.D., 1997. Phlorotannins versus other factors affecting epiphyte abundance on the kelp Ecklonia radiata. Oecologia 109, 461–473. Jormalainen, V., Honkanen, T., 2008. Macroalgal chemical defenses and their roles in structuring temperate marine communities. In: Amsler, C.D. (Ed.), Algal chemical ecology. Springer-Verlag, Berlin/Heidelberg, pp. 57–89. Jormalainen, V., Wikström, S.A., Honkanen, T., 2008. Fouling mediates grazing: intertwining of resistance to multiple enemies in the brown alga Fucus vesiculosus. Oecologia 155, 559–569. Koivikko, R., Loponen, J., Pihlaja, K., Jormalainen, V., 2007. High-performance liquid chromatographic analysis of phlorotannins from the brown alga Fucus vesiculosus. Phytochem. Anal. 18, 326–332. Kubanek, J., Lester, S., Fenical, W., Hay, M.E., 2004. Ambiguous role of phlorotannins as chemical defenses in the brown alga Fucus vesiculosus. Mar. Ecol. Prog. Ser. 277, 79–93. Lau, S.C.K., Qian, P.Y., 1997. Phlorotannins and related compounds as larval settlement inhibitors of the tube-building polychaete Hydroides elegans. Mar. Ecol. Prog. Ser. 159, 219–227. Littler, M.M., Taylor, P.R., Littler, D.S., 1986. Plant defense associations in the marine environment. Coral Reefs 5, 63–71. Lüder, U.H., Clayton, M.N., 2004. Induction of phlorotannins in the brown alga Ecklonia radiata (Laminariales, Phaophyta) in response to simulated herbivory – the first microscopic study. Planta 218, 928–937. Martínez, E.A., 1996. Micropopulation differentiation in phenol content and susceptibility to herbivory in the Chilean kelp Lessonia nigrescens (Phaeophyta, Laminariales). Hydrobiologia 327, 205–211. Maschek, J.A., Baker, B.J., 2008. The chemistry of algal secondary metabolism. In: Amsler, C.D. (Ed.), Algal chemical ecology. Springer-Verlag, Berlin/Heidelberg, pp. 1–24. Matlock, D.B., Ginsburg, D.W., Paul, V.J., 1999. Spatial variability in secondary metabolite production by the tropical red alga Portieria hornemannii. Hydrobiologia 399, 267–273. Menezes-Széchy, M.T., de Paula, J.C., 2010. Phaeophyceae. In: Forzza, R.C. (Ed.), Catálogo de plantas e fungos do Brasil, vol. 1. Andrea Jakobsson Estúdio/Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, Rio de Janeiro, pp. 404–408. Paul, V.J., Van Alstyne, K.L., 1988a. Chemical defense and chemical variation in some tropical Pacific species of Halimeda (Chlorophyta, Halimedaceae). Coral Reefs 6, 263–270. Paul, V.J., Van Alstyne, K.L., 1988b. Use of ingested alga diterpenoids by Elysia halimedae Macnae (Opisthobranchia: Ascoglossa) as antipredator defenses. J. Exp. Mar. Biol. Ecol. 119, 15–29.
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Pavia, H., Toth, G., 2000. Inducible chemical resistance to herbivory in the brown seaweed Ascophyllum nodosum. Ecology 81, 3212–3225. Pavia, H., Cervin, G., Lindgren, A., Aberg, P., 1997. Effects of UV-B radiation and simulated herbivory on phlorotannins in the brown alga Ascophyllum nodosum. Mar. Ecol. Prog. Ser. 157, 139–146. Pereira, R.C., Da Gama, B.A.P., 2008. Macroalgal chemical defenses and their roles in structuring tropical marine communities. In: Amsler, C.D. (Ed.), Algal chemical ecology. Springer-Verlag, Berlin/Heidelberg, pp. 25–55. Pereira, R.C., Yoneshigue-Valentin, Y., 1999. The role of polyphenols from tropical brown alga Sargassum furcatum on the feeding by amphipod herbivores. Bot. Mar. 42, 441–444. Pereira, R.C., Soares, A.R., Teixeira, V.L., Da Gama, B.A.P., Villac¸a, R., 2004. Variation in chemical defenses against herbivory in Southwestern Atlantic Stypopodium zonale (Phaeophyta). Bot. Mar. 47, 202–208. Plouguerné, E., Hellio, C., Cesconetto, C., 2012. Within-thallus variation in polyphenolic content and antifouling activity in Sargassum vulgare. J. Appl. Phycol. 24, 1629–1635. Plouguerné, E., Hellio, C., Cesconetto, C., Thabard, M., Mason, K., Véron, B., Pereira, R.C., Da Gama, B.A.P., 2010. Antifouling activity as a function of population variation in Sargassum vulgare from the littoral of Rio de Janeiro (Brazil). J. Appl. Phycol. 22, 717–724. Ragan, M.A., Glombitza, K.W., 1986. Phlorotannins, brown algal polyphenols. In: Round, F.E., Chapman, D.J. (Eds.), Progress in Phycological Research. Biopress, Bristol, pp. 129–241. Rohde, S., Molis, M., Wahl, M., 2004. Regulation of anti-herbivore defence by Fucus vesiculosus in response to various cues. J. Ecol. 92, 1011–1018. Schoenwaelder, M.E.A., Clayton, M.N., 1999. The presence of phenolic compounds in isolated cell walls of brown algae. Phycologia 38, 161–166. Sieburth, J.M., Conover, J.T., 1965. Sargassum tannin, an antibiotic which retards fouling. Nature 208, 52–53. Steinberg, P.D., 1989. Biogeographical variation in brown algal polyphenolics and other secondary metabolites: comparison between temperate Australasia and North America. Oecologia 78, 373–382. Steinberg, P.D., 1994. Lack of short-term induction of phlorotannins in the Australasian brown algae Ecklonia radiata and Sargassum vestitum. Mar. Ecol. Prog. Ser. 112, 129–133. Steinberg, P.D., Van Altena, I.V., 1992. Tolerance of marine invertebrate herbivores to brown algal phlorotannins in Temperate Australasia. Ecol. Monogr. 62, 189–222. Swanson, A.K., Druehl, L.D., 2002. Induction, exudation and the protective role of kelp phlorotannins. Aq. Bot. 73, 241–253. Targett, N.M., Arnold, T.M., 1998. Predicting the effects of brown algal phlorotannins on marine herbivores in tropical and temperate oceans. J. Phycol. 34, 195–205. Targett, N.M., Arnold, T.M., 2001. Effects of secondary metabolites on digestion in marine herbivores. In: McClintock, J.B., Baker, B.J. (Eds.), Marine chemical ecology. CRC Press, Boca Raton, pp. 391–411. Targett, N.M., Coen, L.D., Boettcher, A.A., Tanner, C.E., 1992. Biogeographic comparisons of marine algal polyphenols: evidence against latitudinal trend. Oecologia 89, 464–470. Toth, G.B., Pavia, H., 2002. Lack of phlorotannin induction in the kelp Laminaria hyperborea in response to grazing by two gastropod herbivores. Mar. Biol. 140, 403–409. Van Alstyne, K.L.K.L., Pelletreau, K.N., 2000. Effects of nutrient on growth and phlorotannin production in Fucus gardineri embryos. Mar. Ecol. Prog. Ser. 206, 33–43. Van Alstyne, K.L., McCarthy III, J.J., Hustead, C.L., Duggins, D.O., 1999. Geographic variation in polyphenolic levels of Northeastern Pacific kelps and rockweeds. Mar. Biol. 133, 371–379. Vergés, A., Paul, N.A., Steinberg, P.D., 2008. Sex and life-history stage alter herbivore responses to a chemically defended red alga. Ecology 89, 1334–1343. Wikström, S.A., Pavia, H., 2004. Chemical settlement inhibition versus postsettlement mortality as an explanation for differential fouling of two congeneric seaweeds. Oecologia 138, 223–230. Yates, J.L., Peckol, P., 1993. Effects of nutrient availability and herbivory on polyphenolics in the seaweed Fucus vesiculosus. Ecology 74, 1757–1766.
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Polyphenols from Stypopodium zonale (Phaeophyceae): Intrapopulational variation, induction by simulated herbivory, and epibiosis effects Glaucia Ank, Bernardo Antonio Perez da Gama, Renato Crespo Pereira ∗ Universidade Federal Fluminense, Departamento de Biologia Marinha, 24020-140, Outeiro de São João Batista, s/n◦ , Centro, Niterói, Rio de Janeiro, Brazil
a r t i c l e
i n f o
Article history: Received 21 August 2012 Received in revised form 14 June 2013 Accepted 20 June 2013 Available online 10 July 2013 Keywords: Chemical defense Phlorotannins Stypopodium zonale Tropical macroalga
a b s t r a c t Phlorotannins are exclusive chemical components of brown macroalgae active against herbivores and fouling, but several dynamic aspects of these chemicals remain underexplored or unexplored, such as intra-populational variability, response to wounding damage in tropical macroalgae, and correlation to levels of coverage by epibionts on the macroalgal surface. In this study, we investigated (i) the variation of phlorotannin content in response to artificial damage (induction experiments), (ii) the relationship between phlorotannin contents and epibiosis levels (low, medium, or high percentage of cover on the macroalga Stypopodium zonale), and (iii) the variation of phlorotannins content within-population of this tropical macroalga. Clipping elicited an increase in phlorotannin concentration only after 2 days. In addition, we detected higher phlorotannin concentrations in individuals exposed to low and medium epibiosis coverage and a lower concentration of these chemicals in individuals with a high level of epibiosis. We detected a broad variation in phlorotannin content among S. zonale individuals, from 0.63 to 3.24% (dry weight). These results suggest that the varying levels of phlorotannins found in the tropical macroalga S. zonale may result from different levels of exposure to herbivores in nature. Similarly, our results suggest that S. zonale phlorotannins may be effective antifoulants. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Several species of macroalgae are more or less susceptible to herbivory due to the production of a diverse array of defensive chemicals, encompassing species from Antarctic polar waters (Amsler et al., 2008) to those from the tropics (Pereira and Da Gama, 2008). Among brown macroalgae (Phaeophyceae), quantitative phlorotannin levels, large polymers of phloroglucinol (1,3,5-tryhydroxybenzene) are often associated with a defensive property against herbivores (Targett and Arnold, 1998, 2001). Although most frequently associated with its concentration (Steinberg and Van Altena, 1992; Pereira and Yoneshigue-Valentin, 1999), the extent to which brown algal phlorotannins affect palatability to herbivores also seems to depend upon compound specificity or molecular structure (Boettcher and Targett, 1992). However, the effectiveness of phlorotannins as feeding inhibitors is still controversial (see Amsler and Fairhead, 2006), but some studies have demonstrated grazing reduction or inhibition (e.g., Geiselmann and McConnell, 1981; Pavia and Toth, 2000). Others
∗ Corresponding author. E-mail addresses: [email protected], [email protected] (R.C. Pereira). 0304-3770/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquabot.2013.06.007
have shown negligible responses or even increased herbivory (Steinberg and Van Altena, 1992; Kubanek et al., 2004). In addition to a defensive property against herbivores, phlorotannins from brown macroalgae play a role also as an antifouling agent. For example, phlorotannins from Fucus sp. have the potential to inhibit settlement of the barnacle Balanus improvisus (Wikström and Pavia, 2004), and those from Sargassum tenerrimum may inhibit larval settlement of the polychaete Hydroides elegans (Lau and Qian, 1997). Those from Sargassum vulgare inhibit the attachment of the mussel Perna perna (Plouguerné et al., 2010). In fact, macroalgal phlorotannins may be multifunctional or multiecological chemicals with putative roles in herbivore deterrence (Pereira and Yoneshigue-Valentin, 1999), antifouling (Plouguerné et al., 2012), as antioxidants (Cruces et al., 2012), and in UV protection (Maschek and Baker, 2008); they also have a primary function as cell wall components (Schoenwaelder and Clayton, 1999). The amount of phlorotannins may be variable, constituting up to 15% of the dry weight of brown macroalgae (Ragan and Glombitza, 1986), which can be influenced by a number of environmental factors. For example, these chemicals may be regulated by temporal variability in physical factors such as salinity (Ragan and Glombitza, 1986), nutrient regime (Van Alstyne and Pelletreau,
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2000), UV exposure (Swanson and Druehl, 2002), and water movement (Dayton, 1985). In addition, grazers or mechanical wounding may also increase phlorotannins levels as part of defense or healing mechanisms (Hammerstrom et al., 1998; Pavia and Toth, 2000; Hemmi et al., 2004). The evaluation of phlorotannins from brown macroalgae as a defense mechanism by simulating single wounding events has yielded conflicting or mixed results. In the order Fucales, wounding simulations induced (Yates and Peckol, 1993) or did not induce (Steinberg, 1994; Pavia et al., 1997) responses in phlorotannin content. For kelps, rapid induction of phlorotannins was detected in 4 out of 5 species from Washington, San Juan Island (Hammerstrom et al., 1998), and Australian Ecklonia radiata (Lüder and Clayton, 2004), but did not induce a response in other kelps (Steinberg, 1994; Martínez, 1996; Toth and Pavia, 2002). In general, phlorotannins occur in high concentrations (>2% DW) in temperate brown macroalgae from Australasia (Estes and Steinberg, 1988), but they are found in smaller amounts in North American species belonging to the same order (Steinberg, 1989; Hay and Steinberg, 1992). Studies on the ecological roles of these compounds are therefore better explored in temperate rather than tropical environments. To our knowledge, no previous papers studied either the variation in contents of these compounds or the wounding effects in tropical brown macroalgae, where grazing pressure is constant and intense, and where strongly selecting species are chemically defended (Pereira and Da Gama, 2008). The hypotheses tested in the present study are as follows: (1) Artificial damage simulating herbivory induces a higher phlorotannin concentration in this brown macroalga; (2) phlorotannins in Stypopodium zonale vary with epibiosis level; and (3) there is a natural variation of phlorotannins in different individuals of S. zonale from the same population. 2. Materials and methods 2.1. Study site and organisms The tropical brown macroalga S. zonale (J. V. Lamouroux) Papenfuss is found in abundance along the sublittoral of the Brazilian coast (Menezes-Széchy and de Paula, 2010). Specimens of this macroalga were collected at Praia do Forno, Armac¸ão de Búzios (22◦ 45 S, 41◦ 52 W, Southeastern Brazil, Rio de Janeiro), in shallow waters about 3 m deep. In February 2011, 40 adult S. zonale individuals with no signs of herbivore attack or excessive epibiosis were collected by hand during free diving and transported to the Chemical Ecology Laboratory at Universidade Federal Fluminense. All individuals were collected with thallus intact, including holdfast, and there was no physical damage on them. From these 40 individuals, 10 were carefully cleaned to remove epibionts and kept in aquaria for 3 days to acclimatize to laboratory conditions for further use in the simulated herbivory experiment. The remaining 30 individuals were separated by their distinct levels of epibiosis, then cleaned and frozen for later lyophilization. During another collection event in January 2012, 32 adult S. zonale individuals were collected at the same site for evaluation of the intra-populational phlorotannin contents. The same collection procedure was used to avoid damage to the thallus. The samples from this collection were transported to the laboratory, where they were carefully cleaned and immediately frozen for further lyophilization. 2.2. Extraction After lyophilization, S. zonale specimens were ground into powder and submitted to extraction of phlorotannins. To maximize the extraction of these chemicals, lipids were removed from the samples prior to the extraction using 1 mL hexane for 3 min, 3 times, as
described previously (Koivikko et al., 2007). The extraction used 10 mL acetone:water (7:3) for 100 mg of alga (dry weight) over 2 h under magnetic agitation, centrifuged for 10 min at 3500 rpm and filtered. After removal of the acetone at room temperature, an aqueous residue that was centrifuged for 10 min at 3500 rpm was obtained; volume was measured, and the residue was frozen for analysis of phlorotannin content. 2.3. Quantification of phlorotannins In order to quantify the phlorotannin content in S. zonale, we used the Folin–Ciocalteau (FC) method, where the FC reagent 1 N (Sigma–Aldrich) was added to an aliquot of diluted extract, and after 3 min, sodium carbonate 20% and water were added to the solution. The reaction was kept in the dark for 45 min, after which phlorotannins were quantified in a spectrophotometer Schimadzu UV1800 at 750 nm using a standard calibration curve obtained with phloroglucinol (r2 = 0.99). Three aliquots of each extract were prepared and analyzed. 2.4. Induction of phenolic production by simulated herbivory To understand the effect of herbivory on the phlorotannin content, we simulate herbivory in 10 S. zonale individuals kept in aquaria after collection for acclimatization to lab conditions (salinity = 35 + 1; temperature = 20 + 1 ◦ C). Artificial damage simulated attacks of herbivores. After 3 days, each S. zonale individual was divided in two fragments, each possessing about 15.0 g wet weight – one control fragment and one treatment. Both the control and treatment suffered one cut each, so any possible effect of this damage was the same in both. Using the same individual in treatment and control allows us to affirm that observed differences in phlorotannin contents would be only due to the treatment as there would be no genetic variation in phlorotannin content or tissue growth. The treatment fragments were submitted to simulate herbivory by removing 10 small circles (5 mm diameter) with a metal tube from the thallus of S. zonale. The injury was as similar as possible to a grazer’s damage (e.g., gastropods). After 2 and 4 days from the clipping event, one piece of both the control and treatment algae were removed for quantification of phlorotannins. 2.5. Epibiosis and phlorotannin content S. zonale individuals were separated according to level of epibionts living on the thallus. We visually established three distinct levels of epibiosis: low (less than 10% of the thallus covered by epibionts), medium (between 10 and 30%), and high (above 30%). Epibionts were not identified at the species level, but they were primarily determined as bacterial biofilm, polychaetes, algae, bryozoans, and hydrozoans. The S. zonale samples were cleaned to remove epibionts, lyophilized, and ground into powder. The powder from the individuals exposed to the same level of epibiosis were joined, homogenized, and extracted (n = 6 extracts per level). Thus, the phlorotannin content corresponded to algal tissue from both thallus parts (holdfasts and blades) of the different S. zonale individuals. 2.6. Statistical analysis The intrapopulational variability was analyzed by histogram distribution of the phlorotannin concentration in the different S. zonale individuals. The results of the simulated herbivory experiments were compared day-by-day with dependent t-tests as control and treatment fragments were obtained from the same individual, characterizing dependence within treatments. Content
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0,7
0,8 0,7 0,6 0,5
Unclipped Clipped
0,4 0,3 0,2 0,1 Day 2
Day 4
Fig. 1. Total phlorotannin content (expressed as % per dry weight) of S. zonale. unclipped (gray bars) and clipped (black bars) in the simulated herbivory experiment (mean + standard deviation). On day 2, “*” refers to significant differences between clipped and unclipped (n = 10; p = 0.038; dependent t-test). On day 4, no significant differences were observed (n = 10; p = 0.25; dependent t-test).
Total phenolic content (% DW)
Total phenolic content (% DW)
0,9
0
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Low 0,6
Medium High
0,5 0,4 0,3 0,2 0,1 0
Levels of epibiosis
Fig. 2. Total phlorotannin content (expressed as % per dry weight) of S. zonale. possessing low (light gray bar), medium (dark gray bar), and high (black bar) levels of epibiosis (mean + standard deviation). The “*” refers to significant difference (n = 6; p = 0.0006; ANOVA).
of phlorotannins and levels of epibiosis were compared by one-way ANOVA. We considered the level of significance to be 5% (˛ = 0.05). 3. Results 3.1. Induction of phlorotannin content in S. zonale by simulated herbivory After 2 days of the clipping procedure, we observed a significant difference in phlorotannin content among control and treatment S. zonale individuals; specimens submitted to damage had significantly higher phlorotannin content than corresponding control individuals (n = 10; p = 0.038; t = −2.31; dependent t-test, Fig. 1). However, we observed an opposite non-significant trend (0.65% in control individuals and 0.50% in treatment, Fig. 1) on day 4. 3.2. Levels of epibiosis and phlorotannin content in S. zonale When the phlorotannin content of S. zonale with different levels of epibiosis were compared, we observed that the specimens of these algae exhibiting a high level of epibiosis had significantly lower amounts of these chemicals (0.368 + 0.022%; p < 0.0001; F = 12.79; ANOVA). However, S. zonale individuals possessing low and medium levels of epibionts were not significantly different in phlorotannin contents (0.540 + 0.051% and 0.526 + 0.098%, respectively; Fig. 2). 3.3. Intrapopulational content of phlorotannins in S. zonale The mean concentration of phlorotannins from 32 S. zonale individuals analyzed was 1.74%, but it varied from 0.63 to 3.24% of algal dry weight (Fig. 3). Despite the wide range of phlorotannin concentration, more than half of the individuals analyzed had values of this chemical around the mean (within 1.4–1.8% of phenolic DW). 4. Discussion 4.1. Induction of phlorotannin content in S. zonale by simulated herbivory Several simulated herbivory experiments have already investigated the effects on phlorotannin concentration (e.g., Yates and
Fig. 3. Intrapopulation variability of phlorotannin concentrations in S. zonale individuals from Praia do Forno, Armac¸ão dos Búzios, RJ, Brazil. Values are expressed in % per dry weight.
Peckol, 1993; Halm et al., 2011), but none involved tropical macroalgae. In this study, we detected higher phlorotannin content in specimens of S. zonale submitted to simulate herbivory when compared with uninjured algae 2 days after wounding. Rapid phlorotannin induction was already described for Agarum fibriatum, Pleurophycys gardneri, Laminaria complanata, and L. groenlandica, which showed higher phenolic content 1–3 days after wounding (Hammerstrom et al., 1998). However, 4 days after the clipping effect, we observed a drastic reduction of phlorotannin content in injured S. zonale. Hammerstrom et al. (1998) also reported rapid relaxation of phlorotannin induction after 7 days, so the relaxation in S. zonale can be considered even faster. This seems an interesting result because faster relaxation after an herbivore attack may be an advantage in tropical environments, where herbivore activity is not seasonally predictable. Mechanical or artificial damage have been used as simulations or substitutes for natural herbivory, but sometimes it fails as only the damage to the tissue itself is represented (e.g., Pavia and Toth, 2000), or produces contradictory results, as observed in Fucus vesiculosus, by decreasing palatability (Hemmi et al., 2004) rather than changing it (Rohde et al., 2004), or even increasing the
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susceptibility to herbivory (Haavisto et al., 2010). However, in this experiment, clipping was sufficient to evidence induced defense in the tropical macroalga S. zonale. S. zonale is known by the production of mixed-origin diterpenes (compounds exhibiting terpenoidic and phenol portions) as a chemical defense against herbivores (e.g., Littler et al., 1986; Hay et al., 1988), including specimens from Brazilian littoral (Pereira et al., 2004). Why does S. zonale produce terpenes as constitutive and polyphenols as inducible defenses? One explanation for the phlorotannin induction is that it is related to the healing process of the wound, and not an anti-herbivore response, as showed by Lüder and Clayton (2004). Further studies will be necessary to investigate the existence of a possible trade-off between biogenetically related compounds (phenolics and mixed-origin diterpenes). This process or change may be also affected by known metabolic turnover (Arnold and Targett, 2000). 4.2. Levels of epibiosis and phlorotannin content in S. zonale The S. zonale individuals exhibiting a high degree of epibiosis – i.e., with more than 30% of the thalli covered by epibionts – had a lower phlorotannin concentration than individuals exhibiting fewer epibionts. At first, this relationship between phlorotannin content and epibiosis on S. zonale could reinforce the experimental evidence that phenolics from brown macroalgae exhibit high antifouling activity. As UV-radiation can also be an inducing factor for phlorotannins (see Cruces et al., 2013), our results could be also due to higher UV absorption on the thallus of individuals with low and medium epibiosis levels, as the high level of epibiosis could decrease the absorption of UV radiation. Although phlorotannins of brown macroalgae have been previously proposed as antifoulants (Sieburth and Conover, 1965), they are water-soluble compounds. Unlike lipophylic terpenoids, they would be less effective defenses against epiphytes (Jennings and Steinberg, 1997). Indeed, the responses of phlorotannin concentration to epibiosis level conflict. For example, a decrease in growth and increase in fouling were observed in F. vesiculosus, but levels of epibiosis were not correlated to phenolic concentrations (Honkanen and Jormalainen, 2005). Phlorotannin concentration and fouling resistance were positively correlated for F. vesiculosus (Jormalainen et al., 2008). However, no such correlation was found for S. vulgare (Plouguerné et al., 2012); instead, there was a rise in antifouling activity accompanied by a drop in total phenolic content. 4.3. Intrapopulational content of phlorotannins in S. zonale We observed broad intrapopulational variability in phlorotannin content in S. zonale individuals. In general, the concentration of phlorotannins in brown macroalgae is extremely variable based on scales, such as time-days to months (see Jormalainen and Honkanen, 2008), between cell layers to within-thallus (e.g., Pereira and Yoneshigue-Valentin, 1999), and geographic (Steinberg, 1989; Targett et al., 1992; Van Alstyne et al., 1999). Because the efficacy of phlorotannins from brown macroalgae as a defensive compound is often concentration-dependent (Pereira and Yoneshigue-Valentin, 1999), and higher concentrations (more than 2.0% of dry weight) are active against herbivores (Ragan and Glombitza, 1986), it is likely that only 25% of the studied S. zonale population could be chemically defended from consumers (i.e., only those individuals possessing more than 2.0% of phlorotannins). But why do S. zonale individuals belonging to the same population produce distinct chemical defense concentrations? Why do all populations not produce large amounts of defensive chemicals against herbivory? For plants, the diversity of chemical phenotypes has been explained by a combination of genetic (Berembaum and Zangerl, 1992), developmental (Bowers and Stamp, 1993),
and environmental (Agrell et al., 2000) factors. However, due to peculiar characteristics of the macroalgae, we can presuppose that life history (Vergés et al., 2008), age structure (Paul and Van Alstyne, 1988b), environmental heterogeneity (Matlock et al., 1999), and limitation on gene flow among the natural population of macroalgae (Faugeraon et al., 2004) can determine the variability and the spatial structure of the secondary chemistry. Despite the known possibility of ontogenetic variability in the production of defensive chemicals in macroalgae (e.g., Paul and Van Alstyne, 1988a), we believe that this is not the reason for the high variability in phlorotannin contents found in the S. zonale individuals studied because only adult specimens were analyzed. However, S. zonale possesses alternating isomorphic life-stages that can be found simultaneously in the field, and this aspect remains a possible interference or explanation of the variable contents of phlorotannins since we may have collected both gametophytes and sporophytes as they are morphologically equal. A new study analyzing individual conditions in the field, like sun exposure, neighboring organisms, and depth could elucidate the reasons for such high intrapopulational variability. 5. Conclusions Phlorotannin content in S. zonale showed intra-populational variability and was higher in individuals submitted to simulated herbivory 2 days after the damage, but after 4 days, there was no difference. Phlorotannins also varied according to epibiosis cover, occurring in lower concentrations in individuals with higher level of epibiosis. Further studies are needed to cast more light on the causes of intra-populational variability in chemical defense; why S. zonale, a known producer of defensive terpenoids, would also produce polyphenols as induced defense after mechanical damage; and the effect of epibiosis on phlorotannins. Acknowledgments The authors thank CNPq and FAPERJ for research grants. GA also thanks CAPES for a doctoral scholarship. We also thank Daneila B. Sudatti, Leonardo M.S. Lima and Ricardo Rogers for help in algae collecting and designing experiments. References Agrell, J., Mcdonald, E.P., Lindroth, R.L., 2000. Effects of CO2 and light on tree phytochemistry and insect performance. Oikos 88, 259–272. Amsler, C.D., Fairhead, V.A., 2006. Defensive and sensory chemical ecology of brown algae. Adv. Bot. Res. 43, 1–91. Amsler, C.D., McClintock, J.B., Baker, B.J., 2008. Macroalgal chemical defenses in polar marine communities. In: Amsler, C.D. (Ed.), Algal chemical ecology. SpringerVerlag, Berlin/Heidelberg, pp. 91–103. Arnold, T.M., Targett, N.M., 2000. Evidence for metabolic turnover of polyphenolics in tropical brown algae. J. Chem. Ecol. 26, 1393–1410. Berembaum, M.R., Zangerl, A.R., 1992. Genetics of physiological and behavioral resistance to host furanocoumarins in Parsnip webworm. Evolution 46, 1373–1384. Boettcher, A.A., Targett, N.M., 1992. Role of polyphenolic molecular size in reduction of assimilation efficiency in Xiphister mucosus. Ecology 74, 891–903. Bowers, M.D., Stamp, N.E., 1993. Effects of plant age, genotype, and herbivory on Plantago performance and chemistry. Ecology 74, 1778–1791. Cruces, E., Huovinen, P., Gómez, I., 2012. Phlorotannin and antioxidant responses upon short-term exposure to UV radiation and elevated temperature in three South Pacific kelps. Photochem. Photobiol. 88, 58–66. Cruces, E., Huovinen, P., Gómez, I., 2013. Interactive effects of UV radiation and enhanced temperature on photosyntesis, phlorotannin induction and antioxidant activities of two sub-Antartic brown algae. Mar. Biol. 160, 1–13. Dayton, P.K., 1985. Ecology of kelp communities. Ann. Rev. Ecol. Syst. 16, 215–245. Estes, J.A., Steinberg, P.D., 1988. Predation, herbivory, and kelp evolution. Paleobiology 14, 19–36. Faugeraon, S., Martinez, E.A., Correa, J.A., Cardenas, L., Destombe, C., Valero, M., 2004. Reduced genetic diversity and increased population differentiation in peripheral and overharvested populations of Gigartina skottsbergii (Rhodophyta, Gigartinales) in southern Chile. J. Phycol. 40, 454–462.
G. Ank et al. / Aquatic Botany 111 (2013) 125–129 Geiselmann, J.A., McConnell, O.J., 1981. Polyphenols in brown algae Fucus vesiculosus and Ascophyllum nodosum: Chemical defenses against the marine herbivorous snail, Littorina littorea. J. Chem. Ecol. 6, 1115–1133. Haavisto, F., Välikangas, T., Jormalainen, V., 2010. Induced resistance in a brown alga: phlorotannins, genotypic variation and fitness costs for the crustacean herbivore. Oecologia 162, 685–695. Halm, H., Lüder, U.H., Wiencke, C., 2011. Induction of phlorotannins through mechanical wounding and radiation conditions in the brown macroalga Laminaria hyperborea. Eur. J. Phycol. 46, 16–26. Hammerstrom, K., Dethier, M.N., Duggins, D.O., 1998. Rapid phlorotannin induction and relaxation in five Washington kelps. Mar. Ecol. Prog. Ser. 165, 293–305. Hay, M.E., Steinberg, P.D., 1992. The chemical ecology of plant–herbivore interactions in marine versus terrestrial communities. In: Rosenthal, G., Berembaum, M. (Eds.), Herbivores: their interaction with secondary plant metabolits: ecological and evolutionary processes. Academic Press, New York, pp. 372–414. Hay, M.E., Duffy, J.E., Fenical, W., 1988. Seaweed chemical defenses: among compound and among-herbivore variance. In: Proc. 6th Int. Coral Reef Congr., vol. 3, pp. 43–48. Hemmi, A., Honkanen, T., Jormalainen, V., 2004. Inducible resistance to herbivory in Fucus vesiculosus – duration, spreading and variation with nutrient availability. Mar. Ecol. Prog. Ser. 273, 109–120. Honkanen, T., Jormalainen, V., 2005. Genotypic variation in tolerance and resistance to fouling in the brown alga Fucus vesiculosus. Oecologia 144, 196–205. Jennings, J.G., Steinberg, P.D., 1997. Phlorotannins versus other factors affecting epiphyte abundance on the kelp Ecklonia radiata. Oecologia 109, 461–473. Jormalainen, V., Honkanen, T., 2008. Macroalgal chemical defenses and their roles in structuring temperate marine communities. In: Amsler, C.D. (Ed.), Algal chemical ecology. Springer-Verlag, Berlin/Heidelberg, pp. 57–89. Jormalainen, V., Wikström, S.A., Honkanen, T., 2008. Fouling mediates grazing: intertwining of resistance to multiple enemies in the brown alga Fucus vesiculosus. Oecologia 155, 559–569. Koivikko, R., Loponen, J., Pihlaja, K., Jormalainen, V., 2007. High-performance liquid chromatographic analysis of phlorotannins from the brown alga Fucus vesiculosus. Phytochem. Anal. 18, 326–332. Kubanek, J., Lester, S., Fenical, W., Hay, M.E., 2004. Ambiguous role of phlorotannins as chemical defenses in the brown alga Fucus vesiculosus. Mar. Ecol. Prog. Ser. 277, 79–93. Lau, S.C.K., Qian, P.Y., 1997. Phlorotannins and related compounds as larval settlement inhibitors of the tube-building polychaete Hydroides elegans. Mar. Ecol. Prog. Ser. 159, 219–227. Littler, M.M., Taylor, P.R., Littler, D.S., 1986. Plant defense associations in the marine environment. Coral Reefs 5, 63–71. Lüder, U.H., Clayton, M.N., 2004. Induction of phlorotannins in the brown alga Ecklonia radiata (Laminariales, Phaophyta) in response to simulated herbivory – the first microscopic study. Planta 218, 928–937. Martínez, E.A., 1996. Micropopulation differentiation in phenol content and susceptibility to herbivory in the Chilean kelp Lessonia nigrescens (Phaeophyta, Laminariales). Hydrobiologia 327, 205–211. Maschek, J.A., Baker, B.J., 2008. The chemistry of algal secondary metabolism. In: Amsler, C.D. (Ed.), Algal chemical ecology. Springer-Verlag, Berlin/Heidelberg, pp. 1–24. Matlock, D.B., Ginsburg, D.W., Paul, V.J., 1999. Spatial variability in secondary metabolite production by the tropical red alga Portieria hornemannii. Hydrobiologia 399, 267–273. Menezes-Széchy, M.T., de Paula, J.C., 2010. Phaeophyceae. In: Forzza, R.C. (Ed.), Catálogo de plantas e fungos do Brasil, vol. 1. Andrea Jakobsson Estúdio/Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, Rio de Janeiro, pp. 404–408. Paul, V.J., Van Alstyne, K.L., 1988a. Chemical defense and chemical variation in some tropical Pacific species of Halimeda (Chlorophyta, Halimedaceae). Coral Reefs 6, 263–270. Paul, V.J., Van Alstyne, K.L., 1988b. Use of ingested alga diterpenoids by Elysia halimedae Macnae (Opisthobranchia: Ascoglossa) as antipredator defenses. J. Exp. Mar. Biol. Ecol. 119, 15–29.
129
Pavia, H., Toth, G., 2000. Inducible chemical resistance to herbivory in the brown seaweed Ascophyllum nodosum. Ecology 81, 3212–3225. Pavia, H., Cervin, G., Lindgren, A., Aberg, P., 1997. Effects of UV-B radiation and simulated herbivory on phlorotannins in the brown alga Ascophyllum nodosum. Mar. Ecol. Prog. Ser. 157, 139–146. Pereira, R.C., Da Gama, B.A.P., 2008. Macroalgal chemical defenses and their roles in structuring tropical marine communities. In: Amsler, C.D. (Ed.), Algal chemical ecology. Springer-Verlag, Berlin/Heidelberg, pp. 25–55. Pereira, R.C., Yoneshigue-Valentin, Y., 1999. The role of polyphenols from tropical brown alga Sargassum furcatum on the feeding by amphipod herbivores. Bot. Mar. 42, 441–444. Pereira, R.C., Soares, A.R., Teixeira, V.L., Da Gama, B.A.P., Villac¸a, R., 2004. Variation in chemical defenses against herbivory in Southwestern Atlantic Stypopodium zonale (Phaeophyta). Bot. Mar. 47, 202–208. Plouguerné, E., Hellio, C., Cesconetto, C., 2012. Within-thallus variation in polyphenolic content and antifouling activity in Sargassum vulgare. J. Appl. Phycol. 24, 1629–1635. Plouguerné, E., Hellio, C., Cesconetto, C., Thabard, M., Mason, K., Véron, B., Pereira, R.C., Da Gama, B.A.P., 2010. Antifouling activity as a function of population variation in Sargassum vulgare from the littoral of Rio de Janeiro (Brazil). J. Appl. Phycol. 22, 717–724. Ragan, M.A., Glombitza, K.W., 1986. Phlorotannins, brown algal polyphenols. In: Round, F.E., Chapman, D.J. (Eds.), Progress in Phycological Research. Biopress, Bristol, pp. 129–241. Rohde, S., Molis, M., Wahl, M., 2004. Regulation of anti-herbivore defence by Fucus vesiculosus in response to various cues. J. Ecol. 92, 1011–1018. Schoenwaelder, M.E.A., Clayton, M.N., 1999. The presence of phenolic compounds in isolated cell walls of brown algae. Phycologia 38, 161–166. Sieburth, J.M., Conover, J.T., 1965. Sargassum tannin, an antibiotic which retards fouling. Nature 208, 52–53. Steinberg, P.D., 1989. Biogeographical variation in brown algal polyphenolics and other secondary metabolites: comparison between temperate Australasia and North America. Oecologia 78, 373–382. Steinberg, P.D., 1994. Lack of short-term induction of phlorotannins in the Australasian brown algae Ecklonia radiata and Sargassum vestitum. Mar. Ecol. Prog. Ser. 112, 129–133. Steinberg, P.D., Van Altena, I.V., 1992. Tolerance of marine invertebrate herbivores to brown algal phlorotannins in Temperate Australasia. Ecol. Monogr. 62, 189–222. Swanson, A.K., Druehl, L.D., 2002. Induction, exudation and the protective role of kelp phlorotannins. Aq. Bot. 73, 241–253. Targett, N.M., Arnold, T.M., 1998. Predicting the effects of brown algal phlorotannins on marine herbivores in tropical and temperate oceans. J. Phycol. 34, 195–205. Targett, N.M., Arnold, T.M., 2001. Effects of secondary metabolites on digestion in marine herbivores. In: McClintock, J.B., Baker, B.J. (Eds.), Marine chemical ecology. CRC Press, Boca Raton, pp. 391–411. Targett, N.M., Coen, L.D., Boettcher, A.A., Tanner, C.E., 1992. Biogeographic comparisons of marine algal polyphenols: evidence against latitudinal trend. Oecologia 89, 464–470. Toth, G.B., Pavia, H., 2002. Lack of phlorotannin induction in the kelp Laminaria hyperborea in response to grazing by two gastropod herbivores. Mar. Biol. 140, 403–409. Van Alstyne, K.L.K.L., Pelletreau, K.N., 2000. Effects of nutrient on growth and phlorotannin production in Fucus gardineri embryos. Mar. Ecol. Prog. Ser. 206, 33–43. Van Alstyne, K.L., McCarthy III, J.J., Hustead, C.L., Duggins, D.O., 1999. Geographic variation in polyphenolic levels of Northeastern Pacific kelps and rockweeds. Mar. Biol. 133, 371–379. Vergés, A., Paul, N.A., Steinberg, P.D., 2008. Sex and life-history stage alter herbivore responses to a chemically defended red alga. Ecology 89, 1334–1343. Wikström, S.A., Pavia, H., 2004. Chemical settlement inhibition versus postsettlement mortality as an explanation for differential fouling of two congeneric seaweeds. Oecologia 138, 223–230. Yates, J.L., Peckol, P., 1993. Effects of nutrient availability and herbivory on polyphenolics in the seaweed Fucus vesiculosus. Ecology 74, 1757–1766.